Synthesis of new precursors for the fabrication of fully organic hybrid nanofibers

Synthesis of new precursors for the fabrication of fully organic hybrid nanofibers Pág. 1 Abstract This project was carried out in the field of biomaterials, in the section regenerative therapies and precisely aimed at tissue engineering. Bone restoration and regeneration are a major importance in the tissue engineering field. Basically, an implant such as scaffolds for bone regeneration has to stimulate osteogenesis and angiogenesis, apart from being bioresorbable as well. Nevertheless, biomaterials for osteogenesis and angiogenesis should be considered not only as a template for cell growth, but also as a delivery agent. Indeed, previous and current studies aimed in this direction, promotes the key role of calcium in blood vessel formation. The interesting achievement is to obtain nanostructured materials by electrospinning with embedded calcium and phosphate compounds, which shall deliver at an appropriate rate. In our case, the study was directed to bioactive glass nanoparticles, whose purpose is to be incorporated in polymeric fibers. In order to reach desired compositions, the particles were synthesized by the sol-gel process. A first glass synthesis was performed to acquire the “G5”, whose composition is known to be as: 44.5% P2O5, 44.5% CaO, Na2O 6% 5% TiO2. However, the main task of this project was to produce new bioactive glass nanoparticles using different precursors: especially calcium propionate and phytic acid to obtain ternary glass particles with a desired composition of 47.5% P2O5, 47.5% CaO, 5% TiO2. Indeed, the challenging nature of the study was to use precursors whose byproducts appear to be biocompatible and biodegradable. The current precursors used, for the fabrication of the regular “G5” particles, contain whether ethanol or 2-methoxyethanol, known to be cytotoxic once released in the body. New precursors whose byproducts after release can be eliminated by the body would an important progress. The difficulty in the control of sol-gel parameters according to the difference of precursors have shown quite broad range of composition which have been compared to the defined G5 glass. Further studies have to be completed in order to define an effective protocol capable of producing these nanoparticles with better control in terms of compositions and morphology, because it is precisely the first features which affect the additional properties of our material, such as pH, calcium release and of course cell proliferation. Pág. 2 Memoria Synthesis of new precursors for the fabrication of fully organic hybrid nanofibers Pág. 3 Table of contents ABSTRACT ___________________________________________________ 1 TABLE OF CONTENTS _________________________________________ 3 1. GLOSSARY ______________________________________________ 5 2. PREFACE ________________________________________________ 7 3. INTRODUCTION ___________________________________________ 9 4. BACKGROUND THEORETICAL _____________________________ 10 4.1. Regenerative medicine and tissue engineering ........................................... 10 4.2. Biomaterials .................................................................................................. 11 4.3. Biomaterials for osteogenesis and angiogenesis ........................................ 14 4.3.1. Nanostructured materials produced by electrospinning .................................. 15 4.3.2. Calcium-phosphate based glasses ................................................................. 17 4.3.3. Sol-gel process ............................................................................................... 19 4.3.4. Controlled release glasses (CRG) .................................................................. 26 4.3.5. Impact on biocompatibility – Cytotoxicity of byproducts .................................. 27 4.4. Objectives ..................................................................................................... 30 5. MATERIALS AND METHODS _______________________________ 31 5.1. Synthesis of the glass nanoparticles ............................................................ 31 5.2. Characterization methods ............................................................................ 38 5.2.1. Scanning Electron Microscopy (SEM) ............................................................ 38 5.2.2. Degradation .................................................................................................... 39 5.2.3. X-Ray Diffraction ............................................................................................. 43 5.2.4. Zeta potential and size measurements of particles......................................... 43 5.3. Cell culture ................................................................................................... 45 5.3.1. Cell seeding .................................................................................................... 46 5.3.2. AlamarBlue® - Proliferation tests .................................................................... 47 6. RESULTS AND INTERPRETATIONS _________________________ 50 6.1. Chemical Composition ................................................................................. 50 6.2. Morphology of the particles (Shape and Size) ............................................. 55 6.3. Zeta potential analysis ................................................................................. 60 6.4. Crystallinity of the particles ........................................................................... 62 Pág. 4 Memoria 6.5. Degradability ................................................................................................ 65 6.6. Cell assays – Proliferation tests .................................................................. 68 7. CONCLUSION ___________________________________________ 71 ACKNOWLEDGEMENT ________________________________________ 73 SPENDING __________________________________________________ 75 ENVIRONMENTAL EVALUATION ________________________________ 78 BIBLIOGRAPHY ______________________________________________ 79 Bibliographical references ..................................................................................... 79 Synthesis of new precursors for the fabrication of fully organic hybrid nanofibers Pág. 5 1. Glossary G5 Glass composition are identified as GX (G standing for Glass and X corresponding to the atomic percentage of titanium in the glass composition) Sol-gel process Chemical synthesis technique for preparing gels, glasses, and ceramic powders. P2O5 Phosphorus pentoxide CaO Calcium oxide TiO2 Titanium dioxide Na2O Sodium oxide Biocompatibility Ability of a material to fulfill its function resulting in an appropriate response of the receiver organism in a specific situation Bioresorbable Gradual degradation in the body over a period of time (do not require mechanical removal) Bioactivity Ability to interact directly with a living tissue Osteoproductive Ability of allowing the colonization of a bioactive surface with osteoblasts – it causes both intra- and extracellular response on its interface Osteoconductive Just a biocompatible surface above which bone cells can migrate – it causes just an extracellular response Scaffold Provide the structural support for cell attachment and subsequent tissue development Angiogenesis The process of developing new blood vessels Osteogenesis The process of laying down new bone material by osteoblasts Electrospinning Technique that use an electric charge to pull very fine fibers from a liquid Pág. 6 Memoria PLA Poly (lactic acid) polymer – molecular formula (C3H4O2)n CaP Calcium phosphate BG Bioglass® - a commercially available family of bioactive glasses HA Hydroxyapatite Ca10(PO4)6(OH)2 Alkoxides A compound formed from an alcohol by the replacement of the hydrogen of the hydroxyl group with a metal Hydrolysis A chemical reaction in which the interaction of a compound with water results in the decomposition of that compound. Condensation A chemical reaction in which two molecules react with the resulting loss of a molecule of water (or other small molecule); the formal reverse of hydrolysis. Hybrid Combination of organic materials and inorganic materials Byproducts Compounds produced during the degradation of the material