Materials Science and Engineering C 39 (2014) 150–160

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Bioactive, mechanically favorable, and biodegradable copolymer nanocomposites for orthopedic applications Sunita Prem Victor, Jayabalan Muthu ⁎ Polymer Science Division, BMT Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram 695 012, Kerala, India

a r t i c l e

i n f o

Article history: Received 21 November 2013 Received in revised form 11 February 2014 Accepted 17 February 2014 Available online 24 February 2014 Keywords: In situ polymerized polymeric nanocomposites Biodegradation Biocompatibility

a b s t r a c t We report the synthesis of mechanically favorable, bioactive, and biodegradable copolymer nanocomposites for potential bone applications. The nanocomposites consist of in situ polymerized biodegradable copolyester with hydroxyapatite (HA). Biodegradable copolyesters comprise carboxy terminated poly(propylene fumarate) (CT-PPF) and poly(trimethylol propane fumarate co mannitol sebacate) (TF-Co-MS). Raman spectral imaging clearly reveals a uniform homogenous distribution of HA in the copolymer matrix. The mechanical studies reveal that improved mechanical properties formed when crosslinked with methyl methacrylate (MMA) when compared to N-vinyl pyrrolidone (NVP). The SEM micrographs of the copolymer nanocomposites reveal a serrated structure reflecting higher mechanical strength, good dispersion, and good interfacial bonding of HA in the polymer matrix. In vitro degradation of the copolymer crosslinked with MMA is relatively more than that of NVP and the degradation decreases with an increase in the amount of the HA filler. The mechanically favorable and degradable MMA based nanocomposites also have favorable bioactivity, blood compatibility, cytocompatibility and cell adhesion. The present nanocomposite is a more promising material for orthopedic applications. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Mimicking nature with respect to the compositional and morphological structure of tissue has been a widely adopted strategy and the ultimate goal in the design of tissue-engineered scaffolds [1]. Though bone-scaffolding materials have been heavily studied in the past two decades. There still exists an unmet clinical need for the development of effective bioactive and biodegradable composite bone scaffolds [2]. Segmental defects remain a significant clinical challenge despite the inherent healing capacity of bone tissue [3]. The current gold standard relies on the use of stabilization with plates and bone regeneration with bone grafts. There are various other strategies being investigated for bone tissue applications. In these, solid scaffolds like poly(propylene fumarate) serve as the stabilization of the defect until the regenerated bone can support the load [4,5]. However, the introduction of porosity, one of the commonly employed strategies in tissue engineering has been known to reduce mechanical properties. So mixing of polymers with ceramics like calcium phosphates to form nanocomposites materials with enhanced mechanical properties has opened out new avenues in bone research [6]. It has long been recognized and established that calcium phosphate like hydroxyapatite constitutes around 60 wt.% of bone and is a crucial biomaterial to design tissue-engineered bone substitutes. HA and related calcium phosphate have been observed to enhance mechanical ⁎ Corresponding author. Tel.: +91 471 2520 212; fax: +91 471 234 814. E-mail address: [email protected] (J. Muthu).

http://dx.doi.org/10.1016/j.msec.2014.02.031 0928-4931/© 2014 Elsevier B.V. All rights reserved.

properties and play a critical role in the osteoconduction and osseointegration of implanted bone grafts [7]. Thus, developing composite materials based on biodegradable polymers and HA has become an intense focus in bone tissue engineering. Such nanocomposites take advantage of the formability of polymers and the bioactivity of HA to enhance the mechanical and biological properties of the fabricated polymer nanocomposites [8–10]. However, nanoparticles tend to aggregate and this behavior influences their mechanical properties. Thus higher filler content could lead to detrimental mechanical properties. One way to overcome this drawback is to use lower filler content to achieve desirable mechanical properties. Of the various polymers actively studied, biodegradable aliphatic polyesters have received particular attention due to their degradability and biocompatibility [11]. Aliphatic polyesters can be synthesized from monomers endogenous to human metabolism. They degrade via hydrolysis into nontoxic carboxylic acid and hydroxyl group short chain oligomers [12]. However, despite the availability of various materials with appropriate biological and structural properties no single material is able to mimic the composition, structure, and properties of bone. Recently nanocomposites have been considered as the best choice for tissue regeneration as they can provide suitable matrix environment, integrate desirable biological properties, exhibit improved mechanical properties and control biodegradability [13,14]. Biodegradability is highly critical and an ideal nanocomposite should maintain its mechanical properties as it degrades until the newly regenerating bone could adequately support the loading. Jayabalan et al. have prepared and evaluated poly(propylene fumarate-co-ethylene glycol) for use as a scaffold for correcting

