glasses for biomedical applications. Such glasses in powder form are envisaged to have applications in novel composite restorations where the following is achievable: dentin cellmediated bioremineralization, dental pulp regeneration and as carrier for therapeutics or antibacterial ions.

The initial aim was to produce soluble porous phosphate glasses using the sol-gel method (phosphate-alkoxide based sol-gel process). Knowing the effect that the variation of Ca content has on the dissolution properties of the glass, a series of glasses where Ca was progressively increased at the expense of Na was produced. The structure of the prepared samples was probed by XRD, XRF and FTIR to confirm the successful synthesis of the target phosphate-based glass compositions. After that a promising methodology was established, attempts were made to replace Ca with Sr. Different Sr sources were used without success due to the difficulty to fully dissolve those precursors in the sol-gel mixture.

Subsequently, the issue of the toxicity of some precursors and solvents used in the sol-gel procedure was recognised. To overcome this obstacle, efforts were made to replace the toxic precursor chemicals with safer ones. Nevertheless, due to the low solubility of some new precursors and the low reactivity of others, the sol-gel process did not proceed in a predictable and reproducible fashion.

At this stage, the sol-gel route was put aside, and two alternative soft and water-based chemical approaches were experimented: the precipitation method and the coacervation process. The first one was found to be unsuitable for our needs for two main reasons: 1) the presence of Na in the composition generated a crystalline material (instead of a glassy amorphous one); 2) the Ca/P ratio of our composition fell in the range of crystalline phase by using this method. In addition, the yield was really low. The second method (coacervation process) was a complete success. The glassy nature of the materials obtained was proved by XRD and XRF and the surface features were tested by BET and SEM. The process was retained for a while as the preferred synthesis route and both the scale-up effect and the possibility to add Sr were analysed. The production scale of the material was increased by 5 times and different Sr sources were tested to find the best one. XRD and XRF analysis proved both the success of the scale-up and the incorporation of the Sr in glass composition.

However, despite the promising results, due to both issues on the reliability of reaching the composition and lack of porosity / surface area meaning, in the end, the meltquench technique was chosen as a definitive and reliable method to produce the above mentioned compositional series. Quaternary phosphate-based melt-quenched derived glasses in the P2O5-CaO-Na2O-SrO system were synthesised in a way that by increasing Sr, Ca decreases. The glasses obtained have the general formula of (P2O5)53-(CaO)(32-x)-(Na2O)15-(SrO)x, where x = 0, 5, 10 or 15 (mol%). Substituting Sr in place of Ca improves the stability and prolongs the degradation of these glasses.

To try to mimic the real architecture of deep dentine tissue and provide for a 3D support for cells, these glasses were mixed as powders with collagen fibres to make scaffolds.

The presence of the collagen as an organic polymer guarantees the three-dimensionality of the structure and provides for an excellent and natural binding sites for cells to easily adhere and proliferate. The glass particles, on the other hand, ensure improved mechanical properties and release over time of important ions such as Ca, P and Sr to trigger and sustain cell differentiation.

In the end, to test the cytocompatibility of such prepared materials, hGFs were seeded in direct contact with the scaffolds and the viability checked at day 2, 4 and 7. As control, cells were seeded in an empty well. Although the control cells showed a high metabolic activity indicating the good health of the cells used, those in contact with the tested scaffolds displayed a significant low viability due to the acidification of the cell medium following glass particles dissolution.

INTRODUCTION

The human tooth is an organ composed of three mineralized tissues (enamel, dentin and cement) and one connective tissue (the pulp). The enamel is the outer layer of the crown.

It is the hardest and the most highly mineralized tissue in the human body. Indeed, 96% of enamel consists of hydroxyapatite (HA) (a calcium phosphate rich mineral). Water and organic materials (mainly proteins such as amelogenin) make up the remaining part. The layer underlying the enamel is the dentine. The structure of dentine consists of microscopic channels called dentinal tubules which radiate outward through the dentine. It is made up of 70% inorganic materials, 20% organic materials (90% of which is collagen 1) and 10% water.

This structure gives to the dentine high permeability leading to more vulnerability towards demineralization. At the crown level, the dental pulp is confined into the pulp chamber. This tissue, located in the centre of crown and root, forms with the dentin a complex called dentinpulp complex. The dental pulp is a highly innervated, vascularized and non-mineralized connective tissue. At the interface between dentin and pulp highly specialized cells called odontoblast are responsible for the synthesis of the dentin. Those cells are large columnar cells forming the so-called odontoblast cell layer: the cell bodies are in the pulp, whereas the cell processes are thrown into the dentinal tubules.

During its life, the tooth undergoes lot of physiopathological processes. Among them the stress caused by microorganisms assumes an important role concerning dental health. In the human mouth more than 500 species of bacteria have been identified and some of them live attached to the tooth surface. Those bacteria are organized in communities called biofilms, better known as dental plaque. Under some circumstances (such as high sugar diet or poor hygiene), the metabolic activity of those bacteria can be detrimental for the tooth.

While metabolising sugars, they produce acids that are secreted in the oral environment. Due to the accumulation of those acids, the pH of the saliva reduces below the HA critical value (5.5) leading to the dissolution of enamel and dentine. Those pathological conditions form the carious process contributing to the development of tooth decay. If the process is not stopped, the exposure of the pulp to the oral environment can be observed due to the loss of the dental mineralized tissues.

The main route to treat caries is the surgical removal of the infected tissues. The tooth is then restored by applying materials to replace damaged tissues. In this regard, different materials are available: metals, alloys, glass ionomer cements (GICs) and resins. Two of the most used metals were titanium and gold. Among the alloys it is possible to include the gold alloys, cobalt-chrome and nickel-chrome alloys and the amalgam. However, despite their long lifespan and good mechanical properties, the lack of aesthetics and the risk of intoxication The two main routes to synthesize bioglasses are melt-quench technique and sol-gel process. In the first case oxides are melted together and rapidly quenched to create the amorphous network; the second is a chemical approach where the precursors polymerize at room temperature to form the glass network. As the MQ has the drawbacks of high temperature processing, the sol-gel route has been extensively researched as an alternative over the last few years. Two of the advantages of the sol-gel compared with melt-quench is the high porosity achievable and the possibility to incorporate bioactive molecules. Due to their high resistance in water, those materials are mainly used as permanent implants and the effects of a long-term exposure to such resistant materials are still debated.

A new generation of (bio)resorbable materials is under studying to create temporal implants. Resorbable means the ability of those materials to completely dissolve in water environment to be replaced by new growing tissues. One of the greatest exponents of this new kind of materials are the phosphate-based glasses. Those glasses have the properties to dissolve in water, alongside, they offer the possibility to tailor the dissolution rate in agreement with the targeted tissue's turnover. Due to this, they can be combined with resorbable organic biopolymers, such as collagen, to mimic the natural 3D architecture of a tissue. In addition, likewise Si-glasses, they can be synthesized both by sol-gel and meltquench.

The aim of this thesis is to develop a new biomaterial by combining collagen scaffolds and PBGs to be placed at the DPJ and recreate, by recruiting cells from the pulp, a new odontoblast-like layer for a cell-mediated dental restauration.

Biomaterials: an introduction

Biomaterials have attracted a great interest in the past few decades as reliable replacements for hard and soft tissue. The most accepted definition of biomaterials is currently the one employed by the American National Institute of Health that describes biomaterial as "any substance or combination of substances, other than drugs, synthetic or natural in origin, which can be used for any period of time, which augments or replaces partially or totally any tissue, organ or function of the body, in order to maintain or improve the quality of life of the individual".

From this definition it seems that biocompatibility is a key property that any biomaterial must possess. The Williams Dictionary of Biomaterials (Williams 1999) defined biocompatibility as ''the ability of a material to perform with an appropriate host response in a specific situation'' 1 . However, this definition appeared incomplete since a list of "nonproperties" has been developed such as nontoxic, non-immunogenic, non-thermogenic, noncarcinogenic, and so on. Thus, the Williams Dictionary of Biomaterials (Williams 2008) updated the definition of biocompatibility: ''the ability of a biomaterial to perform its desired function with respect to a medical therapy, without eliciting any undesirable local or systemic effects in the recipient or beneficiary of that therapy, but generating the most appropriate beneficial cellular or tissue response to that specific situation, and optimizing the clinically relevant performance of that therapy'' 2 .

Globally, biomaterials can be realized from natural or synthetic sources, thus, biomaterials are classified into two categories: biological (natural origin) and biomedical (artificial origin).

Naturally derived biomaterials have some limitations such as risk of infection and batch to batch variability [START_REF] Chai | Bone substitutes: Classification and concerns[END_REF] . On the other hand, biomedical biomaterials can be easily sterilized reducing the risk of infection. In addition, they are more stable over time making them the favoured choice. Among the artificial bioactive materials, of particular interest as well as the subject of this thesis are the bioglasses.

Bioglasses

Generally, the term glass identifies any solid that possesses an amorphous phase and exhibits a (glass) transition temperature when heated towards the liquid state. Therefore, a bioactive glass (or bioglass) is a glass able to elicit a biological response when in contact with the organic matter showing a specific surface reaction.

A great advancement on the formulation and study of bioglasses was achieved when Prof.

L. Hench published papers on his famous BioGlass (also known as 45S5), a melt-quenched derived silica-based glass with composition 45% SiO2, 24.5% CaO, 24.5% Na2O and 6.0% P2O5 in wt% 4 . The bioactivity of this material is attributed to its capacity to form a hydroxyapatitelike (HA) layer at its surface when soaked in simulated body fluid (SBF).

Hench and Thompson classified biomaterials in three categories or generations [START_REF] Hench | Twenty-first century challenges for biomaterials[END_REF] :

First generation or bio-inert materials: the aim was to achieve a suitable combination of mechanical/physical properties to match those of the replaced tissues or organs with no toxic effects (examples are metals and alloys);

Second generation or bioactive materials: they are able to interact with the surrounding tissue to elicit an active response (among these materials, we can count bioceramics, bioglasses, glassceramics and biopolymers). It is commonly accepted that the bioactivity of the material is a function of its ability to grow an HA layer on the surface when interacting with a physiologic fluid;

Third generation or bioresorbable and biodegradable materials: these are materials that linger temporarily in the host, being replaced by a natural growing tissue and are metabolized by the body into non-toxic by-products. An example are the phosphatebased glasses, the subject of this PhD thesis.

Phosphate-based glasses

Phosphorus is element 15 of the periodic table, placed in the group 15 (pnictogen) and respect to the other depends on the oxygen-to-phosphorus ration (O/P) that means, on the relative concentration of other cations in the glass composition [START_REF] Brow | Review: the structure of simple phosphate glasses[END_REF][START_REF] Knowles | Phosphate based glasses for biomedical applications[END_REF] . When the ratio is 2.5, a Q3 structure was found to predominate the network: in this context the glass is made by branched chains and rings in a 3-dimentional structured network. For O/P equal to 3, Q2 prevails and the ultrastructure of the glass appears to be largely made of endless linear chains where cations act as cross-linking agents. When the concentration of cations is so great that the O/P is 3.5, the glass is dominated by pyrophosphate in Q1 configuration. When the O/P fraction reaches the value of 4, free Q0 orthophosphates units are the majority constituents of the structure.

More recently, computational molecular dynamic models have elucidated the position and configuration of modifiers in the phosphate glass network helping to understand the role they play in determining the properties of such glasses [START_REF] Tommaso | Modelling the structural evolution of ternary phosphate glasses from melts to solid amorphous materials[END_REF][START_REF] Hasni | Molecular dynamics modelling of sodium and calcium metaphosphate glasses for biomaterial applications[END_REF][START_REF] Ruiz Hernandez | Molecular dynamics simulations of bioactive phosphate-based glass surfaces[END_REF] . It was argued that the distribution of the cations into the glass bulk is quite homogenous and that they coordinate oxygens in a peculiar manner depending on the type (and so radius) of cation and concentration. Considering the most common glass modifiers Ca and Na in the glass bulk, on average, a Ca 2+ is bonded to 3.9-4.0 chains, while a Na + is only bonded to 3.2-3.3 chains.

These values fall at the surfaces of phosphate glass particles to 2.8-3.0 chains bonded to Ca 2+ and 2.5-2.7 chains bonded to Na + .

Other modifiers can be added to the basic phosphate glass composition with different effects on the structure. In recent times, strontium has attracted a lot of attention as a glass doping agent for its interesting bone therapeutic properties [START_REF] Saidak | Strontium signaling: molecular mechanisms and therapeutic implications in osteoporosis[END_REF][START_REF] Pors Nielsen | The biological role of strontium[END_REF] . In the network Sr bonds to a similar number of phosphate chains as calcium. However, due to its bigger radius, it changes the structure of the glass. Introducing Sr into the glass causes an increase in density that correlates well with the Sr concentration; furthermore, the amount of Q2 phosphorus decreases slightly, resulting in a slight increase in the number of Q1 and Q3 environments [START_REF] Neel | Structure and properties of strontium-doped phosphate-based glasses[END_REF] .

Li (as Na) acts as a strong network modifier. Due to its really small size, when it is added it disrupts the PO double bond at the surface of the glass creating the PO -Li + ionic bond. This results in a changing in the structure from a 3D network of trigonally interconnected tetrahedra to a 1D network of long chains [START_REF] Sistla | Structural study of lithium phosphate glasses by X-ray RDF and computer simulations[END_REF] . Similar effects are found when K is in the glass: it progressively breaks the structure of metaphosphate chains into short pyrophosphate groups (P2O7) [START_REF] Williams | On the mechanisms of biocompatibility[END_REF][START_REF] Chai | Bone substitutes: Classification and concerns[END_REF][4][START_REF] Hench | Twenty-first century challenges for biomaterials[END_REF][START_REF] Mitchell | Use of outer d orbitals in bonding[END_REF][START_REF] Cruickshank | The rôle of 3d-orbitals in π-bonds between (a) silicon, phosphorus, sulphur, or chlorine and (b) oxygen or nitrogen[END_REF][START_REF] Brow | Review: the structure of simple phosphate glasses[END_REF][START_REF] Knowles | Phosphate based glasses for biomedical applications[END_REF][START_REF] Tommaso | Modelling the structural evolution of ternary phosphate glasses from melts to solid amorphous materials[END_REF][START_REF] Hasni | Molecular dynamics modelling of sodium and calcium metaphosphate glasses for biomaterial applications[END_REF][START_REF] Ruiz Hernandez | Molecular dynamics simulations of bioactive phosphate-based glass surfaces[END_REF][START_REF] Saidak | Strontium signaling: molecular mechanisms and therapeutic implications in osteoporosis[END_REF][START_REF] Pors Nielsen | The biological role of strontium[END_REF][START_REF] Neel | Structure and properties of strontium-doped phosphate-based glasses[END_REF][START_REF] Sistla | Structural study of lithium phosphate glasses by X-ray RDF and computer simulations[END_REF][START_REF] Ahmina | Glass-forming region, structure and some properties of potassium manganese phosphate glasses[END_REF] .

Mn, in contrast, breaks the structure of metaphosphate chains (P2O6) 2-into pyrophosphate predominant groups (P2O7) 4-and the POP bonds are depolymerized by the incorporation of distorted Mn units that create POMn bonds [START_REF] Ahmina | Glass-forming region, structure and some properties of potassium manganese phosphate glasses[END_REF] .

The Mg coordination number is around 5-6 and the metaphosphate structure dominates the glass network. Increasing its concentration entails a transition from the open structure (of the metaphosphate glass) to a more compact one which presumably is reflected in the increase of mass density [START_REF] Suzuya | Structure of magnesium phosphate glasses[END_REF] .

Fe or Al-doped phosphate glasses have a metaphosphate to pyrophosphate intermediate structure depending on their relative concentration compared to P. By increasing Al concentration, Q2 groups are disproportionated into Q1 and Q3 groups. The Fe-doped melted glasses Q4 are transformed into Q3 and even Q2 depending on the ionic status of the Fe that determines how rich in oxygens the network is; this in turn depends on the concentration of 25 the Fe [START_REF] Stoch | Structural properties of iron-phosphate glasses: spectroscopic studies and ab initio simulations[END_REF] . This determines that an increase of FeOP or AlOP units crosslink the phosphate chains. Cs acts by shifting Q2 into Q1 and Q0 [START_REF] Mogus-Milankovic | Structure of sodium phosphate glasses containing Al2O3 and/or Fe2O3. Part I[END_REF][START_REF] Mogus-Milankovic | Structure of sodium phosphate glasses containing Al2O3 and/or Fe2O3. Part I[END_REF] .

When Ti is added, Q2 and Q3 species decrease and Q1 units become the dominant phase [START_REF] Silva | Structural characterization of TiO2-P2O5-CaO glasses by spectroscopy[END_REF] .

In Zn-doped phosphate glasses the network is dominated by Q2 chains [START_REF] Sastry | Spectroscopic characterization of manganese-doped alkaline earth lead zinc phosphate glasses[END_REF][START_REF] Sastry | Structural and optical properties of vanadium doped akaline earth lead zinc phosphate glasses[END_REF] . Zn has a coordination number ranging from 4 to 6 and it is present in tetrahedral or octahedral configuration [START_REF] Smith | The structure and properties of ternary zinc phosphate glasses for optical applications[END_REF] .

When Pb is added, phosphate glasses undergo gradual structural changes from metaphosphate, to pyrophosphate and to orthophosphate depending on the P/Pb ratio [START_REF] Ahsan | Infrared study of the effect of P 2 O 5 in the structure of lead silicate glasses[END_REF] .

This suggests a gradual decrease in the number of bridging oxygens and increase in the resonance behaviour of non-bridging oxygens as the mole percentage of metal oxide (PbO) increases in the glass [START_REF] Dayanand | Structural investigations of phosphate glasses: a detailed infrared study of the x(PbO)-(1-x) P2O5 vitreous system[END_REF] .

The presence of copper lead to an increase in the amount of Q3 species, resulting in the formation of non-bridging oxygen as CuO breaks P=O bonds with the formation of P-O-Cu covalent bonds [START_REF] Bae | properties and structure of copper phosphate glasses[END_REF] .

The addition of Ba provides ionic cross-linking between non-bridging oxygens (NBOs), and that results in the decrease of isolated Q0 units. In other words, the decrease of the Q0 content leads to an increase in Q2 units [START_REF] Sastry | Spectroscopic characterization of manganese-doped alkaline earth lead zinc phosphate glasses[END_REF] , [START_REF] Harada | Effect of B2O3 Addition on the Thermal Stability of Barium Phosphate Glasses for Optical Fiber Devices[END_REF] .

Due to the hygroscopic property of the phosphate network, water will react to create P-OH groups that will be part of the structure acting as chain terminator. Due to this, the concentration of Q2 tetrahedra increases and the number of Q3 types decreases [START_REF] Brow | The short range structure of sodium phosphate glasses I. MAS NMR studies[END_REF] .