SEM Scanning Electron Microscopy EDX Energy Dispersive X-ray (Spectroscopy) HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid – chemical buffering agent DRX X-ray Diffraction. Technique for characterization of crystalline phases rMSCs Rat mesenchymal cells AlamarBlue® A method to evaluate the evolution of the cell population TFE Trifluoroethanol D.b.d (Recap chart) Drop by drop Synthesis of new precursors for the fabrication of fully organic hybrid nanofibers Pág. 7 2. Preface The present study was carried out as a Master Thesis internship and includes the six-month internship in laboratory required in order to achieve the first semester of the 5th and final academic year in engineering and materials science at the EEIGM Nancy, European School of Engineering and Materials Science. The internship was performed at the Institute for Bioengineering of Catalonia, located in the Scientific Park of Barcelona – Spain, from September 2013 to February 2014. The Institute for Bioengineering of Catalonia (IBEC) is an interdisciplinary research center focused on bioengineering and nanomedicine, based in Barcelona. It has several areas of research such as:  Cellular Biotechnology  Biomechanics and cellular biophysics  Nanobiotechnology  Biomaterials, implants and tissue engineering  Medical signals and instrumentation  Robotics and biomedical imaging I was involved during this project in the Biomaterials, implants and tissue engineering research program. It concerns the development of new materials and structures for biocompatible replacement, tissue formation and technologies for repairing or replacing tissues and organs. My work took place in the group directed by Dr. Elisabeth Engel, focusing on biomaterials for regenerative therapies, under the supervision of Dr. Oscar Castaño. Pág. 8 Memoria Synthesis of new precursors for the fabrication of fully organic hybrid nanofibers Pág. 9 3. Introduction Tissue engineering and regenerative medicine is an emerging multidisciplinary field involving biology, medicine, and engineering. It is aimed to restore structure and function of damaged tissues and organs by initiating the natural regeneration process. It applies both engineering and biology field in order to develop biological substitutes, which restore, maintain or improve tissue function. As third-generation biomaterials, the use of scaffolds for bone regeneration intent to replace the damaged tissue and activate its regeneration, while being degraded to make room for new tissue. In this way, previous studies suggest that biomaterials for osteogenesis and angiogenesis should be considered also as ion delivery agent, as well as a template for cell growth. Therefore, as we know that calcium plays a key role during the blood vessels formation process, we will try to produce nanostructured materials using the electrospinning technique, in which calcium and phosphate must be integrated and most importantly shall deliver ions at a suitable rate. This practical internship has been realized at the Institute for Bioengineering of Catalonia in the group directed by Dr. Elisabeth Engel under the supervision of Dr. Oscar Castaño. It was oriented towards the production and characterization of nanoparticles for hybrid nanofibers obtained via the sol-gel process using new precursors. This work tries to prevent cytotoxicity of the byproduct organic part of the material once implanted in the body with the use of two major new precursor which are calcium propionate and phytic acid. First of all, attempts will be performed to find suitable methods and protocols in order to obtain convenient compositions and morphology. Characterization standard methods on these CaO-P2O5-TiO2 particles will be carry out so that we can foresee the impacts on the cytotoxicity of our byproducts, as well as an appropriate ions delivery rate and a possible incorporation into the polymeric matrix. This project appears to be quite challenging according to the fact that no previous work relate the desired incorporation of organic/inorganic hybrid nanoparticles into the human body. Pág. 16 Memoria The PLA polymer (polylactic acid) for example, has appeared to be a suitable material for tissue engineering and met almost all the requirements to be used as a scaffold; nontoxic behavior, biodegradable and bioresorbable but so far no bioactivity [28]. In order to get this bioactive behavior, it has to be combined with bioactive molecules or materials such as Bioglass® or more generally phosphate calcium based glasses (osteoconductive and able to bond to bone). When you actually add those CaPs or BG particles, our scaffold is subjected to a degradation process problem, especially about the reduction of acidic degradation. In order to optimize the final scaffold, one technique has shown very good results: electrospinning. Different properties of nanofibers can be obtained or adjusted by controlling the different parameters of the electrospinning process in order to obtain electrospun scaffolds for biomedical applications. A few applications are described: wound dressing, drug delivery, blood vessels, bone tissue engineering, etc... [15] Wound healing: nanofibrous dressings using the electrospinning process have several advantages compared to others processes. Higher surface areas, higher microporous structure, better attraction of fibroblasts to the derma layer (that can release important extracellular matrix components) are properties encountered in wound healing electrospun materials (collagen, polyvynilalcohol PVA, gelatin, chitosan, and more nanofibrous materials). Drug delivery: electrospinning provides a good feature when it comes to choose materials for drug delivery. The desired fibers can be oriented or arranged according to our main needs, they can control the mechanical properties and the biological response of the scaffolds (fibers oriented randomly). One of the very important positive points is that there are several different drug loading methods (coatings, encapsulated drug, and/or embedded drug for example). The attention given especially to those drug delivery systems is based on one essential advantage: reduced toxicity by delivering drugs at a controlled rate. Blood vessels: the basic materials of the extracellular matrix locally around the vascular cells are a combination of type I and type III collagen, as well as elastin, and some proteins. Previous studies have shown artificial artery based on collagen scaffolds; nanomaterials development contributes to improve tissue engineering because of its particular nanostructure similarity to native extracellular matrices. Synthesis of new precursors for the fabrication of fully organic hybrid nanofibers Pág. 17 Bone tissue engineering: natural bone is a biocomposite made of both inorganic (HA crystals) and organic (collagen matrix) materials. In order to follow the matrix role, electrospinning technique appears to support quite easily the production of very fine and continuous nano/micro fibers. PLA matrix and CaPs/BG glasses are in a good example of designed scaffolds in bone tissue engineering, obtained by different ways (sol-gel method for the nanoparticles and electrospinning process for the nanofibers). 