École Sigle

Doctorale

Renée El

Melhem Bât

Pascal Blaise

R Gourdon

M Stéphane

ÉLECTROTECHNIQUE E E A Électronique

M Gérard Scorletti
email: gerard.scorletti@ec-lyon.fr

M Fabrice

Mme Emmanuelle
email: emmanuelle.canet@univ-lyon1.fr

Étage

M Luca

Jean-Yves Buffière
email: jean-yves.buffiere@insa-lyon.fr

M Philippe Boisse

Viviane Polsinelli
email: viviane.polsinelli@univ-lyon2.fr

Brigitte Dubois

J Y Toussaint

M Christian Montes

father, Benjamin Gustavo Emilio

Esteban Emilio

Keywords: apatite, bone substitutes, calcium phosphates, chitosan, composites, gelation, hardystonite, physical hydrogel, 3-D printing apatite, phosphate de calcium, chitosane, composites, gélification, hardystoniste, hydrogel physique, impression 3-D, substituts osseux

Bone substitutes, an approach to attend social demand for bone healing and reparation, are temporary replacements of bone tissue, promote bone formation and growth and finally are bioresorbed. No single material meets these requirements; an alternative is a bioinspired composite material. The objective of this thesis was thus to study the synthesis and properties of two bioceramics/biopolymer composites: chitosan physical hydrogels mineralized with apatite and hardystonite scaffolds impregnated with chitosan physical hydrogels. To obtain the first material, two strategies were developed. The first one consisted in the fabrication of chitosan physical hydrogels and its subsequent mineralization with apatite; the formation of microcapillaries occurred under particular synthesis conditions, and apatite precipitates were found only on the surface of hydrogels. The second strategy consisted in a simultaneous conversion of chitosan-calcium phosphate suspensions into chitosan-apatite hydrogels. The suspensions were prepared by sequential or simultaneous mixing of calcium and phosphate suspensions with chitosan solutions. Smaller and more uniformly distributed mineral aggregates were formed following sequential mixing, attributed to higher homogeneity, lower viscosity and no-presence of chitosan. This enabled the use of these chitosan-calcium phosphate suspensions as inks for 3-D printing. In general, three factors impacted the mechanical properties of mineralized chitosan hydrogels: the base used for gelation (determining the gelation rate: a higher rate preserved chain entanglement, resulting in higher elasticity); the density of physical crosslinks (hence a higher storage modulus) and the ionic strength (that led to chitosan chain disentanglements, thus, low storage modulus). Chitosan hydrogels and mineralized hydrogels were not cytotoxic, having no deleterious effects on osteoblasts proliferation. To fabricate the second material, pre-ceramic ink was 3-D printed and then sintered to form crystalline hardystonite ceramic. Hardystonite scaffolds were impregnated with chitosan solution that was, next, converted to chitosan physical hydrogel. At higher chitosan concentration, viscosity of solution was higher and scaffold impregnation was lower. At higher gelation rate, which depend on base used for gelation, lower weight loss during gelation. Chitosan hydrogel partially filled the pores contributing to bearing of external loads and to energy dissipated by fracture.

i v

Résumé

Les substituts osseux synthétiques représentent une approche pour répondre à la demande actuelle pour la guérison et la réparation osseuse. Ils servent au remplacement temporaire des tissus osseux, favorisent la formation et la croissance et la survie de l'os et sont biorésorbables. Aucun matériau monophasé ne remplissant complètement ces exigences, un matériau composite bioinspiré est une alternative possible. L'objectif de cette thèse était par conséquent d'étudier la synthèse et les propriétés de deux composites biocéramiques/biopolymères : des hydrogels physiques de chitosane minéralisés avec de l'apatite, et une hardystonite architecturée imprégnée par des hydrogels physiques de chitosane. Afin d'obtenir le premier matériau, deux approches ont été développées. La première a consisté à fabriquer des hydrogels physiques de chitosane puis à les minéraliser avec de l'apatite ; la formation de microcapillaires se produit avec des conditions de synthèse spécifiques, et les précipités d'apatite ont été trouvés uniquement à la surface des hydrogels. La seconde approche consiste à convertir des suspensions homogènes contenant le phosphate de calcium et le chitosane en hydrogels de chitosane minéralisés par l'apatite. Les suspensions ont été préparées soit avec un mélange simultané (calcium, phosphate et chitosane sont ajoutés simultanément), soit avec des mélanges successifs de suspensions phosphates de calcium avec les solutions de chitosane. Des agrégats minéraux plus petits avec une distribution plus uniforme ont été formés avec la méthode des mélanges successifs. Cela est attribué à une meilleure homogénéité, une viscosité plus faible et l'absence de chitosane. De manière générale, trois paramètres influencent les propriétés mécaniques d'hydrogels de chitosane minéralisés : la base utilisée pour la gélification (déterminant la vitesse de gélification : une grande vitesse conserve l'enchevêtrement des chaînes, résultant en une meilleure élasticité) ; la densité de la réticulation physique (cela induit un module de conservation plus important) et la force ionique (qui mène au désenchevêtrement des chaînes de chitosane, donc, à un faible module de conservation). Cette compréhension a permis l'utilisation de ces suspensions de phosphate de calcium-chitosane en tant qu'encre pour l'impression 3-D. Les hydrogels de chitosane et les hydrogels minéralisés ne sont pas cytotoxiques, c'est-à-dire qu'ils n'ont pas d'effets néfastes sur la prolifération des ostéoblastes. Pour fabriquer le second matériau, une encre pré-céramique a été imprimée en 3D puis frittée pour former une céramique d'hardystonite cristalline. Les scaffolds d'hardystonite ont été imprégnés par la solution de chitosane, converties ensuite en hydrogels physiques de chitosane. A plus forte concentration de chitosane, la viscosité de la solution était plus grande et l'imprégnation de la matrice plus lente. Avec une vitesse de gélification plus importante, qui dépend de la base utilisée pour la gélification, la perte de poids est plus faible pendant la gélification. L'hydrogel de 1

Introduction

Bone trauma and disease are disorders of growing incidence especially in aged population and under circumstances of obesity and low physical activity in everyday life. Natural self-repair is the main and essential source of bone healing and is enough for small bone defects and cracks. However, some trauma, diseases and such symptoms as chronical pain, physical disability and esthetic problems, require clinical assistance oriented to replace or to favor bone regeneration and healing. To do so, implanting bone extracted from the patient himself would be the most efficient alternative, but it necessitates more than one surgical intervention. Cadaver bones have been used, but it causes problems with immunogenic reactions and the risk of acquiring transmissible diseases from tissues and fluids. Other materials, presenting advantages and limitations, have been used to replace bones: demineralized bone matrix, metals, polymers, ceramics, and composites. The complexity and social importance of bone substitution and healing has led to a growing research area, bone tissue engineering, which purpose is to combine materials, stem cells and bioactive molecules for bone regeneration and healing.

A promising approach is to develop bone substitutes bioinspired of bone structure and composition. A bone substitute would be a material that temporary replaces bone tissue, promotes bone formation and growth, until the material is finally bioresorbed. Hence, for a material to be a potential bone substitute, it should be non-toxic, biodegradable*, osteoconductive*, osteoinductive*, porous and mechanically stable until new bone formation is completed.

Since it is difficult to find a single material that meets all these requirements, an alternative is to combine materials with complementary properties, for example, natural polymers and bioceramics. The objective of this thesis is thus to study the synthesis and properties of ceramic/polymer composites for bone substitution, exploring a wide range of compositions (from 100% mineral to 100% organic), phase distributions (mineral phase as homogeneously dispersed particles in polymer matrix, or as a layer on the polymer matrix, or as a continuous phase impregnated with polymer) and architectures (composites cast in molds or shaped by 3-D printing techniques). To explore these various composites, different options to fabricate a ceramic-polymer material were investigated: (i) formation of a polymer matrix and its subsequent mineralization with a bioceramic; (ii) simultaneous obtaining of the polymer and the bioceramic phases; (iii) beginning with a bioceramic and impregnation by a polymer. Two types of bioceramics and one natural polymer were chosen for their biological properties: hardystonite and calcium phosphates as bioceramics and chitosan as a natural polymer.

-Hardystonite (Ca2ZnSi2O7) is biocompatible; it can be manufactured with a relatively high porosity to increase cell colonization and seems to stimulate in vitro mineralization processes. It presents bending strength and Young modulus close to those of human cortical bone. However, it remains a ceramic and as such is a brittle material. -Among calcium phosphate bioceramics, apatite has a composition and structure close to the inorganic part of extracellular matrix of bones. Apatite integrates with living organisms (osseointegration); it provides a bioactive surface to guide bone remodeling and bone growth (osteoconduction); and, it resorbs under appropriate physico-chemical conditions, allowing new bone growth.

-Chitosan is a non-toxic, bioactive* and a biodegradable natural polymer with high potential of physical and chemical interactions due to its chemical structure. This makes chitosan an easy-to-modulate material with remarkable biological properties. Disadvantages come from poor mechanical properties of chitosan-based materials. Here, chitosan was used in the form of physical hydrogels. Choosing to work on a physical hydrogel allowed us to avoid the use of crosslinking agents that may be toxic for living media. 3D-printed hardystonite scaffolds were chosen as a basis for impregnation with chitosan hydrogel, due to its potentially good biological properties and mechanical properties higher than those of calcium phosphate scaffolds with similar architecture. Although there is some literature on ceramic scaffolds impregnated with chitosan, no research was yet conducted on hardystonite/chitosan composites. One objective of this thesis is the fabrication of hardystonite 3D constructs impregnated with chitosan physical hydrogels, focusing on the relation between the impregnation process, their microstructure and mechanical properties.

Calcium phosphates were chosen to fabricate mineralized chitosan physical hydrogels: indeed, the osteoconductivity of bioactive ceramics (especially hydroxyapatite) is well known; and the composite material could also benefit from the biological properties of chitosan (especially angiogenic* properties).

Chitosan-calcium phosphates composites have been prepared by other research groups as membranes, films, scaffolds, injectable materials, multilayers films and particles. Methods of synthesis have been reported and can be classified in: i) coating method using electrochemical and electrophoretic deposition techniques, favoring the deposition of chitosan and calcium phosphates on metal surfaces; ii) incorporation of chitosan (powder or solution) to calcium phosphate pastes to form calcium phosphates-chitosan cements; iii) mineralization of freeze-dried chitosan scaffold by immersion in solutions saturated with respect to apatite (e.g., simulated body fluid); iv) impregnation of sintered calcium phosphates scaffolds with chitosan solution, obtaining freezedried materials; and v) suspensions obtained by mixing chitosan solution with calcium phosphates particles.

The above-mentioned methods effectively lead to chitosan-calcium phosphates composite materials. However, a major concern is the lack of homogeneity at microscopic level, which induces practical inconveniences in industrial and medical applications. To overcome this drawback, methods of in-situ precipitation of calcium phosphates inside the polymeric matrix have been studied. Frequently, in the last step of in-situ precipitation method, composite material is freeze-dried or dried at room temperature for further use and characterization.

From the analysis of the already tested methods of preparation, it seems that an effort should be devoted to obtain a homogenous dispersion of apatite in a chitosan matrix. It is foreseeable that a hydrogel material may fulfill this objective because hydrogels may be formed from homogenous suspensions of chitosan-calcium phosphates. One objective of the thesis is thus to develop methods of synthesis of a chitosan physical hydrogel mineralized with apatite and to establish relations between synthesis method, chemical composition, microstructure, mechanical and biological properties of the composites, based on physico-chemical interactions between the present species within the system.

The manuscript is organized in three parts that contain six chapters in total, and it ends with general conclusions and outlooks. Chapters 2 to 6 (that describe the results obtained during this research work), were written in a format suitable for journal publication and may therefore contain a few redundant context statements.

Part I, named "Conceptual and experimental frameworks" has a threefold objective: i) to give the reader a necessary context of the research problem, ii) to be a fundamental basis and a support to guide the research to be performed and iii) to help to understand and explain the results of our research experiments. It contains two chapters. The first one, based exclusively on a critical literature review, describes structural and chemical features of the systems studied in this thesis. The second one, based on our experimental results, describes the physico-chemical interactions in the same systems.

Chapter I.1: Critical literature review. Specific topics investigated by critical review of scientific literature include: i) bone tissue composition, structure and properties, justifying the choice of a chitosan-apatite composite material as possible bone substitute; ii) structure and properties of chitosan and calcium phosphates (with an emphasis on apatite); iii) hydrogel properties and chitosan gelation process; iv) the physico-chemical interactions that may occur during the fabrication and in the final composite material. A review of basic aspects of hardystonite as a biomaterial is presented at the beginning of Chapter III.2.

Chapter I.2: Physico-chemical interactions in chitosan-calcium phosphates solutions and suspensions. The scope of this chapter is the study of physico-chemical interactions occurring in systems related with the fabrication of the composite material. The studied systems were: solutions of chitosan, mixed solutions of calcium or phosphate precursor salts with chitosan, and chitosan-calcium phosphates suspensions. The research strategy was to correlate the possible physico-chemical interactions acting in all these aforementioned systems with the nature and the concentration of components, their particle distribution and rheological properties.

Part II, named "Approaches of mineralization" exposes the methods of fabricating composite materials made of chitosan and apatite, keeping in mind two principal goals: i) to prepare materials that, besides preserving the combined biological properties of chitosan and apatite, meet a compromise between sophistication, ease of fabrication, ease of handling and low costs; ii) to investigate the physico-chemical interactions occurring in the systems during the process of obtaining the material. This part of the thesis is developed in two chapters.

Chapter II.1: Mineralization of physical hydrogels of chitosan with calcium phosphate apatite. This work was divided in two stages: first, the fabrication of a physical chitosan hydrogel by gelation with different bases and second, the mineralization of the hydrogel with calcium phosphate particles. In this work, it was intended to make profit of the microstructure within chitosan hydrogels to obtain an even distribution of mineral particles throughout the whole material. Four strategies were developed to mineralize a chitosan hydrogel with calcium phosphates.

Chapter II.2: One-shot synthesis of apatite-chitosan biocomposites: crystallization of apatite simultaneously to polymer gelation. The alternative developed in this chapter to prepare mineralized hydrogels of chitosan is the conversion of homogenous suspensions of calcium phosphate particles into chitosan solutions into hydrogels made of chitosan-apatite. In addition, different nature of salt solutions (sources of calcium phosphates), methods of preparation of chitosan-calcium phosphate suspensions and bases used for gelation were studied as variables that may influence the microstructure, physico-chemical interactions, rheological and biological properties of the resulting materials.

Part III "Architectured composite materials". Two additional fabrication processes based on additive manufacturing are described in this thesis: another method of preparing the target material, using robocasting (a 3D-printing method), and a 3D-printed sintered construct as the ceramic source to prepare a potential bone substitute (hardystonite-chitosan composite).

Chapter III.1: 3D printing of chitosan-calcium phosphate inks: rheology, interactions and characterization. This chapter describes and discusses the robocasting process applied to obtain architectured physical hydrogels of chitosan with intertwined apatite particles. Suspensions of chitosan-calcium phosphates were used as inks. As soon as the extrudate left the nozzle, it was deposited in a solution of alkaline pH, which induced simultaneous gelation of chitosan and recrystallization of calcium phosphates in the form of apatite. The rheological behavior of the inks was explained in terms of interactions between species present in the system.

Chapter III.2: Fabrication and characterization of architectured hardystonite-chitosan composite scaffolds. This chapter deals with the preparation and characterization of hardystonite-chitosan composite materials. The aim was to develop protocols to obtain hardystonite printed scaffolds impregnated with chitosan physical hydrogel and to characterize the effect of impregnation on mechanical properties of the final composite materials.

Finally, the manuscript ends with general conclusions and outlooks of this work.

General conclusions and outlooks. It gathers the main results of this study. This Ph.D. thesis was aimed to develop potential bone substitute materials consisting in organic-inorganic composites, inspired by the natural extracellular matrix of the bone. Two different types of materials were developed: mineralized physical hydrogels of chitosan with apatite and hardystonite scaffolds impregnated with physical hydrogels of chitosan. The relation between synthesis, structure and mechanical properties was investigated and discussed, based on physico-chemical interactions occurring in the different systems.

The scientific scenario presented in this thesis gives a basis to better control physico-chemical, mechanical and biological properties of the obtained bone substitutes.

Finally, outlooks are suggested, both to improve the synthesis methods and to complete the characterization of bone substitutes developed in this work.

My Ph.D. work was performed in two laboratories: Ingénierie des Matériaux Polymères (IMP, UMR CNRS 5223, Université Claude Bernard Lyon 1), and Matériaux: Ingénierie et Science (MATEIS, UMR CNRS 5510, INSA-Lyon). It was supported by the LABEX iMUST (ANR-10-LABX-0064) of Université de Lyon, within the program "Investissements d'Avenir" (ANR-11-IDEX-0007) operated by the French National Research Agency (ANR), and by a "Contrat Doctoral" from the French Ministère de l'Enseignement Supérieur et de La Recherche.

This thesis was highly enriched by collaborative research with Prof. E. Saiz's group (Imperial College London, UK) and Prof. P. Colombo's group (University of Padova, Italy). In particular, experimental work of chapter III.1 was performed at Imperial College London (UK), during a two months stay. It was partly funded by a trust from the Journal of European Ceramics Society. Experimental work described in chapter III.2 was done partially in France and partially at University of Padova, Italy.

Part I: Conceptual and experimental frameworks

Part I has a threefold objective: i) to give the reader a necessary context of the research problem, ii) to expose the fundamental bases that guided the performed research and iii) to provide arguments to support the discussion of the experimental results obtained during my Ph.D. thesis. Part I contains two chapters. The first one, based exclusively on a critical literature review, justifies the choice of the materials studied in this thesis and describes their structural and chemical features. The second chapter, based on our experimental results, investigates the physico-chemical interactions between the different phases of the composite materials under study.

La partie I de ce manuscrit a un objectif triple : elle doit i) présenter au lecteur le contexte de la recherche développée durant mon doctorat ; ii) exposer les bases fondamentales qui ont guidé cette recherche et iii) apporter les éléments nécessaires pour étayer la discussion des résultats décrits dans ce manuscrit.

Cette partie I contient deux chapitres. Le premier chapitre, basé sur une revue critique de la littérature, justifie le choix des matériaux étudiés et décrit leurs propriétés chimiques et structurales. Le deuxième chapitre est quant à lui basé sur des résultats expérimentaux obtenus durant ma thèse et porte sur les interactions physico-chimiques entre les différentes phases présentes dans les matériaux composites étudiés.

Chapter I.1

Critical literature review

Bone trauma and bone disease are disorders of growing incidence in population of all ages [1]. Natural self-repairing is the main and essential source of healing, sufficient for small bone defects and cracks, whereas larger trauma with defect above 1 cm 3 and chronical diseases (possibly associated with pain, physical disability and esthetic problems) require clinical assistance oriented to replace or to favor bone healing and remodeling*. Bone extracted from the same patient would be expected to be the safest alternative, but it has the inconvenience of more than one surgical intervention and limitation in the amount of graft. Other materials, presenting other advantages and limitations, have been used to replace bones or favor bone healing: demineralized bone matrix, metals, polymers, ceramics, and composites combining varied materials in search of synergistic effects. The complexity and social importance of bone substitution and healing has led to a growing research area, bone tissue engineering, whose purpose is to combine materials, stem cells and bioactive molecules for bone regeneration and healing [2,3]. Bone substitution is a promising perspective to contribute to bone regeneration. A bone substitute would consist in a synthetic material that temporarily replaces bone tissue, promotes bone formation and growth, until finally that material is biodegraded. In this context, the objective of the thesis is to develop a material with potential to be used as bone substitute. Selection of materials for bone substitution can be guided by mimicking bone structure and composition. Once the type of material has been chosen to establish a regeneration strategy, the next task at the laboratory level is material development that comprises the establishment of synthesis methods and the characterization of the resulting materials (microstructural, physical and biological properties).

Research in these areas has been very active during the last five decades, as reflected in the increasing scientific publications in the area. Some of these works were taken as references for this PhD research.

Bone tissue

Bone tissue forms the skeleton of vertebrates, which mainly serves functions of structural support to the body, locomotion, protection of internal organs, maintenance of acid-base buffers and ion homeostasis, entrapping of dangerous materials, and hematopoiesis [4][5][6]. Bone also serves as substrate for cartilage and the regeneration of cartilage is nowadays considered in the global idea of the regeneration of the osteo-chondral assembly.

Bone composition and structure

Bone is a connective tissue consisting of cells and a mineralized extracellular matrix (ECM). Approximately 10 % of bone volume is occupied by cells and blood vessels and the remaining 90 % volume, by ECM [4,7].

In addition to different osteogenic precursors, bone cells are of four specialized types, namely osteoblasts, osteocytes, osteoclasts and lining cells. Osteoblasts play a role in forming new bone surfaces by synthesizing and secreting inorganic and organic constituents of ECM. Osteocytes make about 90 % of bone cells and play a role in cell communication and nutrition (they may act as mechanical transducers to direct bone remodeling). Lining cells, thin and elongated, cover and protect most of the surface of bones and have also a role in the signaling of bone remodeling [8]. Osteoclasts are implied in the remodeling process by the function of bone resorption [4,6,7,9].

ECM is formed by two parts, organic and inorganic, ordered in a complex and multiscaled architecture. The organic part is composed of various types of collagen fibrils (mainly collagen of type I) secreted by osteoblasts that account for about 35 % of total weight of bone tissue. The collagen fibrils are assembled in a structure forming concentric rings, called osteons, or Haversian systems. This highly structured pattern results in a network of lacunae and canals that allow for fluids (blood) and ions flow, for lymph vessels and for nerves passing [6,10]. Between 10 to 15 % weight of the organic matrix is non-collagenous proteins (NCP), serum albumin, 2-HS-glycoprotein and trace amounts of many other molecules. In spite of their low content, NCP play an important role in bone cells proliferation, bone cells activity and the physiological process of calcium phosphate formation [6,11,12]. Besides NCP, collagen is active in matrix mineralization [13]. The remaining 65 % of total weight of bone tissue corresponds to the inorganic part composed of calcium phosphate, namely, nonstoichiometric hydroxyapatite*, including several minor elements or ions such as carbonate, 4-6 %; magnesium, 0.5 %; and sodium 0.9 % [6]. In this work, non-stoichiometric hydroxyapatite forms are called apatite. Apatite is the most frequently encountered calcium phosphate in mineralized tissues of vertebrates [12]. Bone presents broad X-ray diffraction peaks, indicating to be a "poorly crystalline" and nanometric structure. Accordingly, ECM can be imagined as a nanostructured array of apatite crystals embedded in a fibril structure of collagen. From the mechanical point of view, it is a system of collagen fibers matrix reinforced by apatite crystals filler.

As in all other tissues, there is a very interdependent relation between cells and ECM. Cells inherently have the capacity to perform specialized functions but only in the appropriate environment. Cells are sensitive to other cells through communication across the ECM, cells are sensitive to active components of ECM [14,15]. Therefore, it is of paramount importance to understand the composition and structure of ECM for the design of bone substitutes. In fact, the objective of this thesis could be rewritten as the advance in the fabrication of a suitable ECM for bone substitution, that is, a bone substitute with mechanical and biological properties that allow interaction with cells to emulate key molecular features of the bone.

Bone structure

Bone is not a homogeneous solid. The structure of bone may take two basic architectures: one is called cancellous, spongy or trabecular, and the second cortical or compact bone. Depending on the bone architecture, the pores inside the bone are filled with lymphatic vessels, marrow cells*, fat cells, nerves, blood vessels, Harversian canals* and osteocytes (Figure 1). [16].

Cancellous and cortical bones have the same ECM composition, but they differ in architecture and density. Cortical bone surrounds the marrow cavity and the trabecular plates of the cancellous bone. Cortical bone is denser than cancellous bone; with 10 % and 90 % porosity, respectively. Cancellous bone is more associated with metabolic functions, whereas cortical bone provides mechanical resistance to the bone [4,17]. The weight composition of adult human skeleton is 80 % cortical bone and 20 % trabecular bone overall [6,10]. Volume, size distribution and interconnection of pores make a porous structure allowing for cell growth, cell migration, nutrient flow, vascularization and ECM production [5,18,19].

Mechanical properties of the bone

Bone is a material of surprisingly high mechanical properties: it is a rigid, non-ductile, and tough material, as shown in the comparisons of mechanical properties of various tissues presented in Figure 2 and in Table 1.

Figure 2. Stress-strain behaviors of different tissues. Bone shows a rigid and non-ductile behavior.

Adapted from [20].

In Table 1, a comparison is shown of some mechanical properties of various bio-and non-biological materials. In this comparison, bone evidences a relative high toughness. Cortical bone 50-151 [21] 100-230 [22] 7-30 [23] 2-12 [23] Dental enamel 10-47 [24,25] 384 [25] 67-84 [25,25,26] 1-2 [27,28] Skin 22 [29] 0.05-0.01 [30] Cartilage 3-5 [31] 0.001-0.01 [31] Bioglass 42 [32] 500 [32] 35 [23] 2 [23] Hydroxyapatite 40-300 [22] 500-1000 [22] 80-120 [23] 0.6-1 [23] Alumina 270-500 [22] 3000-5000 [22] 380-410 [23] 5-6 [33] Measurements of mechanical properties of bone are widely reported in the literature [34][35][36][37][38][39]. However, it is evident that there is a large scattering in the values of bone properties. For example, Table 2 shows a range of stress at break values reported for human cortical bone.

Table 2. Reported values of human cortical bone resistance for different mechanical tests [34][35][36][37][38][39]. For a given mechanical analysis, most of dispersion in the measurements of bone mechanical properties can be explained by the facts that:

-Bone mechanical properties depend on the type of bone under investigation: cortical bone is much harder and more rigid than cancellous bone (Figure 3a). -Bone mechanical properties are subject-dependent: age-dependant (elder persons usually have more fragile bones see Figure 3b). Gender and health-state also have an influence. -In a same person, bone mechanical properties depend on the bone site. -Bone is not an isotropic, elastic and fragile material: it is anisotropic (Figure 4) and viscoelastic. These two points will be discussed more in hereafter.

Bone presents anisotropy, that is, mechanical properties change with the direction of application of load. Bone resistance is much higher in the longitudinal direction (parallel to the axis of long bones, or to the direction of osteons), as shown in Figure 4. This is a clear consequence of its ordered, oriented morphology. Moreover, bone is a viscoelastic material that may behave as an elastic rigid solid, as a ceramic, or may deform permanently (as a fluid), depending on temperature and applied loading rate. [40]. Comparison of stress-strain behavior of the young and the aged human (b). Adapted from [41].

Figure 3. Stress-strain schema showing different mechanical properties of trabecular and cortical bones, which have similar composition but different morphology (a). Adapted from

Figure 4. Anisotropic behavior of femur bone. Comparison of stress-strain behavior of bone at different load application direction. Adapted from [42].

Bone remodeling and healing

Bone remodeling and healing are processes of genetically-controlled development of bone by determining the production of enzymes and structural proteins that regulate signals to influence cell differentiation and activity [43].

i. Bone remodeling By remodeling, new bone is formed to replace the older, through a coupled process of resorption of old bone, performed by osteoclasts, and deposition of new bone, performed by osteoblasts. Remodeling begins before birth and continues during the whole life [4,6,43].

Bone remodeling requires the coordinated activity of four major types of bone cells: bone-lining cells, osteocytes, osteoclasts, and osteoblasts that comprise four overlapped phases (Figure 5) [44]: Phase 1: Initiation/activation of bone remodeling at a specific site; Phase 2: Bone resorption and recruitment of osteoblasts precursors called mesenchymal stem cells; Figure 5. Bone remodeling phases. Adapted from [45].

Phase 1 starts with the recruitment and activation of mononuclear monocytes-macrophages from the blood vessels that interact with bone lining cells [46]. Bone lining cells produce the monocyte/macrophage-colony stimulating factor (M-CSF) and the receptor activator of nuclear factor kB ligand (RANKL). These factors are responsible for the induction of formation of osteoclasts, called the osteoclastogenesis* [47,48]. Therefore, they act as osteoclast precursors leading to the expression of specific genes for formation of mature osteoclasts that activate resorption. The activation of osteoclasts undergoes internal structural changes such as the rearrangement of the acting cytoskeleton and the formation of the sealing zone [44]. The sealing zone area is tightly attached to the bone surface, separating the ruffled border area from the rest of the cell membrane [49].

Phase 2 begins with the formation of osteoclasts [44]. In bone resorption, osteoclasts attach to the bone surface and dissolve the inorganic phase, apatite, and degrade the organic phase, fibrillar collagen, of the bone matrix [46]. Dissolution of apatite is caused by the ability of osteoclasts to generate an acidic extracellular compartment (pH 3~4) between the cell and the bone surface. Six general steps happening simultaneously are described is this process (Figure 6): 1) osteoclasts produce acid intracellular vesicles that move to the ruffled border; 2) vesicles are fused to bone facing plasma membrane to acidify the extracellular matrix. At the same time, when acidic vesicles are fused to the plasma membrane, proton pumps are inserted to ruffled border so that they can continue to pump protons directly from the cytoplasm to the extracellular resorption lacuna; 3) the formation of protons in the cytoplasm is managed by carbonic anhydrase enzymes that catalyze the conversion of carbon dioxide and water to carbonic acid; 4) ion equilibrium in the cytoplasm is insured by the continuous production of protons and bicarbonate ions and by exchange of bicarbonate to chloride by HCO3 -/Cl - exchanger located at the basolateral membrane; 5) the organic (amino acids) and inorganic degradation (Ca 2+ , H2CO3, H3PO4, H2O) products are endocytosed from the ruffled border membrane; 6) afterwards, they are transported in vesicle-like structures through the cell to the plasma membrane domain and released from there into the extracellular space [50]. At this point osteoclasts can migrate towards another zone where resorption is needed or undergo apoptosis (programmed cell death).

Simultaneously with the resorption process, the recruitment of mesenchymal stem cells/osteoprogenitors is also initiated [44].

Figure 6. Cross-section of bone resorbing osteoclast. Schematic representation of the six steps (1)(2)(3)(4)(5)(6) happened during bone resorption. Adapted from [50].

Phase 3 starts as osteoblasts begin to synthesize the osteoid (non-mineralized collagen) and it overtakes bone resorption [44].

Phase 4 involves the mineralization of osteoid. Non-collagenous proteins modulate the formation of bone mineral consisting of crystals of apatite. Enzymes produce by osteoblasts, such as alkaline phosphatase, play an important role in the mineralization process providing the inorganic phosphate necessary for apatite crystallization. After that, osteoblasts can be either trapped in this new bone (becoming osteocytes) or flattened on the surface of the new bone to become bone lining cells [44].

ii. Bone healing By healing, a fractured or broken bone is repaired in the direction of restoring the physical, mechanical and functional conditions prevailing before fracture [51,52]. There are three distinct phases in the process of bone healing: reactive phase, reparative phase and remodeling phase (Figure 7). Adapted from [53].

In the reactive phase, hematoma, inflammatory response and recruitment (induced by signal molecules to regulate bone formation) occur. Blood vessels tear near the site of broken bone and hematoma is formed in clotted blood. Bone cells die of lack of nutrients. Inflammatory cells (macrophages, monocytes, lymphocytes, and polymorphonuclear cells) and fibroblasts (connective cell tissue) form granulation tissue; ingrowth of vascular tissue and migration of mesenchymal cells take place.

In the reparative phase, fibroblasts and osteoblasts enter the area and begin to form new bone. Fibroblasts produce collagen fibers and osteoblasts form spongy bone, which leads to the formation of a soft callus.

In the remodeling phase, excess of ECM is removed, the callus is remodeled by resorption performed by osteoclasts and mineralization is concomitantly performed by osteoblasts. This is a gradual process and it may take months to create bone similar to the original [5,54].

This research work is in the frame of bone healing which includes a bone healing in presence of a bone implant. Our objective is that it could induce/favor all the steps of Figure 7, including the remodeling step, in a physiological process.

Critical analysis of the literature

Once the context of this thesis is described, the question on what material system could be suggested as a bone substitute should be posed. On the basis of the bone composition and structure, bone mechanical properties and bone healing and remodeling, what properties or conditions should a material candidate meet for bone substitution?

A first and basic argument is that nature is in charge of the process of bone remodeling and healing. Cells have natural capability for self-organization, to form complex structures, under the appropriate conditions. Therefore, a general appropriate property of a bone substitute material is that it does not hinder the natural process, but provides a favorable environment for cell function, that is, for cells to live, grow and differentiate and produce their mineralized ECM.

On the other hand, bone substitute material should provide physical support for cells and morphology to favor vascularization and flow of biological fluids. Also, it could be useful that the material provides calcium and phosphate needed in the formation of apatite in the mineralization process.

Once the new bone is formed, the bone substitute material should degrade and be absorbed by the body without any toxicity.

In strict terms, the material with potential for bone substitution should be non-toxic, biodegradable*, osteoinductive*, osteoconductive*, porous and mechanically stable. Beside those basic properties, a compromise should be met between sophistication, ease of handling by the surgeon, ease of production, ease of placing on the market and costs [44].

Those kinds of properties have been addressed in literature [2,5,6,55]. Moreover, a growing number of recent publications propose several approaches, looking for a bone substitute that favors a higher rate of new bone formation: (i) combinations of materials to fulfill the requirements of an appropriate bone substitute; (ii) introduction of stem cells and (iii) bioactive molecules to mimic/favor the natural phenomena involved in tissue development [51,52,56,57].

Materials selection

The literature of bone tissue engineering offers plenty of options of materials under investigation as candidates for bone substitutes [2,5,58,59]. Therefore, the effort will be devoted to review the advantages and disadvantages that each material may present, to evaluate and justify the general options taken in the present work.

The optional materials could be classified as natural polymers, synthetic polymers, ceramics, metals and composite materials [60][61][62][63][64][65].

Natural polymers, such as collagen and chitosan, are originated from living organisms. Chitosan and collagen are non-toxic, bioactive* and biodegradable*. Disadvantages come from poor individual mechanical properties of chitosan or collagen-based materials and the possibility of immunogenic response [66][67][68].

Synthetic polymers, such as poly(lactic acid), poly(glycolic acid), polycaprolactone, poly(lactic-coglycolic acid), and others, can be synthesized in the laboratory by controlled processes to tailor their physical, biological and mechanical properties. However, the operations inherent to synthesis procedures, dissolution, separation, cleaning and remaining of non-reactive monomer, tend to intrinsically create inconveniences of toxicity and problems of non-biocompatibility [63,64].

Ceramics is a class of inorganic, solid materials, composed of metal, non-metal and metalloid atoms joined by ionic covalent bonds. Examples of interest in bone tissue engineering are calcium phosphates, hardystonite and bioglasses. These materials are bioactive*. Bioglasses such as silicate, phosphate and borate, show high bioactivity, both osteoconductivity* and osteoinductivity*. A drawback for ceramics is that they are usually brittle materials [60][61][62][63].

Since it is difficult to find a single material that meets all the appropriate properties of a bone substitute, an alternative is to combine materials, as nature does. Composite materials are the combination of two or more materials: natural polymer, synthetic polymer, metals, bioceramics. One important group of composite biomaterials is that composed of bioceramics -polymer composites.

There are many possibilities to choose a candidate material for bone substitute. But it is possible to pick up from the many possibilities trying to put together a material that meets appropriate properties.

A composite material composed of a natural polymer and calcium phosphate seems to be a good choice [4,5,7] since the original bone is composed the same way. Apatite, whose structure and composition are not very different from that of the mineral part of bone, may enhance the osteoconductivity* of the polymer and provide better mechanical behavior. It may be better to choose apatite rather than hydroxyapatite to provide a material of closer structure to that found in the bone.

As far as natural polymers are concerned, chitosan and collagen are good candidates for this work.

We have chosen chitosan, in principle able to provide angiogenicity* to the composite material.

Chitosan (CS) 1.3.1 Structure and origin

Chitosan is a linear copolysaccharide composed of N-acetyl glucosamine and glucosamine repeating units joined by beta(14) glycosidic bonds. This polysaccharide is mainly obtained by Ndeacetylation of chitin, the second most abundant natural polymer after cellulose. Chitin is found in fungi, mushrooms cell walls, crabs, shrimps, and lobsters cuticles. The main production of chitosan comes from marine crustacean shells wastes [69,70]. The only structural difference between chitosan and chitin is the degree of acetylation (DA) defined as the molar fraction of N-acetyl glucosamine units (Figure 8). As a general rule, the polymer is called chitosan if its degree of acetylation is below 60 % [71]. This value of DA is in fact more rigorously related to the solubility behavior of polymers in aqueous solutions. Chitosan is soluble in weakly acidic aqueous medium, while chitin is not.

Figure 8. N-acetyl glucosamine and glucosamine joined by beta 1->4 glycosidic bond. N-acetyl glucosamine has two hydroxyl groups,-OH; and one N-acetyl group:-NH-C(CH3)=O. Glucosamine

has two hydroxyl groups:-OH, and one free amine group -NH2 that can be protonated in acidic conditions (pH<6.2) Adapted from [START_REF] Hamman | Chitosan Based Polyelectrolyte Complexes as Potential Carrier Materials in Drug Delivery Systems[END_REF].

N-acetyl glucosamine glucosamine

Structural variables

The main macromolecular structural variables of chitosan are the degree of acetylation and the intramolecular distribution of units along the chain, the weight average molar mass Mw and the dispersity of molecular weights (Đ = Mw/Mn). The actual behavior of chitosan is mediated not only by the structural variables but also by external variables such as pH, temperature and the presence of salts in the medium, i.e. ionic strength [START_REF] Schatz | Typical Physicochemical Behaviors of Chitosan in Aqueous Solution[END_REF] (see section 1.7.4.).

Structure-properties relationship

i. Solubility Solubility of chitosan depends upon pH. In acidic aqueous solution, (pH < 6.2), amine groups of glucosamine units are protonated. This protonation allows the polymer dissolution by the establishment of both, electrostatic repulsive forces between polymer chains and hydrogen bonds between water and chitosan. At higher pH, the deprotonation of amine groups, i.e. the decrease in repulsive potential between polymer chains, favors polymer-polymer interactions rather than the polymer-solvent ones. Therefore, chitosan becomes insoluble (Figure 9). The soluble-insoluble transition occurs at pH around 6 to 6.5 depending on the degree of acetylation and molar mass [START_REF] Martino | Chitosan: a versatile biopolymer for orthopaedic tissue-engineering[END_REF][START_REF] Madihally | Porous chitosan scaffolds for tissue engineering[END_REF][START_REF] Dornish | Standards and guidelines for biopolymers in tissueengineered medical products: ASTM alginate and chitosan standard guides[END_REF][START_REF] Fox | The Basic Science of Articular Cartilage[END_REF]. The amine group has a pKa value around 6.5 depending of the DA [START_REF] Sorlier | Relation between the degree of acetylation and the electrostatic properties of chitin and chitosan[END_REF]. [START_REF] Dash | Chitosan-A versatile semi-synthetic polymer in biomedical applications[END_REF].

Figure 9. Solubility of chitosan dependence upon pH

Solubility depends upon structural variables already mentioned in 1.3.2. At DA below 20 %, chitosan shows strong polyelectrolyte behavior (the linear charge density is high) in acidic aqueous solutions that favors expansion of the polymer chain and solubility [START_REF] Schatz | Static Light Scattering Studies on Chitosan Solutions: From Macromolecular Chains to Colloidal Dispersions[END_REF]. The occurrence of entanglements decreases solubility (polymer-solvent interactions) and enhances polymer-polymer interactions. The formation of interchain entanglements occurs when overpassing a critical concentration and molar mass of chitosan [START_REF] Qun | Effects of molecular weight, degree of acetylation and ionic strength on surface tension of chitosan in dilute solution[END_REF][START_REF] Cho | Viscoelastic properties of chitosan solutions: Effect of concentration and ionic strength[END_REF][START_REF] Rinaudo | Characterization of chitosan. Influence of ionic strength and degree of acetylation on chain expansion[END_REF][START_REF] Tsaih | Effect of molecular weight and urea on the conformation of chitosan molecules in dilute solutions[END_REF].

ii.

Biological properties Chitosan is a non-toxic and biodegradable* natural polymer [START_REF] Martino | Chitosan: a versatile biopolymer for orthopaedic tissue-engineering[END_REF][START_REF] Ladet | Multi-membrane chitosan hydrogels as chondrocytic cell bioreactors[END_REF][START_REF] Chedly | Physical chitosan microhydrogels as scaffolds for spinal cord injury restoration and axon regeneration[END_REF][START_REF] Shi | Therapeutic potential of chitosan and its derivatives in regenerative medicine[END_REF][START_REF] Kim | Chitosan and its derivatives for tissue engineering applications[END_REF][START_REF] Anitha | Chitin and chitosan in selected biomedical applications[END_REF]. It is known to promote osteoblast and mesenchymal cells proliferation and in-vivo vascularization [START_REF] Vandevord | Evaluation of the biocompatibility of a chitosan scaffold in mice[END_REF][START_REF] Dutta | Chitin and Chitosan for Regenerative Medicine[END_REF].