Composition

Chemical composition and structure are very interdependent and contribute to determine the properties of that particular material (phosphate glass in this case). In the previous chapter (1.2.1) the structure of Ca-Na-P ternary glass was discussed and the effect of adding different ions (dopants) on the organization of the phosphate glass network was also examined. In this section, their effect on the dissolution rate is investigated.

Another property that has attracted the interest of researchers on phosphate glasses for medical applications is the possibility to tailor the dissolution rate of the glass in agreement with the host tissue by tuning the composition. The ternary system Ca-Na-P is very sensitive to the composition. The reciprocal effect of Ca and Na on the stability of the phosphate glass network has been extensively studied [START_REF] Franks | Development of soluble glasses for biomedical use Part I: In vitro solubility measurement[END_REF] . It is well known that increasing the concentration of calcium at the expense of Na makes the glass network more resistant to the hydrolytic action of the water. It has been proved that increasing the Ca content, and decreasing Na, reduces the solubility of the glass. Meanwhile, increasing Na creates a faster solubility of the network. This phenomenon is understandable and explainable by looking at the structure of such a glass discussed in the previous chapter (1.2.1). The higher coordination number of Ca compared to Na provides for a higher crosslink between the phosphate chains.

In turn, this generates a more compact network then if a high amount of Na was present. A denser structure is more resistant to the penetration of the water resulting in greater stability in aqueous environments. This effect is even more evident considering that Na prefers the surface of the glass rather than the bulk as Ca does [START_REF] Ruiz Hernandez | Molecular dynamics simulations of bioactive phosphate-based glass surfaces[END_REF] . This along with the drop in the coordination number gives an understanding of why by increasing Ca and reducing Na the glass is more stable.

Li and K belong to group 1 of the periodic table like Na however, the ionic size differs.

Li is smaller than Na whereas K has the biggest radius. This affects the field strength of the respective ions and so the depolymerisation (disruption ability) effect that each of them has on the network. Considering all this, it is possible to conclude than the durability of the glasses decreases in the order of Li>Na>K [START_REF] Bunker | Phosphate glass dissolution in aqueous solutions[END_REF][START_REF] Knowles | Investigation of the solubility and ion release in the glass system K2O-Na2O-CaO-P2O5[END_REF] .

Mg [START_REF] Franks | The effect of MgO on the solubility behavior and cell proliferation in a quaternary soluble phosphate based glass system[END_REF] , Sr [START_REF] Fredholm | Influence of strontium for calcium substitution in bioactive glasses on degradation, ion release and apatite formation[END_REF] and Fe 37 (as ferrous) have the same effect as Ca: their presence in the glass composition reduces the glass dissolution rate. In general, it is possible to conclude that replacing alkali with alkaline earth elements enhances the durability of the glass.

Trivalent cations such as Ti [START_REF] Brauer | Effect of TiO2 addition on structure, solubility and crystallisation of phosphate invert glasses for biomedical applications[END_REF] , Al and Fe 37 (as ferric) have been found to show a significant effect on the dissolution rate of the glass network due to a resultant strengthening of the phosphate network. Their occurrence in the glass composition, strongly increases the durability of the glass. Overall, high valence ions decrease the dissolution of the glass by increasing the network connectivity through the ability of each ion to link with 3 or more P-OH (or P-O-) groups.

Putting these considerations all together it is possible to conclude then the hydration energy (defined as the amount of energy released when one mole of ions is combined with water)

for the phosphate glasses decreases in the order Q2>Q1>Q0.

It possible also to add small amounts of other network formers beside phosphate (that remains the main component in any case). The most frequently added network formers are Si and B. They drastically improve the glass durability by increasing the connectivity number of the glass network. In other words, they exert their effect by increasing the average number of bridging anions (bridging oxygen) per network unit [START_REF] Gao | Effect of composition on the release kinetics of phosphate controlled release glasses in aqueous medium[END_REF] .

Fluoride has a singular effect on the dissolution properties of the phosphate glass.

Addition of (F -) reduces chemical dissolution via increasing network connectivity due to its ability to induce re-polymerization via stripping the modifiers (cations) from phosphate glass network [START_REF] Shaharyar | Structure-solubility relationships in fluoride-containing phosphate based bioactive glasses[END_REF] .

Since the main component of phosphate glasses is the phosphate that act as a network former, changing its concentration has a deep impact on the dissolution rate of the network.

Specifically, it was found that by reducing the P2O5 content the durability of the glass increases [START_REF] Döhler | 31 P NMR characterisation of phosphate fragments during dissolution of calcium sodium phosphate glasses[END_REF] . This is due to changes in the structure of the glass network from long phosphate chains more prone to hydrolysis, to shorter chains more resistant to hydrolytic attack.

Synthesis methods

The main route to synthesize phosphate-based glasses is melt-quenching. To overcome the limitations of this technique (see 1.2.3.1), during the last ten years, sol-gel methodologies have been increasingly researched. However, this method also has some issues that require attention. For example, the toxicity of some precursors, solvents used and the volatility of the phosphate precursors. To this end, other techniques have been developed such as precipitation and coacervate processes.

Here, a brief description of those techniques with advantages and disadvantages of each is provided.

Melt-quenching

In melt-quenching a mixture of oxides (or carbonates) is melted together in a kiln at temperatures often higher than 1000 °C. Subsequently, the homogeneous molten mixture is rapidly quenched (cooling) to obtain a solid material. The quenching rate is very crucial: this part has to be performed as fast as possible to avoid the formation of nucleation centres where crystals can develop. If the cooling is made very fast, the melt's disordered state is maintained in the final solid state and a glassy material with 100% amorphous phase is obtained.

A further step can be added to increase the stability of the network of the glass. This process is called annealing. Annealing a glass consists of slowly cooling hot glasses after they have been formed below their glass transition temperature for a given time. This additional thermal treatment removes all the residual internal stresses introduced during the manufacture and increases stability and crack toughness of the material. Such a process is critical for the production of 3-D shaped glass products but not critical for powder glass applications such as those explored in this thesis.

The melt-quench technique has some drawbacks mainly coming from the high temperature and precursors used. The elevated temperature processing removes opportunities for adding any bioactive molecule to the glass pending the decomposition of the substance. The nature of the technique gives a highly compact material with no porosity.

The raw materials used can compromise the purity of the glass (unless highly expensive high purity materials are sought) and in general its homogeneity. On the other hand, the technique presents many advantages. For example, scalability to tonnage level and higher, absolute reliability (in terms of composition and phase), atoxicity, easier to control (fewer process variables to deal with) and it guarantees for high yield in a short time.

Sol-gel

To overcome all those limits, the sol-gel method represents a valid alternative to conventional melt-quenching [START_REF] Pickup | New sol-gel synthesis of a (CaO)0.3(Na2O)0.2(P2O5)0.5 bioresorbable glass and its structural characterisation[END_REF] . The advantages of the method are: room temperature chemistry (low energy required and compatibility in terms of co-mixing with organic actives during processing), high purity and homogeneity, the possibility to explore new compositions otherwise precluded and porosity. Beyond all these attractive properties, the most important advantage is the possibility to fine tune the structure and surface features of the sol-gel glass [START_REF] Brinker | Sol-gel Science: The Physics and Chemistry of Sol-gel Processing[END_REF] . This opens up control of pore size, pore volume, degree of porosity, type of pores and degree of surface roughness (all things are not achievable with melt-quenching). However, this introduces a lot of variables that potentially need to be controlled. Choosing the right precursors, aging time and temperature, pH, type of precursors and order of addition, to name a few, are critical for consistently producing the desired sol-gel material.

When it comes to phosphate glass sol-gel formation, the processing becomes even more challenging: despite the basic unit in the glass network being tetrahedral, as for silica (SiO4) 4-, the monophosphate is not as good a gel former as silica due to different chemical behaviour in solution [START_REF] Durif | Topics in Phosphate Chemistry[END_REF] ; in addition, much less work has been done on the sol-gel synthesis of phosphates as compared with silicates. Phosphoric acid cannot be used because of its high reactivity in water leading to salt precipitation instead of gellation. Phosphate esters have a slow hydrolysis and lead to loss of phosphorus from the composition during calcination due to volatility. Phosphoryl chloride has the same drawback of high phosphorus loss.

Improvements have been seen through the use of alkyl phosphates [START_REF] Livage | Sol-gel synthesis of phosphates[END_REF][START_REF] Carta | Structural studies of bioactive sol-gel phosphate based glasses[END_REF] : they are more reactive and generate less phosphate loss. An alkoxide is the conjugate base of an alcohol, therefore it consists of an organic group bonded to a negativelly charged oxygen atom of the phosphate. These compounds are quite easy to obtain by mixing phosphoric acid with the parental alcohol. In the literature, the two most commonly used phosphate alkoxides are buty-phosphate (as mixture of 1:1 of mono and di-butyl) and ethyl-phosphate (mono-and diester mixture) [START_REF] Lee | Sol-gel synthesis of phosphate ceramic composites I[END_REF] . The latter with a lower boiling temperature and lower polarity effect, ensures shorter drying time and better solubility of the modifier precursors.

To ensure homogeneity into the developing sol-gel system, when adding modifiers, corresponding metal alkoxides are often used. The main modifiers are Ca and Na, usually in the form of calcium methoxyethoxide, 20% in methoxyethanol and sodium methoxide solution 25 (or 30) wt. % in methanol. These are added to the phosphate precursor. NaOCH3 is a strong Lewis base and it is believed to activate P-alkoxides and start the polymerization.

Due to their high reactivity in water, anhydrous conditions are preferred during the preparation of the sol-gel. The extreme toxicity of those precursors and solvents represent the real barrier for scale-up and commercialisation of this technique. In order to get over the problem, researchers have developed safer methods to produce phosphate glasses by soft and wet chemistry (refer to 1.2.3.3, 1.2.3.4 and 1.2.3.5).

The sol-gel process occurs when elements are used to form a sol from which a homogeneous amorphous gel is obtained by transition from a liquid sol to a gel. This process can be divided in different steps.

Preparation of the sol

The first step is the preparation of the sol. It is achieved by mixing components (precursors) in solution to generate a series of successive reactions leading to the formation of the sol. The sol is a colloidal suspension of solid particles that do not adhere to one another, the particles are completely dispersed in the solvent(s).

Colloid formation

When particles start to grow through further aggregation a colloid is formed. Colloids are bigger solid particles dispersed in a liquid with an amorphous or crystalline structure.

Gel formation

During the formation of the gel, particles cluster together to produce a continuous polymeric three-dimensional network containing the fluids.

An important parameter of a gel is the adhesion number (or coordination number) defined as the number of adhesive points of each particle and it depends on the mechanism of gelation. It determines the structure of the gel and its strength: if the adhesion is strong enough the gel does not crack when the water is removed (high adhesion number) otherwise it can crack and form particles / fragments.

Ageing

Ageing, if controlled can be used to strengthen and protect the gel network from cracking during the drying step. Throughout this process more bond formation occurs, and polymers and particles continue to aggregate and attach themselves. Through this process the gel shrinks, and the liquid is pushed out of the pores. This contributes to determining the final structure of the gel.

Drying

In this step the liquid is removed by evaporation. At the very beginning, the more external liquid vaporises determining the volume reduction of the gel; subsequently, due to an increase in capillary pressure, liquid trapped deeper in the gel structure and pores starts to come out and evaporate creating further shrinkage of the network. It is at this stage that cracks are likely to occur.

Thermal treatment

If necessary, the dry gel can undergo thermal treatment with the aim to (i) further densify and stabilize the network and (ii) remove any remaining organics. Failure to perform (ii) properly can lead to carbon entrapment. It is important to note that if an amorphous glassy material is required, the heat treatment should be performed below the glass crystallization temperature. Besides this, temperature ramping, holding time and cooling rate are parameters that have to be optimised accordingly.

Sometimes, a washing step before the thermal treatment might be required to render the dry gel free from organic solvents and by-products of the reaction.

Glass by precipitation

This method [START_REF] Soulié | Development of a new family of monolithic calcium (pyro)phosphate glasses by soft chemistry[END_REF] is based on the possibility to obtain an amorphous solid (pyro)phosphate glass from two liquid solutions. A solution of calcium chloride is added to a pyrophosphate precursor salts solution (tetrapotassium pyrophosphate). A suspension starts to form and ultimately sediments at the bottom of the reaction vessel.

This method has the advantages of being easy to perform and safe, as the only solvent involved is water. In addition, it is a room temperature reaction and fast (no heat treatment required). However, there are several limitations with such a technique that are deemed to be not favourable for further development of useful dental materials. First of all, the phase of the material is strongly dependent on both Ca/P ratio and pyrophosphate salt used. In the first case by increasing the Ca/P ratio the material becomes more and more prone to unwanted crystallization. In the second case, replacing the tetrapotassium pyrophosphate salt with tetrasodium pyrophosphate seems to inhibit the formation of any amorphous solid material. This is due to the different size and ionic strength of the cations Na and K. Thus, there are significant limitations to the range of possible compositions. Furthermore, the yield is very low and Ca-dependent: to increase the yield it is necessary to increase Ca concentration. However, by doing this the Ca/P will increase so the phase of the material will change from amorphous to crystal.

Coacervation process

The coacervation process [START_REF] Pickup | Characterisation of phosphate coacervates for potential biomedical applications[END_REF] generates an aggregation of colloidal droplets held together by electrostatic attractive forces. In general, the coacervation process involves the liquid-liquid phase separation of a homogeneous solution of charged macromolecules resulting in a dense, polymer-rich 'coacervate' phase which coexists with its supernatant. In relation to PBG, the process entails the addition of a high concentrated solution of polyvalent cations (CaCl2, MgCl2 etc.) to an almost saturated solution of sodium polyphosphate (Graham's salt [START_REF] Van Wazer | Phosphorus and its compounds[END_REF] ). When in solution, electrostatic interactions between the long polyphosphate chains and polyvalent cations lead to the formation of the dense coacervate phase. The coacervate phase can be extracted and dried to form a solid glassy material.

The advantages of this route are numerous: It is a safe process as only water is required; a totally amorphous glass material is obtained (the phase is also not dependent on any cation/anion ratio); the reaction is easy and fast to perform (no thermal treatment) and involves a room temperature reaction; there exists the possibility to add additional cations (this widens greatly the range of compositions it is possible to explore). Finally, there is the possibility to control reaction and chain length by adjusting the pH at which the reaction occurs. The only limit is that it is physically impossible to incorporate any anion in the composition due to the repulsive force that is generated between the phosphate and the anion (both are negatively charged).

In addition, it seems that the material obtained with this technique lacks porosity;

however, one study 51 reported a type II hysteresis for a coacervate phosphate glass after thermal treatment. Due to this, and some doubts about the reliability of the actual composition matching the expected one, the melt-quench technique was ultimately chosen as the preferred route to produce the glasses argument of this thesis.

Other methods

Recently an amorphous calcium phosphate porous material has been synthesized through a microwave-hydrothermal method using fructose 1,6-bisphosphate trisodium salt as an organic phosphorus source and CaCl2 52 .

Dissolution behaviour

One of the most important properties of phosphate glasses is their ability to dissolve in aqueous environments: the POP bond of the glass network is broken down by the hydrolytic action of the water and eventually ions (modifiers) spread into the aqueous medium to become available as Mx+ aqueous ions.

According to literature, the network breakage happens in two steps: hydration and hydrolysis [START_REF] Gao | Dissolution mechanism and release kinetics of phosphate controlled release glasses in aqueous medium[END_REF] . In the first one, hydration, an exchange reaction takes place at the glass-water interface between the high mobility ions (mainly Na) and the protons (H3O + ) from the water.

Protons replace Na ions in the glass network and the ions are dissolved in the water. As a consequence of this process, a hydrated layer forms on the surface at the glass-water interface level. During the second step, hydrolysis, the actual breaking of the network takes place due to the cleavage of the POP bonds by the water at the level of the hydrated layer.

As a result, the phosphate glass network is broken down and phosphate chains of different length are released into the water. This step generates decrease of pH and so acidification of the medium.

It is known that three main factors affect the dissolution of the glass network and they are pH, ionic strength of the medium and formulation (directly correlated to the structure).

pH

The dissolution process is strongly dependent on the pH of the media. In particular, hydrolysis is sensitive to the pH. The phosphate glass network is stable around neutral pH [START_REF] Bunker | Phosphate glass dissolution in aqueous solutions[END_REF] , but by reducing the pH the dissolution rate increases dramatically [START_REF] Delahaye | Acid dissolution of sodiumcalcium metaphosphate glasses[END_REF] . This happens because in acidic solutions, protons enhance the POM bond disruption resulting in a higher rate of hydration. The rate of dissolution is also enhanced in basic solutions, but the effect is less pronounced [START_REF] Bunker | Phosphate glass dissolution in aqueous solutions[END_REF] .

Ionic strength of the medium

The dissolution rate of the phosphate glass network decreases with increased ionic strength of the medium the glass is soaked in [START_REF] Cerruti | Effect of pH and ionic strength on the reactivity of Bioglass 45S5[END_REF][START_REF] Icenhower | The dissolution kinetics of amorphous silica into sodium chloride solutions: effects of temperature and ionic strength[END_REF] . It is thought that this is due to saturation or limitation of reactant transport away from the dissolving glass. Also, ion exchange can be a cause of the decrease in dissolution rate. It means that, at the level of the hydrated layer, a saturated micro-domain might be created limiting the exchange reaction. Otherwise, a competition between the ions from the glass to the medium and/or from the medium to the glass might explain the increased resistance of the glass to dissolution.

Glass formulation

The dissolution of the phosphate glass network is a formulation-dependent process.

A pure PBG (without modifiers) is made by PO4 groups forming chains and rings and many

NBOs. Due to the presence of the double bonded oxygen, only 75% of the oxygen is bridging giving 25% of NBOs making the POP links easy to hydrate. The addition of modifiers (M) results

in the replacement of the POP bonds with POM links which consequently increases the durability of the glass. In particular, monovalent and divalent cations occupy "network modifying" positions; whereas, multivalent cations occupy "network forming" positions. The first, by coordinating inter or intra-chains oxygens, densify and stabilize the network. The latter, by substituting the P in the POP bond and creating a cross-link between the NBOs of two phosphate chains, increase the network connectivity giving a great and positive effect to the stability and durability of the network [START_REF] Sharmin | Compositional dependency on dissolution rate and cytocompatibility of phosphate-based glasses: Effect of B2O3 and Fe2O3 addition[END_REF][START_REF] Sharmin | Structure, thermal properties, dissolution behaviour and biomedical applications of phosphate glasses and fibres: a review[END_REF] . (For further details on the effect of the composition see chapter 1.2.2).