4.3.2. Calcium-phosphate based glasses A current direction in the biomaterials field is to move from inert, passive materials to those that degrade and play an important active role in the regeneration process of the tissue. Implant materials are currently being developed in order to support and stimulate a specific biological response inside the body. As described in one paper [16], this new division of materials is often referred as the “3rd generation of biomaterials”, and with no surprise includes largely the phosphate-based glasses. Bioactive glasses, and especially the ones containing silica, have been studied quite largely in various studies as materials for tissue regeneration application. Indeed, these studies have expressed some good opinions for “in vivo” cases. Basically when those bioactive glasses are exposed to physiological fluids, they tend to from a surface apatite layer, which is capable of bonding to collagen synthesize by connective tissue cells (osteoblasts) [17]. One type of bioactive glasses is currently available commercially as Bioglass® (45S5 – composition of 45 wt% SiO2, 24.5 wt% CaO, 24.5 wt% Na2O and 6.0 wt% P2O5. However, silica based bioactive glasses are recently discussed, especially concerning their slow degradation (often taking 1 to 2 years to disappear from the body [18]). Therefore, studies have been undertaken to search for bone repair and had led to phosphate based glasses as interesting alternatives. Biodegradable scaffolds are beneficial products for tissue engineering applications. As an example we can name the CaO-Na2O-P2O5 glasses, which show good properties in the use for hard tissue substitutes or synthetic graft materials. Phosphate glasses can also be doped with a few different metal alkoxides in order to change and modify their physical properties [19]. Pág. 18 Memoria In our case, the synthesis for producing such bioactive phosphate based glasses is performed by using the sol-gel method, which has shown very good results in producing bioactive nanoparticles glasses for the biomedical applications. Synthesis of new precursors for the fabrication of fully organic hybrid nanofibers Pág. 19 4.3.3. Sol-gel process Introduction to sol-gel method: The sol-gel is a low temperature wet-chemical technique for the fabrication of oxide materials. The whole process begins with a chemical solution in order to produce colloidal particles; this is what we call the sol. The usual precursors are inorganic alkoxides and metal chlorides. They undergo hydrolysis and condensation reactions to create the colloid, this sol then evolves (after condensations reactions) towards the formation of an inorganic network containing a liquid phase; the gel. The growth of an inorganic network is due to the formation of M-O-M and M-OH-M bonds (where M is an electropositive element such as Si, Ti, and Al…). A drying step helps to remove the liquid phase and is then able to produce a porous material. A thermal treatment can be applied to enhance polycondensations, which means consolidation and densification of our material structures. One of the very important advantages of the sol-gel method is versatility: the precursor sol can be for example deposited on a substrate, cast with a desired shape, or used to synthetize ultra-fine powders [20]. Advanced theory of the sol-gel process: The first sol-gel polymerization was described by Ebelmen, who introduced it in 1846 as “under the influence of atmospheric humidity a silicon alkoxide changed from a clear liquid into a transparent solid which on heating formed silicon dioxide.” However, the beginning of the sol-gel polymerization started in the 1930’s for the use of the German company Schott, who used it for making glass containers. The sol-gel method then became an important research area for ceramic materials, not only for industrial purposes, because it offers the possibility to tailor your material according to purity, homogeneity, low temperature preparations, and direct molding [21]. Pág. 20 Memoria . The oxide network formation takes place in solution, usually at a temperature close to room temperature. This is a conversion process in solution of metal alkoxides, such as silicon, zirconium, aluminum, titanium alkoxides... Although it is possible to use other derivative, for example chlorides, alkoxides are by far the most widely used because of their moderate reactivity and modularity. Indeed, the choice of the alkyl group enables modulation of the alkoxide reactivity based on the considered final properties for the material [21]. The ability to obtain different rheology (sols, gels or precipitates) is determined by the kinetics of hydrolysis and condensation reactions (Table 2), which is one of the major interests of the sol-gel: Hydrolysis Condensation Rheology Slow Slow Sol Fast Fast Gel precipitate Fast Slow Gel Slow Fast Precipitate Sol-gel polymerization aspect The sol-gel polymerization can be “hydrolytic”, meaning that it requires the addition of water and therefore include one or more hydrolysis steps, or can be “non-hydrolytic” when made without water. The hydrolytic gels case is by far the largest and most widespread. In this case, the sol-gel includes at least one hydrolysis step prior to polymerization. We can describe the reaction in two steps: hydrolysis of the alkoxide, followed by condensation. Table 2 - Different types of products obtained by sol-gel process in terms of hydrolysis and condensation kinetics steps [23] Synthesis of new precursors for the fabrication of fully organic hybrid nanofibers Pág. 21 Hydrolysis During this first reaction, hydroxyl functions are formed around the metal cation, making the precursor even more reactive to the condensation reaction. It is defined by the following chemical equation: M(OR)n + x H2O M(OR)n-x(OH)x + x ROH The hydrolysis (Figure 4) starts by a nucleophilic substitution on the metal atom – step 1 with a proton transfer – step 2. An alkyl group is then removed as an alcohol – step 3. The hydrolysis is more favored as:  The incoming molecule is nucleophilic : δ(H2O) > 0  The metal center is electrophilic : δ(O) << 0 ; δ(M) >> 0  The leaving group is nucleofuge : δ(ROH) >> 0 It should be noted that alkoxides and water are not miscible. Consequently these reactions take place in a common solvent, which generally corresponds to the alcohol generated during the hydrolysis but not necessarily. Indeed, several polar and watermiscible solvents have been used for sol-gel polymerizations. Figure 4: Hydrolysis mechanisms of metal alkoxides M(OR)n [23] Pág. 22 Memoria Condensation After total or partial hydrolysis of alkoxides, those compounds can react with one another; through nucleophilic substitution (OH groups are good nucleophilic groups). It can as a result enable the growth of chains and then a tridimensional inorganic network via the formation of M-O-M bonds. Such a type of propagation reaction is called condensation. This “polymerization process” quite often simultaneous with hydrolysis, can be complicated because several other mechanisms can compete. The relative importance of each mechanism depends on the experimental conditions. In the case of metallic alkoxides, two polycondensations are involved: alkoxolation and oxolation but will not be more detailed in this report. Catalytic mechanisms Acid catalysis: The steps of hydrolysis and condensation happen in acid catalysis through a nucleophilic SN2 substitution mechanism, after protonation of the alkoxide. Figure 5 shows the reaction mechanism of acid catalysis, which is valid for both steps (you can simply replace H2O by SiOH to go from the hydrolysis to the condensation). Base catalysis The mechanism of base catalysis occurs following a slightly different reaction according to the use of a conventional base or fluoride ion. In the first case, there is an attack of the alkoxide by hydroxyl ion as a SN2 mechanism (figure 6), whereas in the second case, silicon derivate is first created followed by an addition of water and as a second step, elimination of water and a fluoride ion. Figure 5 - Reaction mechanism of acid catalysis in the case of silicon alkoxides [23] Synthesis of new precursors for the fabrication of fully organic hybrid nanofibers Pág. 23 pH influence As indicated above, the gel time strongly depends on the type of catalysis. We are able to see on Table 3, the time dependence of the gel according to the pH and the catalyst used. Catalyst pH of the solution Gel time (h) None 5 1000 HF 1.9 12 HCl 0 92 HNO3 0 100 H2SO4 0 106 CH3COOH 3.7 76 NH4OH 10 107 The results show that the “counter ion” has not a significant importance in the case of strong acids, while the pH may be the only important parameter in the other cases. Some studies have been made about the kinetics of gelation as function of pH for various types of acid. The results show that there is an optimum pH of less than 1 or between 3 and 6, which probably corresponds to a compromise between protonation of the leaving group and protonation of the nucleophile. Table 3 - pH and catalyst influence on the gel time [22] Figure 6 - Alkoxide attack by hydroxyl ion in a SN2 mechanism [23] Pág. 24 Memoria An acidic pH catalyzes the hydrolysis while a basic pH catalyzes the condensation. A strong degree of hydrolysis (acidic pH) thus promotes the growth of the network and leads to a polymeric solution. Under acid catalysis the gel formed is called “polymeric