The response of the body to low DA chitosan implants in host tissue is minimal, with little or no fibrous encapsulation [START_REF] Vandevord | Evaluation of the biocompatibility of a chitosan scaffold in mice[END_REF]. Higher DA chitosans induce formation of normal granulation tissue associated with accelerated angiogenesis*, appears to be the typical course of the healing response [START_REF] Martino | Chitosan: a versatile biopolymer for orthopaedic tissue-engineering[END_REF][START_REF] Suh | Application of chitosan-based polysaccharide biomaterials in cartilage tissue engineering: a review[END_REF]. The immune response (macrophages response) seems to stimulate the integration of the implanted material to the host tissue [START_REF] Aimin | Antibiotic loaded chitosan bar. An in vitro, in vivo study of a possible treatment for osteomyelitis[END_REF].

Chitosan has also been reported as antimicrobial to fungi, parasites and bacteria [START_REF] Dash | Chitosan-A versatile semi-synthetic polymer in biomedical applications[END_REF]. The cationic groups of chitosan probably bind the anions present on microorganism cell wall, suppressing biosynthesis and disrupting ion and molecular transport across the wall cell, causing cell starving and ultimately death [START_REF] Martino | Chitosan: a versatile biopolymer for orthopaedic tissue-engineering[END_REF][START_REF] Aimin | Antibiotic loaded chitosan bar. An in vitro, in vivo study of a possible treatment for osteomyelitis[END_REF].

As was mentioned above, the biological properties of chitosan are affected by molecular structure. As the degree of acetylation decreases (higher number of amine groups), biocompatibility and cell adhesion tend to increase, whereas biodegradability tends to decrease [START_REF] Martino | Chitosan: a versatile biopolymer for orthopaedic tissue-engineering[END_REF][START_REF] Schatz | Static Light Scattering Studies on Chitosan Solutions: From Macromolecular Chains to Colloidal Dispersions[END_REF][START_REF] Mao | A preliminary study on chitosan and gelatin polyelectrolyte complex cytocompatibility by cell cycle and apoptosis analysis[END_REF]. As the average molar mass-increases, cell adhesion and antimicrobial properties tend to increase, whereas biodegradability tends to decrease [START_REF] Dash | Chitosan-A versatile semi-synthetic polymer in biomedical applications[END_REF][START_REF] Qun | Effects of molecular weight, degree of acetylation and ionic strength on surface tension of chitosan in dilute solution[END_REF]. These biological properties depend also on the physical form of the chitosan-based material (solution, fibers, gels, etc.) and the implantation site and conditions.

iii.

Characterization techniques of chitosan In the specialized literature, many experimental techniques and calculation methods are used to estimate the structural, physico-chemical and rheological properties of chitosan. A summary of some of those methods is presented in Table 3. 1.4 Calcium Phosphates

Definition

Calcium phosphates are a family of minerals that can be classified, based on the form of phosphate ions, as ortho-(PO4 3-), meta-(PO3 -), pyro-(P2O7 4-), and poly-(PO3)n n-phosphates. They naturally occur in rocks and animal bones [44]. However, since orthophosphates are the most used form of calcium phosphates in bioceramics, this section will focus on this group and in this work they will be called "calcium phosphates".

Calcium phosphates are mainly formed by three elements: calcium, phosphorous and oxygen. Additionally, the chemical composition of many of them includes hydrogen, as in orthophosphate anions (HPO4 2-, H2PO4 -), in H2O groups or forming part of hydroxyl anions (OH -) [44].

The main rationale behind the use of calcium phosphates as bone substitute materials is their chemical similarity to the mineral component of mammalian bones and teeth [109]. Calcium phosphates could be considered as biodegradable and bioactive bioceramics. These properties depend upon crystallinity, composition, particle size, defects, porosity and surface texture [44].

There are many calcium phosphates, as Table 4 shows, but not all of them are used in medicine. Calcium phosphates application in biomedical fields depends upon Ca/P molar ratio and solubility in aqueous solution. Those with Ca/P ratio below 1 are too acid and soluble at physiological pH (pH 7.4) to be implanted. Conditions of pH and temperature defined the range of stability of these materials. For example, dicalcium phosphate dihydrate* (DCPD) is stable at acidic pH, while, hydroxyapatite (HA) and apatite are stable at alkaline pH. On the other hand, α-tricalcium phosphate (-TCP), β-TCP and HA are generally obtained at high temperature (for example by sintering process) while dicalcium phosphate anhydrous (DCPA), DCPD and apatite are obtained at low temperature (for example from precipitation in aqueous medium) [110,111].

Two calcium phosphates are of major interest to this work: hydroxyapatite and dicalcium phosphate dihydrate. Therefore, sections 1.4.4 and 1.4.5 focus on their properties.

Characterization techniques of calcium phosphates

A summary of the characterization techniques of calcium phosphates is shown in Table 5.

Factors affecting the kinetics of calcium phosphates crystallization

The kinetics of phase crystallization/transition determines the effective occurrence or the spontaneous conversion from ACP, DCPD, and OCP to apatite. The kinetics of crystallization of calcium phosphate solutions to apatite is influenced by the concentration of salt in solutions (used as sources of calcium phosphates); the pH of the medium and the presence of ions and molecules that can favor or inhibit crystallization. Transformation of OCP to apatite is retarded by ions like magnesium, citrate and pyrophosphate and accelerated by ions CO3 2-, HPO4 2-, Ca 2+ [114]. The difference in presence of DCPD and OCP in-vivo and in-vitro experiments may be related to the presence of ions in the more complex environment of in-vivo bone formation [115,116].

pH is largely impacting CaP phase formation: a slight acidity favors the presence of DCPD and OCP; on the other hand, apatite is more likely to be formed at neutral or basic condition [115,116]. Indeed, the predominant type of phosphate ions, in equilibrium, in aqueous solution, depends upon pH as reported in Table 6. The spontaneous and immediate precipitation of calcium phosphates, after mixing of supersaturated solutions of calcium and phosphate salts, causes a decrease in pH by changing the distribution of ions OH -and H + , since the global reaction of formation of calcium phosphates releases H + ions [START_REF] Behrens | Handbook of Biomineralization: Biomimetic and Bioinspired Chemistry[END_REF]118,119].

As the medium is buffered at pH between 5 and 9, the predominant phosphate ions in equilibrium are H2PO4 -and HPO4 2-. The divalent phosphate ion, HPO4 2-, has a relative high affinity for Ca 2+ ion, then, favoring the formation of DCPD, CaHPO4.2H2O, which is stable in the pH range of 2 to 6 (Table 4). The formation of DCPD releases H + ions, therefore, tends to decrease pH [118].

By adding an alkaline component to a DCPD solution, the predominant form of phosphate ion in equilibrium changes to PO4 3-. Due to the relative high affinity between Ca 2+ , PO4 3-and OH -ions, apatite is formed, Ca(10-x)(HPO4)(6-x)(OH)(2-x). In turn, the expense of PO4

Hydroxyapatite

Hydroxyapatite, HA, Ca10(PO4)6(OH)2, has a molar ratio Ca/P = 1.67; it is the most stable calcium phosphate at room temperature, 25°C, and pH between 9.5 and 12 (Table 4). In comparison with other calcium phosphates, its solubility in alkaline medium is low. HA can be obtained at high temperature and crystallizes in the space group of P63/m in a hexagonal system with lattice parameters a = b = 9.432 Å, c = 6.881 Å, and γ = 120 [121] (Figure 10).

Figure 10.Crystalline structure of HA and some of the most common ionic substitution occurring in the unit cell [44].

HA is the most used calcium phosphate in dental and skeletal implantology. The reason is that the chemical composition and structure of HA is similar to that of mineral component of bone and dental tissue [44]. As stated in section 1.1.1, HA in bones has a Ca/P ratio less than 1.67 and it presents a poorly crystallized structure of relative small size crystals. The stoichiometric imbalance is attributed to vacancies in the crystalline network and to the facility of different substitutions in both anionic and cationic positions [44] (Figure 10). As was stated above, in this work, non-stoichiometric hydroxyapatite forms are called apatite.

Apatite have remarkable biological properties, it bonds to bone tissue, a unique property of bioactive* materials; it osteointegrates* with bone tissue [44,122]; it presents osteoconduction*; it is a bioresorbable* material. However, apatite is not osteoinductive* [44,113,123,124]. For apatite to become osteoinductive, specific ion substitution for example with silicon can be performed in the synthesis process [44].

Most biological apatite exhibit particular characteristics: i) non-stoichiometric chemical composition due to replacement of PO4 Apatite can be obtained by various methods including precipitation from basic solutions (calcium and phosphate solutions), sol-gel processes, solid-state reaction and others [125][126][127][128][129]. Precipitation from aqueous solutions seems to be a convenient route of synthesis of apatite, since it is performed at low temperature (that of an aqueous solution), water is the only by product, cost is low and yield is high [44].

The reaction to synthesize apatite may take 24 hours or more, depending on temperature and chemical nature of the salt sources of calcium and phosphate ions [114]. The lack of osteoinduction* and the poor mechanical properties of apatite remain to be solved for bone substitute. Thus, a composite material, comprising a polymer and apatite could be promising at least for applications with no high load requirements.

Dicalcium phosphate dihydrate

Dicalcium phosphate dihydrate (DCPD or brushite, CaHPO4.2H2O) has a molar Ca/P ratio of 1.00. It is a stable calcium phosphate phase at pH from 2.0 to 6.0 and it is metastable at pH = 7.4 (physiological pH), (Table 4). DCPD crystallizes in the monoclinic system (space group 1a) with lattice parameters a = 5.812 Å, b = 15.180 Å, c= 6.239 Å and β = 116°25" [134,135] (Figure 11). The biological properties of DCPD have attracted scientific and clinical attention to this material. DCPD, used as a bone substitute under adequate composition and geometry, presents osseointegration*, osteoconduction* and even osteoinduction* properties. DCPD has a strong advantage over other calcium phosphate materials (for example hydroxyapatite): its ability to be resorbed under physiological conditions. However, it has a natural tendency to recrystallize to apatite (stable in physiological conditions) when used as a biomaterial. A fast resorption of DCPD material can only be achieved if its resorption occurs before its conversion to apatite.

Chitosan-calcium phosphates

A composite material composed of (inorganic calcium) phosphates and (organic) chitosan, seems to be a good choice for bone substitute in search of a synergistic effect of combining the properties of the biopolymer and bioceramics that also resembles the composition of bone mineral [142-145].

Synthesis of chitosan-calcium phosphate composite materials

Chitosan-calcium phosphate materials, used in bone tissue engineering, have been prepared as membranes, films, scaffolds, injectable materials, multilayers films and particles [3,[START_REF] Dutta | Chitin and Chitosan for Regenerative Medicine[END_REF]. Methods of synthesis haven been reported and can be classified in: i) coating method [3,146-

i.

Coating method Coatings were performed using electrochemical and electrophoretic deposition techniques, favoring the deposition of hydroxyapatite and chitosan particles. These techniques allow preparing composite materials at room temperature. The presence of hydroxyapatite and chitosan protects the metal surface from corrosion when getting into contact with physiological solutions. Electrochemical deposition has been used to coat titanium surfaces with chitosan solution and DCPD precipitate. The composite chitosan/DCPD was converted to chitosan/HA using sodium hydroxide solution [149]. Coatings on 316L stainless steel substrates and on NiTi shape memory alloys (Nitinol) surfaces have been fabricated by electrophoretic deposition of chitosan/HA composites. Electrodeposition was performed from chitosan solutions or chitosan-heparin solutions containing hydroxyapatite nanoparticles in a mixed ethanol-water solvent [150].

ii.

Incorporation of chitosan to calcium phosphate cements Several approaches of this type have been reported [3,[152][153][154][155][156][157][158][159][160][161][162][163]. The most common consist in the addition of chitosan into a paste of calcium phosphates, which is hardened by raising the pH above 7.4. The final material contains a polymeric matrix load entrapped in an inorganic phase. The addition of chitosan improves injectability, degradation rate, hardening properties and mechanical performance of the resulting materials.

iii.

Mineralization of chitosan scaffold This method involves two major stages: first, the synthesis of an organic polymeric scaffold (pure or modified chitosan) and, second, mineralization of the scaffold in simulated body fluid (the biomimetic way) containing ions (Na + , K + , Mg 2+ , Ca 2+ , Cl -, HCO3 -, HPO4 2-, SO4 2-) and Tris buffer solution or in saturated matrix solutions (calcium and phosphate solutions). The scaffolds can be made in the form of membranes, microspheres, or multilayered materials [164,166].

iv.

Impregnation of calcium phosphates scaffolds with chitosan It has been described as the pre-formation of a porous calcium phosphate scaffold followed by its impregnation within a chitosan solution. Then, the resultant material is neutralized with an alkaline solution and freeze-dried [166,167,181].

v.

Mixing chitosan and calcium phosphates In this method, a suspension is formed by mixing previously prepared calcium phosphate ceramic powder with chitosan solution. Composite materials are usually obtained by freezing and lyophilizing the suspension [3,[START_REF] Dutta | Chitin and Chitosan for Regenerative Medicine[END_REF][169][170][171].

The above-mentioned methods effectively lead to chitosan-calcium phosphates composite materials. However, a major concern is the lack of homogeneity at microscopic level, which induces practical inconveniences in medical and industrial applications. To overcome this drawback, methods of in-situ precipitation of calcium phosphates inside the polymeric matrix have been studied.

vi.

In-situ precipitation In-situ precipitation refers to various methods of preparation of chitosan-calcium phosphates materials that have some common characteristics: i) the process occurs in an acidic aqueous medium solution; ii) the intended result is a homogenous mixture of the inorganic phase in a chitosan matrix; iii) the pH is changed to basic condition; iv) in the last step composite material is freeze-dried or dried at room temperature for further use and characterization [142,167,172-180].

Characterization techniques

From Table 3 and Table 5, it is clear that the required information and techniques to characterize the physico-chemical properties of chitosan and calcium phosphates are very different. Indeed, calcium phosphates are mostly characterized by their properties linked to crystallinity (crystal phases, substitutions, crystallite size) and architecture (porosity, pore size) whereas chitosan is mostly characterized by its macromolecular structure (average molar mass and molar mass distribution, degree of acetylation, acetylation pattern). This will most probably make the characterization of chitosan -calcium phosphate composite difficult, since almost no known characterization techniques are able to provide the necessary information for both materials at the same time. Moreover, some of these techniques may necessitate sample preparation that may affect the results. For example, it is very difficult to characterize the CaP particle size on concentrated chitosan-CaP composite solutions: indeed, they have to remain hydrated, which prevents the use of Scanning Electron Microscopy (drying may artificially increase the particle size); Dynamic Light Scattering cannot be used either because it necessitates further dilution that may solubilize the CaP particles.

Mechanical properties

Mechanical properties of natural bone tissue are higher than those of all developed chitosan-calcium phosphates materials [182]. This is frequently stated as a drawback for using chitosan-calcium phosphates composites as load bearing bone substitutes [183]. This statement would be rigorously true if the materials were intended to last as a substitution of bone [184]. But, in fact, the implanted material envisioned here would be temporary and serves as a support for new bone formation. The implanted material would be resorbed whereas new bone will be the definitive load bearing material.

Biological properties

The advantage of combining an organic natural polymer and inorganic natural ceramic, chitosancalcium phosphates, is the possibility of coming up with a composite material that synergistically integrates the biological properties of chitosan (bioresorbable*, angiogenic*, bacteriostatic*, fungistatic*) with the biological properties of calcium phosphates (osteoconductive*, bioresorbable*) [3,[START_REF] Dutta | Chitin and Chitosan for Regenerative Medicine[END_REF]185].

Biological properties of chitosan-calcium phosphates materials of different composition ratio and different final shape are widely reported in the literature. In fact, the chitosan-calcium phosphate composites have shown to be non-toxic in biological assays with different cell types [3,[START_REF] Dutta | Chitin and Chitosan for Regenerative Medicine[END_REF]166,188]. Bioactivity* has been also reported for these materials, both the inherent bioactivity of calcium phosphates and the stimulation of bone growth due to the presence of chitosan [3,[START_REF] Dutta | Chitin and Chitosan for Regenerative Medicine[END_REF]173,182,189]. Improvements in biodegradability* of calcium phosphates have been reported in the composite material [3,[START_REF] Dutta | Chitin and Chitosan for Regenerative Medicine[END_REF]182]. The composite material has served as a support for tissue infiltration and replacement, simultaneously stimulating cell growth within the tissue [166].

From the analysis of the already tested methods of preparation, it seems that an effort should be devoted to obtaining a homogenous dispersion of apatite in a chitosan matrix. It is foreseeable that a hydrogel may fulfill this objective because hydrogels could be formed from homogenous suspensions of chitosan-calcium phosphates. It would advisable to try a physical hydrogel of chitosan to avoid the use of different cross-linking agents that may result toxic.

Hydrogels

Definition

Hydrogels are polymer networks, formed by physical or chemical crosslinks that can swell in water or release water or aqueous media [START_REF] Dutta | Chitin and Chitosan for Regenerative Medicine[END_REF]190]. Hydrogels have a shear storage modulus G' larger than the shear loss modulus G", typically exhibiting soft, elastic and mechanical stability [START_REF] Dutta | Chitin and Chitosan for Regenerative Medicine[END_REF][190][191][192][193][194][195]. Polymers used to fabricate hydrogels can be natural or synthetic [195,196].

Properties and applications

The properties of a given hydrogel depend upon nature and content of the hydrophilic groups, the type of crosslinking (physical or chemical) and crosslink density. By controlling these variables during the synthesis process, it is possible to tune pore (or mesh) size, network composition and geometry for a given application. Hydrogels can be modified chemically, adding new properties to the material making them suitable for cellular infiltration, proliferation, migration and differentiation [190,196]. Considering the physical and chemical flexibility and the stimuli-responsive properties of hydrogels, osteogenic*, angiogenic* and rapid resorption ability, it is of no surprise that hydrogels have many applications in diverse industries: cosmetics, soil recovering, agriculture, drug delivery and many others, including biomaterials for cell culture and bone substitution. It is also possible to produce hydrogels which composition resembles that of ECM [197,198]. Hydrogel synthesis can be engineered to obtain physical and bioactive properties to resemble extracellular matrix, to be used in medical implants, biosensors and drug delivery devices [START_REF] Dutta | Chitin and Chitosan for Regenerative Medicine[END_REF]124,[191][192][193][194].

Gelation

Gelation is the transition from liquid solution to a gel. The inter and intra molecular chain linking is due to chemical or physical crosslinking [START_REF] Dutta | Chitin and Chitosan for Regenerative Medicine[END_REF]. Chemical crosslinking is due to the formation of chemical bonds (ionic, covalent, metallic bonds) between reacting groups of the polymer chain (hydroxyl, amine, others) that usually requires the use of an external cross-linking agent [190]. In the case of bio applications, it is important to consider the eventual toxicity of the cross-linker [198]. Some polymers undergo a reversible physical gelation, which is defined as the conversion of a sol into a gel, in response to minor changes in their environment such as temperature, pH and ionic strength or concentration. The network is formed through molecular entanglements and/or secondary forces including hydrogen bonding, hydrophobic forces and electrostatic interactions between chemical groups present in the polymer molecule. Physical cross-linked hydrogels are known for their low mechanical properties. But, in turn, they are manageable for designing injectable hydrogels [190,199,200]. Environmental conditions, particularly pH and temperature, usually induce or inhibit the formation of those physical interactions and may induce corresponding changes in the gel state (ex: mechanical properties), swelling or also releasing behavior [START_REF] Dutta | Chitin and Chitosan for Regenerative Medicine[END_REF].

Physical hydrogels of chitosan

Chitosan physical hydrogels can be obtained by the neutralization of aqueous solutions in welldefined conditions [201,202].

As was considered in section 1.3.3, the solution state for chitosan occurs at low pH, as a consequence of the protonation of amine groups. The presence of positive electrostatic charges on the polymer chain allows the solubilization of chitosan by increasing polymer-solvent interactions and also via repulsive forces between chains (Figure 12). The main variables that can play a role in the mechanism of this physical gelation are the charge density of polymer that depends upon pH; the dielectric constant of the solvent medium; temperature, degree of acetylation, molar mass of chitosan, polymer concentration [202,204].

The physical and mechanical properties of the resulting hydrogels depend on the density of hydrophobic interactions, hydrogen bonds, crystallites and remaining chain entanglements within the network [201,202].

Physical gelation of chitosan was chosen in this work because it is a relatively simple method of gelation and it does not need a cross-linking agent. Moreover, the increase in pH necessary for gelation of chitosan solution is in line with obtaining apatite as stable calcium phosphate phase.

Since an objective of this work is to obtain a physical hydrogel of chitosan mineralized with calcium phosphates (apatite), it is important to consider the interactions resulting of the presence of salts (for example, sources of calcium phosphates) that dissociate in solution forming ions that generate electrostatic interactions. The discussion on physico-chemical interactions will give insight into the scientific fundamentals for better understanding of the system under research.

Physico-chemical interactions between chitosan and mineral phases

The versatility of industrial and medical application of chitosan is a consequence of its feasibility for establishing interactions, with itself and with other materials, via hydrogen bonds and electrostatic forces due to the various moieties present in the chain [205]. The actual feasibility of intermolecular interactions is impacted by structural and physicochemical conditions: molar mass, degree of acetylation, pH, ionic strength and temperature.

Chemical groups and physico-chemical interactions

Chitosan presents various chemical groups (Figure 13), each one with specific capability for physicochemical interactions. Hydroxyl group, oxygen, acetyl group and amino groups may form hydrogen bonds; glycosidic rings may induce hydrophobic forces; oxygen and nitrogen may be electron donors to form coordination bonds; ether bond and acetamide group have low reactivity. Acetamide group is bulky, therefore, it may produce steric hindrance* that reduces rotation and increases the stiffness of the chains. Protonated amine group induces repulsive forces between polymer chains and within the polymer chains and electrostatic forces with ions [START_REF] Schatz | Typical Physicochemical Behaviors of Chitosan in Aqueous Solution[END_REF]206].

Molar mass

High molar mass favor entanglements formation provided that a critical polymer concentration is reached above which chains start to overlap with one another. Entanglements could be understood as polymer-polymer interactions that contribute to the viscoelastic properties of chitosan solutions. Therefore, entanglements affect viscosity of solution, strength resistance and energy storing and dissipation of gels [START_REF] Cho | Viscoelastic properties of chitosan solutions: Effect of concentration and ionic strength[END_REF]. The molar mass of the polymer affects conformation of chitosan molecules in solution. Effect of molecular weight on the conformation of chitosan molecules in dilute solution (in hydrochloric acid, ionic strength of 0.01 M) was studied by Tsaih & Chen [START_REF] Tsaih | Effect of molecular weight and urea on the conformation of chitosan molecules in dilute solutions[END_REF]. It was reported that if the weight-average molar mass was higher than 223 kDa chitosan showed a random coil conformation, whereas for weight-average molar mass lower than 148 kDa, conformation was more rod-shape [START_REF] Tsaih | Effect of molecular weight and urea on the conformation of chitosan molecules in dilute solutions[END_REF]. In this work, we will form chitosan hydrogels from high molecular mass chitosan to favor entanglements and obtain hydrogels with suitable mechanical properties.

Degree of acetylation

The degree of acetylation, DA, of chitosan largely impacts the chitosan solution properties, because the polymer apparent charge density decreases when the DA increases.

Ionic strength

As was mentioned above, solutions of chitosan and salt solutions (sources of calcium phosphates) will be mixed to form complex materials.

In an aqueous medium, the inorganic salts dissociate into ions resulting in an increase in the ionic strength of the solution (equation 2), which directly impacts the Debye screening length* (equation 1), which measures the distance of electrostatic effects.

𝜆 𝐷 = (8𝜋𝜆 𝐵 𝐼) -1/2 (1)

𝐼 = 1 2 ∑ 𝑐 𝑖 𝑧 𝑖 2 𝑛 1 (2)
In equation (1), λD stands for Debye length; λB stands for Bjerrum length*; I stands for ionic strength.

In the equation (2), 𝐼 stands for ionic strength; 𝑐 𝑖 is the molar concentration of ion 𝑖; 𝑧 𝑖 is the charge number of ion 𝑖. The sum is taken over all species in the solution.

As a result, when the ionic strength increases, the repulsive electrostatic forces among the polyelectrolyte polymer chain decrease. This directly impacts the conformation of the polymer chain that can evolve from a fully expanded rod-like conformation to a random coil. In other words, the decrease in electrostatic repulsions results in an increase in hydrogen bonding and hydrophobic interactions. Therefore, as ionic strength increases, polymer-polymer interactions are enhanced over those of polymer-solvent, polymer molecules contract, hydrodynamic volume decreases, thus, viscosity decreases [START_REF] Qun | Effects of molecular weight, degree of acetylation and ionic strength on surface tension of chitosan in dilute solution[END_REF][START_REF] Cho | Viscoelastic properties of chitosan solutions: Effect of concentration and ionic strength[END_REF][START_REF] Rinaudo | Characterization of chitosan. Influence of ionic strength and degree of acetylation on chain expansion[END_REF].

The combined effect of all these structural variables (chemical groups, molar mass, degree of acetylation) and conditions (pH, ionic strength) results in the complex behavior of chitosan in solution. These effects could be categorized and grouped in two basic interactions: hydrogen bonds and electrostatic interactions.

Hydrogen bonds

Various types of hydrogen bonds may occur intra and inter chitosan molecular chains, as is shown in Figure 14 [208,209].

Electrostatic interactions i. Interactions with anions

The cationic nature of chitosan allows electrostatic binding with electronegative entities. This property is the base for interactions between chitosan and polyanaions ('Polyelectrolyte complexes) such as anionic glycosaminoglycans (GAG) and proteoglycans, a complex (GAG-chitosan) that could retain and concentrate growth factors secreted by cells [START_REF] Martino | Chitosan: a versatile biopolymer for orthopaedic tissue-engineering[END_REF][START_REF] Madihally | Porous chitosan scaffolds for tissue engineering[END_REF]. Electrostatic bonds may also be used to introduce in the bone substitute different molecules that may have biological activity, for example, drugs favoring mineralization of ECM and healing processes. The cationic nature of chitosan may be even reversed, for example by sulfating the same amine group producing an anionic and water-soluble modified chitosan, usable in biological applications, for example as coagulating agent mimicking heparin [START_REF] Martino | Chitosan: a versatile biopolymer for orthopaedic tissue-engineering[END_REF][START_REF] Dash | Chitosan-A versatile semi-synthetic polymer in biomedical applications[END_REF][START_REF] Suh | Application of chitosan-based polysaccharide biomaterials in cartilage tissue engineering: a review[END_REF][212][213][214].

As was mentioned before, pH is a key factor determining the extent of chitosan protonation, hence electrostatic interactions. In the case of nitrate anion, adsorption increases as pH decreases, since at lower pH, more protons are available to protonate amine groups, thus, increase the electrostatic interaction of protonated amine group (NH3 + ) with nitrate ion. Other cations (Ca 2+ , Cu 2+ , Zn 2+ , Co 2+ …) may form complex on chitosan mainly in its neutralized form (see below).

Chitosan and phosphate ions could have strong interactions depending on the phosphate ion present in solution. Electrostatic interactions may occur between the protonated amine groups and the three forms of orthophosphate ions (monovalent H2PO4 -, divalent HPO4 2-, trivalent PO4 3-). However, only polyvalent phosphate ions can form physical cross-links of polymer chains through the simultaneous interaction with protonated amine groups of different chains, as represented in Figure 16 ii.

Interactions with metal ions Chitosan has the capacity to form coordination bonds with metal ions [218], forming chelation bonds [205,219], due to the presence of electron donor atoms, nitrogen and oxygen. The complex is formed between the free electron pair of the coordinate (nitrogen or oxygen) and the void orbital of the metal [START_REF] Dutta | Chitin and Chitosan for Regenerative Medicine[END_REF]. These interactions may lead to form physical cross-links between polymer chains. Such ability of chitosan for complex formation may play a significant role in forming chitosanhydroxyapatite composites. Hydroxyapatite contains Ca 2+ ions attached to the surface of hydroxyapatite crystals and have a coordination number of 7. Therefore, there might be coordination bonds between the -NH2 of chitosan and Ca 2+ ions of hydroxyapatite, as it is schematized in Figure 17 [211].

Figure 17. Schematic representation of coordination bonds between Ca 2+ ions of hydroxyapatite (HA) and -NH2 of chitosan (----coordination bonds) [211].

The growth of hydroxyapatite crystals is considered to occur by nucleation sites corresponding to the interaction between amine groups of chitosan and calcium group of calcium phosphate compounds [220].

Rheology for materials of interest in this work

Rheological tests can be used to characterize solution and gel samples and to quantify macromolecular behaviors.

In this context, solutions (chitosan and chitosan-salts), suspensions (chitosan-calcium phosphates) and mineralized physical hydrogels were characterized. In all these materials the rheological behavior is dominated by interactions of the constituents [221].

Usually, structured fluids are non-Newtonian, exhibit shear-thinning behavior, that is, their viscosity drops at increasing shear rate; and some other materials will not flow unless the applied stress reaches a certain value called yield stress [221]. Moreover, structured polymer fluids usually exhibit not only a viscous behavior but also an elastic contribution, that is, they are viscoelastic materials [221,222].

Rheological properties of solutions and suspension of chitosan-salts depend on concentration of chitosan, degree of acetylation and ionic strength nature of the ions in solution [START_REF] Cho | Viscoelastic properties of chitosan solutions: Effect of concentration and ionic strength[END_REF]202,222].

As polymer concentration increases, the macromolecules become entangled, the fluids become structured [START_REF] Cho | Viscoelastic properties of chitosan solutions: Effect of concentration and ionic strength[END_REF] thus, the Newtonian behavior range is reduced and more shear-thinning is observed [223]; motion of individual chains gets restricted; therefore, viscosity tends to increase [START_REF] Cho | Viscoelastic properties of chitosan solutions: Effect of concentration and ionic strength[END_REF]223]. Both storage or elastic modulus (G´) and loss modulus (G"), increase with the concentration of chitosan, and tan  = G"/G' decreases, indicating that elasticity is enhanced [START_REF] Cho | Viscoelastic properties of chitosan solutions: Effect of concentration and ionic strength[END_REF]202].

The DA also plays a key factor in determining rheological behavior of chitosan solutions. At DA below 20 %, (polyelectrolyte domain see section 1.7.3), when the DA decreases, the concentration of protonated amine group increases, therefore repulsive forces between polymer chains increase. As a consequence, chitosan molecules become extended, entanglement increases, and again the fluid gets less Newtonian [START_REF] Cho | Viscoelastic properties of chitosan solutions: Effect of concentration and ionic strength[END_REF]202,224].

The ionic strength (see section 1.7.4) also influences rheological behavior of chitosan solutions and suspensions [222]. As was mentioned before, as ionic strength increases, a more compact chain conformation is favored [225]. That is, viscosity decreases and solubility may decrease up to the point of precipitation [START_REF] Qun | Effects of molecular weight, degree of acetylation and ionic strength on surface tension of chitosan in dilute solution[END_REF][START_REF] Cho | Viscoelastic properties of chitosan solutions: Effect of concentration and ionic strength[END_REF][START_REF] Cho | Viscoelastic properties of chitosan solutions: Effect of concentration and ionic strength[END_REF][START_REF] Rinaudo | Characterization of chitosan. Influence of ionic strength and degree of acetylation on chain expansion[END_REF].

In this research project, different analyses will be performed to evaluate the viscosity of solutions or suspensions, to characterize their shear-thinning effect, to understand interactions, and to determine the yield stress and the viscoelastic nature of the composite materials.

Overall conclusions

This critical literature survey is an analytical exercise directed to contribute to the development of this thesis, whose general objective is to develop a biomaterial, with potential for bone substitution, and to elucidate relations between synthesis procedure, structure and properties of the materials.

The final biomaterial is intended to be used for bone substitution. Therefore, it is advisable that the material mimics composition and structure of the bone tissue. Due to the complexity of bone healing and remodeling, it seems more realistic to view the bone temporary substitute as a vehicle for new bone formation, a task to be performed by nature. Indeed, it appears impossible to reproduce the structure of bone at every scale by material processing.

A variety of bone substitute materials have been reported in the literature. Natural, synthetic and composite materials are examples. In order to develop a bio-inspired material and taking into account the need of synergies to meet the requirements of a bone substitute, composite material with inorganic and organic components seems to be a good choice: chitosan and calcium phosphates could be synergistically associated.

Both materials, chitosan and calcium phosphates, have been extensively studied and reported in the literature as bone substitutes.

The gel structure offers an 'open' network, which makes a suitable environment for cell colonization and for delivery of growth factors and bioactive molecules. Consequently, chitosan physical hydrogel as organic part, and apatite (or other calcium phosphate ceramics) as inorganic part, are proposed in developing a bone substitute.

In the synthesis of the composite material, it is important to control the composition (calcium phosphate phase, inorganic and organic proportion), microstructure (crystallinity, crystal size) and distribution of inorganic part in the polymer matrix. Additional guidelines should be considered: to avoid using chemical crosslinking agents to ensure no toxicity; to fabricate a physically and chemically stable composite material; to develop a synthesis process at room temperature and an adequate mixing to obtain apatite particles of small size and poor crystallinity to mimic mineral bone structure and to keep the possibility of incorporating bioactive molecules during the synthesis process.

Up to this point, the proposed research counts on a solid literature support. However, questions remained opened on processing steps and their sequencing to prepare a physical hydrogel of chitosan mineralized with apatite with potential as bone substitute. Those questions focus on the search of preparation methods of the material, the range of accessible composition, the reasons of change in composition and structure occurring along the process; interactions between organic and inorganic parts and relationships between the methods of preparation, the microstructure and the properties of the resulting material.

Chapter I.2

Physico-chemical interactions in chitosan-calcium phosphates solutions and suspensions

This chapter will be submitted as peer-reviewed article with the following co-authors: Solène Tadier, Laurent Gremillard, Emmanuel Gouisset, Alexandra Montembault, Laurent David, Thierry Delair

I. Introduction

A promising approach to deal with healing of damaged bone tissue is the use of bone substitute.

A bone substitute would be a material that temporary replaces bone tissue, promotes bone formation and growth, until the material is finally bioresorbed. Beside those basic properties, a compromise should be met between sophistication, ease of fabrication, ease of handling by the surgeon and costs [1]. Since it is difficult to find a single material that meets all these requirements, an alternative is to combine materials with complementary properties, for example, natural polymers and bioceramics, such as chitosan and calcium phosphates.

Among many applications reported in the literature [2], chitosan-calcium phosphates have been used in wound dressing, drug delivery, space-filling implants and other biomedical applications [3]. Chitosan is a bioresorbable*, angiogenic*, bacteriostatic*, fungistatic*, biocompatible* polymer with high potential for physico-chemical interactions, with itself and with other materials, via hydrogen bonds and electrostatic forces due to its chemical structure [4]. This makes chitosan an easy-to-modulate material with remarkable biological properties. Calcium phosphates have a composition and structure relatively close to the inorganic part of the extracellular matrix of bones. Calcium phosphates present osseointegration*, osteoconductivity* and could be bioresorbed under appropriate physico-chemical conditions [5].

Chitosan-calcium phosphates composites have been prepared as membranes, films, scaffolds, injectable materials, multilayers films and particles [6,7]. Methods of synthesis have been reported and can be classified in: i) coating method using electrochemical and electrophoretic deposition techniques, favoring the deposition of chitosan and calcium phosphates on metal surfaces [6,[8][9][10][11][12][13]; ii) incorporation of chitosan (powder or solution) to calcium phosphate pastes to form calcium phosphates-chitosan cements [6,7,[14][15][16][17][18][19][20][21][22][23][24][25]; iii) mineralization of chitosan scaffold by immersion in solutions saturated with respect to apatite (e.g., simulated body fluid) [26][27][28]; iv) impregnation of calcium phosphates scaffolds with chitosan solution [26,28,29]; and v) suspensions obtained by mixing chitosan solution with calcium phosphates particles [6,[30][31][32][33]. These methods of preparation effectively lead to chitosan-calcium phosphate composite materials; however a major concern is the lack of homogeneity at microscopic level. To overcome this drawback, methods of in-situ precipitation of calcium phosphates within the polymeric matrix have been studied [6].

One alternative, chosen in this work, is to prepare chitosan and calcium phosphate suspensions in water, at acid pH, to favor the homogeneity of the system. Chitosan is soluble in acidic aqueous medium (pH ≤ 6) as a result of the protonation of the primary amine of glucosamine units. Hence, electrostatic repulsive forces between charged polymer chains and hydrogen bonding with water molecules lead to the polymer chain solvation. Calcium phosphates can be prepared from salt solutions, sources of calcium and phosphate ions, which, at acidic pH, favor the precipitation of dicalcium phosphate dihydrate, DCPD (CaHPO4.2H2O), as stable calcium phosphate [34][35][36].

This chapter is framed in a research work oriented to develop chitosan hydrogel-calcium phosphate composites. The scope of this chapter is the study of physico-chemical interactions functioning in systems: chitosan solutions, chitosan-salt solutions, and chitosan-calcium phosphate suspensions. Rheological tests were performed for systems prepared at various concentrations of components. Chitosan-calcium phosphate suspensions were also characterized with respect to phase identification. The research strategy was to correlate possible physicochemical interactions acting in the system with data of composition and rheological behavior. The effect of interactions may be enhanced or diminished by conditions such as pH and concentrations of the different components of the system.

II. Theoretical background

Interactions between species in chitosan solutions alone (polymer, acid and water) and with dissolved salts depend on molar mass, degree of acetylation and concentration of chitosan, ionic strength due to ions present in the system and conditions of pH and temperature. Those interactions impact the rheological behaviors of the systems [37][38][39]. Chitosan molar mass and the degree of acetylation were kept constant in this work. Preparation and characterization of samples were performed at room temperature.

As polymer concentration increases, macromolecules become more entangled, fluids become structured [37] thus, less Newtonian; more shear-thinning is observed [40]; motion of individual chains gets restricted; therefore, viscosity tends to increase [37,41]. The physico-chemical properties of chitosan solution are impacted by the ionic strength of the medium. The increase in ionic strength leads to a reduction of the repulsive electrostatic forces between chitosan chains. Hence, polymer-polymer interactions are enhanced over those of polymer-solvent [42]; disentanglement tends to occur, therefore, the fluid gradually becomes more Newtonian; polymer solubility decreases up to the point that precipitation may occur at high ionic strength [37].

Chitosan may form coordination bonds with calcium ions due to the interaction between the free electron pair of donor atoms in chitosan molecule (nitrogen and oxygen) and the void orbital of calcium ions [4,7,43,44]. These coordination bonds play the role of physical cross-links between polymer chains.

Chitosan and phosphate ions in solution develop strong interactions depending on the charge of the phosphate ions present, which in turn depends on pH. Table 1 shows the predominant orthophosphate ions in equilibrium with their respective pKa and pH range in aqueous solution [45,46].

Table 1. Phosphate ions in equilibrium in aqueous solution. Adapted from [47] Equilibrium reaction pKa pH range H3PO4 ⇄ H3O + + H2PO4 - ~2.12 0 -4

H2PO4 -⇄ H3O + + HPO4 2- ~7.21 5 -9 HPO4 2-⇄ H3O + + PO4 3- ~12.67 10 -14
Electrostatic interactions may occur between the protonated amine groups and the three forms of orthophosphate ions (monovalent H2PO4 -, divalent HPO4 2-, trivalent PO4 3-). However, only polyvalent orthophosphate ions can form physical cross-link of polymer chains through the interaction with protonated amine groups, as represented in Figure 1 [48,49].

Figure 1. Schematic representation of electrostatic interactions between chitosan molecules and phosphate ions.

Adapted from [50].

The divalent phosphate ion is the only one that may form DCPD reacting with calcium ions:

Ca 2+ + 𝐻𝑃𝑂 4 2-+ 2𝑂𝐻 -+ 2𝐻 + → 𝐶𝑎𝐻𝑃𝑂 4 2𝐻 2 𝑂 (1)
The systems of interest in this work are typically called structured fluids, that is, fluids which rheological behaviors are dominated by interactions of the constituents [51].

The fundamental rheological relation links stress and strain. Experimental data could be adjusted to many equations. One of the simplest, useful rheological equation is the power-law (equation 2).