Species released

When a phosphate glass system is soaked in water, several anions and cations are released whose type and kinetics depend on time and concentration [START_REF] Ahmed | Quantification of Anion and Cation Release from a Range of Ternary Phosphate-based Glasses with Fixed 45 mol% P2O5[END_REF][START_REF] Ahmed | Quantification of anions and cations from ternary phosphate based glasses with fixed 50 and 55 mol% P2O5 using ion chromatography[END_REF] .

Cation release

The types of cation released are strictly dependent on the type of modifier oxides included in the composition. The two most common cations used and studied are Na and Ca.

When such a glass is soaked in water, they are released as a consequence of glass network degradation. In particular, Na is the first to be released due to the exchange reaction mentioned previously. Ca is then released as the glass network is broken down more and more.

Other cations like Sr, Li etc. can be leached out if present in the composition. The associated kinetics depend on their concentration, glass soaking time and type of ions.

Valency, field strength charge and coordination number of additional ions influence the overall dissolution rate of the glass network and so the relative leaching speed by determining changes on the structure as discussed previously.

Anion release

Since phosphate is the network former in these glasses, a range of anions can be released during dissolution. Those species have been identified as PO4 3-, P2O7 4-, P3O9 3-and P3O10 5-. P2O7 4-and P3O10 5-are considered branched anions, whereas P3O9 3-is unbranched. In addition, P2O7 4-and P3O10 5-were found to be released at earlier time points; PO4 3-and P3O9 3- required more time to be detected in water, however P3O9 exhibits the highest rate of release.

By comparing the release kinetics of Ca2 + with PO4 3-, P2O7 4-and P3O10 5-and considering that the last two polyphosphates are branched, it was supposed that those anions exerted a chelating action on the Ca ions. On the other hand, due to the unbranched nature of PO4 3- and considering the release rate of Na + , it was thought the kinetics might be linked to Na taking up the residual charge. In addition, P3O9 3-anionic species exhibit the highest rates of release. Putting all these factors together, it seems that P3O9 3-dominates the degradation rates of the glass.

Considering the chelating effect of the phosphate anions towards Ca, and their branched/unbranched nature, a further explanation for the observation that increasing Ca results in a reduced glass dissolution rate supports the previous considerations.

Bioglasses: medical applications

Due to their osteoconductive and osteoinductive abilities, bioglasses (BGs) have attracted attention in tissue engineering, especially for mineralized tissue. When their surface is soaked in a fluid, it undergoes complex ion exchange reactions with the medium leading to the formation of a HA layer. This mechanism can explain the strong ability of BGs to bind bones.

Moreover, this layer plays an important role in favouring cells migration and adhesion.

Bioglasses can be produced by melt-quenching and sol-gel routes. By SG, lot of important therapeutic ions can be incorporated into the glass network: for instance, Cu and Co have to promote angiogenesis; Li and Sr to trigger bone development through the wnt pathway.

Moreover, biogactive glasses have been shown to regulate gene expression and promote cells differentiation. Due to their high resistance in water, the effects of a long-term exposure to such resistant materials are still debated. Thus, PBGs, a new generation of (bio)resorbable materials, are under studying for medical usage.

Phosphate-based glasses for biomedical applications

Phosphate-based glasses have attracted huge interest in the field of biomaterials and tissue engineering due to their properties: chemical similarity with the natural components of bone and teeth (no inflammatory response), water solubility (bioresorbable material) and a controllable degradation profile.

The simplest and easiest phosphate glass is the binary Na-P glass. Despite evidence showing that it does not trigger any inflammatory response 61 , the biocompatibility is absolutely time dependent. Upon long term culture, cells struggle to adhere to the glass due to the advanced state of dissolution [START_REF] Gough | Synthesis, degradation, and in vitro cell responses of sodium phosphate glasses for craniofacial bone repair[END_REF][START_REF] Gough | Long-term craniofacial osteoblast culture on a sodium phosphate and a calcium/sodium phosphate glass[END_REF] . The network solubility seems to be the factor that influences the most the ability of those materials to support cell adhesion and proliferation:

if the glass dissolves too quickly, cells will lose their support and die [START_REF] Salih | Development of soluble glasses for biomedical use Part II: the biological response of human osteoblast cell lines to phosphate-based soluble glasses[END_REF] . In addition, the dissolution rate, in terms of ion release, has a deep effect on the environment: the glass dissolution rate and by-products have been associated with decrease in the pH (due to increased release of HPO4 and PO4 3-species eventually leading to bioincompatibility) and osmotic pressure and this may well influence how cells behave [START_REF] Uo | Properties and cytotoxicity of water soluble Na2O-CaO-P2O5 glasses[END_REF] .

As mentioned before, Ca has a strong effect on the dissolution rate of the glass network and by varying its concentration it is possible to tailor the degradation rate of the glass.

Glasses with low calcium content have higher solubility with a deleterious effect on cells. On the other hand, glasses with high Ca content have lower solubility and better biocompatibility [START_REF] Uo | Properties and cytotoxicity of water soluble Na2O-CaO-P2O5 glasses[END_REF][START_REF] Salih | Development of soluble glasses for biomedical use Part II: the biological response of human osteoblast cell lines to phosphate-based soluble glasses[END_REF] . This could be linked to a more stable and life-compatible pH and/or osmotic pressure associated with a more controllable ion release rate. In addition, Ca was proved to enhance the expression of some important bone marker genes in osteoblast-like cells [START_REF] Fujita | Phosphate provides an extracellular signal that drives nuclear export of Runx2/Cbfa1 in bone cells[END_REF] . Therefore, the most relevant formulation of PBGs for medical applications is the P2O5-CaO-Na2O ternary system typically ranging from 35-55 mol% P2O5 (encompassing ultraphosphate, metaphosphate and polyphosphate glasses) and at least 40 mol% of CaO.

Doping of PBGs for improved biomedical applications

From what has been said so far, it is clear that the behaviour of the phosphate glass strictly depends on its composition and the presence of further elements. It is possible to classify the dopants under three categories: elements with a deep impact on the dissolution behaviour of the glass; elements able to promote differentiation (therapeutic ions); and dopants with antibacterial effect. Using one rather than another or a combination depends on the application.

Other therapeutic ions that can be added to the glass are for example Sr and Zn. Strontium is able to promote osteoblast activity, bone regeneration and inhibit bone resorption by inhibiting osteoclasts [START_REF] Saidak | Strontium signaling: molecular mechanisms and therapeutic implications in osteoporosis[END_REF] , [START_REF] Massera | The influence of SrO and CaO in silicate and phosphate bioactive glasses on human gingival fibroblasts[END_REF] . Additionally, Sr enhances HA formability by increasing Ca absorption at the earlier stages and, because of its high similarity with Ca, it can be incorporated into the HA increasing bone density. For all those reasons, Sr is the good candidate for the purpose of this thesis. Zinc, on the other hand, drastically promotes cell adhesion at the surface by increasing hydrophilicity [START_REF] Salih | Zinc-containing phosphate-based glasses for tissue engineering[END_REF] .

Ti [START_REF] Neel | In vitro bioactivity and gene expression by cells cultured on titanium dioxide doped phosphate-based glasses[END_REF] , Fe [START_REF] Ahmed | Processing, characterisation and biocompatibility of iron-phosphate glass fibres for tissue engineering[END_REF] and Mg [START_REF] Ahmed | Cytocompatibility and effect of increasing MgO content in a range of quaternary invert phosphate-based glasses[END_REF] have a strong effect on the dissolution rate: by varying their concentration in the glass it is possible to increase glass network durability accordingly to the host tissue. The presence of any one of those ions increases the biocompatibility of the glass by providing a more durable support for cells to adhere and proliferate.

As mentioned before, combinations of these ions can be employed to further improve cell response to phosphate glass. Ti and Ca [START_REF] Neel | In vitro bioactivity and gene expression by cells cultured on titanium dioxide doped phosphate-based glasses[END_REF][START_REF] Navarro | Cellular response to calcium phosphate glasses with controlled solubility[END_REF] or Ti and Zn [START_REF] Qaysi | Degradation of zinc containing phosphatebased glass as a material for orthopedic tissue engineering[END_REF] have been proved to strongly enhance a cell response to the glass. It was believed that this was due to the beneficial effect of the ions and a more reactive surface.

Fluoride releasing glasses have been proved to be able to release fluoride in the oral cavity to aid remineralization of carious lesions [START_REF] Pessan | Slow-release fluoride devices: a literature review[END_REF] .

Since phosphate is the main component in phosphate glasses, varying its concentration can have an effect on the dissolution rate of the network and in turn on cell response. By increasing phosphate content in the glass, the cytotoxicity decreases. This is related to a more stable glass network leading to a more stable environment for cells especially in term of pH change [START_REF] Uo | Properties and cytotoxicity of water soluble Na2O-CaO-P2O5 glasses[END_REF] .

Phosphate glasses are hygroscopic materials and wettability or hydrophilicity has been associated with enhanced protein adsorption and, consequently, cell adhesion on biomaterials [START_REF] Shelton | Protein adsorption at the interface between charged polymer substrata and migrating osteoblasts[END_REF] .

Due to their tuneable dissolution rate, another clinical application for phosphate glasses is as a carrier of antibacterial agents. Placed close to an injury, these glasses can release antibacterial ions to help wound-healing. Alternatively, as a coating layer for implants, catheters or tubes where nonsterile biological liquids flow, they can be used to prevent the growth of microorganisms introduced unwittingly as infection during surgical practices [START_REF] Gristina | Biomaterial-centered infection: microbial adhesion versus tissue integration[END_REF] .

Examples of antibacterial ions incorporated into phosphate glasses are Al, Cu and Ga. Al was proved to be effective against S. sanguinis, S. aureus, E. coli and C. albicans [START_REF] Ahmed | Antimicrobial effect of silver-doped phosphatebased glasses[END_REF] . Cu was shown to kill S. sanguinis biofilm [START_REF] Mulligan | The effect of increasing copper content in phosphate-based glasses on biofilms of Streptococcus sanguis[END_REF] and prevent snail hosts of schistosomiasis. Ga has been shown to be effective in inhibiting the growth of both Gram positive (S. aureus and C. difficile)

and Gram negative (E. coli and P. aeruginosa) bacteria [START_REF] Valappil | Controlled delivery of antimicrobial gallium ions from phosphate-based glasses[END_REF] . The most effective element for killing the bacteria has proven to be the Al followed by Ga and Cu. The advantage of using such a method instead of the more conventional antibiotics is to avoid the occurrence of resistant strains.

The recent developments in phosphate-based glasses sol-gel chemistry allows the incorporation of biologically active molecules that are not stable to high temperature (such as proteins, antibiotic and other drugs). The resultant materials have potential to be used in devices to target the delivery of such molecules in the human body and provide controlled, sustained release in situ therapy. Finally, there exists the potential to coat biomedical devices with a sol-gel derived antimicrobial coating via the inclusion of biocidal metal ions such as Ag [START_REF] Zhang | Sol-Gel Synthesis of Al2O3-P2O5 Glasses: Mechanistic Studies by Solution and Solid State NMR[END_REF] and Ga [START_REF] Pickup | Preparation, structural characterisation and antibacterial properties of Gadoped sol-gel phosphate-based glass[END_REF] .

Phosphate glass-based scaffolds and composites

There is much interest in the development of composites for biomedical applications and in particular for hard tissue surgery. The driving force for this interest is due to the possibility of producing materials with a high resistance to fracture (fracture toughness) and also to develop implants with a stiffness close to that found in bone.

The possibility of producing degradable composites has elicited much research and meltquench phosphate-based glasses may be used as an initial reinforcing phase that leads ultimately to completely degradable composites [START_REF] Knowles | Development of a degradable composite for orthopaedic use: in vivo biomechanical and histological evaluation of two bioactive degradable composites based on the polyhydroxybutyrate polymer[END_REF] . The advantage of using these glasses for the production of composites compared to conventional additives such as hydroxyapatite is that more variation in the composition of the glass filler phase is available thus allowing tailoring of the end properties, including mechanical properties and control of the overall dissolution rate of the composite [START_REF] Knowles | Phosphate based glasses for biomedical applications[END_REF] .

Moreover, PBGs could be used in combination with either natural (e.g. collagen) or synthetic polymers (e.g. PCL and PLA) to help the in-growth of vascularisation and the diffusion of nutrient and waste through 3D scaffolds for soft and hard tissue engineering: e.g., the engineering of muscle, ligament, tendon, or bone [START_REF] Knowles | Development of a natural degradable polymer for orthopaedic use[END_REF] .

For the past decade, collagen has been the most widely used biomaterials for biomedical applications, due to its excellent biological features and physicochemical properties. Threedimensional collagen scaffold materials have been designed to mimic one or more of the bone-forming components, to facilitate the growth of vasculature into the material, and to provide an ideal environment for bone formation. The mechanical properties of these porous scaffolds were generally poor, therefore porous collagen scaffolds have been combined with ceramic or glass particles for bone tissue engineering purposes. Based on the advantageous properties of degradable materials, PBGs have been incorporated within the collagen scaffold matrix for medical application. Collagen-based porous scaffolds containing PBGs have been used for skeletal muscle regeneration [START_REF] Shah | Sequential identification of a degradable phosphate glass scaffold for skeletal muscle regeneration[END_REF] , guided nervous tissue regeneration [START_REF] Kim | Phosphate glass fibres promote neurite outgrowth and early regeneration in a peripheral nerve injury model[END_REF] , and for bone regeneration [START_REF] Ferreira | Collagen for bone tissue regeneration[END_REF] .

Whilst sol-gel phosphate glasses might not exhibit significant porosity (possibly as a result of the necessary heat treatment to removes organic solvents), but their use with bioactive polymers may well produce materials with improved mechanical properties for use in tissue engineering reconstruction. Such composites can also be used to produce flexible porous foams, which have potential applications as tissue engineering scaffolds.

In this thesis, a new application for collagen-based scaffolds containing PBGs is proposed:

the possibility to use those materials for dental pulp regeneration and cell-mediated remineralization is suggested.

Summary

In using these materials, it is important to consider how the structure gives rise to physiochemical and biomedical properties and how these properties can be controlled by manipulating the composition. Perhaps the most important property of phosphate glasses is the solubility which determines how fast ions are released from the material, how well cells attach and proliferate and how chemically durable the glass is. It has been shown that varying the concentration of metal ions in the composition can be used to control the solubility. For example, the dissolution rate of Ca-Na-P glass exhibits an inverse trend with respect to the Ca concentration. Measurements show that the Ca ions induce structural changes in the phosphate network where the connectivity varies, giving rise to different solubility. PBGs can be incorporated into collagen scaffolds to create a complete biodegradable material for medical applications.

3 Materials and methods

Materials

A complete list of all the reagents and solvents used for the experiments described in this thesis, together with their manufacturers and abbreviations is provided below:

n-

Methods

Glass synthesis

Sol-gel

The sol-gel glasses were synthesized as follows. Briefly, under constant stirring, n-butyl phosphate (Bt/P) was added dropwise to sodium methoxide (NaOMe) in a vertical three-neck round-bottom flask and left mixing for 1h. Afterwards, the Ca precursor (Camethoxyethoxide, CME) was added dropwise. The resulting ternary mixture was left stirring for 24hrs at room temperature.

The following day, the temperature was raised up to 60 and the sols were left aging under a reflux condenser and constant stirring for one week. After this period, the wet gels were poured into glass beakers and put in the oven at 120 for about 10-15 days. To help with the removal of the solvents and unreacted precursors, the dried gels were washed twothree times with absolute ethanol and filtered. The obtained powders were put in glass Petri dishes and placed in the oven at 120 for 1h to ensure full ethanol evaporation. The dried gels were milled down using a pestle and mortar and calcined in an electric kiln at the following settings: ramp 1 /min up to 250 and holding for 15min. A schematic representation is shown in fig. 1 (adapted from [START_REF] Pickup | New sol-gel synthesis of a (CaO) 0.3 (Na 2 O) 0.2 (P 2 O 5) 0.5 bioresorbable glass and its structural characterisation[END_REF] )

To test the effect of the phosphate precursors, samples were prepared in which ethylphosphate (Et/P) was used instead of Bt/P without altering further the protocol.

To verify the actual applicability of less toxic sources both for Ca and Na, calcium ethoxide (CaOEt) and sodium ethoxide (NaOEt) were used by replacing both Camethoxyethoxide and NaOMt without varying the procedure.

addition was complete to ensure homogeneity. The stirring was then stopped, and the mixture allowed to stand for 1 hr. During this time two layers formed: an aqueous layer and a coacervate layer. The coacervate layer was twice washed with UPW before leaving to stand overnight. After removal of the water layer, a clear, viscous coacervate remained. The coacervate layer was dried at room temperature in a vacuum desiccator containing silica gel for several days to produce a friable powder.

To prove the possibility of incorporating Sr into the glass by using this procedure, at first, CaCl2 was totally replaced by SrCl2 • 6H2O (2M). Subsequently, in order to partially replace Ca with Sr in the composition, the Sr salt was solubilised along with the Ca:

Specifically, 1M of SrCl2 was mixed with 1M of CaCl2 prior addition the Graham's salt.

Melt-quenched glasses

The melt-quenched glasses were prepared as follows (fig. 3).

Glass characterization

The protocols described below were applied to all glasses independently of the synthesis technique.

Semi-quantitative X-ray fluorescence (XRF)

This analysis was performed using a Panalytical Axios XRF Spectrometer. A small amount of material was smeared onto a filter paper and presented to the XRF spectrometer.

The analysis was run using the preinstalled semi-quantitative XRF package.

X-ray diffraction (XRD)

This analysis was performed using a Bruker D8 Advance run in Bragg-Brentano parafocussing mode. Copper K alpha radiation was used throughout. A fixed divergence slit of 0.3 degrees was used, with a fixed 3 degree receiving slit employed prior to the detector. A Lynxeye 1-d detector was used covering a 3-degree two theta window. 192 channels

Fourier Transform Infrared Spectroscopy (FTIR)

The infrared transmittance spectra were recorded using an SAFAS Monaco IR700. The analytical software used was SAFAS SP2000. The glass powders were placed in the centre of the sample holder (on the prism) and a pressure of 12kg was applied prior to measurement.

The infrared spectra were recorded in the range from 4000 to 600 cm -1 in transmission mode.

pH measurement

The milled glass powders were weighed to create a suspension concentration of 1mg/mL: specifically, 25mg of powder was soaked in 25mL of UPW. The system (powder+water) was sealed in a Falcon tube and left shaking in an incubator-shaker at 37 °C, 120 rpm for 2, 6, 24, 48, 168, and 504 hours. For each time point, to remove the glass particles, the solutions were filtered twice using a 0.2μm filter; the filtrates were collected in clean tubes and the pH measured using a Hanna pH-meter.

Inductively coupled plasma (ICP) coupled with mass spectroscopy

After recording the pH, the solutions created as defined in section 2.

