Bioactive organic/inorganic hybrids with improved mechanical performance

New sol–gel functionalized poly-ethylene glycol (PEGM)/SiO2–CaO hybrids were prepared with interpenetrating networks of silica and PEGM through the formation of Si–O–Si bonds. Bioactive and mechanical properties were investigated for a series of hybrids containing varying organic/inorganic ratios and PEG molecular weights. In contrast to the unmodified PEG/SiO2–CaO hybrids, which rapidly dissolved and crumbled, the epoxy modified hybrids exhibited good mechanical properties and bioactivity. The compressive strength and Young's modulus were greater for higher molecular weight PEGM hybrids (PEGM600 compared to PEGM300). Compressive strengths of 138 MPa and 81 MPa were found for the 50 : 50 and 60 : 40 organic/inorganic hybrid samples respectively, which are comparable with cortical bone. Young's modulus values of 800 MPa were obtained for the 50 : 50 and 60 : 40 organic/inorganic hybrids. Bioactivity tests were conducted by immersing the hybrids into simulated body fluid and observing the formation of apatite. Apatite formation was observed within 24 hours of immersion. PEGM600 hybrids showed enhanced apatite formation compared to PEGM300 hybrids. Increased apatite formation was observed with increasing organic/inorganic ratio. 70 : 30 and 60 : 40 hybrids exhibited the greatest apatite formation. All PEGM hybrids samples had good cell viability and proliferation. The 60 : 40 PEGM600 hybrids displayed the optimal combination of bioactivity and mechanical strength. The bioactivity of these hybrids, combined with the enhanced mechanical properties, demonstrate that these materials have significant potential for bone regeneration applications.


