Characterization and In-vitro behavior of Bioactive Glass/Polymeric Composites

Egypt. J. Biophysics. Biomed.Eng. Vol.8. , No.1 ,pp. 25 -43 (2007) Characterization and In-vitro behavior of Bioactive Glass/Polymeric Composites G.T. El-Bassyouni and K. R. Mohamed*

National Research Centre, Biomaterials Department, Behoos Street, Dokki, Giza, Egypt.

CHITOSAN biopolymer characterizes osteoinductive functions and it can be utilized in combination with other bioactive materials that enhances tissue regenerative efficacy and osteoconductivity. Another material of interest is bioactive glass (BG) (45S5) that characterizes its ability to bond to bone and soft tissues very quickly as a result of high level of its surface reactivity. In this study, BG was combined with chitosan to produce novel bio-composites having properties analogous to human cancellous bone. The affinity between BG filler and polymer matrix has been proved by X -ray diffraction (XRD), Fourier transform infrared (FT-IR) and Thermogravimetric analysis (TGA) techniques. The results confirmed that homogeneity and integration between BG filler and polymer matrix especially the composite containing smaller particle size of BG is achieved. Also, the formation of calcium phosphate layer onto the surface of composite was detected post-immersion in simulated body fluid (SBF) for different periods up to 21 days at 37◦C and pH 7.4. Conclusions prove that the presence of smaller particle size of BG filler into the polymeric matrix leads to more effective reinforcement of the composite and increased the bioactivity via high formation of carbonated apatite layer onto the composite surface. These biocomposites are promised for bone replacement and scaffold for tissue engineering applications. Keywords : Biopolymer, Glass, TGA, Chitosan, XRD

Chitosan is obtained by a full or partial deacetylation of chitin, a very abundant natural polymer derived from the outer shells of crustaceans. Due to its numerous and interesting biological properties such as biocompatibility, biodegradability and non toxic properties, chitosan becomes one of the most useful polysaccharides in biomedical area [1]. However, in order to make the polymer adequate to each specific application, modifications of the chitosan structure are critically needed [2]. Chitosan has been used in combination with materials to enhance bone growth such as bone filling pastes for the treatment of periodontal defects or augmentation of edentulous ridges [3]. It is known that it improves osseous healing of defects in femoral coundyl of sheep, stimulates cell proliferation and organizes the hystoarchitectural tissue structure[4] .

The chitosan graft co-polymerization can provide materials with desired properties and is commonly used as a general method for polymer modification [5]. The use of chitosan–based copolymers along with 2-hydroxyethylmethacrylate (HEMA) as a grafting monomer onto a range of polymeric substrates have been reported with increasing success as biomaterials[6,7].Poly-(2-hydroxyethyl methacrylate) (pHEMA) has potentially wide

G.T. El-BASSYOUNI AND K. R. MOHAMED

biomedical applications due to its biocompatibility which allows immobilization of cells or bioactive molecules and has a hardness comparable to bone [8]. Bioactive glass and calcium phosphate are osteoconductive and thus can effectively accelerate bone defect healing. The disadvantage of these bioceramics is their brittle nature which reduces their applications [9]. In order to solve this problem, increasing attention has been paid to composites made of polymer and one or more of ceramic materials including dense and porous hydroxyapatite (HA), tri-calcium phosphate (TCP), bioactive glass (BG) and glass–ceramics, for biomedical applications[10-12]. These polymers including natural collagen, chitosan [13], non-biodegradable polyethylene[14], Polymethyl methacrylate (pMMA)[15], polysulfone and biodegradable α–hydroxyacids[16]. The used composites for bone replacement have been developed using various bioactive ceramics or glass reinforcement in a bio-inert polymer matrix. The mechanical behavior of these composites is dependent upon the interface between the composite components, which in turn affects the interface with the biological system [17] The combination of polymers with a bioactive component takes advantage of osteoconducting properties of bioactive glasses and of their strengthen effect on polymer matrices. The composite is expected to have superior mechanical properties than the neat polymer and to improve structural integrity and flexibility over brittle glasses and ceramics for eventual load–bearing applications [18, 19]. To evaluate the in vitro bioactivity of composites, they were immersed in simulated body fluid (SBF) as a biomimetic medium to achieve direct bond to living bone by the formation of a bone-like apatite layer on the surface after exposure to body fluid [20].