Scanning electron microscopy (SEM)

The powdered samples were coated with a thin layer of gold suitable for examination Light elements such as lithium and boron were not quantified by this analytical technique. Carbon is also omitted.

TGA/DSC analyses

The thermogravimetric analysis (TGA/DSC) was realised in a temperature range from 25°C to 500°C, under air as reactive gas and with a heating rate of 5°C/min. The starting material weight was 15 mg. The device used was the Thermogravimetric Analyzer Mettler Toledo TGA/DSC2.

Scaffold fabrication

Glass milling

To reduce particle size, the glass powders were milled using a Planetary Micro Mill Afterwards, the scaffolds were rinsed with ethanol and deionized water (80:20 ratio) for three times and dried in an oven at 37°C for 24h.

To improve the solubilization of the collagen fibres, some samples were prepared in which 0.02 N of acetic acid (AcOH) was added to the collagen suspension. In addition, to improve the link between the fibres and the glass particles, same scaffolds were prepared with the concentration of the crosslinker increased by 10 times. In the end, four batches were prepared as follows:

Batch 1: without AcOH and 1X of crosslinkers (the control) Batch 2: with AcOH and 1X of crosslinkers

Inductively coupled plasma (ICP) coupled with mass spectroscopy

After recording the pH, the same solutions described in the section 2.2.4.3 were subjected to ICP analysis. Before performing the analysis on the samples, a calibration curve was made in the same way as for the glass particles alone, as described in the section 2.2.2.4.

To fit the calibration curve, the scaffold extracts were diluted twenty times with the 0.6% HNO3 solution and the diluted samples were used for the analysis.

SBF test

170 mg of the scaffolds were soaked in 20 mL of simulated body fluid 89 (SBF) for 1 and 3 weeks at 37 °C, 120 rpm. For each time point, the solutions were filtered twice using a 0.2μm filter. The media were subjected to immediate pH measurement, whereas the scaffolds were dried 24h at 60 °C prior to further analysis.

SEM/EDX

The scaffold samples were analysed, "as received", using SEM in low vacuum mode without any conductive coating. A JEOL JSM6490-LV Scanning Electron Microscope (SEM)

fitted with an Oxford Instruments INCA Energy Dispersive X-ray Analysis system (EDA) was used for examination in accordance with in-house methods M24 and M25. SEM imaging of the scaffold/ fibrous samples was conducted using Backscattered Electron Imaging; using this imaging mode the brightness of a given feature is proportional to its mean atomic number.

Images were taken at different magnifications. Microanalysis results are tabulated on a semiquantitative, normalised, elemental weight basis. Light elements such as lithium and boron are not quantified by this analytical technique. Carbon is also omitted.

The EDX spectra of the scaffolds before and after SBF immersion were collected for 100s at a primary beam energy of 15 keV and at a working distance of 10mm. Objectives: Bioglasses have been widely used in tissue engineering, in particular, for mineralized tissues regeneration. Indeed, they are known for their osteoconductive and osteoinductive properties. Thus, bioglasses seem to be interesting for the regeneration and repair of dental mineralized and periodontal tissues. Several bioglasses have been synthesized whose properties depend on the composition and the synthesis method. The two main methods of elaboration are melt-quench and sol-gel. The latter seems to be promising, allowing the incorporation of bioactive molecules and the control of the porosity. In order to elaborate a new material for dental tissue remineralization, we wanted to have an overview of the literature concerning sol-gel bioglasses applications in dentistry. Thus, the aim of this study is to review the current state of the art of the effects of bioactive glasses produced by sol-gel on cells for dental and periodontal regeneration. The study also discusses associated antibacterial properties. In addition, the methodology and the type of cells used in the included articles to investigate those effects was exanimated in order to underline common points and suggest guidelines.

Results and Discussions:

From an initial 244 articles, after duplicates removal, rejection based on title and abstract and, then full text and addition of articles coming from other sources, 52 papers were included in the manuscript. From those, only 13 have proved both the ability of bioglasses (BGs) to differentiate dental cells at genetic level and their ability of triggering cellmediated mineralization, but only 6 of them showed, along with cells, the antibacterial properties of the glasses. This review shows that sol-gel bioactive glasses are not toxic, can sustain cell proliferation and differentiation at a genetic level and can keep the bacterial population under control. The versatility and the advantages of the sol-gel technique have been demonstrated in terms of applications (coating film, carrier, etc.) and in tailoring the composition to make it more specific in their effects and closer to the real structure of tissues (bioscaffolds). Moreover, the importance of using primary cells instead of cell lines and performing genome analysis was emphasises. Furthermore, by putting together all the data coming from the included studies, an ideal material and a standard protocol are proposed.

Research article: B Bioactivity evaluation of collagen-based scaffolds containing a series of Sr-doped melt-quench derived phosphate-based glasses

Vincenzo Farano, Mark Cresswell, Kerstin Gritsch, Phil Jackson, Nina Attik, Brigitte Grosgogeat, Jean-Christophe Maurin (Accepted: 18 June 2018)

Objectives: Due to their osteoconductive and osteoinductive properties, BGs have been widely used in tissue engineering. However, the effects of a long-term exposure to such resistant materials are still debated. Phosphate-based glasses have been attracting attention due to their possible medical applications arising from unique dissolution characteristics in the human body leading to the possibility of new tissue regeneration. The two main methods of elaboration are melt-quench and sol-gel. However, due to the challenges that the sol-gel route provides, the MQ technique remains the favourite route for those materials. In this study, the effects of increasing Sr concentration at the expense of Ca on phase and solubility in water of a series of Sr-doped PBGs made by melt-quench technique is reported. Afterwards, to mimic the natural 3D structure of tissues, Sr-doped PBG particles were incorporated in collagen based-scaffolds. The influence that the interaction between collagen fibres could have had on the dissolution properties of those particles compare with the row glasses was investigated. The HA formability of such composite materials was tested by soaking the scaffolds in SBF for one and three weeks.

Methods:

The series of S-doped glass with composition (P2O5)53-(CaO)(32-x)-(Na2O)15-(SrO)x, where x = 0, 5, 10 or 15 (mol%) was made by melt-quench technique. The phase was analysed by XRD. To study the dissolution behaviour, the glass particles were soaked in UPW at the concentration of 1mg/mL for several weeks. After, the conditioned media were filtrated and diluted 5 times before the analysis. The glass particles ranging between 0.1 and 10 μm were incorporated into collagen scaffolds at 1:1 wt ratio by freeze-drying and crosslinked using EDC and NHS. Their dissolution kinetics was studied by soaking 170 mg of collagenbased scaffolds in 20 mL of ultra-pure water. To test the HA formability, 170 mg of scaffolds were soaked in 20 mL of simulated body fluid. After drying, the soaked scaffolds were submitted to SEM/EDX analysis.

Results and Discussions:

It was found that Sr did not act as a nucleation sites for crystal growth. However, it influenced the dissolution kinetics of the glasses: by increasing the concentration of Sr in the glass composition, the rate the glass dissolved reduces. It was find as well that the collagen fibres interact with the glass by-products (pyrophosphates and Na 113

Complementary results

Sol-gel glasses

Effect of increasing Calcium (Ca) content

Objectives: Ca content is the parameter that affects the behaviour of fused phosphate glasses the most. In this section the effects of increasing Ca at the expense of Na in a series of sol-gel derived phosphate-based glasses (P2O5 53% -CaO (24+x)% -Na2O (23-x)% with x=0, 8, 16 -mol%) are described.

Methods: For this set of experiments, the glasses were synthesized as follows. Under constant stirring, Bt/P was added dropwise to NaOMe and left mixing for 1h. Afterwards, the Ca precursor (Ca-methoxyethoxide -CME) was added dropwise. Composition, phase and surface features were characterized in agreement with the manual of the instruments.

Results and discussions:

The results indicate that the phosphate network evolves from a 3Dnetwork of phosphate chains linked by Ca to a more disordered system of separated phosphate chains as Ca progressively reduces (or Na increases).

Composition and yield

Table 1 shows the actual composition (in mol%) of the SG glasses. The SG glasses were deficient in P, suggesting losses for volatilisation. Interestingly, the missing percentage of both P and Ca in the samples has been gained by the Na. This perhaps suggests the sodium precursor reacted into the P/Ca/Na oxide structure more readily. Moreover, the table shows the relative yield of the preparations: there is potential for unreacted precursors. The XRD patterns of the tested glasses (fig. 5) showed a broad peak indicating that the materials were totally amorphous despite the progressively increasing Ca levels that might be expected to encourage unwanted crystallisation. It demonstrates elegantly how the sol-gel technique gives products with high amorphous phase purity.

Structure

Further information on the structure and bonding can be gained from infrared analysis. Fig. 6 shows the FTIR spectra recorded for the glasses prepared by sol-gel techniques.

The peaks were assigned as previously described [START_REF] Brow | Review: the structure of simple phosphate glasses[END_REF] . The assignments are listed in table 2 and where possible, the associated Q state is assigned. Based on the shape of the FTIR tracks and in agreement with the previous studies, it is possible to conclude that the atomic scale structure of the three glasses is similar consisting of long chains dominated by Q2 phosphate tetrahedrons.

pH measurement in water

Fig. 7 shows the pH variation after 24 hours of incubation in DI water. The pH dropped down to 3.27, 2.86 and 2.1 considering C24, C32 and C40 glass respectively. It is possible to notice that there is a correlation between the increase of Ca content and the reduction in pH.

Surface features

For the tested glasses, the BET analysis gave a type II hysteresis (fig. 8) typical of nonporous adsorbents 91 . Table 3 summarises pore size, pore volume and surface area of the three materials.

The relationship between Ca content and surface features is shown in fig. 9. No significant difference was found between the total pore volume of C24 and C32, however, in the C40 glass particles it was about 2.3 times larger than the other two glasses. The pore size slightly increases with increasing Ca content. The surface area is directly proportional to calcium: it increases by 1.9 times from C24 to C40, and 2.1 times from C32 to C40. The total mass loss during the analysis was 6.7%, however, those two events alone were responsible of 5.9% of the mass loss. The DSC courve revealed the presence of an exothermic phenomenon between 400 and 460 °C. This phenomenon is often associated to the crystallization and, indeed, an XRD analysis performed on the powder after the thermal analysis (fig. 11) revealed the presence of two main phases, sodium calcium phosphate and calcium oxide phosphate, which were not present before (fig. 1B). Remarkably, no endothermic phenomenon associated to a melting event was detected within the range of temperature considered.

Effect of phosphate precursors

Objectives: The two most commonly used phosphate precursors in sol-gel phosphate glass chemistry are Et/P and Bt/P [START_REF] Livage | Sol-gel synthesis of phosphates[END_REF] . The effects produced on the glass system P2O5 53% -CaO 24% -Na2O 23% (mol%) by using one precursor rather than the other were studied and reported.

Methods: For this study, the glasses were prepared as follows. Bt/P or Et/P was added dropwise to NaOMe and left mixing for 1h. Afterwards, Ca-methoxyethoxide was added.

Composition, phase and surface structure were characterized by using XRF, XRD and BET/BJH.

Results and discussions: By using different phosphate sol-gel precursors it is possible to control the surface characteristics of the glass particles.

Composition and yield

The PBG produced using the Et/P precursor had composition closer to the expected one as compared with the Bt/P precursor. However, the yield seemed to not be significantly improved by the change of the precursor: the yield of the glass produced with Et/P was 5% larger than the glass produced using Bt/P precursor (table 4).

Phase

The broad peak in the XRD patterns measured for the tested glasses (fig. 12) indicated that the materials had an amorphous glassy phase. It is noted that the type of precursors used did not affect the phase in the final material.

Structure

More information on the structure and nature of the bonding can be gained from the infrared analysis. Fig. 13 reports the FTIR spectra of the glasses prepared by sol-gel. The assignments are listed in table 5 along with the related Q states. The spectra of the sol-gel samples displayed the characteristic bands for P-O-P bonding: for the Bt/PBG glass the peaks were identified at 708 cm -1 , 762 cm -1 and 896 cm -1 ;

for the Et/PBG glass the P-O-P bonding peaks were recognized at 698 cm -1 , 722 cm -1 and 896 cm -1 . In both glasses the first peaks represent the P-O-P bond in symmetric configuration, whereas, the second and third peaks characterise the same bond in asymmetric mode. The pairs of the peaks at 1028 cm -1 and 1090 and 1034 cm -1 and 1088 cm -1 were recognised and assigned to the (PO3) 2-bond in the symmetric and asymmetric state respectively for the first and second member of each pair. Further peaks at 1264 cm -1 and 1254 cm -1 belonging respectively to Bt/PBG and Et/PBG glass can be detected and assigned to the asymmetric (PO2) -bond. All the spectra contain Q1 and Q2 units: Specifically, Q1 for the symmetric and asymmetric (PO3) 2-and Q2 for the asymmetric P-O-P and (PO2) -bond.

Based on the shape of the FTIR traces it is possible to conclude that the type of phosphate source used to produce the two glasses did not generate differences in the atomic 123 scale structure of the glasses and in the nature of the bonding. Moreover, it may be concluded that both glasses comprise chains dominated by Q2 units.

pH measurement in water

The aqueous pH dropped to 3.27and 3.20 considering Bt/PBG and Et/PBG glass respectively after 24 hours of incubation in water. It is concluded that the two different precursors lead to no significant difference in pH.

Surface features

For the tested glasses, the BET analyses gave a type II hysteresis (fig. 14) typical of nonporous adsorbents. The change of phosphate precursor affected other aspects of the procedure. The drystep time was significantly reduced when Et/P was used instead of the Bt/P and the delay in the gelation time ensured a more controllable reaction. In addition, a clear and transparent solution was obtained after adding all the precursors (NaOMe first and Ca-methoxyethoxide then) instead of a white milky solution when Et/P was used.

The scale-up effect

Objectives: When production progress from the lab to the industrial scale, retaining the homogeneity and so the target characteristics may be a challenge. In this section, the effects of scaling-up the sol-gel production of P2O5 53% -CaO 24% -Na2O 23% (mol%) glass were studied at a 3-fold increased scale. The findings are reported below. Methods: The sol-gel route was used to produce two different bulks of PBGs. Briefly, under constant stirring, Bt/P was added dropwise to NaOMe and left mixing for 1h. Afterwards, the Ca precursor was added. Composition, phase and surface features were characterized in agreement with the manual of the tools.

Results and discussion: The findings reported here suggested that when the production is scaled, differences between small and large scale could be detected. During the design of any experiment, this possibility should be considered to not waste time and precious resources.

Composition and yield

The up-scaling of the sol-gel production did not bring any variation to the final composition. The XRF analysis (table 7) proved that the actual compositions of both glasses were quite close to each other but equally far from the expected one.

After heat treatment the glass powders were weighed to quantify the yield: the scaledup preparation gave 47% of the expected material, whereas the lower scale gave 76%. The XRF graphs (fig. 16) showed an amorphous phase is replicated in the scaled-up sol-gel glass. This indicates that the size of the production does not affect the phase of the material. respectively to PBG-C24/30 and PBG-C24/100 glass; these peaks were assigned to the asymmetric (PO2) -bond.

All the spectra contain Q1 and Q2 units: specifically, Q1 for the symmetric and asymmetric (PO3) 2-and Q2 for the asymmetric POP and (PO2) -bond.

Based on the shape of the FTIR traces, it is possible to conclude that the scale of production did not result in any modification to the atomic scale structure of the glasses or to the nature of the bonding. Moreover, it may be concluded that both glasses are made by long chains dominated by Q2 unit.

pH measurement

When soaked in UPW for 24h, the glass prepared at the larger scale showed an augmented stability of the network leading to a less dramatic pH decrease: 4.67 for the PBG-C24/100 compared to 3.27 for the PBG-C24/30.

Surface area

For the sol-gel glass samples, the BET analysis gave a type II hysteresis (fig. 18) typical of non-porous materials. Table 9 summarises pore size, pore volume and surface area data for the two glasses.

The relationship between production scale and surface structure is shown in fig. 19. A difference was found between the pore volume of PBG-C24/30 and PBG-C24/100: the pore volume of the higher scale glass was 2.1 times bigger than for the lower scale glass. The pore size increased 1.6 times and the surface area was 1.3 times bigger for larger-scale production All together these results prove that the scale-up of the production may affect some parameters of the material. Factorial Experimental Design (FED) work thus needs to be undertaken when progressing studies from the lab to the industrial scale in order that a robust Standard Operating Procedure (SOP) can be defined.

The FED experiments

Objectives: The initial aim was to produce PBGs by sol-gel route and then apply the glass products in the dental field. Since the materials were intended to be used in humans, issues about the safety and eligibility of some precursors and solvents were raised. Thus, the need to replace them with non-toxic chemicals arose. Changing precursors and/or solvents might lead to changes in the sol-gel structure formation and so the FED was used to identify preferred processing ranges. By fixing some parameters (for instance the glass composition -P2O5 53% -CaO 24% -Na2O 23% (mol%)) and varying others ((i) EtOH to EG ratio, (ii) time under reflux and (iii) temperature under reflux), the FED software was used to understand how those variables affected the sol-gel reaction to find the optimal conditions in terms of sample yield, phase and colour.

Methods:

The yield was determined by directly weighting the sample mass after thermal treatment. The phase was evaluated by XRD (% amorphous phase) and the composition by XRF (deviation from target mol% values for each oxide). The colours underwent a visual examination (subjective scoring on a scale of 1 to 5). The impact that the varying parameters had on yield, phase, colour and composition of the sol-gel preparations was evaluated by using the FED software. Constrains (range of possibility) were imposed and experiments were performed accordingly to the FED software output generated. For each critical response on the final product (phase, yield and colour) a weighting was assigned based on their relative importance.

Results and discussions:

The software identified the optimal performing-region/conditions at the edge of the explored area; moreover, inconsistency between repeated points was encountered. More experiments to explore wider range of possibilities was necessary but due to the lack of time, the trials were not taken forward.

Phase

The phase(s) associated with each of the 11 FED runs were analysed by XRD and data plotted as amorphous phase percentage (wt%). For all the runs, sodium salicylate was recognized as the main crystalline phase. In addition, in the crystalline phases of the runs #2, #5 and #8 the presence of both Na-succinate and Na-maleate was documented.

In depth analysis of the graphs (fig. 20 EtOH/EG ratio. This trend is in good agreement with the trend described in the section 3.2.2.

Considering those findings, it is possible to conclude that EG/EtOH ratio was the variable that affected the most the yield.

Colour

The colour change of the runs after heat treatment (fig. 22) fitted well with the observed trends. All the powders belonging to the first group had a colour varying from brown to light yellow. The others included in the group 2, #4 included, had white colour.

All together those observations lead to the conclusion that colour and phase were linked: while increasing the content of the amorphous phase, the glass powders tended to the brown; the white colour was more typical for highly crystalline materials.