gel” and we obtain after that a gelling open structure. The reactivity of the precursor towards the hydrolysis decreases as it is hydrolyzed. The condensation step, which is kinetically slow, then begins to form chains, which afterward crosslink and form the network. The process leads to the formation of the gel. One the other hand, a low rate of hydrolysis (basic pH) rather promotes nucleation and leads to forming a colloidal solution. In this case, we obtain morphology with spherical particles or block but brittle and opaque, and the pore size can be controlled (in opposition to acid catalysis). The gel formed is in this way called “colloidal gel” and has a large pore structure (clusters). Solvent In addition to the main alcohols (usually methanol or ethanol), several polar solvents (miscible with water) were used for the sol-gel polymerization: formamide, dimethylformamide DMF, tetrahydrofuan THF, dioxane… It is even possible to produce gels in hydrophobic solvents such as tributyl phosphate or dibutylformamide, on the condition that the hydrolysis releases enough alcohol to allow gelation; the residual alcohol is subsequently evaporated while standing at room temperature. Metal alkoxide Precursors M(OR)z , which are called transition metal alkoxides, more specifically those of the d0 transaction metals (e.g. Ti, Zr), are widely used as molecular precursors to produce glasses and ceramics materials. Those alkoxides are usually quite reactive because of highly electronegative OR groups that stabilize M (in its highest oxidation state) and make it very susceptible to nucleophilic attack. Several parameters make metal alkoxides behave differently from group IV silicon alkoxides (Si(OR)4), which remains the most generally used precursors for the sol-gel method. We can isolate a few factors from a previous study [49]: Synthesis of new precursors for the fabrication of fully organic hybrid nanofibers Pág. 25  Transition metals are less stable towards hydrolysis, condensation and any other nucleophilic reaction because they are precisely more electrophilic due to their lower electronegativity.  The coordination of the metal alkoxides can be extended and often have several stable states of coordination.  Its high reactivity requires a very precise control of moisture and hydrolysis conditions in order to form gels rather than precipitates.  The study of the hydrolysis and condensation of transition metal alkoxides is more difficult than in the case of silicon alkoxides generally because of the rapid kinetics of the nucleophilic reactions. Sol-gel process applied to our project Preparing CaO-TiO2-P2O5 glasses by sol-gel method has several convenient points compared to other preparation techniques. The process can be modified to produce porous materials which are a key factor in tissue engineering, where meso- and macrostructure are important in terms of cells growth. The low temperature of the procedure is also an interesting advantage for the application in drug delivery products, where the gel acts as a soluble matrix. Inclusion of a biocompatible polymer in the synthesis in order to produce composite materials with significant properties is also a promising challenge [20]. There are not much recent publications on the direct synthesis of CaO-TiO2-P2O5 via the sol-gel method. 4.3.3.1. Precursors The reason for the lack of publications concerning the preparation of phosphate based glasses via the sol-gel process is, according to a specific study [22], that it is more challenging and strict than the preparation of silicate based glasses by the same process. The basic issue concerns the right phosphorus precursor (hydrolysis of alkyl phosphates is very slow when proceeded via sol-gel), phosphate ions tend to form precipitates rather than network structure based upon P-O-P bonding [21]. This is why this project intent to find suitable precursors for the use via the sol-gel process with one specific purpose: to enhance angiogenesis in tissue regeneration through the formation of organometallic networks which must contain calcium and phosphate and Pág. 32 Memoria  P2O5 alkoxides precursor is obtained by refluxing pure phosphorus pentoxide in absolute ethanol at 78°C. The process needs to be performed under inert atmosphere. The alkoxide precursor solution has a concentration in [P2O5] of 2 mol/L.  TiO2 alkoxide precursor is prepared by the dilution of a Titanium (IV) isopropoxide (95%) in absolute ethanol to obtain a final 2mol/L concentration of [TiO4+] in solution.  Na2O (only used for the first synthesis) is obtained by refluxing metallic sodium in 2-methoxyethanol under inert atmosphere for 24h at 124°C. The final sodium methoxyethoxide has a concentration in [Na2+] of 2mol/L. In order for our desired phosphate-based glass to meet the requirements previously requested, that is to say molar ratio, calculi have been carried out. Molar ratios are exposed in Table 4 and Table 5: Precursor Theoritical molar ratio % Concentration in mol/L CaO 44,5 0.92 P2O5 44,5 2 TiO2 5 1.95 Na2O 6 1.99 Precursor Theoritical molar ratio % Concentration in mol/L CaO 47,5 0.92 P2O5 47,5 2 TiO2 5 1.95 Table 4 - First sol-gel synthesis including Na precursor Table 5 - Major synthesis of the phosphate-based glass desired (5% TiO2 without Na) Synthesis of new precursors for the fabrication of fully organic hybrid nanofibers Pág. 33 First case – G5 nanoparticles preparation with Na According to the desired composition (see Table 6), the synthesis begins with the preparation of a precursor solution in an inert atmosphere. We are using a hermetically sealed balloon flask because such precursors are unstable in the presence of water, and quite reactive (this is why we inject each solution using syringes through the membrane). The different amounts of each precursor, previously calculated, are introduced according to a special order and procedure. We first took the calcium alkoxide precursor solution as a reference and adjusted our volumes in order for the synthesis to have a final ratio of 5% precursor volume and 95% for the solvent. For this first experiment, we used only precursors already prepared by the laboratory team. Details of the calculi are available in the diary book of the laboratory. Precursor Theoretical molar ratio % Concentration in mol/L Volume in mL CaO 44,5 0,92 6,13 P2O5 44,5 2 2,81 TiO2 5 1,95 0,32 Na2O 6 1,99 0,76 We used in that case 10mL in total for the precursors’ solution, which appears to be an amount that we managed to reduce in order to work with basically 5mL for the following preparations. First, the precursors’ solution is carried out by mixing respectively the calcium, sodium and titanium precursors stirred during 1h (under argon inert atmosphere) in order to obtain a homogeneous mixture. Subsequently and according to the 5%-95% ratio of the alkoxide/solvent volumes, we add the 1,4-dioxane, a suitable nonpolar organic solvent which does not dissolve the newly formed nanoparticles [28]. Then the phosphorus precursor is added “a drop at a time” using a syringe and a pump that will control the output Table 6 - Concentrations and volumes of precursors for the first synthesis Pág. 34 Memoria rate of the solution at an approximately rate of 1mL/h, the flask being cool down in water and ice-based bath to avoid undesired exothermic reactions. The hydrolysis and condensations reactions which allow the gel to form are accelerated by introducing an aqueous catalyst in the balloon flask. We used a basic catalyst because it enables the formation of short, highly branched chains and favors the condensation kinetics [34]. The catalyst solution includes water, ammoniac and ethanol with a desired ratio of 60moles of H2O/0,3moles NH3/ 12 moles of ethanol for 1mole of titanium used in the precursor solution. Because in our preparation we only use 0,624.10-3 moles of titanium, we had to adapt our catalyst. The water amount attempts to enhance the hydrolysis which in our case helps to control the balance with the condensation reaction (actually supported by the basicity of the whole solution). As a final step, we transfer our solution into a glass bottle and let the mixture under stirring at a temperature of 75/80°C for 4 days. We can observe a change in the color of the solution, which firstly appears to be yellow orange and turn into white after 48-72h. This effect is expected as it shows the “gelation process”, production of the nanoparticles separated from the liquid phase. To extract our powder from the liquid phase, we performed centrifugations:  First centrifugation at 3000 rpm for 10 minutes at 4°C - separate the liquid phase (dioxane solvent) from the particles.  