𝜏 = 𝐾𝛾̇𝑛 , 𝑙𝑜𝑔 𝜏 = 𝑙𝑜𝑔 𝐾 + 𝑛𝐿𝑜𝑔 𝛾̇ (2)
Where 𝜏 is the shear stress, γ̇ the shear rate, n is the flow exponent and K is a scaling factor also known as the flow consistency index [51,52]. The value of n is an adjusted parameter to fit experimental data to the power-law equation. A physical sense could be and is commonly assigned to its value: if n = 1, it is a Newtonian fluid; the departure of n from 1 is an indicative of less Newtonian behavior; if n < 1, the fluid presents shear-thinning behavior, that is, viscosity drops as shear increases. In this case it is assumed that shear breaks physical or chemical interactions that makes the fluid depart from Newtonian behavior. The presence of chitosan is associated to the presence of chain entanglements that are disentangled as shear rate is applied. Disentanglement makes the fluid less structured. Therefore, it is expected that chitosan solutions, chitosan-salt solutions and chitosan-calcium phosphate suspensions present shear-thinning behavior, that is, n < 1. For n > 1, viscosity increases as shear increases, the fluid is said to present a shear-thickening behavior [51,52].

III. Materials and Methods

A. Materials

Chitosan powder produced from squid pens and supplied by Mahtani Chitosan Pvt. Ltd (batch type 114, No S3 20110121) was characterized with standard procedures [38]. The degree of acetylation, corresponding to the molar fraction of acetylated units within the polymer chains, was close to 5 %; the weight-average molar mass and dispersity were Mw = 550 kg.mol -1 and Ð = 1.9, respectively.

Four salts were used in this study: Ca(NO3)2.4H2O (Sigma-Aldrich, CAS 13477-34-4, assay ≥ 99 %), CaCl2.2H2O (Sigma-Aldrich, CAS 10035-04-8, assay ≥ 99 %), (NH4)2HPO4 (Sigma-Aldrich, CAS 7783-28-0, assay ≥ 99 %) and NH4H2PO4 (Sigma-Aldrich, CAS 7722-76-1, assay ≥ 98 %).

B. Samples preparation

Three kinds of samples were prepared in this work: chitosan solutions, chitosan-salt solutions and chitosan-calcium phosphate suspensions (CS-CaP). CS-CaP suspension refers to a continuous phase of chitosan solution with calcium phosphate particles.

Chitosan solutions of various molar concentrations (mol.L -1 , M) were prepared, 0.02, 0.03, 0.09, 0.14 and 0.15 M (or 0.3, 0.5, 1.5, 2.3 and 2.4 wt % respectively), by adding chitosan powder to acetic acid aqueous solution (acetic acid: Carlo Erba Reagents, CAS 64-19-7, assay 99.9 %). The mass of chitosan powder was calculated taking into account the molar mass of the repeating unit (D-glucosamine and N-acetyl glucosamine), 163.10 g.mol -1 . The amount of acetic acid was adjusted to match the stoichiometric protonation of -NH2 sites of chitosan. The complete dissolution of chitosan was obtained after several five-minute cycles at 3000 rpm, using a speed mixer machine (DAC150.1FVZ-K, Synergy devices Ltd, UK).

The term chitosan-salt solutions refers to chitosan solution in which one of the salt precursors of calcium phosphates is dissolved. To prepare chitosan-salt solutions, a weight of salt powder was added into a 7g of 0.15M chitosan solution and then the system was mixed for three cycles of five-minutes each, at 3400 rpm, using a speed mixer machine. For each salt, concentration was increased from soluble to unstable and heterogeneous system as reported in Table 3. The change in volume of chitosan solution after adding salt powder was considered negligible in the calculus of final molar concentration of chitosan.

CS-CaP suspensions were prepared by adding simultaneously the calcium and phosphate salt solutions into the chitosan solution. To examine the potential role of counter ions of calcium and phosphate salts, two pairs of salts were chosen as sources of calcium and phosphate; pair a) Ca(NO3)2.4H2O and (NH4)2HPO4; pair b) CaCl2.2H2O and NH4H2PO4. In both pairs, concentration of calcium was 2.5 M and concentration of phosphate was 1.5 M. The reasons to choose those concentrations were: i) to have concentrations below the solubility limit of each salt; ii) to maintain the molar ratio of calcium to phosphorous (Ca/P ratio) at 1.67 (equation 3), as in hydroxyapatite (Ca10(PO4)6OH2) and; iii) to obtain a high content of calcium phosphates while keeping chitosan in solution. Each pair of solutions was added to a 0.17 M chitosan solution in an adequate mass proportion to have a weight proportion inorganic/organic of 70/30 (equation 4). Immediately, the system was mixed in the speed mixer for 3 five-minute cycles, at 3400 rpm.

Those pairs of salts were chosen such as to obtain suspensions, CS-a and CS-b, with different pH.

𝑚𝑜𝑙𝑎𝑟 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑐𝑎𝑙𝑐𝑖𝑢𝑚 𝑚𝑜𝑙𝑎𝑟 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑝ℎ𝑜𝑠𝑝ℎ𝑜𝑟𝑜𝑢𝑠 = 𝐶𝑎 𝑃 = 1.67 (3)
𝑀𝑎𝑠𝑠 𝑜𝑓 𝑐𝑎𝑙𝑐𝑖𝑢𝑚 𝑠𝑎𝑙𝑡(𝑔) + 𝑀𝑎𝑠𝑠 𝑜𝑓 𝑝ℎ𝑜𝑠𝑝ℎ𝑎𝑡𝑒 𝑠𝑎𝑙𝑡 (𝑔) 𝑀𝑎𝑠𝑠 𝑜𝑓 𝑐ℎ𝑖𝑡𝑜𝑠𝑎𝑛 (𝑔) = 𝑖𝑛𝑜𝑟𝑔𝑎𝑛𝑖𝑐 𝑜𝑟𝑔𝑎𝑛𝑖𝑐 = 70 30 (4)
Final concentrations after mixing were calculated and reported in Table 4, columns 2, 3 and 5.

Since chitosan concentrations resulted different for the suspension prepared with each pair of salts (0.15 M/2.4 wt % for pair "a" and 0.14 M/2.3 wt % for pair "b"), chitosan solutions were prepared at those concentrations to have a reference for each suspension.

C. Characterization

The pH of solutions and suspensions was measured. For the case of chitosan, chitosan-salt solutions and CS-CaP suspensions a pH-meter FiveGo (Mettler Toledo, Switzerland) was used.

For the case of salt solutions, a pH-meter Corning® 240 was used.

Rheological measurements were conducted at shear rates from 10 -4 or 10 -3 to 100 s -1 , at 25 °C, using a AR2000-ex rheometer (TA Instruments, USA). The steady-state viscosity of chitosan solutions and suspensions was assessed in static mode using aluminum cone-plate geometry (25 mm diameter; 4 °). The minimum gap was fixed at 116 µm. A solvent trap was used to prevent evaporation of water during measurements. The output is a curve of shear stress vs. shear strain or a curve of viscosity vs. shear rate. This test allows stating the rheological model and the shearthinning behavior of the samples, respectively. Triplicate tests were carried out on each sample under the same processing conditions.

The initial ionic strength was calculated with equation ( 5):

𝐼 = 1 2 ∑ 𝑐 𝑖 𝑧 𝑖 2 𝑛 1 (5)
In equation ( 5), I stands for ionic strength; ci for molar concentration of ion i; zi for charge number of ion i. The sum is taken over all species in the solution. Initial ionic strength was chosen as a parameter to compare CS-CaP suspension; although it diminishes along the calcium phosphates precipitation.

X-Ray Diffraction (XRD) analyses were performed using a D8 Advance Bruker AXS diffractometer (CuKα, 40 kV, 40 mA). Scans were acquired in a θ-θ configuration, from 4 ° to 55 ° with a step time of 129 s and a step size of 0.019 °. During scanning, samples were spinning at 30 rpm. Phase identification was carried out by comparison to standard patterns from the International Center for Diffraction Data -Powder Diffraction Files (ICDD-PDF) with the aid of DiffracPlus EVA software (Bruker, AXS). The PDF used for dicalcium phosphate dihydrate (DCPD, CaHPO4.2H2O) identification was 09-077. The obtained diffractograms were normalized with respect to the height of the peak of greatest intensity in the crystalline mineral phase.

Optical microscopy analysis was conducted using a Light Axiophot microscope (Zeiss, Germany). Samples of CS-CaP suspensions were fixed between two glass slides and observed in transmission mode.

IV. Results

A. Chitosan solutions

The results of steady viscosity of chitosan solutions are presented in Figure 2. The curves show a plateau of viscosity at low shear rate and a region of decreasing viscosity as shear rate increases.

As concentration of chitosan increased, the viscosity at low shear rate (plateau) increased, the dropping of viscosity with shear rate became sharper, whereas, the shear rate interval of the plateau diminished (Figure 2a). The value of the Newtonian viscosity was evaluated at low shear rates from the extrapolated plateau and is reported in Table 2. It was observed that, at constant shear rate, the shear stress increased as concentration of chitosan increased (Figure 2b). (Tests conducted at shear rates between 10 -4 or 10 -3 s -1 and 100 s -1 at 25 °C).

The rheology behavior shown in Figure 2b can be adjusted to a power-law equation of the form stated in section II. Values of power-law exponent were calculated and reported in Table 2. As concentration of chitosan increased, the power-law exponent decreased. All chitosan solutions had a pH around 5.2.

Table 2. Data of viscosity plateau and n value of chitosan solution (without salts) at different concentrations.

Chitosan concentration [molL

B. Chitosan-salt solutions

Chitosan-salt solutions of different salt concentrations, thus, at different ionic strengths, containing either phosphate or calcium ions and the respective counterions, were prepared by adding the salts to chitosan solution after complete polymer dissolution (see section III.B). The values of salt and chitosan concentration, ionic strength and pH measured are reported in Table 3, columns 2, 3, 4 and 5, respectively.

Table 3. Data of chitosan-salt solutions and non-homogenous chitosan-salt suspensions. The physical appearance of the chitosan-calcium salt and chitosan-phosphate salt solutions was observed by the naked eye and reported here (Table 3, column 6). In all cases, small aggregates were formed instantaneously at first contact between the powder salt and chitosan solution. At low salt concentration, aggregates re-dissolved by stirring, resulting in a homogeneous solution (Figure 3, left side). The chitosan-salt solution became opaque as concentration of salt increased. At higher concentration, a limit of solubility was reached for each salt. Beyond that limit, aggregates could no longer be re-dissolved by stirring. Phase separation occurred (Figure 3, right side). It was noticeable that, under the salt concentrations range tested in this work, this limit was 10 times higher for the case of calcium salts compared with phosphate salts (Table 3, column 2).

As seen in Table 3, pH values were rather constant with salt concentration, except for the case of chitosan-(NH4)2HPO4 solution, for which a slight pH increase was registered (up to 5.9) in the concentration range from 0.02 M to 0.04 M. On the other hand, the pHs of chitosan-salt solutions were slightly higher than that of chitosan solution (pH = 5.2), except for the case of chitosan-NH4H2PO4 solution, for which it remained the same value (Table 3, column 5).

Figure 3. Pictures of chitosan-Ca(NO3)2 and chitosan-NH4H2PO4 solutions in the soluble interval (left side) and suspension beyond the limit of solubility showing the aggregates formation (right side).

The results of steady shear viscosity of chitosan-salt solutions are presented in Figure 4. A concentration limit can be identified in each case, bellow that limit, rheological data are reproducible, above that limit they are not. Curves of viscosity vs. shear rate (Figure 4: a, c, g, e)

show two regions, similar to the case of chitosan solutions: a plateau at low shear rate and a drop of viscosity beyond a certain shear rate. Curves of shear stress vs. shear rate (Figure 4: b, d, f, h), similar to the case of chitosan solutions, indicate that equation ( 2) was applicable to fit those data.

Values of power-law exponent were calculated and reported in Table 3, column 6. It was observed that the n value was rather invariable with concentration for each salt solution. In the case of chitosan-calcium solutions (chitosan-CaCl2 and chitosan-Ca(NO3)2 solutions), the powerlaw exponent, n, was in the range of 0.30, slightly lower than that for chitosan solution (0.34). For the case of chitosan-(NH4)2HPO4, n value was close to that in chitosan solution reference, whereas, it was higher in the case chitosan-NH4H2PO4 solution.

It was observed that in the case of chitosan-phosphate solutions, at constant shear rate, the shear stress decreased as the concentration of salt increased. In the case of chitosan-calcium solutions, this tendency was not so clear (Figure 4: b, d, f, h). Viscosities in the plateau region of all chitosan-salt solutions were read from Figure 4 reported in Table 3, column 5. In all cases, viscosity variation was small.

Values of viscosity at the plateau were higher for chitosan-salt solutions than that of reference chitosan solution (0.15 M; viscosity: 1115 Pa s), except for the case of chitosan-NH4H2PO4 solution, for which viscosity remained about the same.

C. Chitosan-calcium phosphate suspensions

CS-CaP suspensions were prepared and characterized with respect to mineral phase composition, particle dispersion and rheology behavior. Details of CS-CaP suspensions preparation are given in Section III.B. Table 4 reportes the final concentration of species in CS-a and CS-b suspensions. CS-a and CS-b prepared at room temperature and at acid pH (Table 4), were characterized by XRD.

The peaks corresponding to the principal planes of DCPD were identified: (020) at 2θ = 11.681 °, (021) at 2θ = 20.935 °, (041) at 2θ = 29.258 °, (220) at 2θ = 34.156 ° (Figure 5). DCPD was the only detectable crystalline phase of calcium phosphates in CS-a and CS-b suspensions. No diffraction peaks of chitosan were identified indicating that the amount of crystalline chitosan was below the detection limit. The X-ray diffuse halo observed from approximately 2θ = 24 ° to 36 ° in the diffractograms may be attributed to the presence of water (about 96 wt % of the suspension was water). Optical microscopy of chitosan-DCPD suspensions (Figure 6) showed that they were composed of a dispersed phase, platelet aggregates of DCPD in a continuous phase (chitosan solution). In CS-a, the dimensions of DCPD crystal aggregates were 26 ± 7 µm length, 20 ± 5 µm width.

In CS-b, the aggregate size distribution was broader than in CS-a, with many particles in a smaller size range.

Figure 6. Optical micrographs of CS-a and CS-b suspensions. DCPD crystal aggregates in chitosan solution.

Curves of viscosity vs. shear rate, Figure 7a, show the same two regions found in the case of chitosan and chitosan-salt solutions, a plateau at low shear rate and a viscosity drop above a certain shear rate. Therefore, equation ( 2) was applicable to data from Figure 7b. Values of power-law exponent were calculated and reported in Table 4, column 7. CS-a suspension had a lower n value compared with its chitosan solution reference. On the contrary, n value of CS-b was higher compared with its chitosan solution reference. Data of viscosities in the plateau region showed that viscosity of CS-a was higher than in the reference CS-0.15 M, whereas viscosity of CS-b was lower than in the reference CS-0.14 M (Table 4, column 6). It was observed in Figure 7b that, at a given shear rate, shear stress was higher in CS-a than in the reference CS-0.15 M, whereas shear stress was lower in CS-b than in the reference CS-0.14 M. Salt concentration was lower in CS-a compared with CS-b. CS-a had about the same pH than CS-0.15 M. CS-b had lower pH than CS-0.14 M.

V. Discussion

A. Chitosan solutions

In acidic aqueous solutions, the amine groups of chitosan were mostly protonated. This positive charge induced repulsion with other positive charges on the polymer chains, producing an extended conformation of the macromolecules. Therefore, chitosan chains occupied a relatively large hydrodynamic volume that maximized interactions with solvent molecules via hydrogen bonding.

On the other hand, chitosan chains may form entanglements. One criterion to establish if entanglements occur is to calculate the relation between the viscosity of the polymer solution, 𝜂, and the viscosity of the solvent, 𝜂 𝑠 . If that relation is higher than 50, entanglements occur [53].

𝐼𝑓 𝜂 𝜂 𝑠 > 50 𝑒𝑛𝑡𝑎𝑛𝑔𝑙𝑒𝑚𝑒𝑛𝑡𝑠 𝑜𝑐𝑐𝑢𝑟 (6)
In the case of all systems treated in this work, it is safe to affirm that entanglements occurred since the viscosity of the solvent was in the order of 10 -3 Pa s and the viscosity of polymer systems were in the order of 10 to 10 3 Pa s.

The viscosity of chitosan solution is impacted by the hydrodynamic volume of the macromolecules, polymer-solvent interactions and chain entanglements. It is determined by flow restrictions of solvent and polymer molecules. The mobility of solvent is mostly restricted by the interference of the volume occupied by polymer molecules and by the polymer-solvent interactions via hydrogen bonds.

As chitosan concentration increased, the polymer-solvent interactions and the entanglements increased, which explains the increase in viscosity observed in Figure 2a and Table 2.

The aspect of the viscosity vs. shear rate curves and the values of power-law exponent, lower than 1, are typical of fluids with shear-thinning behavior (the lower the n value, the higher shearthinning behavior) (Figure 2 and Table 2). Shear-thinning is due to the effect of shear on chain entanglements [54][55][56]. As shear rate is applied, disentanglement occurs, the fluid becomes more Newtonian; therefore, viscosity decreases progressively. Physical junctions between groups of chitosan chain may also be broken down under shear, contributing to reduce viscosity [57].

On increasing the chitosan concentration, the number of interactions between polymer chains increased and so, the effect of shear rate was more pronounced: the viscosity plateau was reduced and the n value was lower.

B. Chitosan-salt solutions

Chitosan-salt solutions under analysis contained acidic water, chitosan extended polymer molecules and protonated amine groups, entangled polymer chains and ions formed from the dissociation of salts.

Salt dissociation reactions at the current conditions of pH are:

Ca(NO 3 ) 2 → Ca 2+ + 2NO 3 - (7)
CaCl 2 → Ca 2+ + 2Cl - (8)
NH 4 H 2 PO 4 → NH 4 + + H 2 PO 4 - (9)
H 2 PO 4 -⇄ 𝐻 + + 𝐻𝑃𝑂 4 2-(𝑝𝐾 𝑎 ~7.2) (10)
(NH 4 ) 2 HPO 4 → 2𝑁𝐻 4 + + 𝐻𝑃𝑂 4 2- ( 11
)
𝐻𝑃𝑂 4 2-+ 𝐻 + ⇄ H 2 PO 4 -(𝑝𝐾 𝑎 ~7.2) (12)
As was mentioned in the theoretical background, chitosan-calcium solutions contain Ca 2+ ions that may form coordination bonds between the free electron pair of electron donor atoms in chitosan molecule (nitrogen or oxygen) and the void orbital of Ca 2+ ion. These interactions may form physical cross-links between chitosan chains. Chitosan-phosphate solutions contain the anions HPO4 2-and H2PO4 -. The divalent phosphate anions may generate electrostatic interactions with protonated amine groups, forming physical cross-links between chitosan chains. The monovalent phosphate anions cannot form cross-links between polymer chains.

Consequently, the effect of pH on salt dissociation should be considered. As reported in Table 3, column 5, pH for chitosan-(NH4)2HPO4 was in the range from 5.5 to 5.9, whereas the pH of chitosan-NH4H2PO4 was about 5.3. In this pH range (5.3-5.9), the dissociation of phosphate salts in chitosan-phosphate solutions formed HPO4 2-in a proportion 4 times higher in (NH4)2HPO4 than in NH4H2PO4. The dissociation of (NH4)2HPO4 also caused a higher concentration of NH4 + (equation 11, stoichiometric factor of two). Therefore, higher ionic strength would be expected in chitosan-(NH4)2HPO4 solutions, as effectively reported in Table 3, column 4.

The low solubility of phosphate salt in chitosan solution may be explained by the strong interactions between chitosan and divalent phosphate ions, leading to the precipitation of the chitosan-phosphate complex. Interestingly, we showed that interactions between calciumphosphate ions were stronger than the polymer-ion interactions, irrespective of the nature of the ion. Indeed, by adding calcium (or phosphate) salt to the limit of solubility of chitosan-phosphate (or chitosan-calcium), a re-dissolution of the chitosan-ion aggregates was observed, with the formation of a homogeneous dispersion of small particles. It may also indicate that interactions between chitosan and ions were reversible interactions.

Rheology data of all reported chitosan-salt solutions corresponded to power-law fluids with shearthinning behavior.

In the case of chitosan-(NH4)2HPO4 solution, viscosity was higher by a factor of 2, whereas n value was about the same compared with chitosan solution (Table 3). Viscosity tended to increase with physical cross-linking of polymer chains induced by divalent phosphate ions, and to decrease with higher ionic strength. The resulted higher viscosity reflected that in this case the effect of polymer cross-link was predominant.

As was stated before, chitosan-NH4H2PO4 solutions had a higher proportion of monovalent phosphate ions with respect to chitosan-(NH4)2HPO4 solutions. Thus, the former solution would have a lower ionic strength and lower density of physical cross-links of polymer chains than the latter. The ionic strength would be lower in reference chitosan solutions compared with salt solutions. Compared with chitosan solution, viscosity was the same indicating that tendencies brought by ionic strength and cross-link of polymer chains balanced off. Chitosan-NH4H2PO4 solutions showed higher n value in comparison with chitosan solution, indication of being a more Newtonian fluid than reference chitosan solution. It would mean that, in this case, the tendency provoked by ionic strength was predominant over the effect of polymer chain cross-links.

In the case of chitosan-calcium solutions, for a same initial ionic strength 0.375 M or 0.750 M, the values of pH, viscosity and n were not significantly different in chitosan-Ca(NO3)2 and chitosan-CaCl2, indicating that there was no different effect of counterions nitrate and chloride. With respect to chitosan solutions, pH was about the same, n was slightly lower (0.30 compared with 0.34) and viscosity was higher, by a factor of 4, in chitosan-calcium solutions (Table 3). Higher viscosity may be the effect of physical cross-links of polymer chains due to chitosan-calcium ion interactions. The less Newtonian fluid, indicated by a slightly lower n value, may be also explained as the effect of physical cross-links of polymer chains.

The mentioned concentration limit of chitosan-salt solutions, beyond which the solution became a non-homogenous suspension, that is, the occurrence of de-solvation and phase separation, may be due to an increase in ionic strength or to an increase in physical cross-linking. Both causes may be concurrent.

C. Chitosan-calcium phosphate suspensions

As described in section III.B, chitosan-calcium phosphate suspensions were prepared by simultaneous addition of calcium and phosphate salts solutions into chitosan solution. Hence, CS-CaP suspensions under analysis consisted in an acidic aqueous medium containing i) chitosan in solution, ii) DCPD particles (Figure 5 and Figure 6), iii) remaining phosphate and calcium ions in solution, iii) nitrate or chloride anions depending on the calcium salt used, iv) ammonium cations from the phosphate salts.

As was reported above, a limit of solubility was not achieved in chitosan-calcium phosphate suspensions, even if the final concentration of phosphate salt in the suspensions was already above the solubility limit reported for chitosan-phosphate solution (Table 3). This result could be explained considering the high affinity between calcium and phosphate ions to form calcium phosphates. Based on XRD results, the calcium phosphate phase in the suspensions was DCPD (Figure 5). It means that the resultant system was a suspension composed by a continuous phase of chitosan solution with calcium phosphate particles. Formation of DCPD carried a decrease of ions in solution. The consumption of ions forming DCPD caused a diminution of polymer chain physical cross-links via chitosan-divalent phosphate and chitosan-calcium interactions. However, since the initial Ca/P molar ratio was 1.67 and the stoichiometric ratio Ca/P in DCPD is 1, calcium ions remained in solution impacting ionic strength and being available to form physical cross-links between chitosan chains. Additionally, counterions would also impact the ionic strength. Therefore, ionic strength in chitosan-DCPD suspensions would be higher than in chitosan solution.

On the other hand, DCPD crystals contain Ca 2+ , HPO4 2-ions and water molecules. Chitosan-DCPD interactions may occur in the suspensions: calcium and divalent orthophosphate ions may form physical cross-links between polymer chains and hydrogen bonds may form between water molecules of DCPD crystals and hydrophilic groups in chitosan molecules.

Rheology tests showed that CS-a (Ca(NO3)2-(NH4)2HPO4) and CS-b (CaCl2-NH4H2PO4) suspensions were power-law fluids with shear-thinning behavior. As compared with chitosan solution (CS-0.15 M), CS-a suspension had a higher viscosity (a factor of 3) and lower n (0.28).

As compared with chitosan solution (CS-0.14 M), CS-b suspension had a lower viscosity (a factor of 1/3) and higher n (0.40) (Figure 7).

In the case of CS-a (pH = 5.3), salts dissociated completely, forming calcium ions and divalent phosphate ions (these same dissociation reactions occurred when salt solution was in contact with chitosan solution, pH = 5.5) (equation 7 and 11). Divalent phosphate ions were consumed in various reactions: 1) forming DCPD with calcium ions (equation 13); 2) forming an equilibrium reaction with monovalent phosphate ions (equation 12); 3) forming polymer chain physical crosslinks by electrostatic interaction with protonated amine groups. Low concentration of monovalent phosphate ions and much lower concentration of divalent phosphate ions would be the result of this dynamic. Ionic strength was influenced by mono and divalent phosphate, ammonium ions and nitrate counterions.

Ca(NO 3 ) 2 → Ca 2+ + 2NO 3 - (7)
(NH 4 ) 2 HPO 4 → 2𝑁𝐻 4 + + 𝐻𝑃𝑂 4 2- (11)
Ca 2+ + HPO 4 2-+ 2𝑂𝐻 -+ 2𝐻 + → 𝐶𝑎𝐻𝑃𝑂 4 2𝐻 2 𝑂 ( 13
)
𝐻𝑃𝑂 4 2-+ 𝐻 + ⇄ H 2 PO 4 -(𝑝𝐾 𝑎 ~7.2) (12)
Comparing CS-a and its chitosan solution reference, the former was a less Newtonian fluid due to the presence of DCPD crystal aggregates and polymer chain cross-links. The less Newtonian fluid nature was revealed by a higher shear-thinning behavior (lower n) and by a higher viscosity. Apparently, the ionic strength was not sufficiently high to induce a significant decrease of viscosity by disentanglement of polymer chains.

For the case of CS-b, salts dissociated completely forming calcium and monovalent phosphate ions (equation 8 and 9). At initial pH = 5.2 (chitosan solution), divalent phosphate ions were formed in the equilibrium reaction (equation 10). In this reaction, the pH decreased. The divalent phosphate and calcium ions formed DCPD (equation 13). Divalent phosphate ions were spent forming DCPD as they were generated. Thus, the concentrations of divalent and monovalent phosphate ions were expected to remain low.

CaCl 2 → Ca 2+ + 2Cl - (8)
NH 4 H 2 PO 4 → NH 4 + + H 2 PO 4 - (9)
H 2 PO 4 -⇄ 𝐻 + + HPO 4 2-(𝑝𝐾 𝑎 ~7.2) ( 10
)
Ca 2+ + HPO 4 2-+ 2OH -+ 2H + → 𝐶𝑎𝐻𝑃𝑂 4 2𝐻 2 𝑂 (13)
As pH decreased, pH < 4, the phosphate ion equilibrium was no longer between the divalent and monovalent phosphate ions but between monovalent phosphate ions and phosphoric acid (equation 14). The prevalent presence of monovalent ions (98 % compared to phosphoric acid) contributed to increase the ionic strength of the system without any possibility to form physical cross-links or to be transformed into divalent phosphate ions.

H 2 PO 4 -+ 𝐻 + ⇄ H 3 PO 4 (𝑝𝐾 𝑎 ~2.1) (14)
Comparing CS-b and its chitosan solution reference, the former was a more Newtonian fluid reflected in lower viscosity and less shear-thinning behavior. As stated in the theoretical background, at high ionic strength, repulsive electrostatic forces between chitosan chains are reduced; polymer chain entanglements decrease, then viscosity and shear-thinning behavior decrease.

VI. Conclusions

The systems analyzed in this work were chitosan, chitosan-calcium or chitosan-phosphate solutions and chitosan-DCPD suspensions.

The basic interactions derived from composition and pH of the systems were: i) repulsions between polymer chains due to protonated amine groups in the polymer chains; ii) hydrophilic interactions polymer-solvent via hydrogen bonding; iii) polymer-polymer interaction expressed in entanglement of polymer chains; iv) coordination bonds between polymer and calcium ions that may lead to physical cross-linking of chains; v) electrostatic interaction of divalent phosphate ions with protonated amine groups that may form physical crosslinks between polymer chains; vi) affinity between calcium and phosphate ions to form calcium phosphate phases (DCPD at pH from 4 to 6) and, vii) screening of electrostatic interactions due to the ionic strength in the system.

All tested systems were power-law fluids with shear-thinning behavior.

In the case of pure chitosan solutions, viscosity plateau and shear-thinning behavior increased with chitosan concentration since at higher concentration, more chain entanglement occurs.

Viscosity plateau in chitosan-salt solutions and chitosan-DCPD suspensions depended on two opposite tendencies: increase with the presence of DCPD particles and with physical cross-link of polymer chains, decrease with ionic strength. Shear-thinning behavior of chitosan-salt solutions and chitosan-DCPD suspensions depended on two opposite tendencies: cross-link tends to increase shear-thinning behavior whereas ionic strength tends to decrease it. Chitosan-calcium solutions resulted in less Newtonian fluids than chitosan solution alone due to the chitosan-calcium ion interactions that may form physical cross-links between chitosan chains.

The interactions chitosan-phosphate ions depend on the type of phosphate ion in solution. The monovalent phosphate ions cannot form physical cross-links between chitosan chains, whereas divalent phosphate ions can. Chitosan-phosphate solutions would be less Newtonian fluids as concentration of divalent phosphate ions increases.

A limit of solubility of salt in chitosan solution was achieved when ionic strength and/or density of chitosan chain physical cross-links were high enough; then chitosan molecules became insoluble in acid aqueous solutions. This upper limit was lower in chitosan-phosphate than chitosan-calcium solutions, indicating stronger physico-chemical interactions in chitosanphosphates systems.

Affinity between calcium and phosphate ions to form DCPD was higher than affinity chitosancalcium and chitosan-phosphate interactions. Therefore, the formation of physical cross-links between chitosan chains and the ionic strength of chitosan-calcium phosphates suspensions were lower than in chitosan-salt solution (calcium or phosphate) due to the formation of DCPD particles.

The pair of salts used to form DCPD affects the physico-chemical interactions in the system. In the case chitosan-Ca(NO3)2-(NH4)2HPO4 suspension, at pH = 5.3, equilibrium monovalentdivalent phosphates ions occurred. Polymer chain physical cross-links, relatively weak ionic strength and the presence of DCPD led to a relatively high structured fluid (less Newtonian). In the case chitosan-CaCl2-NH4H2PO4 suspension, at pH = 3.8, equilibrium monovalent phosphate ion-phosphoric acid occurred; equilibrium monovalent-divalent phosphate ions could not occur.

The high ionic strength caused by remaining monovalent phosphate ions and the relative low density of polymer chain physical cross-links led to a less structure fluid, more Newtonian, even in comparison with chitosan solution alone.

The study of the rheological behavior and the possible physico-chemical interactions that may occur in the homogenous chitosan-calcium phosphate suspensions gave valuable information to set up methods of preparation of chitosan hydrogel-calcium phosphate composites. The concentrations of calcium salt, phosphate salt and chitosan solutions used to come up with a homogenous suspension were established with this work. Since the nature of the salts used to form calcium phosphates have an impact on the rheological behavior of chitosan-DCPD suspensions, it seems interesting to study the effect of salts of different chemical composition on rheological properties of chitosan hydrogel-calcium phosphate composites.

On the other hand, the method of mixing salts and chitosan solutions may also impact the rheological properties of the chitosan hydrogel-calcium phosphate composites. Besides, simultaneous addition of salt solutions into chitosan solution, other mixing methods could be tested; for example, precipitate first the calcium phosphates and then add it into chitosan solution.

Crystal aggregate size and chitosan-ions interactions may be impacted.

Part II: Approaches of mineralization

Part II exposes the methods developed during my Ph.D. work to fabricate chitosan-apatite composites, keeping in mind two principal goals: i) to prepare materials that resembles some features of extracellular matrix: composition, calcium to phosphorus atomic ratio of the inorganic phase, and dispersion of poorly crystalline apatite in a continuous biopolymer matrix; ii) to investigate the physico-chemical interactions occurring in these complex materials during their fabrications. This part of the thesis is developed in two chapters. The first one reports the fabrication of chitosan physical hydrogels gelled with different bases (sodium hydroxide, ammonia vapors, calcium hydroxide), and, once gelled, their subsequent mineralization with apatite particles. Composite materials were characterized with respect to chemical composition and microstructure.

The second chapter describes methods of preparation of mineralized hydrogels by the simultaneous conversion of homogenous suspensions of calcium phosphate particles in chitosan solutions, into chitosan physical hydrogels with intertwined particles of apatite, by increasing the pH. Different variables were studied: nature of salts used as sources of calcium phosphates, methods of preparation of chitosan-calcium phosphate suspensions and bases used for gelation. Discussion is presented on the effects of these variables upon microstructure, physico-chemical interactions, rheological and biological properties of the resulting materials.

La partie II de ce manuscrit expose les méthodes d'élaboration développées au cours de ce travail de thèse pour préparer des matériaux composites chitosane -apatite, tout en gardant à l'esprit deux buts majeurs : i). préparer des matériaux qui ont des caractéristiques proches de celles de la matrice extra-cellulaire : composition, rapport atomique calcium sur phosphore et répartition d'une phase apatitique peu cristalline à l'intérieur d'une matrice continue de biopolymère ; ii). étudier les interactions physico-chimiques existant dans ces systèmes complexes

I. Introduction

Hydrogels are polymer networks, mechanically stable, that can swell or release water or aqueous media [1]. Physical hydrogels of chitosan can be obtained by the neutralization of polymer aqueous solutions in well-defined and controlled conditions [2,3]. The solution state of chitosan occurs at low pH (pH < 6.2), and it is due to the presence of protonated amine groups. This protonation allows the polymer dissolution by the establishment of both, electrostatic repulsive forces between polymer chains and hydrogen bonds between water and chitosan. Physical gelation can occur at neutral or basic pH (pKa ~ 6.5) and it is due to a modification of the balance between hydrophilic and hydrophobic interactions within the solution [2]. Such gelation occurs when the solution is in contact with a base, e.g., ammonia vapors [2] or sodium hydroxide solution [3]. The process of gelation follows progressively within the solution, showing a sol-gel transition layer, also called "gel front". No external crosslinking agent is necessary for this gelation process but caution should be taken to have and preserve a minimum chain entanglement density to avoid chitosan precipitation [2].

The gelation process and the microstructure of the formed hydrogel depend on the solution characteristics (charge density of the polymer, dielectric constant of the solution, degree of acetylation (DA) corresponding to the molar fraction of N-acetyl glucosamine units, weight average molar mass (Mw), polymer concentration) and on the base used to increase the pH [4,5].

The microstructure can generally be described by the presence of a dense layer of approximately 200 μm deep, formed at the interface between the base use for gelation and the chitosan solution (Figure 1). This layer is compact and homogenous and presents strong mechanical properties [6,7]. Below this dense layer, the presence of capillaries has been reported in specific conditions [3,5,6]. These capillaries are oriented microstructures parallel to the direction of the "gel front" 66 [5,6] (Figure 1). Those capillaries may result from convective flows near the "gel front" during the gelation process [5,6].

Using ammonia vapors as a base, capillaries of 4-6 μm diameter have been evidenced by Small Angle Laser Light Scattering (SALS) (for chitosan hydrogel of polymer concentration 1 wt%, DA 1.5%, Mw 515 kg.mol -1 , 7.7 mol.L -1 NH4OH solution) [5]. When a solution of sodium hydroxide is used as a base, capillaries of 10-50μm diameter have been observed by SALS and Confocal Laser Scanning Microscopy (CLSM) (for chitosan hydrogel of polymer concentration between 1 and 2.5 wt%, DA 4.0%, Mw 570 kg.mol -1 , 1mol.L -1 NaOH) [3,6]. The objective of this work is to design materials for bone substitutes. One possibility is to fabricate chitosan physical hydrogels and then induce the mineralization of chitosan hydrogels with calcium phosphates (CaPs). This bio-inspired composite material would combine the biological properties of chitosan (bioresorbability*, angiogenic*, bacteriostatic* and fungistatic* properties [8][9][10][11]) and calcium phosphate apatite (bioresorbability and bioactivity*) [12]. The properties of the apatite phase and of the chitosan hydrogel in the final composite depend on the physico-chemical conditions during fabrication (e.g., pH and temperature) [12,13].

Several authors have already investigated the fabrication of such chitosan-calcium phosphates composites. Examples of methods of fabrication are: i) incorporation of chitosan (powder or solution) in calcium phosphate paste to form calcium phosphate-chitosan cements [14][15][16][17][18][19][20][21][22][23][24][25][26]; ii) mineralization of freeze-dried chitosan scaffold in simulated body fluid or in saturated solutions [27][28][29]; and iii) impregnation of calcium phosphates scaffolds with chitosan [27,29,30].

Frequently, in the last step of fabrication methods, composite materials are freeze-dried or dried at room temperature for further use and characterization [14].

The above-mentioned methods effectively lead to composite chitosan-calcium phosphate materials. However, a major concern is the lack of homogeneity at microscopic level, which may induce practical inconveniences in medical and industrial applications [14]. In our work, we intend to make profit of the possible presence of the capillaries in chitosan hydrogels to favor the diffusion across the hydrogel and obtain even distribution of mineral particles throughout the whole material. Interestingly, capillaries may also favor the invasion of cells and hence provide

Capillaries

increased biological properties to the hydrogel: the cellular invasion may lead to a fast neotissue formation progressively replacing all the volume of the hydrogel implant [3,6].

In this work, four strategies were developed to mineralize chitosan hydrogels with calcium phosphate ceramics. The resulting microstructure and the nature of inorganic phase of composites were then investigated. As reported previously [3,5,6], the microstructure of hydrogel depended on the base used. The impact of this microstructure in the mineralization with calcium phosphates was studied.

II. Materials and Methods

A. Physical hydrogels of chitosan (non-mineralized)

Fabrication of hydrogels

Physical hydrogels were obtained by the neutralization of chitosan solutions with different bases. Table 1 sumarizes the experimental conditions investigated in this work.

Chitosan powder produced from squid pens and supplied by Mahtani Chitosan Pvt. Ltd (batch type 114, No S3 20110121) was characterized with standard procedures [2]. The degree of acetylation, corresponding to the molar fraction of acetylated units, was close to 5%; the weightaverage molar mass and dispersity were Mw = 550 kg.mol -1 and Ð = 1.9, respectively.

Chitosan solutions were prepared by adding chitosan powder in acetic acid aqueous solution (acetic acid: Carlo Erba Reagents, CAS 64-19-7, assay 99.9%). The mass of chitosan powder was calculated taking into account the molar mass of the repeating unit (D-glucosamine and N-acetyl glucosamine), 163.10 g.mol -1 . The amount of acetic acid was adjusted to match the stoichiometric protonation of -NH2 sites of chitosan. The complete dissolution of chitosan was reached in about 18 h under magnetic stirring. Chitosan solutions were then poured in Petri dishes of 3.5 cm in diameter and allowed to rest for 6 h to eliminate air bubbles. Chitosan hydrogel disks were prepared in two sizes for further characterization: i) small hydrogels: 1 mm height, 14 mm diameter (for Confocal Laser-Scanning Microscope characterization) and ii) larger hydrogels: 5 mm height, 35 mm diameter (for mineralization strategies).

Three bases were used: sodium hydroxide solution, calcium hydroxide suspension and ammonia vapors: (i) Hydrogels denoted as H1 in this work were obtained by pouring (~ 4 mL of) a solution of 1 M NaOH (Carlo Erba Reagents, CAS 1310-73-2, assay ≥ 97%) on a 1.5 wt% chitosan solution contained in the Petri dish. The contact time between NaOH solution and chitosan solution was 1 h, this time was sufficient for having a complete gelation (from the top to the bottom of the gels); (ii) hydrogels denoted H2 were prepared by pouring (~ 4 mL of) a suspension of 0.5 M Ca(OH)2 (Sigma-Aldrich, CAS 1305-62-0, assay ≥ 96%) on a 1.5 wt% chitosan solution.