Bioglass 45S5 was chosen in this study as bioactive phase because it has the greatest bioactivity and can stimulate osteoblast function faster than hydroxyapatite [21]. In the present study, a BG having different particle sizes were combined with pHEMA grafted chitosan co-polymer to obtain novel bio-composites that overcome the particle migration problem when they are used as bone filling. Also, it was intended to highlight the effect of incorporation of both matrices on the mechanical properties and the bioactivity of the produced composites.

Materials and methods

2.1. Materials The polymer material used in this study was chitosan (High molecular weight and Brookfield viscosity: 800,000 cps) obtained from chemical company and its particle size was in the range from 250 to 355 µm., 2-hydroxyethylmethacrylate (HEMA) was also purchased from same company and cerric ammonium nitrate initiator (CAN) was provided from another company to graft HEMA monomer onto chitosan polymer.

The bioactive material used was a melt-derived bioactive glass powder (45S5) and prepared according to Hench et al. [22] because it has bioactive behavior [23]. Its nominal composition in weight percentage: 45: SiO2, 24.5: Na2O, 24.5: CaO and 6: P2O5, it was melted in a platinum crucible at 1350 ◦C for 2 hours. The melt was rapidly quenched in distilled water to prevent phase separation. Glass fragments were ground in a ball mill and the produced powder was

submitted to size classification by sieving to obtain two particle sizes of (150–300 µm)

CHARACTERIZATION AND IN-VITRO BEHAVIOUR OF BIOACTIVE…

denoted as BG1 filler and (700-900 µm) denoted as BG2 filler .

2.2. Methods 2.2.1. Preparation of co-polymer Chitosan (0.1g) was dissolved in 7.5 ml of 3% acetic acid solution, and then 2.5 ml of HEMA monomer and 0.1 g cerric ammonium nitrate (CAN) were added to achieve the grafting process. The mixture solution (1) was kept in a water bath at 40◦C for 3 hours to obtain a homogenous co-polymer mixture. The co-polymer mixture was left overnight at room temperature, and then the co-polymer matrix was washed with hot ethanol with stirring for 2 hours to remove the homopolymer which is not interacted with chitosan polymer. The co-polymer mixture was filtrated, collected and dried at 60°C overnight. The pHEMA grafted onto chitosan is denoted as the co-polymer sample. The grafting percentage (G %) was calculated according to the following equation [24] .

G % = (g-go / go) x 100 Where g: weight of the grafted chitosan, go: weight of the original chitosan. 2.2.2. Preparation of co- polymeric / bioglass composites The two composites containing bioactive glass (BG) having different particle sizes BG1 filler (150–300 µm) and BG2 filler (700-900 µm) were prepared. Fixed weight (1.5 g) of each filler size was loaded, well dispersed and mixed separately into the copolymer mixture (1) from the above experiment after 2.5 hours from the co-polymerization process at 40◦ C for 30 minute in a water bath. Mixing process is important to improve the dispersion of BG particles into the co-polymer solution. The produced composites were washed, filtered, dried and collected at 60◦C overnight. Two composites were denoted as BG1 composite and BG2 composite. Also, the yield grafting percentage of the composite in the presence of BG filler was calculated according to the previous equation. .

2.2.3. Characterization Phases were identified using X-ray powder diffractometer (Philips PW 1730) with Cu Kα target. All measurements were performed at room temperature within the range of 2θ = 4-50◦ at a scanning speed of 2◦/min. The diffractometer was operated at 40 KV/25 mA. The FT-IR spectra were measured for the prepared samples using KBr pellets made from a mixture of powder for each sample. The FT-IR spectra were measured from 400 to 4000 cm-1 using a Nexus 670, Nicloet FT-IR spectrometer. The thermal properties of the investigated samples were evaluated using thermogravimetric analysis (TGA). These assays were carried out using a Perkin–Elmer, 7 series thermal analyzer in nitrogen atmosphere at a heating rate of 10◦C/ min. over the temperature range of 50-800◦C. The prepared samples were dried and analyzed pre-and post-immersion for 21 days in SBF using FT-IR and scanning electron microscopy (SEM-EDAX), JXA 840A Electron Probe Microanalyzer. For SEM, the substrates were mounted on metal stubs and coated with gold before being examined.