Addition of ethanol to “wash” the particles  Five repeated “washing” centrifugations  Put the centrifugation tubes with the particles collected in the stove at 80°C – drying process. The powder obtained is then supposed to be mixed with a biodegradable polymer solution (PLA, gelatin) in order to be processed into fibers by electrospinning for example. Synthesis of new precursors for the fabrication of fully organic hybrid nanofibers Pág. 35 Second case – G5 nanoparticles preparation without Na (Main part of the project) In this case, the mechanisms involved are the same as in the previous one, and the desired morphology of the particles stays the same as well. However, the glasses produced this time have some different compositions. Indeed compounds of calcium (Ca), and phosphate (P) have been synthesized through various alkoxide precursors in order to test their respective influence on the hydrolysis and therefore on the final composition and morphology of the oxide. We performed several syntheses, involving the following parameters:  Concentrations  Temperature  Time  Ratio precursors/solvent Precursors used: As calcium (Ca) precursors Calcium oxide CaO Calcium propionate C6H10CaO4 D-Pantothenic acid hemicalcium salt C18H32CaN2O10  Calcium oxide CaO: Same preparation as mentioned before in the first experiment.  Calcium propionate C6H10CaO4 (Figure 11): We used it as dry powder (97% pure) solubilized in propionic acid under inert atmosphere at the very beginning of the synthesis. Stirring for 1h to dissolve it as much as possible before introducing other precursors. We weighted (according to the calculi) 0,6589g of calcium propionate and put it inside the balloon flask and then we added the propionic acid inside under stirring. We waited 1h after injection of the titanium precursor. Figure 11 - calcium propionate salt (a) and its propionic acid (b) formula (from Sigma-Aldrich) Pág. 36 Memoria  Calcium D-pantothenate salt C18H32CaN2O10 (Figure 12): It is a white crystalline powder associated to the pantothenic acid, which is better known as vitamin B5. It is slightly soluble in alcohol but easily in water, as it is a hygroscopic compound. D-pantothenic acid may have a role in controlling keratinocyte proliferation and differentiation. According to other current studies, a dissolution using the trifluoroethanol (TFE) could be a good option but has to be very carefully prepared (40min at 40°C), with a precise control of the temperature. Otherwise and in our case, the dissolution was directly followed by solidification. As phosphorus (P) precursors Phosphorus pentoxide P2O5 Phytic acid C6H18O24P6 (in 50% water solution and in salt powder)  Phosphorus pentoxide P2O5: Same preparation as mentioned before in the first experiment.  Phytic acid C6H18O24P6 in 50% water solution: This precursor is already prepared by Sigma Aldrich in a 205mL glass bottle. We introduced the 1,04mL “drop by drop” using the pump at a rate of 1mL/h; important precipitation occurs quite quickly.  Phytic acid sodium salt hydrate C6H18O24P6: It is a very fine white powder. After several researches and tests in order to dissolve it (water, Phosphate buffered saline PBS, and temperature control), we were unable to find a good procedure to use it as an alternative precursor compared to the one at 50% water. Figure 12 - D-Pantothenic acid hemicalcium salt formula (from Sigma Aldrich) Synthesis of new precursors for the fabrication of fully organic hybrid nanofibers Pág. 37 The titanium (Ti) precursors used in the following preparation is the same as in the previous experiments: Titanium dioxide TiO2. As seen on the previous projects, the amount of titanium is the content that critically affects the satiability of the glass network, and for that actually modifies the rate of degradation of the glass. After past experiences on different compositions for the titanium amount [34], it has been experimented that a 5%-titanium ratio is preferred. We then decided to keep this direction and make a 5%-titanium amount glass adjusting the other compounds for all our experiments (Table 7): Precursor Theoretical molar ratio % CaO 47,5 P2O5 47,5 TiO2 5 The syntheses were performed following to the same procedure, which was obviously adapted according to the specifics of each “new tested” precursor.  Injection of the precursors of calcium, titanium, and then phosphorus “drop by drop” (which is the one who initiates the hydrolysis)  We let the solution under stirring and water and ice bath until complete hydrolysis, when the solution turns into white.  Centrifugation to separate the particles from the solvent and then five times more to “wash” them using ethanol. In those cases, we programmed the centrifuge for 10 minutes at 10000rpm or even 15000rpm in order to properly separate the nanoparticles.  Put the centrifuge tubes in the stove at 80°C for approximately 4 to 6 hours.  Grind the powder to remove agglomerations and to homogenize the particles. As it has been mentioned before, we tried to adapt the preparation according to our different precursors, especially because we have been using both solid salt powder precursors as well as solution (more or less effective depending on solubility of the products selected). Table 7 - Composition molar ratio theoretical Pág. 38 Memoria 5.2. Characterization methods The first characterization method that we used was to show the morphology and the chemical composition of the particles. 5.2.1. Scanning Electron Microscopy (SEM) The scanning electron microscopy SEM is a powerful observation of surface topography technique. It is mainly based on the detection of secondary electrons emerging from the surface under the impact of a very fine brush of primary electrons sweeping the observed surface and provides images with a resolving power often less than 5 nm and a great depth of field. The SEM uses, in addition, the others interactions of the primary electrons with the sample: emergence of the backscattered electrons, absorption of the primary electrons, photon X emission, and sometimes the interaction with photons which are close to the visible spectrum. Each of these interactions often gives us significant informations of the topography and/ or the surface composition. This technique is used to form an almost parallel brush, fine (up to few nanometers), strongly accelerated by adjustable voltages from 0.1 to 30 kV, to focus on the area to be examined and to scan it gradually. Suitable detectors, specific electrons detectors (secondary, backscattered, sometimes absorbed...), supplemented by photon detectors allow to gather significant signals when scanning the surface and to form several meaningful images. In our case, Scanning Electron Microscopy and Energy Dispersive Spectrometry analysis we performed on a Quanta 200 scanning electron microscope equipped with an EDX energy dispersive spectrometer. Working under high vacuum and high voltage, the samples had to be covered with a layer of graphite to prevent negative charges from accumulating in the surface of the material, which could then deforms the electron beam and alters its effective energy. The energy dispersive spectrometry allows us to know the chemical composition of the samples. Synthesis of new precursors for the fabrication of fully organic hybrid nanofibers Pág. 39 To prepare our powder samples, we disperse a very small amount of the nanoparticles on the sample holder (which has been divided on 4 different parts). Below (Image 1) is presented an example of graphite sample holder with powder nanoparticles. Scanning electron microscope field emission (FE-SEM) We performed our observations on a FEI Nova NanoSEM. With this equipment, unlike the SEM, we were able to work under low vacuum system, which means using lower voltage (the graphite coating is not necessary in this case). Thus, we have achieved observations of our powders with higher magnifications. 