The contact time between Ca(OH)2 suspension and chitosan solution was 1 h or 64 h for smaller and larger disks respectively; (iii) chitosan solutions were also gelled with NH3 vapors: to obtain a saturated atmosphere, 25 mL of NH4OH solution (Sigma-Aldrich, CAS 1336-21-6, assay 28-30%) were left for 18 h inside a tight 5 L desiccator, close to the Petri dish containing the chitosan solution to be gelled. Two concentrations of NH4OH solution were used: 1 M (hydrogels H3) and 68 7.7 M to prepare hydrogels H4 to H7 from different chitosan concentrations: 1.3, 1.5, 2 and 3.5 wt% (Table 1).

In all cases, once gelled, hydrogels were washed several times with reserve osmosis water until pH in the final washing water was neutral, around 7. Hydrogels were stored in reserve osmosis water to prevent drying prior to the step of mineralization and further characterizations.

Characterization of hydrogels

The microstructure of the hydrogels was investigated by inverted Confocal Laser-Scanning Microscope (CLSM, Zeiss LSM 510, USA) powered by an argon laser (λ=488 nm) and with the following settings: oil immersion, 40 x lens and 1.3 of numerical aperture. The smaller hydrogel samples (1 mm in height and 14 mm in diameter) were placed directly between two glass slides. A few drops of water were added to maintain the hydration state of the hydrogels during observation. No fluorescent stain was used; the obtained micrographs were artificially colored in green to distinguish the hydrogels of chitosan from empty spaces and porosities [6].

Micrographs were taken at about 350 μm deep from the top of the hydrogel. The diameter of capillaries and their density distribution were estimated by analyzing three micrographs (evaluation surface of 126 μm 2 each) for each hydrogel, using the software ImageJ [32]. Results are reported in Table 1.

Dehydration and rehydration tests

In search of establishing protocols of mineralization of hydrogels, dehydration and rehydration studies of hydrogels H1 and H3 were performed (H2 was not included because of difficulties in handling the samples). Samples of H1 and H3 were dehydrated with the purpose of favoring the salt aqueous solution intake during rehydration. The dehydration of three samples of each hydrogel was performed in a drying-oven at a mild temperature of 37 °C, to avoid damaging the hydrogel structure. Hydrogels were left on a grid support during dehydration in such a way that their bottom (as defined in Figure 1) was in contact with the grid support. The weight of the samples was registered versus time to study the kinetics of dehydration.

Rehydration was carried out by complete immersion of the dehydrated hydrogels in water (reference samples) or salt aqueous solution at room temperature. Hydrogels were immersed under the same configuration in salt solutions in such a way that their bottom was in contact with the bottom of beaker. The salt solutions used were 5 M Ca(NO3)2.4H2O (Sigma-Aldrich, CAS 13477-34-4, assay ≥ 99%) of density ca = 2.18 g.mol -1 and 3 M (NH4)2HPO4 (Sigma-Aldrich, CAS 7783-28-0, assay ≥ 99%) of density p = 1.40 g.mol -1 . The flask, containing the immersion bath (salt solution or water) and the hydrogel, was closed with parafilm M® during the procedure. Rehydration was performed until final constant weight. The evolution of the weight of samples was registered versus time to study the kinetics of rehydration.

The weight percentage lost or gained during each step of the process was calculated with equation (1).

% 𝑤𝑒𝑖𝑔ℎ𝑡 𝑔𝑎𝑖𝑛𝑒𝑑 𝑜𝑟 𝑙𝑜𝑠𝑠 = 𝑤𝑒𝑖𝑔ℎ𝑡 𝑎𝑓𝑡𝑒𝑟 𝑡ℎ𝑒 𝑐𝑜𝑛𝑠𝑖𝑑𝑒𝑟𝑒𝑑 (𝑠𝑡𝑒𝑝) -𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑤𝑒𝑖𝑔ℎ𝑡 𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑤𝑒𝑖𝑔ℎ𝑡 * 100 (1)
The percentage weight gained or lost of solution (equation 1) may be affected (i) by the different densities of the three rehydration media and (ii) by the fact that H1 and H3 have different weights due to the different gelation route. Therefore, the volume of solution intake was normalized with the following equation ( 2):

𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 𝑖𝑛𝑡𝑎𝑘𝑒 𝑝𝑒𝑟 𝑔𝑟𝑎𝑚 𝑜𝑓 ℎ𝑦𝑑𝑟𝑜𝑔𝑒𝑙 = 𝑤𝑒𝑖𝑔ℎ𝑡 𝑔𝑎𝑖𝑛𝑒𝑑 𝑑𝑢𝑟𝑖𝑛𝑔 𝑟𝑒ℎ𝑦𝑑𝑟𝑎𝑡𝑖𝑜𝑛 (𝑑𝑒𝑛𝑠𝑖𝑡𝑦 𝑜𝑓𝑟𝑒ℎ𝑦𝑑𝑟𝑎𝑡𝑖𝑜𝑛 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛) * (𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑤𝑒𝑖𝑔ℎ𝑡) (2)
The pH of the salt solutions before and after the rehydration step was measured using the pHmeter Corning® 240 (USA).

B. Mineralized hydrogels of chitosan

Mineralization of hydrogels

Three main strategies to mineralize H1 and one strategy to mineralize H2 were tested, as schematized in Figure 2.

Strategy 1 consisted of two steps: preparation of a calcium phosphate (CaP) suspension and immersion of hydrogel H1 in the CaP suspension already precipitated. For the preparation of the suspension: 20 mL of a 0.3 M H3PO4 solution (phosphoric acid: Sigma-Adrich, CAS 7664-38-2, assay 49-51 %) were added to 20 mL of 0.5 M Ca(OH)2 under magnetic stirring. The concentration of salt solutions was imposed by the low solubility of calcium hydroxide in water at room temperature (solubility: 1.73 g.L -1 in water at 20 °C). The pH of the suspension was adjusted to 7.4 using few drops of 7.7 M NH4OH or drops of 1 M NaOH (pH was kept at 7.4 to preserve the hydrogel state). The hydrogel H1 was then immersed into the CaP suspension under continuous agitation for 24 h. Strategy 2 consisted in immersion for 48 h of hydrogel H1 in 5 M Ca(NO3)2.4H2O a solution, with pH adjusted to 10 using 1 M NaOH solution, followed by immersion into a solution of 3 M (NH4)2HPO4 under NH3 vapors for 62 h. In this strategy, alkaline environment was maintained in each step since the pH of calcium salt solution and the atmosphere during immersion in phosphate salt solution were controlled using NaOH solution and NH3 vapors respectively.

In strategy 3a, the hydrogel H1 was 30 % dehydrated at 37 °C (equation 1); the dehydrated sample was immersed for 8 h in 5 M Ca(NO3)2.4H2O solution followed by immersion for 24 h in 3 M (NH4)2HPO4 solution.

In strategy 3b, the hydrogel H1 was 30 % dehydrated at 37 °C; the dehydrated sample was immersed for 47 h in 5 M Ca(NO3)2.4H2O solution. The calcium-loaded hydrogel was then 10 % dehydrated at 37 °C. Finally, the sample was immersed for 24 h in a solution of 3 M (NH4)2HPO4.

In strategy 4, the hydrogel H2 was immersed without prior washing in a 0.3 M (NH4)2HPO4 solution, for 24 h (H2 already contained calcium since it was prepared using Ca(OH)2 both, as calcium source and as a base for gelation process).

The initial concentrations of salts used for mineralization strategies were chosen in such a way that the calcium salt was soluble in water, and keeping the Ca/P molar ratio at 1.67 (molar ratio of stoichiometric hydroxyapatite, Ca10(PO4)6OH).

In all cases, the mineralized hydrogels were washed several times by immersion in reverse osmosis water and renewing the water every 10 minutes until the pH of the washing water was stabilized to 7.

Characterization of mineralized hydrogels

The microstructure of mineralized hydrogels was investigated by X-ray micro-tomography (μ-CT, Phoenix v│Tome│x, General Electric, USA, equipped with a Varian Paxscan detector (1920x1536 pixels) and an X-ray source of 1-4 μm). The resolution was set between 3 and 13 μm/voxel. Their composition and crystallinity were investigated by X-Ray Diffraction (XRD) analysis of the surfaces (both top and bottom of the hydrogels, as defined in Figure 1) using a D8 Advance Bruker AXS diffractometer, Germany (CuKα radiation, 40 kV, 40 mA). Scans were acquired in a θ-θ configuration, from 4° to 55° with a step time of 129 s and a step size of 0.019°. During scanning, samples were rotated at 30 rpm. Phase identification was carried out by comparison of the acquired patterns with standard patterns from the International Center for Diffraction Data -Powder Diffraction Files (ICDD-PDF) with the aid of DiffracPlus EVA software (Bruker, AXS). The PDF used in this work were: 44-1481 for Ca(OH)2 and 09-0432 for apatite. Chitosan characteristic reflections were identified based on previous studies [33]. The obtained diffractograms were normalized with respect to the height of the peak of greatest intensity in the crystalline mineral phase.

III. Results

A. Physical hydrogels of chitosan

Characterizations

Immediately after gelation began, a "gel front" was observed to progress from top to bottom of the sample, but a thin dense layer formed on the top of the sample. Thus, once gelation was completed, the top and bottom of the gel were easily distinguishable.

Hydrogels obtained by gelation under NH3 vapors were more opaque than the others; hydrogels obtained with NH3 vapors and with NaOH solution were easier to handle than those obtained with Ca(OH)2 solution. In Figure 3, the general aspect of chitosan hydrogels is shown. Figure 4 shows the CLSM analysis of hydrogels of chitosan (artificially colored in green). Capillaries were found in hydrogels obtained by gelation with NaOH (H1) and Ca(OH)2 (H2), whereas no capillaries were found in hydrogels obtained by gelation with NH3 vapors, neither by CLSM nor by μ-CT, independently of the initial concentration of chitosan solution (hydrogels H4 to H7). Capillaries started to be detected about 100-200 μm below the top of the hydrogel sample, forming oriented microstructures, parallel to the direction of "gel front", as it was expected (Figure 1). Size and density of capillaries were different depending on the base used for the gelation: smaller (12 ± 4 µm vs. 29 ± 9 µm diameter) and denser (0.6 ± 0.12 capillaries/µm 2 vs. 8.10 -2 ± 4.10 -2 capillaries/µm 2 ) capillaries were found in hydrogels H1 than in hydrogels H2 (Table 1). Capillaries were cylindrical in shape and seemed more homogeneously distributed in H1 compared with hydrogels H2 (Figure 4).

Dehydration and rehydration tests

A preliminary study showed that the dehydration stage led to the shrinkage of the hydrogels. Moreover, it was more difficult to fully rehydrate the hydrogels when the dehydration and shrinkage were strong. In fact, when hydrogels were 50 % dehydrated, they could recover up to 70 % of their initial weight by rehydration; whereas, if dehydration was stopped at 30 %, hydrogels could nearly recover 90 % of their initial weight during rehydration. Consequently, for further tests, 30 % dehydration was chosen.

H2

Weight change of samples was recorded versus time during both dehydration and rehydration (Figure 6). For all hydrogels, loss of water during dehydration occurred linearly with time, reaching approximately 30 % dehydration (H1: 29.2 ± 0.6 %; H3: 29.7 ± 0.9 %) in about 195 minutes (Figure 6a). An anisotropic deformation of hydrogel was observed no matter the base used: the bottom of the hydrogel always shrank more than its top. This could be due to the presence of the thin dense layer that apparently limited the shrinkage (Figure 1) on the top of the hydrogel.

Concerning the rehydration stage, it was observed that all hydrogels behave qualitatively in the same manner: after a quick intake of salt solution or water, hydrogels mass tended to stabilize over time. In general, most of rehydration occurred in less than 100 minutes (Figure 6b).

Initial (before dehydration) and final (after rehydration) weights of hydrogels are reported in Table 2, together with the rehydration conditions (salt nature, concentration of rehydration medium and pH of the respective medium). Except for the hydrogel H1-Ca, a net weight lost was observed after the entire process. Additional tendencies also appeared in Table 2: i) the initial weight was systematically higher in H1 hydrogels than in H3 hydrogels; ii) weight recovery during rehydration was higher in H1 hydrogels than in H3 hydrogels; iii) weight recovery was systematically higher in hydrogels rehydrated with solutions of calcium than in hydrogels rehydrated with solutions containing orthophosphate ions; iv) hydrogels rehydrated with reserve osmosis water showed the highest weight lost percentage of solution intake after rehydration.

Considering the volume intake, normalized with respect to the sample weights, i) whatever the rehydration medium, a higher volume of solution diffused in H1 than in H3; ii) for H1, the same volume intake was calculated for all rehydration medium; iii) whereas for H3, the volume of osmosis water was significantly higher than the volume intake of the two salt aqueous solutions.

B. Mineralization of hydrogels

Hydrogels H1 were the ones, which were selected for the main mineralization strategies, since they exhibited large capillaries potentially easing the diffusion of solutions and mineral particles (Table 1).

Strategy 1: hydrogels H1 immersed in a CaP suspension

As shown in Figure 2, the first step of strategy 1 was to prepare a CaP suspension. Evidences of the formation of a suspension of CaP were: i) the precipitate formation immediately after mixing the salt solutions; ii) the peak, although slight, which may correspond to the (211) family planes at 2θ = 31.774 ° and (112) at 2θ = 32.902 ° of apatite, visible in the X-ray diffractogram of CaP suspension (Figure 7). Sedimentation of some precipitates was observed during the XRD acquisition, which can also account for the low intensity of the apatite peaks. After immersion in the CaP suspension, the weight of hydrogel H1 augmented by 11 %. However, part of the weight gained was lost during washing; thus, the net final weight increase was only 3 % after washing. Observing the hydrogel after washing (Figure 8), solid particles remained on the surface of the hydrogel. This was corroborated by μ-CT images that showed the presence of small aggregates, which absorbed the X-rays, more than the hydrogel part (yellow arrows in Figure 9). However, no XRD peak indicated the presence of apatite in the final washed hydrogel (Figure 10, H1: strategy 1).

Strategy 2: hydrogels H1 immersed in ionic solutions

The weight of hydrogel H1 augmented by 32 % after immersion in Ca(NO3)2 solution and to 35 % with respect to the initial weight after immersion in (NH4)2HPO4 solution. Then, after washing step there was a net weight reduction of 14 % with respect to initial weight. The pH of Ca(NO3)2 solution after the immersion remained stable at pH = 10, whereas the pH of (NH4)2HPO4 solution increased up to pH = 12 due to the NH3 atmosphere (Figure 2). A thin transparent membrane appeared surrounding the hydrogel after the second immersion (Figure 11); this membrane mostly disappeared after washing (Figure 12).

Strategies 3a and 3b: dehydration then rehydration of hydrogel H1 in ionic solutions

To also mineralize the bulk of the hydrogels, the diffusion of calcium and phosphate ions through the hydrogels should be eased. Thus, a preliminary dehydration step was implemented, to favor the intake and diffusion of ionic solutions. The highest gain of water or solution during

cm cm

500 µm chitosan rehydration occurred in hydrogels H1 (Table 2); thus, these hydrogels were selected for mineralization strategies 3a and 3b.

Hydrogels H1 of initial weight (3.7 ± 0.1) g were first dehydrated, then immersed in Ca(NO3)2 solution at pH 3.7 ± 0.1. This immersion led to an increase of pH up to 5.5 ± 0.1 in 8 h (strategy 3a) and further to 6.5 ± 0.1 in 47 h (strategy 3b). The weight of sample increased by 11 % in 8 h (strategy 3a) and by 3 % in 47 h (strategy 3b) (Table 3). In strategy 3a, samples were directly immersed in the second solution, (NH4)2HPO4, whereas in strategy 3b, the hydrogels were dehydrated a second time before immersion in the second solution. In both cases, the pH of (NH4)2HPO4 solution remained constant at 8.2 for 24 h during the immersion of samples (Table 3). The weight of hydrogels increased by 35 % in strategy 3a and by 32 % in strategy 3b. After washing, the weight of samples reduced back: the net weight loss was 14 % with respect to initial weight in strategy 3a and 19 % with respect to initial weight in strategy 3b (Table 3).

Table 3. Changes in weight and in pH during strategies 3a and 3b.

Strategy 3a Strategy 3b

Initial weight of hydrogel H1 weight after the 1 st dehydration After immersion in the (NH4)2HPO4 solution (in 3a and 3b strategies), the hydrogels also appeared to be surrounded by a thin membrane (Figure 14) that mostly disappeared during washing (Figure 15).

Strategy 4: Ca(OH)2 solution used both as a base and as a source of calcium

For the 4 th strategy, hydrogel H2 was used, i.e. the solution of chitosan was gelled with a Ca(OH)2 suspension (Figure 2); then, H2 was immersed in a (NH4)2HPO4 solution. The use of Ca(OH)2

µm

Chitosan hydrogel

µm

Chitosan hydrogel solution both as source of calcium and as a base may offer at least two advantages: there was no need for a first immersion in a calcium solution since calcium was already present and distributed into the gelled hydrogel; also, less counterions were present in the system: only the ammonium counterions from the phosphate salt would remained to be washed in the last step of the process.

XRD analysis of the mineralized H2 surfaces (top and bottom) allowed identifying the peaks corresponding to the principal planes of apatite, (002) at 2θ = 25.879 °, (211) at 2θ = 31.774 ° and (112) at 2θ = 32.902 °(Figure 10, H2: strategy 4). μ-CT slices showed that deposits of an absorbent phase, probably CaP apatite mineral, partly covered the surface (top and bottom) of the hydrogel (Figure 18). Again, no evidence was found of CaP mineral within the hydrogel by μ-CT observations.

IV. Discussion

The discussion will address three main points of the study: the first one will be related to the processing of chitosan physical hydrogels, the second one to the mineralization of chitosan hydrogels with analysis of pH, weight and precipitation of apatite.

A. Physical hydrogels of chitosan (non-mineralized)

Characterization of hydrogels obtained in different physico-chemical conditions

The base used for gelation provides the hydroxyl needed to deprotonate the NH3 + groups of chitosan in solution. As mentioned above, as gelation proceeds, a sol-gel transition was observed to progress from top to bottom of the sample, faster if sodium hydroxide is used as base for gelation, in comparison with ammonia vapors. A "gel-front" was observed immediately after contact between the base used for gelation and the chitosan solution. The speed of the ¨gel-front¨ was observed to decrease as the "gel front" advances since diffusion of NaOH across a thicker hydrogel layer is necessary for the "gel-front" progression [6].

500 µm 500 µm 500 µm 500 µm 500 µm 500 µm 500 µm 500 µm 500 µm

Chitosan hydrogel

The higher speed of the gelation front using sodium hydroxide could be explained by the more rapid availability of hydroxyl ions when i) using a strong base rather than a weak base and ii) ions were already dissociated in solution (compared to alkaline vapors, which first needed to dissolve in the chitosan solution). The immediate formation of a thin dense layer at the interphase between alkaline medium and chitosan constitutes an additional resistance to diffusion of the base to reach the chitosan solution. Such hindered diffusion results in a gradient -property gel, the top layer being more entangled due to immediate gelation, and the bulk hydrogel at larger depths being softer and more disentangled after slower neutralization kinetics [6,7].

In the case of Ca(OH)2, a strong base, availability of OH -anions for gelation was limited by the low solubility of Ca(OH)2 in water. For 0.5 M Ca(OH)2 used as base for gelation, a sediment was for deposited at the base-chitosan interphase constituting an additional mass transfer resistance. Therefore, the observed speed of "gel-front'' advance was slower in comparison with sodium hydroxide solution. Contact time for complete gelation depended on diffusion rate of the base for gelation and on sample size dimensions: 1 h was needed for a hydrogel of 1mm height and 35 mm of diameter, whereas it was 64 h for a hydrogel of 5 mm height, 35 mm diameter.

The presence of capillaries in chitosan hydrogels obtained by gelation with NaOH solution under the conditions tested was already reported [3,6]. The size, shape and density distribution of capillaries found in this study were comparable with those found previously [3,6].

No evidence of capillaries was found in hydrogels obtained using ammonia vapors under the conditions tested (chitosan concentration).

Dehydration and rehydration of hydrogels

Dehydration was accompanied by irreversible shrinkage of hydrogels (H1 and H3); such shrinkage was accentuated with dehydration percentage. Shrinkage in turn seemed to affect the capacity of hydrogel for fluids intake during the rehydration step: as shrinkage increased, the physical crosslink density of chitosan chain increased partly irreversibly, rendering more difficult the medium intake during rehydration.

Figure 6 shows that rehydration (at room temperature) occurred faster than dehydration (at 37°C).

During rehydration, intake of water or salt solutions was probably driven by hydrophilic interactions (chitosan and water) and electrostatic interactions (chitosan and ions in solution) and the water depletion in dehydrated hydrogels. Table 2 shows that the volume of solution intake during rehydration, with respect to initial weight of hydrogel, depends on both the base used for gelation and, for H3, of the rehydration medium. It may be concluded that hydrogels gelled with NaOH solution (H1) had a higher volume of solution intake than those gelled with NH3 vapors (H3). These results may be related to the existence of large capillaries in the case of H1 hydrogels, since these capillaries may favor the diffusion of fluids. In the H3 hydrogels, the absence of large capillaries did not facilitate the solution intake. In this case, hydrophilic and electrostatic interactions may become predominant: a significant difference was evidenced between rehydration with water and rehydration with ions containing solutions. However, the volume intake for (NH4)2HPO4 rehydration solution was about the same as for Ca(NO3)2 rehydration solution. It is to be noted that, due to the acidic pH of the Ca(NO3)2 solution, one cannot exclude that some chitosan may have re-dissolved during rehydration in this medium, thus impacting the weight measured after rehydration.

Difference in volume of solution intake may be explained considering the chitosan-water and chitosan-ions interactions: i) Between chitosan and water, there are hydrophilic interactions via hydrogen bonds between chemical groups of chitosan (hydroxyl group, oxygen, acetyl group and amino group) and water. ii)

Between chitosan and Ca(NO3)2 solution, two types of interactions may be expected.

Chitosan may form coordination bonds with calcium ions due to the interaction between the free electron pair of donor atoms in chitosan molecule (nitrogen and oxygen) and the void orbital of calcium ions [34][35][36]. These interactions may lead to physical cross-links between polymer chains. Besides, the pH of the calcium solution was 3.9 (Table 2). This acid condition may have caused some protonation of chitosan chains and dissolution of part of the hydrogel of chitosan (pKa of chitosan ~ 6.5); in this case, protonated amine groups would be available to form electrostatic interactions with NO3 -ions. Protonated amine groups could also create repulsive forces between polymer chains; therefore, opening of the microstructure may be favored, thus leading to a large intake of Ca(NO3)2 solution into the gel. iii)

Between chitosan and (NH4)2HPO4 solution, electrostatic interactions could occur between the few protonated amine groups present in chitosan hydrogel and the orthophosphate ions. Moreover, the valence of the orthophosphate ions that may interact with chitosan depends on the pH of the medium. Indeed, divalent phosphate ions can form physical cross-link of polymer chains through the interaction with protonated amine groups [35,37]. In this case, the pH of (NH4)2HPO4 solution was 8.2, therefore the orthophosphate ions in equilibrium equation is [38,39]:

𝐻𝑃𝑂 4 2-+ 𝐻 + ⇄ H 2 PO 4 - (4)
Based on the pKa value of 7.21 for this equilibrium, the divalent orthophosphate ions (HPO4 2-) were present in a proportion of 91 %, while monovalent orthophosphate ions (H2PO4 -) corresponded to the remaining 9 % of total orthophosphate ions.

As the highest volume intake in chitosan hydrogels occurred in water, it may indicate that hydrophilic interactions were the driving force for rehydration (Table 2).

B. Mineralization of hydrogels

Analysis of pH during the mineralization step

The pH had a marked influence along all steps to obtain a hydrogel of chitosan mineralized with apatite: pH < 6.2 was a required condition to prepare a solution of chitosan, whereas pH > 6.5 was necessary both for the gelation of chitosan (specifically under physico-chemical conditions tested in this work) and for obtaining a stable apatite phase. pH changes were observed during the performance of mineralization strategies. pH of Ca(NO3)2 solution increased after the immersion of H1 hydrogels. Initially, pH was 3.7 that corresponds to [H + ] = 2*10 -4 mol.L -1 . After immersion, pH values depended on time: after 8h immersion (strategy 3a), pH was 5.5, whereas after 47 h immersion (strategy b), pH was 6.5 (Table 3). These pH values correspond to [H + ] = 3*10 -6 mol.L -1 and [H + ] = 3*10 -7 mol.L -1 respectively. It would be expected that [H + ] were consumed protonation of amine groups. Based on the degree of acetylation and the mass of chitosan in the hydrogel, the concentration of amine groups, available for protonation, was about 3*10 -4 mol.L -1 . It was clear that amine groups were partially protonated since there were more amine groups to be protonated than [H + ] to protonate. Moreover, not all of the available [H + ] was consumed.

In strategies 2, 3 and 4, the spontaneous precipitation of calcium phosphates causes a decrease in pH by changing the distribution of OH -and H + ions, since the global reaction of formation of calcium phosphates generally releases H + ions [40][41][42]. As was mentioned before, pH plays a role in determining the exact phase of the CaP precipitate. Thus, during mineralization tests, the control of the pH was mandatory: the medium was kept alkaline, at pH > 6.5, therefore apatite was the most stable, less soluble, calcium orthophosphate phase.

Evolution of weight of samples during mineralization

In strategies 2, 3a and 3b, after the washing step, there was a weight loss of about 16% with respect to initial weight of chitosan in hydrogels. This may indicate that several species were washed out the mineralized hydrogels: water, ions coming from the salt solutions, particles of calcium phosphate deposited on the surface of the hydrogel and even maybe re-dissolved chitosan. To explain the washing out of chitosan, it should be considered that during the immersion of hydrogels, loaded with calcium, in (NH4)2HPO4 solution (at a pH of 8.2), two phenomena occurred: i) the appearance of a thin membrane surrounding the hydrogel and later removed by the washing step (Figure 11 and Figure 14) and; ii) the precipitation of calcium phosphates onto the hydrogel surface, which may have led to a local decrease of pH. This local decrease in pH on the surface of hydrogel would lead to the protonation of amine groups in chitosan molecules (pKa ~ 6.5), thus to local re-dissolution of chitosan.

In strategy 1, the first step was to prepare a suspension of CaP particles. The decrease of pH due to the liberation of H + ions during the precipitation of CaP was avoided for the immersion of hydrogels: indeed, the pH of the CaP suspension was adjusted and maintained at 7.4 before and after the immersion of the hydrogel in this suspension. Therefore, the local decrease of pH on the hydrogel surface could not happen. Consequently, after immersion in the CaP suspension, no-thin membrane was observed after washing step; a net weight loss was not registered. Nevertheless, no mineralization occurred due to the poor diffusion of preformed particles in the hydrogel bulk.

Strategies 3a and 3b included a dehydration step. The aim of the dehydration step was to favor Ca(NO3) solution intake during rehydration. But, contrary to what was aimed, the weight of H1 decreased with immersion time in Ca(NO3) solution: after 8 h of immersion, the gain of weight was 11 % (strategy 3a, Table 3) and remained constant up to 23 h (Table 2, consistent with the rehydration behavior illustrated in Figure 6b) but decreased to 3 % after 47 h (strategy 3b, Table 3). In the same time, the pH of the Ca(NO3) solution increased from 3.9 to 5.5 in 8 h and further to 6.5 in 47 h (Table 3). Since the gain of weight decreased whereas the pH increased, it is more than probable that the immersion in calcium solution provoked a partial re-dissolution of chitosan hydrogels.

All in all, the dehydration step did not increase the intake of any of the solutions: indeed, the gain of Ca(NO3) solution was significantly higher in strategy 2 (32 % in 48 h of immersion, without prior dehydration) than in strategy 3 (3 % in 47 h of immersion). And, regarding the immersion in (NH4)2HPO4 solution, the gain of weight was about the same in strategies 2 (without any dehydration), 3a (with only one dehydration) and 3b (with two dehydrations) (~34 %).

Formation and distribution of apatite

For all four strategies, the mineralization of hydrogels resulted in the presence of apatite on the external surface of hydrogels but no evidence of mineralization was found deep in the hydrogel bulk. One hypothesis is that no apatite was found inside the hydrogel due to mass transfer resistance. The latter would be caused by two reasons: i) the difficulty for diffusion through the hydrogels; ii) the precipitated apatite on the surface of hydrogel may prevent calcium and phosphate ions to enter further into the hydrogel.

In the strategy 1, CaP suspension (probably made of apatite particles since planes (211) and (112) were identified by XRD analysis, Figure 7) was formed before immersion of H1 hydrogel. There were evidences of deposits of apatite on the surface of the hydrogel after its immersion in CaP suspension. However, it seemed that most of apatite was washed out since no evidence of apatite was found by XRD. It may indicate that most of the apatite remained in suspension and only a small amount rested on the hydrogel surface without strong enough adherence.

In the strategy 2, mineralization of H1 hydrogel was done by consecutive immersion in Ca(NO3) and (NH4)2HPO4 solutions. For the second immersion, in (NH4)2HPO4, as H1 loaded with Ca(NO3) was in contact with (NH4)2HPO4 solution, a rapid precipitation occurred forming CaP mineral. Therefore, a CaP precipitate was formed all around the hydrogel, probably plugging the microstructure of hydrogel preventing the entrance of ions in the bulk. Evidence was found of the presence of apatite on the surface of chitosan hydrogel at the end of the process (Figure 10, H1: strategy 2). Analogous phenomena may have occurred in implementing the strategies 3a and 3b.

Based on XRD analysis of the mineralized hydrogels surfaces (Figure 10), it seemed that apatite was more crystalline in strategy 2 than strategies 3a and 3b reflected in better-defined peaks. It may be explained because the strategy 2 implemented (Figure 2): i) a longer duration of immersion in (NH4)2HPO4 solution (62 h) than in strategies 3a and 3b (24 h); ii) an alkaline environment that was maintained in each step of the process (the pH of calcium salt solution and the atmosphere during immersion in phosphate salt solution were controlled using NaOH solution and NH3 vapors respectively).

Comparing strategy 3a and 3b, apatite was more crystalline in strategy 3b than in 3a. It seemed that the longer immersion time in Ca(NO3) solution and the additional dehydration step in strategy 3b, impacted the apatite crystallinity. However, the weight gained after the immersion in (NH4)2HPO4 solution (final step) was about the same in both strategies (Table 3).

In the strategy 4, diffusion resistance during the gelation step probably arose due to the appearance of sediment of Ca(OH)2 at the interface between chitosan gel-Ca(OH)2. The sediment may be converted to apatite as entering in contact with (NH4)2HPO4 solution. This formed apatite preserved the resistance to diffusion inside the hydrogel. In this strategy, it seemed that apatite crystallinity was about the same as observed in strategy 2 (Figure 10, H2: strategy 4).

V. Conclusions

Chitosan physical hydrogels were obtained by gelation of chitosan aqueous solution with various bases: sodium hydroxide solution, calcium hydroxide suspension and ammonia vapors. In all cases, hydrogels were predominantly XRD -amorphous. The final microstructure depended on the gelation route: capillaries were found in hydrogels obtained by gelation with sodium hydroxide solution and calcium hydroxide suspension, no capillaries were detected in hydrogels obtained by gelation with ammonia vapors occurring with a much slower kinetics. As in previous studies [3,6], capillaries were oriented parallel to the direction of the gelation base within the solution but they were smaller and denser in hydrogels gelled with sodium hydroxide.

Calcium hydroxide suspension was used both as a source of calcium to form calcium phosphates and as a base for gelation. This dual use of calcium hydroxide makes unnecessary the immersion of the hydrogel in a different calcium solution and minimizes the presence of counterions in the system. However, the low solubility of calcium hydroxide in water limits its use to low concentration, affecting the gelation rate (speed of the "gel-front" advance) and the mechanical properties (handling) of the obtained hydrogels. Moreover, the deposit of sediments at the interface between chitosan hydrogel-calcium hydroxide suspension may constitute a mass transfer resistance. These difficulties remained to be solved, for instance by tuning the properties of the calcium hydroxide powder (e.g., particle size).

Four different strategies were implemented to mineralize physical hydrogels of chitosan: immersion of hydrogel in calcium phosphate suspension; immersion of hydrogel in solutions of calcium and phosphate; dehydration and rehydration of hydrogels in calcium and phosphate solutions; suspension of calcium hydroxide to gel chitosan solution and to provide calcium ions followed by immersion of calcium loaded hydrogel in phosphate solution.

Dehydration kinetics was similar in hydrogels obtained with sodium hydroxide solution and NH3 vapors. However, volume of solution intake during rehydration procedure depended on both, the gelation route and the rehydration medium. Hydrogels obtained with sodium hydroxide showed capillaries that may serve as micro ducts facilitating flow of liquid solutions. The differences in volume intake with rehydration medium were explained considering physico-chemical interactions chitosan -water and chitosan -ions in solution. The higher volume intake of water compared with intake volume of salt solutions was explained as resulting of stronger interactions chitosan-water than chitosan-salt ions, confirming the hydrophilic property of chitosan hydrogel network.

The condition of pH had a marked influence on all steps of obtaining a mineralized hydrogel of chitosan.

In some mineralization strategies, a weight loss of about 16 % occurred during the final step corresponding to the washing of hydrogel. This weigh loss was attributed to washing out of counterions of dissolved salts, apatite resting on the surface of the hydrogel and even probably chitosan locally dissolved, due to partial protonation of amine groups by H + ions released from the reaction of calcium and phosphate salts during precipitation of apatite.

The common result of mineralization of hydrogels, under all four strategies, was the appearance of apatite on the external surface of hydrogels and no evidence of mineralization inside the hydrogels. The absence of apatite inside the hydrogels may be due to the difficult diffusion of ions through the hydrogels probably because of physico-chemical interactions among ions and between ions and chitosan.

A more in-depth study of the mineralization inside the hydrogel will be performed to assess whether or not there was a presence of nano particles of apatite (possibly amorphous particles) in the matrix. Characterization techniques such Infrared microscopy and Small angle X-ray scattering could be used with this purpose.

The reported experiments and results of mineralization of chitosan physical hydrogel call for research paths to fabricate mineralized hydrogels with a homogenous dispersion of calcium phosphate within the hydrogel. Another approach to overcome the lack of homogeneity would be the preparation of homogeneous chitosan-calcium phosphate suspensions and then their conversion into chitosan hydrogels mineralized with apatite.

Introduction

A promising approach to deal with healing and replacement of damaged bone tissue is the use of a synthetic bone substitute. Bone substitutes are materials that temporary replace bone tissue, promote bone formation and growth, until finally they are bioresorbed*. Beside those basic properties, a compromise should be met between sophistication, ease of fabrication, ease of handling by the surgeon and costs [1]. It would be difficult to find a single material that meets all these requirements. One alternative is to fabricate a composite material, each component with its own mechanical and biological properties that, combined synergistically, approximates to those required for a bone substitute. One important group of those composite materials is formed by calcium phosphates and polymers. Various polymers have been used: collagen, chitosan, gelatin, poly(lactic acid) and poly(glycolic acid). Overall, hydroxyapatite-polymer composites show better physical, bio and mechanical properties for scaffolds than both, pure polymer or pure bioceramics component [2][3][4][5][6][7][8][9][10]. From that variety of materials, chitosan and apatite seem to be an appropriate choice for their properties. Indeed, chitosan is a bioresorbable, angiogenic*, bacteriostatic*, fungistatic*, polymer with high potential for physico-chemical interactions, with itself and with other materials, via hydrogen bonds and electrostatic forces due to its chemical structure [11]. This makes chitosan an easy-to-modulate material [12,13] with remarkable biological properties [14][15][16][17]. Apatite has a composition and structure close to the inorganic part of the extracellular matrix of bones. It osseointegrates*; it is osteoconductive*; and, it dissolves under appropriate physico-chemical conditions, allowing new bone growth [18]. The physico-chemical conditions during fabrication of apatite determine their properties [19]. Chitosan-calcium phosphates composites have been prepared as membranes, films, scaffolds, injectable materials, multilayers films and particles [20]. Methods of synthesis have been reported and can be classified in: i) coating method using electrochemical and electrophoretic deposition techniques, favoring the deposition of chitosan and calcium phosphates on metal surfaces [20][21][22][23][24][25][26]; ii) incorporation of chitosan (powder or solution) to calcium phosphate pastes to form calcium phosphates-chitosan cements [20,[27][28][29][30][31][32][33][34][35][36][37][38]; iii) mineralization of chitosan scaffold by immersion in solutions saturated with respect to apatite (e.g., simulated body fluid) [39][40][41]; iv) impregnation of calcium phosphates scaffolds with chitosan solution [39,41,42]; and v) suspensions obtained by mixing chitosan solution with calcium phosphates particles [20,[43][44][45][46].

The above-mentioned methods effectively lead to chitosan-calcium phosphate composite materials; however, a major concern is the lack of homogeneity at microscopic level, which induce practical inconveniences in medical and industrial applications. To overcome this drawback, methods of in-situ precipitation of calcium phosphates within the polymeric matrix have been studied [20]. In-situ precipitation refers to various methods of preparing chitosancalcium phosphates materials that have some common characteristics: i) the process occurs in an acidic aqueous medium solution; ii) the intended result is a homogenous mixture of the inorganic phase in a chitosan matrix; iii) the pH is changed to basic condition; iv) in the last step, composite material is freeze-dried or dried at room temperature for further use and characterization [39,47,[48][49][50][51][52][53][54][55][56].

One alternative, chosen in this work, is to prepare suspensions of chitosan and calcium phosphate and then, to induce gelation of chitosan and recrystallization of calcium phosphates into apatite.

The interest for hydrogel viscoelastic state relies on the feasibility of having a homogeneously dispersed two phase material that, additionally, may provide a network structure that makes a suitable environment for cell colonization and for delivery of growth factors and bioactive molecules [12,57].

Thus, the global objectives of our work are: i) to develop methods of obtaining physical hydrogels of chitosan with intertwined particles of apatite; ii) to determine their final chemical composition and microstructure; iii) to find correlations between methods of preparation, chemical composition, microstructure and physico-chemical interactions that may explain the rheological and biological properties of the material.

The first step to obtain mineralized hydrogels of chitosan was to prepare solutions of salts that were the sources of calcium phosphates. The second step was the preparation of the suspension composed of chitosan and calcium phosphates, followed by simultaneous gelation of the suspension and recrystallization of calcium phosphates in apatite. Chitosan is soluble in acidic aqueous solution (pKa of chitosan ~ 6.5). Also at acid conditions, pH between 2 and 6, crystalline calcium phosphate is stable under the structure of dicalcium phosphate dihydrate, DCPD, CaHPO4.2H2O [18,58]. Gelation of chitosan solution occurs when the solution is in contact with a base, e.g., ammonia [57] or sodium hydroxide [12], and is due to a modification of the balance between hydrophilic and hydrophobic interactions within the polymer solution [57]. No external crosslinking agent is necessary for this gelation route. On the other hand, DCPD is not stable at pH > 6.5; it transformes to apatite [18,58], the most stable calcium phosphate at room temperature and pH between 6.5 and 12. Therefore, the chitosan-calcium phosphates suspension could be converted into a physical hydrogel of chitosan with intertwined particles of apatite, by adjusting the suspension above a pH of 6.5.

In this research work, nature of salts, methods of preparation of chitosan-calcium phosphates suspensions and gelation route were studied as variables that may influence microstructure, physico-chemical interactions and rheological properties of the obtained materials.

Materials and Methods

A. Materials

Chitosan powder produced from squid pens and supplied by Mahtani Chitosan Pvt. Ltd (batch type 114, No S3 20110121) was characterized with standard procedures [57]. The degree of acetylation, corresponding to the molar fraction of acetylated units within the polymer chains, was close to 5 %; weight-average molar mass and dispersity were Mw = 550 kg.mol -1 and Ð = 1.9, respectively.

Solutions of chitosan of concentrations 0.17 mol.L -1 (M) (2.8 wt%) and 0.15 M (2.4 wt%) were prepared by adding chitosan powder to acetic acid aqueous solution (acetic acid: Carlo Erba Reagents, CAS 64-19-7, assay 99.9 %). The mass of chitosan powder was calculated taking into account the molar mass of the repeating unit (D-glucosamine and N-acetyl glucosamine), 163.10 g.mol -1 . The amount of acetic acid was adjusted to match the stoichiometric protonation of -NH2 sites of chitosan. The complete dissolution of chitosan was obtained after several five-minute cycles at 3000 rpm, using a speed mixer machine (DAC150.1FVZ-K, Synergy devices Ltd, UK).