Introduction
Bone is the most widely transplanted material, with the exception of blood. 1 Each year more than 2.2 million bone gra operations are performed to repair bone defects in orthopaedics and dentistry. 2,3Bioactive glasses represent one of the most promising synthetic bone replacement materials; these materials have the potential to be more bioactive than calcium phosphate-based materials.Bioactive glasses, developed by Larry Hench in 1969 show class A bioactivity; they bond to bone and stimulate new bone growth even away from the glass-bone interface. 4,5elt-derived Bioglass ® (46.1% SiO 2 , 24.4% Na 2 O, 26.9% CaO, and 2.6% P 2 O 5 mol%) is used commercially in products such as Novabone ® and NovaMin ® for repairing bone defects and re-mineralizing dental enamel. 5,6Under physiological conditions the glass slowly dissolves releasing controlled amounts of Ca, P and Si.The soluble Ca and P then precipitate onto the surface to form an amorphous calcium phosphate layer which then crystallizes into apatite (the mineral component of bone). 7Studies have shown that the controlled release of Ca, P and Si can stimulate gene transcription in osteoblasts. 8,9The glass surface also supports cell adhesion and proliferation.There are however limitations; Bioglass ® requires very high processing temperatures ($1400 C 10 ), is difficult to process into scaffolds, bres or coatings, and has a very small processing window which can result in crystallization.In addition, melt-quench glasses have low surface areas and low porosity which is not ideal for cell ingrowth or vascularization.Sol-gel derived bioactive glasses have been developed to overcome many of these obstacles. 113][14] The sol-gels process has greater versatility; bioactive particles, foams, coatings and bres can all be prepared using this methodology.][17] Apatite formation occurs in the macropores resulting in a much greater degree of apatite growth. 18,19Despite their excellent bioactivity and acceptable compressive strength their poor tensile strength and brittle nature has severally limited their applications.
Sol-gel derived glasses can be combined with organic polymers to improve their mechanical strength and toughness. 20,21ioactive glass particles can be dispersed in a polymer matrix to form composites. 22 However, the bioactive particles are generally covered by the polymer matrix and are therefore not in direct contact with body uid before polymer degradation, resulting in slow bonding to bone tissue in vivo. 23In addition, the bioactive glass particles are usually on the micrometre-scale, thus likely to cause inhomogeneity.Alternatively, inorganic/ organic hybrids can be synthesised by introducing the polymer at an early stage e.g.around the hydrolysis stage.Organic/ inorganic hybrids show similar improvements in mechanical properties compared to the composites but also show much better bioactivity due to the mixing of glass and polymer on molecular scale.Therefore, hybrids have attracted much attention owing to these advantages.
Hybrids can be classied as class I and class II hybrids according to the interaction between inorganic chains and organic chains.In class I hybrids inorganic chains entangle with organic chains with only weak (hydrogen and/or van der Waals bonding) interactions, whereas by modifying the polymer to react with the inorganic phase strong covalent bonds can be formed (class II hybrids). 249][30] For example Salinas et al. 31 have prepared dense and homogeneous crack-free monoliths of PDMS-modied CaO-SiO 2 system.These hybrids are silicate networks covalently bonded to PDMS which show apatite formation when immersed in simulated body uid (SBF).It was also shown that their elastic modulus decreased with PDMS content but their apatite forming ability increased with calcium content. 32Biodegradable silica/poly(3-caprolactone) (PCL) have also been prepared, in which hydroxyl groups at either end of the PCL polymer chains were modied with 3-isocyanatopropyl triethoxysilane (IPTS), resulting in a polymer end capped with a triethoxysilyl group.The end capped PCL then was introduced into a sol to synthesize bioactive glass/PCL hybrids. 33,34Bioactive silica-poly(gglutamic acid) hybrids have also been obtained by covalently bonding the g-PGA and bioactive silica through an organosilane coupling agent, glycidoxypropyl trimethoxysilane (GPTMS), 24 and calcium was conrmed to be incorporated in the silica network by using calcium alkoxide precursor.However, sometimes the resulting hybrids are not sufficiently hydrophilic for ion exchange with the surrounding physiological uids.Hydrophilic organic components have been introduced to prepare organic/inorganic hybrids.For example, natural polymer gelatin was functionalized using GPTMS, which was then introduced into a sol of hydrolyzed TEOS to covalently link gelatin to silica with -Si-O-Si-.Then the wet gels were freeze dried to form porous scaffolds, the mechanical properties of which are similar to the cancellous bones. 35Granqvist et al. have developed PEG/siloxane bres by functionalising PEG end groups with 3-isocyanatopropyl triethoxysilane. 25The functionalised PEG was incorporated into the siloxane network through covalent bonds resulting in bres with much higher elasticity compared with non-functionalised bres.The bres were shown to be effective substrates for the nucleation and growth of bone-like apatite.
Traditionally calcium has always been incorporated in solgels in the form of calcium nitrate which then requires the solgel to be heated >400 C to burn off the toxic nitrates and for calcium to be incorporated into the silicate network. 19,36Nitrates are therefore unsuitable for hybrid materials containing polymers which decompose at high temperatures.However, it has recently been shown that the precursor calcium methoxyethoxide (CME) is suitable introducing calcium into hybrids. 36,37alcium has been shown to enter the silicate network at processing temperatures as low as 60 C when using CME.
The purpose of the present study was to prepare homogeneous organic/inorganic hybrid materials for bone repair using low molecular weight (300, 600) functionalized PEG.The inorganic phase consisted of (SiO 2 ) 70 -(CaO) 30 (S70C30, the archetypal bioactive sol-gel).The organic phase consisted of polyethylene glycol (PEG), a biocompatible polymer (and bioresorbable for low molecular weight polymers 38 ) which is completely soluble in both water and ethanol, easy to modify and can be incorporated into typical sols for sol-gel hybrid synthesis.PEG ends were rst modied with epoxy ethane, then ammoniapropyltriethoxysilane was added to react with epoxy ethane to form PEG end capped siloxane (PEGM).The organic/ inorganic hybrid materials were prepared by introducing PEGM into the sol-gel process, using TEOS and calcium methoxyethoxide as precursors, so that the silica network and organic polymer form interpenetrating networks at the nanoscale.The aim was to investigate whether this system can improve the mechanical properties of the hybrid materials whilst retaining the apatite forming bioactivity of the inorganic sol-gel phase.
The preparation was carried out in a 4 step process as shown in Fig. 1.
Calcium methoxythoxide preparation.One gram of Ca metal was added into 24 mL of methoxyethanol under reux for 24 h at 80 C. 39 Calcium methoxyethoxide solution was obtained from the supernatant uid aer centrifuging (Ca-methoxyethoxide, 17% in methoxyethanol).
Modifying PEG with EO (PEG-EO).PEG (50 mmol) was dissolved in 50 mL of tetrahydrofuran.Epichlorohydrin (200 mmol) and NaH (2.1 g, 60% in mineral oil) were added and the medium was stirred under reux (66 C) for 24 h.Aer the solution cooled to room temperature, 30% H 2 SO 4 in methanol was added to neutralize the solution.The solvent and excess epichlorohydrin were then removed under reduced pressure. 1H NMR spectra were recorded at 25 C on an Avance III 400 spectrometer to conrm the EO modication.Chemical shis are reported in parts per million (ppm) and a reference standard of CDCl 3 (7.26ppm) was used.FTIR spectra were recorded for PEG, PEG-EO and PEGM using a Bruker Equinox 55 spectrometer to monitor and conrm the reaction process.
Preparation of organic/inorganic hybrid material.Under constant stirring PEGM were rstly mixed with TEOS, then Camethoxyethoxide was added at different organic/inorganic molar ratios of 70 : 30, 60 : 40, 50 : 50 and 40 : 60 whilst maintaining the inorganic composition of S70C30.Aer 30 min, water (H 2 O/TEOS ¼ 2, molar ratio) was added drop wise into the solution and the solution gels within an hour.The resultant gels were aged at ambient temperature for two days then dried in an oven, rstly at 40 C for two weeks and then at 120 C for a further week.Finally the samples were dried at 280 C for 1 h to ensure full condensation and complete evaporation of the solvent.A control sample of PEG600 with a 60 : 40 organic : inorganic ratio was prepared using unmodied PEG to act as a reference. 29Si solid state MAS NMR spectroscopy experiments were carried out on the dried materials using a Bruker Avance III 400 MHz instrument providing 29 Si Larmor frequencies of 79.30 MHz. 4 mm MAS BB probes spinning at 12 kHz were used.A 2.5 ms pulse ($60 tip angle) was used, and the recycle delay was 90 s. 29 Si NMR spectra were referenced using tetramethylsilane (d Si ¼ 0 ppm).