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bone defects [15]. Orthopedic load bearing applications require a nonporous biodegradable composite for binding the fractured bone and sustaining the compression load. Subsequently, bone growth and remodeling occur by gradual biodegradation. A recent comprehensive review [16] on different copolyester matrices lists out various copolyesters developed for various bone applications. Some of the drawbacks of these traditional copolyester matrices include poor mechanical properties, poor stability, nontunable degradation profiles and nonmineralization. In the present investigation, we report the synthesis and physiochemical and biological evaluation of mechanically favorable, bioactive and biodegradable in situ polymerized biodegradable copolyester with HA. To the best of our knowledge, the in situ synthesis of this novel copolyester has been reported for the first time. This in situ synthetic procedure led to homogenous distribution and could facilitate better mechanical properties with less amount of filler content. 2. Methods 2.1. In situ polymerization of biodegradable carboxy terminated poly(propylene fumarate) (CT-PPF) polyester with hydroxyapatite

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containing 3 wt.% of HA and 5 wt.% of HA with respect to the total weight of the polymer have been abbreviated as 3CT-PPF and 5CT-PPF respectively. The molecular weight of the CT-PPF resins was determined using a Waters HPLC system with Styragel-HR-5E/4E/2/0.5 columns in series with the mobile phase tetrahydrofuran. 2.2. In situ polymerization of biodegradable poly(trimethylol propane fumarate-co-mannitol sebacate) (TF-Co-MS) copolyester resin with hydroxyapatite TF-Co-MS resin with HA incorporated in situ was synthesized by refluxing and vacuum-condensation methods. Maleic anhydride, trimethylol propane and HA were mixed and refluxed at 140 °C for half an hour under nitrogen atmosphere. Sodium acetate and morpholine were added to catalyze the polymerization reaction (Scheme 2). The reaction mixture was then cooled to room temperature and subsequently mannitol and sebacic acid were added. Refluxing at 140 °C was continued for another 30 min. The above mixture was then subjected to vacuum condensation at 180 °C for 5 min. The reaction product obtained was dissolved in acetone and then washed with aqueous methanol to remove any unreacted reactants. The polymer resin was reprecipitated in ether, filtered, and dried

CT-PPF resin with HA incorporated in situ was synthesized by refluxing and vacuum-condensation methods and by modifying and using previously established protocols [15]. Maleic anhydride, 1,2propanediol and hydroxyapatite were mixed and refluxed at 148 °C, followed by vacuum condensation at 185–190 °C for 15 min. Sodium acetate and morpholine were added to catalyze the above polymerization reaction (Scheme 1). The reaction product obtained was dissolved in acetone and then washed with 25% aqueous methanol to remove any unreacted reactants. The polymer resin was reprecipitated in ether, filtered, and dried using a rotary evaporator. Hydroxyapatite nanoparticles used as particulate filler in the synthesis mentioned above were initially prepared by co-precipitation technique. Calcium chloride was dissolved in 100 ml of distilled water. 20 ml of fresh conjugate base prepared with 5.4 wt.% trisodium citrate was then introduced. The solution was stirred for 15 min before 2.2 wt.% of 100 ml disodium hydrogen phosphate was added drop wise from a burette. The reaction was allowed to proceed under stirring for 24 h. The resulting suspensions obtained were washed with distilled water, centrifuged, and lyophilized. The nanoparticles obtained were collected and stored for further use. The phase analysis was carried out by X-ray powder diffraction (XRD) method (Bruker AXS, X-ray diffractometer, reflection mode, Japan) using CuKα radiation with tube voltage 40 KV and 35 mA of tube current. The filler content in the final CT-PPF resin was varied to prepare two different batches of resin, containing 3 wt.% and 5 wt.% of filler content with respect to the total weight of the polymer resin. The CT-PPF resins

Scheme 1. In situ polymerization of biodegradable carboxy terminated poly(propylene fumarate) (CT-PPF) polyester with hydroxyapatite.

Scheme 2. In situ polymerization of biodegradable poly (trimethylol propane fumarateco-mannitol sebacate) (TF-Co-MS) copolyester resin with hydroxyapatite.