Composition

The compositions of the most promising runs (#3, #9, #8 and #10) were determined by XRF (table 10). The runs #9 and #10 showed the closest composition to the target one.

Glasses by precipitation

Objective: Due to its relative ease of execution, room temperature reaction and non-toxic chemicals used, this methodology was considered as a promising new approach to produce phosphate glasses by a soft and wet chemistry route.

Method: A calcium chloride solution was added to a pyrophosphate solution using a peristaltic pump at a constant volumetric flowrate (7.5 mL min -1 ). After the complete addition of the calcium reagent, the solutions were stirred for five minutes, and the final colloidal solutions centrifuged at 5000 rpm for 10 min. The resulting gel was washed two times with UPW and heated at 70 °C for 7 days.

Results and discussions:

The methodology has some intrinsic limitations like the narrow range of compositions allowed and low yield that strongly limit the applicability of this method for producing desired phosphate glass compositions. As such, it was deemed unsuitable for the purpose of this thesis.

From the article [START_REF] Soulié | Development of a new family of monolithic calcium (pyro)phosphate glasses by soft chemistry[END_REF] containing the procedure adopted in this work, it is evident that the Ca/P ratio represents an important factor in this methodology because by increasing this ratio the obtained material becomes more and more prone to unwanted crystallinity. In addition, the concentration of the precursors and so the volume of the solutions might have potential to affect the outcome.

In order to validate the procedure and verify the technical feasibility of this method to be adapted to our glass composition (P2O5(48%)-CaO(28%)-Na2O(24%) -mol%) and scale, 6 sets of experiments were planned and organized as follows:

RUN 1: the concentration of the precursors and the volume of the solutions were kept the same as described in the article. The expected yield was 3.45g RUN 2: A scale-up trial was performed increasing the yield up to 10g. In this case, the concentration of the sources was maintained as described. However, due to the increase in yield, the volumes of the solutions were bigger (from 200mL up to 578mL

for the Na4P2O7 and from 20mL of Ca up to 55mL) RUN 3: A trial corresponding to the PYG-00 composition described in the paper but using Na4P2O7 (instead of K4P2O7) -{[(Ca 2+ )1-x(H + /Na + )2x]2[(P2O7 4-)1-y(PO4 3-)4y/3]}n(H2O))

RUN 4: Matching the PYG-01 formulation described in the article but using Na4P2O7

(instead of K4P2O7) -{[(Ca 2+ )1-x(H + /Na + )2x]2[(P2O7 4-)1-y(PO4 3-)4y/3]}n(H2O))
RUN 5: The PYG-00 composition described in the paper using K4P2O7 -{[(Ca 2+ )1-

x(H

+ /K + )2x]2[(P2O7 4-)1-y(PO4 3-)4y/3]}n(H2O))
RUN 6: The PYG-01 formulation described in the article using K4P2O7 -{[(Ca 2+ )1-

x(H + /K + )2x]2[(P2O7 4-)1-y(PO4 3-)4y/3]}n(H2O))

pH measurement
For runs 1 and 2 the pH of each step was measured as undertaken in the article. The values are reported in table 11. Those values were in good agreement with the expected ones.

Yield

After the drying step, all the materials appeared white, brittle and in a fine granular form. The yields of the RUNS were recorded by weighing the powders and are reported below in table 12. The yields of the RUNS 5 and 6 were 6% and 15% respectively and as expected they matched RUNS 3 and 4. The yield of the reactions was very low representing a problem in terms of economically viable production.

From the graph shown in fig. 23 it is evident that the yield was Ca-dependent.

Phase

It seems that the phase of the materials obtained by this procedure is Ca-dependent.

By increasing the Ca content (or increasing the Ca/P molar ratio) the material becomes more prone to crystallization. The Ca/P molar ratio of the composition under investigation here is 0.292 which falls within the crystallization range.

The XRD analysis of the RUNS 1 to 4 showed a crystallization phase corresponding to calcium di-sodium pyrophosphate tetra-hydrate (CaNa2(P2O7)•4H2O). In contrast, RUNS 5 and 6 were totally amorphous. After the reaction, the material obtained was a white flocculated mixture (a curd) of coacervate Sr-phosphate. The weight recovered was 1.24 g (corresponding to a 67% of the yield) and 99 wt% of amorphous phase (fig. 26).

In the second of the Sr formulations, Ca was partially replaced by Sr in the composition. The SrCl2 • 6H2O (1 M) is very soluble in water and when mixing with CaCl2 (1M)

and added to the (NaO₃P)n solution two different coacervate phases were recognized: one oily/gel-like and one flocculated. This was due to the different size and weight of the doping ions (Ca and Sr). The final material weighed 1.0g (corresponding to about 50% of the expected yield) with 100 wt% amorphous phase (fig. 27). Furthermore, XRF data (Appendix 2) confirmed the incorporation of the Sr (as SrO) in the glass network.

All together these results proved that it is possible to use the coacervation methodology to incorporate Sr into a phosphate glass composition without affecting the phase of the final materials.

Different composition

To check if the phase of the material obtained with this process is Ca dependent (or Ca/P dependent), another glass formulation was run. The XRD pattern (fig. 28) shows the amorphous halo typical of glass materials. It is therefore possible to conclude that the coacervate technique gives amorphous materials over a wide range of compositions. This means that the technique is adaptable and can be used to tailor the formulation of the material in agreement with our need to dope the glass with other ions.

Cytocompatibility assay

Fig. 33 shows the vitality of hGFs seeded in contact with the scaffolds in comparison with the ctr for 7 days. The cells in contact with the tested materials displayed a vitality ranging between 20 and 30 % of the control; in addition, the incubation time seems to be not a determining factor.

Melt-quench vs Sol-gel

Objective: In this section a comparison of the same glass composition ((P2O5(53%)-CaO(32%)-Na2O(15%) -mol%) produced both by sol-gel and melt-quench is described.

Methods:

The sol-gel glass was synthesized by adding, under constant stirring, Bt/P to NaOMe. After 1h, the Ca precursor (Ca-methoxyethoxide -CME) was added dropwise.

The melt-quenched glass was prepared by placing a homogeneous blend of raw materials in a quartz crucible and heating it at 1300 for about four hours. After, the molten mixtures were quickly quenched by pouring onto a steel plate. After quenching, the obtained glass was milled to a particle size ranging between 20 and 200 μm using a TEMA Mill machinery model T 750 k.

Results and Discussions:

The methodology used can impact the surface features of the same material. This might determine drastic changes in its behaviour (water solubility for example).

This becomes particularly important when it comes to medical applications: different topology (rough surface rather than smooth, for instance) can trigger different cell response

Composition and yield

Table 13 shows the actual final composition of both SG and MQ glasses with respect to the target values. It is possible to notice that the MQ glass delivers a composition closer to that expected when compared to the SG glass. The MQ glass is deficient in P, suggesting losses from volatilization. Moreover, the table shows the relative yield of both preparations: the MQ technique provides for a higher yield than SG for the tested composition. The only losses in MQ arise from glass adhered to the refractory pot used for melting; for SG, there is potential for unreacted precursors to be lost during processing to isolate and purify the solid phases.

Phase

The XRD patterns of both MQ (Fig. 33 B) and SG (Fig. 33 A) glasses show a broad peak indicating that the materials are totally amorphous. It demonstrates elegantly how both techniques equally give high quality materials. incubation. A deeper observation revealed a subsequent small decrease in ion concentration over time -more evident for P whilst barely visible for sodium and calcium. This may be due to salt precipitation. Also, in this case, the relative content of the ions in solution reflected their concentration in the glass formulation: it should be notice that Na and Ca had the same kinetics due to their almost equally content in the actual composition.

pH measurement in SBF Fig. 37 shows the variation of pH as a function of the MQ and SG incubation time.

Surprisingly, the pH of the media became more basic, up to 8.04 and 8.13 for SG and MQ respectively after three weeks. One can notice that between the tested glasses, there is no significant difference in the pH trend development over time.

The green triangle and the violet "X" represent, respectively, the pH of the SBF for the MQ glass and SG glass recorded after 24h of incubation when 10 times the glass concentration is used in the dissolution test (that means 10mg/mL instead of 1mg/mL). Remarkably, despite such a high glass powder concentration, the pH remained quite stable without reaching a critical value. In addition, a difference between the two glasses is detectable in this case: the MQ glass demonstrated a more stable and neutral pH than the SG glass.

Comparison of pH variation between water and SBF

Sol-gel phosphate glasses

Effect of increasing Ca content on the microstructure Table 1 summarizes the actual composition (with respect to the expected values reported in section 3.1.1) of three phosphate-based glasses produced by sol-gel where Ca content increases progressively at the expense of Na with the P concentration fixed. From the data obtained by XRF it is possible to notice that for all three glasses, P content was lower than expected. This difference may be explained by one or a combination of the following possibilities:

1. Under the conditions trialled the phosphoric esters have low reactivity so it may be reasonable to think that some of the butyl-P molecules did not take place in the network forming condensation reactions [START_REF] Livage | Sol-gel synthesis of phosphates[END_REF] 2. The semi-quantitative nature of the analysis itself 3. The greater volatilization of the phosphate in the SG glass system due to the low density of the SG powder [START_REF] Carta | A structural study of sol-gel and melt-quenched phosphate-based glasses[END_REF][START_REF] Carta | Structural studies of bioactive sol-gel phosphate based glasses[END_REF] 4. A possible arrangement between phosphate and Ca may have subtracted those ions from the sol-gel polymerization 5. The subsequent washing step may have washed away soluble CaP species along with any unreacted precursor.

However, the aim to increase Ca content was reached. In addition, all three glasses gained Na in their composition. This might be explained considering the role that NaOMe plays in the reaction. NaOMe is a strong Lewis base and it has the task to react and activate P precursors. Its action is proved by the fact that when Na is added, the mixture heats up and the solution turns into a white, thick and milky blend. This fits well with the proposed mechanism and, due to this important role, it is the first precursor to be added. It is known that in sol-gel chemistry the order of addition matters in determining structure and composition.

The yield of phosphate sol-gel product reduces with increasing Ca; in other words, it reduces by reducing the relative amount of sodium precursor used in the reaction. Again, this can be explained considering the previous discussion: a reduced amount of sodium precursor results in less P precursor being activated, and as such less reactive network former is generated and subsequently fewer condensation reactions take place resulting in a lower glass product yield.

Ca is present in the sol-gel mixture as free ions and it is eventually incorporated into the glass network by coordinating the NBOs of the P tetrahedra in the chains.

XRD analysis as shown in Fig. 1 revealed that all products were totally amorphous, despite the increased Ca/P ratio. (It may have been expected that an increased Ca/P ratio would result in the preferential formation of stable crystalline calcium phosphate phases).

This can be explained considering the random distribution of the ions within the network.

The FTIR spectra (fig. 2) reveal no differences in the atomic bonding of the network components attributable to the fact that the P content (as the only network former present) is equal across the three tested glasses.

Fig. 3 shows that after 24 hrs of incubation, the pH of the water dropped in a Cadependent manner. Overall the drop of the pH is due to the presence of phosphate ions in the media as a result of the hydrolysis of the P-O-P phosphoester bonds in the glass network.

Whereas, the changing composition-dependent trend could be explained by the fact that by increasing Ca content, the amount of sodium being leached from the glasses is also reduced.

This has a consequent impact on the ability to buffer the phosphate-induced acidification of the media.

The hysteresis loops of the glasses are shown in fig. 4. They are typical of non-porous materials [START_REF] Thommes | Physisorption of gases, with special reference to the evaluation of surface area and pore size distribution (IUPAC Technical Report)[END_REF] . The relationship between Ca content and surface features is reported in the graphs in fig. 5. A clear correlation between Ca content and surface topography is evident.

The increase in surface area could be due to both the significant increase in pore volume and comparable pore size. The direct correlation between Ca content and surface area could be explained considering that Ca ions have higher coordination number compared to Na and act as an inter-chains cross-likers, whereas Na is linked to just one chain [START_REF] Ruiz Hernandez | Molecular dynamics simulations of bioactive phosphate-based glass surfaces[END_REF][START_REF] Christie | Structures and properties of phosphate-based bioactive glasses from computer simulation: a review[END_REF] . This results in a greater disorder of the phosphate network for the sample containing the lower level of Ca. It means that the sol-gel system evolves from a 3D-network of phosphate chains linked by Ca to a more disordered system of separated phosphate chains as Ca progressively reduces (or Na increases).

Due to its "average" Ca content, the sample C32 was chosen to be evaluated for its thermal properties (fig. 6). The shape and the temperature of the first broad peak (corresponding to the first mass loss event) was likely due to the volatilization of structural water, phosphates, by-products, maybe unreacted precursors and/or some residual solvent molecules. This is also supported by the fact that it represents the biggest mass loss event in agreement with the high molecular weight of some chemicals used for the reaction. The second mass loss event overlaps with the crystallization phenomenon observed. Due to this and its shape, the two phenomena were thought to be correlated. The cause could be due to the volatilization of some structural components of the glass network and/or further organics.

The following XRD analysis performed on the powder after TGA (fig. 7) shows that two main crystal phases appeared: sodium calcium phosphate and calcium oxide phosphate.

Phosphate precursor effect on the sol-gel chemistry and surface features Table 4 shows that when butyl-phosphate (Bt/P) was replaced with ethyl-phosphate (Et/P), the actual composition of the glass was closer to that as expected. A deeper observation reveals that the content of P in the Et-PBG was bigger than in the Bu-PBG, whereas, the Na follows the opposite trend. In contrast to what was observed in the presence of the Bt/P (see section 4.1.1), when NaOMe was added to Et/P, the blend appeared clear and transparent, moreover, the gelling time was extended. These findings can be explained considering the different reactivity of the two phosphate precursors. It is possible to speculate that the relative lower reactivity of the ethyl precursor allows the Na precursor to activate and recruit more P precursors. This in turn ensures the target composition is achieved and with less material loss. Ca is present as free ions able to coordinate the chains. The yield seems to not be affected by the replacing of P precursor. From what said, it seems that the ethyl-P is a better precursor.

Fig. 8 shows that the final phase of the materials is independent of which phosphate precursor is used. This is important because it is possible to tailor the physical characteristic of the sol-gel products based on the intended application by modifying the precursors used without altering the phase.

The FTIR spectra reported in fig. 9 show no significant differences between the glasses despite the different phosphate precursors used. A slightly bigger definition for the peaks at 896 and 722 cm -1 (vas(POP) and vs(POP) respectively) can be observed for the glass made by using the Et/P. This could be explained considering the higher P content in this glass compared to the Bt/PBG (table 4).

The drop of the water pH after 24 hrs of glass incubation did not show any difference despite the different P precursors used. This suggests that the dissolution properties of the material are independent of the phosphate precursor used.

A type 2 hysteresis (fig. 10) typical of non-porous materials was obtained for both glasses after BET analysis, independent of the phosphate precursor used to carry out the solgel reaction. The relationship between textural features and P precursor is shown in fig. 11.

The difference in surface area could be due to the big increase in pore volume and pore size in Et/PBG glass. The influence of the phosphate precursors on the surface profile of glass particles may be explained by considering the alkoxide groups of the two precursors used for this study. The butyl group has four atoms of carbon in its structure, whereas, the ethyl group has two atoms of carbon. The steric influence of the two groups is different, being bigger for the butyl group as compared with the ethyl group. Therefore, the use of different phosphate sol-gel precursors reveals differences in the properties of the phosphate glass products and the kinetics of the sol-gel reactions taking place. A possible reaction mechanism could be suggested where the butyl is displaced and replaced faster than the ethyl. In addition, due to the smaller size, the ethyl remains trapped easily into the network during the formation, explaining the bigger values (pore size and pore volume and surface area) found.

The scale-up effect

The XRF-obtained composition of the materials produced at lower and higher reaction scales (table 7) did not show any significant difference. That means that the scale of the reaction process did not compromise the material in terms of composition. On the contrary, the yield seems to be compromised: the higher scale gave the lowest yield. This could be a consequence of the volume of the reaction and the dilution effect: the volume of the higher scale reaction was 500 mL compare to the 200 mL of the lower scale. Since a temperature of 60 °C is necessary to obtain the complete gelation, a difference in temperature could have been created between the edge of the flask (closer to the heat source) and the core of the reaction (far from the heat source). This might have dispersed the reactant subtracting them from being activated and being incorporated in the sol-gel network. However, the phase of the glasses was preserved despite the scale (fig. 12).

From the spectra reported in fig. 13 it is possible to gather the production scale did not determine any change in the atomic structure of the PBGs.

The difference in the drop of the water pH after 24 hrs of incubation is interesting. It is known that Na prefers the surface of the glass rather than the bulk [START_REF] Ruiz Hernandez | Molecular dynamics simulations of bioactive phosphate-based glass surfaces[END_REF] . A bigger reaction volume could have allowed a better mixing and so ensure the ions occupy easily their favourite position. Ions on the surface are more prone to be released and exchanged with the media rather the ones in the bulk. Due to this scenario, the faster release of bigger quantities of Na of the higher scale glass compare to the smaller one could explain the pH values detected (3.27 and 4.67).

Fig. 14 shows a non-porous hysteresis loop for the two scale glasses under study. The relationship between scale production and surface features is reported in fig. 15. The higher surface parameters could be explained considering the reaction volume: the phosphate chains had more space to build the network and this entailed for bigger pores of the existed ones.

The FED experiments

Despite the sol-gel route gave good glass materials which were fully characterized, the need to use less hazardous precursors driven by the intended (biological and clinical) endapplication stimulated the research of more suitable alternative precursors (Appendix 3). In this context the FED software was used in an attempt to optimize the experimental set-up of a new route of sol-gel reaction involving alternative and more safety chemicals.

Figs. 16 and 17 (left hand) show respectively the trend of phase and yield from the 11 FED runs. As described in the previous sections (3.2.1 and 3.2.2), it is possible to assemble the runs in two separate groups based on the EG/EtOH ratio. The highest yield and amorphous phase content (wt%) was given by the runs with high EtOH content. This can be explaining considering the viscosity of the solvents: 0.983 and 16.9 mPa.s for EtOH and EG respectively.

It is well-known that the viscosity of the media affects the reaction by reducing the reaction rate. The increasing concentration of the EtOH had the effect of reducing the viscosity of the reaction media favouring the sol-gel reaction.

The temperature, rather than the time under reflux, seemed to play a synergic role: the increasing temperatures had the effect to further reduce the viscosity of the medium encouraging more efficient sol-gel reactions.

Another parameter to be affected by the temperature and the "mixing effect" (but not related with viscosity) is the density of the reaction medium. In this this case both increasing EtOH concentration and increasing temperature had the effect of reducing the density of the medium favouring the reactions. Considering this, it is possible to conclude that the viscosity rather than the density or a combination of both were responsible for the observed trends. Therefore, it is not surprising that the closest composition to the target belongs to the runs #10 and #9 (table 10).