Bioactivity testing
Bioactivity was tested in vitro by immersing 150 mg monoliths in 100 mL of simulated body uid at 36.5 AE 0.5 C. SBF was prepared by dissolving reagents NaCl, NaHCO 3 , KCl, K 2 HPO 4 $3H 2 O, MgCl 2 $6H 2 O, CaCl 2 , and Na 2 SO 4 in deionized water. 40The solution was buffered to pH 7.4 with Tris-(hydroxymethyl)-aminomethane ((CH 2 OH) 3 CNH 2 ) and hydrochloric acid to give a resultant ion concentration similar to human plasma.Aer immersing in SBF for different times the samples were washed gently with pure water and le to dry in a desiccator without heating.Apatite formation was evaluated using scanning electron microscopy (SEM), Energy Disperse Xray Spectroscopy (EDXS), Fourier transform infrared (FTIR) and X-ray diffraction (XRD).
SEM/EDXS.Following immersion the samples were dried and then coated with a thin layer of gold (Au) by sputtering (SCD 500).The surface development was observed using a JEOL-6700 instrument operating at a voltage of 15 kV.
XRD.Following immersion the samples were dried and then nely powdered.X-ray diffraction spectra were collected using a Rigaku (D/MAX 2500) instrument with Cu Ka radiation (l ¼ 1.54 Å), operated at 40 kV and 200 mA.Data was collected for 2q values between 10 to 60 with a step size of 0.02 .
FTIR.Fourier transform infrared spectra were collected in the range 2000-400 cm À1 on a Bruker Equinox 55 instrument.All samples were diluted with dry KBr, ground down to ne powders and pressed into pellets.Measurements were made at ambient temperature.

Mechanical testing
Compressive mechanical properties of the specimens were evaluated using general a purpose-testing machine (Ins-tron3365, Instron Co., Canton, MA, USA) at 23 AE 2 C and 50 AE 5% relative humidity conditions.Specimens were cut from homogeneous regions of the organic/inorganic hybrids to form blocks measuring 4 mm Â 4 mm Â 8 mm.These samples were positioned between parallel plates and compressed with a crosshead speed of 0.5 mm min À1 and a 5.0 kN load cell.A minimum of ve repeats were taken per sample to provide an average.The mean and standard deviation were reported.The Young's modulus was determined from the slope of the initial linear elastic portion of the stress-strain curve.Compressive strength was taken as the maximum strength of the sample until failure and elongation to failure was the percentage strain at fracture.