2.2.4 Testing 2.2.4.1 Mechanical The co-polymer and both composites were tested to determine the effect of particle size on mechanical properties. The molding process was only carried out to prepare the samples for the mechanical testing via casting the copolymer or BG filler/co-polymeric solutions after directly the co-polymerization process into the mould and dried at 60 ºC overnight. The Wolpert hardness was determined using Wolpert Hardness Tester HT 2004, DIN 53 456. Compressive strength was measured for the prepared composites using Zwick Material Prufugi 1425, Germany. The average value was taken for three samples to insure the results. The number of the measured samples was three, shape of the sample was cylindrical (1 cm × 1 cm), and load cell was10 KN and crosshead speed was 10 mm/min. 2.2.4.2 Water absorption (W.A%) For water-uptake measurements, all the samples were weighed before being immersed in distilled water (at room temperature). After immersion for different periods, the samples were carefully removed from the media and immediately weighed for the determination of the wet weight as a function of the immersion time. The average value of three samples was taken for W.A% determination at each period [25]. The W.A% was calculated according to the following equation .

W.A% = [(Wf – Wi )/ Wi ] x 100 Where Wi is the initial weight of the sample, and Wf is the final weight post-immersion.

2.2.4.3 In vitro tests Standard in vitro bioactivity tests were carried out to evaluate the formation of an apatite layer on the surface of the co-polymer and composites. In order to study the bioactivity, each sample was soaked in SBF for each investigated composite at different periods, The SBF solution was proposed by Kokubo et al. [26] at body temperature (37◦C) and pH = 7.4 for several periods [27] . The SBF has a composition similar to human blood plasma and has been extensively used for in vitro bioactivity tests[28]. After the immersion periods end, the solutions were analyzed by spectrophotometer (UV-2401PC, UV-VIS Recording spectrophotometer) using biochemical kits (Techo Diagonstic) to detect the total calcium ions (Ca2+) at λ= 570 nm and phosphate ions (PO4

3-) concentration at λ= 675 nm. The average value of three tests was taken for Ca2+ and PO4

3- ions determination at each period. The immersed samples were removed from the SBF solution then abundantly rinsed using deionized water in order to remove the soluble inorganic salts and to stop the reaction.

Results and discussion

3.1. The co-polymer and composite samples The yield of grafting percentage of pHEMA grafted onto chitosan was 26.94 % which is higher than the corresponding value of BG1 composite (24.72 %) and lower than that of BG2 composite (27.16 %). This may prove that small filler particle size has reduced the grafting percentage as a result of scission of co-polymer chains by smaller particle size of BG1 filler. While the coarse bioactive glass (BG2) is used as filler it shows better grafting percentage. This result coincided with Wang et al. [29] findings as they found that the co-polymer float

around the filler of large particle size indicating that no chemical bond exists between the co-polymer matrix and filler.

CHARACTERIZATION AND IN-VITRO BEHAVIOUR OF BIOACTIVE… 3.2. Characterization 3.2.1 Phase analysis The XRD patterns of chitosan, co-polymer, BG filler, BG1 and BG2 composites are shown in Fig.1. Figs. 1a and b show that the patterns of chitosan into the co-polymer structure are broad hump with lower intensity and some shift to lower angle compared to the original chitosan (Card No: 39-1894) proving chemical interaction between chitosan polymer and HEMA monomer. This result is coincided with the reported work that the wide peak assigned to chitosan was sharper and broader after grafting process [30]. For BG filler and both composites, the peak of BG filler appears as a broad hump in the range of 2θ (25–35)◦ and is still persists in both composites (BG1 and BG2) with slight lower peaks intensity for BG1 composite compared to BG2 composite. The chitosan peaks of copolymer disappear in both composites due to effect of the BG particles on the copolymer matrix and amorphous nature for BG and copolymer matrices (Figs.1b, c and d). This proves that the effect of coating and integration was enhanced in case of the smaller particle size composite compared to the larger particle size composite, and the stability of filler into the composite

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Fig. 1. XRD analysis of the samples

3.2.2 FT-IR analysis The FT-IR spectra of co-polymer, BG filler, BG1 and BG2 composites are shown in Fig.2. The transmittance behaves inversely relationship with the optical density (O.D). After the loading of the BG filler particles onto the co-polymeric matrix, for BG1 composite, the optical density (O.D) of co-polymer bands such as OH: 3440 cm-1, C-H: (749 and1384 cm-1), amide: (1642 and 1560 cm-1), C=O: 1722 cm-1, C-O: 1162 cm-1 and C-CO: 1248 cm-1 [31] were reduced compared to original co-polymer proving effect of BG particles having the smaller size onto these sites. Also, the O.D of phosphate and/or Si-O-Si at 1060 cm-1 and OH at 3460 cm-1 appearing into the spectrum of BG1 composite were reduced compared to original BG filler denoting effect of coating with the polymer layer. For BG2 composite, the majority of bands characterizing the copolymer structure and BG filler into this composite except phosphate and/or Si-O-Si groups were nearly disappeared in comparison with the original copolymer, BG filler and BG1 composite proving high effect of two matrices on each other especially the composite having higher size of BG on copolymer bands. This observation reveals the enhancement of coating process for the smaller particle size filler as in BG1 composite and, on the other hand, it indicates the separation of both matrices for the larger particle size filler as in BG2 composite [32].