In the light of what has been said, one can suppose that the optimal experimental condition could have been met by keeping the EG as low as possible and rising the temperature as high as possible. This thought was corroborated by the 3D-graphs (figs. 16 and 17 -right hand -fig. 18) that placed the optimal region at the very edge of the graph.

However, this position is unfavourable and makes the prediction unreliable. In fact, the run #1 (the best obtained) was repeated twice with different results. This lack of reproducibility was supported also by the run #3 which is present twice (run #10) in the experiments as a check point. Due to those difficulties, the sol-gel route was put aside to explore other methods more reliable and safety.

Glasses by precipitation

The precipitation method [START_REF] Soulié | Development of a new family of monolithic calcium (pyro)phosphate glasses by soft chemistry[END_REF] was explored as a novel technique to obtain glassy materials by soft and wet chemistry. However, this methodology revealed some intrinsic limitations that make it not so suitable for our scopes. One of those was the extreme low yield. Two aspects of such a limitation were the high amount of waste material generated and the impossibility to run a complete set of experiments on the same preparation. In addition, the yield was strictly Ca dependent (table 12 and fig. 19): this was quite logic since the phosphate was constant and the Ca increased progressively. This revealed another limitation of within the procedure: the Ca/P ratio. It was shown that by increasing the Ca/P ratio (in other words increasing the Ca content) the tendency to produce crystalline products is increased. This imposes a limit to the range of compositions for which it is possible to make an amorphous product with this method. For instance, the Ca/P molar ratio of the targeted composition is 0.292 and when synthesis of this material by this new precipitation route was attempted the product was found to be highly crystalline. Comparing with the sol-gel preparation, the yield seems to follow the opposite trend: it reduces by increasing Ca. This may be due to the low Na precursors that recruit less phosphate precursors to make the glass.

In addition, the sol-gel route seems to overcome the Ca/P issue.

A further limitation is given by the additional ions to Ca and P it is possible to incorporate in the composition. Keeping the Ca/P in the range of amorphous phase, by replacing K (as describe in the original method) with Na a crystal phase appeared. This might be due to the different size and ionic strength of the two ionic species. This limits further the application of this methodology to produce the compositions of interest for this study.

Coacervate phosphate glasses

To overcome the limitation described before (section 4.3) this new approach [START_REF] Pickup | Characterisation of phosphate coacervates for potential biomedical applications[END_REF] was taken into consideration. In order to verify that the Ca/P ratio was not a restriction for this process, besides the composition described in the article, our own formulation was made.

Both resulted in amorphous products and gave high yields (fig. 20 and fig. 24). The composition studied in the article was scaled up by five times to check whether this would affect the phase purity of the product. The obtained yield was very high (80%) and XRD showed that the product glass was amorphous (fig. 21). The observed sodium chloride residue could have been due to an excess of ions coming from the augmented number of precursors and maybe, with the scale-up, a more extensive washing protocol is required.

The next step was to further verify the flexibility of the composition by fully replacing Ca with Sr or at least adding it at the expense of Ca. On replacement of Ca with Sr in the process, it was observed that the coacervate product went from being oily and gel-like to becoming flocculated and particle-like. This change in product consistency is likely to be due to the differing ionic radii of the calcium and strontium species involved in the reaction. [START_REF] Van Wazer | Phosphorus and its compounds[END_REF] .

Nevertheless, the material obtained was still totally amorphous (fig. 22). The joint presence of Ca and Sr did not affect the phase of the material, XRD again showed that this material was totally amorphous (fig. 23). During the reaction the two stages were recognised due to the co-existent in the solution of both Sr and Ca. In both cases, XRF data confirmed that the Sr was incorporated in the composition as oxide.

Fig. 25 reports a non-porous material type 2 isotherm for the coacervate phosphate glass. This may be explained considering that in the silica sol-gel the porosity comes from the removal of water resulting in the formation of pores and inter-particle voids. Since coacervation is an aggregation process due to the immiscibility of two phases that silica solgel mechanism does not happen, and pores are not formed. However, this result contrasts with W. Kopp, 2012 [START_REF] Kopp | Calcium polyphosphate coacervates: effects of thermal treatment[END_REF] : for the same material he published a type 4 hysteresis typical of mesoporous materials. This difference may be explained by considering some differences in the methodology. First, in this work the drying step was performed under vacuum and at room temperature instead of 60 °C for 24 hours. In addition, in Kopp's paper, this step was followed by a thermal treatment at 500 °C for 24 hours which is missing in my case. Maybe the thermal treatment is the cause and explanation for the (meso)porosity (by removing water as for silica)?.

Altogether, these results confirm that the coacervate technique gives a non-porous glassy material formed by smooth polygonal particles (fig. 30).

Collagen-based bioscaffold containing melt-quenched derived Sr-doped phosphate glass particles

The initial scope of the project leading to this thesis was to produce porous phosphate glasses by sol-gel technique for dental applications. After some failed attempts, the successful protocol described in the section 2.2.1.1 was established. However, despite the initial expectations, the obtained glasses were non-porous.

The ultimate goal was to incorporate these materials in biocompatible scaffolds for human use, therefore, the presence of hazardous solvents (MtOH and 2-methoxyethanol) and precursors (Ca-2-methoxyethoxide) made this route infeasible and alternative precursors/solvents were explored. Table 1 in the Appendix 3 shows all the attempts made to find valid substitutes (like water or EtOH as solvents and acetate-based precursors) to obtain the PBGs via sol-gel process. After all, nothing really promising came, so the sol-gel route was put aside, and other room temperature methodologies were considered.

The first method explored was the precipitation method, however due to the aforementioned limitations, it proved to be unsuitable for our scope.

The coacervate process was highly promising but also in this case the materials obtained were non-porous.

Due to tight deadlines and still having obtained non-porous materials, the wet chemistry route to produce PBGs was laid aside in favour of the melt-quench technique which is more reliable in terms of composition and phase and gives easily high yield.

Considering all those facts together, in the end, phosphate glasses produced by meltquench technique were chosen to be combined with collagen fibres to make porous scaffolds.

The Col1 fibres are not soluble in water undergoing a rehydration process rather than a proper solubilization. This might introduce limitations for the crosslinkers to react with collagen functional groups and interact with the particles. A weaker interaction between the fibres and the glass particles might affect some important mechanical properties.

To verify if an augmented solubility of the collagen fibres and/or a tighter link between the fibres and the glass particles could have improved some mechanical properties of the scaffolds, a compression test was carried out on the four experimental groups described in the section 3.6.1. From the graph illustrated in fig. 31 it is possible to recognize some trends:

1. the presence of the AcOH (G1 vs G2) did not affect the properties of the scaffolds; 2. the increased concentration of cross-linkers seems to improve the resistance to compression only when the AcOH is present in the collagen suspension (G3 vs G4); more crosslinker seems, on the other hand, to reduce the mechanical properties of the scaffolds when only water is present (G1 vs G3 vs G4). From this, it is possible to conclude that the conditions in G4 gave the best scaffold performer. This could be explained considering that the AcOH could have made available more functional groups on fibres to react with the crosslinkers. However, the big standard deviation between the groups made those differences non-statistically significant (P=0.76, n=5). The presence of such big errors was to be attributed to the spongelike nature of the materials and maybe its non-homogeneity due to the methodology used:

within the same bulk there might have been microdomains where the concentration of glass particles or the aggregation/configuration of the fibres is bigger/different than other parts.

For those reasons the ctrl group (G1) was chosen for the next experiments.

To check the biocompatibility of the scaffolds, hGFs were seeded in direct contact with the materials for 7 days. Fig. 32 shows that, for the whole experiment, the vitality of cells was low compare with the ctr (understood as cells seeded in an empty well): those data suggest that cells were in a state of suffering. The reason beyond this unwelcome condition is to be found within the scaffolds. As described in materials and methods section, the Col to PBG particles ratio was 1:1, that means that 50% of the bulk of each scaffold was made up of phosphate glasses. It is known that those glasses dissolve very quickly in water environments releasing great amount of phosphate ions which entails the acidification of the medium the glasses are soaked in. Thus, it is possible to conclude that the acidification of the cell medium below the physiological pH due to the release of phosphate ions following the hydrolysis of the glass network was the cause of such low values. This was confirmed by the phenol red (normally presents in the cell media as pH indicator): as soon as the medium was in contact with the scaffolds, it exhibited a transition to yellow indicating pH way below 6.0.

Melt-quench vs Sol-gel

Table 13 compares the actual composition the MQ glass and SG glass with respect to the expected composition. The difference between the actual and the expected composition for the SG preparation may be explained by one or a combination of the following possibilities, as described earlier in section 4.1.1: 1. The phosphoric esters are poorly reactive so it may be reasonable to think that some of the butyl-P molecules did not react [START_REF] Livage | Sol-gel synthesis of phosphates[END_REF] ; 2. the semi-quantitative nature of the analysis itself; 3. the greater volatilization of the phosphate in the SG glass system due to the lower density of the SG powder compared with MQ powder [START_REF] Carta | A structural study of sol-gel and melt-quenched phosphate-based glasses[END_REF] ; 4. a possible arrangement between phosphate and Ca may have subtracted those ions from the sol-gel polymerization; 5. the subsequent washing step may have washed away soluble CaP species along with any unreacted precursor. These considerations (along with the bigger number of steps that the SG material went through) may also explain the difference in yield between SG and MG.

The relative phases are shown in fig. 33: both methodologies gave totally amorphous materials due the random and non-ordered arrangement of the atoms in the networks.

By comparing the FTIR spectra of the two glasses (fig. 34), it is interesting to note that the intensity of the bands at 1200 and 1100 cm -1 is greater for MQ glass. The same can be said for the bands at 770 and 725 cm -1 . Those differences suggest a more connected network for the MQ glass and a more open structure for the SG glass (also maybe to the fact that there is a higher amount of P2O5 in the MQ glass sample). However, based on the shape of the FTIR peaks and in agreement with the previous studies [START_REF] Carta | A structural study of sol-gel and melt-quenched phosphate-based glasses[END_REF] , it is possible to conclude that at the atomic scale, the structure of the two glass samples is similar.

Independently of the preparation method, fig. 35 shows that both glasses reduce the aqueous pH due to the presence of phosphate ions as results of the dissolution of the POP bond in the glass network.

The drastic difference of the release kinetics between MQ and SG glasses (fig. 36) may be explained considering both the relative density of the two networks and the dissolution mechanism. As reported [START_REF] Carta | A structural study of sol-gel and melt-quenched phosphate-based glasses[END_REF][START_REF] Carta | Structural studies of bioactive sol-gel phosphate based glasses[END_REF] , despite the similar atomic scale structure of the glasses coming from sol-gel and melt-quenching, the latter presents a higher density: tighter cross-linking

Conclusion and future perspectives

In the presented thesis, different methods to produce bioactive glasses have been reported. The synthesised glasses were characterised in terms of structural properties including XRD, BET, SEM and FTIR spectroscopy that indicated the successful production of phosphate-based glasses. The composition was confirmed by XRF and dissolution behaviour by ICP.

Initially, the effects of increasing Ca at the expense of Na on sol-gel phosphate glasses were studied. However, there were some limitations: such phosphorus loss (4 mol%), lack of porosity and mainly toxicity of some solvents/precursors. To overcome the phosphorus loss, butyl phosphate was replaced with ethyl phosphate for sol-gel synthesise the ternary P2O5-CaO-Na2O glass systems. Thus, the effects of replacing the precursors were analysed. The XRF revealed an increased phosphorus content.

In an attempt to find a safer sol-gel route, FED software was used to plan experiments with alternative precursors. The lack of reproducibility required the evaluation of alternative glass synthesis methods. The precipitation method was the first to be explored but the high limitation imposed by Ca/P ratio to obtain amorphous materials, ability to use alternative ions and very low yield obliged the exploration of alternative approaches.

The coacervation process was revealed to be promising and without limits. The feasibility of the method was validated by synthesizing different compositions, scales and introducing different ions. However, due to the lack of porosity and apparent lower reliability in reaching a target composition, in the end, the melt-quench technique was chosen to prepare glasses with the general formula of (P2O5)53-(CaO)32-X-(Na2O)15-X-(SrO)X, where X = 0, 5, 10 or 15 mol% for dental application. The degradation studies revealed that the dissolution rate of these glasses reduced as Sr was substituted in place of Ca because of the enhanced cross-linking effect of strontium and gave an indication of how the solubility of the glasses can be controlled for various biomedical applications.

Subsequently, the MQ glasses were incorporated in collagen-based scaffolds for dental pulp regeneration application and cell-mediated remineralization. The effect of collagen crosslinking and improved collagen solubility was studied: the compression test revealed no significant improvement for the tested conditions. Scaffolds were presented as highly porous materials with interconnected channels to potentially favour nutrient and oxygen flow to the regenerating host tissue. In water they were found to degrade indicating the possibility to be replaced by natural tissue (a property called bioresorbability). The collagen fibres were proved to interact with the glass by-products altering the dissolution rate of the glasses. This is an interesting property because this ion retention could facilitate crystal growth on the surface. In fact, one of the tested scaffolds was able to grow an HA-like layer after immersion in SBF for one week.

A comparison between the different methods described and used in this thesis has been made. The methodology used determined changes in some key features of the materials like particle shape, dissolution behaviour and surface topography which may generate different cell response. The application should be the driving force for choosing the most suitable method.

From this thesis it is evident that health, safe, repeatability and yield were the driving forces for determining the most suitable process. Considering the characterizations made on the materials coming from the explored methods, it is clear that the coacervation process seems to be the most promising route. The use of non-toxic precursors, the relatively fast execution, the high yield and the repeatability of the methodology, besides its flexibility in terms of compositional range make it a worthy route for future experimentations. This direction of research should focus on optimised thermal treatments to achieve porous materials and further doping with Ag for possible antibacterial effect and other ions for bioactivity, cytocompatibility and so on. Since, from the knowledge of the author, no commercial products for medical and in particular for dental application are on the market, possible incorporation of the materials coming from this route in a marketable product might be considered.

Another key point is the composition which determines all the final properties of the material. Considering the findings discussed in the present thesis and the literature review exposed in the introduction, a phosphate glass with high concentration of Ca, between 40 and 50 mol%, and a P content ranging around 40-45 mol% should guarantee a glass network stable enough to support cell attachment and proliferation, thus allowing them to create a natural extracellular matrix that would replace the glass during longer-term dissolution.

ppm measured coming exclusively from the SBF. A peak followed after 1 day to progressively reduce during the next 3 weeks. The Sodium presented even more different kinetics. Very little Na appears to dissolve / diffuse from the powder during the initial 24 hours of experiments. This holds for both in SG and MQ glasses. However, after 24 hours there is a divergence. For the MQ glass, its concentration rose for 2 days and 1 week to drop at week 3;

in contrast a completely opposite kinetic trend was exhibited for the SG glass (a drop concentration between 2 days and 1 week of experiment and a rise at the third week to reach the same Na concentration of the MQ Na).

During the experiment, a visual change in the powder was recognised after 1 and 3 weeks for both the MQ and SG glasses. The powders resembled flakes rather than a scattered powder. The materials were collected and submitted for the XRD to check if any change in the phase had occurred. After 1 and 3 weeks of incubation, the only phase detectable in all cases was NaCl, the amount found being both glass and time dependent. For SG glass the amount of NaCl accumulated on the surface was 1.1 wt% and 13.0 wt% 1 and 3 weeks of incubation respectively. Whereas, for MQ glass 2.4 wt% and 5.8wt% of NaCl formed on the surface after 1 and 3 weeks of incubation respectively. This may explain the trend for Na seen in figure 1, namely that lower concentrations of Na in solution correlate to increased NaCl crystal formation

N.B.

The data above must be taken into consideration carefully because: 1) the dilution of the conditioned SBF media for ICP analysis was performed using only ultrapure water without nitric acid (0.6% was usually used) which is needed to dissolve eventual precipitated salts; 2) the blank (the SBF alone without powder) was considered as a sample so was not subtracted out to level the background noise (which explains why, for example, we have more than 4000 ppm of Na and about 120 ppm of Ca associated exclusively with the media); 3) the concentration used (1mg/mL) might be too low to be actually measured.

APPENDIX 3

Here below is a complete table (table 15) summarizing all the attempts made to define an optimised sol-gel protocol. Optimisation was based on the use of alternative precursors (safer and less toxic compare to the established ones as well as offering high % yield in the final product) to produce phosphate glasses. The problems encountered, and the reason of the failures are documented. Where available, pictures of the obtained produced are shown.

Figure 15 .Figure 16 .Figure 17 .Figure 18 .Figure 19 .Figure 20 .Figure 21 .Figure 22 .Figure 23 .Figure 24 .Figure 25 .Figure 26 .Figure 27 .Figure 28 .Figure 29 .Figure 30 .Figure 31 .Figure 32 .Figure 33 .Figure 34 . 143 Figure 35 .Figure 36 .Figure 37 .Figure 38 .Figure 39 .Figure 40 .Figure 41 .Figure 42 .