Cell evaluation
Preparation of hybrids extracts.Organic/inorganic hybrids were soaked in a-MEM, preheated to 37 C, extracting vehicle ratio was 3 cm 2 mL À1 , as specied by ISO 10993-12:2007.Organic/inorganic hybrids, which were previously sterilized under UV light for 12 h.Aer 1, 3 and 7 days at 37 C under sterile conditions, a-MEM was ltered to eliminate hybrids, and these extracts (E1, E2 and E3, respectively) were used as culture medium aer adding 10% fetal bovine serum (FBS) Cell culture.A preosteoblast cell line (MC3T3-E1; ATCC, CRL-2593, Rockville, MD, USA) was used to investigate the cell compatibility in vitro.The MC3T3-E1 cells were cultured in a humidied incubator under an atmosphere containing 5% CO 2 This journal is © The Royal Society of Chemistry 2015 at 37 C. Dulbecco's Modied Eagle Medium (a-MEM) supplemented with fetal bovine serum (FBS, 10%), penicillin (100 mg mL À1 ) and streptomycin (100 mg mL À1 ) was used as the culture medium.Cells were subcultured every 5 days and maintained at 37 C in a humidied incubator with 5% CO 2 .
Cell viability assays.A Cell Count Kit-8 (CCK-8, Beyotime, Jiangsu, China) was employed to quantitatively evaluate cell viability. 41MC3T3-E1 was seeded on 96-well culture plates at a density of 1.7 Â 10 4 cells per mL, aer cell adhesion was veri-ed; the culture medium was replaced by different extracts (E1, E2 and E3) of the organic/inorganic hybrids.The cells incubated in a-MEM without organic/inorganic hybrids extract were used as a control.The cells were incubated in the culture plates for 3 and 6 days.Then, the CCK-8 solution (20 mL per well) was added and incubated for 4 h at 5% CO 2 and 37 C, 100 mL of the reacted reagent from each well was transferred to 96-well plates, and the absorbance at 450 nm was determined using a micro-plate spectrophotometer (MD, SpectraMax M2, USA).Six parallel replicates of each sample at each time point were prepared so that statistics could be performed.

Degradation studies
Hybrids (75 mg) in triplicates were immersed in 50 mL of SBF at 36.5 AE 0.5 C and stirred at 120 rpm.At various time points (1, 4, 24, 72, 168, 336, 504 and 672 h), the hybrids were removed from their respective containers and dried under vacuum prior to weighing.To obtain the rate of weight loss, the initial weight (M 0 ) of each sample was measured as well as the weight loss at time t (M t ) to give a weight loss%: weight loss% ¼ (M 0 À M t )/M 0 Â 100%.pH values of the extracts were recorded aer 1, 3 and 6 days immersion.

Statistical analysis
The one-way analysis of variance (ANOVA) was used to evaluate signicant differences between means in the measured data.Each experiment was repeated three times.All quantitative data are presented as mean AE standard deviation.

Sample characterisation
PEG end groups were successfully modied with epoxy (EO) as shown in Fig. 2. The 1 H NMR spectra conrms the addition of the epoxy functional groups.The spectra are assigned as follows: 1 42 The reaction process of PEG-EO to PEGM and corresponding FTIR spectra are shown in Fig. 3.A feature at 914 cm À1 appears for PEG-EO which is consistent with an epoxy group. 43,44The epoxy peak then disappears for the PEGM as expected (see Fig. 3a bottom).Clear amorphous monoliths were produced as shown in Fig. 4. The orange colour emanates from the CME precursor which is a dark red/brown colour as previously reported.