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using a rotary evaporator. The filler content in the final TF-Co-MS resin was varied to prepare two different batches of resin, containing 3 wt.% and 5 wt.% of filler content with respect to the total weight of the polymer resin. The TF-Co-MS resins containing 3 wt.% of HA and 5 wt.% of HA with respect to the total weight of the polymer have been abbreviated as 3TF-Co-MS and 5TF-Co-MS respectively. The molecular weight of the TF-Co-MS resins was determined similar to the CT-PPF resin.

10 mm at a speed of 5 mm/min ignoring the first 2 mm length. The samples (dimension 2 cm × 1 cm and known width) were wetted in distilled water for 24 h and cleaned prior to the contact angle measurements. Experiments for the copolymer nanocomposites CNV3, CMA3, CNV5 and CMA5 have been done in triplicate and reported with standard deviation. 3.4. Evaluation of surface morphology

2.3. Preparation of copolymer nanocomposites The 3CT-PPF resin was crosslinked with 50 wt.% of N-vinyl pyrrolidone (NVP) or methylmethacrylate (MMA) monomer by weight using dibenzoyl peroxide as an initiator (2% w/w). The second polymer resin 3TF-Co-MS was then added and mixed well. Further N,Ndimethylaniline (0.2% w/w) was added with rapid stirring. The mixture was then molded using the appropriate molds for different tests. Curing occurred after leaving the mixtures at room temperature within an hour. It was then heat-treated under nitrogen atmosphere at 140 °C for 24 h. The copolymer nanocomposites prepared using NVP and MMA have been coded as CNV3 and CMA3 respectively. Similarly, copolymer nanocomposites were prepared using the polymer resins 5CT-PPFand 5TF-Co-MS with the method mentioned above. Subsequently the copolymer nanocomposites prepared using NVP and MMA have been coded as CNV5 and CMA5 respectively. The copolymer nanocomposite CNV3 consists of 3CT-PPF resin, 3TFCo-MS resin, and crosslinker NVP. Copolymer nanocomposite CMA3 consists of 3CT-PPF resin, 3TF-Co-MS resin, and crosslinker MMA. Copolymer nanocomposite CNV5 consists of 5CT-PPF resin, 5TF-Co-MS resin and crosslinker NVP. Copolymer nanocomposite CMA5 consists of 5CT-PPF resin, 5 TF-Co-MS resin and cross linker MMA. 3. Characterization 3.1. Determination of molecular weight of polymer resins 1% solution of CT-PPF and TF-Co-MS in tetrahydrofuran (THF) was used for the analysis. 50 μl of the sample was injected to the HPLC system (Waters) connected with 600 series pump and 2414 refractive index detector. The styragel column used was (HR-5E/4E/2/0.5) connected in series. Mobile phase (THF) was pumped at a flow rate of 1 ml/min and the relative calibration was done with polystyrene standards (Mp-100000, 9130, and 162). 3.2. Determination of chemical composition Fourier transform infrared spectra were recorded as diffused reflectance spectra using a Thermo Nicolet, 5700, Germany spectrometer. The FT-IR spectra of HA and the HA containing polymer resins were obtained in the region 400 to 4000 cm−1. To avoid water vapor and CO2 bands, the instrument was continuously flushed with nitrogen. The chemical composition of the HA containing polymer resins and the copolymer nanocomposites were further complimented by their characteristic bands as analyzed using Raman spectroscopy (Witec-Alpha 300, USA) using the 514 nm laser line with a laser power of 40 mW. The distribution among the different constituents of the copolymer composite was viewed by Raman spectral imaging with a microscopic lateral resolution based on the original spectrum obtained. The spectral mapping has been performed with 100 × 100 points in an area of 1 μm × 1 μm. The laser used was 532 nm with an integration time of 0.5 s and a 600 g/mm grating. 3.3. Contact angle measurements The advancing and receding contact angles of the nanocomposite samples were determined in water by the Wilhelmy method using a KSV sigma 701 tensiometer. The samples were immersed to a depth of