5.2.2. Degradation One of the important parameter of our phosphate-based glasses is absorbability. In order to control and to predict our powder behavior “in the body” conditions, we have to study the degradation rate. Indeed the release of calcium, phosphorus and sodium is studied so we can know how they behave (how fast and in which amount they degrade in the body). Previous studies have shown that calcium have a key role in blood vessels formation, thanks to its angiogenic properties. It is then quite important to have a good knowledge on how it is released. To do so we carried out two important assays: Ca release and pH tests. Image 1 - Sample holder covered with graphite / ready for SEM analysis Pág. 40 Memoria These tests require a special procedure. For the measurements to be relevant, in agreement with our “in the body” conditions, we prepared our samples and let them in the sterilizer at 37, 5°C during the whole time of the assays. We used well-plate to prepare our powder samples. After having selected three different nanoparticles powders, based on their shape and chemical composition, we placed them as described below (Figure 13): We put on each slot, 15 micrograms of powder, covered by a cellulose acetate filter and a Teflon® ring (so that it keeps the filter from moving and the particles from going through). Therefore we submerged each sample with 750 microliters of a HEPES solution with a pH of 7.4 to stay under conditions close to reality. (Figure 14) Figure 13 - Preparation of nanoparticles powder for pH and Ca release assays Synthesis of new precursors for the fabrication of fully organic hybrid nanofibers Pág. 41 5.2.2.1. Ca2+ release The equipment used is a pH & ion meter GLP22+ as shown in the next figure. The red electrode is used to control the pH of our solutions and the green one to measure the evolution of conductivity of free ions, in our case Ca2+, and then give us directly the concentration of ions. This involves first a calibration of the engine; we measure the conductivity of five solutions containing five different calcium concentrations; 10-5, 10-4, 10-3, 10-2 and 10-1 mol/L. With this calibration, we obtain the logarithmic ratio between concentration and conductivity after plotting the following curve line (Figure 15). Figure 15 - Calibration curves for the two calcium release tests performed Figure 14 - Components of assays preparation Pág. 48 Memoria dilution from the highest number of cells. We used respectively 80.000/40.000/20.000/10.000/5.000/2.500/1.125/0 cells per well (approximately 500 µL), as described in the following scheme (Figure 21). The calibration well-plate was incubated for 3 hours. The next step is the addition of AlamarBlue® solution, starting with a 50 µL removal of the incubation medium, and then addition of 50 µL of AlamarBlue® in a dark environment, so that the samples are partially protected from direct light. After three hours and thirty minutes of incubation, we put three volumes of each well into another 96 well-plate. The results are given by a micro-plate reader. Figure 21 - number of cells par well Figure 20 - Calibration curve recta patron Synthesis of new precursors for the fabrication of fully organic hybrid nanofibers Pág. 49 Pág. 50 Memoria 6. Results and Interpretations 6.1. Chemical Composition As mentioned before, the aim was to achieve a final compound with a controlled release rate. In order to do so, several parameters are required, of which composition is a major part. 47.5 CaO - 47.5 P2O5 – 5 TiO2 was the desired composition for our bioactive glass nanoparticles without Na. The following compositions of the different nanoparticles synthesis were obtained through the EDX energy dispersive spectrometry during the SEM sessions (Figure 22). Figure 22 - Composition results of nanoparticles synthesized Synthesis of new precursors for the fabrication of fully organic hybrid nanofibers Pág. 51 According to both the table 8 and the previous diagram, we can first say that the dispersion of the data show the difficulty of preventing sol-gel results with different precursors. Even tough, careful calculi have been carried out; the slightest change in one parameter of the synthesis can demonstrate its tendency to completely affect the final composition. However, we are able to distinguish at least a tendency depending on whether we used the calcium propionate or the phytic acid precursors. As we can see on the diagram, the final composition of particles, when using the calcium propionate precursor, seems to be quite centralized with very close molar ratio difference between one another. But modifications have to be applied, in the concentrations and volumes studies, to obtain a higher amount of P2O5 and adjust CaO and TiO2 levels. On the other hand, the phytic acid precursor has shown much broader range of compositions. After having performed the first synthesis using the phytic acid, during which an important precipitation has been observed. Without studying the composition with the EDX spectrometer, further syntheses were carried out trying to reduce this precipitation phenomenon. Looking at the results it was obviously a mistake, as the molar ratio for P2O5 Composition %molar ratio obtained Number Type of nanoparticles P2O5 CaO TiO2 1 G5 Ca Pro 12h 32,91 63,49 3,6 2 G5 Ca Pro 6h 33,17 64,05 2,78 3 G5 Ca Pro 24h 33,28 63,93 2,79 4 G5 Ca Pro (0,6589*2g) 32,72 63,7 3,58 5 G5 Ca Pro 30°C 33,29 63,59 3,12 6 G5 Ca Pro 40°C 8,99 77,15 13,86 7 G5 Ca Pro 5/95 29,3 44,6 26,1 8 G5 Ac.Fit 07/10/13 (1,04ml) 46,57 46,01 7,42 9 G5 Ac.Fit (0,1ml) 30,92 68,61 0,47 10 G5 Ac.Fit (0,01ml) 12,7 77,65 9,65 11 G5 Ac.Fit (1,04ml) 5/95 42,8 50,9 6,3 12 G5 Ac.Fit (1,04ml) 2,5/97,5 27,23 61,42 11,35 Table 8 - Contents table of nanoparticles obtained/ composition %molar ratio (for details of each samples – see next tables 9 and 10 Pág. 52 Memoria clearly decreases, which is quite logical according to the dilution applied to the phytic acid precursor. After a review of the previous data, it appears we have at least two final nanoparticles with a relatively good composition, as the difference between the results and the theoretical desired composition:  G5 Ac.Fit 07/10/13 (1,04ml)  G5 Ac.Fit (1,04ml) 5/95 The 2 following tables (9-10) show the different procedures and results obtained: Synthesis of new precursors for the fabrication of fully organic hybrid nanofibers Pág. 53 %mol obtained 63,48CaO - 32,91P2O5 - 3,6 TiO2 64,04CaO - 33,17P2O5 - 2,78 TiO2 63,93CaO - 33,28P2O5 - 2,79 TiO2 63,7 CaO - 32,72 P2O5 - 3,51 TiO2 0 63,59 CaO - 33,29 P2O5 - 3,12 TiO2 %molar theoretic al 47,5CaO - 47,5P2O5 - 5 TiO2 47,5CaO - 47,5P2O5 - 5 TiO2 47,5CaO - 47,5P2O5 - 5 TiO2 47,5CaO - 47,5P2O5 - 5 TiO2 47,5CaO - 47,5P2O5 - 5 TiO2 47,5CaO - 47,5P2O5 - 5 TiO2 Proportion precursors/s olvent 20%G5 - 80%Dioxane 20%G5 - 80%Dioxane 20%G5 - 80%Dioxane 20%G5 - 80%Dioxane 20%G5 - 80%Dioxane 20%G5 - 80%Dioxane Temperature Ambiant 20°C + 13°C Dioxane/Phosphore Ambiant 20°C + 13°C Dioxane/Phosphore Ambiant 20°C + 13°C Dioxane/Phosphore Ambiant 20°C + 13°C Dioxane/Phosphore Ambiante 20°C + 13°C Dioxane/Phosphore 30°C Aging time 12h 6h 24h 24h 24h 24h Volumes 3,22 ml d'acid propionic, 0,177ml de Ti, 20 ml de Dioxane, 1,61 ml de P2O5 (d.b.d) 3,22 ml d'acid propionic, 0,177ml de Ti, 20 ml de Dioxano, 1,61 ml de P2O5 (d.b.d) 3,22 ml d'acido propionic, 0,177ml de Ti, 20 ml de Dioxane, 1,61 ml de P2O5 (d.b.d) 3,22 ml d'acid propionic, 0,177ml de Ti, 20 ml de Dioxano, 1,61 ml de P2O5 (d.b.d) 3,22 ml d'acido propionic, 0,177ml de Ti, 20 ml de Dioxane, 1,61 ml de P2O5 (d.b.d) 3,22 ml d'acid propionic, 0,177ml de Ti, 20 ml de Dioxane, 1,61 ml de P2O5 (d.b.d) Concentrations 0,6589g de Calcium Propionate (Ca(C2H5COO)2), [P2O5] = 2M, [TiO2] = 1,95M, Acid propionic 99% 0,6589g de Calcium Propionate (Ca(C2H5COO)2), [P2O5] = 2M, [TiO2] = 1,95M, Acid propionic 99% 0,6589g de Calcium Propionate (Ca(C2H5COO)2), [P2O5] = 2M, [TiO2] = 1,95M, Acid propionic 99% 1,31g de Calcium Propionate (Ca(C2H5COO)2), [P2O5] = 2M, [TiO2] = 1,95M, Acid propionic 99% 0,33g de Calcium Propionate (Ca(C2H5COO)2), [P2O5] = 2M, [TiO2] = 1,95M, Acid propionic 99% 0,6589g de Calcium Propionate (Ca(C2H5COO)2), [P2O5] = 2M, [TiO2] = 1,95M, Acid propionic 99% G5 Ca Pro 12h G5 Ca Pro 6h G5 Ca Pro 24h G5 Ca Pro (0,6589*2g) G5 Ca Pro (0,6589/2g) G5 Ca Pro 30°C Table 9 - Recap chart 1 Pág. 54 Memoria %mol obtained 44,6 CaO - 29,3 P2O5 - 26,1 TiO2 46,01CaO - 46,57P2O5 - 7,42 TiO2 68,61 CaO - 30,92 P2O5 - 0,47 TiO2 77,65 CaO - 12,7 P2O5 - 9,65 TiO2 50,9 CaO - 42,8 P2O5 - 6,3 TiO2 61,42 CaO - 27,23 P2O5 - 11,35 TiO2 %molar theoretic al 47,5CaO - 47,5P2O5 - 5 TiO2 47,5CaO - 47,5P2O5 - 5 TiO2 47,5CaO - 47,5P2O5 - 5 TiO2 47,5CaO - 47,5P2O5 - 5 TiO2 47,5CaO - 47,5P2O5 - 5 TiO2 47,5CaO - 47,5P2O5 - 5 TiO2 Proportion precursors/s olvent 5%G5 - 95%Dioxano 20%G5 - 80%Dioxane 20%G5 - 80%Dioxane 20%G5 - 80%Dioxane 5%G5 - 95%Dioxano 2,5%G5 - 97,5%Dioxano Temperature Ambiant 20°C + 13°C Dioxane/Phosphore Ambiant 20°C + 13°C Dioxane/Phosphore Ambiant 20°C + 13°C Dioxane/Phosphore Ambiant 20°C + 13°C Dioxane/Phosphore Ambiante 20°C + 13°C Dioxane/Phosphore Ambiante 20°C + 13°C Phosphore Aging time 24h 24h 24h 24h 24h 24h Volumes 3,22 ml d'acid propionic, 0,177ml de Ti, 20 ml de Dioxane, 1,61 ml de P2O5 (d.b.d) 3,69 ml de CaO, 0,2 ml de Ti, 20 ml de Dioxano, 1,04 ml de Phytic acid (d.b.d) 3,69 ml de CaO, 0,2 ml de Ti, 20 ml de Dioxano, 0,1 ml de Phytic acid (b d.b.d) 3,69 ml de CaO, 0,2 ml de Ti, 20 ml de Dioxano, 0,01 ml de Phytic acid (b d.b.d) 3,69 ml de CaO, 0,2 ml de Ti, 95 ml de Dioxano, 1,04 ml de Phytic acid (d.b.d) 1,845 ml de CaO, 0,1 ml de Ti, 97,5 ml de Dioxano, 0,52 ml de Phytic acid (d.b.d) Concentrations 0,6589g de Calcium Propionate (Ca(C2H5COO)2), [P2O5] = 2M, [TiO2] = 1,95M, Acid propionic 99% [CaO] = 0,92M, [TiO2] = 1,95M, Phytic acid (50%H2O) [CaO] = 0,92M, [TiO2] = 1,95M, Phytic acid (50%H2O) [CaO] = 0,92M, [TiO2] = 1,95M, Phytic acid (50%H2O) [CaO] = 0,92M, [TiO2] = 1,95M, Phytic acid (50%H2O) [CaO] = 0,92M, [TiO2] = 1,95M, Phytic acid (50%H2O) G5 Ca Pro 5/95 G5 Ac.Fit 07/10/13 (1,04ml) G5 Ac.Fit (0,1ml) G5 Ac.Fit (0,01ml) G5 Ac.Fit (1,04ml) 5/95 G5 Ac.Fit (1,04ml) 2,5/97,5 Table 10 - Recap chart 2 Synthesis of new precursors for the fabrication of fully organic hybrid nanofibers Pág. 55 6.2. Morphology of the particles (Shape and Size) As we already said earlier on this project, the size and shape of the particles have an important effect on the way they interact with the desired matrix during the elaboration process; in our case we assume it is PLA and we want to it those fibers to be produced by electrospinning. After consulting several studies [41], it has been demonstrated that most favorable shape would be a spherical shape. Indeed it presents the minimum contact surface between the phases, meaning a decrease of the surface tension. In this way, we can assume a better wettability between the nanoparticles and the polymeric matrix. Through SEM and FE-SEM sessions, we are able to observe our nanoparticles. According to the images (Image 3), nanoparticles obtained using the calcium propionate Image 3 - Calcium propionate nanoparticles imaging by SEM: a) G-5 calcium propionate 24h aging. b) G-5 calcium propionate 1,31g. c) G-5 calcium propionate 30°C. d) G-5 calcium propionate 40°C. Pág. 56 Memoria powder precursor show the difficulty in the control of getting perfect spherical shape. Using higher magnification (Image 3-a), we can distinguish partially round particles in the first plan, but with no real homogeneous tendency due to a general important agglomeration. Image 3-b confirms the very high amount of particles agglomerates when increasing the initial amount of calcium propionate (1,31g of initial powder instead of 0,656g). However in Image 3-c.d, the images suggest plate shape particles rather than spherical. This can be explained by a strong dependence on the growth speed, which leads to an anisotropic behavior (especially for crystalline particles). SEM showed in previous studies [42] that bioactive glass particles in the nanoscale range had a tendency towards agglomeration. Most studies about bioactive glass nanoparticles concerns ternary systems with silicon dioxide SiO2, which present a focus on the synthesis of irregularly spherical particles presented as aggregate. One paper suggests controlling the morphology of these particles using lactic acid in the sol-gel process [43]. Besides the shape, particles sizes influence the interaction with the PLA matrix as well. It has been already demonstrated that the decrease in particles size from the micro scale to the nano scale promote the embedding between the matrix and the particles. The sizes of our particles were measured by both SEM images (using the ImageJ software) and Zetasizer measurements for the most interesting and satisfactory ones. Graphic 1 shows the results obtained for the calcium propionate: Graphic 1 - Size measurements by ImageJ – calcium propionate Synthesis of new precursors for the fabrication of fully organic hybrid nanofibers Pág. 57 When using the phytic acid precursor, it appears on the SEM imaging (Image 4); we still have a recurrent issue concerning the agglomeration. However, very fine and perfectly spherical nanoparticles were found in the sample of the G-5 Phytic acid with ratio precursors/solvent 20-80%, see image 4-b. Contrary to what has been observed with the calcium propionate, the shape of the particles obtained with the phytic acid show better ellipsoidal-spherical behavior. According to some studies on bioactive glasses, smaller nanoparticles can be obtained by controlling carefully the rate of polycondensations, meaning slowing it down, through several parameters [44] (accurate control of temperature during the synthesis, pH, decreasing aging time, molar ratio of solvent/precursors). Unfortunately, the results don’t seem to reach a common stance as the control of all the Image 4 - Phytic acid nanoparticles imaging by SEM: a) G-5 Phytic acid 20%-80%. b) G-5 Phytic acid 20%-80% higher magnification. c) G-5 Phytic acid 5%-95%. d) G-5 Phytic acid 0,1ml. jjhfj sojdvndihfbvidkcvkdncv Pág. 64 Memoria Those results are in accordance with our expectations and totally logical as we did not perform any heat treatment after the synthesis. Therefore we obtained a glassy structure, which will clearly suits the desired rate of degradation, rather than a crystalline structure, which would take more time to degrade. In the case of G5-Phytic acid 0,1 mL (Graphic 8), we can observe several peaks at 2Ɵ=28,33 and 51°, suggesting that a calcium phytate crystalline phase was encountered in the powder. The very low amount of titanium can be the reason, as we have a final composition of almost binary calcium phosphate glass. The use of the phytic acid as the phosphorus precursor shows definitely a good advantage: previous studies [47] have shown that a controlled hydrolysis of the glass sol precursor is relevant towards the obtaining of typical amorphous structure for embedding calcium-phosphate nanoparticles. This makes the phytic acid a suitable phosphorus precursor for our particles synthesis as it has a self-catalysis behavior towards hydrolysis and condensation in the sol-gel process [32]. Graphic 8 - X-ray diffraction spectrum G5 - Phytic acid 0,1ml Synthesis of new precursors for the fabrication of fully organic hybrid nanofibers Pág. 65 6.5. Degradability The main purpose of these nanoparticles is the release of ions. When immersed in an aqueous medium, the release of calcium, phosphorus, and titanium ions occur, it suggests a change in the pH value of the medium, which can affect the parameters that we try to control. This release is studied as it is important to have knowledge on how fast and in which amount these ions pass from the glass through the body, especially for our major goal, which is precisely the release of calcium (as we mentioned before, important for angiogenic properties [48]). Graphic 9 - pH measurements (48h to 9 days) – Test n°1 Graphic 10 - pH measurements (48h to 7 days) – Test n°2 Pág. 66 Memoria In order for living cells to survive, a specific pH range has to be defined. This pH value is known as the physiological pH and is close to 7,4. Consequently, it means that values far from this 7.4 pH may induce rejection from the cells. The graphic 9 and 10 show the results obtained for different powder samples: G5 particles using phytic acid with a ratio solvent/precursor of 5/95% and 2,5/97,5% express the most acidic behavior after 48h, with values included between 3,75 and 5,50 until 72h after immersion, which is obviously too low in comparison with the physiological pH. However these results can be directly correlated to