Structural characterisation
Fig. 5 shows the 29 Si MAS NMR spectra of (a) PEGM300/TEOS (calcium free) (b) the PEGM/TEOS/CME hybrid and (c) the unmodied PEG300/TEOS/CME hybrid.The modied PEG hybrids (samples a and b) both show the presence of T n and Q n species (T n and Q n correspond to the structure of CSi(OSi) n (R) 3Àn and Si(OSi) n 0 (R) 4Àn 0 respectively, where R is a nonbridging oxygen).In contrast the unmodied PEG hybrid (sample c) only showed the presence of Q n species.Results of the Gaussian tting of T n and Q n distributions of the 29 Si MAS NMR spectra are summarised in Table 1.The ratio of T 3 /T tot and Q 4 /Q tot in Table 1 show the degree of condensation of the PEGM and silica network respectively.For the TEOS-derived silica network, 29 Si exhibit several overlapping contributions associated with Q n units: three resonances at $À95, $À101 and $À110 ppm were assigned to Q 2 , Q 3 and Q 4 units respectively.In addition to these Q n resonances, the 29 Si MAS NMR spectra of sample a and b showed two peaks at $À60 and $À65 ppm characteristic of T 2 and T 3 units resulting from the hydrolysis/ condensation of the terminal triethoxysilane groups of PEGM.Despite poly-condensation between PEGM chains not being excluded, the high occurrence of T 3 units suggests that PEGM is successfully graed to the BG network, thereby coupling the organic and inorganic parts of the hybrid to form a Class II hybrid.Sample a had higher T 3 /T tot and Q 4 /Q tot values compared to samples b and c, which suggests that the silica species were highly condensed.

Bioactivity
Apatite formation was observed on the monolith's surface aer immersion in SBF.Fig. 6 illustrates a representative SEM image showing both hemispherical and needle like apatite formation.Needle like formations are consistent with previous reports of apatite formation on bioactive sol-gels. 45Apatite formation occurs within 24 hours of immersion and become denser with increasing time.
X-ray diffraction results are shown in Fig. 7-9.Fig. 7 illustrates the formation of apatite as a function of SBF immersion time for PEGM600 with an organic/inorganic molar ratio of 60 : 40.HA diffraction peaks appear aer only 24 hours and then increase in magnitude aer 3 and 7 days conrming apatite formation on the surface of these materials.Monoclinic HA is shown for reference standard. 46Fig. 8 and 9 show the apatite diffraction peaks as a function of organic/inorganic ratio for the PEGM600 and PEGM300 hybrids respectively.
The intensity of the apatite diffraction peaks increases with increasing organic/inorganic ratio for both PEGM600 and PEGM300 hybrids.It is evident that the 60 : 40 and 70 : 30 hybrids show the greatest bioactivity (apatite formation).For all organic/inorganic ratios the PEGM600 spectra shows signicantly more apatite formation demonstrating that PEGM600 hybrids are more bioactive compared to their equivalent PEGM300 counterparts.
EDXS results are shown in Fig. 10 as a function of SBF immersion time.Prior to immersing in SBF, the hybrid surface consisted of Si, C and O as expected.Within a day of immersion in SBF the hybrid surface is rapidly covered with a calcium phosphate layer as expected, in addition the Si and C signal from the bulk hybrid is signicantly attenuated by the Ca/P surface formation.The Ca and P signals continue to increase with time (3 and 7 day immersion) and the Si signal is further attenuated.
FTIR spectra are shown in Fig. 11.The pronounced band $1090 cm À1 and the smaller peak $790 cm À1 are attributed to Si-O-Si vibrations. 47The broad band $3440 cm À1 and the sharper peak $1630 cm À1 are attributed to O-H stretching. 47eaks at $459, 567 and 607 cm À1 are attributed to phosphate  This journal is © The Royal Society of Chemistry 2015 absorption bands. 45These peaks evolve as a function of immersion time in SBF further supporting the formation of phosphates on the hybrids surface.

Mechanical testing
Compressive mechanical testing was performed on the dried hybrid monoliths to test the strength and exibility of the hybrids.Fig. 12 shows the stress-strain behaviour and effect of organic/inorganic ratio on Young modulus of hybrids, and Table 2 presents the corresponding Young's modulus and compressive stress data for PEGM300 and PEGM600 samples as a function of organic/inorganic ratio.The elongation to failure was greatly increased to 14-35% for the hybrids materials compared to 4.2% for sol-gel glasses (70S30C monoliths, sintered at 800 C), 48 indicating improved strain to failure and therefore increased toughness.Two clear trends are also observed; rstly the mechanical properties increase with increasing inorganic ratio.The Young's modulus and compressive strength are greatest for the 50 : 50 hybrid with the overall trend 50 : 50 > 60 : 40 > 70 : 30 and this trend is observed for both the PEGM300 and PEGM600 hybrids.The second point is that Young's modulus and compressive stress are greater for the PEGM600 hybrid compared to its equivalent PEGM300 hybrid (for the same organic/inorganic ratio).The PEGM600 hybrid with a 50 : 50 organic/inorganic ratio was therefore found to have the best mechanical properties with 60 : 40 PEGM600 having very similar Young's modulus values.The unmodied PEG hybrid had very poor mechanical properties and easily crumbled, it was therefore not possible to measure the modulus or strength of these samples.PEG rapidly dissolved from the unmodied composite resulting in just the inorganic phases.Strong covalent links between the organic and inorganic chains are therefore essential in order to obtain tough composites which degrade uniformly acting as a single phase material.