 15161718192021222324252627282930313233341433536373839404142 Figure 15. Variations in pore volume, pore size and surface area as function of phosphate precursors 124

TSPP

  : scanning electron microscopy/energy-dispersive X-ray spectroscopy SG: sol-gel Sr: strontium SrAc: strontium acetate Ti: titanium TGA/DSC: thermal gravimetric analysis/differential scanning calorimetry the development of a new series of Sr-doped phosphate-based

(

  the amalgam contains mercury) have limited their use over time being replaced by safer and highly aesthetic materials. Cements are widely used to fill the cavity: GICs are one of the most representative of this category. They are white, biocompatible and offer the possibility to release fluorite by being recharged after each brush. Composite resins are made by a resin (methacrylate-based polymers) and a filler (glass-like powder dispersed in the resin). Different fillers have been developed and the silica-based glass (also called bioglass or bioactive glass) fillers are the most used. Bioglasses have the interesting property of making grow on the surface a HA layer if soaked in a high concentrated ionic solution (such as the saliva) miming the natural organisation of the tooth mineralised tissues.

period 3 ;

 3 Its electron configuration is [Ne] 3s2 3p3. To comply with the octet rule, phosphorus can bond to four atoms of oxygen forming the phosphate ion where one out of the four oxygens is linked by a double bond. This determines that the phosphate ion has five resonance structure due to the delocalization of the electrons. All those physical-chemical properties of the phosphate are at the base of the atomic structure and particular properties of the phosphate-based glasses.The structurePhosphate glasses are inorganic polymers based upon the tetrahedral phosphate anions formed by the sp3 hybridisation of the phosphorus outer-shell electrons (3s2 3p3) with oxygen atoms. The remaining fifth electron is promoted to the 3d orbital where it creates strong π-bonding molecular orbitals with oxygen 2p electrons 6 , 7 . Based on the covalent oxygen bonds, phosphate tetrahedra are present in glass structures in different anionic forms classified in agreement with the Qi terminology, where "i" represents the number of bridging oxygens (POP bonds)[START_REF] Brow | Review: the structure of simple phosphate glasses[END_REF] : in particular, Q0 is the orthophosphate, Q1 the pyrophosphate, Q2 the metaphosphate and Q3 the ultraphosphate configuration. By considering the glass network, Q1 represents the end unit, Q2 the middle unit and Q3 the branching unit; Q0 can be considered an isolated/free unit dispersed in the network. The prevalence of one unit with

  A batch calculation was performed to determine the wt% of the raw materials needed to deliver the final target oxide formulation (P2O5)53-(CaO)(32-x)-(Na2O)15-(SrO)x, where x = 0, 5, 10 or 15. The raw materials were weighed out and manually mixed to generate a homogeneous blend. Quartz (QZ) crucibles were pre-heated at 1150 and the mixed powders were poured in little by little. When all the powders were in the crucible, the temperature was raised up to 1300

Figure 2

 2 Figure 2 Schematic representation of the coacervate process to produce phosphate glasses

Figure 3

 3 Figure 3 Route to produce melt-quenched phosphate-based glasses

  2.2.4 were sent for ICP analysis. Before performing the analysis on the samples, a calibration curve was made using the following ICP standards: phosphorus standard for ICP (Sigma), sodium standard for ICP (Sigma), strontium standard for ICP and calcium standard for ICP (Sigma). A 0.6% HNO3 solution was made (from a 65% solution, ACROS Organics) by diluting the acid in water. Next, the aforementioned standards were diluted to concentrations of 5, 10, 20, 30, 40, 50 and 70 ppm in the 0.6% HNO3 solution. To comply with the calibration curve, the glass extracts were diluted five times with the 0.6% HNO3 solution prior to analysisSurface features (BET/BJH)The surface area and pore volume parameters were determined by nitrogen adsorption/desorption isotherms at 77 K in a Belsorp Mini II Surface Area analyser. The samples were degassed at 200°C for 4 hours prior to measurement. The Brunauer-Emmett-Teller (BET) equation was used for specific surface area quantification whilst the Barret-Joyner-Halenda (BJH) approach was used for the mean pore size and pore volume determination.

  of the surface and morphology of the particles in Secondary Electron mode. A JEOL JSM6490-LV Scanning Electron Microscope (SEM) fitted with an Oxford Instruments INCA Energy Dispersive X-ray Analysis system (EDA) was used for examination in accordance with in-house methods M24 and M25. Images were taken at different magnifications. Microanalysis results were tabulated on a semi-quantitative, normalised, elemental weight basis.

PULVERISETTE 7

 7 premium line by Fritsch. 100 g of 1mm ZrO2-beads and 15g of glass powder were each placed into two zirconia-lined jars and sealed (zirconia-lined lid fitted with a polymer "O" ring). One cycle of milling was performed at 750 rpm for 5 min at room temperature. Subsequently, 15 g of isopropanol was added to each vessel and, after resealing, 5 cycles of milling at 750rpm for 5 min (each interrupted by an equal number of pauses of 10 min to reduce temperature build-up) were performed at room temperature. The wet glass powders were filtered using a 50μm sieve to separate the beads from the milled glass. The glasses were dried overnight at 37 °C and the particle size distribution was measured with a Malvern Mastersizer 3000. The size of the particles chosen for the scaffold preparation described in this article ranged between 0.1 and 10 μm.Scaffold elaborationHomogeneous collagen suspensions of 40 mgmL -1 were prepared by mixing the collagen fibres in ultra-pure water under continuous magnetic stirring. Then, glass particles were added to the collagen suspensions at a 1:1 wt ratio. After 5 min of vigorous stirring, the homogeneous mixtures were loaded into Petri dishes, frozen using liquid nitrogen and lyophilized under vacuum for 2 days. Finally, the scaffolds were chemically cross-linked overnight using 20 mM of N-(3-(dimethylamino)propyl)-Nʹ-ethylcarbodiimide hydrochloride (EDC) and 8 mM of N-hydroxysuccinimide (NHS) in 80:20 (vol%) ethanol/deionized water.

Batch 3 :

 3 without AcOH and 10X of crosslinkers Batch 4: with AcOH and 10X of crosslinkers The four groups were submitted for compression testing Scaffold characterization Mechanical test: compression All specimens (n=4 for each batch) were held at ambient temperature and humidity. Each specimen in turn was placed on a powder compaction test fixture of bore diameter 30 mm in an Instron universal testing machine. The loaded sample fixture was placed on the 5 kN capacity load cell platen. The load cell was tared before the loading piston was placed on the sample. Once the loading piston was positioned, a preload of 5 N was applied. The crosshead was adjusted to bring the loading piston into contact with the upper platen. The position measurement was then tared using the Bluehill software and the test initiated. The test parameters were: Crosshead speed = 1 mm/min Output statistics: Load in N at 2 mm extension F(2) Position change in mm at 250N X(250) Surface features (BET/BJH) The surface area and pore volume parameters of the collagen-based scaffold were determined by nitrogen adsorption/desorption isotherms at 77 K in a Belsorp Mini II Surface area analyser. The sample was subjected to prolonged degassing at 40°C prior to measurement. The Brunauer-Emmett-Teller (BET) equation was used for the specific surface area quantification while Barrett-Joyner-Halenda (BJH) was used for mean pore size and pore volume determination. pH measurement in water 170 mg of collagen-based scaffolds were soaked in 20 mL of ultra-pure water and monitored for pH. The soaked scaffolds were sealed in a Falcon tube and left shaking (120rpm) in an incubator-shaker at 37 °C for 2, 6, 24, 48, 168, and 504 hours. For each time point, to remove debris, the solutions were filtered twice using a 0.2μm filter; the conditioned waters were collected in clean tubes and the pH measured using a Hanna pH-meter.

  Cytocompatibility assay3.2.4.7.1 Cells seedingHuman gingival fibroblasts (hGFs) were used to assess the cytocompatibility of the collagen-based scaffolds containing Sr-doped melt-quenched derived phosphate glasses(18.4 mm of diameter x 9.8 of height for a total of 0.45 g each).Cells at passage 6 were defrosted and placed into two 75 cm 2 Corning® CellBIND® Surface cell culture flasks to let them grow. During this period the complete DMEM medium (10% FBS and 1% Pens-Strep) was changed every 2 days. When cells reached a confluence of about 90%, they were harvested by trypsinization with 0.25% trypsin without EDTA incubated for 3 min at 37 °C. After, the trypsin reaction was stopped by adding complete medium and cells were collected by centrifugation at 1200 rpm for 5 min. Cells were seeded at a density of 10 5 cells/scaffold by applying 300 μL of cell suspension on the samples at 37°C for 20 minutes. This step ensured that cells penetrated the scaffolds without felling in the gaps between the scaffolds and the walls of the wells. Afterwards, 2 mL of medium was added to cover the samples. As control, hGFs were seeded in an empty well at the concentration of 10 5 cells/well in a 12-well plate.of them according to their importance: the phase was chosen as the most critical parameter followed by yield, composition and colour. The final desirability plot combines the four outcomes. Having set preferred ranges for each outcome the desirability plot shows the variable combinations delivering the best desirability.Experimental setupTo begin, EtOH and EG were mixed in a three-neck round-bottom flasks. Subsequently, the ethyl-P precursor was added to the blend and left stirring for about 10 min to ensure a homogeneous mix. Afterwards, NaAc was added and left stirring for 1h. The last precursor, CaAc, was then added and the flasks sealed. The flasks were kept sealed under constant stirring for 24h at room temperature before being immersed in the water thermal bath. At this point a condenser was attached. The temperature of the thermal bath and the time under reflux were chosen in agreement with the FED outputs.Immediately after the reactions were completed, the wet gels were filtered using a 500mL VWR® Vacuum Filtration Systems with 0.20 μm pore size of the membrane. The materials obtained were collected in glass Petri dishes and calcined in an electric kiln at the following setting: ramp 1 /min up to 250 and holding for 15min.

Figure 4

 4 Figure 4 Experiments generated by the FED software

Methods:

  The research was performed by consulting three databases (PubMed, ScienceDirect and Web of Science) to identify relevant published literature using the following terms (in Title/Abstract): ((((Bioglass*) OR glass*)) AND (((((dent*) OR odonto*) OR tooth) OR teeth) OR periodont*)) AND ((sol-gel) OR mesoporous). The research was performed considering five years' window time (from 01/01/2012 to 31/08/2017) and not language restriction was imposed. The Preferred Reporting Items for Systematic Reviews and the Meta Analyses (PRISMA) statement was used as a guideline for the review methodology.

Figure 5 .

 5 Figure 5. XRD graphs of C24 (A) C32 (B) and C40 (C) glasses

Figure 7 .

 7 Figure 7. pH variation of the water as a function of Ca increment after 24 hours of incubation

Figure 8 .

 8 Figure 8. Adsorption and desorption isotherms of the tested glass samples

Figure 9 .Figure 10 .

 910 Figure 9. Variations of pore volume, pore size and surface area as function of Ca increment

Figure 11 .

 11 Figure 11. XRD analysis on the C32 glass powder after TGA

Figure 12 .

 12 Figure 12. 1 XRD graphs of Bt/PBG (A) Et/PBG (B) glasses

Figure 13 .

 13 Figure 13. Infra-red spectra in the range 600-3000 cm -1

Figure 14 .

 14 Figure 14. Adsorption and desorption isotherms of the Bt/PBG and Et/PBG glasses

Figure 15 .

 15 Figure 15. Variations in pore volume, pore size and surface area as function of phosphate precursors

Figure 16 .

 16 Figure 16. XRD graphs of C24/30 (A) and C24/100 (B) sol-gel glass samples

Figure 18 .

 18 Figure 18. Adsorption and desorption isotherms of the C24/30 (left) and C24/100 (right) sol-gel glasses

Figure 19 .

 19 Figure 19. Relationship between surface features and production scale

  ) revealed that it is possible to group the experiments into two sets: runs #1, #11, #10, #9 and #8 and runs #6, #2, #3, #5 and #7. Run 130 #4 in the middle acts as a linking point. The first group belongs to all the runs characterized by a low EtOH/EG ratio; the second group belongs to all the runs characterized by a high EtOH/EG ratio.Considered together, the results show that the EtOH/EG ratio was the main influencing factor.YieldAfter the heat treatment, the powders were weighed, and their final yield recorded and plotted (fig.21) as a percentage of the expected product (20g).

Figure 20 .Figure 21 .

 2021 Figure 20. Bi-dimensional (left) and tri-directional (right) representation of the trend of the phases in relationship with the variations of solvents ratio, time and temperature

Figure 22 .

 22 Figure 22. Tri-directional representation of the colour range in relationship with the variations of solvents ratio, time and temperature

Figure 23 .

 23 Figure 23. Yield of the four materials as a function of the quantity of Ca

Figure 26 .

 26 Figure 26. XRD of the full Sr phosphate glass obtained by coacervation process

Figure 27 .Figure 28 .Figure 31 .

 272831 Figure 27. XRD graph of the coacervate-derived Sr-doped phosphate glass

Figure 32 .

 32 Figure 32. Vitality of hGFs seeded in contact with the scaffolds in comparison with the ctr

Figure 33 .

 33 Figure 33. XRD graphs of sol-gel (A) and melt-quenched (B) glasses

Figure 36 .

 36 Figure 36. Cumulative release (ppm) of P (A), Ca (B) and Na (C) ions as a function of time (h) for MQ and SG glasses

Fig. 38

 38 Fig. 38 compares the pH variation of both SG and MQ glasses both in water and SBF during 3 weeks of incubation. It is possible to notice that for both glasses the water pH dropped quickly to 4.27 and 3.51 respectively after 2 hours and it stabilized during the time course. The same glasses in SBF acted differently: the pH remained quite stable during whole the experiment becoming even slightly more basic (8.04 and 8.13 for SG glass and MQ respectively after three weeks).

Figure 37 .Figure 38 .

 3738 Figure 37. Variation of SBF pH as a function of the incubation time

7. 2 APPENDIX 2

 22 Below (fig. 40 and 41), the XFR data of the Sr-doped phosphate coacervate glasses is shown to indicate the successful incorporation of the Sr in the glass networks.

Figure 41 .

 41 Figure 41. XRF (wt%) of the full Sr phosphate glass obtained by coacervation process

Figure 42 .

 42 Figure 42. Composition (wt%) obtained by XRF of the coacervate-derived Sr-doped phosphate glass

Table 1 .

 1 Actual composition (mol%) and yield of the phosphate-based glasses prepared by

	sol-gel

Table 2 .

 2 Infrared peak positions (cm-1) and Q assignment of the observed bands of sol-gel

	glass samples

Table 3 .

 3 Pore volume, pore size and surface are of the glasses after BET analysis 118

Table 4 .

 4 Composition (in mol%) obtained by XRF of the phosphate glasses produced by solgel using different phosphate precursors 121

Table 5 .

 5 Infrared peak positions (cm-1) and assignment of the observed bands for sol-gel glass samples 122

Table 6 .

 6 Pore volume, pore size and surface area of the glasses obtained by BET analysis 124

Table 7 .

 7 Composition obtained by XRF of the sol-gel glass preparations 125

Table 8 .

 8 Infrared

peak positions (cm-1) and assignment of the observed bands of sol-gel glasses 126

Table 9 .

 9 Pore volume, pore size and surface area of the glasses after BET analysis 128

Table 10 .

 10 Compositions (in mol%) by XRF of the most promising FED-derived runs 132

Table 11 .

 11 pH progress during the different steps of the protocol 133

Table 12 .

 12 Yield of the four materials obtained by precipitation

Table 13 .

 13 Composition in mol% of sol-gel and melt-quenched glasses

Table 14 .

 14 Infrared peak positions (cm-1) and assignment of the observed bands of sol-gel and melt-quenched glass samples

Table 15

 15 Procedures tested to define an optimized sol-gel protocol

	ABBREVIATIONS
	AcOH: acetic acid
	Al: silver
	Ba: barium
	BET: Brunauer-Emerett-Teller test
	BJH: Barrett-Joyner-Halenda analysis
	BOs: bridging oxygens
	Bt/P: butyl-phosphate
	Ca: calcium
	CaAc: calcium acetate
	CaOEt: calcium ethoxide
	CME: calcium methoxyethoxide
	Co: cobalt

Table 1 .

 1 Actual composition (mol%) and yield of the phosphate-based glasses prepared by sol-gel

	Name P 2 O 5 CaO Na 2 O Yield (%)
	PBG-C24 49	24	27	77
	PBG-C32 49	30	21	50
	PBG-C40 49	38	13	37

Table 3 .

 3 Pore volume, pore size and surface are of the glasses after BET analysis

Glasses Pore volume [cm 3 g -1 ] Pore size [nm] Surface area [m 2 g -1 ]

	C24	0.014651	10.537	5.5619
	C32	0.013949	11.065	5.0427
	C40	0.032437	12.129	10.698

Table 4 .

 4 Composition (in mol%) obtained by XRF of the phosphate glasses produced by sol-gel using different phosphate precursors

	Glasses P 2 O 5 CaO Na 2 O Yield (%)
	Bt/PBG 49	24	27	78
	Et/PBG 56	24	20	83

Table 5 .

 5 Infrared peak positions (cm -1 ) and assignment of the observed bands for sol-gel glass samples

	Samples	vas(PO2) -/Q2	Peaks (cm -1 )/Q states vas(PO3) 2-/Q1 vs(PO3) 2-/Q1 vas(POP)/Q2 vs(POP) vs(POP)
	Bt/PBG	1264	1090	1028	896	726	708
	Et/PBG	1254	1088	1034	896	722	698

Table 6

 6

	vole of Et/PBG was 3.2 times bigger than Bt/PBG glass. The pore size increases by 1.8 times
	when ethyl-phosphate was used as precursor instead of butyl-phosphate. The surface area
	was 1.8 times bigger in Et/PBG glass particles.

summarises pore size, pore volume and surface area of the two materials. The relationship between phosphate precursors and surface features is shown in fig.

15.

A great difference was found between the pore volume of Bt/PBG and Et/PBG: the calculated pore

Table 6 .

 6 Pore volume, pore size and surface area of the glasses obtained by BET analysis

	Bt/PBG	0.014651	10.537	5.5619
	Et/PBG	0.047313	18.711	10.115
	4.3.1.2.6 Other considerations

Glasses

Pore volume [cm 3 g -1 ] Pore size [nm] Surface area [m 2 g -1 ]

Table 7 .

 7 Composition obtained by XRF of the sol-gel glass preparations

	Glasses P 2 O 5 CaO Na 2 O Yield (%)
	C24/30	49	24	27	77
	C24/100 48	23	29	47
	4.3.1.3.2 Phase

Table 9 .

 9 Pore volume, pore size and surface area of the glasses after BET analysis

Glasses Pore volume [cm 3 g -1 ] Pore size [nm] Surface area [m 2 g -

				1 ]
	C24/30	0.014651	10.537	5.5619
	C24/100	0.030392	16.702	7.2788

Table 10 .

 10 Compositions (in mol%) by XRF of the most promising FED-derived runs

	Runs	P2O5 (53%)	CaO (32%)	Na2O (15%)
	#10	50%	38%	12%
	#9	50%	38%	13%
	#3	44%	52%	4%
	#8	45%	46%	8%

Table 11 .

 11 pH progress during the different steps of the protocol

				pH measurement
	Samples	CaCl2 solution	Na4P2O7 solution	After mixing (Ca + P solution)	After centrifuge	After water washing
	RUN 1	8.26	10.48	10.05	10.04	9.21
	RUN 2	8.30	10.52	10.53	10.04	9.41

Table 12 .

 12 Yield of the four materials obtained by precipitation

Samples RUN 1 RUN 2 RUN 3 RUN 4 RUN 5 RUN 6

	Yield (%)	37	32	7	15	6	15

Table 13 .