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CH C-O

CHCH3 C=O CH/OH OH

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PO43- CH3 C=O OH

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Fig. 2. FT-IR spectra of a) co-polymer, b) BG filler, c) BG1 and d) BG2 composites

3.2.3 Thermal analysis (TGA) . To evaluate the thermal stability of the prepared samples that were assessed using TGA analysis. Fig.3 shows the weight loss curves of BG filler, copolymer and the two composites. The initial thermal destruction of copolymer occurred at temperature less than 220◦C and recorded weight loss (6.941%) (Fig.3a). It shows an almost complete weight loss at 387◦C with total weight loss of 98.53 % accompanying the release of all CO2, NH3 gases and water molecules proving high grafting % between chitosan polymer and HEMA monomer [33] . For BG1 composite, the thermal decomposition started at 199◦C and its weight loss was 3.6% proving the release of absorbed water (Fig.3c). The total weight loss was 43.40 % as a result of complete destruction of co-polymer layer which is attached onto the BG filler particles with the release of rest organic matter. On the other hand, the TGA curve (Fig.3d) for BG2 composite shows the release of absorbed water at 214◦C with weight loss (5.93%) and record complete decomposition at 394◦C with weight loss (84.35 %). The last weight loss (9.1%) at 489 ◦C is due to removal of the rest of organic matter. The total weight loss became 99.38 %

coinciding with the highest grafting (27.16 %). Hence, the BG2 composite curve had similarity with copolymer curve associated with the increase in grafting percentage, which suggests that grafted branches chain grow with the

increase of grafting percentage denoting phase separation of both matrices [2]. Therefore, the attached copolymer layer onto the BG particles was calculated via the difference between the weight loss percentage of BG (0.44) and BG1 or BG2 composite. Hence, the attached copolymer for BG1 composite recorded 42.96 % while recorded 98.943 % for BG2 composite. This result proves that the attached copolymer layer onto BG particles was lower for small size of particles composite as in BG1 composite compared to large size of particles composite as in BG2 composite which manifests growing of grafting yield[2] and separation of two matrices. This result is not in the favor of the formation of good composite.

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W t.loss= 43.40 %BG 1 com posite

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Fig.3. TGA analysis of the samples

3.3 Testing 3.3.1 Mechanical It was notified that hardness of BG1 composite (27.22) and BG2 composite (11.60) was enhanced compared to the original copolymer (7.86) especially BG1 composite proving effect of small particle size. The reason of this behavior is that a smaller particle size had a greater surface area which resulted in polymer/filler interaction and adhesion. Also, the compressive stress for BG1 composite (4.45 MPa) and BG2 composite (2.16 MPa) was lower than that of the neat co-polymer (8.74 MPa) and these values are still located within the range of

compressive stress of human cancellous bone (2-12 MPa) [35]. As a result, the smaller particle size of BG filler into the copolymer leads to more effective reinforcement and therefore, a

CHARACTERIZATION AND IN-VITRO BEHAVIOUR OF BIOACTIVE… stiffer composite. This observation could be attributed to the fact that the ductile behavior is reduced with the composite containing small size of BG particle [11,34]. 3.3.2 Water absorption (W.A %) Fig.4 shows that BG1 composite has lower W.A % values compared to copolymer and BG2 composite at all periods proving its higher stability in water. The copolymer and BG2 composite had nearly the same high grafting % and high W.A % while BG1 composite had nearly the same low grafting % and low W.A %. Thus, it could be concluded that there is a direct proportional relationship between grafting and water absorption %. This result proves that the presence of small particles of BG fillers into the copolymer affected content of OH groups which is responsible for affinity to water molecules within the composite and reduced W.A% as in BG1 composite due to the formation of a temporary BG barrier preventing water permeating into the copolymer matrix [36].

0 5 1 0 1 5 2 0 2 50

B G 1 c o m p o s i te

B G 2 c o m p o s ite

C o - p o ly m e r

T im e ( d a y s )

Fig.4. Water absorption (W.A%) of the samples in pure water for different periods.

