Keywords: spray drying, hydroxyapatite, UHMWPE, selective laser sintering, bone tissue engineering

In this work, an investigation on the manufacturing of 3D parts using composite biomaterials, additive manufacturing and targeting bone tissue applications is presented. The employed manufacturing process was selective laser sintering (SLS) and the feedstock powder was commercial UHMWPE and self-synthesized hydroxyapatite (HA). First, a step-by-step approach to use spray drying (SD) for obtaining HA particles with suitable morphological characteristics for SLS processing was employed. Varying processing parameters it was possible to produced SDHA particles the within mean diameter range of 15 to 25 µm. A major influence of atomization pressure variation was identified, a greater pressure value resulted in smaller particle size. Desirability function was employed to determine the optimal SD processing parameters and the selected parameters have considered the processing efficiency.

The SDHA powder was mixed, at 5 and 10% weight content, with sieved UHMWPE and several SLS processing parameters were evaluated. Powder characteristics were assessed, samples' dimensional and mechanical properties were measured, likewise, preliminary in vitro essays were conducted. Warping effect and dimensional variation were observed with the employed parameters combination. Results indicated that the addition of SDHA particles have favoured the processability and biological response, nevertheless, a greater amount of SDHA has hindered samples' mechanical properties so a compromise must be found.

TABLE OF FIGURES

CHAPTER 1 General Introduction

General Introduction

Personalized medical devices have improved quality of life and welfare, also favouring faster recovering and being useful for a wide range of applications. Over the past decades, great development has been done in devices for bone repairs and substitution. Bone tissue engineering is a field of study that comprises techniques and treatments to overcome the limitations of conventional treatments of bone diseases [1]. Composite materials have been used in different parts of the human body (Figure 1), the implanted devices must match the local mechanical properties requirements and bone morphology.

Progress in biomedicine, material science, surgical techniques and other related fields have favoured faster healing and minimally invasive procedures, increasing patient's welfare.

The employed materials must avoid pathological reactions after implantation and ideally restore the functionality of the damaged area.

Essentially, human bone is a complex vascularized structure, composed of organic collagen fibrils and inorganic calcium phosphate (Ca-P) crystals [3]. In terms of proportion, the Ca-P phase comprises to 65-70%, water to 5-8% and the remaining portion is the organic phase of the bone [4]. Figure 2 illustrates the bone composition and its morphological features from the macro to the nanostructure, whereas the portion of compact and spongy bone can vary according to the bone's location the body. The optimization of bone structure is interrelated with Wolff's law: "Mechanical stress placed upon a bone is responsible for determining the architecture and the external form of the bone" [5]. Consequently, bones can be considered as an anisotropic material and this aspect must be considered in the product development for the medical device. Fabricating a device using the combination of a matrix composed of a more flexible material (i.e., polymers) and the addition of a mineral filler (i.e., Ca-P) is suitable to mimic the bone structure and chemical compatibility. Therefore, the feasibility of manufacturing complex geometries, combining composite biomaterials, matches with the requirements of implantable devices used for human tissue recovery. Additive manufacturing (AM) is a breakthrough technology that allowed significant progress for tissue engineering applications, and still reveals promising solutions for custommade bone scaffolds [6][7][8][9]. Powder bed fusion, frequently referred to as selective laser sintering (SLS) is one of the commercially available AM processes [10]. In this process, particulate materials are fused layer by layer via heat supplied by an infrared laser source, creating 3D parts that were originally designed using computer-assisted design (CAD) tools.

The SLS fabricated parts are supported by a powder bed during manufacturing. It can achieve printing resolution up to 0.09 mm [11], which is dependent on the type of material employed. On the other hand, during SLS processing high temperatures can be achieved and the manufactured parts are characterized by a rough surface finish [12]. Nevertheless, SLS can be considered one of the most versatile AM techniques in terms of different material usage and structural stability. When using the SLS technique multiple parameters influence the outcome of the product. The parameters involved in this AM technique include, but are not limited to the following: part bed temperature, laser energy density, layer thickness, scan speed and hatch distance affect the mechanical and structural properties of fabricated parts [13]. Currently, many researchers are trying to find a correlation between these parameters, their adjustments, and material selection [13,14]. However, while different properties vary amongst the material, the correlation between the laser parameters and materials are currently being investigated. A challenging aspect of this technique is establishing material's processing within the optimal sintering window, which varies amongst different materials.

SLS processing with proper materials selection can contribute to enhancing the scaffold's final properties, particularly bioactivity [15,16]. When processing composite materials (i.e., bioceramic fillers in a polymeric matrix) an association of properties can be achieved, enhancing the fabricated piece performance. Either, in terms of particle size, a substantial difference between each material is beneficial, allowing the filler to occupy voids in the interstices of matrix particles.

Concerning polymer SLS feedstock, there are preferable powder characteristics that improve the sinterability and final properties of the fabricated piece [17,18]. Related literature has indicated better processing and geometry accuracy when using spherical particles and narrow particle size distribution with an average size below 150 µm or equivalent to the laser beam diameter [17,19].

Polyethylene (PE) is one of the most common plastic in use and its variations have been used alone or in combination with other types of materials for medical devices. In terms of implantable devices, it is illustrated in Figure 2 different applications of the PE are shown: face bone replacement material, finger joint, tendon/ligament and total hip/knee replacement.

Concerning ultra-high-molecular-weight polyethylene (UHMWPE), it has also been used in different industries due to its outstanding physical and mechanical properties, such as: chemical inertness, lubricity, impact resistance, and abrasion resistance [20]. Its low friction coefficient and chemical inertness contributed to extensive use in orthopedics, mainly as bearing material in artificial joints. Given the widespread use of UHMWPE for medical applications, its processability using SLS equipment was also investigated. Rimell and Marquis [21] observed considerable shrinkage-induced warpage while fabricating multilayer parts of UHMWPE. Using a precise combination of processing conditions, including preheated powder Goodridge et al. [22] have successfully produced multilayer parts with relative accurate dimensions. Similarly, using optimized SLS processing parameters, Song et al. [23] minimized the warpage effect, achieving 83.25% density and tensile strength up to 14.1 MPa.

Ca-P materials [24,25] such as hydroxyapatite (HA) and beta-tricalcium phosphate (β-TCP) are widely used as bone substitutes and are commercially found in a few different geometries (e.g., granules and sticks). Manufacturing customized scaffolds from these materials remains a challenge, therefore, the use of SLS processing also shows to be advantageous.

When compared to HA, β-TCP is degraded and absorbed quite rapidly by the body often occurring before new bone formation, therefore, discrepancies can be generated between new bone formation and material absorption [26]. Advantages of HA [Ca10(PO4)6(OH)2] is its chemical similarity with natural bone, possessing non-immunogenic properties, good biocompatibility and bone conductivity, it is considered one of the most suitable materials for bone scaffold fabrication [27]. In terms of biomedical applications, HA can correspond to different needs and has been used as an implant coating, drug delivery system and as bone scaffold and filler [28]. According to its application, appropriate HA morphological features must be fulfilled and specific processing techniques are required.

Spherical HA particles can be obtained by different techniques [29][30][31][32][33][34] in a diverse range of particle sizes. However, process scalability remains a challenge and not all techniques are capable of producing particles in the size range of 15 to 25 µm to be used as filler in a polymeric matrix.

Spray drying (SD) consists of the transformation of a fluid material into dried particles.

SD had shown remarkable development in the last decades, being used by different industries [35]. SD commercial equipment may differ in terms of configuration. Rotary, hydraulic and pneumatic nozzle atomizers are commonly used [15]. The variables that affect how the spray is mixed with the hot gas depend upon the type of gas flow: co-current, countercurrent, or mixed flow [36]. Spray-dried HA (SDHA) powder has been successfully employed for distinct biomedical applications, using different manufacturing techniques [37][38][39][40][41][42]. Moreover, SD enables advantageous powder characteristics that are desirable for 3D printing use [35].

Although SDHA powder morphology indicates suitability for SLS processing, publications concerning this subject are scarce [43].

Regarding the considerable use of HA and UHMWPE materials for implantable devices, along with the benefits of spray drying into particle's morphological features and the combination of UHMWPE/HA properties, the present work investigated the addition of SDHA particles into the UHMWPE matrix for SLS processing. First, the spray drying process was assessed for the feasibility to obtain narrow particle size distribution and mean diameter within the range of 15 to 25 µm. The produced SDHA powder was further added to UHMWPE, varying the SLS processing parameters to produce rectangular samples for mechanical and dimensional properties assessment. Finally, based on the sample's properties, the chosen SLS parameters were used to produce cylindrical samples for biological analysis.

General objective

The general objective of this work is to produce and characterize UHMWPE/SDHA samples using the SLS technique, aiming at bone tissue applications.

Specific objectives

For achieving general objectives, the specific objectives were established:

• Synthesis and characterization of stoichiometric nano-sized HA powder.

• Investigate the suitability of the spray drying process to produce spherical HA particles within the range of 15 to 25 μm.

• Select SLS processing parameters for fabricating UHMWPE/SDHA samples and evaluate if mechanical properties are appropriate for bone tissue applications.

• Preliminary biological essays to evaluate SLS fabricated samples biocompatibility.

CHAPTER 2

Manuscript 1

Introduction to Chapter 2

This chapter contains information found in the related bibliography concerning SLS processing of polymers/calcium phosphate biocomposites. Bone tissue is composed of a mineralized structure with a flexible matrix. The combination of polymer and calcium phosphates are a suitable match in terms of desirable properties. Implantable devices for bone tissue engineering can be obtained by different fabrication processes. However, AM facilitates custom-made implants, enables multi-material usage and is capable of producing complex geometries that mimic bone structure.

Among the available AM techniques, SLS was chosen because of its capability of processing different categories of powder materials, further, it allows the fabrication of complex geometries with interconnecting pores, it is industry scalable and concurrently fabricating tailored pieces.

Nevertheless, material selection and its particle's characteristics must meet some requirements for achieving proper SLS processability. Either, SLS processing parameters are broadly variable and affect the proprieties of the fabricated pieces. A literature review allows acquiring relevant information concerning the target topic, endorsing choices of possible research paths.

Polymer/calcium phosphate biocomposites manufactured by selective laser sintering: an overview

Henrique Schappo

Abstract

Additive manufacturing (AM) drove important advancements for implantable devices for tissues regeneration. Selective laser sintering (SLS), also known as powder bed fusion (PBF) is a laser-assisted AM method, capable of producing well-defined pieces using different types of material targeting diverse applications. Regarding bone tissue engineering, calcium phosphates are considered as reference materials for their biocompatibility and bone regeneration capability. Likewise, certain polymers are also widely used for implantable devices due to their processability, adequate properties and bioabsorption. This review article assessed original research articles available between the years 2000 and 2020, that have been processed by SLS polymer/Ca-P composites mixtures. Considering the reviewed articles, it was possible to elaborate an overview of the state of the art and future trends. Important aspects of processing are discussed, indicating powder mixture approaches, employed materials and processing parameters details, as well as biological assessment. Facts and information are provided along with this review article for a comprehensive understanding of the involved proceedings for polymer/Ca-P implantable devices.

Keywords: polymer; calcium phosphate; selective laser sintering; bone tissue engineering.

Introduction

Personalized parts are preferred in different applications, being greatly helpful in medicine. It is recognized that custom-made medical devices have improved quality of life and welfare, also favouring faster recovering. In terms of implantable devices, there are many advantages in using patient-match components for bone-related pathologies, particularly oncologic reconstruction [1]. Further, clinical essays have shown that bone-related injuries have considerable benefits when treated using osteoconductive customized implants [2], [3]. These achievements can rely upon the use of additive manufacturing (AM), a breakthrough technology wherein characteristics, still arise opportunities for significant progress in the field of bone tissue engineering.

Bone tissue consists of 30% organic collagen fibrils and 70% inorganic calcium phosphate crystals [4]. Its structure is composed of overall 80% cortical bone and 20% trabecular bone, having respectively, less than 10 vol% and 50-90 vol% porosity [5], [6]. Since bone is a complex mineralized structure with a flexible matrix, the combination of biopolymers and bioceramics is particularly interesting for bone replacing applications. Moreover, these materials have characteristics like biocompatibility and osteoconductivity that are highly desirable [7], [8]. Towards ceramic materials capable of favouring bone tissue formation, calcium phosphates (Ca-P) are considered an overcome and have been used for many years [9], [10]. This group of material was successfully mixed with biopolymers and processed by SLS, however, different mixture methods and performance outcomes can be found in the bibliography. The use of a polymeric matrix together with Ca-P materials, provide benefits in terms of mechanical properties and favours processability. Further, bioabsorbable polymers ensure mechanical stability and can be degraded along with bone tissue recovering.

Selective laser sintering, also know as powder bed fusion by ISO/ASTM 52900 [11] is among the commercially available AM techniques. This process fuses particulate materials layer by layer, via heat supplied by an infrared laser source, allowing the creation of complex 3D parts. SLS is considered one of the most versatile in terms of material usage, structural stability and, through optimized parameters, it is possible to achieve the desired mechanical properties for tissue engineering scaffolds [12]. The performance of SLS fabricated parts is highly depended on powder materials characteristics and process parameters. Using suitable SLS processing parameters it is possible to enhance cell infiltration, nutrients and metabolites throughout the manufactured scaffold [13]. This fabrication method enables to mimic the bone structure, favouring faster healing of the damaged bone tissue.

This overview presents information from research articles that have processed, through SLS, a combination of polymer and calcium phosphates materials, targeting bone tissue engineering applications. Powder preparation methods, materials selection, processing and final proprieties of the fabricated piece will be outlined and discussed. Finally, future perspectives of polymer/Ca-P biocomposites processed by SLS will be summarized.

Methodological approach

The adopted search strategy was to use the Scopus database to find publications associated with the keywords displayed in Table 1. Keywords were selected to compass a vast number of articles indexed by a reputed scientific database. The star keys were used in favour of searching singular and plural forms of the keywords in the database.

The articles found were assorted in lists for checking duplicates and adequacy. The selection criteria were original research articles that fabricated samples by SLS, using polymer and calcium phosphates composite mixture and having publication year between 2000 and 2020. Among a total of 650 articles, 63 were selected and analyzed; review articles, books and conference releases were not considered.

Materials

Polymer matrix

Aliphatic polyesters are among the most used biopolymers for medical use, thanks to characteristics such as non-toxicity, biocompatibility and biodegradability [14]. Not surprisingly, this category of polymers was the most used among analyzed articles as shown in Figure 1. The majority of reviewed articles have used a polymer as the predominant material of the polymer/Ca-P mixture, whereas around 10% (6 articles) have used less than 20% of polymer content. Medical devices fabricated by SLS requires two main material's characteristics, one related to application demands and the other concerning its processability. Only a few polymers have suitable processing for the SLS technique, limiting the range of commercially available powders options and industrial-scale production of implantable devices [15], [16]. Polyamide (PA) is commercially available and is already well known in the SLS industry. However, in our research, we have found only 3 articles using it. This might be attributed to its low poor cell seeding behaviour [17] and author's greater interest in using bioabsorbable polymers.

The most used polymers were polycaprolactone (PCL) and poly(lactic acid) (PLA) with ~30% of usage each. The largest use of these polymers are probably due to their easy access and proven track record as tissue compatible. Owning a slow degradability (2 to 4 years), PCL is preferable for long-term implantable devices, nonetheless, its degradation rate, bioactivity and mechanical properties can be modified with the incorporation of Ca-P materials [18].

Poly(lactic acid) (PLA) is also approved by Food and Drug Administration (FDA) for human body applications. Its body degradation rate depends on the polymer's characteristics and the implant location. According to the literature, its absorption time may vary from 40 weeks to 6 years [19]. As a consequence of the polymeric chain optical activity, PLA is available in three different variations: D,D-lactide (not employed in the reviewed articles); L,Llactide (accounting for ~24% of usage) and D,L-lactide (~6% of usage) [20].

Figure 2 displays SEM images of widely used polymer materials, particle's morphology distinction is a result of the applied preparing method. Figure 2a shows pure PCL spherical particles that were produced using a modified S/O/W emulsification solvent evaporation method [21]. This method also allows obtaining the microspheres with HA in the particle's surface (up to 20 wt% content). In Figure 2b, PLLA particles were obtained using cryogenic milling, this method forms irregular morphologies, and in this case, the ceramic particles are added after the milling process.

Ca-P powders

Ca-P materials have substantial chemical similarity with bones, consequently, their biocompatibility, biodegradability and osteoconductivity are suitable for bone tissue applications [23]. Specific chemical formulations differ in each type of Ca-P material.