Four aqueous solutions of salts were prepared as sources of calcium and phosphate ions. More precisely, two pairs of solutions were studied: pair a, Ca(NO3)2.4H2O (Sigma-Aldrich, CAS 13477-34-4, assay ≥ 99 %) and (NH4)2HPO4 (Sigma-Aldrich, CAS 7783-28-0, assay ≥ 99 %); pair b, CaCl2.2H2O (Sigma-Aldrich, CAS 10035-04-8, assay ≥ 99 %) and NH4H2PO4 (Sigma-Aldrich, CAS 7722-76-1, assay ≥ 98 %). The pairs of salts were chosen such as, after mixing (formation of calcium phosphate suspensions), each pair had different pH (Figure 1). In both pairs, concentration of calcium was 2.5 M and concentration of phosphate was 1.5 M. The reasons to choose those concentrations were: i) to have concentrations below the solubility limit of each salt; ii) to maintain the molar ratio of calcium to phosphorous (Ca/P ratio) at 1.67, as in hydroxyapatite (Ca10(PO4)6OH2) and; iii) to obtain a high content of calcium phosphates while keeping chitosan in solution.

Two bases were used to induce chitosan gelation: a solution of sodium hydroxide or vapors of ammonia, NH3. A solution of 1 M NaOH (Carlo Erba Reagents, CAS 1310-73-2, assay ≥ 97 %) was obtained by dissolving soda pellets in water. An atmosphere of NH3 vapors was obtained by leaving 25 mL of 3 M NH4OH solution (Sigma-Aldrich, CAS 1336-21-6, assay 28-30%) inside a tightly closed 5 L desiccator.

B. Methods of preparation of mineralized hydrogels

Chitosan physical hydrogels mineralized with apatite were prepared by first, preparing a chitosancalcium phosphate (chitosan-CaP) suspension and second, gelation of this suspension. In this work, chitosan-CaP suspension refers to a suspension composed by a continuous phase of chitosan solution with calcium phosphate particles.

90

Chitosan-CaP suspensions were prepared by two methods, 1) sequential and 2) simultaneous, using the pairs of salts a or b. The gelation of suspensions was then performed using solution of sodium hydroxide or vapors of ammonia.

Preparation of chitosan-CaP suspension by sequential method

Two steps were performed sequentially: preparation of a CaP suspension (calcium phosphate particles in aqueous medium) and then mixing of this suspension with a chitosan solution, to achieve a chitosan-CaP suspension (Figure 1). Practically, a volume of calcium salt solution was poured in equal volume of phosphate salt solution and immediately mixed in a speed mixer for 5 five-minute cycles, at 3000 rpm. The resulting CaP suspension was then poured into the solution of chitosan to reach an inorganic/organic weight proportion of 70/30 (1). The mixture was finally immediately mixed in a speed mixer for 3 five-minute cycles, at 3400 rpm.

Preparation of chitosan-CaP suspension by simultaneous method

In this method, the calcium and phosphate salt solutions were added simultaneously into the chitosan solution (Figure 1), in a weight proportion of inorganic/organic of 70/30 (equation 1). Immediately, the system was mixed in the speed mixer for 3 five-minute cycles, at 3400 rpm.

Gelation of the resulting chitosan-CaP suspensions

The resulting chitosan-CaP suspensions, independently of the method of preparation, were poured into Petri dishes and allowed to rest for 1h to eliminate air bubbles. Then, the suspensions were put in contact with the base used to induce gelation of chitosan and formation of apatite, using one of two methods. Either 1 M NaOH solution was poured directly into the Petri dish containing the suspension and left for 15 h, or the Petri dish containing the suspension was kept in a desiccator under ammonia atmosphere (provided by 3 M NH4OH solution) for 22 h. The contact time between the base (NaOH solution or NH3 vapors) and the suspension was sufficient for having a complete gelation of the chitosan-CaP suspensions. Reference, non-mineralized chitosan hydrogels were also prepared from chitosan solution, using the same bases. The concentration of the reference chitosan solution was 2.4 wt %, which was the resulting concentration of chitosan solution after mixing the inorganic and the organic parts. Table 1 summarizes the nomenclature of samples prepared for this study.

Table 1. Nomenclature of materials prepared and characterized in this work.

Pair of salts

Method of preparation of suspension

Acronym of suspensions Base Acronym of mineralized hydrogels

a ---- a * ---- ---- b ---- b * ---- ---- a Sequential S1 NaOH M1 a Sequential S1 NH3 M2 a Simultaneous S2 NH3 M3 b Sequential S3 NaOH M4 ---- ---- ---- NaOH Ref1 ---- ---- ---- NH3 Ref2
a * : suspension of calcium phosphates obtained from pair of salts a. b*: suspension of calcium phosphates obtained from pair of salts b.

The resulting chitosan hydrogels or mineralized chitosan hydrogels (pH ~ 12) were washed with deionized water until neutrality, at pH around 7.

Chitosan hydrogels or mineralized chitosan hydrogels were prepared in two sizes: 10 mm diameter plates of 3 mm height, and 35 mm diameter plates of 5 mm thickness (Figure 2). The smaller samples were used for cell proliferation test (described below), whereas bigger samples were used (as hydrogels or as freeze-dried hydrogels) for other characterization tests.

Samples preparation

When necessary, the samples were sterilized in deionized water for 20 min at 121 °C using Getinge 22K7 autoclave (USA), and stored in deionized water at room temperature for further characterization.

Freeze-dried samples were prepared by freezing samples in a refrigerator overnight at -20 °C. Then, the frozen samples were left for two days in a vacuum pump at -85 C at pressure of 0.081 mbar using a Lyo Labconco freeze-drier (USA). If necessary, for some of the characterizations, freeze-dried samples were ground using a spatula.

C. Characterizations

Table 2 summarizes the characterization techniques used in this work to determine the chemical composition, microstructure, rheological and biological properties of the samples. The pH of solutions of salts and suspensions of calcium phosphates was measured using pH-meter Corning® 240 (USA). For solutions of chitosan and for suspensions chitosan-calcium phosphates, a FiveGo pH-meter (Mettler Toledo, Switzerland) was used to measure pH. The obtained diffractograms were normalized with respect to the height of the peak of greatest intensity in the crystalline mineral phase.

The crystal size was determined by Rietveld refinement analysis of X-ray diffractograms using Diffract. Topas software Bruker (AXS). Analyses of each sample were done three times; the standard deviation is reported.

Thermogravimetric analysis in air atmosphere (60 mL/min) was performed using a TGA system coupled with a gas controller (GC200, Mettler Toledo, USA). Samples were heated from ambient temperature to 100 °C at a heating rate of 20 °C/min; then, the temperature was maintained constant at 100 °C for 10 minutes, followed by a heating ramp up to 700 °C, at a rate of 5 °C/min. The percentage of weight loss was registered and the first derivative of thermogravimetric curves was calculated to highlight the temperature of fast gravimetric variations. Residues of samples were kept for further characterization by XRD and Rietveld refinement analysis.

Fourier transform infrared spectroscopy (FTIR) was carried out using a Thermo Scientific Nicolet iS50 FT-IR spectrometer (USA). Samples were characterized in transmission mode at ambient temperature; from 400 to 4000 cm -1 (ATR analysis) with 4 cm -1 of resolution and 64 scans.

Microstructure

Optical microscopy analysis was conducted using a Light Axiophot microscope (Zeiss, Germany). Samples were fixed between two glass slides and observed in transmission mode. Size of crystal aggregates was measured using ImageJ software [60] in three 64 μm 2 micrographs of each sample. Scanning electron microscopy (SEM) analysis was done using a SUPRA 55 VP SEM (Zeiss, Germany), working at 5 kV. Prior to SEM, samples were coated with gold for 40 s, at 30 mA at 5cm distance between the source and the sample.

Rheology

Rheological measurements were conducted at shear rates from 10 -4 or 10 -3 to 100 s -1 , at 25 °C, using an AR2000-ex rheometer (TA Instruments, USA). The steady-state viscosity of chitosan solutions and suspensions was assessed in static mode using aluminum cone-plate geometry (25 mm diameter; 4 °). The minimum gap was fixed at 116 µm. A solvent trap was used to prevent evaporation of water during measurements. The output is a curve of shear stress vs. shear strain or a curve of viscosity vs. shear rate. This test allows stating the rheological model and the shearthinning behavior of the samples, respectively.

Rheological measurements were also carried out in dynamic mode at room temperature by using an ARES 4800 rheometer (TA Instruments, USA), operating with a plate-plate geometry (diameter 25 mm). The samples were cut using a 25 mm-diameter punch. The test consisted of applying an oscillatory strain to the sample. The strain amplitude was chosen to ensure that the measurements were carried out within the linear viscoelastic region, so that the storage (or elastic) modulus G' and the loss (or viscous) modulus G" were independent of the strain. The normal force sensor of the rheometer was used to measure the first contact between the sample and the upper plate. The gap varied around 5mm; the storage and loss moduli were then measured from a constant-strain frequency sweep within angular frequency ranges of 0.05 to 100 rad.s -1 .

Rheology analyses were carried out in triplicate on three samples obtained with the same processing conditions.

Biological behavior

Sterilized mineralized chitosan hydrogels and chitosan hydrogels references were incubated in a complete culture medium for two weeks to stabilize mass transfer between culture medium and the samples under test.

The proliferation rate of Mg-63, osteoblast-like cells originally isolated from a human osteosarcoma (cell line culture passage number 43) (ATCC, ref CRL-1427), was evaluated by culturing them in contact with mineralized hydrogels chitosan hydrogels references in 24 wellcell culture plates (Corning, USA, ref 3524). 30 µL per well of a cellular suspension of 150000 cells per mL was prepared using a cell counter (Millipore Scepter purchased at Dutscher, ref: 053750). The Scepter cell counter uses the Coulter principle of impedance-based particle detection to reliably and accurately count every cell in the sample, in RPMI 1640 medium (Roswell Park Memorial Institute) (stable L-glutamine, phenol red) supplemented with 10 % fetal bovine serum (FBS) (Dutscher, ref P040637100) and 5 % antibiotics/antimycotic solution (Dutscher, ref SV30079.01). Cells were cultured at 37 °C in a humified atmosphere containing 5% CO2. The cell viability was carried out using the PrestoBlue® reagent (Invitrogen, Carlsbad, CA): the Resazurine, which is blue and non-fluorescent and it is reduced by the metabolic mithochondrial activity of the cells in resorufine, a pink and fluorescent product, easily detectable by fluorimetry. After 7, 10 and 14 days, the culture medium was discarded and replaced by 1 mL of the culture medium without FBS, antibiotics and phenol red but with 10 % PrestoBlue put on each well. The samples were incubated 1.5 h at 37 °C under a humidified atmosphere of 5 % CO2.

Then the plate was stirred and 100 μL of each well were transferred in a 96 black-well plates. The fluorescence intensity values were collected at excitation and emission wavelength of 535 and 615 nm respectively, in fluorimeter Infinite Pro 200 (Tecan, Männedorf, Switzerland) to infer the cell proliferation. After each measurement, cells were rinsed two times with RPMI, then 2 mL of medium were added and the plate incubated until the next measure (10 and 14 days). Each assay was done in triplicate. Blank reference consisted of 10 % PrestoBlue diluted in RPMI without dye, and it was used to correct fluorescent values of the samples. Additionally, for each sample, a 96 plastic control (cell culture treated polystyrene) was included to verify the normal behavior of cells, and it was referred to as "control".

Results

A. Chitosan-CaP suspensions

As described in section IB, suspensions of chitosan-CaP were prepared by two methods: sequential and simultaneous.

By the sequential method, two CaP suspensions were first prepared by solutions of salts from pair a or b (Figure 1). In preparing CaP suspension a*, as soon as the solution of Ca(NO3)2 was dropped into the solution of (NH4)2HPO4, a stable, milky and homogeneous suspension was formed, with a final pH of 4.7. In the case of b*, as soon as the solution of CaCl2 was dropped into the solution of NH4H2PO4, a milky but unstable suspension appeared, with a final pH of 1.7; the unsteadiness of the suspension manifested by fast sedimentation. CaP suspensions a* or b* were added into a chitosan solution (pH = 5.5) to form chitosancalcium phosphate suspensions, S1 and S3 respectively (Figure 1). The resultant pH in S1 and in S3 were 5.3 and 3.8 respectively.

To investigate the crystalline mineral phase of CaP suspensions (a* and b*) before adding them into the chitosan solution, the suspensions were freeze-dried and analyzed by XRD (Figure 3). In suspension a*, the principal identified mineral phases were dicalcium phosphate dihydrate (DCPD, CaHPO4.2H2O) and dicalcium phosphate anhydrous (DCPA, CaHPO4); whereas in suspension b*, the principal identified mineral phases were calcium hydrogen chloride phosphate hydrate (CaClH2PO4.2H2O) and ammonium chloride (NH4Cl). By the simultaneous method, suspension S2 was prepared from solution of salts pair a: Ca(NO3)2 and (NH4)2HPO4 salts were added directly into the chitosan solution (pH = 5.5). The final pH of S2 was 5.1 (Figure 1).

The crystalline phase in the chitosan-CaP suspensions (S1, S2 and S3) was investigated by XRD.

According with the XRD results (Figure 4), the peaks corresponding to the principal planes of DCPD, (020) at 2θ = 11.681 ° and (021) at 2θ = 20.935 ° were identified. DCPD was the only detectable crystalline phase of calcium phosphates in the chitosan-CaP suspensions (S1, S2 and S3). No diffraction peaks of chitosan were identified indicating that the amount of crystalline chitosan was below the detection limit. The X-ray diffuse halo observed from approximately 2θ = 24 ° to 36 ° in the diffractograms may be attributed to the presence of water (about 96 wt % of the suspension was water).

Figure 4. X-ray diffractograms of chitosan-DCPD suspensions (without freeze-drying): S1 (sequentially method and pair a), S2 (simultaneously method and pair a) and S3 (sequentially method and pair b). The blue arrows correspond to DCPD characteristic planes.

Optical micrographs of the chitosan-DCPD suspensions (Figure 5) showed that they were composed of a dispersed phase, platelet aggregates of DCPD, in a continuous phase (chitosan solution). The dimensions of aggregates were 11 ± 3 µm length, 5 ± 0.4 µm width for S1, 20 ± 5 µm length; 14 ± 5 µm width for S2 and 21 ± 2 µm length, 13 ± 2 µm width for S3.

Size of DCPD aggregates seemed to be dependent on the method of preparation and the pair of salts used. Comparing S1 and S2 (Figure 1, same salts but different methods of preparation), it was observed that the sequential method (S1) led to the formation of smaller DCPD aggregates and more uniform distribution than the simultaneous method (S2). In the case of S1 and S3 ((Figure 1, same method of preparation different salts), it was observed that using the pair a (Ca(NO3)2 and (NH4)2HPO4) led to the formation of smaller DCPD aggregates than using the pair b (CaCl2 and NH4H2PO4). The steady viscosity of chitosan solutions and chitosan-DCPD suspensions was assessed. The results are presented in Figure 6.

Figure 6. Rheological measurements on chitosan solutions and chitosan-DCPD suspensions S1 (sequentially method and pair a), S2 (simultaneously method and pair a) and S3 (sequentially method and pair b). Viscosity vs. shear rate in (a) and shear stress vs. shear rate in (b). (Tests

conducted at shear rates between 10 -4 or 10 -3 s -1 and 100 s -1 at 25 °C).

The rheology of chitosan solution and chitosan-DCPD suspensions (curves shown in Figure 6) may be described by a power-law equation:

𝜏 = 𝐾𝛾̇𝑛 (2)
In equation ( 2), 𝜏 stands for shear stress, γ̇ for shear rate, n for the power-law exponent and K for a flow consistency index. Shear-thinning behavior is typically exhibited by power-law fluids, those whose rheological behavior can be adjusted by equation ( 2) with exponent n  1 [61]. At lower n value, the shear-thinning behavior is higher. The values of power-law exponent and of the Newtonian viscosity at very low shear rate (plateau) are reported in Table 3. Suspensions S1, S2, S3 and chitosan solutions showed shear-thinning behavior. Viscosity of S1 and S2 were higher than that of the reference chitosan solution, whereas, viscosity of S3 was lower than that of reference chitosan solution (Table 3). Values of power law exponent, n, were slightly lower in S1 and S2 and higher in S3 compare with chitosan solution. It may indicate that S1 and S2 were more structured fluids, whereas S3 was less structured fluid compared with chitosan solution without calcium phosphates. These differences in rheological behavior will be explained by the physico-chemical interactions between the species and they will be discussed in section III.

B. Mineralized hydrogels of chitosan

Mineralized hydrogels M1 and M4 were formed by neutralization of S1 and S3, respectively, using NaOH solution; mineralized hydrogels M2 and M3 were formed by neutralization of S1 and S2, respectively, using NH3 vapors (Table 1and Figure 1). Rheological data of chitosan hydrogel references and mineralized hydrogels were gathered in Figure 7. Average values of storage and loss moduli at low angular frequency are reported in Table 4.

Figure 7. Variation of G' and G'' vs. angular frequency for chitosan hydrogels references (Ref1 and Ref2

) and mineralized hydrogels. (Tests conducted from 100 to 0.05 rad.s -1 , at controlled strain of 0.25 %, at 25 °C). The resulting materials (M1, M2, M3 and M4) were hydrogels since all of them exhibited storage modulus (G') higher than loss modulus (G'') and moduli had a pronounced plateau along a range of angular frequency (Figure 7 and Table 4). Those are the conditions that define a gel-like material [62]. It was also observed that inorganic part in the hydrogel of chitosan reinforced the elastic nature of the material: G' in M1 > G' in Ref1 (NaOH); G' in M2 and M3 > Ref2 (NH3 vapors), except for M4 obtained from the gelation of S3 where G' was about the same of G' in Ref1. The discussion of the low G' in M4 will be given in section III. The crystalline phases of mineralized hydrogels and chitosan hydrogel reference were identified by XRD (Figure 8). Transformation from DCPD to apatite was confirmed by XRD of mineralized hydrogels (Figure 8). Characteristics planes of apatite appeared, (002) at 2θ = 25.879 °, (211) at 2θ = 31.774 ° and (112) at 2θ = 32.902 °, whereas characteristic planes of DCPD were not detected. It was noted that a peak appeared at around 2θ = 20 °, compatible with planes (220) and (200) of crystalline chitosan. This peak was more visible in the hydrogels gelled with NH3 (Ref2, M2 and M3). It may indicate higher crystallization of chitosan under the experimental conditions used to obtain them. XRD of chitosan hydrogels and mineralized hydrogels after sterilization showed the same two crystalline phases, chitosan and apatite, with a better definition of peaks compared with those of unsterilized samples (Figure 9).

Figure 8. X-ray diffractograms of the surface of chitosan hydrogels references and mineralized hydrogels. The red arrows correspond to apatite characteristic planes and the gray squares correspond to chitosan characteristic planes. Ref 1, M1 and M4 after 15 h of contact with NaOH solution; Ref 2, M2 and M3 after

22 h of contact with NH3 vapors.

Figure 9. X-ray diffractograms of the surface of sterilized chitosan hydrogels references and mineralized hydrogels. The red arrows correspond to apatite characteristic planes and the gray squares correspond to chitosan characteristic planes.

Optical micrographs were taken to investigate distribution of the mineral phase within the organic phase. In Figure 10, it was observed that, in all mineralized hydrogels, aggregates of apatite were distributed in a matrix of chitosan hydrogel. Chitosan hydrogel references and mineralized hydrogels were subjected to freeze-drying (to eliminate water) and grinding, prior to investigate the microstructure of samples by SEM. The micrographs are shown in Figure 11 and Figure 12. From SEM micrographs, aggregates of apatite were clearly forming platelets in M1 (Figure 12), whereas the apatite platelets were not clearly observed for hydrogels M2, M3 and M4, at least in the observed zone of the sample. In all mineralized hydrogels, the apatite crystals forming aggregates were very small, in the nanometric scale. The size of crystals forming aggregates observed in SEM micrographs was determined from XRD data of freeze-dried grounded samples. The results are shown in Table 5. Nanometric scale of crystals was confirmed, size ~20 nm. The presence of chitosan in chitosan hydrogel references and of chitosan and apatite in mineralized hydrogels was confirmed by FTIR analysis, and these results are revealed in Figure 13 and Table 6. Regarding the mineralized hydrogels, the broadening of the band corresponding to PO4 3-at 1024 cm -1 in apatite (g in Figure 13) would indicate interactions between phosphates groups and chitosan [63]. In the case of M4, the band "g" was narrower, which may indicate that this mineralized hydrogel may have fewer interactions between PO4 3-ions and chitosan molecules. The presence of the characteristic bands (d and h in Figure 13) of CO3 2-ions in mineralized hydrogels would indicate an apatite with substituted ions (chemical formula Ca10(PO4,CO3)6OH) [63].

Hydrogel

Table 6. The assignments of FTIR bands or range bands of chitosan hydrogels references and mineralized hydrogels [63][64][65][66]. Freeze-dried chitosan hydrogels and mineralized hydrogels were analyzed by TGA to estimate the global inorganic/organic weight proportion of mineralized hydrogels (Figure 14). [67]. The resultant weight percentage after thermal treatment in mineralized hydrogels corresponded to CaP.

Figure 14. TGA curves in the temperature range of 50 o C to 700 o C for chitosan hydrogels references and mineralized hydrogels. Three regions were identified: 1) elimination of bonded water (H2O), which is physically adsorbed and/or weakly hydrogen-bonded to chitosan molecules, temperature < 100 °C; 2) depolymerisation of chitosan chains, decomposition of pyranose ring through dehydration and deamination and finally ring-opening reaction, temperature between ~230-400 °C; and finally 3) chain scission with the formation of volatile products, temperature > 400 °C

In mineralized hydrogels, the weight loss did not reach ~100 % since in this temperature range, only the organic part, chitosan, decomposed, whereas the weight of mineral component remained.

In chitosan hydrogels references the weight loss did not reach exactly 100 % because at the end of the thermal treatment a coal residue was found (1-3 wt % of initial material). The small weight of coal residue suggested that: i) the yield of thermal degradation in oxidized conditions was high and, ii) it may indicate possible cross-linking structures that were formed through oxygen bridges, which would break up at temperatures > 600 °C. Three main regions of weight loss were found during the increase of temperature, that roughly correspond to: 1) elimination of bonded water (H2O), which is physically adsorbed and/or weakly hydrogen-bonded to chitosan molecules, temperature ≤ 100 °C; 2) depolymerisation of chitosan chains, decomposition of pyranose ring through dehydration and deamination and finally ring-opening reaction, temperature between ~230-400 °C; and finally 3) chain scission with the formation of volatile products above 400 °C [67].These three regions were evidenced in chitosan hydrogels and mineralized hydrogels and allowed to identify the inorganic and organic phases (Figure 14).

The data of weight proportion are reported in Table 7. From the weight of salts initially added to prepare the suspensions, the chemical reactions that may occur (equations 13 and 17), and the weight of apatite found by TGA, it was possible to estimate the yield of conversion of calcium and phosphate salts to apatite that was 79 % for M1; 70 % for M2; 84 % for M3 and 88 % for M4. The residue of mineralized hydrogels after TGA was analyzed with respect to inorganic composition and crystal size. XRD of that residue (Figure 15) showed that apatite was stable through the TGA test. No chitosan was found. For comparison of data reported in Table 5 (before and after TGA), it can be deduced that parameters "a" and "c" remained rather constant, whereas size of crystals tended to increase by thermal treatment during TGA.

Figure 15. X-ray diffractograms of the residue of mineralized hydrogels after TGA. The red arrows correspond to apatite characteristic planes.

Once the different systems were characterized with respect to their composition and microstructure, tests were performed on cell proliferation. The results are shown in Figure 16. It was observed that: 1) Cell proliferation tended to be higher in Ref1 than Ref2; 2) Cell proliferation tended to be higher in the reference than in the mineralized hydrogels (Ref1 > M1, M3; Ref2 > M2, M3); 3) Cell proliferation tended to be slightly higher in M1 compare with M2, M3 and M4.

Discussion

To facilitate the discussion of the results, a schematic representation of the different synthesis methods of mineralized hydrogels, the pH of each step and the identified crystal mineral phase is presented in Figure 1.

Physico-chemical interactions

The discussion is centered in correlating experimental results of characterization of chitosan-CaP suspensions and mineralized hydrogels, with the physico-chemical interactions that may occur in the systems under the conditions of the performed experiments. The strategy of analysis here was 1) to describe the expected interactions and the events that may occur during the preparation of suspensions and mineralized hydrogels; 2) derive the effects of those interactions on properties related with microstructure and rheological properties and 3) compare those deductions with the experimental results reported in section II.

Chitosan-CaP suspensions

Considering the used salts, the resulting pH in each case and the XRD analysis (Figure 4), suspensions (S1, S2, S3) are expected to contain chitosan in solution, DCPD particles (the stable calcium orthophosphate in acidic aqueous media), Ca 2+ ions and may also contain phosphate ions. S1 and S2 contain counterions NO3 -and NH4 + and S3 contains counterions Cl -and NH4 + .

i. Comparison sequential (S1) vs. simultaneous (S2) methods of preparation

In the first step of the sequential method (preparation of CaP suspension, a*), the pH of a* was 4.7 (Figure 1). At this pH, salts of calcium and phosphate dissociate forming calcium, nitrate, ammonium and divalent phosphate ions:

Ca(NO 3 ) 2 → Ca 2+ + 2NO 3 - (3) (NH 4 ) 2 HPO 4 → 2𝑁𝐻 4 + + 𝐻𝑃𝑂 4 2- (4)
Divalent phosphate ions can react: 1) forming DCPD with calcium ions; 2) decomposing to form monovalent phosphate ions in an equilibrium reaction:

Ca 2+ + 𝐻𝑃𝑂 4 2-+ 2𝑂𝐻 -+ 2𝐻 + → 𝐶𝑎𝐻𝑃𝑂 4 2𝐻 2 𝑂 ( 5
)
𝐻𝑃𝑂 4 2-+ 𝐻 + ⇄ H 2 PO 4 -(𝑝𝐾 𝑎 ~7.2) (6)
XRD of freeze-dried suspension a* (Figure 3) showed that DCPD was the principal mineral phase ((020) at 2θ = 11.681 ° and (021) at 2θ = 20.935 °). Besides DCPD, other mineral components were identified such as DCPA, which could be formed during sample preparation (freeze-drying procedure and storage under dry atmosphere).

DCPD crystallization occurred without chitosan in a medium at high mixing speed (5 five-minute cycles, at 3000 rpm) and low viscosity. Homogenous distribution of calcium and phosphate ions would be rapidly reached in the entire system volume. Under these conditions, many and well distributed number of DCPD crystal seeds would be born, grew and formed DCPD aggregates. Due to the high number of DCPD aggregates, the size would be relatively small. Most of divalent phosphate ions, HPO4 2-, were expected to form DCPD, although, some remained in the system since there was an equilibrium with monovalent phosphate ions, H2PO4 -. DCPD has a relation Ca/P = 1; since the salts were added to have a proportion Ca/P = 1.67; more than the stoichiometric excess of calcium ions remained in the calcium phosphate suspension a*.

The second step of the sequential method was the mixing with the chitosan solution (pH = 5.5). Divalent phosphate ions may physically cross-link chitosan chains by electrostatic interaction with protonated amine groups [68,69]. Moreover, calcium ions may also form physical cross-links of chitosan chains by coordination with electron donor atoms (nitrogen and oxygen) in chitosan molecule [70,71].

At this point, it would be expected that most of divalent phosphate ions were consumed in the formation of DCPD and in electrostatic interactions with chitosan. Monovalent phosphate ions were expected to remain in low concentration since the only source was divalent phosphate ions.

In the simultaneous method (S2), both salt solutions were added directly into the chitosan solution. The same type of reactions was expected to occur as in the sequential method. However, the dynamic of the process would be different. Dissociation of salts, formation of DCPD, electrostatic interactions between ions and chitosan, and formation of physical cross-links of polymer chains occurred simultaneously. DCPD formed in competition with chitosan-calcium and chitosan-divalent phosphate ion interactions and with spatial interference due to the presence of chitosan molecules. Compared to the sequential method, viscosity would be higher and the homogeneity of distribution of calcium and phosphate ions would be lower. The relative heterogeneity of the ion distribution would lead to the formation of lower number of DCPD crystal seeds; therefore, the size of DCPD aggregates would be relatively larger. On the other hand, higher density of physical cross-linking of polymer chains could be expected because chitosan, divalent phosphate and calcium ions are in the system since the beginning of simultaneous mixing, that is, at high concentration of species.

Size of crystal aggregates and physical cross-link density affect viscosity of the chitosan-CaP suspensions in opposite tendencies: i) larger crystal aggregates tend to decrease viscosity (compare to smaller crystal aggregates that tend to increase viscosity) and; ii) physical cross-link density of polymer chains tends to increase viscosity. Experimental results showed that S1 and S2 had about the same viscosity (Figure 6 and Table 3).

Those results would mean that, in this case, tendencies balanced off or that, differences in crystal aggregate size and physical cross-link density in S1 and S2 were not big enough to affect viscosity significantly. Discussion about the mineralized hydrogels obtained from S1 and S2 (M2 and M3 respectively) could clarify the issue.

ii. Comparing different salts Two suspensions, S1 and S3, were prepared by sequential method, using pair of salts a and b respectively (Figure 1). The formation of S1 was described above. The discussion here will be on the formation of S3.

In the first step of the sequential method (preparation of CaP suspension, b*), the pH of b* was 1.7 (Figure 1). At this pH, salts of calcium and phosphate dissociate forming calcium, chloride, ammonium and monovalent phosphate ions:

CaCl 2 → Ca 2+ + 2Cl - (7) NH 4 H 2 PO 4 → NH 4 + + H 2 PO 4 - (8)
Different reactions occur: i) equilibrium between monovalent phosphate ions and phosphoric acid and ii) formation of calcium hydrogen chloride phosphate hydrate:

H 2 PO 4 -+ 𝐻 + ⇄ H 3 PO 4 (𝑝𝐾 𝑎 ~2.1) (9)
𝐶𝑎 2+ + 𝐶𝑙 -+ 𝐻 2 𝑃𝑂 4 -+ 𝑂𝐻 -+ 𝐻 + → 𝐶𝑎𝐶𝑙𝐻 2 𝑃𝑂 4 𝐻 2 𝑂 ( 10
)
The formation of CaClH2PO4.H2O in calcium phosphate suspension b* was confirmed by XRD (Figure 3).

The second step of the sequential method was the mixing with the chitosan solution (pH = 5.5).

Chitosan solution increases the pH of the system S3, then, CaClH2PO4.H2O is no longer stable and re-dissolves; therefore phosphate ions are formed:

𝐶𝑎𝐶𝑙𝐻 2 𝑃𝑂 4 𝐻 2 𝑂 → 𝐶𝑎 2+ + 𝐶𝑙 -+ 𝐻 2 𝑃𝑂 4 -+ 𝑂𝐻 -+ 𝐻 + (11) H 2 PO 4 -⇄ 𝐻 + + HPO 4 2-(pK a ~7.2) ( 12
)
pH is also adequate to form DCPD:

Ca 2+ + HPO 4 2-+ 2OH -+ 2H + → CaHPO 4 2H 2 O (13)
The pH in S3, pH = 3.8, results from mixing chitosan solution (pH = 5.5), CaP suspension b* (pH = 1.7) and the release of protons by both the dissolution of CaClH2PO4.H2O and the formation of divalent phosphate ions (equations 11 and 12). At this pH (3.8), the equilibrium of monovalent phosphate ions with phosphoric acid occurs (equation 9), thus, the formation of DCPD is less favored, although it remains stable in the pH range from 2 to 6. Ionic strength would be higher in S3 than S1 because in S3, monovalent phosphate ions remain in the system without possibility of forming DCPD whereas, in S1, monovalent phosphate ions are continuously consumed to form divalent phosphate ions involved in the formation of DCPD (equations 5 and 6).

As ionic strength increases, the fluid becomes less structured (more Newtonian), that is, shearthinning behavior is less pronounced and viscosity is lower.

Comparison with experimental results show agreement with those deductions: 1) CaClH2PO4.H2O and DCPD were found in calcium phosphate suspension b* (Figure 3), and in S3 (Figure 4) respectively; 2) viscosity was lower in S3 than in S1 and even than in chitosan solution reference (Table 3); 3) S3 showed less shear-thinning behavior (characterized by a higher n value, see Table 3).

Mineralized hydrogels

The simultaneous conversion of chitosan-DCPD suspensions into chitosan-apatite hydrogels was reached by an increase from pH  6 to pH > 9 (Figure 1). Solution of sodium hydroxide and vapors of ammonia were used to reach the required alkaline condition, as described in section IB.

At pH > 6, DCPD becomes unstable and de-protonation of NH3 + in chitosan molecules occurs:

𝐶𝑎𝐻𝑃𝑂 4 2𝐻 2 𝑂 → Ca 2+ + 𝐻𝑃𝑂 4 2-+ 2𝑂𝐻 -+ 2𝐻 + (14)
𝑂𝐻 -+ 𝑐ℎ𝑖𝑡𝑜𝑠𝑎𝑛 -𝑁𝐻 3 + → 𝑐ℎ𝑖𝑡𝑜𝑠𝑎𝑛 -𝑁𝐻 2 + 𝐻 2 𝑂 (𝑝𝐾 𝑎 ~6.5) (15)
In alkaline environment (pH > 9) divalent phosphate ions are in equilibrium with trivalent phosphate ions:

𝑂𝐻 -+ 𝐻𝑃𝑂 4 2-⇄ 𝑃𝑂 4 3-+ 𝐻 2 𝑂 (𝑝𝐾 𝑎 ~12.7) (16)
Apatite, stable at pH > 6.5, is formed:

10𝐶𝑎 2+ + 6𝑃𝑂 4 3-+ 2𝑂𝐻 -→ 𝐶𝑎 10 (𝑃𝑂 4 ) 6 (𝑂𝐻) 2 (17)
However, the experimental conditions, room temperature and aqueous media, are not favorable to obtain the high crystallinity phase of hydroxyapatite, instead, the less perfect crystal, nonstoichiometric hydroxyapatite, which corresponds to apatite.

Interactions chitosan-apatite may appear by coordination bonds between calcium ions and chitosan [61] and by electrostatic interactions between phosphate ions and chitosan [68,69].

Gelation depends upon availability of hydroxyl anions, which are provided by the base. Sodium hydroxide is a strong base and rapidly and completely dissociates in solution, forming sodium cations and hydroxyl anions. Ammonia vapors first dissolve in acidic water (chitosan-DCPD suspension) and then, ammonia being a weak base, partially dissociates forming ammonium cations and hydroxyl anions. Under these conditions, hydroxyl anions are available more rapidly for gelation using sodium hydroxide solution than using ammonia vapors. Thus, in the case of gelation induced by NaOH, the diffusion of the base and the neutralization are faster as compared to gelation induced by NH3 vapors [13]. The mobility of the chitosan polymer chains decreases more rapidly in the case of NaOH, preserving a higher amount of chitosan chain entanglements [13].

Moreover, considering the tendency of XRD chitosan peaks (Figure 8, Ref1 and Ref2), broader and less defined in the case of Ref1-NaOH hydrogels compared with those in Ref2-NH3 hydrogels, it may be deduced that crystallinity and crystal size would be smaller in the case of Ref1-NaOH hydrogels. At smaller crystal size, the interphase area crystal phase-polymer phase would be higher; therefore more interphase junctions (ties) would be expected, reinforcing the elastic nature of the hydrogel.

Recrystallization to apatite in presence of NaOH solution needed less time than in the presence of NH3 vapors. In fact, after 15 h, apatite was observed by XRD in the case of mineralized hydrogels obtained with NaOH (Figure 8, M1 and M4) and not in the case of those obtained with NH3, for which 22 h were necessary, (Figure 8, M2 and M3).

i. Effect of the nature of the base Suspension S1, prepared by sequential method, was gelled using sodium hydroxide and vapors of ammonia as bases to form mineralized physical hydrogels M1 and M2 respectively (Figure 1). Therefore, the only difference during the formation of M1 and M2 is the rate of neutralization due to the use of different bases. The rate of neutralization would impact the recrystallization of apatite and the mobility of chitosan chains. When using sodium hydroxide, the rate of neutralization was higher, time for apatite growing crystals was then shorter, and therefore, a lower crystal size of apatite should be obtained.

As was stated before, mobility of chitosan polymer chains decreases more rapidly using sodium hydroxide; then, chitosan chain entanglements are mostly preserved. Thus, even in the presence of apatite, the preservation of a higher amount of polymer chain entanglements, would allow the formation of stiffer hydrogels. Accordingly, M1 would have higher G´than M2.

Experimental results showed that: 1) M1 and M2 were mineralized hydrogels, with a discontinuous phase of apatite dispersed in a continuous matrix of chitosan (Figure 10 and Figure 11); 2) M1 had higher G´than M2 (Figure 7 and Table 3), which is coherent with our analysis.

ii. Effect of the method of preparing chitosan-DCPD suspensions M2 was prepared from S1 (sequential method) and M3 from S2 (simultaneous method) (Figure 1), using the same pairs of salts (a) and gelation base (NH3). As was mentioned above, both suspensions (S1 and S2) were found to have about the same types of reactions, similar viscosity and similar shear-thinning behavior (about the same n value, Table 3). However, S2 was expected to have higher physical cross-link density of chitosan chains; therefore, higher G´would be expected in M3 than in M2.

Experimental results confirmed that effectively, M3 had higher G´than M2 (Figure 7 and Table 3).

iii. Effect of salts M1 and M4 were prepared from S1 and S3 (Figure 1). Suspensions were prepared by sequential method and the base used was NaOH. S1 was found to be a more structured fluid than S3 (Table 3). As was described above, based on the chemical reactions that occurred in S1 and S3, the ionic strength in S3 would be higher than is S1. The ionic strength is a dominant factor determining the rheological state of the system. The ionic strength may induce a significant decrease of viscosity by disentanglement of polymer chains. In fact, the viscosity of suspension S3 was lower due to higher ionic strength in comparison with S1. Thus, the resulting M4 hydrogel from S3 showed lower G' than M1 from S1 (Figure 7 and Table 3).

Conclusions

Mineralized hydrogels of chitosan were obtained by gelation of chitosan-calcium phosphate suspensions.

A. Chitosan-calcium phosphate suspensions

The nature of salts used as sources of calcium and orthophosphate ions, and the method of mixing salt and chitosan solutions (sequential or simultaneous) were the synthesis variables in the preparation of suspensions. The resulting acidic suspensions (pH < 6), before gelation, were composed of a chitosan solution with dispersed aggregates of DCPD crystals and residual ions of dissolved salts.

Under those conditions of composition and pH, several interactions were likely to occur: i) repulsion between polymer chains due to protonated amine groups in the polymer chain; ii) hydrophilic interactions polymer-solvent due to hydroxyl, amine and protonated amine groups of chitosan; iii) polymer-polymer interaction expressed in entanglement of polymer chains; iv) physical cross-linking of chains due to coordination bonds between polymer and calcium ions; v electrostatic interactions of divalent phosphate ions with protonated amine groups that may form physical crosslinks between polymer chains; vi) affinity between calcium and phosphate ions to form calcium phosphate phases (DCPD at pH from 4 to 6) and, vii) screening of electrostatic interactions due to the ionic strength of the suspension.

The size and distribution of DCPD crystal aggregates varied with the method of preparing the suspensions: they were smaller and uniformly distributed with the sequential method while they were larger and less uniformly distributed with the simultaneous method. This difference of microstructure was attributed to the environment prevailing in the formation of calcium phosphate suspensions, of relatively low viscosity, high homogeneity and in absence of chitosan in the case of the sequential method.

The ionic strength had a pronounced effect on rheological properties of chitosan-calcium phosphate suspensions. Ionic strength impacted the density of entanglement of polymer chains, which in turn influenced its shear-thinning behavior, its moduli (storage, G' and loss, G'') and the viscosity of the system. When the ionic strength was higher, lower chitosan chain entanglements occurred, therefore the shear-thinning behavior, the moduli, the yield stress and the viscosity tended to decrease.

B. Physical hydrogels of chitosan mineralized with apatite

The pH of the chitosan-DCPD suspensions was increased to basic condition (pH ~ 12) using NH3 or NaOH. This change in pH induced two effects at the same time: the gelation of the suspension and the transformation of mineral phase to form apatite. Thus, the chitosan-DCPD suspension was transformed into a physical hydrogel of chitosan with intertwined apatite particles.

Different events were expected to occur during the process of gelation: i) DCPD was no longer stable: it dissolved and recrystallized forming the stable phase, apatite; ii) amine groups were deprotonated, thus electrostatic repulsive forces between charged polymer chains decreased; iii) chain junctions (inter and intra chains) formed physical cross-links leading to the formation of a hydrogel; iv) electrostatic interactions between chitosan and orthophosphate ions formed physical cross-links of polymer chains; and v) coordination bonds between chitosan and calcium ions also formed physical cross-links of polymer chains.