The surface morphology of the copolymer nanocomposites was examined and analyzed by environmental scanning electron microscopy (ESEM) analysis. The freeze-dried nanocomposites were viewed under low vacuum condition. The samples were then shock frozen in a liquid nitrogen bath for 1 min. The frozen samples undergo brittle fracture and their cross sections were analyzed by SEM. 3.5. Mechanical evaluation Three specimens have been used for a sample in all the mechanical testing. Statistical analysis was performed using Student's t-test. All values are reported as the mean with standard deviation. Tensile strength of the copolymer nanocomposites was determined using the universal automated mechanical test analyzer (Instron, model 3345) connected with a long travel extensometer. Samples were cut to dumb bell shape using die (ISO 527-2 type 5A). Tensile strength was tested with a load cell of 100 N at 25 °C with a crosshead speed of 10 mm/min. The stress–strain data were recorded. The compressive properties of the copolymer nanocomposites were determined using an Instron model series IX automated materials 7.43.00 testing system at a cross head speed of 5 mm/min. The nanocomposites were molded into cylindrical pellets 6 mm in diameter by 12 mm high for compression testing. The shore hardness of the nanocomposites was measured using a durometer. The values were determined by the penetration of the durometer indenter foot into the sample. The force was applied for 60 s and the indentation hardness was read on the scale. 3.6. Monitoring of degradation behavior In-vitro degradation studies of the nanocomposites were carried out by incubation in media for a maximum duration of three months. The media used were Ringer solution and phosphate buffered saline solution. Nanocomposites of known weight were incubated in tubes containing the buffer media. Each tube containing buffer with the composite sample was maintained in a constant temperature water bath. The weight loss was determined by measuring the change in weight after lyophilizing the sample at regular intervals of time. Further, the media were also analyzed for changes in pH and conductivity at intervals of 30 days with a pH-conductivity meter (CyberScan PC 510). All the experiments were performed in triplicate, by running three parallel independent measurements simultaneously. The change in mechanical properties was tested by tensile test as described above. The tensile strength of CMA3 and CMA5 was determined after immersion in 28 days in PBS. 3.7. In-vitro evaluation of bioactivity The assessment of the in-vitro bioactivity has been carried out in stimulated body fluid (SBF), which has an ionic composition similar to that of the human blood plasma [17]. The ratio of the copolymer composite weight to SBF solution volume was 1.5 mg/mm3. The samples have been put into a polyethylene bottle covered with a tight lid. The bottles have been placed in a constant temperature water bath at 37 °C for 2 h and two weeks respectively without refreshing the SBF solution. After soaking, the samples have been removed from the SBF and characterized by AFM and Energy Dispersive X-ray Analysis (EDAX) respectively.

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3.8. Blood compatibility studies Blood was collected in tubes containing 3.8% sodium citrate at a blood:anticoagulant ratio of 9:1. The blood components utilized for hemolysis and aggregation studies were separated as follows. The red blood corpuscles (RBCs) were obtained by centrifuging fresh blood at 700 rpm. White blood corpuscles (WBCs) and platelet rich plasma (PRP) were separated by centrifuging fresh blood after layering with histopaque to form two layers for 15 min at 750 rpm. The composite samples were extracted in sterile 1 ml PBS for 48 h and 100 μl aliquots were employed for aggregation and hemolysis studies.

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3.9.1. MTT cell viability assay MTT cell viability assay was carried out as per ISO 10993-5 using L929 mouse fibroblast cell culture. PBS treated samples were incubated in culture medium overnight at 37 °C. This medium was then utilized for culturing the L929 cells. 1 ml cell suspension containing approximately 5 × 104 cells were seeded on to a 24 well tissue culture plate and 1 ml of the above medium was added and incubated at 37 °C and 5% CO2 for 72 h. Quantification of surviving fibroblast cells after incubation was done by MTT assay and Neutral Red (NR) assay [18]. 3.10. Assessment of cell adhesion and proliferation by direct contact method

3.8.1. RBC aggregation studies Aggregation tests were carried out by incubating 100 μl of the PBS extract with 100 μl dilute RBC suspension (diluted with PBS (1:4)) for 20 min at 37 °C. Normal saline and PEI were used as the negative and positive controls respectively. The cells were then observed through a phase contrast microscope (Leica DMIRB, Germany) at a magnification of 40×.

The direct contact assay was used to evaluate the cell adhesion and cell proliferation on the nanocomposites. The composite samples of 1 cm diameter was placed over a sub-confluent layer of L929 cells and allowed to proliferate for 24 h in a CO2 incubator. Empty wells were used as a control. After the incubation, the samples were viewed under an inverted phase contrast microscope attached with an imaging camera.

