Nanostructured calcium silicates glasses and organic-inorganic hybrid scaffolds studied in this thesis have been synthesised using the sol-gel procedure. This involved the use of alkoxides as precursors which has the advantage that homogeneous, high purity, sol-gel products can be obtained at low temperature and the structural and textural properties can be tailored by manipulating the sol-gel parameters (see Chapter 1). The samples have been synthesised by Dr Julian R. Jones’ group at Imperial College London. The nanostructured sol-gel silicate glasses have been obtained with different nominal compositions: 100S (100 mol% SiO2),
70S30C (70 mol% SiO2, 30 mol% CaO) and 58S (60 mol% SiO2, 36 mol% CaO, 4
mol% P2O5). Different calcium oxide sources have been used: calcium nitrate
tetrahydrate (Ca(NO3)2·4H2O), calcium chloride (CaCl2) as inorganic sources and
calcium methoxyethoxyde (CME-Ca(OCH2CH2OCH3)2) as an alkoxide source. Class
I and Class II hybrid samples were synthesised, where in the former only weak Van der Waal’s forces, hydrogen bonding and/or ionic bonding exist and in the latter strong covalent bonding is present due to the use of the organosilane GPTMS as a coupling agent. The polymers used as organic networks are gelatin and poly(γ- glutamic acid) (γ-PGA). The structure of alkoxides used in the sol-gel process is shown in Figure 3.13. CH3CH2O Si OCH2CH3 OCH2CH3 OCH2CH3 Si OCH3 OCH3 OCH3 H3C Si CH3 OCH2CH3 CH3 O O CH3CH2O P OCH2CH3 OCH2CH3 O Tetraethylorthosilicate TEOS Trimehylethoxysilane TMES 3-Glycidoxypropyltrimethoxysilane GPTMS Triethylphosphate TEP Ca(OCH2CH2OCH3)2 Calcium methoxyethoxide CME
3.5.1.Sol-gel synthesis of 70S30C bioactive glasses
Synthesis of traditional 70S30C consists of five stages: mixing, casting, ageing, drying and stabilisation. During the mixing stage TEOS was hydrolysed in a 0.2mol/l nitric acid (HNO3) for 1 h. The mole ratio H2O/TEOS=12 and volume ratio
H2O/HNO3=6. Calcium nitrate, Ca(NO3)2·4H2O was added to the solution in a
proportion to obtain the composition 70 mol% SiO2, 30 mol% CaO on the final
product. The sol was then cast in 60 ml polymethyl pentene (PMP) moulds. The sol was left in the mould for 72 h at room temperature, when the poly-condensation reaction start to progress. Gelation and ageing of the gels took place at 60 °C for 72 h using a heating rate of 5 °C/min. The drying of the gels was carried out by loosening the mould lids and heating with a three-stage schedule: to 60 °C at 0.1 °C/min and holding for 20 h; heating to 90 °C at 0.1 °C /min and holding for 24 h; heating to 130 °C at 0.1 °C/min and holding for 40 h. The dried gels were cooled to room temperature at 0.1 °C/min and then transferred into an alumina crucible for stabilisation, which followed a three-stage schedule: heating to 300 °C from room temperature at 1 °C/min and holding for 2 h; heating to 600 °C at 1 °C/min and holding for 5 h; furnace cooling to room temperature. The process flow chart is presented in Figure 3.14.
H2O+ HNO3 TEOS Calcium nitrate
Mixing
SOL Gelation GEL
Ageing WET GEL MONOLITH Drying Stabilisation GLASS H2O+ HNO3 TEOS Calcium nitrate Mixing
SOL Gelation GEL
Ageing
WET GEL
MONOLITH Drying
Stabilisation
GLASS
Figure 3.14. Process flow chart for the synthesis of 70S30C sol-gel derived glasses.
New materials were synthesised by adding trimethylethoxysilane (TMES) at different stages of the process. Two groups of samples modified with TMES (TMS- 70S30C) were synthesised: TMS-A series, where the TMES was added to the sol early in the process, soon after hydrolysis completed and before casting. In TMS-B samples, the TMES was added 3 days after casting the sol after some condensation had taken place, but prior to complete gelation. For both TMS-A and TMS-B,
specific amounts of TMES was added to 35 ml of traditional 70S30C sol (T-70S30C, 70S30C without TMES) and vigorously agitated for 1 h. Sub-groups within the TMS-A and TMS-B samples were divided according to the TMES amounts added, which determined the TMES:TEOS ratio (TT ratio). Details are shown in Table 3.4.