3.3.3 In-vitro test Measurement of calcium and phosphate ions in SBF after withdrawal of the samples Calcium ions (Ca++) Standardized in vitro bioactivity test procedure was carried out to evaluate the formation of the apatite layer on the surface of the bioactive glass after immersion in SBF [37]. The in-vitro bioactivity result represents the possible change in Ca++ and PO4

3- ions concentration in SBF solution after soaking the samples for different periods (1-21 days). Fig.5 shows lower levels of Ca++ concentration for co-polymer sample compared to control at all periods proving deposition of calcium ions onto the copolymer surface denoting interaction between calcium ion in SBF and chitosan into the copolymer. It is known that the polymer

matrix influences the formation of the HA nanocrystalites by specific organic-inorganic interactions between components by complexation of Ca++ with some functional groups

(amino, acetylamino and OH) from chitosan [38]. Level of Ca++ ions concentration after one day for BG1 composite was high compared to control SBF proving release of Ca++ into the media from the composite containing CaO in its structure. Then, the concentration of Ca++ was slightly reduced after 3, 7, 15 and 21 days compared to one day period but it is still higher than control. This proves the continuous release of Ca++ ions with longer time till 21 days as a result of effect smaller particles of BG filler into the composite. For BG2 composite, it behaves the same trend for Ca++ ions concentration and its corresponding values were lower at all intervals compared to BG1 composite (Fig.5). This indicates higher release of Ca++ ions from BG1 compared to BG2 composite due to the higher surface exposure of filler component in the former composite. Therefore, the particle size and surface area of the BG filler presented into the composite affect Ca++ release into the SBF medium.

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BG2 composite

Control

Copolymer

T ime/days

Fig.5. Calcium ions concentration pre- and post-immersion of the samples in SBF at different periods compared to control.

Phosphate ions (PO4

3-) Fig.6 shows phosphate ions at different periods for the prepared samples post-immersion. The PO4

3-concentration at all periods record lower values for copolymer samples compared to control denoting deposition of phosphate ions onto the surface of copolymer. The PO4

concentration records lower values at 1 and 3 days for BG1 composite compared to the control and copolymer showing deposition of PO4

3- ions onto the composite surface while, its concentration at other periods nearly diminished proving high deposition of PO4

3- ions on the surface. This result is in the favour of formation of bone-like apatite layer onto the composite

surface. For the BG2 composite, the concentration of PO43- records higher values compared to

BG1 composite but still lower than control values showing also high deposition of PO43-onto

CHARACTERIZATION AND IN-VITRO BEHAVIOUR OF BIOACTIVE… the surface. This finding confirmed the idea that the presence of small particle size of BG filler within the copolymer enhances the deposition of phosphate ions which is in favour of formation of apatite layer onto the composite surface. In this context, it was reported that the interaction between amino groups of chitosan polymer and phosphate groups of octacalcium phosphate (OCP) was performed [39]. So, we could expect the occurrence of interaction between phosphate ions in SBF and the BG composite surface, therefore, the presence of BG filler within copolymer had the ability to promote the formation of calcium phosphate (Ca-P) layer especially for BG1 composite.

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B G 1 c o m p o s i t e

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C o p o l y m e r

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Fig.6. Phosphate ions concentration pre- and post- immersion of the samples in

SBF for different periods compared to control 3.3.4 Characterization of samples after their withdrawal from SBF 3.3.4.1 FT-IR analysis For co-polymer, the optical density (O.D) values of the most of co-polymer bands are enhanced in post-immersion treatment compared to pre-immersion indicating their involvement in the biolayer formation (Fig.7). For BG filler, the O.D of OH groups of water molecules recorded at 3449, 1643 and 1514 cm-1 are increased at 7 days post-immersion compared to pre-immersion showing formation of biolayer on its surface Also, another band at 775 cm-1 which is assigned to Si-OH group shows the same behavior. The O.D of phosphate bands at 473, 932 and 1036 cm-1 are increased especially at 7 days while they decreased at 21 days, this is in the favour of the formation of apatite layer (Fig.8). This result is coincided with those reported[40] ,that apatite-forming ability is occurred by decreasing soaking time in SBF . For BG1 and BG2 composites, the O.D of OH and phosphate groups are enhanced post-immersion compared to pre-immersion especially at 21 days for BG1 composite and at 7 days for BG2 composite proving biolayer and deposition of phosphate ions onto the surface, this result is in the favour of the formation of apatite layer (Figs.9,10). New bands appeared at

1400-1450 cm -1 post-immersion which is assigned to carbonate groups proving formation of carbonated apatite layer (Fig.9). On the other hand, the O.D of C=O and C-O groups are

enhanced especially at 21 days for BG1 composite and at 7 days for BG2 composite showing its involvement into the composite structure, on the other hand, other bands such as CH3 and C-CO in BG1 composite spetrum and CH3 in BG2 composite spectrum were disappeared post-immersion denoting its masking (Figs.9,10).