Concerning the most used Ca-P materials, we can rank hydroxyapatite (HA) followed by tricalcium phosphate (TCP); other Ca-P materials accounted for almost 8% of the total. Only 3 articles have used modified versions of HA, also, 3 articles have used a combination of bioceramics.

The major use of pure HA is probably related to significant benefits and performance advantages, when using HA as a bone repair and bone replacement material [24]. TCP degradation in the human body is faster than HA so that its benefits were explored by a few authors. The combination of HA + β-TCP is useful for certain types of implants by gathering properties of each material [25], [26].

Figure 3 illustrates Ca-P powders used in the reviewed articles, it can be seen that individual nano-HA needle-like particles tend to agglomerate into irregular clusters. Particle geometry influences powder spreading and the best results are achieved with spherical particles [27]. In this sense, it is preferable to use polymeric/Ca-P particles with spherical morphology for SLS processing such as those shown in Figure 3b.

Additives

Biocomposites that are employed for medical applications must have high stability, therefore, surface properties might be improved to enhance material's compatibility [30]. Our findings revealed that additives among polymer/Ca-P composites are mainly used to allow SLS processability, interfacial bonding and to bring supplementary properties for the fabricated device. Carbon/gold nanoparticles can be used as a sensitizing agent to absorb the laser radiation energy, making possible the processability of polymer/Ca-P composites by singlemode fibre laser [31]- [34].

When composites are processed by SLS, employed materials should possess interfacial bonding to achieve better mechanical properties. Shuai et al. [35] have evaluated the addition of 2-Carboxyethylphosphonic acid (CEPA), a phosphonic acid coupling agent, when processing hydroxyapatite (HAP) and poly-l-lactic acid (PLLA). Results have shown an increase of scaffold's tensile strength and modulus up to 2.79 times when using surfacemodified HA particles with CEPA.

An example of adding supplementary properties to the fabricated device was seen in the work of Tan et al. [36] where they have successfully used PLLA/HA/metformin scaffolds for bone repair and tumour cells inhibition. Liu et al. [37] have modified PCL/HA scaffolds with vascular endothelial growth factor (VEGF) and results have shown excellent cytocompatibility and greater blood vessel formation. The addition of mesoporous silica Santa Barbara Amorphous-15 (SBA15) into PLLA/HA scaffolds have improved hydrophilicity, cell proliferation and enhanced mechanical properties [38].

Although SLS can be applied to fabricated devices for drug delivery [39], our research did not find polymer/Ca-P composites with incorporated drugs. However, this possibility is cited by Duan et Wang [40] who have successfully fabricated scaffolds using Ca-P/PHBV nanocomposite microspheres loaded with bovine serum albumin (BSA), demonstrating the viability of incorporating biomolecules on the SLS powder. Nonetheless, SLS technology is already successfully used for processing drugs agents for pharmaceutical purposes [41].

The use of additives has shown great potential to provide benefits and supplementary properties for the fabricated scaffolds. Still, many aspects that should be deeply investigated such as the interaction between materials, properties preservation after laser irradiation and biological response.

Manufacturing process 4.1 SLS equipment and powder processability

Around 30% (18 articles) have stated that the SLS equipment was developed in-house, this small amount is probably due to the relative complexity level of hardware assembly and software communication. At the same time, SLS equipment construction requires time and expertise. Not all authors provided enough information related to SLS equipment (20%), while more than a half (32 articles) have informed to use a commercially available machine. Some authors have described modifications on commercial machines, these modifications were mainly to reduce the quantity of powder consumption.

Most available commercial SLS are capable of using inert gas during SLS processing, likewise, in-house SLS equipment might also be constructed to provide this feature. This feature avoids unwanted chemical reactions and degradation. Despite its benefits, we have found only one manuscript [42] that claimed to use nitrogen during processing. The lack of use of inert gas during Ca-P/polymer SLS processing is probably due to the high cost and minor benefits.

Notwithstanding, inert gas during SLS processing is desirable while fabricating metal devices [43].

The laser source has great importance for the type of material to be processed. The main laser source in the reviewed articles was CO2 with a wavelength of 10.6 µm, representing 73% of the total. This observation can be related to the greater efficiency of CO2 lasers when processing oxide ceramics and polymers [41]. Representing 8% of usage, fibre laser (λ = 1.06 μm) were used for surface selective laser sintering (SSLS), whereas a small amount (0.1 wt%) of carbon/gold nanoparticles is added in the powder mixture. It will absorb laser radiation energy, avoiding internal overheating of polymer particles and minimize degradation.

Surprisingly, not all authors have provided information related to which laser type was used, these articles represented 19% of the total.

According to the laser type and material's characteristics, there are key aspects that directly influences powder's processability by SLS, as shown in Figure 4. Although Schmid et al. [44] have elaborated this diagram examining polymers, many properties can also be considered when processing Ca-P by SLS. Intrinsic characteristics (optical, thermal and rheological) are related to the molecular structure, being more difficult to change. On the other hand, extrinsic properties (related to powder and particles) are versatile to modify [44].

Employment of adequate SLS processing parameters preserves material's characteristics, also enabling the achievement of expected biological response. Further, the scaffold's surface characteristics, microporosity and mechanical properties are affected by the processing parameters [45]. The absence of sufficient laser energy will lead to poor structural stability (lack of neck formation between particles), in contrast, excessive energy will cause heating that might deteriorate polymers and cause geometric distortion. The importance of suitable processing parameters is reinterred by many authors' in-depth analysis of its consequences on the sample's final properties. Subsequently, most of the reviewed articles applied the design of experiments (DOE) and/or other statistical tools for selecting the optimal processing parameters. Supplementary analysis using scanning electron microscopy (SEM) images, Fourier-transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) are often used to verify variations on morphological and chemical features as a consequence of different processing parameters.

Polymer/Ca-P powder mixture and preparation methods

In terms of desirable powder characteristics for proper SLS processing, it is preferable to have a wide sintering window (difference between melting and crystallization onset temperatures), good flowability and relatively narrow particle size distribution (preferably with high sphericity). For polymer particles, the thermal stability, melting and solidification behaviours are particularly important during the SLS process.

Concerning Ca-P materials, particle's morphology and size distribution can be adjusted using distinct powder preparation methods, however, crystal growth formation is highly dependent on which synthesis method is applied. Consequently, it is preferable to use a synthesis method that will approach the most to the desired particle's characteristics, possibly, eliminating a further additional process.

Sadat-Shojai et al. [46] have reviewed HA synthesis methods, relating the different synthesis methods with obtained powder characteristics. Among diverse approaches to synthesize HA nanoparticles, wet methods (e.g., chemical precipitation and hydrothermal) have the advantage of more precise control over the morphology and size of particles [46]. From our study, we observed that the majority of authors have used commercially available Ca-P powders and only a few have synthesized it themselves, this disparity might be justified by the high cost of synthesis related apparatus.

Alternately, no information about self-synthesis was identified for polymers, all authors stated to use commercially obtained materials. Polymers were delivered in pellets or powders, normally requiring an additional process (e.g., milling, sieving) before mixing with Ca-P powder. According to which method, substantial differences in morphological features can be observed. Figure 2a shows SEM images from PCL microspheres prepared by the solid-in-oilin-water (S/O/W) emulsification solvent evaporation method [21]. Figure 2b shows PLLA cryogenic milled particles [22]. All articles that have used poly(hydroxybutyrate-cohydroxyvalerate) (PHBV), employed the oil-in-water (O/W) method to obtain polymeric spherical particles. However, other articles in the literature have used SLS to solely process PHBV without further particle modification [47], [48].

Attrition forces present during the milling process can significantly increase powder's temperature, therefore, polymers with a low melting point may benefit from cryogenic milling such as PCL [22], [49]- [55]. As well as to prevent temperature increasing, liquid nitrogen was used during the milling of PA12/HA pellets compounded by a twin-screw extruder [56].

Although the milling process may not provide homogenous spherical particles (Figure 2b), it remains the most convenient and fast method for particle size reduction. As a result, we have observed that the most common process used to achieve polymer desirable particle size was milling followed by sieving.

Towards polymer and Ca-P powder mixture procedure, two principal methods were identified: physical blending and solvent evaporation. Table 2 contains information related to employed materials, mixture methods and their sub-types. Concerning physical blending, several authors did not provide enough details while describing the method, for these cases, we have considered the sub-type as "general".

Among physical blending, general mixing is predominant, probably due to its simplicity, equipment accessibility and efficacy, representing around 49% of all preparation methods. Secondly, the solid-in-oil-in-water (S/O/W) emulsion solvent evaporation process represented 14% of the utilized methods. Wet methods (around 12%) consist in diluting and mixing the particles using a liquid carrier, the most used liquid was ethanol. The remaining methods used physical blending and were categorized as detailed by authors (i.e., use of extrusion compound, rotatory tumbler, V or Y type mixer).

Composite mixture Type of blending Sub-type Sources

PMMA + TCP Physical blending General [57], [58] PCL + TCP Physical blending General [49], [52], [54], [59], [60] Rotary tumbler [61], [62] PCL + HA Solvent evaporation S/O/W [21], [37], [63] Physical blending General [51], [55], [64]- [66] V-type mixer [22], [53] Rotary tumbler [67] PDLA + TCP Physical blending Wet method [START_REF] Gayer | Influence of the material properties of a poly(D,L-lactide)/β-tricalcium phosphate composite on the processability by selective laser sintering[END_REF] PDLA + HA Solvent evaporation S/O/W [START_REF] Lin | Selective laser sintered nano-HA/PDLLA composite microspheres for bone scaffolds applications[END_REF] Physical blending General [31], [33] PLLA + TCP Physical blending General [34], [START_REF] Liu | Mechanical properties' improvement of a tricalcium phosphate scaffold with poly-L-lactic acid in selective laser sintering[END_REF]- [START_REF] Shuai | Inhibition of phase transformation from β-to αtricalcium phosphate with addition of poly (L-lactic acid) in selective laser sintering[END_REF] PLLA + HA Physical blending Wet method [35], [36] General [START_REF] Woźna | The influence of different composite mixtures (PLA/HA) manufactured with additive laser technology on the ability of S. aureus and P. aeruginosa to form biofilms[END_REF], [START_REF] Zhang | Development of porous scaffolds using selective laser sintering of polylactic acid/ hydroxyapatite for bone tissur engineering[END_REF] Physical blending Supercritical CO2 [START_REF] Antonov | Formation of a bioactive material for selective laser sintering from a mixture of powdered polylactide and nano-hydroxyapatite in supercritical carbon dioxide[END_REF] PLLA + HA + MET Physical blending Wet method [36] PLLA + CHA Solvent evaporation S/O/W [START_REF] Duan | Three-dimensional nanocomposite scaffolds fabricated via selective laser sintering for bone tissue engineering[END_REF] Physical blending Rotary tumbler [START_REF] You | Selective Laser Sintering of Poly(L-Lactide)/Carbonated Hydroxyapatite Nanocomposite Porous Scaffolds for Bone Tissue Engineering[END_REF] SBA + HA + PLLA Physical blending Wet method [38] pDA + HA + PLLA Physical blending Wet method [START_REF] Feng | In Situ Generation of Hydroxyapatite on Biopolymer Particles for Fabrication of Bone Scaffolds Owning Bioactivity[END_REF] PLGA + HA Physical blending General [START_REF] Shuai | Development of composite porous scaffolds based on poly(lactide-co-glycolide)/nano-hydroxyapatite via selective laser sintering[END_REF] PLGA + HA + TCP Physical blending General [26] PEEK + HA Physical blending General [32], [START_REF] Tan | Fabrication and characterization of three-dimensional poly(ether-ether-ketone)/-hydroxyapatite biocomposite scaffolds using laser sintering[END_REF] Roller mixer [START_REF] Tan | Scaffold development using selective laser sintering of polyetheretherketonehydroxyapatite biocomposite blends[END_REF] PEEK + TCP + PLLA Physical blending Wet method [START_REF] Feng | A Multimaterial Scaffold With Tunable Properties: Toward Bone Tissue Repair[END_REF] PEEK + PGA + HA Physical blending Wet method [START_REF] Shuai | Characterization and bioactivity evaluation of (polyetheretherketone/polyglycolicacid)-hydroyapatite scaffolds for tissue regeneration[END_REF] PA + HA Physical blending General [17], [29], [START_REF] Ramu | Optimization of the configuration of porous bone scaffolds made of Polyamide/Hydroxyapatite composites using Selective Laser Sintering for tissue engineering applications[END_REF] Extrusion [56] Y-mixer [START_REF] Dabbas | Viscoelastic Properties and Creep-Fatigue Behavior of PA2200/HA Composites Manufactured by Selective Laser Sintering[END_REF], [START_REF] Dabbas | Selective Laser Sintering of Polyamide/Hydroxyapatite Scaffolds[END_REF] a-PC + HA Physical blending Planet ball [28] HDPE + HA Physical blending Y-mixer [START_REF] Salmoria | Functional graded scaffold of HDPE/HA prepared by selective laser sintering: Microstructure and mechanical properties[END_REF] Extrusion [42], [START_REF] Hao | Rapid manufacturing of bioceramic/polymer composite implants by selective laser sintering[END_REF] PHBV + Ca-P Solvent evaporation S/O/W [40], [START_REF] Duan | Three-dimensional nanocomposite scaffolds fabricated via selective laser sintering for bone tissue engineering[END_REF], [START_REF] Duan | Surface modification of threedimensional Ca-P/PHBV nanocomposite scaffolds by physical entrapment of gelatin and its in vitro biological evaluation[END_REF]- [START_REF] Duan | Optimized fabrication of Ca-P/PHBV nanocomposite scaffolds via selective laser sintering for bone tissue engineering[END_REF] EP + HA + TCP Physical blending General [25] EP + PA + TCP Physical blending General [START_REF] Liulan | Design and Fabrication of Bone Tissue Engineering Scaffolds via Rapid Prototyping and CAD[END_REF] EAM + HA Physical blending Wet method [START_REF] Zhang | Computer-aided design and computeraided manufacturing hydroxyapatite/epoxide acrylate maleic compound construction for craniomaxillofacial bone defects[END_REF] Our findings have also revealed other articles that have used a small amount of polymer in a ceramic matrix. In one of these articles, the effect on scaffold's mechanical properties when adding 0.5 to 3 wt% of PLLA on a β-TCP matrix was investigated by Liu et al. [START_REF] Liu | Mechanical properties' improvement of a tricalcium phosphate scaffold with poly-L-lactic acid in selective laser sintering[END_REF]. Their findings revealed that the addition of 1 wt% has improved values of compressive strength and fracture toughness, as a consequence of β to α-TCP phase transformation inhibition. The occurrence of this phenomenon, using 1 wt% PLLA, was further studied by Shuai et al. [START_REF] Shuai | Inhibition of phase transformation from β-to αtricalcium phosphate with addition of poly (L-lactic acid) in selective laser sintering[END_REF].

In contrast, unsuccessful processing of TCP particles coated with a thin film of PLA polymer was reported by Antonov et al. [34].

The majority of authors have used pure polymer for comparison purposes and to outline SLS processing parameters. Properties contrasts between pure polymer and Ca-P filled scaffolds are often used for benchmark and performance correlation. Although the use of pure polymer can provide valuable information for processing parameters window, Ca-P filler addition might influence the optimal SLS parameters which have a great influence on the scaffold's final properties.

Processing parameters and characteristics of fabricated specimens

Each powder material has its intrinsic characteristics, requiring fine adjustment of SLS processing parameters for adequate properties. Changes such as type of SLS machine, type of composite powder, particle size and morphology, might imply better tuning of processing parameters. This should be considered for processing parameters selection and comparison with the related bibliography.

A summary of the reviewed articles is displayed in Table 3, which gathers information on polymer/Ca-P combination, type of mixture, SLS equipment and processing parameters. Some authors did not provide enough data in their manuscripts to allow the completion all the related information.

Table 3 contains all the polymer/Ca-P compositions and proportions that were tested by the authors, although not all proportions were satisfactorily processed and had favourable properties. Likewise, not all processing parameters values indicated by authors were suitable, occurrence of polymer degradation or low sintering degree was often reported by authors until optimal values were found. When a wide range of parameters was tested, authors were able to better investigate its influence on scaffold's densification, mechanical properties and bioactivity. Our findings also revealed a few publications have used directly optimal processing parameters that were established in previous works.