Cell viability test
As shown in Fig. 13, good cell proliferation (96-157%) was observed in the presence of the three extracts (E1, E2 and E3).Good cell viability was observed for all the extracts, no signicant difference in viability was observed for the different extracts aer 3 days.However aer 6 days the cell viability was higher for the both hybrids compared to the control.The cell proliferation for the PEGM600 hybrids extracts is slightly higher than PEGM300 hybrids (Fig. 13a).The cell proliferation increased with culture time in each group (Fig. 13b).

Degradation studies
A preliminary evaluation of the degradation behaviour of the hybrids was conducted through mass loss upon immersion in SBF solution.Results are summarised in Fig. 14.Mass loss increased rapidly for both samples at during the rst week before plateauing at $43% at 3 weeks.These results are  Table 1 Chemical shifts and relative proportions of T n and Q n species in Class II PEGM/bioactive silica hybrids synthesised (a) without calcium (b) with calcium methoxyethoxide, (c) unmodified PEG/bioactive silica hybrids.The errors in the chemical shift (d) are AE0.5 ppm, the intensities are AE1% and the ratios are AE0.05 favourable compared to other organic/inorganic hybrids.For example, Russo et al. report mass losses as high as 76% at 3 weeks for dPEG : GPTMS with a 1 : 2 ratio and losses of 52% at 3 weeks for dPEG : GPTMS with a 1 : 4 ratio. 49able 3 presents the pH data for the PEGM600 60 : 40 and PEGM300 60 : 40 sample as a function of immersion time.It can be seen that the pH value of the extracts are stable with only a small increase in pH.The pH of these hybrids are lower than values reported for NovaBone (7.64) and S70C30 scaffolds (8.19) aer immersion for 72 h. 50It has been reported that higher pH value may be detrimental for cell growth.The PEG modied hybrids exhibit lower pH values compared to the same inorganic composition (S70C30) this indicates that PEG moieties may help stabilize the inorganic phase of the hybrid.