the amount of phosphorus contained in the different samples: particles containing high amount of phosphorus (typically more than 40% of molar ratio) are the ones of which the pH values stay under 6 for the first 5 to 6 days. With a molar ratio of only 12.7% in P2O5, we can observe that the values of pH for the G5-Phytic acid 0,01mL are above 8 since the beginning of the assays. The pH differences depend directly on the composition of our powders indeed. This is due to the reactions between the ions liberated from the G5 and the HEPES medium. Liberation of P2O42- leads to a low acidic pH (formation of H+ ions) and simultaneously liberation of Ca2+ promotes an increase of the pH (due to formation of HO- ions). This is why a good balance of both entities will lead to a suitable pH for the cells. Graphic 11 - Ca release measurements Synthesis of new precursors for the fabrication of fully organic hybrid nanofibers Pág. 67 We performed calcium release measurements as observed in the graphic 10. The calcium has to be released at a suitable rate in order to promote the formation of new blood vessels. For timetable issues, we only carried out these assays on a 7 days basis. The graphic 11 clearly expresses a high release of Ca2+ during the 3 first days for the powder samples with important calcium molar ratio (G5-Phytic acid 5/95% and G5- Phytic acid 0,01mL especially). We decided for the second test to perform this assay on a “regular” G5 with sodium content in order to get a comparison with our produced particles. It appeared that the release for the G5 with sodium is more homogenous in time. After having calculated the cumulative calcium release data, we were able to obtain the release rate for each material: Rate of release (mmol.L-1/day) G5-Phytic acid 0,01mL 1,87 G5-Phytic acid 5/95% 1,21 G5-Phytic acid 20/80% 1,17 G5 with Na 1,76 The table 12 shows that the rate of calcium released is, as expected, composition dependent. Besides, the G5-Phytic acid 0,1mL is a good example of the important role of the titanium content. According to our EDX results, G5-Phytic acid 0,01mL expressed a higher amount of TiO2 (molar ratio 9.65%) for only 12% of P2O5, which is directly linked to its apparent “control” release of Ca2+. We know for a fact that the titanium is the stabilizer of the vitreous network in our case, which is why the 5% or more required amount of TiO2 has to be precisely respected in order to get a suitable ions release. However such a low amount of P2O5 is due to the low quantity of precursor injected during the synthesis, cells assays will not respond as expected towards a possible reduced toxicity of our products, the main compound being the CaO with a molar ratio of 77,65%, as we know obtained with the 2-methoxyethanol. The release of calcium when the hybrid material is obtained by electrospinning would be worth considering, as we know that calcium release can reach levels of cytotoxicity Table 12 - Rate of release of particles samples for 7 days Pág. 68 Memoria in the case of some studies [50]. But as the results show no calcium release higher than 10 mmol/L, it does not seem to have any issues once embedded in the polymeric matrix. 6.6. Cell assays – Proliferation tests After having calculated the calibration curve from the AlamarBlue® product, as mentioned before, we were able to analyze the data obtained for our different powder samples on rat mesenchymal cells (Graphic 12): As we know how difficult it was to control the pH from our powder samples, as well as the release of calcium, it is without surprise that we attest the non-effective aspect on the cells assays, at least for the G5-phytic acid 0,01mL and for the G5-phytic acid 5/95%. The low pH is precisely the reason for such low values in the case of the G5-phytic acid 5/95%, combined with the fact that the calcium precursor used with phytic acid was obtained in 2- methoxyethanol and as we know is cytotoxic. When the pH of the sample is higher (above 7) , in the case of G5-phytic acid 0,01mL for example, we can observe that it reaches more than twice the number of cells than with the G5-phytic acid 5/95%, even though the very low Graphic 12 - Proliferation test 3/7 days using AlamarBlue Synthesis of new precursors for the fabrication of fully organic hybrid nanofibers Pág. 69 amount of P2O5 misrepresent what we could have expected. The same interpretation can be done for the G5 with sodium. However we have to notice still an important difference compared to the control, which is obviously due to the toxicity of the byproducts of both 2- methoxyethanol and ethanol from the precursors used. Pág. 70 Memoria Synthesis of new precursors for the fabrication of fully organic hybrid nanofibers Pág. 71 7. Conclusion Throughout this project, the sol-gel method was used to produce glass nanoparticles with different precursors. The chemical compositions of the glasses were adjusted according to the initial standards and previous studies. It appeared that the use of different precursors in order to prevent cytotoxicity was possible, we carried out interesting syntheses to obtain our types of G5 nanoparticles with a theoretical composition of 47,5% CaO - 47,5% P2O5 and 5% TiO2. These particles were suggested to have a spherical shape and to be positively charged in surface for a further good impregnation with PLA matrix for example. SEM imaging allowed establishing the G5 nanoparticles size range from 200 nm to 700 nm depending on the different samples, and the use of the Zetasizer attested a positive surface charge for the particles obtained with the phytic acid precursor. Besides, the effects of G5 nanoparticles in medium for cell proliferation assays were measured. pH and AlamarBlue® tests indicated that the phosphate ions release from the breaking network induced an significant acidification effect on the medium. A better control of the synthesis using the calcium propionate precursor has to be considered, as very low quantities of particles were produced, too insufficient for performing most of the regular characterization methods. Unfortunately the cell proliferation assays did not meet our desired expectations, which is mostly due to very broad results in terms of composition. Once the synthesis parameters will be more precisely controlled, better morphology and compositions of particles will allow us to fully understand and interpret the non-toxic feature of byproducts released by these new precursors. Thus, further and deeper studies regarding these precursors would be of significant interest. Despite a lack of publications concerning this step of organic byproducts release in the body, it would be in my opinion interesting to do some research towards the concurrent use of both calcium propionate and phytic acid precursors in the same synthesis (obviously paying attention the pH) and towards the possibilities to use only one precursor for both calcium and phosphorus entities as well. For instance, phytic acid calcium salt (currently available at Sigma-Aldrich) would be very interesting in this way, but sadly too expensive. Synthesis of new precursors for the fabrication of fully organic hybrid nanofibers Pág. 73 Acknowledgement First, I would like to thank the IBEC laboratory, and especially the biomaterials for regenerative therapies group directed by Elisabeth Engel, for giving me the opportunity to do this project. Furthermore, I would like to express my gratitude to my supervisor Oscar Castaño for the useful comments, remarks and engagement through the learning process of this project. Also I would like to thank all the people who have willingly shared their precious time with me, for any kind of questions, comments or observations. Pág. 80 Memoria JONATHAN C. KNOWLES, MARK E. SMITH AND ROBERT J. NEWPORTA, New sol–gel synthesis of a (CaO)0.3(Na2O)0.2(P2O5)0.5 bioresorbable glass and its structural characterization [17] L. L. Hench and J. K. West, Life Chem Rep., 1996, 13, 187. [18] E. S. TADJOEDIN, G. L. DE LANGE, D. M. LYARUU, L. KUIPER AND E. H. BURGER, Clin. Oral Implants Res., 2002, 13(4), 428 [19] E. A. ABOU NEEL, I. AHMED, J. PRATTEN, S. N. NAZHAT ANDJ. C. KNOWLES, Biomaterials, 2005, 26, 2247 [20] ENSANYA A. ABOU NEEL, DAVID M. PICKUP, SABEEL P. VALAPPIL, ROBERT J. NEWPORT AND JONATHAN C. 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