The energy density (ED) indicates the amount of energy provided that reaches the bulk material and it can be represented by Energy density = Laser power/(Scan speed x Scan spacing) [START_REF] Williams | Advances in modeling the effects of selected parameters on the SLS process[END_REF]. Although ED information can be helpful while comparing processing parameters, only 14% of the authors have explicit values in the manuscript. On the other hand, 17% of the reviewed articles have provided less than 2 parameters information displayed in Table 3.

Omitting processing parameters information might be due to secure sensitive information, however, it imposes restrictions for a benchmark with other articles.

In terms of equipment, not every SLS machine was equipped with a powder bed heater (which allows controlling the powder bed temperature), this feature is highly recommended to minimize gradient temperature and avoid curling and shrinkage on the fabricated pieces.

Another important feature, but hardly used due to high costs, is a sealed chamber with an inert atmosphere that minimize polymer degradation during processing.

One important factor, that not every author has provided, is the layer thickness used for the sample's fabrication. The layer thickness must be in accordance with powder's particle size, for example, when using an average particle size of 150 µm, the minimal value for layer thickness must be 150 µm. Thereby, each layer is filled with at least 1 particle in height, this will guarantee proper particle's cohesion, better densification and more homogeneous fabricated layers.

Customized implant devices may have several geometry variations for an appropriate anatomical fit, whereas the type of material, pore size and scaffold design must be selected accordingly with implant location and the type of tissue to be restored [START_REF] Qu | Biomaterials for bone tissue engineering scaffolds: A review[END_REF]. According to Yang et al. [START_REF] Yang | The design of scaffolds for use in tissue engineering. Part I. Traditional factors[END_REF] cell's diameter dictates the minimum pore size to promote bone regeneration, scaffold's suitable pore size is in the range of 100-350 µm. Moreover, it is known that geometries with a controlled interconnected porous structure have great importance and are desirable for bone tissue engineering applications, in this sense, authors generally fabricate porous samples, but varying their architecture.

The fabricated specimens were generally cylindric or rectangular, probably due to enable mechanical properties measurement. A few authors have used ASTM D695 and D638 standards for compressive and ensile testing. For biological analysis, it was common to produce porous/solid cylindrical disks within the range of 5 mm to 15 mm diameter and 0.6 mm to 5 mm height. From our research, it was possible to observe that articles published by the same group/authors mostly have fabricated the same geometry, possibly for results comparison.

Fabrication of personalized implants (i.e., Figure 5 left) is one of the purposes of using AM techniques, allowing to better treat large bone defects as reported by Zhang et al. [START_REF] Zhang | Computer-aided design and computeraided manufacturing hydroxyapatite/epoxide acrylate maleic compound construction for craniomaxillofacial bone defects[END_REF]. A wide range of fabricated geometries was observed between the reviewed articles, demonstrating the versatility of SLS processing.

Certain authors have reported the occurrence of contraction/distortion on the fabricated pieces during the SLS process, affecting the overall quality of the scaffolds. To address that, a solid base was incorporated for the scaffold (see Figure 5) [40], [START_REF] Duan | Three-dimensional nanocomposite scaffolds fabricated via selective laser sintering for bone tissue engineering[END_REF], [START_REF] You | Selective Laser Sintering of Poly(L-Lactide)/Carbonated Hydroxyapatite Nanocomposite Porous Scaffolds for Bone Tissue Engineering[END_REF], [START_REF] Zhou | Selective laser sintering of References[END_REF]. This phenomenon was not mentioned by Du et al. [21] while fabricating PCL and HA scaffolds with similar dimensions. Biocompatibility is essential for implantable devices, however, essays related to this aspect were only performed in a small majority (51%) of analyzed articles. Bioactivity analysis is often costly and needs expertise for obtaining reliable results, this might be one of the reasons for only almost half of the reviewed articles to employ these analyses. Among them, only 5 articles (8%) have performed in vivo implantation, in which, two have implanted scaffolds in rabbits [63], [START_REF] Feng | In Situ Generation of Hydroxyapatite on Biopolymer Particles for Fabrication of Bone Scaffolds Owning Bioactivity[END_REF], one in sheep [54] and one in humans [64].

Animal in vivo study is an important phase for data assessment for further human trials.

Animal implantation studies must be conducted following up-to-date legal provisions, guarantee animal welfare and also have approval of the Animal Experimental Ethics Committee. Within the reviewed articles, two types of animals were used: rabbits and sheep Du et al. [63] had successful subchondral bone regeneration with osteochondral repair in rabbits, while Feng et al. [START_REF] Feng | In Situ Generation of Hydroxyapatite on Biopolymer Particles for Fabrication of Bone Scaffolds Owning Bioactivity[END_REF] stated that the implanted scaffold (pDA/HA/PLLA) have significantly promoted new bone tissue formation, repairing the created bone defect on rabbit's radial bone. Lohfeld et al. [54] have conducted in vivo study using mountain sheep, a critical size 20 mm cylindrical defect was created in the middle of the diaphysis of the right tibia.

PCL/TCP scaffold was placed in the created bone defect, findings revealed promotion of bone regeneration, scaffold degradation and also undesired inflammatory reaction.

Human scaffold implantation and experiments must follow legal and regulatory requirements, therefore most published articles did not conduct them. In the case of Rasperini et al. [64] a large periodontal osseous defect was treated using 96% PCL + 4% HA scaffolds, although authors stated that this case was unsuccessful in the long term, they foresee advancements related to this field of interest. Zhang et al. [START_REF] Zhang | Computer-aided design and computeraided manufacturing hydroxyapatite/epoxide acrylate maleic compound construction for craniomaxillofacial bone defects[END_REF] have appointed postsurgical infection when implanting 78% HA + 22% EAM scaffolds for treating various complex irregular craniomaxillofacial defects. Results showed that HA/EAM scaffold adhered to the defect area, not showing migration and no foreign body rejection was noticed.

In vitro experiments do not require Ethics Committee approval and provides valuable information to asset the biological response of the fabricated devices. From the reviewed articles, most authors have used commercially available kits to analyze cell culture, viability, proliferation, seeding and attachment. As expected, a greater content of Ca-P material resulted in greater bioactivity. An augmented carbonate apatite layer in the sample's surface was generally cited by authors moreover, the presence of PO4 3-and CO3 2-functional groups were also identified. When samples' weight loss was measured, near-linear weight loss behaviour along time was observed. Finally, the use of additives has demonstrated better results in terms of biocompatibility and osteogenic differentiation.

Conclusion

Polymer-CaP scaffolds produced by SLS have shown to be a viable solution for bone tissue regeneration applications. SLS processing allows producing complex and functionalized scaffolds, addressing new possibilities for customized implants. However, taking into consideration that only 63 articles were selected to be reviewed from the 650 articles found in the database, it demonstrates how incipient is the combination of polymer/Ca-P for scaffolds fabrication.

Among the materials used, PCL and PLLA, HA and TCP were the major selection. A few articles have used more than 3 materials in the composite mixture, mainly to add beneficial properties such as increase processability or functionalization. The use of certain additives can improve material's sinterability as well as bring supplementary properties for scaffolds, for example, to promote bioactivity.

Selection of mixing technique is important to maintain powder's homogeneity and also must take into account material's properties to avoid degradation. SLS processing parameters have shown great influence on scaffold's properties, therefore, the use of DOE and statistical tools are recommended. Home-made and commercial SLS equipment may have significant differences in performance, but both can achieve complex geometries in the fabricated devices.

Biocompatibility essays are fundamental for achieving better bone regeneration, however, these essays were not applied in many of the reviewed articles.

Advancements in bulk material preparation, use of additives, scaffold's geometry, as well as processing parameters optimization, may bring important advancements for polymer/Ca-P implantable devices.

Conclusions of Chapter 2

The preceding review article has provided substantial information in terms of SLS processing of polymer/Ca-P. Therefore, the outcomes and observations from the authors were considered for the further steps of the present work.

Physical powder mixture has shown to be effective and propitious for homogeneous mixing of the composite powders, thus, it was chosen as a method for mixing UHMWPE/SDHA powders. No concense was found regarding the geometry of fabricated samples, most authors have used rectangular and cubic forms. The selected geometries for the present work considered former dimensions already employed in our research group. Cylindrical forms were selected due to the better fitting inside cell-culture dishes.

CO2 laser source was proven to be suitable for polymer/Ca-P composites processing accounting for the majority of users. The use of commercially available SLS machines have the advantage of not requiring hardware/software expertise for building it, however, the self-made machines are more cost-effective. This work was conducted using an experimental selfdeveloped SLS machine, available at NIMMA (UFSC) laboratory facilities.

Finally, the choice of HA from the available calcium phosphate materials is well endorsed by the available publications. TCP was also widely employed in the reviewed articles, but its faster degradability and absorption within the body, its use should be in accordance with new bone growth and formation to avoid poor local stability. The lack of UHMWPE employment as a polymer of choice might be justified because of the challenges towards SLS processing.

The combination of UHMWPE and HA particles were not revealed among the selected keywords and database. Therefore, with the purpose to investigate the state of the art and related bibliography of SLS processing of UHMWPE and HA, the following keyworks were searched using Google Scholar: UHMWPE, hydroxyapatite, "selective laser sintering".

Among the 339 results revealed, only 10 met the following selection criteria: original research articles that were processed by SLS, using UHMWPE (with or without HA) powder as feedstock for fabricating implantable devices. Among the selected 10 articles, none of them has used the combination of UHMWPE and HA or SDHA, revealing a research opportunity of these widely used biomaterials.

CHAPTER 3

Manuscript 2

Introduction to Chapter 3

The following chapter contains experiments conducted at IRCER (Institut de Recherche sur les Céramiques), being the first experimental published article. It describes the method employed for investigating the adequacy of the spray drying method for producing HA particles suitable for SLS processing. Synthesized HA particles have a needle-like morphology and tend to agglomerate, deleterious characteristics for the SLS processing. Ideally, the HA particles added into the UHMWPE matrix would be spherical and with a size compatible to occupy voids among polymeric particles. The spray drying technique showed the potential to produce HA spherical particles with a mean diameter in the range of 15 to 25 µm. Further, this technique is scalable and can be used by industry.

A large number of experiments were conducted to achieve the summarized results

shown in the article. This methodology allowed a better understanding of the spray drying machine and its particularities. Understanding machine limitations and performance have great importance for larger powder production.

Introduction

Customized fabricated parts are required in a wide range of applications, being particularly important in medicine. The feasibility of manufacturing complex geometries, combining different materials, matches the requirements of implantable devices used for human tissue recovery. Tissue engineering (TE) seeks to restore, maintain, or improve damaged tissues through the combination of scaffolds, cells, and biologically active molecules into functional tissues [1].

Human bone is a complex vascularized structure, composed of organic collagen fibrils and inorganic calcium phosphate (CaP) crystals [2]. CaP materials [3,4] such as hydroxyapatite (HA) and beta-tricalcium phosphate (β-TCP) are widely used as bone substitutes and are commercially found in a few different geometries (e.g., granules and sticks). Great interest is found on synthetic HA due to its strong affinity to hard tissues, also, HA is the most stable Ca-P compound in physiological conditions [5]. However, the fabrication of customized scaffolds from these materials remains a challenge, especially in terms of patient's implant fitting.

Additive manufacturing (AM) is a breakthrough technology that allowed significant progress for tissue engineering applications, and still reveals promising solutions for custom-made bone scaffolds [6][7][8][9].

Powder bed fusion, frequently referred to as selective laser sintering (SLS) is one of the commercially available AM processes [10]. In this process, particulate materials are fused layer by layer via heat supplied by an infrared laser source, creating 3D parts that were originally designed using computer-assisted design (CAD) tools. SLS does not need any support during manufacturing and presents high resolution and fast processing. On the other hand, SLS is carried out at high processing temperatures and the manufactured parts are characterized by a rough surface finish [11]. Nevertheless, SLS can be considered one of the most versatile AM techniques in terms of material usage and structural stability. Moreover, through optimized parameters, it is possible to achieve the desired mechanical properties for TE scaffolds [12].

SLS processing with proper materials selection can contribute to enhancing the scaffold's final properties, particularly bioactivity [13,14]. When processing composite materials (i.e., bioceramic fillers in a polymeric matrix) a substantial difference between material's particle size is beneficial, allowing the filler to occupy voids in the interstices of matrix particles.

Concerning polymer SLS feedstock, there are preferable powder characteristics that improve the sinterability and final properties of the fabricated piece [15,16]. Related literature has indicated better processing and geometry accuracy when using spherical particles and narrow particle size distribution with an average size below 150 µm or equivalent to the laser beam diameter [15,17]. Spherical HA particles can be obtained by different techniques [18][19][20][21][22][23][24] in a diverse range of particle sizes. However, process scalability remains a challenge and not all techniques are capable of producing particles in the range of 15 to 25 µm.

Spray drying (SD) consists of the transformation of a fluid material into dried particles.

SD had shown remarkable development in the last decades, being used by different industries [25]. SD commercial equipment may differ in terms of configuration. Rotary, hydraulic and pneumatic nozzle atomizers are commonly used [15]. The variables that affect how the spray is mixed with the hot gas depend upon the type of gas flow: co-current, countercurrent, or mixed flow [26]. Spray-dried HA (SDHA) powder has been successfully employed for distinct biomedical applications, using different manufacturing techniques [27][28][29][30][31][32]. Moreover, SD enables advantageous powder characteristics that are desirable for 3D printing use [25].

Although SDHA powder morphology indicates suitability for SLS processing, publications concerning this subject are scarce [33]. Similarly, comprehensive information about how to produce SDHA powders for SLS processing is rarely reported in the literature. This paper proposes a screening method for producing suitable HA powders for SLS processing.

Materials and methods

Materials and compositions

Suspensions to be spray-dried were prepared with nano-sized HA synthesized in-house by hydrothermal route. Synthesis further details can be found elsewhere [34]. Methylcellulose (Methocel A15 Premium LV, Dow Chemical) was added as a binder to form an aqueous suspension using deionized water, according to the compositions in Table 1.

Spray drying parameters

A mini spray drying machine (Buchi B-290) was used. As displayed in Figure 1, the slurry mixture is pumped through a pneumatic external mixing nozzle (2) and sprayed by the spray gas (1) into the drying chamber (4). The drying gas (3) is heated and serves as a carrier for the spray-dried particles that will be deposited in the recipient (5) under the cyclone (6).

Larger particles that were not carried by the drying gas can be recuperated under the drying chamber. Smaller particles will be retained in the filter (7).

Some spray drying parameters were kept constant in all experiments: inlet temperature of hot air 170 °C, volume flow of hot air aspiration 39 m³ h-1, slurry feed 21 mL.min-1, and atomization flow 45 mmHg. The intention is to use spray dried HA particles as a filler in a polymeric matrix. In this sense, better homogeneity and cohesion within materials can be achieved when HA particles are smaller and can be located in the voids of polymeric particles. Therefore, to obtain spherical HA particles, possessing an average diameter of 15 to 25 µm and narrow particle size distribution, two SD parameters were varied slurry feed and atomization pressure. A factorial design experiment with two factors and two levels was carried out (Table 2). The optimal point among the experimental results originated from factorial design was determined using a desirability function. Results were ranked by preferable attributes taking into consideration the D50 value, (D90-D10)/D50 and particle morphology.

Characterization of powder and suspension

Morphological analysis was performed using a scanning electron microscope (SEM, JEOL IT300LV), after coating the samples with platinum (Agar Scientific). Departure HA powder was analyzed, without coating, using a field emission gun microscope (FEI, Quanta to experimental data. After spray drying, the sprayed powder was submitted to thermal treatment using ramps of 3 °C/min up to 500 °C, for binder removal, and 10 °C/min up to 1000 °C, with a hold at this temperature for 1 h, for densification (LHT 04/17, Nabertherm GmbH).

Results and discussion

Raw powder characteristics

The synthesized HA powder had a specific surface area of around 80 m² g-1. As a means to reduce its reactivity, the powder was heat-treated at 650 °C for 30 min resulting in a surface area of 34 m² g-1. This heat treatment allows eliminating synthesis residues (i.e., nitrate and ammonium ions), providing a homogeneous specific surface and reproducible characteristics of the departing powder. Further, it contributes to a more stable suspension and minimizing agglomeration between HA particles. Heat treatment and SD processing did not affect the phase compositions, being the same as pure HA according to diffraction pattern (Figure 2). As revealed on SEM images (Figure 3) and PSD curve (Figure 4) the nano-sized HA particles (D50 value 0.13 µm) tend to agglomerate; therefore, the suspension for SD processing must be carefully prepared.