The microstructure and rheological properties of mineralized hydrogels were compared with respect to the nature of the base, the method of preparing the chitosan-calcium phosphate suspensions prior to gelation and the salt sources of calcium phosphates used. Three factors strongly impacted the rheological properties of the mineralized hydrogels: 1) the neutralization rate of protonated amine groups of chitosan molecules; 2) the density of physical cross-link of polymer chains in chitosan-calcium phosphate suspensions, prior to gelation and; 3) the ionic strength of chitosan-calcium phosphate suspensions. At faster neutralization rate (using sodium hydroxide), chain entanglements are preserved, leading to a mineralized hydrogel with relatively high storage modulus. Difference in method of preparing suspensions impacted the physical cross-link density due to interactions between polymer chains and calcium or phosphate ions. Higher density of physical cross-links in chitosan-calcium phosphates suspensions obtained from simultaneous method led to mineralized hydrogels with relatively high storage modulus.

Difference in pair of salts to prepare chitosan-calcium phosphate suspensions resulted in difference in ionic strength before and during gelation. Higher ionic strength in chitosan-calcium phosphate suspensions, obtained from CaCl2 and NH4H2PO4 salts sources, led to chitosan chains disentanglements reflected in a relatively low storage modulus in mineralized chitosan hydrogels.

Chitosan hydrogels and mineralized hydrogels were tested for osteoblast cell proliferation. Whatever the material, the measured fluorescence (proportional to the quantity of cells) gradually increases with time. This indicates that the chitosan hydrogels and mineralized hydrogels have no deleterious effects on cell proliferation. However, further experiments are needed to understand the results obtained in this work. It seems necessary to study the phases stability and the possible interactions between the samples and the components of the culture medium that will impact the microstructure, biological and rheological properties of chitosan and mineralized hydrogels.

Part III reports the fabrication processes of the potential bone substitutes developed in this Ph.D. work, based on additive manufacturing methods: using robocasting (a 3-D printing method), and a 3-D printed sintered construct as the ceramic source to prepare hardystonite-chitosan composites. Part III contains two chapters. The first one is on fabrication of architectured physical hydrogels of chitosan with intertwined apatite particles, via robocasting. Chitosan-calcium phosphates homogeneous suspensions were used as inks. As soon as the extrudate left the nozzle, a simultaneous gelation of chitosan and recrystallization of calcium phosphates in the form of apatite was induced by immersion of the extrudate in alkaline bath. This chapter also details results on the rheology of inks, interactions in the systems and characterization of final materials.

The second chapter deals with fabrication and characterization of architectured hardystonitechitosan composite scaffolds. The impregnation of hardystonite scaffolds by chitosan physical hydrogel impacted the mechanical properties of the composite; this study opens another route for the fabrication of architectured composites for bone repair.

La partie III expose l'élaboration et la mise en forme des substituts osseux développés dans cette thèse, par des méthodes de fabrication additive : d'une part en utilisant le « robocasting » (une méthode d'impression 3D) et d'autre part, à partir d'une céramique déjà frittée et architecturée servant de support pour l'obtention de composites chitosane -hardystonite. Ce travail de thèse a été grandement enrichi par des collaborations avec le groupe du

3-D printing of chitosan-calcium phosphate inks: rheology, interactions and characterization

This chapter will be submited as peer-reviewed article with the following co-authors: Solène Tadier, Laurent Gremillard, Eduardo Saiz, Eric Maire, Laurent David, Thierry Delair, Alexandra Montembault

I. Introduction

Worldwide occurrence of bone disorders is a social problem of major concern due to the growing incidence in aged population and under current life circumstances of obesity and low physical activity. During the last four decades, bone tissue engineering has been a constantly growing new research area, whose purpose is to propose solutions for bone substitution and regeneration by a synergistic combination of biomaterials, cells and factor therapy [1].

Bone substitute would be a material that temporary replaces bone tissue, thus ensuring its mechanical function, while promoting bone formation and growth and at the same time biodegradation of the implanted material. Resulting from these requirements, a compromise should be met between sophistication, ease of fabrication, ease of handling by the surgeon and costs [2].

Since it is difficult to find a single material that meets all the properties of a bone substitute, an alternative is to combine materials with complementary properties, for example, bioceramics and biopolymers [1,[3][4][5][6][7][7][8][9]. A composite material composed of calcium phosphates (inorganic, bioceramics) and chitosan (organic, biopolymer) may provide a good substrate for the growth of bone tissue [10][11][12][13].

Chitosan is a non-toxic and biodegradable* natural polymer [14][15][16][17][18][19]. It is known to promote osteoblast and mesenchymal cells proliferation and in-vivo vascularization [20][21][22]. Chitosan has also been reported as antimicrobial to fungi, parasites and bacteria [23].

Apatite is a calcium phosphate bioceramic family, which composition and structure is close to the inorganic part of the extracellular matrix of bones. Apatite osseointegrates*; it is osteoconductive* [2,24,25]; and it dissolves under physiological conditions, allowing new bone growth [2].

Chitosan-calcium phosphates materials have been prepared as membranes, films, scaffolds, injectable materials, multilayers films and particles [22,26]. Among these final physical forms, scaffolds mimic the extracellular matrix of bone by providing a template for cell attachment and bone tissue formation in-vivo [27][28][29][30]. Chemical composition, pore size, pore volume and mechanical strength are the critical parameters defining the performance of bone substitute scaffolds [30]. Three dimensional (3-D) porous microstructures that can be fabricated by different methods including chemical/gas foaming [31], solvent casting, particle/salt leaching [32,33], freeze drying [34], super-critical CO2 drying [35], thermally induced phase separation [36], and foam-gel [37]. However, pore size, shape, and pore interconnectivity at different scales (30-500 m) cannot be fully controlled using all these processing methods [30]. To overcome this drawback, scaffolds have been designed and fabricated using additive manufacturing (AM) approaches, a range of technologies that use computer aided design and automation to build pre-defined structures through a layer-by-layer process [38], and enabling a control of the pore morphology in the micron-scale and above.

Additive manufacturing comprise a range of technologies, including laser additive manufacturing, stereolithography, inkjet printing and extrusion based prototyping [38]. Robocasting technique is an extrusion based prototyping system. Ink is pumped from a syringe to be delivered through a nozzle. A robot determines the nozzle position where ink is extruded drawing out the shape of the printed object [30]. An ink to be used for robocasting should meet two basic conditions: 1) it should flow under mild stress to avoid excessive pumping, but at the same time; 2) it should recover enough stiffness just after extrusion, when the stress is released, to retain the shape and to solidify to bear its own weight and the weight of successive layers extruded on top [39][40][41]. This means that a precise control of rheological properties of the ink is crucial in this technique [41][42][43], through the extrusion and solidification steps. One approach to reach these properties is the use of chemical crosslinking agents in the ink preparation [41]. However, chemical crosslinking agents could complicate the control of ink properties and moreover, they may be toxic to the patient. Besides using chemical crosslinking agents, another approach to reach the rheological properties is to use calcium phosphates powders already synthesized combined with chitosan solution [42]. In all of these approaches, a freeze-drying step is included to end up with a dried scaffold [41][42][43].

An alternative is to form a chitosan physical hydrogel with intertwined particles of calcium phosphates. Such intertwining would also contribute to crosslinking of the network, and would occur by topological and physical interactions still in line with the idea to avoid toxic crosslinkers. Sources of calcium phosphates in this case could be aqueous solutions of calcium and phosphate salts. This approach resembles the physiological mineralization process of bones, since calcium phosphate formation also occurs in a hydrated polymeric matrix. These conditions at low temperatures should come up with a poorly crystalline inorganic phase as it is found in bones [44,45].

Concerning the organic chitosan pseudo-matrix, the interest for hydrogel state raises the issues to have a homogeneously dispersed two phase material that, additionally, may provide a scaffold structure that makes a suitable environment for cell colonization and/or for possible delivery of bioactive molecules [46,47].

Thus, the general objective of this work is to obtain physical hydrogels of chitosan with intertwined apatite particles, via robocasting. To this aim, as soon as the liquid extrudate leaves the nozzle, it was induced, simultaneously, the gelation processes of chitosan and the recrystallization of calcium phosphates in the form of apatite, in-situ in the formed hydrogel.

Printing inks are comprised of acid aqueous solutions of chitosan and salts of calcium and phosphate that needs proper formulation and characterization.

The specific objectives of this work were thus: 1. To prepare, characterize rheological features and test inks composed of chitosan and calcium phosphates, to find a processing window for robocasting. 2. To set up an experimental protocol to fabricate scaffolds of mineralized chitosan hydrogels by 3-D printing-robocasting. 3. To correlate results of chemical composition and structure of inks to investigate possible synergistic physico-chemical interactions (between ions, mineral phase and chitosan chains).

Results showed that inks prepared in this work were shear-thinning fluids and, depending on composition, could even present a yield stress. The rheology behavior was explained in terms of interactions mainly due to hydrogen bonds, electrostatic forces in addition to the viscoelastic properties of the chitosan solution.

II. Applied background for the formulation of chitosan/calcium phosphate inks

The starting materials to fabricate inks are solutions of chitosan, of calcium salt and of phosphate salt, to form suspensions of chitosan and calcium phosphates. Dissolution of chitosan occurs at pH < 6, in an acid aqueous medium, due to electrostatic repulsions between polymer chains induced by protonated amine groups and hydrophilic interactions [48]. The presence of calcium and phosphate salts in solutions at low pH, favors the precipitation of dicalcium phosphate dihydrate, DCPD (CaHPO4.2H2O), as stable calcium phosphate [2,49,50], thus, forming a suspension of chitosan and DCPD particles, that becomes the ink for robocasting in this work.

Classically, inks for robocasting should meet rheological characteristics required by the conditions of flow during operation. Flow along the syringe and through the nozzle occurs at high shear rate. To avoid high pressure pumping, inks should have shear-thinning behavior, that is, viscosity should decrease as shear rate increases. On the other hand, after leaving the nozzle, flow should stop to avoid shape distortion of the filament due to its own weight and to the weight of successive layers extruded on top. To retain filament shape, inks should have yield stress, that is, a material property for which flow would occur only above a minimum stress. Therefore, an ink for robocasting should have shear-thinning behavior and yield stress [51,52]. Another way contributing to avoid shape distortion of the filament is to introduce a rapid change in the structure of the material to stop flow after leaving the nozzle, for example, a gelation of the material [42,51].

Rheological characteristics of chitosan-calcium phosphate suspensions depend upon the physicochemical interactions occurring between chitosan chains, particles and ions, thus, they may be impacted by shear, pH and temperature in addition to the macromolecular structure of chitosan and the microstucture of inorganic particles [53]. Such interactions are mainly hydrogen bonds, electrostatic forces (attractive and repulsive) and polymer chain entanglements [53]. Interactions between intertwined particles of calcium phosphate and chitosan also may affect rheological properties of inks: calcium ions and electron donor atoms (nitrogen and oxygen) of chitosan molecule form coordination bond [54]; phosphate ions and amine groups form electrostatic interactions [55,56]. These interactions may induce chitosan chain cross-links. Chitosan is soluble in acidic aqueous medium (pH ≤ 6) since, at this pH, primary amine groups become protonated, hence, solvation of polymer chains occur by the effects of electrostatic repulsive forces between charged polymer chains and hydrogen bonds of hydrophilic groups with water molecules [47]. Salts, sources of calcium and phosphate ions, dissociate in water forming ions that impact interactions and contribute to the ionic strength of the medium, which in turn, may screen electrostatic interactions [57]. Screening weakens the repulsive forces between polymer chains; chitosan chains become more flexible and tend to disentangle [57]. If the suspension is subjected to flow, disentanglement may occur by the application of high shear strain [58]. As concentration of chitosan increases, polymer chain entanglement increases [47]. Thus, entanglements tend to increase with concentration of chitosan and to decrease due to screening effect or when shear rate is applied [57,59].

As a result, interactions between species (chitosan chains, ions in solution an calcium phosphate particles) in the ink tend to form a physical network that determines the rheological behavior of the so-called structured fluids, which, usually are non-Newtonian, and non-linear viscoelastic fluids [58], due to the impact of shear on the network. The non-linear rheological behavior of chitosan-calcium phosphate suspensions is thus a key parameter to be related with the 'printability' of given ink.

The fundamental rheological relation is between stress and strain. Experimental data could be adjusted to many equations. One the simplest, even useful rheological equation is the power-law (equation 1).

𝜏 = 𝐾𝛾̇𝑛 𝑜𝑟, 𝑙𝑜𝑔 𝜏 = 𝑙𝑜𝑔 𝐾 + 𝑛𝐿𝑜𝑔 𝛾̇ (1)
Where 𝜏 is the shear stress, γ̇ the shear rate, n is the flow exponent and K is a scaling factor also known as the flow consistency index [51,58]. The value of n is an adjusted parameter to fit experimental data to power-law equation. A physical sense could be and is commonly assigned to its value: if n = 1, it is a Newtonian fluid; the departure of n from 1 is an indicative of less Newtonian behavior; if n < 1, the fluid presents shear-thinning behavior, that is, viscosity drops as shear increases. In this case it is assumed that shear breaks physical or chemical interactions that makes the fluid depart from Newtonian behavior; it is said that the fluid presents shear-thinning behavior. The presence of chitosan is associated to the presence of chain entanglements that are disentangled as shear rate is applied. Disentanglement makes the fluid less structured. Therefore, it is expected that suspensions of chitosan and calcium phosphates present shear-thinning behavior, that is, n < 1. For n > 1, viscosity increases as shear increases, the fluid is said to present shear-thickening behavior [51,58].

123

The extrudate must be converted from a chitosan -DCPD suspension into a physical hydrogel of chitosan mineralized with apatite. Gelation of chitosan solutions can occur at neutral or basic pH, due to a modification of the balance between hydrophilic and hydrophobic interactions within the system [46,47]. The increase of the pH in proper physico-chemical conditions leads to the formation of physical hydrogels. Protonated amine groups become deprotonated; therefore, repulsion between polymer chains is reduced. Weakening of repulsion between polymer chains allows for the formation of junctions between groups of polymer chains, constituting the physical cross-linkers of the hydrogel [48]. On the other hand, at pH  6.5, apatite is the most stable calcium phosphate at room temperature [2,60].

III. Materials and Methods

A. Ink preparation and formulation

Inks were prepared as acidic aqueous suspensions of calcium phosphate particles in a chitosan solution.

Chitosan powder produced from squid pens and supplied by Mahtani Chitosan Pvt. Ltd (batch type 114, No S3 20110121) was characterized with standard procedures [47]. The degree of acetylation, corresponding to the molar fraction of acetylated units within the polymer chains, was close to 5 %; the weight-average molar mass and dispersity were Mw = 550 kg.mol -1 and Ð = 1.9, respectively.

Chitosan acetate solutions of various molar concentrations (0.15 M (2.4 wt %), 0.17 M (2.8 wt %) and 0.19 M (3.1 wt %) were prepared by adding chitosan powder to acetic acid (Carlo Erba Reagents, CAS 64-19-7, assay 99.9 %) aqueous solutions. The mass of chitosan powder was calculated taking into account the molar mass of the repeating unit (D-glucosamine and N-acetyl glucosamine), 163.10 g.mol -1 . The amount of acetic acid was adjusted to match the stoichiometric protonation of -NH2 sites of chitosan. Chitosan solution was mixed in a Thinky ARE-250 planetary mixer at 2000 rpm in mixing cycles of 5 minutes until complete dissolution.

Salt solutions were prepared by dissolving Ca(NO3)2.4H2O (Sigma-Aldrich, CAS 13477-34-4, assay ≥ 99 %) and (NH4)2HPO4 (Sigma-Aldrich, CAS 7783-28-0, assay ≥ 99 %) in water. Initial concentrations are reported in Table 1. Calcium and phosphate solutions were added simultaneously into chitosan solutions, and mixed in the planetary mixer at 2000 rpm in mixing cycles of 5 minutes until having of chitosan-calcium phosphate homogenous suspensions.

Chitosan solutions and salt solutions were mixed in such volume proportions as to obtain different proportions of inorganic to organic components (in/or) while maintaining the calcium to phosphorous molar ratio constant to 1.67 (i.e. the same value as in stoichiometric hydroxyapatite Ca10(PO4)6OH2). Thus, the chitosan salts solutions were formulated according to the ratios defined in equations ( 2) and (3). Three chitosan solutions of different polymer concentration, without added salt, were prepared as references. The compositions of inks are reported in Table 1. Most of the ions present in the solution resulted from dissociation of salts. Since dissociation of salts is dependent on pH, this parameter was systematically recorded. In our case, pH of chitosancalcium phosphates suspensions was measured as 5.5, therefore, the calcium nitrate was fully dissociated, and the major ionic form of the phosphate ions was HPO4 2-.

𝑀𝑎𝑠𝑠
The initial ionic strength was calculated for all inks, and reported in Table 1. After mixing, the ionic strength should decrease as calcium phosphates precipitate. Since it is difficult to calculate and measure the ionic strength after/during calcium phosphates precipitation, the initial ionic strength was chosen as a parameter to compare inks.

B. Rheological behavior of chitosan-calcium phosphate inks

The rheological measurements of inks were carried out using a TA Instruments Discovery HR-1 rheometer with 40 mm parallel plate geometry, 1mm gap and a solvent trap to prevent drying.

Three kinds of tests were performed on each ink prepared:

1. Amplitude sweep test between 1-100 % of strain, in dynamic conditions, at 0.1 rad.s -1 and at 25 °C. The output of the test is the evolution of moduli (G', G") vs. oscillation strain. This test allows the determination of the linear viscoelastic region (LVR), above which the rheological behavior changes with strain due to the disruption of the network structure. Besides the LVR evaluation, the test allows to identify if ink has yield stress provides an estimated value. 2. Time test at a strain of 0.1 % and angular frequency of 0.1 rad.s -1 were performed up to 300 s. The output of the test is of the evolution of storage and loss moduli, G' and G" vs. time. We used such time tests to determine if at the material is in a steady state. Besides information on stability of the system, this test serves to characterize the rheological behavior of the material, elastic or solid-like if G' > G", or viscous liquid-like, if G" > G'. 3. Flow ramp tests were conducted in continuous mode at shear rates of 10-1000 s -1 . The output is either a shear stress vs. shear strain rate or a viscosity vs. shear rate graph. This test allows to characterize the non-linear behavior and quantify the shear-thinning behavior of the ink.

C. Printing and characterizations

The printing machine was a Robocasting system (3DInks, USA). A 3D CAD model of 8 mm x 8 mm was designed using the RoboCAD software (3DInks, USA). A syringe barrel of 3 cm 3 was filled with different inks. To avoid air bubbles in the ink, it was slowly transferred to a second syringe. Conical nozzle of 400 m internal diameter was used to print all scaffolds.

Printing was performed on a flat and smooth glass substrate immersed in a bath containing sodium hydroxide hydro-alcoholic solution (water 70/ethanol 30) (the range of NaOH concentration used was between 0.05 M and 0.40 M). Immediate gelation of ink was induced by this alkaline bath, while the presence of ethanol made the bath less dense than the ink and prevented the extrudate from floating (as occurred with a bath containing only sodium hydroxide water solution). The temperature of the system was controlled by means of a custom-built enclosure and a convection heater set to 23 °C while the humidity was measured and varied from 65 to 85 %. Printed hydrogel scaffolds were washed several times with distilled water until pH in the final washing water was constant, around 7. They were stored in water for further characterization.

The architecture of scaffolds was studied by X-ray micro-tomography using a Phoenix v│Tome│xmicro-tomographer (μ-CT) (General Electric, USA) equipped with a Varian Paxscan detector (1920x1536 pixels), and with voxel size between 1 and 4 μm.

X-ray diffraction (XRD) analyses were performed using a D8 Advance Bruker AXS diffractometer operated at 40 kV and 40 mA, with CuKα radiation (1.54060 Å) Scans were acquired in a θ-θ configuration, from 4 ° to 55 ° with a step time of 129 s and a step size of 0.019 °. Phase identification was conducted by comparison to standard patterns from International Center for Diffraction Data -Powder Diffraction Files (ICDD-PDF) with the aid of DiffracPlus EVA software (AXS, Bruker). The PDF used for apatite and dicalcium phosphate dihydrate (DCPD) identification were 09-0432 and 09-077 respectively. The obtained diffractograms were normalized with respect to the height of the peak of greatest intensity in the crystalline mineral phase.

XRD was performed on chitosan-calcium phosphate suspensions. XRD and Scanning Electron Micoscopy (SEM) analysis were also performed on freeze-dried scaffolds. Scaffolds were first immersed for freezing in liquid nitrogen for 1 minute, then the frozen samples were left overnight in a vacuum pump at -86 °C at pressure of 0.008 mbar. SEM analyses were performed on gold-coated freeze-dried scaffolds, using imaging with secondary electrons in a JEOL JSM-6010 SEM (USA) at 20 kV acceleration voltage. Element maps showing the distribution of elements were obtained by Energy Dispersive Spectroscopy (EDS) using a Silicon Drift Detector (SDD) coupled to the SEM. Optical microscopy analysis was conducted using a Light Axiophot microscope (Zeiss, Germany). Samples of chitosan-calcium phosphate suspensions were fixed between two glass slides and observed in transmission mode. Size of crystal aggregates was measured using ImageJ software [61] in three 64μm 2 micrographs of each sample.

IV. Results

A. Chemical composition and microstructure of inks

All inks prepared in this work were chitosan-calcium phosphate suspensions and had (Table 1) had a pH about 5.5.

The reaction schema for DCPD formation under the conditions of this work could be represented as follows:

𝐶𝑎(𝑁𝑂 3 ) 2 → 𝐶𝑎 2+ + 2𝑁𝑂 3 - (4)
(NH 4 ) 2 HPO 4 → 2𝑁𝐻 4 + + 𝐻𝑃𝑂 4 2- (5)
Divalent phosphate ions can react: 1) forming DCPD with calcium ions; 2) decomposing to form monovalent phosphate ions in an equilibrium reaction:

𝐶𝑎 2+ + 𝐻𝑃𝑂 4 2-+ 2𝑂𝐻 -+ 2𝐻 + → 𝐶𝑎𝐻𝑃𝑂 4 2𝐻 2 𝑂 (6)
𝐻𝑃𝑂 4 2-+ 𝐻 + ⇄ H 2 PO 4 -(𝑝𝐾 𝑎 ~7.2) (7)
It was observed that immediately after mixing salt solutions and chitosan solution, the system was no longer transparent but rather white indicating the formation of DCPD particles (Figure 1).

The crystalline phase in the chitosan-calcium phosphate suspensions was investigated by XRD.

According with the XRD results (Figure 2), the peaks corresponding to the principal planes of DCPD, (020) at 2θ = 11.681 °, (021) at 2θ = 20.935 °, (041) at 2θ = 29.258 °, (-221) at 2θ = 30.506 °, (220) at 2θ = 34.156 °, (022) at 2θ = 37.105 ° were identified. DCPD was the only detectable crystalline phase of calcium phosphates in the chitosan-calcium phosphate suspensions.

No diffraction peaks of chitosan were identified indicating that the amount of crystalline chitosan was below the detection limit. The X-ray diffuse halo observed from approximately 2θ = 24 ° to 36 ° in the diffractograms may be attributed to the presence of water (about 96 wt % of the suspension was water.

127 Optical micrographs of inks (chitosan-DCPD suspensions) (Figure 3, example for ink 6) showed that they were composed of a dispersed phase, platelet aggregates of DCPD, in a continuous phase (chitosan solution). The dimensions of aggregates were 20 ± 5 µm length and 14 ± 5 µm width. Most of divalent phosphate ions, HPO4 2-, were expected to form DCPD, although, some should remain in the system due to the equilibrium with monovalent phosphate ions, H2PO4 -, reaction (6). DCPD has a relation Ca/P = 1; since the salts were added to have a proportion Ca/P = 1.67, it is safe to affirm that calcium ions remained in the chitosan-DCPD suspension. On the other hand, DCPD crystals contain Ca 2+ , HPO4 2-ions and water molecules. Chitosan-DCPD interactions may occur in the suspensions: calcium and divalent orthophosphate ions may form physical cross-links between polymer chains and hydrogen bonds may form between water molecules of DCPD crystals and hydrophilic groups in chitosan molecules.

B. Rheology study

Rheology analysis of inks allows determining if the tested solutions meet the requirements for robocasting with both shear-thinning and yield stress evidence. We can discuss the rheological behavior invoking the physico-chemical interactions between chitosan and ions in solution.

Linear viscoelastic region

LVR, see section III.B, can be determined by measuring moduli, G' and G" as function of strain. Figure 4 shows the results for the set of inks tested.

Observing the region of constant moduli for each ink sample and for references of chitosan solutions. The LVR strain limit at 0.1 rad.s -1 was below 10 % in all cases. Thus all other rheological tests we performed at strains below 10 %.

µm

Time sweep experiments

Other important consideration when testing suspensions is to find out if the system is in steady state. Figure 5 shows the results of oscillation time test. It may be observed that moduli remained rather constant over time for all tested samples indicating stability. Values of loss modulus G" and storage modulus G' are presented in Table 2, column 3 and 4, respectively. From these data, it can be deduced that inks 5, 6, 7, 8, 9 and Ref3 are solid-like (G' > G"), while inks 1, 2, 3, 4, Ref1 and Ref2 are liquid-like (G' < G''). Thus, the chitosan concentration largely impacted the viscoelastic properties; an apparent gel state is obtained at the highest concentration at 3.1% w/w. Adding calcium and phosphate salts decreased the values of viscosities, elastic and loss shear moduli, as a result of salt screening effects.

Shear-thinning behavior

The stress-strain-rate curve of chitosan -calcium phosphate inks were well described by a powerlaw model, as shown by equation ( 1), with flow exponent n  1 [58].

𝜏 = 𝐾𝛾̇𝑛 𝑜𝑟, 𝑙𝑜𝑔 𝜏 = 𝑙𝑜𝑔 𝐾 + 𝑛𝐿𝑜𝑔 𝛾̇ (1)
Where 𝜏 is the shear stress, γ̇ the shear rate, n is the flow exponent and K is a scaling factor also known as the flow consistency index [51,58].

Figure 6 shows the rheological curves for tested inks. Curves were fitted to equation ( 1) to estimate values of K and n. Values of n much lower than 1 were calculated for all inks, indicating a high shear-thinning behavior. Table 2, column 5, shows the results. Shear-thinning behavior can be also observed from viscosity vs. shear rate curve. It is characterized by a decrease of viscosity as shear rate increases, as shown in Figure 7. Thus, all inks tested met the condition of shear-thinning for robocasting. The shear-thinning behavior of chitosan solutions is well known [62][63][64]. This effect has been attributed to polymer chain disentanglements and/or interchain junction breakdown under shear [65].

Yield stress

In cases of inks 5, 6, 7, 8, 9 and Ref3 (Figure 8), the ink was solid-like at low oscillation amplitudes and there was a point where G' declined; indicative of a breaking of the solid structure to become a fluid, therefore, the material has yield stress. For the case of ink 8 as example, it may be observed that up to a strain of about 6 %, G' was constant and higher than G", indicating a solid-like material. Above that critical strain ( 𝛾 𝑐 , equation 8), storage modulus declined and the material gradually changed from solid-like to liquid-like behavior, the liquid-like behavior being obtained at a strain around 100% (red square in Figure 8). The storage modulus and the critical strain gc delimiting the linear viscoelastic region (determined in Figure 8) should be empirically related to an ideal yield stress that separating the transition from solid-like to liquid-like behaviors (see Figure 8). In this view, we determined the yield stress 𝜎 𝑦 according to equation ( 8) [58].

𝐺 ′ (𝛾 < 𝛾 𝑐 ). 𝛾 𝑐 = 𝜎 𝑦 (8) As an example, in the case of ink 8, storage modulus was 148 Pa, the critical strain was estimated to be 6 %; therefore, the estimated value of yield stress was ~ 9 Pa. In analogous manner, for inks that show a well-defined yield stress, values of critical strain and storage modulus were measured from curves of storage and loss moduli vs. oscillation strain, and the corresponding values of yield stress were calculated with equation (8). The results are shown in Table 2, in column 6.

C. 3-D printing of scaffolds

To design the robocasting processing, three major key-points must be addressed: ink rheological properties (stability, shear-thinning behavior and yield stress), machine parameters and printing environment.

All inks (chitosan-calcium phosphate suspension) were prepared at acid pH, ~5.5, they were stable, presented shear-thinning behavior and suspensions with high concentrations of chitosan presented yield stress, as shown in the preceding section.

A bath of sodium hydroxide was chosen as printing environment. The basic medium favored mainly two goals. The first one was the retention of filament shape after extrusion thanks to gelation process, regardless of yield stress (that is, retention of filament shape by a change of material structure from suspension to hydrogel). The second goal, concurrent to gelation process, is the immediate phase transformation of the mineral phase in the filament, from DCPD to apatite. These facts support the final aim of 3-D printing of mineralized chitosan physical hydrogel with intertwined apatite particles.

Machine parameters in robocasting are nozzle diameter (D, μm); distance between layers in vertical direction (z, μm); filament distance in direction on the printing plane (Dx, Dy, μm); speed of ink deposition by extrusion from the nozzle tip (S, mm.s -1 ), distance between nozzle tip and substrate (Zero, mm) [52,66]. Such machine parameters were fixed after a few trial experiments. A set of printing parameters was chosen as shown in Table 3. In particular, the z distance between two successive layers was set between 32.5 % and 35 % of nozzle diameter (0.4 mm) to favor a good adhesion between the printed layers, whereas the usual value found experimentally in robocasting of other different material inks is in the range of 70-80 % of nozzle diameter.

As a result, scaffolds from all inks tested were successfully printed up to five layers (final with of printed sample: 2 mm) (Figure 9). However, printing more than five layers was difficult, since the printed filament had not the necessary stiffness to support the weight of the layers printed above. Thus, further optimization of ink formulation and alkaline bath are needed.

Table 3. Printing parameters for this study.

Printing parameters Value

Internal nozzle diameter, D 400 µm

D. Characterization of printed scaffolds

Scaffolds were characterized to determine their composition and microstructure, more precisely to identify the calcium phosphate phase formed and the spatial distribution of inorganic and organic phases.

Printed scaffolds were thus studied by X-ray micro-tomography. As an example, Figure 10 shows a well-defined grid-like sample observed in a μ-CT image of scaffold fabricated with ink 5. The gray scale used allows distinguishing lighter zones that correspond to calcium phosphate aggregates in a darker matrix corresponding to chitosan hydrogel. Black channels are empty spaces (with air). The calcium phosphates aggregates were rather homogenously distributed within the polymeric matrix in all scaffolds.

From XRD analyses of freeze-dried printed scaffolds, apatite was identified as inorganic crystalline phase (Figure 11). An X-ray diffuse halo (between 8 °and 22 °) can be observed, and it was attributed to chitosan; other peaks corresponded to the reflection lines of apatite, (002) at 25.879 °, (211) at 31.774 ° and (112) at 32.902 °. Peaks were broad and not very well defined indicating poorly crystalline apatite, in spite of inorganic/organic ratios up to 80/20. XRD of scaffold made from ink 1 shows no peaks of apatite but an X-ray diffuse halo (between 24 ° and 34 °) of somewhat amorphous structure. However, the X-ray diffuse halo may indicate the presence of amorphous inorganic phase. These results could be explained considering that this scaffold was made from the ink with the lowest calcium and phosphate concentration and the lowest inorganic/organic ratio (Table 1); therefore, the amorphous phase dominates the diffractogram in comparison with the other scaffolds.

135 EDS analyses allowed to detect the presence of elements on the surface of freeze-dried scaffolds. Signals for carbon, oxygen, phosphorous, calcium and gold were found, as shown in Figure 13, whereas no signal was found for nitrogen (Figure 13a). Signal corresponding to nitrogen seemed to be masked by carbon and oxygen signals. Distribution of each element detected on the surface of scaffold seemed to be homogeneous (Figure 13, c-f).

V. Discussion

Questions arise related with the order of magnitude of shear rate and viscosity of ink under the conditions of robocasting used in this work (Table 3). We estimated such processing parameter values from capillary rheology equations for non-Newtonian fluids. Equation ( 9) is valid for flow in pipes/capillaries for fluids of any rheology:

𝜏 = ∆𝑃 2𝐿 𝑅 ( 9
)
Where 𝜏 is the shear stress; ∆𝑃/𝐿 is the pressure drop per unit length of nozzle; 𝑅 is the radius of the nozzle. Volumetric flow in pipes can be calculated with the Hagen-Poiseuille equation, modified in the case of a power-law fluid (10):

𝑄 = 𝜋𝑅 3 1 𝑛 + 3 ( ∆𝑃 𝑅 2𝐾𝐿 ) 1 𝑛 (10)
In equation (10), 𝑄 is the volumetric flow and the other parameters were as defined in section IVB.

Volumetric flow can also be calculated with flow velocity of fluid (speed ink deposition), S, and nozzle diameter, equation (11):

𝑄 = 𝑆 𝜋𝑅 2 (11)
Combining equations ( 1), ( 9), (10) and (11), the shear rate can be classically calculated according to (8)

𝛾̇= 𝑆 ( 1 𝑛 + 3) 𝑅 (12)
Applying equation (12), taking a value of n = 0.2, from Table 2, S = 10 mm.s -1 and R = 0.2 mm then, the shear rate of flow along the nozzle is evaluated to be γ̇ = 400 s -1 . At this shear rate, a typical shear stress would be τ = 1100 Pa (Figure 6). The pressure drop per unit length can be calculated from equation (9). The result is ∆P L = 11000 Pa/mm. Such parameters are quite acceptable for extrusion across the nozzle with length, L = 30 mm. From Figure 7, viscosity can be read at γ̇ = 400 s -1 and can be extrapolated at a shear rate close to rest (when filament leaves the nozzle), γ̇ = 1 s -1 . Table 2 (columns 7 and 8) shows the calculated values of viscosity for all ink tested at those shear rates.

Due to shear thinning, viscosity at the exit of the nozzle was much higher than along the nozzle. However, it was not high enough in all inks to keep shape of the printed scaffold (viscosity of at least 600 Pa s would have been required [51]), therefore, a printing bath that favors the retention of shape of the extruded filament was necessary.

In the following, our discussion will focus on finding correlations between composition of inks (Table 1) and their rheological behavior, based on data reported in Table 2.

The rheological features of the prepared inks (chitosan-DCPD suspensions) depend fundamentally upon composition, pH and temperature. For the case, all suspensions were at about the same pH = 5.5 and all processes and tests were performed at room temperature. Concentration of species (chitosan and dissolved ions), and inorganic to organic ratio (in/or) were the variables of composition.

In the chitosan concentration range used in this work, viscosity of chitosan solutions and viscosity of inks were hundreds of times higher than viscosity of solvent (water), therefore, it would be safe to expect chain entanglements in all inks tested. Density of chain entanglement is higher as chitosan concentration is higher. Chain entanglement makes the fluid more structured, that is, less Newtonian. The corresponding effect of chain entanglement on rheological properties would be, effect 1: increase in chain entanglement density. At higher chain entanglement density, the fluid would be more solid-like, would show more shear-thinning behavior, would have higher storage modulus, there would be higher probability of showing yield stress and would have higher viscosity [62][63][64].

Inorganic to organic ratio (in/or) is directly related with the mineral content (DCPD precipitate) of the system and with the concentration of dissolved ions in the system. As the mineral content increases, the fluid becomes more structured, more solid-like, with the corresponding effect on rheological properties, effect 2: increase in mineral content. As mineral content increases, the fluid would be more shear-thinning behavior, would have higher storage modulus, there would be higher probability of showing yield stress and would have higher viscosity [53]. On the other hand, concentration of dissolved ions determines the ionic strength of the in the system. Screening of electrostatic interactions occurs as an effect of ionic strength. The screening of electrostatic interactions reduces the repulsive forces between chitosan polymer chains. Therefore, the fluid becomes less structured, the non-Newtonian nature decreases, more fluid-like with the corresponding effect on rheological properties, effect 3: increase in ionic strength. At higher ionic strength, elastic properties (G'), shear-thinning behavior and viscosity decrease [53]. It should be noticed that in/or ratio provokes to opposite tendencies: as in/or ratio increases, mineral content and ionic strength increase. As in/or ratio increases, structured fluid nature increases by increasing mineral content and decreases by increasing ionic strength. The observed rheological features would be the result of these two opposite tendencies.

Experimental results could be discussed with these bases:

The effect of chitosan concentration alone can be analyzed in data of references Ref1, Ref2 and Ref3 (Table 1). As chitosan concentration increased, chain entanglement density increased. Therefore, effect 1 would be expected. In fact, the chitosan solution changed from liquid-like at concentrations of 0.15 M and 0.17 M, to solid-like fluid at concentration of 0.19 M (Table 2). Yield stress was observed only in the most concentrated sample. Viscosity increased as chitosan concentration increased (Table 2). It was also observed (Figure 14) that chitosan solution of all tested concentration showed shear-thinning behavior and that shear-thinning behavior was higher as chitosan concentration increased (the absolute value of slope of viscosity vs. shear rate curve increased with chitosan concentration). That is, as the chitosan concentration increased, the fluid became more structured. Inks with the same chitosan concentration and increasing in/or ratio would be expected to have higher mineral phase content and higher ionic strength. For the higher mineral content, effect 2 is expected; for the higher ionic strength, effect 3 is expected. Set of inks 1, 2, 3 and 4 have chitosan concentration of 0.15 M and increasing in/or ratio (Table 1). Experimental results showed an increase in ionic strength and a decrease in storage modulus and viscosity (Table 2). Therefore, the effect of ionic strength dominated on the effect of mineral content. The set of samples were liquid-like fluids (G" > G'). None of the samples showed yield stress. The value of n, indicative of shear-thinning, showed no clear variation, that is, shearthinning behavior was about the same for set of inks of the same chitosan concentration.

Set of inks 5, 6 and 7 (analogous to inks 1, 2, 3 and 4) have chitosan concentration of 0.17 M (Table 1). Experimental results showed that, in this case also, the effect of ionic strength dominated on the effect of mineral content. But for the set with higher chitosan concentration, fluids were solid-like (G' > G") and yield stress appeared, effect 1 was evidence. However, yield stress decreased as ionic strength increased. That is, yield stress appeared at higher chitosan concentration and its value diminished as ionic strength increased (Table 2). No significant change in shear-thinning behavior in the set 5, 6 and 7 was observed. However, compared with the set 1, 2, 3 and 4, it may be deduced that as chitosan concentration increase from one set to another, shear-thinning behavior increased (lower n values observed at higher chitosan concentration, Table 2). Set of inks 8 and 9, at chitosan concentration 0.19 M and decreasing in/or ratio and ionic strength, showed that experimental results led to the same discussion of set 5, 6 and 7 (Table 2).

Inks with the same in/or ratio and increasing chitosan concentration and ionic strength would have increasing chain entanglement and higher screening of electrostatic interactions. For the higher chain entanglement density, effect 1 is expected; for higher ionic strength, effect 3 is expected. Sets of inks 2, 5; 3, 6 and 4, 7, have the same in/or ratio, and increasing chitosan concentration and ionic strength in each set (Table 1). Experimental results showed that storage modulus, yield stress and viscosity increased, whereas n value decreased. That is, the fluids became more structured, effect 1 dominated on effect 3. That is, the dominant factor determining rheological properties was chitosan concentration. The fluids showed more shear-thinning behavior as chitosan concentration increased.

VI. Conclusions

The experimental protocol set up for 3-D printing by robocasting was adequate to fabricate scaffolds from chitosan-calcium phosphate suspensions at pH of 5.5, and to convert the extrudate into a physical hydrogel of chitosan with intertwined particles of apatite.

Characterization of composition and microstructure lead to conclude that tested scaffolds, obtained by robocasting in this work, were physical hydrogels of chitosan with intertwined particles of calcium phosphate in the form of apatite. Apatite was poorly crystalline, forming aggregates in platelet shape of nanometric scale with a rather uniform distribution within the polymer matrix of chitosan.

Properties of inks used in this work depend upon two composition variables in the preparation of the suspensions: chitosan concentration and inorganic to organic ratio (in/or ratio). Chitosan concentration determines the formation of polymer chain entanglements. The in/or ratio determines the mineral content and the ionic strength in the suspensions.

As chitosan concentration increases, the fluid becomes more structured; that is, less Newtonian, shear-thinning behavior, solid-like nature and viscosity increase. Chain entanglement and mineral content also make the fluid less Newtonian; ionic strength makes the fluid more Newtonian.