3.8.2. Hemolysis Hemolysis assay of the samples was carried out as reported in literature. 100 μl of the PBS extract was mixed with 100 μl dilute RBC suspension and incubated at 37 °C for 3 h. Normal saline and water were used as the negative and positive controls respectively. The positive and negative controls were both mixed with equal volumes of RBC suspension. After incubation it was subjected to centrifugation at 3000 rpm for 10 min. The optical density (OD) was then measured at 541 nm by UV–Vis spectrophotometer (Varian). Hemolysis of the various samples was calculated as follows

3.10.1. Live/dead assay L929 cells were cultured onto the composite for 5 days. After rinsing with PBS, cultures were treated with a mixture of acridine orange (100 μg/ml) and ethidium bromide (100 μg/ml) and viewed immediately under an epifluorescence microscope (Optika SRL) using blue filter for acridine orange and green filter for ethidium bromide [19]. Under the microscope, living cells were clearly detected on the membrane with bright green cell nuclei; dead cells were stained red in live/dead assay. Two images were taken from the same field using both filters and the images were merged by imaging software Photoshop 8 CS software.

Hemolysisð% Þ ¼ ODsample −ODð–Þ

control =ODðþÞ control −ODð−Þ control :

3.9. Evaluation of cytocompatibility The mouse lung fibroblast cell line, L929 was selected for in vitro studies. The cells were cultured in basic medium composed of Dulbecco's Modified Eagle's Medium with glucose (HiMedia), supplemented with 10% fetal bovine serum (FBS), penicillin, streptomycin and sodium bicarbonate. The cells were maintained at 37 °C at 5% CO2. The medium was replaced with fresh medium once in 3 days. The cells were trypsinized and subcultured after attaining around 80% confluence.

3.11. Statistical analysis The experiments were carried out with 5 or 6 samples from each group and the results were presented as means ± standard deviations. Statistical analysis was implemented with one-way ANOVA using online calculator, Statistics Calculator version-3 beta. The level of significance was set at p b 0.05 for all calculations. 4. Results and discussion In this study, we have prepared biodegradable and bioactive nanocomposites consisting of in situ polymerized biodegradable copolyester with HA. Mechanical characteristics and biocompatibility are crucial in

Fig. 1. TEM micrograph of HA (a) and XRD pattern of HA (b).

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the selection of materials for bone applications. Incorporation of HA into the matrix has been shown to improve both biological responses as well as material mechanical properties. The bright field TEM microphotograph and XRD pattern of the HA nanoparticles are given in Fig. 1. The bright field TEM microphotograph exhibits distinct, homogenous, high aspect ratio needle like particles (Fig. 1a). The XRD patterns (Fig. 1b) reveal characteristic peaks at 2θ = 26.12°and 32.13° corresponding to those expected from the HA structure (JCPDS 09-432) [20]. The average crystalline size of the HA particles has been estimated using Scherrer formula with (002) diffraction peak. The line broadening of the (002) peak was used to evaluate the crystalline size since the peak is well resolved and d002 values are related to crystal size in the broader aspect of hydroxyapatite crystallites. The average crystalline size of the HA particles has been found to be 30 nm. The cell parameter calculations indicated that the Ca/P ratio is around 1.65 indicating that the HA used in the present studies is nonstoichiometric in nature [21]. Although naturally occurring stoichiometric HA has a Ca/P ratio of 1.67, the physiologically occurring HA is always calcium deficient, due to the incorporation of hydrogen phosphate ions. Furthermore it has also been reported that calcium-deficient hydroxyapatite plays an important role in bone remodeling and bone formation and is very efficient in inducing the precipitation of bone-like apatite [22]. So the HA

Fig. 2. (a) FTIR spectra of 3TF-Co-MS and 3CT-PPF resins. (b) FTIR spectra of CNV3 and CMA3 copolymer nanocomposites.