Effect of suspension formulation

Suspension composition has major importance because its characteristics will influence the spray drying process as a whole. For this step, the trial and error experimentation method is applicable when no information is found in the literature. However, there are aspects of fundamental importance to be considered (e.g., maximal solid content, slurry viscosity) that can be easily found in the related bibliography. Moreover, machine manufacturers can provide elementary information about recommended processing characteristics. Early trial and error experiments, together with literature information, have provided a point of departure in terms of mixture preparation, solid content, binder selection, and processing parameter values.

First, the binder selection must consider the powder's final application (in this case, bone tissue applications). Therefore, after processing, it must maintain the original HA chemical composition. In previous essays, the performance of different binders: corn starch (Roquette, ref: 764071), polyvinylpyrrolidone (grade 30 and 90 from BASF), and methylcellulose (Methocel A15-LV Premium) were analyzed. Methylcellulose demonstrated a larger average particle size, better process efficiency, and requiring lower concentrations.

Spray-dried HA (SDHA) powder, after heat treatment at 1000 °C for 1 h, has matching phase compositions to heat-treated HA as shown in Figure 2, confirming the suitability of methylcellulose in terms of not modifying powder composition.

Once the type of binder is selected, the amount of HA must be defined. Adding too much HA in the suspension will prevent good homogeneity and induce solid deposition.

Moreover, it will increase viscosity and difficult spray drying processing (i.e., nozzle blocking).

On the other hand, a low solid concentration will result in smaller particle size [36]. Previous essays indicated the use of around 20 wt% HA content for the proposed screening method and particle size objectives. To guarantee suspension homogeneity, it is recommended to agitate with a magnetic stirrer for at least 10 h before starting the spray drying process. While processing, continuously stirring is required to maintain the mixture uniform and with constant viscosity [37].

SD process is highly dependent on the suspension's characteristics and its viscosity has a great influence on the obtained powder. As detailed in Figure 5, the amount of methylcellulose has a large effect on the suspension's rheology. Table 2 provides the values obtained from the slurry rheological analysis, Herschel-Bulkley model was used given its fitting with experimental results. Yield stress values were similar between SD01-SD02 and SD03-SD04, indicating two types of behaviour according to binder amount. All the suspensions showed pseudoplastic shears thinning behaviour, implicating in a viscosity decrease with increasing shear rate. This is an important characteristic that guarantees suspension uniform viscosity along with all the SD processing, under constant agitation. Large viscous forces will need more energy for breaking the droplets, resulting in larger droplets [38]. This phenomenon was observed when processing SD01, where occurred slurry droplets deposition in the drying chamber.

PSD analyses were performed for all suspensions (Figure 6). SEM images (Figure 7) suggest that the high amount of methylcellulose binder in suspensions SD01 and SD02 formed binder agglomerates in the particles, causing defects after sintering that can be observed by the empty spaces in the particles (forming doughnut-like particles). Contrastingly, heat-treated SD04 powder presented some particles with an insufficient amount of binder, preventing proper cohesion between HA departing particles (also evidenced by smaller values on PSD) and restricting the formation of appropriate morphology.

According to rheological analysis and PSD values, SD03 and SD04 have shown the most suitable results. When also considering morphological characteristics of spray-dried granules, SD04 did not have a sufficient amount of binder to guarantee proper cohesion of nanosized HA particles, therefore, SD03 was chosen to proceed with the following steps.

Effect of spray drying parameters

Nozzle cap position is important to avoid excessive droplets deposition on the drying chamber wall, which can result in lower process efficiency. In order to verify any influence of spray width on the particle's morphology, particle measurement and SEM images were conducted. Particle size distribution (Figure 8) and morphology have shown to be similar between the nozzle's four positions (SD05 to SD08), hence, these attributes were not directly affected by the changes in the spray angles, neither they were sufficient to validate an optimal nozzle position.

Considering the similarity in morphology and particle size distribution, position II was chosen because SD06 had around 5% more process yield (powder recover quantity) when compared to the others. The final properties of spray-dried particles are highly dependent on processing parameters, as well as equipment's features and configurations. Although it is possible to adjust particle characteristics by changing the processing parameters, it is necessary to establish a suitable departing suspension to achieve the targeted goals in terms of particle. Afterwards, the heat treatment process will affect the particle density, crystallinity, and mechanical integrity [39]. Considering that heat treatment is a post-process of spray drying, not directly influencing the particle size distribution and morphology, it will not be investigated here. In terms of particle-size distribution and process yield, key parameters are suspension's solid content, spray drying slurry feed, and atomization pressure; yet, other factors like nozzle diameter, inlet temperature, aspirator velocity, and drying gas humidity also influence the obtained particles [26,39,40]. As mentioned before, an early trial and error method should be applied to comprehend the basic correlations between all the processing factors. In-depth analysis for investigating the influence of different processing variables should be done using statistical tools and it is recommended to perform at least 3 replicates for establishing any conclusions. Particle size information is displayed in Figure 8 and Table 3. It has been reported in the related bibliography that a higher atomization pressure decreases the particle size [36,39,41].

This observation is in accordance with our results, when comparing SD09 and SD11 (same slurry feed and SD11 with higher atomization pressure), SD11 possesses lower particle values for D10 and D50. In the same manner, SD12 has D10, D50, and D90 particle sizes lower than SD10 (same slurry feed and SD12 with higher atomization pressure). On the other hand, from our results, it was not possible to observe the association between larger particle sizes with increases in slurry feed rate reported in the literature [39,41]. Analyzing the curves in Figure 9, it can be observed similarities between SD09-SD10 and SD11-SD12, this might indicate a minor influence of slurry feed in our experiences. Probably, a greater difference between slurry feed values would evidence variances in particle size. Nevertheless, it is important to highlight that these comparisons are only illustrative, for representative conclusions it would be necessary more replicates, experimental error determination, and the use of statistical analysis of variance (ANOVA). SEM images (Figure 10) have shown similar spherical morphology for the respecting spray-dried powder and, as expected, a greater population of bigger particles were observed on SD09 and SD10. Introducing satisfaction index for the responses D50, (D90-D10)/D50 and particle morphology, desirability values were obtained (Table 4). Individual desirability of D50 and morphology were calculated using 𝒟 = (𝑌𝑖 -𝐿)/(𝑇 -𝐿) equation, where Yi is the response, L the lower value, and T the target value. Conversely, for (D90-D10)/D50 desirability a minimized result was targeted, therefore, 𝒟 = (𝑈 -𝑌𝑖)/(𝑈 -𝑇) equation was used (U means upper value). The applied ranges of values were: i) D50: 100% desirability if value greater than 25 and 0% desirability if value less than 15; ii) (D90-D10)/D50: 100% desirability if value less than 1 and 0% desirability if value greater than 3; iii) particle morphology: 100% desirability if value greater than 5, 0% desirability if value less than 1. For particle morphology, SEM images were analyzed and for each sample, it was given a 1 to 5 score (5 is the most suitable morphology). Higher values of desirability were observed on SD09 and SD10 mainly due to D50 and (D90-D10)/D50.

Although global desirability pointed out SD09 and SD10 as the most suitable powder characteristics, all SD powders achieved the proposed morphology and size objectives. The variation of two processing parameters has shown the capability of adjusting particle size characteristics.

Conclusions

This screening method contemplates the main steps for powder preparation suitable for SLS. Spray-dried HA particles were successfully produced within the proposed morphology.

Binder and HA content of the departing slurry have an important effect on viscosity. Therefore, special attention must be given to preparing the departing suspension. Variations on spray drying processing parameters have shown the possibility of tailoring particle size and distribution. For a proper analysis of the processing parameters variation, statistical tools must be used. PSD and SEM images have indicated suitable particle characteristics for SLS processing, although additional studies must be conducted to confirm SLS processability. The

Conclusions of Chapter 3

Results achieved in the published article were satisfactory in terms of particle size and morphology. However, deficiency in terms of powder production (low process efficiency) has motivated deeper research for improvement. In this sense, different processing parameters values were evaluated to achieve greater spray-dried powder production.

Table 5 displays the employed parameters to attempt higher levels of efficiency, in this occasion 3 replicates were performed, all using the favourable suspension formulation (20.32%

of HA, 1.63% of MC and 78.05% of H2O) and nozzle position. The fixed parameters were airflow temperature of 170 o C and aspiration at 100%, suspensions were prepared following the same procedure and agitation time. Results have shown that two parameters combination with favourable efficiency: i) slurry feed of 50% and an atomization pressure of 60 mmHg and ii) slurry feed of 30% and an atomization pressure of 60 mmHg, the respective rounds are highlighted in the table in light green (i) and darker green (ii). It can be noticed that i) possess greater D50 and (D90-10)/D50 values, along with lower efficiency, therefore, the chosen parameters for producing SDHA for the SLS experiments were ii) slurry feed of 30% and an atomization pressure of 60 mmHg.

Once the parameters were selected, a greater amount of prepared suspension was spraydried for producing SDHA to use with UHMWPE in the SLS machine. When processing larger amounts of suspension, it was possible to achieve higher amounts (up to 44%) of process efficiency, this can be attributed to inherent process loss that is more evidenced when processing lower amounts of suspension.

The collected SDHA powder was also submitted to thermal treatment, before SLS processing, using ramps of 3 °C/min up to 500 °C, for binder removal, and 10 °C/min up to 1000 °C, holding this temperature for 1 h. This heat treatment was chosen to slowly remove the binder in the first ramp (avoid morphological changes in the particles). The second ramp was intended to enhance particle densification and, at the same time, to prevent HA phase change (that occurs above 1100 °C).

CHAPTER 4 Manuscript 3

Introduction to Chapter 4

The following chapter (to be submitted to publication) consisted in the final part of the impeding homogeneous powder spreading of the following layer, causing critical defects in the sample. Limitations on the lens system (galvanometric mirror system) were also observed, whereas laser speed greater than 1,000 mm/s fails to keep pace with the designed piece geometry.

Visual inspection and manual handling were used to assess samples' fabrication while varying the processing parameters. This phase was essential for better knowledge for SLS processing UHMWPE powder, enabling to establish a departure point in terms of processing parameters.

Introduction

Additive manufacturing (AM) techniques introduced significant advancements for tissue engineering applications and it is still revealing favourable solutions for customized bone scaffolds [1][2][3][4]. Selective laser sintering (SLS), also called as powder bed fusion, [5] is a powerbased AM technique that uses a laser beam as a heating source to fuse particles layer by layer to create 3D parts that were conceived using computed assisted design (CAD) tools. SLS is versatile in terms of structural stability and material usage, but spherical particles and good flowability characteristics are preferable for suitable processing [6]. Besides the wide use of metallic materials, the combination of biomaterials as feedstock for SLS has been intensively researched due to its interest in bone tissue applications [7][8][9]. Moreover, through optimized parameters, it is possible to achieve the desired mechanical properties for tissue engineering scaffolds [10].

Bone is a complex tissue with intricate hierarchical architectures, composed of organic collagen fibrils and inorganic calcium phosphate (CaP) crystals [11,12]. According to the bone location in the skeleton, functions and mechanical requirements will change; therefore, its characteristics and geometry vary accordingly.

For proper bone tissue treatment and recovery, it is highly desired that the applied materials induce osteoconduction and osteoinduction [13]. Osteoconduction means that the material surface is able to support the adhesion, proliferation and differentiation of bone cells.

On the other hand, osteoinduction implies releasing molecular inducers such as growth factors and cytokines for mesenchymal precursors to differentiate into the osteoblastic lineage. Either osseointegration is highly desirable to guarantee bone-to-implant anchorage and it is particularly important for oral applications [14]. The bioceramics, more specifically calcium phosphates (CaPs), have been widely used as bone substitutes due to compositional similarities and excellent biocompatibility [15]. More recently, these materials also gained attention in view of their versatility and tailored attributes, allowing the employment of growth factors and drug delivery systems [16]. Among the available CaPs, hydroxyapatite (HA) and tricalcium phosphate (TCP) are the most commonly used in bone tissue engineering [13]. HA [Ca10(PO4)6(OH)2] has a proven track record associated with bone tissue and dental applications, being commercially available in different shapes such as powder, granules and sticks. Its chemical and structural properties, similar to the natural bone, minimize body rejection of implanted devices and promotes bone formation through osteoconduction. The difficulty of processing and inherent brittleness restrain applications with bioceramics as medical devices [17]. Thus, adding polymers while processing bioceramics by AM techniques is beneficial, especially in terms of providing supplementary characteristics to the fabricated bioproducts [18].

UHMWPE is a biocompatible polymer, widely used for implantable devices and it is being particularly important for bearing replacement in the knee, hip and shoulder joints. Its powder is produced using the Ziegler process [19] and the requirements for medical-grade purity are specified in ASTM F648 [20] and ISO standard 5834 [21]. To consolidate UHMWPE into different forms, elevated temperatures and pressures are necessary due to its high melt viscosity. Therefore, processing techniques like injection moulding and screw extrusion are not practical [22]. Hence, UHMWPE devices are mainly obtained by machining, which implies size and shape limitations. SLS provided advancements related to devices for tissue engineering. Its versatility allows the processing of different kinds of materials and composites, including UHMWPE. Rimell and Marquis [23] reported remarkable shrinking while producing sheetlike UHMWPE structures, and suggested the improvement of starting powders. When optimizing processing parameters, as well as providing accurate preheating to the feedstock powder, Song et al. [24] effectively produced UHMWPE sample parts. Kalil et al. [25] manufactured rectangular samples in which length and width shrinking was evident, also mentioning dimensional inaccuracy as a major challenge. Fumed silica particles as an anticaking agent were applied in UHMWPE in order to reduce sintering difficulties; however, mechanical properties decreased [26]. Thus, research is needed to enhance UHMWPE processability and properties of the fabricated devices.

UHMWPE/HA composites processed by SLS are poorly investigated in the literature, possibly due to the processing complexity. While SLS has the capability of processing powder materials, whereas UHMWPE can provide the strength and structure, HA can provide the osteoconductivity. Finally, the powder preparation for fulfilling SLS feedstock requirements might provide suitable processability. Production of rounded HA particles can be performed through different techniques [27][28][29][30][31][32][33]. Essentially, spray drying (SD) is a process that transforms fluid feedstock into dried particles, being employed in different kinds of industry. This process is scalable and capable of producing spherical granules with adaptive size, according to the hardware setup [34]. Further, spray-dried HA (SDHA) has been effectively employed in manufacturing processes for biomedical applications [35][36][37][38][39][40]. In terms of 3D printing use, SD powder possesses desirable characteristics that allow fabricating precise products with complex geometry [41]. Although SDHA powder morphology indicates suitability for SLS processing, publications concerning this subject are scarce [42]. Nevertheless, the challenge remains to produce polymer/ceramic spray-dried composite granules with a suitable mechanical behaviour [43]. In this work, SDHA was mechanically mixed with UHMWPE powder for further SLS processing. Processing parameters were evaluated to find the most beneficial combination in terms of processability and mechanical strength. Preliminary in vitro biological analyses were conducted for assessing cell proliferation and viability.

Materials and methods

Materials and compositions

HA particles, synthesized by hydrothermal route [44], were used in an aqueous suspension (with deionized water) for the spray-drying process. Methylcellulose (Methocel A15 Premium LV, Dow Chemical) was used as a binder. The spray-dried HA powder (SDA)

was mixed with a commercially manufactured ultra-high-molecular-weight polyethylene (UHMWPE) powder (Ticona, GUR 1020).

Three different powder mixtures were used for the SLS processing, varying SDHA content and identified as follows: UH (100 wt% UHMWPE); UH5HA (95 wt% UHMWPE + 5 wt% SDHA) and UH10HA (90 wt% UHMWPE + 10 wt% SDHA). Powders were mechanically mixed for 4 h using 3D dry powder mixer equipment.

Powder characterization

Powder particle size and distribution (PSD) were measured by laser scattering (Partica LA-950V2, Horiba). Scanning electron microscopy (SEM, JSM-6390, JEOL) was employed to analyse pre-SLS powder's morphology and fabricated samples surface, both coated with a thin gold layer. Morphological analysis of spray-dried HA (SDHA) particles was performed by SEM (IT300LV, JEOL), after platinum coating (Agar Scientific). Synthesized HA particles and SLS samples submitted to bioactivity essays were observed using a field emission gun microscope (Quanta 450, FEI). Particle flow and apparent density were determined using a Hall flowmeter. Concerning the particle's flow, a passing time of 50 g of each powder combination was taken 3 times and the average value was considered. For the apparent density calculations, a standard 25 cm 3 cylinder was filled and weighed.