Table 3.4. The dosage of trimethylethoxysilane per 35 ml of normal 70S30C of TMS-A and TMS-B sub-groups (TT represents the mole ratio of TMES/TEOS).
Samples Time point of addition TMES per 35 ml sol TT Ratio
TMS-A4 Before casting 4 ml 1:4 TMS-B1 After casting 1 ml 1:16 TMS-B2 After casting 2 ml 1:8 TMS-B3 After casting 3 ml 3:16 TMS-B4 After casting 4 ml 1:4
TMS-A samples were found to have separated into two components, termed upper and lower components. TMS-B glasses were more homogeneous.
3.5.2.Sol-gel synthesis of 58S bioactive glasses
Glasses composed of 60 mol% SiO2, 36 mol% CaO, 4 mol% P2O5 were
prepared by the sol-gel method employing two different pathways: via an inorganic route and via an alkoxide route. Calcium nitrate and calcium methoxyethoxide (CME) have been used as the calcium sources in the inorganic and alkoxide routes, respectively. TEOS and triethylphosphate (TEP) have been used as precursors for the SiO2 and P2O5. The inorganic route has been similar with the synthesis procedure
described for the S70C30 glasses in the section 3.5.1. First, the sol preparation from TEOS, TEP and Ca(NO3)2•4H2O in acidified water took place. The precursors have
been mixed in proportion for glass composition required when the hydrolysis of alkoxides takes place. After the sol was cast in the moulds for completion of gelation, was followed by aging at 60°C for 3 days and drying of the gel at 130°C. Thermal stabilisation took place at 700°C. High temperature samples were achieved by sintering at 800 ºC, after stabilisation stage.
CME is an alkoxide which was synthesised by reacting calcium metal with 2- methoxyethanol under an argon atmosphere at 80˚C for 24 h. The ratio of the reaction was 1 g calcium metal to 24 ml 2-methoxyethanol. The resultant solution was centrifuged to remove unreacted calcium metal and the CME solution was obtained with a concentration of 0.001 mol/ml.
The glass synthesis with CME started with mixing TEOS and CME under nitrogen atmosphere. After 1h of being stirred, the sol was hydrolysed by introducing it inside a hermetic container together with water and gelling at 25 ºC for 3 days. The gel obtained was aged and dried at 60 ºC. The samples were taken through the full sol-gel heat treatment (dried at 60~130 ºC, stabilised at 700 ºC, sintered at 800 ºC after stabilisation) to compare with conventional sol-gel glasses prepared via the inorganic route.
A portion from the sol was poured into polymethyl propylene moulds and gelled by hydrofluoric acid (HF). The ratio of HF (0.5 %) to sol is 1.5 ml to 50 ml.
All the samples were grind into particles of sizes between 38-90 µm before characterisation. The names of the samples were represented by 58S “calcium precursor” followed by the temperature, e. g. 58S Nitrate (CME) 700. Where HF has been used as catalyst was specified after the temperature (58S Nitrate 700 HF).
3.5.3.Organic-inorganic nanocomposite scaffolds
3.5.3.1.Silica-gelatin hybrid scaffolds
Class I and class II silica-gelatin have been synthesized. Gelatin (Bovine) was dissolved in 10 mM hydrochloric acid (HCl) at a concentration of 50 mg/ml. This solution was functionalised by addition of an appropriate amount of GPTMS as a coupling agent to covalently link gelatin to silica, to give C-factors (the mole ratio of GPTMS/gelatin) ranging between 0-2000. The functionalised gelatin solution was left to mix for 14 h before being added as a sol-gel precursor. TEOS, the sol-gel precursor for silica, was hydrolysed separately by adding in the following order: deionised water, HCl (1 N) and tetraethyl orthosilicate (TEOS). The mole ratio of H2O/TEOS was 4 and HCl was added to catalyse TEOS hydrolysis at a volume ratio (water/HCl) of 3. The solution was stirred for 1 hour to allow hydrolysis of TEOS, resulting in a colloidal solution (a sol) of Si(OH)4 (see eq. 1.1) before adding the
functionalised gelatin solution, which was mixed for a further 1h.