The analyzed relations between composition and rheological properties of inks tested here, open further research directions to find quantitative correlations (composition, properties), to tune composition parameters to specific ink behavior. Another point for further research is to secure enough stiffness of the extrudate at the nozzle exit to maintain sample shape. Options to be tested could be: use of physical crosslinking or even mild chemical crosslinking; increasing yield stress by changing composition and structural parameters. Another approach could be to change from gelation in an alkaline liquid bath to gelation in an ammonia vapors saturated atmosphere.

Chapter III.2

Fabrication and characterization of hardystonite-chitosan composite scaffolds

This chapter will be submitted as peer-reviewed article with the following co-authors: Solène Tadier, Laurent Gremillard, Eric Maire, Laurent David, Thierry Delair, Alexandra Montembault, Hamada Elsayed, Paolo Colombo

I. Introduction

Worldwide occurrence of bone disorders and healing conditions is a social problem of major concern due to the growing incidence in aged population and under current life circumstances of obesity and low physical activity. During the last four decades, bone tissue engineering has been a constantly growing new research area, whose purpose is to propose solutions for bone substitution and regeneration by a synergistic combination of biomaterials, cells and growth factors [1].

Several materials in different shapes have been used for bone tissue engineering. Materials include bioactive glasses, bioceramics, inorganic filler reinforced biopolymer composites as well as polymer-coated or infiltrated ceramic and organic-inorganic composites; shapes include scaffolds, pellets, cements pastes and others [2]. Among composite scaffolds, impregnation of ceramics with polymers has been widely reported in the literature [1][2][3][4][5][6][7][8]. Ceramic scaffolds have mostly been impregnated with synthetic polymers such as polycaprolactone (PCL), poly-DL-lactide (PDLLA) or poly(lactic-co-glycolic acid) (PLGA); more recently, polymers of natural origin, such as gelatine, silk, alginate, collagen and chitosan, have been used [2]. The addition of a soft biodegradable polymer to inorganic scaffolds may provide a toughening or strengthening effect besides the possibility of becoming a vehicle for controlled in-situ delivery of bioactive molecules, for example, growth factors [2] or adhesion peptides [9,10].

This work deals with the preparation and characterization of hardystonite (HT) and chitosan (CS) composite material. The aim is to develop protocols to obtain HT scaffolds impregnated with CS physical hydrogel and find out the effect of impregnation on mechanical properties of composites.

A few studies of scaffold impregnation by chitosan have been reported in the literature [11][12][13][14], but no article concerning hardystonite scaffold impregnation with chitosan physical hydrogel was found.

Hardystonite and chitosan were chosen for their biological properties [18][19][20], which are of interest for the conception of a bone substitute material.

Hardystonite (Ca2ZnSi2O7) is a bioceramic of interest for biomedical use because of its biological properties [15][16][17]. It has been shown to increase proliferation and osteogenic differentiation of mesenchymal stem cells [17,18]. Furthermore, it have been proven to stimulate expression of alkaline phosphatase, osteocalcin and collagen type I when in contact with human osteoblast-like cells [16,18]. The bending strength of dense HT is in the 50-140 MPa range, reaching the range of human cortical bone (50-150 MPa), and Young's modulus in the range of 20-40 GPa also close to that of cortical bone (7-30 GPa) [19,20]. In order to increase cell colonization and cytocompatibility, different kind of bioceramics, including HT, have been manufactured with relatively high porosity [21], thereby limiting the strength of the material. In comparison with hydroxyapatite (Ca5(PO4)2OH), hardystonite has shown improved biocompatibility, bending strength and fracture toughness [15,18,19,22].

Chitosan is a copolymer of N-acetyl-glucosamine and glucosamine, mainly obtained by deacetylation of chitin. Chitin is one of the most abundant polysaccharide on earth, found in particular in the exoskeleton of crustaceans and the endoskeleton cephalopods [23,24]. It can be regarded as a waste from fish industry and, as such, chitin/chitosan extraction is a valorization of this by-product. Chitosan is a non-toxic and biodegradable* natural polymer [25,26]. It is known to promote osteoblast and mesenchymal cells formation and in-vivo vascularization [27][28][29].

Chitosan-based hydrogels have promising properties for fabrication bone substitutes, such as biodegradability*, antimicrobial activity and low toxicity [26,[30][31][32].

The physical gelation of a chitosan solution can occur at neutral or alkaline pH and it is due to a modification of the balance between hydrophilic and hydrophobic interactions within the solution [33]. Such gelation occurs when solution is in contact with a base, e.g., ammonia [33] or sodium hydroxide [34]. The increase of the pH in proper physical chemical conditions leads to the formation of physical hydrogels. No external crosslinking agent is necessary for this gelation process.

The solution state of chitosan occurs at pH < 6, and it is due to the presence of protonated amine groups that allow for hydrophilic interactions, making chitosan soluble in acid aqueous medium [35][36][37][38].

Here HT scaffolds were prepared by 3-D printing of preceramic-filled systems of specific geometry, followed by formation of hardystonite ceramics (ceramization), according with a preceramic processing route reported in a previous work [18]. HT scaffolds were impregnated with solutions of chitosan, followed by gelling of the solutions to obtain hardystonite-physical hydrogel of chitosan composites, HT-CS.

Crystalline phase identification and porosity of HT and HT-CS structures were characterized.

Fracture surface of HT-CS scaffolds were analyzed to investigate the presence of chitosan in the pores, and possible polymer-ceramics interactions. Fracture behavior (in particular compressive strength) was assessed in HT and HT-CS scaffolds. It was found that the fabricated HT scaffold was a ceramic of high purity that presents both micro and macro porous that were filled with physical hydrogel of chitosan. In the HT-CS system, chitosan hydrogel partially filled the pores contributing to bearing of external loads and to energy dissipation by fracture.

II. Materials and Methods

A. Preparation of 3-D hardystonite scaffolds

The preparation of 3-D Hardystonite scaffolds (HT scaffolds) includes the steps of: i) design and preparation of printing ink, ii) printing of a preceramic-filled system of specific geometry and iii) formation of hardystonite ceramics (ceramization).

Ink was designed and prepared to produce pure hardystonite upon heat treatment. A commercially available silicone resin, MK (Silres MK polymethylsilsesquioxane, in form of powder <100 μm, Wacker Chemie, Germany) was dissolved in isopropanol alcohol under stirring. To modify ink rheology and provide a suitable pseudo-plastic behavior, silicone resin and fumed silica (FS, Aerosil R106, Evonik, Essen, Germany) were mixed in a weight ratio of 90MK:10FS. Active fillers, powders of calcium carbonate, CaCO3 (< 10 µm; Bitossi, Italy) and zinc oxide, ZnO (d50 = 14 µm; Sigma Aldrich, Germany) were added in the appropriate molar ratio to lead to hardystonite ceramic as a final product after ceramization. 30 wt% hardystonite powder, previously synthesized under the same procedure, was added to the mixture to improve final quality, as will be explained later. Preceramic-filled system was mixed in an agate jar with agate balls (diameter ~1 cm) by a planetary ball mill for 4h at 400 rpm. The resulting mixture was the designed ink for printing.

A printer equipped with a syringe (Powerwasp Evo, Wasp, Massa Lombarda, Italy) was used to fabricate HT scaffolds [39] whose geometry was 15  5  5 mm 3 , with 1 mm spanning length between the center of two contiguous filaments in the same layer, and layer thickness of 0.35 mm. Ink was placed into a syringe and extruded at room temperature through a conical nozzle (D = 0.41 mm, Nordson Italia S.p.A., Milano, Italy).

High purity hardystonite ceramics were obtained by heating the preceramic-filled system in air, for 1h at 1200 °C. During ceramization, calcium carbonate decomposed releasing gaseous carbon dioxide that may cause cracks in the ceramics. The crack formation was minimized through two actions. First, addition of 30 wt% hardystonite powder, previously synthesized under the same protocol, grounded and sieved to < 45 µm, to the ink preparation reduced the volume of CO2 produced by calcium carbonate decomposition. Second, a multistep thermal treatment was performed, with low heating rates and holding stages at the temperature of the highest weight loss [39].

B. Preparation of hardystonite-chitosan physical hydrogel composite.

Preparation of hardystonite-chitosan composites (HT-CS) involved three steps: preparation of chitosan solution, impregnation of hardystonite with chitosan solution and finally gelation to form the chitosan physical hydrogel Chitosan powder produced from squid pens and supplied by Mahtani Chitosan Pvt. Ltd (batch type 114, No S3 20110121) was used. It was characterized before use as previously described in [33]. The degree of acetylation, corresponding to the molar fraction of acetylated units, was close to 5 146 %; the weight-average molar mass and the dispersity were Mw=550 kg.mol -1 and Ð=1.9, respectively.

Three chitosan solutions were prepared (at 0.5, 1.5 and 2.5 wt% or 0.03, 0.09 and 0.15 M respectively) by adding chitosan powder to an acetic acid aqueous solution (acetic acid: Carlo Erba Reagents, CAS 64-19-7, assay 99.9 %). The mass of chitosan powder was calculated taking into account the molar mass of the repeating unit (D-glucosamine and N-acetyl glucosamine), 163.10 g.mol -1 . The amount of acetic acid was adjusted to match the stoichiometric protonation of -NH2 sites of chitosan. The complete dissolution of chitosan was obtained after several cycles of fiveminute speed mixing at 3000 rpm, using a speed mixer machine (DAC150.1FVZ-K, Synergy devices Ltd, UK).

Impregnation of HT scaffolds with chitosan solutions was performed following five different protocols (varying vacuum pressure, concentration of chitosan, and base used for gelation) (Table 1). HT-scaffold of known weight and chitosan solution of known volume were kept separately in a single desiccator and outgassed for 1h under controlled pressure using a vacuum pump. While still under vacuum, the HT scaffold was dropped into the chitosan solution and left to rest for 25 min. After vacuum release, the resulting sample was dropped into a second chitosan solution of known volume, at atmospheric pressure. The whole system was then kept under the same vacuum pressure as in the first impregnation, for 1.42 h. Then gelation was performed either by immersion during 1 h in a 1 M NaOH (Carlo Erba Reagents, CAS 1310-73-2, assay ≥ 97 %), or exposition for 22 h to an ammonia atmosphere provided by a 1M NH4OH solution (Sigma-Aldrich, CAS 1336-21-6, assay 28-30 %). After gelation, HT-CS scaffolds were washed several times until measuring a pH ~ 7 in the washing water. The HT-CS scaffolds were weighted at each step of the process. Samples were labeled HT-CS-x, with x being the protocol number used. To investigate the effect of chitosan hydrogel on mechanical properties of HT-CS scaffolds, the chitosan weight content and concentration were calculated in each step of formation of HT-CS scaffolds. The calculation procedure was the following:

Each sample was weighted before impregnation (m0), after first impregnation (m1), after second impregnation (m2), and after gelation (mg). After each step, sample was weighted three times and the average was taken. Weight of chitosan solution gained after each impregnation and in gelation:

𝑤𝑒𝑖𝑔ℎ𝑡 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 𝑔𝑎𝑖𝑛𝑒𝑑 𝑎𝑓𝑡𝑒𝑟 1𝑠𝑡 𝑖𝑚𝑝. (𝑔) = 𝑚 𝑠𝑜𝑙 1-0 = 𝑚 1 -𝑚 0 (1) 𝑤𝑒𝑖𝑔ℎ𝑡 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 𝑔𝑎𝑖𝑛𝑒𝑑 𝑎𝑓𝑡𝑒𝑟 2𝑛𝑑 𝑖𝑚𝑝. (𝑔) = 𝑚 𝑠𝑜𝑙 2-1 = 𝑚 2 -𝑚 1 (2) 𝑤𝑒𝑖𝑔ℎ𝑡 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 𝑔𝑎𝑖𝑛𝑒𝑑 𝑎𝑓𝑡𝑒𝑟 𝑔𝑒𝑙𝑎𝑡𝑖𝑜𝑛 𝑎𝑛𝑑 𝑤𝑎𝑠ℎ𝑖𝑛𝑔 (𝑔) = 𝑚 𝑠𝑜𝑙 𝑔-2 = 𝑚 𝑔 -𝑚 2 (3)
Weight of chitosan, CS, gained after each impregnation:

𝐶𝑆 𝑔𝑎𝑖𝑛𝑒𝑑 𝑖𝑛 1𝑠𝑡 𝑖𝑚𝑝. (𝑔) = 𝑚 𝐶𝑆 1-0 = 𝑚 𝑠𝑜𝑙 1-0 * [𝐶𝑆 1𝑠𝑡 ] (4)
𝐶𝑆 𝑔𝑎𝑖𝑛𝑒𝑑 𝑖𝑛 2𝑛𝑑 𝑖𝑚𝑝. (𝑔) = 𝑚 𝐶𝑆 2-1 = 𝑚 𝑠𝑜𝑙 2-1 * [𝐶𝑆 2𝑛𝑑 ] (5)
Both, initial concentration of chitosan for first and second impregnation, affected the actual concentration resulting after the second impregnation:

𝐶𝑆 𝑟𝑒𝑠𝑢𝑙𝑡𝑖𝑛𝑔 𝑎𝑓𝑡𝑒𝑟 2𝑛𝑑 𝑖𝑚𝑝. (𝑤𝑡%) = [𝐶𝑆 𝑟 ] = 𝑚 𝐶𝑆 1-0 + 𝑚 𝐶𝑆 2-1 𝑚 𝑠𝑜𝑙 1-0 + 𝑚 𝑠𝑜𝑙 2-1 * 100 (6)
For further calculations that concentration, [𝐶𝑆 𝑟 ], is considered as the concentration of chitosan in the system subjected to gelation and as the final concentration in HT-CS scaffold. Therefore, the weight of chitosan gained during gelation corresponds to:

𝑚 𝐶𝑆 𝑔-2 = 𝑚 𝑠𝑜𝑙 𝑔-2 * [𝐶𝑆 𝑟 ] (7)
Knowing the weight of chitosan in the sample and the weight of HT scaffold, a CS/HT ratio can be calculated:

𝐶𝑆 𝐻𝑇 = 𝑚 𝐶𝑆 1-0 + 𝑚 𝐶𝑆 2-1 + 𝑚 𝐶𝑆 𝑔-2 𝑚 𝐻𝑇 (8)

C. Characterizations

Morphology of HT scaffolds was investigated by X-ray micro-tomography (μ-CT, Phoenix v│Tome│x, General Electric, USA, equipped with a Varian Paxscan detector (1920x1536 pixels) and an X-ray source of 1-4 μm). The resolution was set between 2-5 μm/voxel. Open porosity of HT scaffolds and pore interconnection size were measured using a mercury porosimeter (AutoPore IV, Micromeritics Ltd, Norcross, GA, USA).

Dimensions of HT scaffolds were measured after ceramization by means of a digital caliper (values were averaged after 3 measurements in different regions of the sample), and scaffolds were weighted using an analytical balance. Then, density of the ceramic part was determined geometrically. Apparent and true densities of 3-D hardystonite scaffolds were measured by means of a Helium gas pycnometer (AccuPyc 1330, Micromeritics, Norcross, GA, USA) on samples in bulk and powder form. The total porosity of the scaffolds was then calculated.

Rheological measurements on chitosan solutions were conducted at shear rates from 10 -4 or 10 -3 to 100 s -1 , at 25 °C, using AR2000 ex rheometer (TA Instruments, USA). The steady-state viscosity of chitosan solutions was assessed in static mode using aluminum cone-plate geometry (25 mm diameter; 4°). The minimum gap was fixed at 116 µm. A solvent trap was used to prevent evaporation of water during measurements.

X-ray diffraction analysis on the surface of HT and HT-CS scaffolds were performed using a D8 Advance diffractometer (CuKα, 40 kV, 40 mA) (Bruker AXS, Karlsruhe, Germany). Scans were acquired in a θ-θ configuration, from 4° to 55° with a step time of 129 s and a step size of 0.019 . Phase identification was carried out by comparison to standard patterns from the International Center for Diffraction Data -Powder Diffraction Files (ICDD-PDF) with the aid of DiffracPlus EVA software (Bruker). To identify Hardystonite phase the PDF #35-0745 was used. The obtained diffractograms were normalized with respect to the height of the peak of greatest intensity in HT phase

The microstructures of HT and HT-CS freeze-dried scaffolds were analyzed by scanning electron microscopy (SEM, Supra55VP (Zeiss, Germany) working at 1.5 kV) and optical stereo microscopy (Light Axiophot, Zeiss) in reflection mode, on fracture surfaces. Prior to these analyses, scaffolds were freeze-dried to remove water without disturbing the hardystonite structure: they were frozen in a deep-freezer overnight, then left for two days at -86 C under 0.080 mbar absolute pressure.

Compression tests of HT-CS-x scaffolds were performed using a mechanical universal testing machine (Instron 1121 UTM, Danvers, USA), at cross-head speed of 0.5 mm/min, at room temperature. At least ten samples were measured for each formulation, and the results were reported as mean ± standard deviation.

III. Results

A. HT scaffold

XRD taken on the scaffold surface is shown in Figure 1. The characteristic planes corresponding to hardystonite were identified: (111) at 2θ=23.928°, (201) at 2θ=28.910°, (211) at 2θ=31.930° and (310) at 2θ=36.274°. The micro, macro and total porosities of HT scaffold structure were determined. The geometrical density of the HT scaffold was calculated from its external shape and its weight, leading to a total porosity of 72 vol%. A μ-CT reconstruction of HT scaffold structure is shown in Figure 2a. A regular macroporosity was observed as well as indications that only limited sagging of the filaments occurred after printing. μ-CT displayed in Figure 2b shows that there was a very good adhesion between struts and no significant large size porosity was present inside the struts. However, obvious longitudinal cracks were observed at the center of the struts. Evidently, some further optimization of ink composition and processing conditions are needed to completely eliminate these defects from the HT scaffold. Intra-struts porosity could not be detected by µ-CT. Mercury intrusion porosity analysis (Figure 3) indicated that the micropores in the struts had size ranging from 0.2 to 0.5 µm. The total volume of micropores was 0.12 mL/g, corresponding to a micro-porosity of 41 vol%, thus, to an inter-strut porosity of 31 vol%. No information on interstruts porosity morphology could be obtained from MIP, since the inter-strut pores are too large to be detectable by this technique.

B. Impregnation of HT scaffolds and formation of HT-CS scaffolds

The rheological curves of chitosan solutions, Figure 4, correspond to pseudo-plastic, shear-thinning fluids. It can be observed that viscosity of chitosan solution strongly depend upon chitosan concentration, as previously reported [39]. In the worst case, the impregnation of HT scaffolds occurred by free diffusion flow, that is, at low shear rate. Thus, we focused on the Newtonian viscosity. The latter was evaluated from plateau and was reported in Table 2.

Table 2. Newtonian viscosity of chitosan solutions at different concentrations. Table 3 shows the results of weighting the scaffold samples during the processes of impregnation and gelation to form HT-CS scaffolds.

Shear rate (s -1 )

Data reported in Table 3 and the procedure described in section IIB were used to calculate the changes of weight during the impregnation and gelation processes, the final chitosan concentration in the hydrogel immersed in the scaffold and the weight ratio CS/HT. Results are shown in Table 4. Notice that there were negative changes of weight during the process of gelation indicating loss of solution.

153 Table 4. Changes in weight for HT and HT-CS-x scaffolds; the final chitosan concentration in HT scaffold and the CS/HT ratio.

C. Characterization of HT-CS scaffolds

Once the scaffolds were impregnated, the external surfaces were analyzed by XRD to investigate the effect of chitosan on composition and phase stability. Figure 5 shows the diffractograms for HT before and after impregnation following all protocols. It was observed that the characteristic planes of hardystonite were identified in all cases, (111) at 2θ=23.928°, (201) at 2θ=28.910° and (211) at 2θ=31.930°, except in HT-CS-1, the only one sample in which solution of sodium hydroxide was used as base for gelation. It is also observed that no chitosan peaks appeared.

Figure 5. X-ray diffractograms of external surfaces HT and HT-CS-x scaffolds.

A picture of HT-CS-1 fracture surface was taken to illustrate the dense layer of physical chitosan hydrogel covering the surface of HT scaffold (Figure 6). Additionally, a picture of HT-CS-3 fracture surface is shown as an example of HT-CS scaffolds obtained by ammonia vapors as a base used for gelation (Figure 6).

mm mm HT-CS-1 HT-CS-3

Micrographs of optical microscopy of fracture surface of freeze-dried scaffolds are reported in Figure 7, corresponding to HT and HT-CS-x scaffolds. There was a clear difference between HT and HT-CS-x scaffolds. Chitosan appeared as a layer covering the fracture surface and homogeneously filling the macropores of HT-CS-x under all impregnation conditions. SEM observations (Figure 8 and Figure 9) allowed analyzing the presence of chitosan in strut microporous for different impregnation protocols.

Cross section of struts (named with the letter a) and micropores on fracture surface (named with the letter b) are shown in Figure 8 and Figure 9. In a clear contrast with HT scaffold, a layer of chitosan was observed covering the fracture surface of the struts and partially filling the micropores of HT-CS-x scaffolds. Analysis of stress-strain data of HT-CS-x and HT-H2O (Figure 10 and Table 5) allowed estimating the effect of CS/HT ratio on mechanical properties such as maximum stress before plastic flow, the corresponding strain at maximum stress, the work at maximum stress (calculated as the area under the stress-strain curve) and the apparent Young´s modulus (Figure 11). To facilitate the analysis, it has been included the CS/HT ratio of the CS-HT-x scaffolds in Table 5. σmax; strain at maximum stress: ε@σmax; work at maximum stress: W@σmax; apparent Young´s modulus: E.

IV. Discussion

HT-CS-x scaffolds had a weight ratio CS/HT ranging from 0.84 to 2.16. Chitosan appeared as a film around the struts and inside the strut microporosity.

A. Impregnation of HT scaffolds

The weight of chitosan gained during scaffold impregnation was expected to depend on concentration and viscosity of chitosan solution and on apply vacuum pressure. As chitosan concentration increases, the chitosan weight impregnated would increase. As viscosity of chitosan solution increases, the impregnation weight of solution would diminish. It was expected that higher vacuum pressure, impregnation weight of solution would be higher. In these regards, some trends derived from data reported in Table 4 are discussed below.

Effect of chitosan solution concentration

Protocols 3 and 4 have equal conditions for first impregnation but, for second impregnation, higher concentration of chitosan in protocol 4 (2.5 wt%) than in protocol 3 (1.5 wt%). Results showed about the same gain of chitosan solution weight and chitosan solute during first impregnation. For second impregnation, gain of solution weight was twice higher in protocol 3 than in protocol 4. On the other hand, in the second impregnation, the gain in chitosan weight was only 1.2 times higher in protocol 3. The lower proportion of weight of solute with respect to weight of solution gained could be explained considering that at lower chitosan concentration, the viscosity is lower thus the impregnation weight is higher, but it contains lower weight of solute per unit weight of solution.

Protocols 3 and 5 have the same vacuum pressure; concentration of chitosan is higher in protocol 5 for first impregnation and equal for second impregnation. It was observed that during first impregnation, weight gain for protocol 5 was higher, 1.72 times for weight of solution and 5.17 times for weight of chitosan. Although viscosity was higher in protocol 5, the dominant effect was the high concentration of chitosan, 3 times more mass of chitosan per unit weight of solution. On the contrary, during second impregnation, weight gain for protocol 3 was higher, 3.7 times for weight of solution and for weight of chitosan. To explain these results, it should be considered that viscosity of the solution already impregnating (1 st impregnation) the scaffolds was higher for protocol 5 than for protocol 3. Therefore, resistance for further impregnation was higher in protocol 5.

After gelation, samples under protocols 2 and 3 resulted to have about the same concentration of chitosan, [𝐶𝑆 𝑟 ], and they had about the same loss of solution during gelation. On the other hand, the same samples, (HT-CS-2 and HT-CS-3), had 2 times higher loss of weight of solution than HT-CS-4 during gelation. These results could be explained considering that HT-CS-2 and HT-CS-3 had lower concentration of chitosan solution compared with HT-CS-4. It means that when concentration of chitosan was lower, then the viscosity was lower and the weight lost during gelation was higher.

Effect of vacuum pressure

In the first impregnation, protocols 2 and 3 have the same chitosan concentration (0.5 wt%), and different vacuum pressure, 80mbar for protocol 2 and 50mbar for protocols 3. The resulting weight increase was in the same range, 95 % in both cases. It may indicate that for the first impregnation, in the applied interval of vacuum pressure and chitosan concentration, vacuum pressure had a weak effect on impregnation. In the second impregnation, protocols 2 and 3 have the same chitosan concentration (1.5 wt%) and higher vacuum pressure in protocol 3. Results showed a slightly higher, but not significant, weight gained in protocol 3. This small difference was reflected in a slightly higher CS/HT weight ratio (1.1 times) in protocol 3.

Effect of the nature of the base used for gelation

Protocols 1 and 4 differ only in the base used for gelation, sodium hydroxide for protocol 1 and vapors of ammonia for protocol 4. During gelation, sample under protocol 4 lost over 11 times more chitosan solution than in protocol 1.

Gelation depends upon availability of hydroxyl anions, which are provided by the base. Sodium hydroxide is a strong base that rapidly and completely dissociates in solution forming sodium cations and hydroxyl anions. Ammonia vapors first dissolve in acidic water (chitosan solution) and then, ammonia being a weak base, partially dissociates forming ammonium cations and hydroxyl anions. Under these conditions, hydroxyl anions are available more rapidly for gelation using sodium hydroxide solution than using ammonia vapors. Thus, in the case of gelation induced by sodium hydroxide, the diffusion of the base and the neutralization are faster as compared to gelation induced by ammonia vapors. In fact, it was observed an almost instantaneous formation of an opaque layer in the case of gelation with sodium hydroxide, whereas it took longer time with ammonia vapors (difference of ~ 10 minutes). The opaque layer constituted a mass transfer resistance for solution to flow, but did not prevent the overall gelation process to be faster with NaOH.

B. Microstructure of scaffolds

XRD of HT-CS-x surfaces showed well-defined crystalline structures of pure hardystonite, except for HT-CS-1 (Figure 5). The absence of peaks of chitosan was indicative of poor crystallization of chitosan in relation with hardystonite. The unexpected result in the case of HT-CS-1 in which neither chitosan nor hardystonite appeared in the diffractogram could be explained as due to the layer of chitosan hydrogel covering the surface. This layer was thick enough to screen the crystal structure in XRD analysis (Figure 6). In fact, to the naked eye, this layer was more opaque and thicker in this case, compared with scaffolds in which ammonia vapors were used as a base for gelation (Figure 6).

Layers of chitosan covering the fracture surface and filling the macro pores of HT-CS-x scaffolds were observed by optical microscopy and by SEM (Figure 7, Figure 8 and Figure 9). Observation of fracture surface of HT scaffold by SEM allowed identifying a number of holes that may indicate empty micropores (Figure 8-HT-a). In contrast, those holes were rarely found in HT-CS-5 fracture surface that corresponds to the scaffold with highest charge of chitosan (Figure 9-HT-CS-5a). Moreover, the frequency of those micropores diminished as content of chitosan increased, from HT-CS-2 to HT-CS-5. Observation of micropores on fracture surface showed chitosan filaments that filled and bridged micropores in contrast with HT scaffold where no filaments were observed (Figure 8-HT-CS-xb and Figure 9-HT-CS-xb). The frequency of those filaments can be observed to increase with the content of chitosan in the HT-CS-x scaffold.

C. Mechanical properties

HT-CS-2 to HT-CS-4 scaffolds, gelled with the same base, showed no significant differences neither in CS/HT ratio nor in mechanical properties. Comparing those samples with HT-H2O, no significant difference was found with respect to fracture strength. It seemed to indicate that the chitosan content in those samples was not enough to impact fracture strength significantly.

Comparing the mean calculated value of properties in HT-H2O and the group HT-CS-2 to HT-CS-4 scaffolds, gelled with ammonia vapors, there seemed to show a correlation between increasing chitosan content and improving capacity for energy dissipated by fracture: higher deformation and more energy for deformation in presence of chitosan gel (strain at maximum stress, work at maximum stress). Considering that even the most concentrated chitosan hydrogels in the scaffolds are made of more than 97 % water, poro-mechanics considerations should be taken into account.

Indeed, considering 0.2 to 0.5 µm pores filled with water (Figure 3), a surface tension of 72•10 -3 J•m -1 for water at room temperature and a contact angle of water on hardystonite of ~33 ° [40], applying Jurin's law one can calculate a capillary pressure around 0.5 MPa. This gives an order of magnitude of the pressure necessary to expulse water from the pore, thus of the additional resistance brought by the filling of the pores by water. This resistance could be even higher if water is immobilized by the physical hydrogel. However, this explanation needs more evidence to be completely satisfactory.

Comparing HT-CS-2 to HT-CS-4 scaffolds (about the same chitosan content) with HT-CS-5 scaffolds (higher chitosan content) showed that a significant increase in chitosan content resulted in improvement of capacity for energy dissipation by fracture: higher deformation and more energy for deformation (higher strain at maximum stress, more work at maximum stress, lower Young´s modulus).

Comparing HT-CS-1 to HT-CS-4, having no significant differences in chitosan content (CS/HT ratio) but differing in base used for gelation, results showed no significant differences in mechanical properties, although, their seemed to be a tendency of improving stiffness (higher maximum stress and higher Young´s modulus) and capacity for energy dissipation by fracture (higher strain at maximum stress, more work at maximum stress) by using sodium hydroxide in gelation.

HT-CS-1 and HT-CS-5 scaffolds differ in base used for gelation (sodium hydroxide solution and ammonia vapors respectively) and in chitosan content (higher in HT-CS-5). Comparing the mean calculated value of properties, stiffness was higher and capacity for energy dissipation by fracture was remarkable lower in HT-CS-1. It seemed to indicate that stiffness increased by using sodium hydroxide for gelation. This might be explained considering that the elastic modulus (G´ in the rheological curve) of chitosan physical hydrogel is about 1.5 times higher for hydrogel obtained with sodium hydroxide solution (4.0 ± 0.4 kPa) than for hydrogels obtained with vapors of ammonia (2.6 ± 0.3 kPa), at the same concentration of chitosan [33,41]. In the case of gelation with sodium hydroxide, diffusion of hydroxyl groups and neutralization were faster as compared with gelation using ammonia vapors. Therefore, chain mobility decreases more rapidly by gel formation with sodium hydroxide, preserving chitosan chain entanglements, thus, resulting in higher G´ for the hydrogel [41]. The remarkable high capacity for energy dissipation by fracture in HT-CS-5 could be caused by the significant higher chitosan content. Considering standard deviation of data, the only clear tendency was the higher capacity for energy dissipation by fracture related to high chitosan content.

Comparing HT-H2O and HT-CS-1 scaffolds, it was not possible to find a clear trend on change in mechanical properties with chitosan content due to the relative high standard deviation in value of strain at maximum stress. However, comparing with the mean calculated value of properties, there seemed to show an improvement of stiffness and capacity for energy dissipation by fracture with the presence of chitosan hydrogel. Moreover, this tendency was more accentuated in the case of HT-CS-1 than in the group HT-CS-2 to HT-CS-4, that is, in the case of using sodium hydroxide solution instead of ammonia vapors for gelation.

V. Conclusions

HT and HT-CS scaffolds prepared in this work showed a well-defined crystalline structure corresponding to pure hardystonite. HT showed macro and micro porosity that makes this bioceramic suitable for impregnation with chitosan solutions in the concentration range from 0.5 wt% to 1.5 wt%.

Scaffolds gained and lost weight of chitosan solution during immersion and gelation respectively. Weight gain during impregnation of HT-scaffolds depended on two related variables of the chitosan solution: concentration and viscosity. As chitosan concentration was higher, viscosity increased, thus mass transfer resistance increased, leading to low impregnation of scaffolds and, during gelation, to low weight loss. On the other hand, as chitosan concentration increased, so did the density of the solution, therefore, weight gained per unit of weight of chitosan solution impregnated, increased. Rate of gelation seemed to affect mass transfer resistance due to formation of a dense layer in the "gel-front". In the case of sodium hydroxide solution as base used for gelation, the rate of gelation was higher; consequently the weight loss was lower.

In HT-CS, capacity for energy dissipation by fracture and stiffness increased as chitosan content increased in the scaffold. With respect to base used for gelation, stiffness was higher for HT-CS scaffolds gelled with sodium hydroxide solution as compared to HT-CS scaffolds gelled with ammonia vapors. By caution, considering dispersion of data (standard deviation), the only clear tendency was the higher capacity for energy dissipation by fracture related to high chitosan gel content.

The presence of water filling the pores of scaffolds added mechanical resistance to the samples. This resistance could be even higher if water was immobilized by the chitosan hydrogel. Moreover, chitosan filaments bridged the cracks and therefore might have improved the stiffness and energy dissipation by fracture of HT-CS.

Thanks to the collaboration with the group of Prof. P. Colombo, the work described in this chapter permitted to explore a new approach to obtain architectured biocomposites. The results and the processes obtained here could be, in a future study, adapted to impregnate 3-D scaffolds made of calcium phosphate materials.

General conclusions and outlooks

This Ph.D. thesis was aimed to develop potential bone substitute materials consisting in inorganic-organic composites, inspired by composition and microstructure of the natural extracellular matrix of bone tissue. Different synthesis methods, a wide range of compositions (from 100% mineral to 100% organic) and of inorganic-organic phases distribution (mineral phase as homogeneously dispersed particles in polymer matrix, or as a layer on the polymer matrix, or as a continuous phase impregnated by the polymer) were explored. In the fabrication of bioceramic/biopolymer composites the relation between synthesis method, chemical composition and mechanical properties was investigated and discussed, based on physico-chemical interactions occurring in the different systems. Two composite materials were prepared: chitosan physical hydrogels mineralized with apatite, and hardystonite scaffolds impregnated with chitosan physical hydrogels.

To obtain mineralized chitosan physical hydrogels, two mineralization approaches were developed. The first one consisted in the fabrication of chitosan physical hydrogels and their subsequent mineralization with apatite; the second approach consisted in a simultaneous conversion of chitosan-calcium phosphate suspensions into chitosan-apatite hydrogels. Chitosancalcium phosphate suspensions were also used as inks for 3-D printing of the same composite material.

In the first mineralization approach, the general protocol to obtain mineralized hydrogels of chitosan was to gel a solution of chitosan, then, to mineralize the preformed hydrogel. The final microstructure of the hydrogel depended on the gelation route: capillaries were found within hydrogels obtained by gelation with sodium hydroxide solution and calcium hydroxide suspension, whereas no capillaries were detected within hydrogels gelled with ammonia vapors. Capillaries were oriented parallel to the direction of the flux of the gelation base within the solution; they were smaller and denser in hydrogels gelled with sodium hydroxide. The obtained chitosan hydrogels were then mineralized using four different strategies: i) immersion of hydrogel in calcium phosphate suspension; ii) immersion of hydrogel in salt solutions of calcium and phosphate; iii) dehydration and rehydration of hydrogels in calcium and phosphate solutions; iv) contact between a suspension of calcium hydroxide and a chitosan solution both to gel the chitosan and to provide calcium ions, followed by immersion in phosphate solution.

The common result of mineralization of hydrogels, under all four strategies, was the formation of apatite on the external surface of hydrogels without any evidence of mineralization inside the hydrogels. The absence of apatite inside the hydrogels may be due to the difficult diffusion of ions through the hydrogels probably because of physico-chemical interactions among ions and between ions and chitosan.

The lack of homogeneity of the materials obtained using the first approach led us to explore the second approach: the conversion of a homogenous chitosan-calcium phosphate suspension into a mineralized chitosan physical hydrogel.

The nature of salts used as sources of calcium and orthophosphate ions, and the method of mixing salt and chitosan solutions (sequential or simultaneous) were the synthesis variables in the preparation of suspensions. The resulting acidic suspensions (pH < 6), before gelation, were composed of a chitosan solution with dispersed aggregates of DCPD crystals and residual ions of dissolved salts. Under those conditions of composition and pH, several interactions were likely to occur: i) repulsions between polymer chains due to protonated amine groups in the polymer chain; ii) hydrophilic polymer-solvent interactions due to hydroxyl, amine and protonated amine groups of chitosan; iii) polymer-polymer interaction expressed in entanglement of polymer chains; iv) coordination bonds between polymer and calcium ions that may lead to physical cross-linking of chains; v) electrostatic interactions of divalent phosphate ions with protonated amine groups that may form physical crosslinks between polymer chains; vi) affinity between calcium and phosphate ions to form calcium phosphate phases (DCPD at pH from 4 to 6) and, vii) screening of electrostatic interactions due to the ionic strength of the suspension. The size and distribution of DCPD aggregates varied with the method of preparing the suspensions: they were smaller and uniformly distributed with the sequential method while they were larger and less uniformly distributed with the simultaneous method. This difference of microstructure was attributed to the environment prevailing in the formation of calcium phosphate suspensions, of relatively low viscosity, high homogeneity and in absence of chitosan in the case of the sequential method.

The ionic strength had a pronounced effect on rheological properties of chitosan-calcium phosphate suspensions. Ionic strength impacted the density of entanglement of polymer chains, which in turn influenced its shear-thinning behavior, its moduli (storage, G' and loss, G'') and the viscosity of the system. When the ionic strength was higher, lower chitosan chain entanglements occurred, therefore the shear-thinning behavior, the moduli, the yield stress and the viscosity tended to decrease.

The pH of the chitosan-DCPD suspensions was then increased to alkaline condition (pH~12) using ammonia vapors or sodium hydroxide solution. This change in pH induced two effects at the same time: the gelation of the suspension and the transformation of mineral phase into apatite. Thus, the chitosan-DCPD suspension was transformed into a physical hydrogel of chitosan with intertwined apatite particles. Apatite was poorly crystalline, forming aggregates of nanometric platelets with a rather uniform distribution within the polymer matrix of chitosan. Different events were expected to occur during the process of gelation: i) DCPD was no longer stable: it dissolved and recrystallized forming the stable phase, apatite ii) amine groups of chitosan were deprotonated, thus electrostatic repulsive forces between charged polymer chains decreased; iii) chain junctions (inter and intra chitosan polymer chains) formed physical crosslinks leading to the formation of a hydrogel; iv) electrostatic interactions between chitosan and orthophosphate ions formed physical cross-links of polymer chains; and v) coordination bonds between chitosan and calcium ions also formed physical cross-links of polymer chains.

In general, three factors impacted the mechanical properties of mineralized chitosan hydrogels: the base used for gelation (determining the gelation rate: a higher rate preserved better chain entanglement, resulting in higher elasticity); the density of physical crosslinks (hence a higher storage modulus) and the ionic strength (that led to chitosan chain disentanglements, thus, low storage modulus). Chitosan hydrogels and mineralized hydrogels were not cytotoxic, having no deleterious effects on osteoblasts proliferation.

The second material studied in this thesis is a hardystonite scaffold impregnated with chitosan hydrogel. The hardystonite scaffolds were obtained using a 3-D printing technology: pre-ceramic ink was 3-D printed and then sintered to form crystalline hardystonite ceramic. Hardystonite scaffolds were impregnated with chitosan solution that was, next, converted to chitosan physical hydrogel. At higher chitosan concentration, viscosity of polymer solution was higher and scaffold impregnation was lower. During gelation, a weight lost was observed. This weight loss was lower at higher gelation rate, which depended on base used for gelation. Chitosan hydrogel partially filled the pores contributing in some extent to bearing of external loads and to energy dissipation.

This thesis advanced on research paths that remain open for optimization of process synthesis and for further validation of the reported achievements.

Besides the fabrication and comprehension of materials, achievements were made on the characterization of their physico-chemical properties. Indeed, calcium phosphates and chitosan are materials, which are very sensitive to their environment: their properties can be altered when the sample preparation or when the characterization protocols are not fully optimized. Specifically, alterations of the phases themselves, and modifications of their interactions, may be experienced due to drying of the materials, to high-energy beams (such as X-ray or electron beams), to slight changes in their environments (temperature, pH…) and so on. Therefore, the characterization of the physico-chemical properties and of the microstructure of the biocomposites in their native state was extremely challenging. However, characterization was performed and advances were made in this direction such as: i) protocols for sample preparation adapted to different characterization means and protocol for sterilization; ii) characterization of the mechanical properties by rheology tests; iii) design of a specific reactor for in-situ gelation of composites under X-ray diffraction.

New research directions will assess the potential of the obtained materials as bone substitutes.