The thermal behaviour of UH, UH5HA and UH10HA powders was analysed by differential scanning calorimetry (DSC, Jade, Perkin Elmer). Samples were sealed in an aluminium capsule and heated from 0 to 220 ºC at 10 ºC/min with isothermal mode and nitrogen gas at 20 ml/min.

Synthesized HA and SDHA particles were analysed by X-ray diffractometry (XRD, D8

Advance, Bruker), with a scan range from 27° to 40°, 2θ of 0.02° and acquisition time of 5 s.

Moreover, a wider scan range, from 10° to 40°, 2θ of 0.02° and acquisition time of 1 s was applied for UHMWPE and SDHA powders, as well as for UH, UH5HA and UH10HA. EVA software and Powder Diffraction Files (PDF: 00-09-0432) from the International Center for Diffraction Data (ICDD) were used to identify the XRD phases.

Spray drying of HA powder

In order to determine the formulation of spray-drying suspension and its processing parameters, a screening method was employed as described in previous work [34]. In the present study, the suspension to be spray-dried contained, in weight, 20.32% HA, 1.63% methylcellulose and 78.05% deionized water. The employed spray-drying equipment was Büchi B-290 and the processing parameters were inlet temperature 170 °C, aspiration 100%, atomization pressure 60 mmHg and slurry feed 8.75 ml min -1 . After spray drying, the granules were submitted to thermal treatment using ramps of 3 °C/min up to 500 °C, for binder removal, and 10 °C/min up to 1000 °C with a hold of 1 h for densification in a resistive oven (LHT 04/17, Nabertherm). The obtained SDHA particle characteristics were D10 = 9.05 ± 0.14 µm; D50 = 17.15 ± 0.44 µm and D90 = 29.90 ± 1.15 µm.

SLS processing

SLS processing was conducted using a SLS equipment (Alkimat) with a CO2 laser source (λ = 10.6 μm), 100 W maximum power, 200 μm spot diameter and a simple internal heating system.

To establish appropriate SLS processing conditions, the following parameters were varied: laser speed (LS), laser power (LP), hatch spacing (HS), layer thickness (LT) and powder bed temperature (BT). Visual observation and sample integrity were considered for suitable parameters selection. After that, laser power and scan speed were varied according to a factorial plan (Table 1), keeping the following parameters constant: hatch spacing (200 µm), layer thickness (200 µm) and powder bed temperature (100 o C). Parameters presented in Table 1 were applied to fabricate rectangular prismatic samples with 35 mm × 5 mm × 1.40 mm (length × width × height). To guarantee better homogeneity and powder spreading on each fabricated layer, the rectangular samples were positioned perpendicularly to the powder's roller. The most suitable SLS processing parameters were chosen according to the sample characterization and used to fabricate cylindrical discs of 10 × 5 mm (diameter × height) for biological analysis.

Low cooling rates induce homogeneous shrinking and minimum distortion of components [45]. Therefore, to minimize shrinking by thermal gradients, samples were kept at around 100 o C for 30 min (machine heating system) after SLS processing. Subsequently, the heating system was switched off and samples were held in the fabrication chamber until samples' temperature achieved 60 o C (in around 30 min). Finally, the surrounding powder was removed using a brush and the samples were taken from the polyamide base using a razor blade knife (to avoid physical efforts in the samples).

SLS samples characterization

SLS samples mechanical behaviour was measured through a single cantilever mode on dynamic-mechanical analysis equipment (Q800, TA Instruments). Single cantilever mode essay is suitable for thermoplastic materials and bar shaped samples. The stress-strain curves were obtained at a strain rate of 2 mm/min and a constant temperature of 37 o C, mechanical properties were measured using 3 different samples for each SLS fabrication parameters. Samples dimensional variation was quantified using a digital Vernier calliper, also using 3 samples. SEM images and EDS analysis (TM3030, Hitachi) were performed to verify particle coalescence/dispersion and chemical composition.

Biological analysis

For routine cell culture, murine calvaria pre-osteoblast MC3T3-E1 cells subclone 14 (ATCC, United States) were cultured in ascorbic acid-free alpha modified Eagle's medium (α-MEM; Gibco, United States) containing 2 mM L-glutamine and supplemented with 10% fetal calf serum (ThermoFisher, United States) 100 UI/ml penicillin and 100 μg/mL streptomycin (Gibco, United States), incubated at 37°C under 5% humidified CO2 atmosphere (complete culture medium). For routine cultivation, the cells were seeded in T75 flasks (Sarstedt, Germany). The complete culture medium was replaced every 2 to 3 days and cells were split when confluence reached 70-80 % of the culture area.

Prior to the cell culture, the different samples were sterilized by exposition to UV light under the culture laminar flow hood (ThermoFisher) for 30 min each side. Because the UH, UH5HA and UH10HA samples were floating in the complete culture medium, they were immobilized at the bottom of the culture wells (24-well culture plate, Sarstedt, Germany) by using a fibrin gel moulded around the UHs samples. Briefly, after having placed the samples in the wells, were added subsequently in the bottom of the well: 100 µl of a bovine fibrinogen solution (MP Biomedicals) in PBS 1X (Gibco) at 6 mg/ml, 100 µl of a complete culture medium and 100 µl of an aqueous solution of bovine thrombin (Merck) 25 U/ml. The solidification of the gel was starting immediately but the fibrin gel polymerization was let to complete for 30 min at 37 °C. Afterwards, the samples were covered by a complete culture medium and equilibrated for 1 h at 37 °C.

For each experiment, cells were rinsed in PBS 1X, detached by incubation in Tryple 1X

(Gibco) for 3-5 min at 37 °C and centrifuged at 300 g for 5 min at room temperature. The pellet was resuspended in a complete culture medium, the cells were numbered on a Malassez chamber and seeded at the density of 25,000 cells/cm 2 on the samples in a complete culture medium (1 ml in 24-well culture plates). The cells were cultured at 37 °C under a 5 % humidified CO2 atmosphere for the desired duration.

The cell metabolic activity was evaluated using an MTT assay. Briefly, after 24 h and 48 h of culture, 1.2 mM of MTT was added to the culture medium of the cells at the surface of the tested materials and let to incorporate during 2 h at 37 °C. The cell medium containing the soluble MTT is then discarded, 100 µl of DMSO (Sigma Aldrich) are added on top of the samples to lyse the cells and dissolve the water-insoluble formazan crystals resulting of the MTT reduction by cell enzymes such as succinate dehydrogenases. The lysis is completed by 15 min of agitation on a rotative plate, in the dark, at room temperature. Afterwards, 25 µl of the supernatant are taken in triplicate and distributed into a 96 well plate (Sarstedt, Germany)

for OD reading at 595 nm on a plate reader (FluoSTAR Optima, BMG Labtek, Germany). Cells seeded over glass coverslips (ThermoFisher) in a complete culture medium or complete culture medium containing 100 µM of H2O2 were respectively used as control of viable and dead cells.

After 24, h of culture, viable cells were stained with 10 µM Calcein Red-Orange AM (Molecular probes). The dye was added to the culture media and incubated for 30 min at 37 °C.

Cells were then rinsed in complete culture medium 3 times, then in PBS 1X and fixed in PFA 4 % for 10 min. They were again rinsed 3 times for 5 min in PBS 1X. Nuclei were stained by incubation in a Hoescht 33342 20µM (ThermoFisher, United States) solution in PBS 1X for 5 min and cells were rinsed again 3 times for 5 min in PBS 1X. Subsequently, samples were observed under AxioImager M2 microscope (Carl Zeiss, Germany), pictures were processed using the Zen blue software (Carl Zeiss, Germany). For each experiment, the analysis was performed at 10 different fields of the sample's surface. Finally, the pictures taken were analysed by ImageJ software (NIH, http://rsb.info.nih.gov/ij/). The cell density was evaluated by counting the number of cells attached to the pellet by surface area unit (cm 2 ).

For each assay, three independent experiments were performed. Statistical analyses were done using GraphPad Prism 9. Data were assayed for normality using the Shapiro-Wilk test. According to the data set and normality test results, the Kruskal-Wallis test was performed, followed by a Dunn's post hoc test. Differences were considered as significant for p < 0.05.

Results and discussion

Feedstock powder characteristics

The synthesized HA powder is composed of nano needle-like structures (Figure 1).

These structures tend to agglomerate in the form of irregular clusters that have low flowability, which is unfavourable for SLS processing. SDHA particles have the advantage of being more spherical and show a homogenous particle size dispersion (Figure 2). SEM images (Figure 3) also revealed that UHMWPE is composed of small particles agglomerations and interconnected by fibrils. This feature has already been documented in the literature, being a consequence of Zeigler Natta catalyst production route, not being the most favourable morphology for laser sintering [46,47] SDHA have a lower particle mean diameter than UHMWPE, so they are capable of filling voids left between UHMWPE particles and increase apparent density (Table 2). The lower flow time of UH5HA and UH10HA can be related to the smaller HA particles, which act as a lubricant and facilitate particles flow. It can be observed a greater value of (D90-D10)/D50 ratio along with great HA content, this is related to the increase of HA small particles in the mixture. In addition, PSD graphs (Figure 4) of UH5HA and UH10HA have shown bimodal behaviour, associated with the considerably different particle sizes from UHMWPE and SDHA. In a previous study [34], we did not identify phase modifications from synthesized HA to SDHA, and concerning UH, UH5HA and UH10HA samples there were also no phase divergences. XRD curves (Appendix I) intensities were according to HA's content, whereas UH10HA sample has shown greater intensity at HA's diffraction pattern due to the higher content.

Powder mixtures of UH (100% UHMWPE), UH5HA (5% HA) and UH10HA (10% HA) were also analysed by DSC. As seen in Figure 5, the powder thermal behaviour has changed with the addition of SDHA in the powder mixture as the crystallization temperature has dropped ~7.65 o C and the melting temperature dropped ~4.70 o C. These changes, as well as the lower and wider pics observed for UH5HA and UH10HA, can be attributed to different powder composition. Additionally, HA (ceramic) has a different heat transfer coefficient than UHMWPE (polymer), which alters the heat transfer kinetics.

The SLS sintering window can be defined between onset points of crystallization temperature (Tc) and melting temperature (Tm). However, this sintering window is an idealized representation (considering cooling/heating rates of 10 o C/min), being a completely different circumstance of the hardly controllable temperature changing rates during the SLS processing [6]. Nevertheless, DSC graphs (Figure 5

SLS processing and parameters

While fabricating pieces by additive manufacturing techniques, piece design and building orientation must be considered. Component design specifications must be according to the manufacturing capabilities and limitations of the intended AM technique [48]. Further, building orientation has a significant effect on the properties of fabricated pieces by SLS [49][50][51]. Figure 6 exhibit how samples were disposed of in the fabrication chamber. One of the reasons for this positioning was because rollers for powder feed moves along the x-axis (red line), providing more homogenous powder disposal. Another important reason is the building orientation, whereas the selected placement provides the larger surface perpendicular to the build direction. This means that the laser beam is exposed to a greater surface area, favouring cohesion between subsequent layers. The energy density (ED) can be used to quantify the amount of energy that is placed on the feedstock powder during the SLS processing. It can be calculated using Eq. ( 1), being expressed in J/mm As seen in Figure 5, the UHMWPE polymer possesses a narrow SLS sintering window (~3.40 o C), therefore, slight changes in processing parameters are preferable. However, considering SLS equipment limitations for small changes in processing parameters, we have chosen to investigate a wider variety of parameters and the possible outcomes of parameters combination.

Some of the employed ED were not suitable for the sample fabrication due to low particle cohesion or, in contrast, excessive energy application leading to polymer degradation (smoke/flame with laser beam passage). Concerning UH samples values of ED of 0.500; 0.625 and 0.667 J/mm 3 did not achieve a proper particle cohesion, therefore, samples were not rigid enough to conduct accurate mechanical and dimensional measurements.

Diversely, when processing UH5HA and UH10HA samples with 1.042 and 1.389 J/mm 3 flame was produced with a laser beam passage on the powder bed, therefore, polymer degradation was evident, and the samples were unconsidered. Interestingly, the ED value of 1.111 J/mm 3 , which is greater than 1.042 J/mm 3 , did not show flames during processing but some smoke was produced. This might be attributed to the longer exposure time (lower laser speed) of the laser beam in the powder bed. Although smoke is also considered indicative of polymer degradation, it was chosen to go forward with these samples for mechanical and dimensional analysis.

Fabricated sample's properties

The fabricated samples were submitted to SEM analysis, images revealed homogeneous SDHA powder dispersion in the polymeric matrix. The UH samples that were processed with low ED (i.e. 0.500; 0.625 and 0.667 J/mm 3 ) evidenced poor particle cohesion and sintering neck formation was not observed. The EDS technique has confirmed the presence of SDHA particles along with the UHMWPE matrix as indicated in Figure 7, chemical composition is displayed in Table 4. It can be observed the substantial difference in phosphorous, calcium and carbon content in the measured points. Additionally, SDHA particles kept their morphological features after SLS processing, while UHMWPE particles fused between each other. Dimensional variation and warpage effects after processing has been evidenced by diverse studies while processing polymers [58][59][60][61][62]. Further, substantial warpage and dimensional variation are frequently cited by authors that have processed UHMWPE by SLS [23,24,63]. Dimensional variations can be attributed to inhomogeneous shrinkage as a result of inhomogeneous temperature distribution during SLS processing, leading to piece distortion due to stress forces [64]. For customized implantable devices, dimensional accuracy is essential.

Eventual piece shrinkage must be considered in the project in the fabrication phases. In our study, pieces were fabricated in the same position of the coordinate plane (Figure 6), whereas axis x refers to piece width, axis y to length and axis z to thickness. The employed SLS equipment had limitations related to layer thickness accuracy and homogeneity. Therefore, sample thickness values were not taken into consideration for discussing dimensional results.

Thus, for assessing dimensional variation only the length and width of the fabricated pieces were compared with the nominal values. A total of 3 samples from each powder composition (UH, UH5HA and UH10HA) were measured using vernier callipers; in length, only 1 measure was taken on each sample, and in width, 3 measures were taken (2 in the extremities and 1 in the middle).

Appendix II contains the arithmetic mean values and standard deviation for the measured values of each powder mixture. Regarding length and width measures, it can be observed that a greater ED implied greater mean dimensions. This can be related to the fusion of adjacent particles that originally were not supposed to be fused by the programmed laser track. When compared to the nominal length dimension (35 mm), it can be observed certain degree of shrinkage in all the ED values, probably due to material shrinkage. Contrastingly, width values have shown greater variation between the different ED values, in some cases, exceeding the nominal value (5 mm). In those instances, a greater ED value was applied to the samples and the increased dimensions can be related to a higher warping effect [65].

Table 5 contains the mean area variation of fabricated pieces, where negative values denote piece shrinkage. For UH samples, lower area variation was observed with ED of 1.389 J/mm 3 , followed by ED of 1.042 J/mm 3 and the only positive area variation was the ED of 1.389 J/mm 3 . Concerning UH5HA samples, relative uniform area variation values were observed with the different parameters combinations whereas ED is 0.833 J/mm 3 and shown positive area variation with ED of 1.111 J/mm 3 . UH10HA samples have shown the greatest area variation among the different ED values employed, this might be related to the more heterogeneous composition due to higher SDHA amount, like UH5HA that had bigger area variation when compared to UH samples. Stress versus strain graphs from the fabricated samples are illustrated in Figure 8 and Stress versus strain graphs have also shown discrepancies in mechanical behaviour for the same group of samples, leading to great deviations in the ultimate strength values. The employed equipment for mechanical analysis considers the cross-section area (width × thickness) for plotting stress versus strain graphs, therefore, considering the low accuracy related to sample's thickness, this might justify disparities between stress-strain curves among the same samples. Other than that, inconsistences in the microstructure (e.g., voids, micro-fissures, agglomeration of SDHA particles) may also lead to weakening polymeric matrix resulting in atypical mechanical behaviour. Some processing parameters combination of UH and UH5HA samples achieved strength values that are comparable to cortical bone [1]. Better mechanical properties might be achieved using a more precise parameters combination or posttreatment methods, such as heat treatment and hot isostatic pressure [66]. To select suitable processing parameters to fabricate cylindrical samples for biological analysis, the selection criteria were in accordance with mechanical properties. High ultimate strength and stress-strain homogenous curve behaviour were considered essential. Therefore, for UH samples the chosen ED of 0.833 J/mm 3 (LS of 450 mm/s and LP of 15 W), for UH5HA samples the ED of 0.833 J/mm 3 (LS of 600 mm/s and LP of 20 W) and for UH10HA ED of 0.625 J/mm 3 (LS of 600 mm/s and LP of 15 W).