At higher C-factors, the total organic content is increased substantially due to the organic contribution from GPTMS. Two volume ratios (functionalised gelatin/hydrolysed TEOS) were used in this study: 60/40 and 85/15 referred to as 30 wt% and 60 wt% respectively, representing the final weight percentages of gelatin in the 0 C-factor materials. Once the functionalised gelatin solution has mixed for 1
hour in the sol, hydrofluoric acid (HF, 5 vol%) was added to the sol at sol/HF volume ratio of 100/1 to catalyse the inorganic condensation reaction. Sols were cast into moulds, where they gelled as monoliths and were aged for 1 week to allow for further condensation and then dried at 60 ˚C for 120 h.
3.5.3.2.Silica-calcium oxide- (γ-PGA) organic-inorganic hybrids
Silica-calcium oxide-poly(γ-glutamic acid) (γ-PGA) organic-inorganic hybrids were synthesised starting from different calcium precursors: calcium chloride, CaCl2 and the calcium salt form of γ-PGA. In all samples, the mole ratio of
Si/Ca was 70/30 to obtain the inorganic part in the same composition as the 70S30C (70 mol% SiO2, 30 mol% CaO) bioactive glasses. Calcium nitrate has not been used
in the synthesis of the hybrids, as the removal of toxic nitrate by-products takes place over 400ºC [1]. At this temperature the polymer can be removed from the system.
Figure 3.15 shows the schematic reactions of the hybrid synthesis using CaCl2 as calcium precursor.
OH O H O N H H CH3CH2O Si OCH2CH3 OCH2CH3 OCH2CH3 Si OCH3 OCH3 OCH3 O O O H O O N H H O O N H H O O OH Si OCH3 OCH3 H3CO n γ -PGA + GPTMS DMSO 80oC x y n Functionalised polymer Functionalisation of γ -PGA + H2O + HCl Hydrolysis of TEOS + (CaCl2 + H2O) Addition of calcium Organic-inorganic hybrid TEOS
Figure 3.15. Schematic of the reactions for the hybrid synthesis.
γ-PGA was first functionalised with the organosilane GPTMS in dimethyl sulfoxide (DMSO) at 80 ºC for 12 h in a dry N2 atmosphere, where the carboxylic acid group
esterification. During the functionalisation process the inorganic functional group trimethoxysilane on GPTMS had hydrolysed and partially condensed to other GPTMS molecules forming Si-O-Si bridging oxygens. Part of DMSO was removed by rotary vacuum evaporation. The second step in the synthesis of the hybrid was the addition of CaCl2 solution to the functionalised polymer (Figure 3.15). The solution
was stirred for 5 minutes. The inorganic sol was prepared by the hydrolysis of the silica precursor, TEOS, in acidic conditions, using a solution of 1N HCl as catalyst, in a separate beaker and mixed for 1 h. The hydrolysis ratio H2O/TEOS (R) was 6.
The two solutions were added together to create the hybrid sol (Figure 3.15). A portion from the hybrid sol (10 ml) was poured into polymethyl propylene moulds and gelled by HF (0.6 ml, 5 vol%). The gelled hybrids were sealed and aged at 60 oC for 3 days and dried at 60 oC for 4 days. The wt% ratio of inorganic/organic constituents was 60/40 and the glutamic acid:GPTMS mole ratio (XEC) was in the
range 1 to 100. SiO2- (γ-PGA) hybrids corresponding to XEC of 2 and 50 have been
synthesised following the same procedure, but without adding the CaCl2.
The synthesis of organic-inorganic hybrids starting from the calcium salt form of γ-PGA (Figure 3.16) instead of CaCl2, has been performed following the
same procedure as above (Figure 3.15).
O NH O O H H - 1/2Ca2+ n O NH O O H H - 1/2Ca2+ n
Figure 3.16. Calcium salt form of γ-PGA.
1 mol of GPTMS was added for every 2 moles of polymer with 40% polymer in the inorganic matrix.