Those research paths are briefly summarized hereafter: i) Use of calcium hydroxide both as source of calcium to form calcium phosphates and as a base for gelation to form the chitosan hydrogel. It could minimize the presence of counterions in the system. ii)

Determination of the kinetics of gelation and kinetics of apatite crystallization, under the conditions of synthesis processes, as tools that eventually may serve to control synthesis processes and microstructure of target materials.

iii)

Thorough understanding of the mechanisms of physico-chemical interactions acting in the systems and further characterization of the microstructure of the target materials, using additional and complementary characterization techniques: characterization of the interaction between ions, mineral crystals and chitosan by Fourier-transform infrared spectroscopy; determination of chemical substitutions in apatite crystals by Infrared and Raman spectroscopy techniques; evolution of materials and of the characteristics of the crystalline phases, both during synthesis (e.g., during gelation of the hydrogels) and once the composites have been fabricated with time-resolved Infrared spectroscopy and with in-situ X-ray diffraction (in particular, this latter will be eased by the reactor, which has been designed in the frame of this Ph.D. work); determination of ions concentration in solution and in biocomposites by inductively coupled plasma atomic emission spectroscopy and characterization of gradients of microstructure and physico-chemical properties by small angle X-ray scattering and by FT-Infrared microscopy. iv)

Adjusted rheological properties of chitosan-calcium phosphate suspensions used as inks for robocasting to develop printing procedures in air, by different strategies: variation of inorganic to organic ratio; variation of chitosan concentration; introducing mild cross-links of chitosan chains by adding non-toxic cross-linking agents. v)

In-vitro and in-vivo biological tests of chitosan hydrogels, mineralized chitosan hydrogels and hardystonite-chitosan hydrogel scaffolds, and investigation of the interactions occurring between these materials and the cell culture medium. Bone substitutes, an approach to attend social demand for bone healing and reparation, are temporary replacements of bone tissue, promote bone formation and growth and finally are bioresorbed. No single material meets these requirements; an alternative is a bioinspired composite material. The objective of this thesis was thus to study the synthesis and properties of two bioceramics/biopolymer composites: chitosan physical hydrogels mineralized with apatite and hardystonite scaffolds impregnated with chitosan physical hydrogels. To obtain the first material, two strategies were developed. The first one consisted in the fabrication of chitosan physical hydrogels and its subsequent mineralization with apatite; the formation of microcapillaries occurred under particular synthesis conditions, and apatite precipitates were found only on the surface of hydrogels.

The second strategy consisted in a simultaneous conversion of chitosan-calcium phosphate suspensions into chitosan-apatite hydrogels. The suspensions were prepared by sequential or simultaneous mixing of calcium and phosphate suspensions with chitosan solutions. Smaller and more uniformly distributed mineral aggregates were formed following sequential mixing, attributed to higher homogeneity, lower viscosity and no-presence of chitosan. This enabled the use of these chitosan-calcium phosphate suspensions as inks for 3-D printing. In general, three factors impacted the mechanical properties of mineralized chitosan hydrogels: the base used for gelation (determining the gelation rate: a higher rate preserved chain entanglement, resulting in higher elasticity); the density of physical crosslinks (hence a higher storage modulus) and the ionic strength (that led to chitosan chain disentanglements, thus, low storage modulus). Chitosan hydrogels and mineralized hydrogels were not cytotoxic, having no deleterious effects on osteoblasts proliferation. To fabricate the second material, pre-ceramic ink was 3-D printed and then sintered to form crystalline hardystonite ceramic. Hardystonite scaffolds were impregnated with chitosan solution that was, next, converted to chitosan physical hydrogel. At higher chitosan concentration, viscosity of solution was higher and scaffold impregnation was lower. At higher gelation rate, which depend on base used for gelation, lower weight loss during gelation. Chitosan hydrogel partially filled the pores contributing to bearing of external loads and to energy dissipated by fracture.

MOTS-CLÉS

Figure 1 .

 1 Figure 1. Bone structure showing the two basic architectures: spongy and compact bone [16].

  accessible à l'adresse : http://theses.insa-lyon.fr/publication/2018LYSEI010/these.pdf © [S.S. Ramírez Caballero], [2018], INSA Lyon, tous droits réservésPhase 3: Osteoblast differentiation and activation; Phase 4: Mineralization of organic portion of the bone matrix formed prior to complete development of bone, called osteoid, and completion of bone remodeling.

6

  Cette thèse est accessible à l'adresse : http://theses.insa-lyon.fr/publication/2018LYSEI010/these.pdf © [S.S. Ramírez Caballero],[2018], INSA Lyon, tous droits réservés

Figure 7 .

 7 Figure 7. Bone healing scheme illustrating the phases of the process: reactive (a), reparative (b, c)and remodeling (d). Adapted from[53].

Figure 11 .

 11 Figure 11.Crystallographic structure of DCPD. Adapted from [136].

Figure 12 .

 12 Figure 12. Schematic representation of the mechanism of chitosan gelation by increasing pH; an indicator dye was used. Adapted from [203].

Figure 13 .

 13 Figure 13. Moieties of the molecular structure of chitosan. Purple circle: ether group; red circle: protonated amine group; orange circle: hydroxyl group; green circle: acetyl group; blue circle: acetamide group. Adapted from [207].

Figure 14 .

 14 Figure 14. Schema of Hydrogen bonds in chitosan molecule: -𝑂𝐻 ⋯ 𝑂 -(a); -𝐶 = 𝑂 ⋯ 𝐻𝑂 -(b); -𝑂𝐻 ⋯ 𝑂𝐻 -(c); -𝐶 = 𝑂 ⋯ 𝐻𝑁 -(d). Adapted from [210].

Figure 15 .

 15 Figure 15. Schematic representation of hydrogen bonds between hydroxyapatite and chitosan (---hydrogen bonds) [211].

  [215,216].

Figure 16 .

 16 Figure 16. Schematic representation of electrostatic interactions between chitosan molecules and phosphate ions. Adapted from [217].

  Cette thèse est accessible à l'adresse : http://theses.insa-lyon.fr/publication/2018LYSEI010/these.pdf © [S.S. Ramírez Caballero],[2018], INSA Lyon, tous droits réservés

Figure 2 .

 2 Figure 2. Rheological measurements on chitosan solutions. Viscosity vs. shear rate in (a) and shear stress vs. shear rate in (b).

Figure 4 .

 4 Figure 4. Results of viscosity vs. shear rate and shear stress vs. shear rate for (NH4)2HPO4 (a) and (b), NH4H2PO4 (c) and (d), Ca(NO3)2 (e) and (f), and CaCl2 (g) and (h) respectively. Tests conducted at shear rates of 10 -4 or 10 -3 -100 (s -1 ) at 25°C.

  Cette thèse est accessible à l'adresse : http://theses.insa-lyon.fr/publication/2018LYSEI010/these.pdf © [S.S. Ramírez Caballero],[2018], INSA Lyon, tous droits réservés

Figure 5 .

 5 Figure 5. X-ray of chitosan-calcium phosphate suspensions obtained at room temperature. Blues arrows indicate the principal planes of DCPD: (020) at 2θ = 11.681 °, (021) at 2θ = 20.935 °, (041) at 2θ = 29.258 °, (220) at 2θ = 34.156 °.

  accessible à l'adresse : http://theses.insa-lyon.fr/publication/2018LYSEI010/these.pdf © [S.S. Ramírez Caballero], [2018], INSA Lyon, tous droits réservés

Figure 7 .

 7 Figure 7. Rheological measurements of chitosan-calcium phosphate suspensions and reference chitosan solutions (tests conducted at shear rates of 10 -4 or 10 -3 -100 (s -1 ) at 25 °C). Viscosity vs. shear rate in (a) and shear stress vs. shear rate in (b).

  Cette thèse est accessible à l'adresse : http://theses.insa-lyon.fr/publication/2018LYSEI010/these.pdf © [S.S. Ramírez Caballero],[2018], INSA Lyon, tous droits réservés

Figure 1 .

 1 Figure 1. Schematic representation of the microstructure of chitosan hydrogels and "gel front" direction.

  of the hydrogel "Bottom" of the hydrogel Direction of the "gel front"Dense layer"Bottom" of the hydrogel "Bottom" of the hydrogel

Figure 2 .

 2 Figure 2. Schematic representation of the four strategies used in this work to mineralize physical hydrogels of chitosan (filled green cylinders represent hydrogels of chitosan; the empty green cylinder represents a not-yet-gelled chitosan solution).

Figure 3 .

 3 Figure 3. Aspect of the top of a chitosan hydrogel sample gelled using sodium hydroxide solution (H1).

Figure 4 .

 4 Figure 4. Confocal microscopy micrographs of hydrogels H1, H2 and H4. Micrographs were taken at ~350 μm deep from the top of the hydrogel.

Figure 5 .

 5 Figure 5. X-ray diffractograms of the top of hydrogels H1, H2 and H3. The gray square identifies the chitosan characteristic planes (220) and (200); the gray arrow identifies the calcium hydroxide characteristic plane (001).

Figure 6 .

 6 Figure 6. Dehydration kinetics of hydrogels of chitosan H1 and H3 (a) and rehydration kinetics in different solutions containing osmosis water (w), calcium (Ca) or orthophosphate ions (P) (b).In this study n=3 and error bars represents the standard deviation. The exact compositions of the rehydration solutions are recalled in table 2.

Figure 7 .

 7 Figure 7. X-ray diffractogram of the CaP suspension prepared in strategy 1. The indicated peaks may correspond to apatite planes (211) and (112).

Figure 8 .

 8 Figure 8. Picture of mineralized H1 hydrogel obtained from immersion in a suspension of calcium phosphate (strategy 1, after washing step).

Figure 9 .

 9 Figure 9. Picture showing a zoom of the top of the mineralized H1 (strategy 1) obtained from a μ-CT image (resolution of 13 μm/voxel). Yellow arrows show small aggregates on the top of the hydrogel.

Figure 10 .

 10 Figure 10. X-ray diffractograms of the top of chitosan hydrogels H1 or H2 mineralized with the different strategies (1-4). Gray square identifies the chitosan characteristic planes (220) and (200); red arrow identifies the apatite characteristic planes (002) at 2θ=25.879 °, (211) at 2θ=31.774 ° and (112) at 2θ=32.902 °.

Figure 11 .

 11 Figure 11. Picture of hydrogel H1 immersed in 3M (NH4)2HPO4 solution (strategy 2).

Figure 12 .

 12 Figure 12. Picture of hydrogel H1 (strategy 2)after several washings with reverse osmosis water.

Figure 13 .

 13 Figure 13. Picture showing a zoom of the top of the mineralized H1 obtained from a μ-CT image (resolution of 13 μm/voxel). Yellow arrows show aggregates on the top of the hydrogel that may correspond to apatite.

( 3 .

 3 7 ± 0.1) g (2.6g ± 0.

Figure 14 .

 14 Figure 14. Picture of hydrogel H1 immersed in in 3M diammonium phosphate solution.Example taken from strategy 3a.

Figure 15 .

 15 Figure 15. Picture of hydrogel H1 after washing step. Example taken from strategy 3a.

Figure 16 and

 16 Figure16and Figure17show precipitates of mineral phase on the top of hydrogels. The lighter zones, indicated by yellow arrows, probably corresponded to apatite. It is noticeable that precipitates were not homogenously distributed on the hydrogel surfaces. No evidence was obtained of apatite inside the hydrogel.

Figure 16 .

 16 Figure 16. Picture showing a zoom of the transverse section of μ-CT image of H1 from strategy 3a (resolution of 13 μm/voxel). Yellow arrows show aggregates on the top of the hydrogel that may correspond to apatite.

Figure 17 .

 17 Figure 17. Picture showing a zoom of the transverse section of μ-CT image of H1 from strategy 3b (resolution of 3 μm/voxel). Yellow arrows show aggregates at the surface of the hydrogel that may correspond to apatite.

Figure 18 .

 18 Figure 18. Picture showing the transverse section of μ-CT image of H2 from strategy 4 (resolution of 6 μm/voxel). Yellow arrows show aggregates located at the surface of the hydrogel that may correspond to apatite.

Figure 1 .

 1 Figure 1. Schematic representation of the different synthesis methods of mineralized hydrogels.The irregular yellow shapes represent the aggregates of mineral crystals.

Figure 2 .

 2 Figure 2. Pictures of two sizes of samples. Mineralized hydrogel, M1, in (a) and hydrogels of chitosan, Ref1 (Table1), in (b).

Figure 3 .

 3 Figure 3. X-ray diffractograms of CaP suspensions a* in (a) and b* in (b) without chitosan. In (a), the blue and red arrows correspond to DCPD and DCPA characteristic planes respectively; in (b), the rose and yellow arrows correspond to CaClH2PO4.2H2O and NH4Cl characteristic planes respectively.

98 Figure 5 .

 985 Figure 5. Optical microscopy micrographs of S1 (sequentially method and pair a), S2 (simultaneously method and pair a) and S3 (sequentially method and pair b) suspensions.

Figure 10 .

 10 Figure 10. Optical microscopy micrographs of chitosan hydrogel (Ref2) and mineralized hydrogels.

Figure 11 .

 11 Figure 11. SEM micrographs of chitosan hydrogels (Ref1 and Ref2) and mineralized hydrogels (M1 and M2).

Figure 12 .

 12 Figure 12. SEM micrographs of M3 and M4.

Figure 13 .

 13 Figure 13. FTIR spectra of chitosan hydrogels references and mineralized hydrogels. Black arrows indicate the range bands or bands that chitosan and apatite have in common; Gray arrows correspond to chitosan bands and Black dash arrows correspond to apatite bands. (Test conducted from 400 to 4000 cm -1 with 4 cm -1 of resolution and 64 scans).

  accessible à l'adresse : http://theses.insa-lyon.fr/publication/2018LYSEI010/these.pdf © [S.S. Ramírez Caballero], [2018], INSA Lyon, tous droits réservés

  accessible à l'adresse : http://theses.insa-lyon.fr/publication/2018LYSEI010/these.pdf © [S.S. Ramírez Caballero], [2018], INSA Lyon, tous droits réservés

Figure 16 .

 16 Figure16. Cell proliferation measured by the PrestoBlue method on osteoblasts Mg63 culture with chitosan hydrogels references and mineralized hydrogels, after 7, 10 and 14 days. Additionally, a plastic control (cell culture treated polystyrene) was included to verify the normal behavior of cells, and it was referred to as "control". Fluorescence units are proportional to the number of cells.

Figure 1 .

 1 Figure 1. Picture of ink 6 (chitosan-DCPD suspension) before characterization.

Figure 2 .

 2 Figure 2. Example of X-ray diffractogram of ink 6 (chitosan-DCPD suspension). Black arrows identify the DCPD characteristic planes: (020) at 2θ = 11.681 °, (021) at 2θ = 20.935 °, (041) at 2θ = 29.258 °, (-221) at 2θ = 30.506 °, (220) at 2θ = 34.156 °, (022) at 2θ = 37.105 °.

Figure 3 .

 3 Figure 3. Example of optical microscopy micrograph of ink 6 (chitosan-DCPD suspension).

Figure 4 .

 4 Figure 4. Results of amplitude sweep test (between 1-100 % of strain, at 0.1 rad.s -1 ) for the different inks and references with (a) liquid-like behavior and (b) solid-like behavior.

Figure 5 .

 5 Figure 5. Results of oscillation time test (strain of 0.1 % and frequency of 0.1 rad.s -1 during 300 s) for ink and references with (a) liquid-like behavior and (b) solid-like behavior.

Figure 6 .

 6 Figure 6. Shear stress vs. shear rate curve for inks and reference solutions. Data obtained from flow ramp tests conducted at shear rates of 10-600 s -1 .

Figure 7 .

 7 Figure 7. Shear-thinning behavior: viscosity decreases as shear rate increases. Data obtained from flow ramp tests conducted at shear rates of 10-1000 s -1 .

Figure 8 .

 8 Figure 8. Results of amplitude sweep test (between 1-100 % of strain, at 0.1 rad.s -1 ) for inks and references with solid-like behavior. The red square in indicates the transition point from solidlike to liquid-like. The black arrow indicates the critical strain in ink 9.

Filament

  between layers, z 130-140 µm Speed of ink deposition, S 9-10 mm.s -1

Figure 9 .

 9 Figure 9. Picture of mineralized chitosan physical hydrogel scaffold fabricated from ink 5. The scale bar is 8 mm long.

Figure 10 .

 10 Figure 10. Picture showing the transverse section of scaffold fabricated with ink 5 obtained from μ-CT image (resolution of 3.5 μm/voxel).

Figure 11 .

 11 Figure 11. Examples of X-ray diffractograms of fabricated scaffolds. Gray arrows identify the chitosan X-ray diffuse halo (between 8 ° and 22 °); black arrows identify the apatite characteristic planes (002) at 2θ = 25.879 °, (211) at 2θ =31.774 ° and (112) at 2θ = 32.902 °.

Figure 12 .

 12 Figure 12. SEM micrographs of scaffolds fabricated with ink 1. (a) Well-defined grid of the scaffold and (bd) calcium phosphate aggregates embedded in chitosan filaments. Blue arrows indicate the calcium phosphate aggregates in chitosan filaments.

Figure 13 .

 13 Figure 13. EDS results of analysis of scaffold fabricated with ink 1. The element analyzed is presented in (a), the analyzed region is presented in (b), the map of each element found is presented from (c) to (f).

Figure 14 .

 14 Figure 14. Slope estimation from viscosity vs. shear rate curve for Ref1 (a), Ref2 (b) and Ref3 (c); where shear-thinning behavior increases as concentration of chitosan increases. Data obtained from flow ramp tests conducted at shear rates of 10-1000 s -1 .

  accessible à l'adresse : http://theses.insa-lyon.fr/publication/2018LYSEI010/these.pdf © [S.S. Ramírez Caballero], [2018], INSA Lyon, tous droits réservés

Figure 1 .

 1 Figure 1. X-ray diffractogram of HT scaffold surface. Red points indicate the characteristic planes of hardystonite: 111) at 2θ=23.928°, (201) at 2θ=28.910°, (211) at 2θ=31.930° and (310) at 2θ=36.274°.

Figure 2 .

 2 Figure 2. a) 3-D reconstruction; b) Cross-section of HT-scaffold obtained from μ-CT (resolution of 4 μm/voxel).

Figure 3 .

 3 Figure 3. Mercury intrusion porosity of HT scaffold.

Figure 4 .

 4 Figure 4. Rheological measurements on chitosan solutions. (Tests conducted at shear rates between 10 -4 or 10 -3 s -1 and 100 s -1 at 25°C).

Figure 6 .

 6 Figure 6. Pictures of HT-CS-1 and HT-CS-3 scaffold after fracture.

Figure 7 .

 7 Figure 7. Optical microscopy micrographs of fracture surface of HT and HT-CS-x scaffolds.

Figure 8 .Figure 9 .

 89 Figure 8. SEM micrographs of HT, HT-CS-1 and HT-CS-2 scaffolds. Column a: cross section of struts; column b: detail of the fracture surface of struts.

Figure 10 .Figure 11 .

 1011 Figure 10. Stress vs. strain curves for HT-H2O and HT-CS-x scaffolds Figure 11. Schematic representation of the analysis performed on stress -strain curves of each sample. Maximum stress:σmax; strain at maximum stress: ε@σmax; work at maximum stress: W@σmax; apparent Young´s modulus: E.

  'UNIVERSITE DE LYON OPEREE AU SEIN DE L'INSA LYON NOM : Ramírez Caballero DATE de SOUTENANCE : 07/02/2018 Prénoms : Silvia Stella TITRE : Composites made of bioceramic and chitosan physical hydrogel as potential bone substitutes NATURE : Doctorat Numéro d'ordre : 2018LYSEI010 Ecole doctorale : Matériaux de Lyon Spécialité : Biomatériaux composites RESUME :

Table 1 .

 1 Mechanical properties comparison of bone with other materials.

	Material	Tensile Strength (MPa)	Compressive Strength (MPa)	Elastic Modulus (GPa)	Fracture Toughness (MPa m 1/2 )

Table 3 .

 3 Experimental techniques for determining the structural, physico-chemical and rheological properties of chitosan and chitosan solutions.

	Property	Chitosan material	Technique	Reference
	Molar mass
	Viscosity average	Solution	Capillary Viscometer	[81,83,96]
			(Ubbelohde Viscometer)	[73]
	Weight-average molar mass and distribution	Solution	Static Light Scattering Size Exclusion Chromatography	[97]
	Degree of acetylation	Solution	Potentiometry (titration)	[98,99]
		Solution or solid	1 H-NMR Spectroscopy	[83,100,101]
		Solution or solid	FTIR spectroscopy	[81,102]
		Solution or solid	UV spectroscopy	[99]
	Protonation	Solution	pH method (titration)	[82,103,104]
	Water content	Solution or solid	TGA	[105]
	Root-mean-square gyration radius	Solution	Static Light Scattering	[73]
	(RGz)
	Second virial coefficient A2	Solution	Intrinsic Viscosity	[106]
	Conductivity	Solution	Conductivity-meter	[82]
	Turbidity	Solution	UV-visible spectrometry	[82]
	Surface tension	Solution	Maximum bubble pressure (MBP)	[107]
	Rheology	Solution or gel	Oscillatory shear flow	[108]
		Solution	Rotational (Brookfield type)	[108]
			viscometer

Table 4 .

 4 Some calcium orthophosphates ordered according to the Ca/P molar ratio. Adapted from[44,109].

	Name	Acronym	Chemical formula	Ca/P molar ratio	Solubility at 25 °C (g/L)	pH stability range of precipitates in aqueous solutions at 25 °C
	Dicalcium phosphate anhydrous	DCPA	CaHPO4	1.00	~0.048	[a]
	Dicalcium phosphate dihydrate	DCPD	CaHPO4.2H2O	1.00	~0.088	2.0-6.0
	Amorphous calcium phosphate	ACP	CaxHy(PO4)z•nH2O	1.0-2.2	[b]	5-12
	Octacalcium phosphate	OCP	Ca8(HPO4)2(PO4)4•5H2O	1.33	~0.0081	5.5-7.0
	α-tricalcium phosphate	α-TCP	α-Ca3(PO4)2	1.5	~0.0025	[c]
	β-tricalcium phosphate	β-TCP	β-Ca3(PO4)2	1.5	~0.0005	[c]
	Non-stoichiometric hydroxypatite	Ap or apatite	Ca(10-x)(HPO4)(6-x)(OH)(2-x)	1.5 -1.67	~0.0094	6.5-12
	Stoichiometric hydroxyapatite	HA	Ca5(PO4)3OH	1.67	~0.0003	[c]

[a] Stable at temperatures above 100 °C; [b] Cannot be measured precisely; [c] These compounds cannot be precipitated from aqueous solutions.

Table 5 .

 5 Summary of characterization techniques of calcium phosphates[112,113].

	Property

Table 6 .

 6 Phosphate ions in equilibrium. Adapted from[117].

	Equilibrium reaction	pKa	pH range
	H3PO4 ⇄ H + + H2PO4 -	~2.12	0 -4
	H2PO4 -⇄ H + + HPO4 2-	~7.21	5 -9
	HPO4 2-⇄ H + + PO4 3-	~12.67	10 -14

  During this curing time, microstructure may change from ACP to DCPD to apatite. Other intermediate calcium phosphate may form such as OCP [130,131]. Materials made of apatite usually present a porous microstructure that results in brittleness, low impact resistance, low tensile strength (6 to 20 MPa) in comparison with cortical bones, although,

compressive resistance is in the range than that of cortical bones (up to 170 MPa)

[132]

. Mechanical properties are a major weakness of apatite as possible bone substitute; however, tissues surrounding an implantation of apatite (bone, tendons and muscles) may contribute to bear loads, making apatite usable

[133]

.

  The presence or addition of ions like magnesium, has been reported to slow down transformation of DCPD to apatite phase [137-141]. DCPD has been tested in-vitro, in-vivo and clinically for a series of orthopedic and dental applications, as well as for other biotechnological uses, such as drug delivery, cancer therapy and the development of biosensors [137-141].

Table 4 .

 4 Data of chitosan-calcium phosphate suspensions.

	1	2	3	4	5	6	7
	Sample	Final [Ca] molL -1	Final [P] molL -1	Final [CS] molL -1	pH	Newtonian viscosity (Pa s)	n
	CS-a	0.176	0.106	0.15	5.3 ± 0.1	3705 ± 370	0.28 ± 0.01
	CS-0.15M*	0	0	0.15	5.2 ± 0.1	1115 ± 120	0.34 ± 0.01
	CS-b	0.242	0.145	0.14	3.8 ± 0.1	230 ± 25	0.40 ± 0.01
	CS-0.14M**	0	0	0.14	5.3 ± 0.1	700 ± 70	0.36 ± 0.01

*CS-0.15M is the chitosan solution reference for CS-a **CS-0.14M is the chitosan solution reference for CS-b

  Différents paramètres ont été étudiés, tels que la nature des sels précurseurs de la phase phosphocalcique, le protocole d'élaboration des suspensions ou encore les milieux alcalins utilisés pour leur gélification. L'analyse et la discussion des résultats mettent en lumière l'influence de ces paramètres sur la microstructure, les interactions physicochimiques, les propriétés rhéologiques et biologiques des matériaux ainsi élaborés.

	lors de leurs synthèses.
	Cette partie du manuscrit contient deux chapitres. Le premier porte sur l'élaboration d'hydrogels
	physiques de chitosane gélifiés par différents milieux alcalins (vapeurs d'ammoniaque,
	hydroxydes de sodium ou de calcium). Une fois gélifiés, ces hydrogels ont été minéralisés par de
	l'apatite phosphocalcique. La composition et la microstructure des matériaux composites ainsi
	obtenus ont toutes deux été caractérisées.
	Le second chapitre décrit des procédés d'élaboration d'hydrogels minéralisés à partir de

suspensions homogènes de particules de phosphates de calcium dans des solutions de chitosane ; les deux phases sont alors converties en apatite et gélifiées simultanément par l'augmentation du pH de la suspension, et ce afin d'obtenir des hydrogels physiques de chitosane contenant des particules d'apatite finement dispersées.

Table 1 .

 1 Chitosan physical hydrogels prepared in this study contacting a chitosan acetate with different bases, at different base concentrations and chitosan concentrations. Hydrogels exhibited a capillary structure in specific conditions.

		Experimental conditions	Capillaries characteristics (~350 μm deep)
		Concentration of		Concentration	Capillaries	Average	Density of
	Sample	chitosan solution	Base	of the base	presence	diameter	capillaries
		(wt%)		(mol.L -1 )	(CLSM)	(μm)	(cap/μm 2 )
	H1	1.5	NaOH	1	Yes	12 ± 4	0.6 ± 0.1
	H2	1.5	Ca(OH)2	0.5	Yes	29 ± 9	8.10 -2 ± 4.10 -2
	H3	1.5	NH3	1	No [31]
	H4	1.3	NH3	7.7	No
	H5	1.5	NH3	7.7	No
	H6	2.0	NH3	7.7	No
	H7	3.5	NH3	7.7	No

Table 2 .

 2 Dehydration followed by rehydration experiments: experimental conditions and results.

								% Weight
	Sample	Base used for gelation	Initial weight (g) ± 0.1	Weight after 30% dehydration (g) ± 0.1	Rehydration medium	pH of the medium ± 0.1	Weight after rehydration (g) ± 0.1	gained or lost after dehydration-rehydration	Volume of solution intake per gram of hydrogel (mL/g) (eq. 2)
								(eq. 1)
	H1-w	NaOH	3.8	2.7	H2O	7.4	3.4	-11	0.18
	H1-Ca	NaOH	3.8	2.7	5M Ca(NO3)2	3.9	4.2	11	0.18
	H1-P	NaOH	3.9	2.7	3M (NH4)2HPO4	8.5	3.7	-5	0.18
	H3-w	NH3	3.3	2.3	H2O	7.4	2.8	-15	0.15
	H3-Ca	NH3	3.3	2.3	5M Ca(NO3)2	3.8	3.0	-9	0.10
	H3-P	NH3	3.2	2.3	3M (NH4)2HPO4	8.7	2.8	-13	0.11

Table 2 .

 2 Type of samples and characterization techniques.

	Sample	Composition	Crystal size	Microstructure	Rheology	Biological behavior
	Freeze-dried
	suspensions of calcium phosphates	XRD
	(a*, b*)
	Suspension of chitosan-calcium phosphates (S1, S2, S3)	XRD		Optical microscopy	Measurements of steady viscosity (static mode)
					Measurements
	Solution of chitosan				of steady
	(CS solution)				viscosity (static
					mode)
	Mineralized
	hydrogels (M1, M2, M3, M4)	XRD		Optical microscopy
	Sterilized				Rheological	Cell
	mineralized	XRD			measurements in	proliferation
	hydrogels				dynamic mode	test
	Ground freeze-dried mineralized hydrogel	XRD, TGA, spectroscopy FTIR	refinement Rietveld	SEM
	Residue after thermogavimetric analysis	XRD	Rietveld refinement

DCPD a) NH 4 H 2 PO 4 P 4 ON 6 NH 4 PO 3 NH 4 NO 3 DCPA

									CaClH 2 PO 4 *H 2 O	NH 4 Cl
																b)
	Intensity (a.u)							Intensity (a.u)
	8	12	16	20	24	28	32	36	8	12	16	20	24	28	32	36
				2(°)  Cu				8	12	16	20 2 (°)  Cu	24	28	32	36

Cette thèse est accessible à l'adresse : http://theses.insa-lyon.fr/publication/2018LYSEI010/these.pdf © [S.S. Ramírez Caballero],

[2018]

, INSA Lyon, tous droits réservés

Table 3 .

 3 Data of viscosity plateau and n value of chitosan solution and suspensions of chitosancalcium phosphates.

	Sample	Newtonian viscosity (Pa s)	n	pH
	CS solution	2163 ± 200	0.323 ± 0.006	5.5 ± 0.1
	S1	3804 ± 350	0.285 ± 0.006	5.3 ± 0.1
	S2	3381 ± 300	0.293 ± 0.007	5.1 ± 0.1
	S3	324 ± 30	0.404 ± 0.013	3.8 ± 0.1

Table 4 .

 4 Viscoelastic parameters of chitosan hydrogels references (Ref1 and Ref2) and mineralized chitosan hydrogels at low angular frequency. (Average values and uncertaintiesobtained after measurements on three samples).

	Sample name	G' (kPa)	G" (kPa)
	Ref1	4.0 ± 0.4	0.4 ± 0.02
	Ref2	2.6 ± 0.3	0.2 ± 0.02
	M1	5.6 ± 0.5	0.5 ± 0.05
	M2	3.7 ± 0.4	0.4 ± 0.06
	M3	6.6 ± 0.3	0.6 ± 0.03
	M4	4.1 ± 0.3	0.4 ± 0.05

Table 5 .

 5 Parameters

	Sample name	Lattice	Lattice parameter a a =

and size of apatite crystals in freeze-dried ground samples and in TGA residues. The uncertainties of lattice parameters were lower than 10 -2 .

b (Å) Lattice parameter c (Å) Crystal size (nm) Freeze-dried grounded samples

	M1	Hexagonal	9.43	6.90	16.17 ± 0.64
	M2	Hexagonal	9.44	6.88	23.60 ± 1.47
	M3	Hexagonal	9.43	6.89	27.47 ± 1.22
	M4	Hexagonal	9.43	6.89	19.37 ± 0.64
			TGA residue of samples
	M1	Hexagonal	9.40	6.89	19.50 ± 0.55
	M2	Hexagonal	9.43	6.88	22.23 ± 1.61
	M3	Hexagonal	9.43	6.89	29.23 ± 0.40
	M4	Hexagonal	9.42	6.89	22.00 ± 0.36

Band or range name Band or range value (cm -1 ) Chitosan assignments Apatite assignments

	a	3000-3600	NH	OH
	b	2871	CH2
	c	1500-1750	NH2 amino, amine deformation, amine I	OH
	d	1427	CH	CO3 2-
	e	1370	CN
	f	1150	COC
	g	1024		υ3PO4 3-
	h	890	CO	CO3 2-
	i	1024	C=O
	j	670-460		υ2PO4 3-, υ4PO4 3-

Table 7 .

 7 Results of weight percentages in chitosan hydrogels references and mineralized hydrogels.

	Sample	H2O	Chitosan	Apatite	Apatite/chitosan	Apatite/chitosan weight
	name	wt %	wt %	wt %	weight ratio expected	ratio experimental
	Ref1	7	90	0	0	0
	Ref2	8	91	0	0	0
	M1	6	56	37	0.84	0.66
	M2	5	59	35	0.84	0.59
	M3	5	55	39	0.84	0.71
	M4	5	47	47	1.13	1.00

7 th day 10 th day 14 th day Cette

	16000
	4000 8000 12000 Fluorescence Units
	0	Ref1 Ref2 M1	M2	M3	M4 Control

thèse est accessible à l'adresse : http://theses.insa-lyon.fr/publication/2018LYSEI010/these.pdf © [S.S. Ramírez Caballero], [2018], INSA Lyon, tous droits réservés

  This thesis was highly enriched by collaborative research with Prof. E. Saiz's group (Imperial College London, UK) and Prof. P. Colombo's group (University of Padova, Italy). In particular, experimental work of chapter III.1 was performed at Imperial College London (UK), during a two months stay. It was partly funded by a trust from the Journal of European Ceramic Society (JECS Trust). Experimental work described in chapter III.2 was done partially in France and partially at University of Padova, Italy.

  Cette partie III contient également deux chapitres. Le premier des deux porte sur la fabrication d'hydrogels physiques de chitosane contenant des particules d'apatite finement dispersées, architecturés par robocasting. Pour cela, des suspensions homogènes de phosphates de calcium dans du chitosane ont été utilisées comme encres. Dès que l'encre est poussée hors de la canule, la gélification du chitosane et la recristallisation des particules de phosphate de calcium en apatite sont induites de manière simultanée par l'immersion de l'extrudat dans un bain alcalin. Sont également détaillées dans ce chapitre : la rhéologie des encres, les interactions dans ces systèmes, et la caractérisation des matériaux architecturés obtenus. Le deuxième chapitre présente la fabrication et la caractérisation de composites architecturés hardystonite-chitosane. L'imprégnation des substrats en hardystonite par des solutions de chitosane, gélifiées par la suite, permet d'améliorer de manière significative leurs propriétés mécaniques et ouvre une nouvelle voie d'obtention de composites architecturés pour réparation osseuse.

	Professeur
	E. Saiz (Imperial College London, GB) et le groupe du Professeur P. Colombo (Université de
	Padoue, Italie). En particulier, le travail expérimental rapporté dans le chapitre III.1 a été réalisé
	dans le cadre d'un séjour de deux mois à l'Imperial College. Cet échange a été en partie financé
	grâce à une bourse du « Journal of European Ceramic Society » (JECS Trust). Les travaux

décrits dans le chapitre III.2 ont été quant à eux réalisés en partie en France et en partie à l'Université de Padoue, Italie.

Table 1 .

 1 Inks prepared in this work.

	𝑜𝑓 𝑐𝑎𝑙𝑐𝑖𝑢𝑚 𝑠𝑎𝑙𝑡(𝑔) + 𝑀𝑎𝑠𝑠 𝑜𝑓 𝑝ℎ𝑜𝑠𝑝ℎ𝑎𝑡𝑒 𝑠𝑎𝑙𝑡 (𝑔) 𝑀𝑎𝑠𝑠 𝑜𝑓 𝑐ℎ𝑖𝑡𝑜𝑠𝑎𝑛 (𝑔)	=	𝑖𝑛𝑜𝑟𝑔𝑎𝑛𝑖𝑐 𝑜𝑟𝑔𝑎𝑛𝑖𝑐	(2)

Table 2 .

 2 Correlation between composition and rheological behavior for all ink tested.

	1	2	3	4	5	6	7	8
	Ink	Ionic strength mol.L -1	G" (Pa)	G' (Pa)	n	𝛔 𝐲 (Pa)	Viscosity (Pa s) 𝛄̇= 𝟏 𝐬 -𝟏	Viscosity (Pa s) 𝛄̇= 𝟒𝟎𝟎 𝐬 -𝟏
	1	0.303	74	69	0.19		50	2.7
	2	0.455	59	45	0.16		40	2.4
	3	0.682	60	46	0.16		45	2.3
	4	0.850	52	37	0.18		35	2.1
	5	0.510	114	127	0.14	19.8	150	3.2
	6	0.761	102	110	0.14	11.2	65	3.0
	7	0.943	92	93	0.14	9.4	60	2.8
	8	1.314	125	144	0.13	8.9	85	3.5
	9	0.866	159	188	0.21	18.5	95	3.6
	Ref1	-----	72	56	0.16		50	2.5
	Ref2	-----	101	91	0.20		75	3.3
	Ref3	-----	175	200	0.23	8.24	200	3.5

Table 1 .

 1 Impregnation protocols of HT scaffolds with chitosan solutions

	Impregnation Protocol	Vacuum (mbar) pressure	1 st [CS1st] wt% impregnation	2 nd [CS2nd] wt% impregnation	Gelation (1 M) method	Sample
	1	50	0.5	2.5	NaOH	HT-CS-1
	2	80	0.5	1.5	NH4OH	HT-CS-2
	3	50	0.5	1.5	NH4OH	HT-CS-3
	4	50	0.5	2.5	NH4OH	HT-CS-4
	5	50	1.5	1.5	NH4OH	HT-CS-5

Table 3 .

 3 Weight results for HT and HT-CS-x scaffolds during the impregnation process.

	Protocol	𝒎 𝟎 (𝒈)	𝒎 𝟏 (𝒈)	𝒎 𝟐 (𝒈)	𝒎 𝒈 (𝒈)
	1	0.269 ± 0.015	0.507 ± 0.037	0.600 ± 0.133	0.588 ± 0.119
	2	0.277 ± 0.015	0.541 ± 0.067	0.663 ± 0.082	0.561 ± 0.053
	3	0.266 ± 0.022	0.530 ± 0.045	0.664 ± 0.064	0.564 ± 0.048
	4	0.268 ± 0.022	0.521 ± 0.057	0.608 ± 0.075	0.544 ± 0.078
	5	0.257 ± 0.026	0.712 ± 0.102	0.748 ± 0.078	0.626 ± 0.068

Table 5 .

 5 Analysis of stress -strain data for HT-H2O and HT-CS-x scaffolds, and CS/HT ratio.

	Sample	Max stress (MPa)	Strain at max stress (%)	Work at max stress (%MPa)	Apparent Young's modulus (MPa)	CS/HT ratio
	HT-H2O	3.05 ± 0.46	7.27 ± 2.04	7.34 ± 2.38	207 ± 87
	HT-CS-1 4.73 ± 0.72	9.98 ± 0.89	15.85 ±2.96	196 ± 89	1.16 ± 0.46
	HT-CS-2 2.86 ± 0.78	8.00 ± 0.62	9.15 ± 2.29	134 ± 42	0.84 ± 0.02
	HT-CS-3 3.03 ± 0.63	8.13 ± 0.66	9.74 ± 2.02	149 ± 53	0.94 ± 0.04
	HT-CS-4 3.57 ± 0.71	9.07 ± 0.93	11.62 ± 2.00	171 ± 38	0.93 ± 0.01
	HT-CS-5 3.88 ± 1.20 23.19 ± 0.73	39.88 ± 0.83	97 ± 4	2.16 ± 0.13

  : apatite, bone substitutes, calcium phosphates, chitosan, composites, gelation, hardystonite, physical hydrogel, 3-D printing Laboratoire (s) de recherche : MATEIS, UMR CNRS 5510, INSA-Lyon ; IMP, UMR CNRS 5223, Université Claude Bernard Lyon 1

Directeur de thèse: Laurent Gremillard Président de jury : Eduardo Saiz Composition du jury : Christèle Combes, Catherine Le Visage, Solène Tadier, Alexandra Montembault, Laurent Gremillard

Cette thèse est accessible à l'adresse : http://theses.insa-lyon.fr/publication/2018LYSEI010/these.pdf © [S.S. Ramírez Caballero],[2018], INSA Lyon, tous droits réservés

Acknowledgements

I would like to express my gratitude to my Director, Laurent Gremillard, and co-directors, Solène Tadier, Alexandra Montembault, Thierry Delair and Laurent David for giving me the opportunity to do the master and Ph.D. with them. Thank you for enriching this work by scientific and nonscientific contributions. A special thanks to Laurent, Solène and Alexandra for their constant

Appendix B: calculation of shear rate in cylindrical ducts

Equation ( 1) is derived from application of the equation of Hagen-Poiseuille to the rheological curve of Newtonian fluids:

In equation ( 1), 𝛾̇ stands for shear rate, Q for volumetric flow rate and R for the nozzle radius of cylindrical shape.

In Chapter 5, equation ( 2) was used to calculate the shear rate. This equation takes into account the value of n, the exponent of the power law fluid.

In equation ( 2), S stands for speed fluid extrusion. Equation ( 2) is the same equation ( 1) for a Newtonian fluid, n = 1.

For n = 1,

Now,

Then,

Therefore, replacing S from the equation ( 5) in to the equation (3):