Biological assays

Preliminary biological assays were conducted to evaluate differences in the biological response of the sample with different SDHA content. Pure dense HA disks (HA), produced using uniaxial presage using 0.5 ton and 10 mm matrix and heat treatment at 1200 o C (30 o C/min ramps), were used as a standard to compare results with the UH, UH5HA and UH10HA samples.

In a first approach, was evaluated the metabolic activity of the cells seeded at the different material surfaces and cultivated for 24 h and 48 h. The results are presented in Figure 9. Because the HA samples were smaller than UH, UH5HA and UH10HA samples, the values were normalized on the available geometric surface. The results were expressed as a percentage of activity relative to the control, which is HA, as a biocompatible material. At 24 h, considering the variations related to the measurements, all the values were similar except for the cells that have grown at the surface of UH. While not significantly different from HA (p = 0.01), the metabolic activity measured for UH is low and may be associated either with a defect in initial cell attachment or potential toxicity. At 48 h, no significant difference was detected whatever the considered sample even for the UH sample. This would indicate that, on one hand, at 24 h the cell had likely some difficulties to adhere to the material with a possible delay for adhesion explaining at less in part the result obtained at 48 h. On the other hand, cell growth would be not affected in this condition. Preliminary MTT results performed for 72h of cell culture no exhibit an alteration in cell viability with results comparable to those obtained for HA samples.

To go further and evaluate the initial attachment of the cell an in situ observation of the cells at the sample surfaces was performed after 24h of culture (Figure 10) and allowed to estimate the cell density (Figure 11 and Figure 12). The first observation is that most of the cells observed, regardless of the samples nature on which they were growing, were positive for calcein (red staining) so viable cells. The density is apparently higher on HA as confirmed by the quantitative analysis with more than 40,000 cells/cm 2 on HA against fewer than 10,000 cells/cm 2 on UH samples. However, it is important to note that the surface topography on UH samples is more uneven than on HA disks, with peaks and valleys, possibly with cells outside the focal plane of the microscope objective. Hence, the cellular density at the surface of those materials may have been a little underestimated. That would explain the discrepancy with MTT results, in which no difference in terms of cellular metabolic activity was detected when HA was compared with UHs samples. Fluorescence microscope images also revealed the influence of the sample's surface roughness and morphology on cells.

While HA disposed of homogeneous cell dispersion along the sample surface, UH samples have shown low cellular density. To go further, whereas on the relatively flat surface of HA disks when compared to UH samples, cells exhibited rounded nuclei (Hoescht33342 channel, left panel on Figure 12) with a spread morphology and presence of extensions, at the surface of the composite samples, the nuclei were smaller and elongated and it was observed that cells tended to allocate along the asperities (Figure 12). This can be related to the greater energy amount required for cells to allocate in curved surfaces [67]. Due to the important role of the cytoskeleton in cell behaviour, the topography of the UH samples may certainly impact bone cell biology that needs further experiments to be assessed. Finally, it should be reported that on the border of the UH samples (highest than those of HA), some residues of the fibrin gels used to immobilize the samples in the culture well bottoms were found. Fibrin being a protein of the extracellular matrix, cells can adhere easily to it. On fibrin above the material surface, cells were well spread with numerous extensions that contrasted with the morphology of the cells directly growing on composite disks (Figure 13) and did not follow any asperities.

Conclusions of Chapter 4

Results have shown the capability of the SLS process to fabricated rectangular and cylindrical samples of UHMWPE/SDHA composites. The addition of SDHA particles have ameliorated powder flowability and increased the SLS sintering window. Indeed, SLS processing of UHMWPE is challenging, however, a better UHMWPE powder preparation and the employment of precise SLS equipment would be favourable for better results.

Mechanical properties have shown the great influence of SDHA content on strength values, moreover, SLS processing parameters also play an important role. For a better understanding of mechanical performance and the suitability of UHMWPE/HA composites to fabricate implantable devices, further mechanical assays should be conducted. Ultimately, pieces simulating the desired design should be fabricated, evaluating dimensional variation and mechanical properties.

Preliminary biological assays confirmed the non-toxicity and the support pre-osteoblast attachment of UHMWPE after SLS processing, either, sample surface topography has influenced cell behaviour. Nevertheless, SDHA particles were also revealed to be advantageous for UHMWPE processing and their use in more suitable SLS processing polymers should be explored. This would expand the potential of use to other types of implants. Considerable dimension variation and narrow sintering window are the main drawbacks when SLS processing UHMWPE. The addition of SDHA has slightly contributed to a wider sintering window, however, UHMWPE is still highly influenced by thermal gradients while processing by SLS. To assess this issue, an accurate selection of processing parameters and precise SLS equipment is required.

Mechanical properties of the SLS fabricated samples have shown great dependency on processing parameters, moreover, lower ultimate strength is associated with higher SDHA content. Still, values compatible with cortical bone were achieved for most of the fabricated samples, except for UH10HA. The SLS fabricated samples were shown to be non-toxic, seem not to inhibit cell growth and were able to support pre-osteoblast attachment but with lower efficiency than pure HA dense disks. Surface finishing of the samples has impacted bone cell biology and further experiments must be assessed.

Future works should investigate the influence of SDHA on other polymers, preferably those which have better SLS processability such as polyamide (PA), polycaprolactone (PCL) and polylactide acid (PLA). Likewise, evaluate the feasibility and usefulness of manufacturing pieces using different contents of SDHA powder. Supplementary biological assays with more replications should also be conducted for a better understanding and more representative results. Finally, doped HA particles have shown very promising results and benefits for biological use; therefore, the spray drying process and SLS should also be evaluated.

RESUMO EXPANDIDO SINTERIZAÇÃO SELETIVA A LASER DE BIOCOMPÓSITOS UHMWPE/HA

PARA APLICAÇÕES EM TECIDO ÓSSEO

Introdução

Em virtude da disponibilidade de implantes personalizáveis, com geometrias complexas e combinação de diferentes tipos materiais, esses dispositivos promovem recuperação mais rápida, aumentando a qualidade de vida dos pacientes. Consequentemente, processos de manufatura aditiva (MA) têm sido estudados para solucionar diferentes problemáticas relacionadas à regeneração de tecidos ósseos. Isso se faz necessário quando há diagnóstico de patologias, como osteoporose, tumor, infecção, fraturas e desordens genéticas, que prejudicam ou impossibilitam o processo de recuperação natural e necessitam de intervenção cirúrgica.

Para tal, a técnica mais utilizada e com altas taxas de sucesso é o enxerto ósseo autógeno, no qual se retira um bloco ósseo do próprio paciente, com dimensões compatíveis ao local a ser efetuado o reparo. Dentre os fatores limitantes desse método, destaca-se a impossibilidade de recuperação de grandes defeitos ósseos, necessitando-se de outras técnicas para estes casos. Amplamente utilizados na regeneração do tecido ósseo, arcabouços tridimensionais porosos, conhecidos como scaffolds, são estruturas que mimetizam a matriz extracelular do tecido ósseo, servindo de alternativa para o enxerto ósseo.

O tecido ósseo humano possui uma complexa estrutura vascularizada, sendo composto de uma porção orgânica, com fibrilas de colágeno, e outra inorgânica, contendo cristais de fosfato de cálcio. Nesse sentido, a combinação de biopolímeros e biocerâmicos mostra-se oportuna devido à associação de propriedades fortemente desejadas, especialmente de biocompatibilidade e osteocondutividade. Biocerâmica, em particular hidroxiapatita (HA), já é amplamente utilizada para implantes ósseos, por possuir características de osteocondução e biocompatibilidade. Entretanto, desafios no processamento desse tipo de material dificulta a obtenção de implantes personalizados.

Polímeros biocompatíveis propiciaram soluções para implantes devido às características de maior processabilidade. Diversos polímeros são empregados como matéria-prima, como é o caso do polietileno de ultra alto peso molecular (Ultra High Molecular Weight Polyethylene, UHWMPE), utilizado há décadas para implantes de quadril, joelho e ombro. Considerado de difícil processamento devido ao alto peso molecular por métodos convencionais, a MA se apresenta como alternativa para obtenção de peças, como por exemplo via sinterização seletiva a laser, que viabiliza o processamento deste material para obtenção de implantes customizáveis.

O processamento via sinterização seletiva a laser (Selective Laser Sintering, SLS) consiste na obtenção de peças 3D a partir da energia fornecida por um feixe de laser, que sinteriza as partículas depositadas sucessivamente em camadas. Esse processo é favorecido caso os materiais possuam uma combinação de características, sendo altamente desejável a preparação da matéria prima a ser utilizada.

Um dos processos que podem ser utilizados para modificar propriedades morfológicas das partículas de HA é a atomização (ou spray drying, SD), que permite a obtenção de partículas esféricas e com tamanho controlado. Partículas de HA obtidas por spray drying (SDHA) apresentam características morfológicas mais apropriadas para serem processadas via SLS, quando comparadas com partículas de HA sintetizadas.

Dessa forma, julga-se que a combinação de biocompósitos UHMWPE/SDHA utilizando o processo de SLS, seja apropriada para fabricação de dispositivos implantáveis para regeneração de tecido ósseo.

Objetivos

O objetivo geral deste trabalho é produzir e caracterizar amostras de UHMWPE/SDHA obtidas pelo processo de SLS.

Para tal, os objetivos específicos do trabalho proposto contemplam:

• Síntese e caracterização de pó nanométrico de HA estequiométrico.

• Investigar a aptidão do processo de spray drying para obter partículas de HA com característica morfológica esférica e gama de tamanho entre 15 e 25 μm.

• Selecionar parâmetros de processamento apropriados para o processamento de amostras UHMWPE/SDHA e verificar se propriedades mecânicas são apropriadas para uso em tecidos ósseos.

• Realizar ensaios biológicos para avaliar a biocompatibilidade das amostras.

Metodologia

A primeira etapa do trabalho envolveu a síntese e preparação do pó de HA. A síntese de HA foi efetuada por meio do processo de co-precipitação, o protocolo consiste na adição controlada de uma solução de fosfato (fosfato diamônico (NH4).2HPO4) em uma solução de cálcio (obtida a partir da dissolução de nitrato de cálcio: Ca(NO3)2.4H2O). A quantidade de reagentes foi calculada para obter-se uma razão molar de Ca/P = 10/6. A síntese foi realizada em atmosfera de argônio, mantendo-se pH = 9 e temperatura de 85 o C, com tempo de maturação de 100 minutos. Após, o preparado foi centrifugado e lavado com água destilada, secando-se a O processo de síntese resultou pós nanométricos de HA, então submetidos a um processo de spray drying para produzir partículas com formato esférico e distribuição controlada de tamanho de partícula. Para encontrar os parâmetros ideais de processamento, considerando o objetivo de se obter partículas esféricas homogêneas, com diâmetro médio de 15 a 25 µm, foram variados os parâmetros de fabricação por meio de um planejamento fatorial e de uma função de desejabilidade. O equipamento de spray drying utilizado possibilitou a variação dos seguintes parâmetros: temperatura de entrada, velocidade de aspiração, taxa de alimentação e pressão de atomização. Além desses, pode-se variar a concentração da suspenção inicial, em termos de quantidade de HA e de ligante (metilcelulose). As partículas de HA recuperadas após o processo de spray drying (SDHA) foram submetidas a um tratamento térmico com rampa de 3 °C/min até 500 °C para remoção do ligante, seguido por uma rampa de 10 °C/min até 1000 °C, mantendo-se esta temperatura por 1 h. Métodos de caracterização como microscópio eletrônico de varredura (JEOL IT300LV e Quanta 450), difração de raios X (D8 Advance, Bruker), espectroscopia de infravermelho por transformada de Fourier, medidas de reologia (AR1500 TA Instruments, US) e análise de distribuição de tamanho de partícula por espalhamento a laser (Partica LA-950 V2, Horiba) foram empregados para as partículas de HA e SDHA.

A segunda parte do projeto contemplou a fabricação de amostras de UHMWPE/SDHA, por meio do processo de SLS, visando aplicações em engenharia de tecido ósseo. Amostras com dimensões de 35 × 5 × 1,40 mm, foram fabricadas utilizando 3 quantidades diferentes, em massa, de SDHA: 0% (UH); 5% (UH5HA) e 10% (UH10HA). Parâmetros de processo foram investigados, utilizando-se um planejamento fatorial com ponto central para avaliar a variação dos parâmetros de potência e velocidades do laser.

As amostras retangulares foram submetidas a análises mecânicas (Q800, TA Instruments) e medidas dimensionais por meio de paquímetro. A partir dos resultados de comportamento mecânico, selecionaram-se parâmetros de processamento para fabricação de amostras cilíndricas (10 × 5 mm) para efetuar análises biológicas de viabilidade e proliferação celular. curvas de tensão-deformação, havendo queda de propriedades mecânicas com o aumento da proporção de SDHA na amostra. No entanto, maiores concentrações de SDHA favoreceram resultados de viabilidade e proliferação celular nas amostras.

Considerações Finais

Os resultados obtidos na primeira etapa apontam viabilidade de produção e modificação de características morfológicas apropriadas para sinterização seletiva a laser a partir de partículas nanométricas de hidroxiapatita. Na segunda etapa do trabalho, constatou-se a difícil processabilidade do pó de UHMWPE. Assim, o aumento da porção de SDHA nas amostras prejudica propriedades mecânicas. No entanto, a adição das partículas de SDHA demonstrouse benéfica em relação à processabilidade dos materiais e em relação a atividade e proliferação celular.

Palavras

Résultats et discussion

Les étapes du travail ont abouti à la rédaction et à la soumission de trois articles scientifiques liés aux activités proposées dans l'accord de cotutelle de thèse. Une revue bibliographique depuis la base de données Scopus, en utilisant des mots clés pertinents par rapport à la thématique étudiée, a permis la rédaction d'un article permettant d'avoir une vue globale de la fusion en lit de poudre de composites polymère/phosphate de calcium.

Conclusion

Les résultats obtenus démontrent la faisabilité de la production par atomisation de particules d'hydroxyapatite nanométriques de morphologie contrôlée adaptées au processus de frittage sélectif par laser. L'élaboration de composite UHMWPE /HA par SLS, il a été constaté qu'une quantité importante de SDHA dans les échantillons altère les propriétés mécaniques.

Cependant, l'ajout de particules de SDHA permet d'améliorer la processabilité SLS des matériaux ainsi que la biocompatibilité des composites.

Mots-clés : spray drying ; UHMWPE ; hydroxyapatite ; frittage sélectif par laser ; ingénierie du tissu osseux.

1 Figure 1 :

 11 Composite materials and their applications in different body parts, adapted from Ramakrishna et al. [2]. .............................................................................................................. 19

Figure 2 : 20 Figure 3 : 2 Figure 1 : 33 Figure 2 : 35 Figure 3 :

 220321332353 Figure 2: Hierarchical macro to nanostructure and composition of a typical human bone [4]. .................................................................................................................................................. 20 Figure 3: Simplified SLS scheme [12]. ................................................................................... 21

Figure 4 : 40 Figure 5 : 3 Figure 1 : 73 Figure 2 : 75 Figure 3 : 76 Figure 4 : 76 Figure 5 : 79 Figure 6 : 80 Figure 7 :

 440531732753764765796807 Figure 4: Interconnection between polymer's properties for SLS processing [44]................. 40 Figure 5: Customized bioresorbable scaffold for periodontal repair (left) [64]. Tetragonal porous scaffold (L x W x H = 8 x 8 x 15.5 mm³ and pores size 1 x 1 x 1 mm³) with solid base (L x W x H = 9 x 9 x 3 mm³) with different processing parameters (right) [40]. .................... 47

Figure 8 :

 8 Figure 8: PSD of HA densified particles (after heat treatment at 1000 °C for 1 h): SD05 (pos. I), SD06 (pos. II), SD07 (pos. III), SD08 (pos. IV). ................................................................. 82

Figure 9 : 84 Figure 10 : 4 Figure 1 :Figure 2 :Figure 3 :Figure 4 :Figure 5 :Figure 6 :Figure 7 :Figure 8 :Figure 9 :Figure 10 :Figure 11 :Figure 12 :Figure 13 : 1 : 2 : 3 : 3 Table 1 : 2 : 80 Table 3 : 83 Table 4 : 5 : 4 Table 1 :Table 2 : 3 :

 9841041234567891011121312331280383454123 Figure 9: PSD dispersions of HA particles SD09, SD10, SD11, SD12, after heat treatment at 1000 °C for 1 h. ........................................................................................................................ 84 Figure 10: SEM images of HA particles SD09, SD10, SD11, and SD12, heat-treated at 1000 °C for 1 h. ................................................................................................................................. 85

Figure 1 :

 1 Figure 1: Composite materials and their applications in different body parts, adapted from Ramakrishna et al. [2].

Figure 2 :

 2 Figure 2: Hierarchical macro to nanostructure and composition of a typical human bone [4].

Figure 3 :

 3 Figure 3: Simplified SLS scheme [12].

Figure 1 :

 1 Figure 1: Proportion of each polymer and Ca-P employed.

Figure 2 :

 2 Figure 2: SEM images of polymer powders a) PCL microspheres [21] and b) PLLA irregular particles [22].

Figure 3 :

 3 Figure 3: SEM of calcium phosphates used in SLS of biocomposites: a) nanostructured HA [28] and b) spherical HA powder [29].