 13 Composition

		51	34	15	81
	SG/PBG-C32	49	30	21	50

in mol% of sol-gel and melt-quenched glasses Glass/mol% P2O5 (53%) CaO (32%) Na2O (15%) Yield (%) MQ/PBG-C32

Table 15

 15 Procedures tested to define an optimized sol-gel protocol

Sol-gel PBG-C32: P2O5 (53%) -CaO (32%) -Na2O (15%) -mol% NON-aqueous system

	Precursors Ethyl-P CaAc NaAc Butyl-P CaAc NaAc	H2O Solvent( s) EtOH/EG (60/40) EtOH/EG (60/40)	AcOH No Catal yst Heati ng (°C) No 250 No 250	No Reflux Yes (100℃ /6 days) Yes (100℃ /6 days	No Drying Yes Yes (100℃/7 Yes (100℃/7 days) days)	Salts precipita tion Problem s Milky solution CaAc not complete ly dissolved Milky	Not record ed Yield (g) Not record ed Not record ed	Pictures
	Precursors Ethyl-P CaOEt NaOEt (21%in EtOH) Ethyl-P CaOEt NaOEt (21%in EtOH) Ethyl-P CaOEt NaOEt (21%in EtOH) Ethyl-P CaAc NaAc CaAc NaAc (15g final yield instead of Ethyl-P Ethyl-P CaAc NaAc Ethyl-P CaAc NaAc Ethyl-P CaAc NaAc Precursors Ethyl-P CaAc NaAc Ethyl-P CaAc NaG Ethyl-P CaAc NaAc Butyl-P CaAc NaG Ethyl-P CaAc NaG Butyl-P CaAc NaAc Butyl-P CaAc CaAc NaAc (Inverted order: CaAc first) Ethyl-P NaG	Solvent( s) EtOH EtOH EtOH EtOH/EG (60/40) EtOH/EG (60/40) H2O H2O H2O Solvent( s) EtOH/EG (60/40) EtOH/EG (60/40) EtOH/D MFA (60/40) EtOH/EG (60/40) EtOH/D MFA (60/40) EtOH/D MFA (60/40) EtOH/EG (60/40)	Catal yst No AcOH No Heati ng (°C) 250 AcOH No No 250 No 250 NH4O H No AcOH 250 NH4O H 250 Factorial Experimental Design (FED) Reflux Drying Problem s Yes yes (120℃/1w eek) Ca poorly soluble yes yes (120℃/1w No No Ca poorly soluble (100℃ /6 days) No n of the run below: CaAc not well days) days) /6 (100℃/7 (100℃ Yes Yes Yes Yes repetitio eek) Ca poorly soluble No No No reaction Yes yes (100℃/1w eek) Salts precipita tion Yes yes (100℃/1w eek) Salts precipita tion Catal yst Heati ng (°C) Reflux Drying Problem s No 250 Yes (100℃ /6 days) No The No 250 Yes (100℃ /6 days) Yes Yes (100℃/7 days) No 250 No No Yes solution Yes CaAc not (100℃ (100℃/7 complete /6 days ly days) dissolved Yes (100℃ /6 days) Yes Yes (100℃/7 days) No 250 Yes Yes Yes No No Yes (100℃ /6 Yes (100℃/7 days) CaAc not complete ly dissolved days (100℃ /6 (100℃/7 days) days) No 250 days) days) /6 (100℃/7 (100℃ Yes Yes Yes	Yield (g) Not record ed Not record ed 0.44 ed record Not Not record ed Not record ed Not record ed Not record ed Yield (g) 7.79g Not record ed Not record ed Not record ed Not Not record ed record ed ed record Not	Pictures Pictures
	20)					Fust dissolved
	Ethyl-P			Yes		reaction
	Butyl-P CaOEt NaOEt (21%in EtOH) Butyl-P CaOEt volume) Ethyl-P CaAc NaAc CaAc NaAc Ethyl-P CaAc NaAc the increase (Keeping the EG/EtOH ratio (60%: 40%),	EtOH EtOH EtOH/EG (100/0) EtOH/EG (80/20) EtOH/EG (60/40)	No AcOH No No No 250 No 250 No 250	No No (100℃ /6 days) Yes (80℃/ days) 4 Yes (100℃ /6 days)	No No No No Yes (100℃/7 days)	between butyl-P and NaOEt so absolutel y no solubility of CaOEt Fust reaction butyl-P between	Not record ed ed record Not 0.26 5.07 Not record ed

Discussion

ABSTRACT 2 LITERATURE REVIEW

Glass by precipitation

Calcium and pyrophosphate reagent solutions were prepared separately by dissolving CaCl2•2H2O and Na4P2O7 or K4P2O7 in ultra-pure water (UPW). The calcium chloride solution was then added to the pyrophosphate solutions using a peristaltic pump at a constant volumetric flowrate (7.5 mL min -1 ) for all samples. After the complete addition of the calcium reagent, the solutions were stirred for five minutes, and the final colloidal solutions centrifuged at 5000 rpm for 10 min. The resulting gel was washed two times with UPW. The resulting gels were poured into a Petri dish and heated at 70 °C for 7 days.

Aliquots of the solutions were collected during each step in order to measure the pH.

Coacervate-based phosphate glasses

The samples were prepared, as shown in fig. 2 (adapted from [START_REF] Pickup | Characterisation of phosphate coacervates for potential biomedical applications[END_REF] ), by slowly adding a 2 M solution of the calcium chloride to an equal volume of a 4 M solution of sodium polyphosphate (Graham's salt) at a rate of 5 mL/min. The sodium polyphosphate solution was stirred during this addition and the resultant mixture stirred for a further hour after the

Cells viability test

Cells viability was checked after 2, 4 and 7 days by using AlamarBlue. Briefly, the dye was added to each well at the dilution of 1:10 and left to incubate overnight at 37 °C. The following morning, a drop of 100 μL of media containing the dye from each condition was placed in a flat bottom 96 well plate and the absorbance was checked without cover using NanoQuant infinite M200PRO microplate reader (by Tecan) setting 600 nm as reference wavelength and 570 nm as measurement wavelength. The calculated difference between measurement and reference was used to compare the cell viability between the control and the samples.

After the measurement, the scaffolds and the control were washed with PBS and a fresh medium was added.

The experiment was performed in duplicate.

Use of Factorial Experimental Design (FED) in sol-gel composition optimisation

Designing of experiments

An article was published [START_REF] Cai | Microstructural characteristics and crystallization of CaO-P 2 O 5-Na 2 O-ZnO glass ceramics prepared by sol-gel method[END_REF] in which a Zn-doped phosphate glass ceramic was produced by a sol-gel method in which a mixture of 40% EtOH and 60% EG (by volume) was used as the reaction medium. Inspired by the paper, the Design Expert "Statease" factorial experimental design software package was used to create a series of experiments in which the impact of (i) temperature, (ii) EtOH:EG ratio and (iii) time of the sol-gel reaction on final material properties could be appraised. For each of the three variables, ranges were imposed: for the volume ratio the window was from 100% EtOH and 0% EG to 0% EtOH and 100% EG; for the temperature under reflux the range was from 60 to 100 °C; the time under reflux was varied between 2 to 6 days. Having set these parameters, the software was used to create a restricted number (11) of representative experiments to run (the output-fig. 4). Based on the outcome from the trials, the FED software was used to calculate which variable (or variable combinations) were most influential on the chosen outcomes. Four responses were considered: phase, yield, colour and composition. A different weighting was assigned to each ions) altering the dissolution profiles of the glasses. Furthermore, the EDX analysis revealed the presence of deposits rich in Ca and P on the surface of the scaffolds after soaking them in SBF. The Ca/P ratio of such precipitate was 1.68 (mol%) in good agreement with the HA ratio being 1.67 (mol%). For those reasons, their possible application in restorative dentistry is proposed.

The spectra of the sol-gel samples reveal the characteristic bands associated with P-O-P bonding: for the C24 glass the peaks were identified at 708 cm -1 , 762 cm -1 and 896 cm -1 ; for the C32 glass the POP bonding peaks were recognized at 705 cm -1 , 776 cm -1 and 898 cm - 1 ; for the C40 the same peaks were assigned to 702 cm -1 , 720 cm -1 and 908 cm -1 . In all the three cases the first peaks represent the POP bond in symmetric configuration, whereas, the second and third peaks characterise the same bond in asymmetric mode. The pairs of peaks at 1028 cm -1 and 1090 cm -1 , 1028 cm -1 and 1088 cm -1 and 992 cm -1 and 1086 cm -1 were recognised and assigned to the (PO3) 2-bond in the symmetric and asymmetric state respectively for the first and second member of each pair. Further peaks at 1264 cm -1 , 1254 cm -1 and 1256 cm -1 belonging respectively to C24, C32 and C40 glass can be detected and assigned to the asymmetric (PO2) -bond. All the spectra contain Q1 and Q2 units: especially, Q1 for the symmetric and asymmetric (PO3) 2-and Q2 for the asymmetric POP and (PO2) -bond. The infrared spectra (fig. 17) provided more information on the structure and on the nature of the bonding of the glasses prepared by sol-gel at a different scale. The peaks were assigned as described by Brow, R. at al 2000 [START_REF] Brow | Review: the structure of simple phosphate glasses[END_REF] . The assignments are listed in table 8 and the related Q states were given where it was possible. The spectra of the sol-gel samples had the characteristic bands of the P-O-P bonding:

for the PBG-C24/30 glass the peaks were identified at 708 cm -1 , 726 cm -1 and 896 cm -1 ; for the PBG-C24/100 glass the P-O-P bonding peaks were recognized at 701 cm -1 , 758 cm -1 and 890 cm -1 . In both glasses the first two peaks represent the P-O-P bond in symmetric configuration, whereas, the third peaks characterise the same bond in asymmetric mode. The pairs of the peaks at 1028 cm -1 and 1090 and 1019 cm -1 and 1086 cm -1 were recognised and assigned to the (PO3) 2-bond in the symmetric and asymmetric state respectively for the first and second member of each pair. Further peaks at 1264 cm -1 and 1252 cm -1 belonging

Coacervate-based phosphate glass

Objective: Investigate a novel approach to produce phosphate glasses with wet and soft chemistry at room temperature with non-toxic precursors and solvents.

Methods: The samples were prepared by slowly adding a 2 M solution of calcium chloride to an equal volume of a 4 M solution of sodium polyphosphate (Graham's salt) at a rate of 5 ml/min under constant stirring. Then, the coacervate layer was washed twice with UPW before leaving to stand overnight. After removal of the water layer, the coacervate layer was dried at room temperature in a vacuum desiccator.

Results and discussions:

This methodology seemed to be promising, allowing to explore a wider range of compositions and incorporate further ions into the structure. Moreover, it ensured high yield.

To test this procedure, four different trials were performed:

1. Attempts were made to re-produce the (Na2O)0.15(CaO)0.35(P2O5)0.50•yH2O-mol% composition of glass described in a literature article [START_REF] Pickup | Characterisation of phosphate coacervates for potential biomedical applications[END_REF] 2. Scale-up of the composition mentioned in 1. at a x5 scale 3. Sr-doping by fully and partially replacing Ca in trial 1.

4. Preparation of a different composition (P2O5(53%)-CaO(32%)-Na2O(15%) -mol%)

Repeating work described in the article

The coacervate phosphate glass studied by D. M. Pickup, et al. 2013 was reproduced as described in the "Materials and methods" section. In agreement with the phase description in the article, fig. 24 shows that the material obtained was totally amorphous to indicate the possibility to obtain a fully amorphous glass material by using the coacervate process.

Scale-up

The same composition was scaled up by 5 times. The material obtained was a bulk of 10.08 g (corresponding to about 80% of the expected yield) and containing 95 wt% of amorphous phase as detected by XRD (fig. 25). The crystal phase recorded was assigned to

NaCl. This proved that the production can be scaled-up without significantly affecting either the yield or the phase (in term of quality) of the material.

Sr-doping

Before doping the glass, to check the real possibility to incorporate Sr by using this procedure, Ca was fully replaced by Sr using SrCl2 • 6H2O (2M) as source.

Surface features

To further characterize and understand the properties of the material obtained by this method, the coacervate glass powder was submitted for BET/BJH and SEM analysis. Visualized by microscopy (fig. 30), the material appeared made by particles of irregular shape and different size with sharp edges and well-defined sides. It is possible to notice that faces are smooth with some smaller particles standing above.

Structure

The FTIR spectra of the glasses (fig. 34) prepared by sol-gel (blue line) and meltquenching (red line) gave further information on the structure and on the bonding in both glass networks. The peaks were assigned and listed in table 14. The associated Q state was also given. The spectrum for the sol-gel sample presents the characteristic band of the P-O-P bonding at 705, 776 and 898 cm -1 ; in particular, the first two in symmetric configuration, the last in asymmetric mode. In addition, two further peaks at 1028 and 1088 cm -1 can be identified and assigned to the (PO3)2-in symmetric and asymmetric stretch mode respectively.

The peak at 1254 cm -1 was recognized as asymmetric (PO2) -. For the MQ glass sample, the P-O-P bond can be recognized both in symmetric and asymmetric configuration (624, 768 and 144 890 cm -1 ); the asymmetric and symmetric (PO3) 2-at 1090 and 1031 cm -1 respectively. The (PO2) -in asymmetric arrangement at 1266 cm -1 can be recognized.

All the spectra contain Q1 and Q2 units: especially, Q1 for the symmetric and asymmetric (PO3) 2-and Q2 for the asymmetric POP and (PO2) -.

pH measurement in water Fig. 35 shows the pH variation over time. After 2 hours of incubation, the water pH dropped to 4.27±0.06 and 3.51±0.04 for SG-PBG and MQ-PBG respectively. After this time, the pH had stabilized for both glasses.

Dissolution in water

Both glasses were found to degrade in water but with different kinetics (Fig. 36). The MQ glass showed a linear degradation during the first 24 hours for all the three ions; after this, a plateau phase was reached. The concentration of the ions in the water reflected their content in the glass formulation: the leached phosphate had the highest value because of its highest content in the glass, then the Ca and Na the lowest. In addition, the ion content influenced the release kinetics of the three ions during the linear phase: P had the highest rate followed by Ca and then Na. The SG glass, instead, presented a fast, burst release: the content of ions in the medium (water) reached the highest level already after two hours of

Microstructure comparison

From the SEM images (fig. 39) of the glass particles it is possible to notice that their surface topologies and shape were significantly different. The particles of the glass produced by sol-gel had a rough and jagged surface. Whereas, the surface of the particles of the meltquenched glass were smooth. Also different was the overall shape of the particles: more rounded for the sol-gel glass particles but polygonal for the melt-quenched. In addition, on the surface of the sol-gel particles, needle-like structures were visible absent in the meltquench preparation. results in a further difficulty for the water to penetrate the glass network and hydrolyse the POP bonds. In addition, sol-gel glasses contain a significant amount of OH groups, compared with MQ glasses, which act as chain terminating groups [START_REF] Pickup | New sol-gel synthesis of a (CaO) 0.3 (Na 2 O) 0.2 (P 2 O 5) 0.5 bioresorbable glass and its structural characterisation[END_REF] and strongly reduce the connectivity of the network. Moreover, it has been reported that the presence of OH groups reduces the chain length 100 : the bridging action of the modifiers is weaker resulting in less connectivity between the chains. The combination of these factors determines a faster protonation of the most external NBOs that causes the instant hydrolysis of the network and the burst release of the modifier ions.

The pH trend (fig. 37) of the SBF towards basic values after the glasses were soaked for three weeks could be explained by considering that the SBF, due to the presence of the tris-HCl, is a buffered system: it is able to subtract protons from the media stabilizing the pH.

Due to this, the Na (and the Ca) coming from the glasses overtakes the "acidification power" associated with the phosphates leached from the dissolving networks leading to a more basic value. This is supported by the 24 hours single points measured where despite the concentration of the powder is 10 times higher, the pH did not reach critical values. The difference between MQ and SG could be explained taking into consideration the concepts expressed immediately above.

By comparing the development of the water and SBF pH (fig. 38) one can observe that the same glass material can act differently in different solutions based on the properties of the solution itself. This can be explained considering what just said: buffer system, composition, ionic strength and so on.

The SEM images at different magnitudes of both MQ and SG/PBGs (fig. 39) show the different texture of the particles. The different surface feature can be explained considering the different approaches. In the sol-gel method (bottom-up approach) nanoparticles are built by progressive aggregation 101 , whereas, in the melt-quench technique (top-down) particles are fused together generating more compact units. This provides the explanation of the different surface topologies observed between the two tested glasses coming from the two different methods and adds further support to what was observed for the dissolution behaviour.

Considering the previous discussion, it is possible to draw the conclusion that the surface features of the same material compositionally can be modified by changing the production route. This might determine drastic changes in the behaviour of the materials (water solubility for example). This becomes particularly important when it comes to medical applications: different topology (rough surface rather than smooth, for instance) can trigger different cell response or different degradation timing and this can be used to tailor the material in agreement to the application or the host tissue.

Melt-quench vs Sol-gel vs coacervation

Based on the samples with the same composition (P2O5(53%)-CaO(32%)-Na2O(15%) -mol%) it possible to compare the data coming from the figures 5B and 28; 4B and 29A; 30 and 39 in order to underline differences and similarities between three different glass processing approaches: melt-quench, sol-gel and coacervation process. The first group of figures refers to the phase of the obtained materials: all the three methods were equally able to give totally amorphous glassy materials. The second group of figures analyses some surface parameters of the particles: the BET performed on the glass particles produced by sol-gel and coacervation gave a type 2 isotherm typical of non-porous materials. For the melt-quench derived glass particles the BET was not performed because it is known that such a technique generates non-porous materials. The biggest difference was revealed by SEM: whereas coacervation and melt-quench generate glass particles with the same topography (smooth surface and polygonal shape), the sol-gel route generates less well-shaped and defined particles with irregular edges and rough surfaces. Thus, it is possible to argue that the three materials might behave differently under the same condition. This further supports and is

supported by what has been discussed in the previous paragraph (4.7). In terms of yield, the sol-gel route proved to be inferior.

It is possible to conclude that the choice of a particular processing method should be done considering the safety of the process, how the production route may affect the properties of the materials and considering the future applications.

From the cytocompatibility test performed arises that, when it comes to composites, the ratio between the different constituents is an important feature of their formulation. In the case examined in this thesis, it is evident that the 1:1 ratio between col and PBG particles is too high leading to an unfavourable environment for cells. In the future, composite scaffolds where the glass particles content is way lower than collagen fibres content could be explored.

Alternately, methods that increase the stability of the glasses (e.g. annealing) might be taken into consideration.

7 Appendix 167

APPENDIX 1

This section reports a comparison study on the dissolution behaviour of a single phosphate glass composition (P2O5 (53%) -CaO (32%) -Na2O (15%) -mol%) produced by solgel versus melt-quench routes. In both trials the test involved three weeks of incubation in SBF at 37 .

From fig. 39, it is possible to notice that the three ions dissolve into the SBF with different kinetics. In the MQ system, the phosphate was found to dissolve almost linearly for the first two hours to reach a plateau; whereas, in the SG system we have a burst release during the first 5 min of incubation immediately followed by a plateau. As explained in the section 4.7, it might be due to 1. lower density of the network 2. higher quantity of OH groups 3. reduced chain length

weaker network connectivity

Calcium release suggested similar kinetics between the two glass systems. It seems that there was almost no dissolution at all during the first 6 hours of incubation with the ̴ 120