Discussion
PEG was successfully modied, via an epoxide functionalization, to yield PEGM/70S30C bioactive hybrids with covalent (Si-O-Si) bonds linking the organic and inorganic phases.A series of hybrids containing different organic/inorganic molar ratios and PEG molecular weights were prepared and characterised.In all cases the modied PEGM hybrids showed signicantly improved mechanical properties compared to the unmodied PEG hybrids.The improvement in mechanical properties was attributed directly to the covalent bonding between the organic and inorganic phases for the modied PEG hybrids.The present samples exhibited favourable bioactivity and mechanical properties compared to equivalent materials previously reported in the literature.In previously studies, Liu et al. prepared PEG/ SiO 2 -CaO-P 2 O 5 hybrid xerogels by introducing PEG during the hydrolysis process to provide a molecular scale interaction between the organic and inorganic phases.The PEG molecular chains were found to penetrate into the SiO 2 network forming a semi-IPN structure.Since there was no covalent bonding between inorganic and organic phases, although a signicantly increase in both the Young's modulus (320 to 420 MPa) and hardness was achieved compared to the pure inorganic    Rhee has developed poly(3-caprolactone)/silica hybrids for bone repair applications, with covalent bonding between inorganic and organic phases. 33These materials show good apatite forming ability and Young's moduli of 200-600 MPa.Russo et al. have recently developed silica/bis(3-aminopropyl) polyethylene glycol(dPEG) inorganic/organic hybrids by using 3glycidopropyltrimethoxysilane (GPTMS) as a coupling agent to provide the covalent links. 49The strain to failure increased from 4.2% for the sol-gel glass to 8-15% for the hybrids and compressive strengths of 15-20 MPa were reported which are comparable to cancellous bone (4-12 MPa) but low compared to cortical bone (100-230 MPa).In the present study we are achieving compressive strength values of 138 and 81 MPa for PEGM600 50 : 50 and 60 : 40 hybrids respectively.In addition to the covalent bonding, the improvement in mechanical properties achieved in our study might be partly due to the choice of molecular weight of polymer segments used.Previous studies have employed polymers with molecular weights greater than 1000. 33,49However, the polymer phase is mechanically weaker in compression compared to the inorganic phase.In the present study, PEG600 was found to be better than PEG300 suggesting there is also a lower limit for optimised polymer weight.However, we also note that it was not possible to form monoliths from higher molecular weight PEG (2000, 3000, 4000 and 6000).Therefore, an optimal molecular weight of polymer must to be chosen, in order to maximise the toughness of these hybrids.
Apatite formation was observed for 50 : 50, 60 : 40 and 70 : 30 PEGM600 and 60 : 40 and 70 : 30 PEGM300 hybrids.For a given organic/inorganic ratio the apatite formation of PEGM600 hybrids were consistently higher than the equivalent PEGM300 hybrids; this is attributed to the higher the molecular weight of the polymer.Apatite formation increased with increasing organic content with 60 : 40 and 70 : 30 (organic/ inorganic molar ratio) showing the most promising formation.PEG is hydrophilic and relatively exible, the increase in apatite formation with PEG molecular weight and PEG content are therefore likely to have resulted from better penetration of SBF into the material.This would enable the simulated uid to react more quickly with the bioactive inorganic phases.
The 60 : 40 PEGM600 hybrids were the most promising materials demonstrating both good apatite formation and good mechanical properties.Young's modulus values $800 MPa are higher than those of cancellous bone and signicantly higher than non-functionalised PEG hybrids and other values reported in the literature.Compressive strength of $80 MPa is similar to values required for compact bone (100 MPa).The excellent biocompatibility and aqueous solubility of PEG molecules, as well as the bioactivity of the inorganic components, coupled with the improved mechanical properties mean these hybrids exhibit great potential for bone regeneration applications.
The hybrids demonstrate good biocompatibility for both cell viability and proliferation.The hybrids exhibit an initial rapid weight loss, which provides the essential release of Ca and Si that is necessary for apatite and to stimulate cellular activity. 8,52er the initial weight loss observed during the rst week which peaked at 37% the weight-loss plateaus and only a further 4% weight-loss is observed during the subsequent 3 weeks.Apatite   formation counteracts further weight-loss aer the rst week such that the total weight loss aer 4 weeks in only 41%.
No signicant difference was observed between the PEGM300 and PEGM600 samples.This conrms that the molecular weight has little effect on the degradation of hybrids, and the degradability may be controlled by inorganic phase.During the rst week, the mass loss was slightly lower for the PEG300M hybrid; this may be attributed to the higher hydrophilicity and the higher cross-link density of lower molecular weight polymer.

Conclusions
Functionalised PEGM/70S30C bioactive hybrids were successfully prepared via sol-gel synthesis.PEGM was successfully used as a cross-linking agent between organic and inorganic components at the molecular level.This resulted in strong covalent Si-O-Si bonds between the organic and inorganic phases which signicantly improved the mechanical properties of the resultant hybrids.A corresponding increase in the apatite forming ability was observed for increasing molar fraction of the organic phase; at 50% or below the apatite formation was signicantly reduced.Hybrids obtained from higher molecular weight PEGM600 had a higher compressive stress and showed enhanced apatite formation compared to the equivalent PEGM300 hybrid.The 60 : 40 PEGM600 hybrids were the most promising materials demonstrating good apatite formation, cell proliferation and viability and good mechanical properties.These organic/inorganic hybrid materials exhibited improved mechanical properties and excellent apatite forming ability, conrming their potential use as bone regeneration material.

Fig. 2
Fig. 2 (a) 1 H NMR spectra of PEG (top) and the modified PEG-EO polymer (bottom), (b) illustrates the reaction process of PEG to PEG-EO.

Fig. 3
Fig. 3 (a) Reaction process illustrating the modification of the PEG-EO end groups, (b) FTIR spectra of PEG, PEG-EO and PEGM.The epoxide group of PEG-EO appears at 914 cm À1 .

Fig. 14
Fig. 14 Degradation assay into SBF solution as a function of soaking time for hybrid materials obtained from PEGM600 and PEGM300 with the organic/inorganic ratio of 60 : 40.

Table 3
pH value of samples after immersion in a-MEM