Figure 4 :

 4 Figure 4: Interconnection between polymer's properties for SLS processing [44].

Shuai

  et al. have combined PEEK/PGA to unify the advantages of each polymer and adding different amounts of HA content, which were able to improve elastic modulus, bioactivity, cell attachment and proliferation of the fabricated scaffolds. Also using PEEK, Feng et al.[START_REF] Feng | A Multimaterial Scaffold With Tunable Properties: Toward Bone Tissue Repair[END_REF] have fabricated scaffolds containing PEEK/β-TCP/PLLA, in vivo bone defect repair results have proven the efficiency of bone formation and regeneration. Targeting to reduce the low degradability time of HA, Gao et al.[26] have fabricated specimens containing HA/β-TCP (50/50 w/w). The addition of 1 wt% of PLLA or PLGA introduced a liquid phase during SLS processing, this favours HA/β-TCP grain boundary sliding and rearrangement, leading to better densification and mechanical properties.

Figure 5 :

 5 Figure 5: Customized bioresorbable scaffold for periodontal repair (left) [64]. Tetragonal porous scaffold (L x W x H = 8 x 8 x 15.5 mm³ and pores size 1 x 1 x 1 mm³) with solid base (L x W x H = 9 x 9 x 3 mm³) with different processing parameters (right) [40].

Figure 1 :

 1 Figure 1: Scheme of BUCHI B-290 mini spray drying [35].

  450). Particle size and distribution (PSD) were determined by a laser scattering (Partica LA-950V2, Horiba). The specific surface area was measured by nitrogen adsorption (ASAP 2020, Micromeritics) and calculated according to the Brunauer-Emmet-Teller equation. X-ray diffraction (XRD, D8 Advance, Bruker) was carried out with a scan range from 27° to 40°, 2θ of 0.02° and acquisition time of 5 s. For XRD phase identification, EVA software was employed using HA's diffraction pattern (Powder Diffraction Files -PDF: 00-09-0432) from International Center for Diffraction Data (ICDD). The rheological behaviour of HA slurries was assessed with a concentric rheometer (AR1500 TA Instruments, US) using a 40 mm parallel plate. Rheology flow sweep measurements were performed on suspensions 20 °C, namely 10 points over stress rates from 0.1 to 40 Pa. The Herschel-Bulkley model was fitted

Figure 2 :

 2 Figure 2: XRD curves of pure HA treated at 650 °C for 0.5 h and SDHA treated at 1000 °C for 1 h.

Figure 3 :

 3 Figure 3: SEM-FEG images of heat-treated departing HA powder, indicating the tendency of the agglomeration (left). Close-up of agglomerated nano-sized particles (right).

Figure 4 :

 4 Figure 4: PSD curve of heat-treated departing HA powder.

  Although the suspensions have similar dispersion curves, SD01 and SD02 indicate a larger volume of particles around 50 µm area, suggesting that the suspension is not well homogenized. As detailed in Table 2, this bimodal distribution reflects the values of D10, D50, D90, and its ratio. Particle size values of D10 mean that 10% of particles have smaller diameters than the D10 value, in the same way, D50 and D90 represent the 50% and 90% portions. (D90-D10)/D50 ratio provides information related to the particles' distribution width, in which, a wider distribution is observed with high ratio values. The values obtained by the (D90-D10)/D50 ratio enhance the significant distribution behaviour of SD01-SD02 versus SD03-SD04. It is possible that suspensions SD01 and SD02 have not completely dissolved the binder, remaining agglomerates. Individual nanosized HA particles tend to gather together (Figure 3), accentuating agglomeration effects.

Figure 5 :

 5 Figure 5: Viscosity measurements of HA suspensions SD01, SD02, SD03, and SD04.

Figure 6 :

 6 Figure 6: PSD curves of suspensions SD01, SD02, SD03, and SD04.

Figure 7 :

 7 Figure 7: SEM images of HA particles from SD01, SD02, SD03, and SD04, after heat treatment at 1000 °C for 1 h.

Figure 8 :

 8 Figure 8: PSD of HA densified particles (after heat treatment at 1000 °C for 1 h): SD05 (pos. I), SD06 (pos. II), SD07 (pos. III), SD08 (pos. IV).

Figure 9 :

 9 Figure 9: PSD dispersions of HA particles SD09, SD10, SD11, SD12, after heat treatment at 1000 °C for 1 h.

Figure 10 :

 10 Figure 10: SEM images of HA particles SD09, SD10, SD11, and SD12, heat-treated at 1000 °C for 1 h.

Figure 1 :

 1 Figure 1: FEI images from departing HA powder: need-like nanostructures, scale bar: 1 µm (left) and irregular cluster agglomerations, scale bar: 20 µm (right).

Figure 2 :

 2 Figure 2: SEM images of SDHA particles, scale bar: 50 µm (left). FEI detailed images of SDHA particles, scale bar: 10 µm (right).

Figure 3 :

 3 Figure 3: SEM images from Sample A. Various particles, scale bar: 500 µm (left) and individual particle, scale bar: 20 µm (right).

Figure 4 :

 4 Figure 4: PSD of UH, UH5HA and UH10HA powder mixtures.

  ) show clear diverse thermal behaviour and SLS sintering windows. The calculated values for the sintering window, using data from DSC were ~3.40 o C for UH; ~14 o C for UH5HA; and ~13.25 o C for UH10HA. Khalil et al. [47] have confirmed different values for UHMWPE sintering windows, according to distinct heating and cooling rates. Therefore, the disparate thermal behaviour from UH toward UH5HA and UH10HA can affect the SLS processing behaviour.

Figure 5 :

 5 Figure 5: DSC runs from Sample A and B (left). DSC runs for UH, UH5HA and UH10 powder mixtures (right).

Figure 6 :

 6 Figure 6: Sample's arrangement in the print bed, lines represent z (blue), y (green) and x (red) axes.

Figure 7 :

 7 Figure 7: SEM image of UH10HA sample with respective points of EDS analysis.

Figure 8 :

 8 Figure 8: Selected stress-strain graphs with ED of 0.833 J/mm³ for UH, UH5HA and UH10HA.

Figure 9 :

 9 Figure 9: Metabolic activity of cells grown at the surface of UH, UH5HA, UH10HA and HA during 24 h and 48 h. The results were normalized on the geometric available surface and expressed as a % of control (HA). Statistical analysis: Kruskall-Wallis by Dunn's post hoc test. ns: non-significant (p > 0.05), *: p≤0.05.

Figure 10 :

 10 Figure 10: In situ observation of cell growing onto the surface of respectively UH (A to C), UH5HA (D to F), UH10HA (G to I) and HA (J to L). A, D, G and J: nuclei stained by Hoescht33342 (blue); B, E, H and K: viable cells are stained by calcein (red); C, F, I and L: merge. Scale bar: 100 µm. Arrows point to cells growing along asperities.

Figure 11 :

 11 Figure 11: Cell density at the surface of materials after 24 h of cell culture. Statistical analysis: Kruskall-Wallis followed by Dunn's post hoc test; ns: non-significant (p > 0.05); *: p≤0.05.

Figure 12 :

 12 Figure 12: In situ observation of cell growing onto the surface of respectively UH (A to C), UH5HA (D to F), UH10HA (G to I) and HA (J to L). A, D, G and J: nuclei stained by Hoescht33342 (blue); B, E, H and K: viable cells are stained by calcein (red); C, F, I and L: merge. Scale bar: 50 µm.

Figure 13 :

 13 Figure 13: Cells on fibrin on UH sample. Scale bar: 100 µM. Arrows point to visible fibrin fibres.

CHAPTER 5

 5 General Conclusions and Final Considerations UHMWPE/HA composite samples were successfully produced by the selective laser sintering (SLS) technique, whereas the UHMWPE served as a flexible matrix to add SDHA particles to enhance bioactivity. Some of the fabricated rectangular samples achieved mechanical strength values compatible with the range of cortical bone. Likewise, the biological response of in vitro assays has shown the capability of using SLS-manufactured UHMWPE/HA composites to be used for implantable devices. The submitted review article (Chapter 2) has extracted information from the bibliography regarding SLS processing of polymer/Ca-P composites. This work has summarized relevant information from different stages of SLS processing: material selection, mixture, employed equipment and processing parameters. Results have shown the novelty of using SDHA particles in composite mixtures as SLS feedstock. Likewise, morphological features of spray-dried particles demonstrate suitable characteristics for SLS processing. It was possible to observe the capability of the SLS technique to produce controlled porosity devices, in different ranges of size and materials combination. With the appropriate combination of materials and processing parameters, it is possible to fabricate diversified implantable devices. Chapter 3 presented a screening method for the spray drying process intending to produce suited particles for SLS feedstock. Experiments indicated the importance of the suspension preparation and the selection of proper processing parameters. Finally, additional experiments have increased powder production efficiency without losing the desirable morphological features. In this work, spherical HA particles in the range of 15 to 25 μm were obtained with good process efficiency, demonstrating the aptitude of the spray drying process for preparing SLS feedstock materials.Further bibliographic research, detailed at the end of Chapter 2, has demonstrated challenges associated with processing UHMWPE and its limitations. These aspects were evidenced in Chapter 4 while processing UHMWPE and UHMWPE/SDHA powders.

  100 o C. O pó obtido foi submetido a um tratamento térmico de 650 o C durante 30 minutos (rampa de 10 o C/min) para remover subprodutos da reação.

  -chave: spray drying; UHMWPE; hidroxiapatita; sinterização seletiva a laser; engenharia de tecido ósseo. La synthèse a abouti à des poudres de HA nanométriques, qui ont ensuite été soumises à un procédé d'atomisation afin de produire des particules de forme sphérique avec une distribution granulométrique contrôlée. L'objectif était d'obtenir des particules sphériques de distribution granulométrique homogène et d'un diamètre moyen de 15 à 25 µm. L'étude des paramètres procédés a été faite à l'aide d'un plan factoriel et les conditions optimisées ont été mises en évidence grâce à une fonction de désirabilité. D'une part, l'équipement d'atomisation utilisé a permis de faire varier les paramètres suivants : température d'entrée, vitesse d'aspiration, débit d'alimentation et pression d'atomisation. D'autre part, la concentration de la suspension initiale, c'est-à-dire la quantité de poudre d'HA et de liant (méthylcellulose), a été optimisée. Les particules d'HA obtenues après le processus d'atomisation (SDHA) ont consolidé par traitement thermique avec une rampe de 3 °C/min jusqu'à 500 °C pour éliminer le liant, suivi d'une rampe de 10°C/min jusqu'à 1000 °C en maintenant cette température pendant 1 heure. Les particules d'HA et de SDHA ont été caractérisées par microscopie électronique à balayage (JEOL IT300LV et Quanta 450), diffraction des rayons X (D8 Advance, Bruker), spectroscopie infrarouge à transformée de Fourier et granulométrie laser (Partica LA -950 V2, Horiba). Des études rhéologiques ont également été conduites (AR1500 TA Instruments, US). La deuxième partie du projet comprenait la fabrication d'échantillons UHMWPE/SDHA, via le procédé SLS. Des échantillons mesurant 35 mm × 5 mm × 1,40 mm ont été fabriqués en utilisant 3 pourcentages massiques différents de SDHA : 0 % (UH), 5% (UH5HA) et 10% (UH10HA). Les paramètres procédés ont été étudiés à l'aide d'un plan factoriel à point central afin d'évaluer l'influence de la puissance et de la vitesse du laser. Les propriétés mécaniques des échantillons rectangulaires obtenus ont été évaluées (Q800, TA Instruments) et des mesures dimensionnelles réalisées en utilisant un pied à coulisse digital. Les paramètres permettant d'obtenir les résultats mécaniques les plus probant ont ensuite été utilisés pour la réalisation d'échantillons cylindriques (10 mm × 5 mm) destinés aux analyses biologiques de viabilité et de prolifération cellulaire.

  Dans la première étape du travail, la possibilité d'obtenir des particules d'HA de morphologie sphérique, une distribution granulométrique uniforme et un diamètre moyen de 15 à 25 µm par atomisation a été vérifiée. Il a été constaté que la modification des paramètres du procédé, notamment la vitesse d'alimentation et la pression d'atomisation, influencent directement la taille des particules obtenues. De même, lors de la préparation de la solution initiale, il est important de prêter aux caractéristiques rhéologiques de la suspension (viscosité trop élevée) et à la quantité de solide ajoutée afin de limiter la formation d'agglomérats d'HA qui rendent le procédé irréalisable. La deuxième étape du travail a porté sur la fabrication d'échantillons composites UHMWPE / particules de SDHA. Différentes compositions de poudres ont été mélangées mécaniquement : UH (100% UHMWPE), UH5HA (95% UHMWPE avec 5% SDHA) et UH10HA (90% UHMWPE avec 10% SDHA). Des échantillons rectangulaires (35 mm × 5 mm × 1,40 mm) ont été fabriqués, en faisant varier les paramètres de processus (puissance et vitesse du laser) par un plan factoriel. Les propriétés mécaniques et la variation dimensionnelle ont été évaluées. Les paramètres les plus appropriés pour la fabrication d'échantillons cylindriques (10 mm × 5 mm) ont été sélectionnés. Des analyses de viabilité cellulaire et de prolifération ont été effectuées. Les résultats de l'analyse dimensionnelle ont montré une contraction et une distorsion des échantillons fabriqués, un phénomène également observé dans la bibliographie. Il a été constaté qu'une densité d'énergie plus élevée permet une meilleure cohésion des particules situées en périphérie de l'échantillon, contribuant à des dimensions plus grandes. Les tests mécaniques ont été représentés à l'aide de courbes contrainte-déformation. Une altération des propriétés mécaniques lié à une augmentation de la proportion de SDHA dans l'échantillon a été observées. Cependant, des concentrations plus élevées de SDHA ont favorisé la viabilité et la prolifération cellulaire dans les échantillons.

Table 4 :

 4 EDS results of each element found on different points of UH10HA sample. ......... 112

Table 5 :

 5 Mean area variation from nominal values of fabricated samples. ........................... 113

Table 6 :

 6 Mean values and deviation of ultimate strength for fabricated samples. ................ 115

		LIST OF ABBREVIATIONS, ACRONYMS AND SYMBOLS
	AM	Additive manufacturing
	a-PC	Aliphatic polycarbonate
	BIS-GMA	Bisphenol A glycidyl methacrylate
	C	Carbon
	CAD	Computer-assisted design
	Ca-P	Calcium phosphates
	CF	Carbon fiber
	C-HA	2-Carboxyethylphosphonic acid-modified hydroxyapatite
	CHA	Carbonated hydroxyapatite
	GF	Glass fibers
	HA	Hydroxyapatite
	HDPE	High-density polyethylene
	KF	Kevlar fibers
	LCP	Liquid crystalline polymer
	PA	Polyamide
	PBF	Powder bed fusion
	PC	Polycarbonate
	PCL	Polycaprolactone
	PDLA	Poly(d-lactic acid)
	PEA	Polyethylacrylate
	PEEK	Polyetheretherketone
	PEG	Polyethylene glycol
	PELA	Block co-polymer of lactic acid and polyethylene glycol
	PET	Polyethyleneterephthalate
	PGA	Poly(glycolic acid)
	PHB	Polyhydroxybutyrate
	PHBV	Poly(3-hydroxybutyrate-co-3-hydroxyvalerate)
	PHEMA	Poly(2-hydroxyethyl methacrylate)
	PLGA	Poly(lactic-co-glycolic acid)
	PLLA	Poly(l-lactic acid)
	PMA	Polymethylacrylate
	PMMA	Polymethyl methacrylate
	PP	Polypropylene
	PS	Polysulfone
	PTFE	Polytetrafluoroethylene
	PU	Polyurethane
	S/O/W	Solid-in-oil-in-water
	SBA	Santa barbara amorphous
	SDHA	Spray-dried HA
	SLS	Selective laser sintering
	SR	Silicone rubber

This thesis is divided into five chapters. Chapter 1 provides a general overview of the current work along with its objectives. The Chapters 2 to 4 are the manuscripts that resulted from this PhD thesis. Chapter 2 presents a literature review of polymer/calcium phosphate biocomposites manufactured by selective laser sintering. Chapter 3 provides a screening method for producing suitable spray-dried HA powder for SLS applications. Chapter 4 details SLS processing of spray-dried HA and UHMWPE powder composites. Finally, Chapter 5 contains the general conclusions and final considerations.