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C H C = O O H /C HO H

c ) 2 1 d a y s

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Fig.7. Copolymer spectra of pre-and post-immersion in SBF

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P O 43 -

P O 43 - a b s o r b e d H 2 O

c ) 2 1 d a y s )

Fig.8. Bioactive glass (BG) spectra of pre-and post-immersion in SBF

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a ) B G 1 c o m p o s ite (p re - im m e rs io n )

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Fig.9. BG 1 composites spectra of pre-and post-immersion in SBF

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Fig.10. FT-IR of BG 2 composites spectra of pre-and post-immersion in SBF G.T. El-BASSYOUNI AND K. R. MOHAMED

3.3.4.2 Surface microstructure

For copolymer pre-immersion (Fig.11a), SEM plate shows smooth surface with irregular lines showing formation of co-polymer having fibrous structure of chitosan, on the other hand, post-immersion (Fig.11b), SEM plate indicates the appearance of many spherical particles containing minute pores on the copolymer surface indicating formation of apatite layer and EDAX analysis confirmed this result via the value of Ca/P ratio (1.42) which is close to the ratio of hydroxyapatite (1.67) (Fig.11c). Therefore, one can expect that the presence of copolymer within the BG filler will lead to the enhancement of nucleation of apatite layer onto the composite surface. SEM plates observation for of BG filler post-immersion in SBF indicated that the surface morphology changes upon immersion and formation of few spherical particles were distributed onto the surface of BG particles showing some precipitation of calcium phosphate (Fig.11d).

For BG1 composite pre-immersion, SEM analysis reveals the presence of numerous fine grains which look like protrusions (created by distribution of bioactive glass of small particle size) between which the co-polymer could flow during the extrusion process (Fig.11e). At higher magnification, SEM plate (Fig.11f) illustrates that open pores are easily seen with smooth surface proving coating effect, and the open pores on the surface enabled a rapid reaction and formation of calcium phosphate precipitation [23]. Post-immersion, bone-like apatite layer appears as thick layer containing spherical shapes having minute pores accumulated to each other onto the composite surface indicating effect of immersion in SBF (Fig.11g) .

For BG2 composite pre-immersion, SEM analysis reveals the presence of particles of large particle size distributed on the surface of the copolymer proving its floating on the surface (Fig.11h). SEM image post-immersion portrays that BG2 composite shows the growth of an apatite layer on the material surface with some open pores denoting effect of immersion. But it is clear that BG particles are still distinguished underneath the formed apatite layer [9] (Fig.11i). It was notified that the formation of bioactive apatite layer onto the surface of copolymer, BG filler and both composites were achieved especially the copolymer and BG1 composite containing smaller particles size of BG in comparison to BG filler and BG2 composite having higher particles size.

Fig.11. SEM morphology of Copolymer (a) pre- and (b,) post-immersion and (c) its

EDAX analysis, BG filler (d) post-immersion, BG1 composite (e, f) pre-and (g)

post-immersion, and BG2 composite (h) pre-and (i) post-immersion in SBF for 21

Conclusions

• The grafting % was reduced for BG1 composite containing smaller particle size as a result of scission of co-polymer chains while it was enhanced for BG2 containing larger particle size because the particles floats around the copolymer leading to their separation.

• Phase, FT-IR and TGA analyses proved integration, interaction and coating between two matrices especially BG1 composite.

• Mechanical properties showed that the copolymer containing smaller particle size of BG filler leads to more effective reinforcement and stiff composite

• In vitro bioactivity and FT-IR post-immersion confirmed stability of composite in SBF, the formation of carbonated apatite layer onto the surface of composite at longer period (21 days) as in BG1 composite and at early period (7days) as in BG2 composite.

• SEM-EDAX analysis showed the formation of thick apatite layer onto BG1 composite surface compared to BG2 composite and BG filler.

• Finally, it is notified that fabrication of novel bio-composites containing BG and copolymeric matrix having properties comparable to human cancellous bone such as bioactivity, mechanical properties and the formation of carbonated apatite layer will enhanced for their uses in bone replacement and scaffold for tissue engineering applications.

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