  1,2 , Karine Giry 2 , Gean Salmoria 1 , Chantal Damia 2 , Dachamir Hotza3

Table 1 :

 1 Details of search strategy for each database.

	Database Keywords

Table 2 :

 2 Polymer/Ca-P composite mixtures, type of blending and number of publications.

Table 3 :

 3 Polymer/Ca-P mixtures and processing parameters.

							Range of processing parameters
	Materials (wt/wt)	Type of mixing	SLS equipment	Laser source	Energy density	Laser spot size	Scanning Speed	Laser power	Bed temperature	Layer thickness	Reference
					[J/mm²]	[µm]	[mm/s]	[W]	[°C]	[µm]
	100% PMMA
	5% β-TCP + 95% PMMA
	10% β-TCP + 90% PMMA	General	Built in-house	CO2	0.15	540	450-480	34-38	-	-	[57]
	15% β-TCP + 85% PMMA
	20% β-TCP + 80% PMMA
	100% PCL
	10% HA + 90% PCL	S/O/W	HRPS-IV	CO2	-	200	1200-2000	6-10	45	200	[21]
	20% HA + 80% PCL
	100% PCL 10% β-TCP + 90% PCL 50% β-TCP + 50% PCL	General	Sinterstation 2500plus	CO2	-	410	-	7	49	110	[49]
	100% PMMA
	5% β-TCP + 95% PMMA
	10% β-TCP + 90% PMMA	General	Built in-house	CO2	0.10-0.16	540	250-812	13-42	100	500	[58]
	15% β-TCP + 85% PMMA
	20% β-TCP + 80% PMMA
	99% β-TCP + 1% PLLA	General	Built in-house	CO2	0.20-0.60	500	250	20	-	100	[73]

  porous tissue engineering scaffolds from poly(L-lactide)/carbonated hydroxyapatite nanocomposite microspheres," J. Mater. Sci. Mater. Med., vol.19, no. 7, pp. 2535-2540, 2008.

Table 1 :

 1 Compositions of spray-drying HA aqueous suspensions.

	Sample	Hydroxyapatite	Methylcellulose	Deionized water
		(wt%)	(wt%)	(wt%)
	SD01	20.00	3.20	76.80
	SD02	20.16	2.42	77.42
	SD03	20.32	1.63	78.05
	SD04	20.49	0.82	78.69

Table 2 :

 2 Rheological

	Sample	Yield Stress (Pa)	Viscosity (Pa•s)	Rate index	D10 (µm)	D50 (µm)	D90 (µm)	(D90-D10)/D50 (µm)
	SD01	8.47	1.70	0.67	2.18	5.43	55.65	23.08
	SD02	8.56	1.07	0.66	2.13	5.88	46.99	19.32
	SD03	5.91	0.51	0.65	2.24	5.10	12.42	3.26
	SD04	5.92	0.18	0.70	2.22	4.29	8.36	1.83

values obtained from the Herschel-Bulkley model and respective D10, D50, D90, and ratio measures for HA suspensions.

Table 3 :

 3 Factorial design parameters and values and respective D10, D50, D90 mean size and size ratios, after heat treatment at 1000 °C for 1 h. The (-) signal represents lower and (+) higher values of the two levels factorial design.

	Run Sample	Atomization pressure (mmHg)	Slurry feed (mL min -1 )	D10 (µm)	D50 (µm)	D90 (µm)	(D90-D10)/D50 (µm)
	1	SD09	45 (-)	21.00 (-)	13.92	23.67	38.15	1.04
	2	SD10	45(-)	25.50 (+)	13.90	23.12	36.53	0.96
	3	SD11	60 (+)	21.00 (-)	9.71	19.02	39.52	2.11
	4	SD12	60 (+)	25.50 (+)	9.01	16.85	29.24	1.37

Table 4 :

 4 Samples and respective desirability responses.

			Analyzed responses	Individual desirability
	Sample	D50 (µm)	(D90-D10)/D50 (µm)	Particle morphology	D50	(D90-D10)/D50	Particle morphology	Global desirability
	SD09	23.67	1.04	4	86.70%	97.33%	75.00%	85.86%
	SD10	23.12	0.96	4	81.20% 100.00%	75.00%	84.76%
	SD11	19.02	2.11	3	40.20%	26.00%	50.00%	37.39%
	SD12	16.85	1.37	3	18.50%	75.33%	50.00%	41.15%

Table 5 :

 5 Factorial design rounds and obtained results.

	Round	Slurry Feed (mL min -1 )	Atom. Press. (mmHg) D50 (µm) (D90-D10)/D50 Efficiency (%)
	1	9.5 (-)	45 (-)	19.92	1.70	10.85
	2	15 (+)	45 (-)	18.94	1.24	17.99
	3	9.5 (-)	45 (-)	22.76	1.24	11.96
	4	15 (+)	45 (-)	23.31	1.12	6.47
	5	15 (+)	60 (+)	19.40	1.20	24.35
	6	15 (+)	60 (+)	19.23	1.49	17.39
	7	9.5 (-)	60 (+)	16.75	1.33	21.60
	8	9.5 (-)	60 (+)	17.62	1.44	26.57
	9	15 (+)	45 (-)	23.33	1.11	9.45
	10	9.5 (-)	45 (-)	22.75	1.22	16.05
	11	15 (+)	60 (+)	19.57	1.51	19.38
	12	9.5 (-)	60 (+)	17.09	1.44	26.38

  doctorate, mainly conducted at NIMMA (Núcleo de Inovação em Moldagem e Manufatura Aditiva) but the biological analyses were conducted at IRCER (Institut de Recherche sur les Céramiques) with the help of Amandine Magnaudeix, Adeline Dumur and Emeline Renaudie.

Similar to spray drying equipment, a large number of experiments were conducted to comprehend equipment performance and limitations. Initial trials for SLS processing UHMWPE powder have considered values found in the related bibliography. The varied parameters were powder bed temperature, layer thickness, laser power and speed. It was possible to notice that layer thickness greater than 200 µm caused excessive distortion,

Table 1 :

 1 Factorial plan for SLS sample fabrication. The (-) signal represents lower, (0) central and (+) higher values.

	Run Laser power (W) Laser speed (mm/s)
	1	25 (+)	450 (+)
	2	25 (+)	600 (0)
	3	25 (+)	750 (-)
	4	20 (0)	450 (+)
	5	20 (0)	600 (0)
	6	20 (0)	750 (-)
	7	15 (-)	450 (+)
	8	15 (-)	600 (0)
	9	15 (-)	750 (-)

Table 2 :

 2 SLS feedstock powder characteristics.

	Sample	Flow time (s)	Apparent Density (g/cm 3 )	D10 (µm)	D50 (µm)	D90 (µm)	(D90-D10)/D50
	UH	49.09	10.20	85.97 138.12	213.30	0.92
	UH5HA	44.21	10.46	85.02 146.78	223.44	0.94
	UH10HA	40.02	10.63	71.90 140.28	217.66	1.04

  3 [52][53][54][55][56][57], whereas LP is laser powder (W), LT is layer thickness, LS is laser speed (mm/s) and HS is hatch spacing (mm) -also known as scan spacing. Values of LT and HS were fixed in 200 µm during all experiments, Table3indicates the respective ED for each combination of SLS parameters displayed in Table1, where the lighter colour represents lower ED values.

	𝐸𝐷 =	𝐿𝑃 𝐿𝑇 × 𝐿𝑆 × 𝐻𝑆	(1)

Table 3 :

 3 Energy density (ED) for SLS sample fabrication.

			Laser speed (mm/s)
	ED (J/mm³)	750	600	450
	Laser	15	0.500	0.625	0.833
	power	20	0.667	0.833	1.111
	(W)	25	0.833	1.042	1.389

Table 4 :

 4 EDS results of each element found on different points of UH10HA sample.

		Carbon (wt%) Oxygen (wt%) Phosphorus (wt%) Calcium (wt%)
	Point 1	26.73	28.15	12.00	33.10
	Point 2	26.69	25.78	13.75	33.78
	Point 3	31.40	23.60	10.90	34.08
	Point 4	88.79	8.70	-	2.50

Table 5 :

 5 Mean area variation from nominal values of fabricated samples.

				UH (%)			UH5HA (%)		UH10HA (%)
	Laser speed (mm/s)
			750	600	450	750	600	450	750	600	450
		15	-	-	-4,83	-6,36	-4,85	-1,25	-9,90	-4,30	3,99
	Laser power (W)	20	-	-7,33	-4,15	-6,32	-0,66	5,74	-2,63	2,20	13,34
		25	-7,20	-2,20	1,30	-1,91	-	-	4,14	-	-

Table 6

 6 indicate the arithmetic mean ultimate strength and deviation values for each parameter combination. All results are presented in Appendix III. A total of 3 samples of each ED were submitted to mechanical analysis and most samples had homogenous curve behaviour up to around 10% strain. After this point, a different curve pattern was observed suggesting the occurrence of a different deformation mechanism in the polymeric chain. It can be observed that the strain at break value systematically decreases with higher SDHA content. Although SEM images have shown good interparticle bonding of the UHMWPE matrix and homogeneous SDHA dispersion into the matrix, the low chemical affinity between UHMWPE and SDHA phases contributes to lower mechanical properties. Further, a higher weight content of SDHA particles will corroborate to fewer UHMWPE particles' contact area and, ultimately, leading to less particle cohesion and a more heterogeneous microstructure. Facilely evidenced in UH10HA samples, the reduced area under the stress-strain curves denotes that SDHA particles have induced a reduction in toughness.

Table 6 :

 6 Mean values and deviation of ultimate strength for fabricated samples.

  , but no phase modification was observed by XRD. Biological essays demonstrated the biocompatibility of the employed UHMWPE/HA composite mixture. The SLS fabricated samples showed to be non-toxic, seem to not inhibit cell growth and were able to support pre-osteoblast attachment. It was observed that SDHA content and the selected SLS processing parameters have a great influence on the mechanical properties of the fabricated pieces. The mean ultimate strength values varied from 16.51 to 83.34 MPa and samples revealed greater dimensional measures when using higher energy density values. Further work is necessary to assess and verify correlations between the processing parameters and achieved results. Moreover, due to the small sintering window of UHMWPE/SDHA powders it is necessary to employ SLS machine with high precision of processing parameters. Finally, the UH polymers were not toxic, seem not to inhibit cell growth and were able to support pre-

	4. Conclusions
	Selective laser sintering of UHMWPE has inherent difficulties related to material
	properties and powder characteristics; nevertheless, feedstock preparation has shown prominent
	450 x 25
	results. The addition of spray-dried hydroxyapatite (SDHA) particles to UHMWPE increased (ED = 1.389 -	-
	J/mm 3 )
	the powder flowability and apparent density. Further, DSC results showed that the thermal
	behaviour was affectedosteoblast attachment. No difference was observed between the rates of HA incorporation into
	UH samples.

Innovation Laboratory for Molding and Additive Manufacturing (NIMMA), Federal University of Santa Catarina (UFSC), 88040-900 Florianópolis, SC, Brazil

University of Limoges, CNRS, IRCER, UMR 7315, F-87000 Limoges, France

Interdisciplinary Laboratory for the Development of Nanostructures (LINDEN), Federal University of Santa Catarina (UFSC), 88040-900 Florianópolis, SC, Brazil Manuscript under review in the Journal Progress in Additive Manufacturing.

Innovation Laboratory for Molding and Additive Manufacturing (NIMMA), Federal University of Santa Catarina (UFSC), 88040-900 Florianópolis, SC, Brazil

University of Limoges, CNRS, IRCER, UMR 7315, F-87000 Limoges, France

Interdisciplinary Laboratory for the Development of Nanostructures (LINDEN), Federal University of Santa Catarina (UFSC), 88040-900 Florianópolis, SC, BrazilManuscript to be submitted.

Acknowledgements ............................................................................................................... References.............................................................................................................................. Appendix I ............................................................................................................................. Acknowledgements This study was financed in part by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior -Brasil (CAPES) -Finance Code 001, and Fundação de Amparo à Pesquisa e Inovação do Estado de Santa Catarina (FAPESC). The authors thank the French Agence Nationale de la Recherche in the scope of the LabExSigmaLim (ANR-10-LABX-0074-01 Sigma-LIM). The authors are grateful to the Service Commun de Caractérisation des Matériaux de Limoges (CARMALIM) and more especially to Yann Launay for SEM acquisitions, Marguerite Bienia and Mickäel Lacroix for rheological measurements. The authors also thank Emeline Renaudie for her assistance in the elaboration of calcium phosphate ceramics, and Geoffroy Rivaud for the support in spray drying. Acknowledgements This study was financed in part by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior -Brasil (CAPES) -Finance Code 001 and the support of Fundação de Amparo à Pesquisa e Inovação do Estado de Santa Catarina (FAPESC/287/2018). The authors thank the French Agence Nationale de la Recherche in the scope of the LabExSigmaLim (ANR-10-LABX-0074-01 Sigma-LIM). The authors are grateful to the Service Commun de

Screening Method for Producing Suitable Spray-dried HA powder for SLS application

Henrique Schappo 1,2 , Karine Giry 2 , Chantal Damia 2 , Dachamir Hotza 3

Resultados e Discussão

As etapas do trabalho resultaram na elaboração de artigos relacionados às atividades propostas na convenção de cotutela. Uma revisão bibliográfica a partir das bases de dados Scopus, utilizando palavras-chave pertinentes ao tema estudado, possibilitou elaborar um artigo com uma visão global da fusão em leito de pó de compósitos de polímeros/fosfato de cálcio.

Na primeira etapa do trabalho, verificou-se a possibilidade de obter partículas de HA com morfologia esférica, distribuição de tamanho de partículas uniforme e com diâmetro médio de 15 a 25 µm. Verificou-se que a modificação dos parâmetros de processo, especificamente taxa de alimentação e pressão de atomização, influenciam diretamente no tamanho das partículas obtidas. Igualmente, na preparação da solução inicial, deve-se atentar para as características reológicas e quantidade de sólido adicionado de modo que não haja aglomerados de HA ou alta viscosidade que impossibilite o processamento.

A segunda etapa do trabalho investigou a fabricação de amostras da mistura de UHMWPE com adição de partículas de SDHA. • La synthèse et la caractérisation de poudre stoechiométrique d'HA nanométrique.

• L'étude de la capacité du procédé d'atomisation à produire des particules d'HA sphériques avec une gamme de taille comprise entre 15 et 25 μm.

• De sélectionner les différents paramètres de fabrication par SLS adaptés à la fabrication d'échantillons UHMWPE/SDHA et évaluer si les propriétés mécaniques sont appropriées pour des applications en substitution osseuse.

• D'évaluer in vitro la biocompatibilité des échantillons.

Méthodologie

La première étape du travail impliquait la synthèse et la préparation de la poudre de HA.

La synthèse de HA a été réalisée par coprécipitation en voie liquide. Le protocole consiste en l'ajout contrôlé d'une solution de phosphate diammonique (NH4).2HPO4) à une solution de calcium, obtenue à partir de la dissolution de nitrate de calcium (Ca(NO3)2.4H2O). La quantité des réactifs a été calculée pour obtenir un rapport molaire de Ca/P = 10/6. La synthèse a été réalisée sous atmosphère d'argon, en maintenant pH = 9 et une température de 85 °C, avec un temps de maturation de 100 minutes. Ensuite, la préparation a été centrifugée et lavée avec de l'eau distillée puis séchée à 100 °C. La poudre obtenue a été soumise à un traitement thermique à 650 °C pendant 30 min, avec une rampe de 10 °C/min, pour éliminer les sous-produits de réaction.