used in the present study could promote better bioactivity and facilitate in bone formation. The molecular weight of the 3CT-PPF was 1153 g/mol (Mn) and 1363 g/mol (Mw) and the polydispersity was 1.18. The molecular weight of the 5CT-PPF was 1458 g/mol (Mn) and 1520 g/mol (Mw) and the polydispersity was 1.04. The molecular weight of the 3TF-CoMS synthesized was 982 g/mol (Mn) and 999 g/mol (Mw) and the polydispersity was 1.02. The molecular weight of the 5TF-Co-MS synthesized was 1024 g/mol (Mn) and 1100 g/mol (Mw) and the polydispersity was 1.07. The polymers and the copolymer nanocomposites were characterized by FTIR and Raman techniques respectively. The IR spectrum (Fig. 2a) of 3CT-PPF resin shows an intense strong band at 1712 cm−1 for stretching frequency of ester carbonyl group. The presence of peak at 3434 cm−1 reveals that the end groups are carboxyl groups. The peak around 2930 cm− 1 is due to stretching frequency of CH2. The mild peak for unsaturated double bonds (C_C) between carbon atoms of fumarate linkage was observed at 1644 cm−1 and 982 cm−1. Other pertinent peaks observed were methylene scissoring and methyl asymmetric bend in the 1455 cm− 1 region and C\O stretch at 1262–1297 cm− 1 and 1159 cm− 1. The spectrum further shows the presence of characteristic phosphate stretching bands at about 900– 1200 cm−1 and phosphate bending at 562 cm−1 and 603 cm− 1 [23] of HA. The presence of the carbonate bands at 864 cm− 1 and 1412 cm − 1 in all the spectra is due to the presence of carbonate ions in the HA [23]. Similar IR spectral responses were also observed with 5CT-PPF resin. The IR spectrum of 3TF-Co-MS resin shows a band at 1697 cm−1 for stretching frequency of ester carbonyl group and an intense broad band for hydroxyl group of alcohols at 3734 cm− 1. The peak around 2910 cm−1 for the stretching frequency of CH2 has also been observed. The spectrum further shows the presence of characteristic phosphate stretching bands at about 900–1200 cm−1 and phosphate bending at 562 cm− 1 and 603 cm− 1 [22] of HA. Similar IR spectral responses were also observed with 5TF-Co-MS resin. The IR spectrum (Fig. 2b) of copolymer nanocomposites CNV3 and CMA3 shows characteristic peaks at 1644 cm− 1 for unsaturated double bonds (C_C) between carbon atoms of fumarate linkage and 3450 cm−1 for carboxyl end groups. The reduction of peak intensity of the peak at 1644 cm−1 indicates the cross linking with monomer. The unsaturated polyesters, CT-PPF and TF-Co-MS undergo crosslinking with the monomer NVP and MMA through their unsaturated double bonds in the presence of initiator and accelerator by a free radical mechanism to form CNV3 and CMA3 respectively. The FTIR analyses confirm the successful crosslinking in copolymer nanocomposites. Similar IR spectral results have been obtained with the CNV5 and CMA5 copolymer nanocomposites. The parent polymeric resins are acidic in nature, owing to which the crosslinking is fast with high setting temperatures and short setting times. The setting time and temperatures of the exothermic process during crosslinking were 120 s, 52 °C; 300 s, 50 °C; 130 s, 53 °C and 320 s, 52 °C for the CNV3, CMA3, CNV5 and CMA5 copolymer nanocomposites respectively. The structures of these polymers and copolymer nanocomposites were further complimented by Raman studies. The Raman spectra of the polymers 3CT-PPF and 3TF-Co-MS and the copolymer nanocomposites CNV3 and CMA3 are represented in Fig. 3a. The strongest Raman active phosphate mode appears in the spectrum at 970 cm−1 for hydroxyapatite (HA) as evident in all the spectra. The additional bands at 1730 cm−1, 1450 cm−1and 2950 cm−1 have been attributed to the presence of ester groups and CH2 and CH3 stretching vibrations respectively. The peaks at 1375 cm− 1 and 3100 cm−1 observed in the spectra of CT-PPF and TF-Co-MS stand for C_C bending and stretching vibrations respectively. These bands are not observed in CNV3 and CMA3 since these copolymer nanocomposites have no C_C moiety in them. Similar results have been obtained for the CNV5 and CMA5 copolymer nanocomposites. The distribution

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Fig. 3. Raman spectra of 3TF-Co-MS, 3CT-PPF, CNV3 and CMA3 samples (a) and Raman spectral imaging of the individual molecular constituents in the nanocomposite CMA3 (b) (red represents the range 1730–1750 cm−1 of the ester groups and blue represents the peak 960 cm−1 of the phosphate group of HA). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

among the different constituents of the CMA3 copolymer composite was visualized by Raman spectral imaging technique (Fig. 3b). The image clearly reveals a uniform homogenous distribution of the individual molecular constituents in the nanocomposites (red represents the

range 1730–1750 cm− 1 of the ester groups and blue represents the peak 960 cm− 1 of the phosphate group of HA). Thus it can be seen that the HA nanoparticles have been uniformly distributed in the copolymer nanocomposites. This homogenous distribution of HA

Fig. 4. SEM image of as prepared copolymer nanocomposite surface of CNV3 (a), CMA3 (b), along with fracture morphology of CNV3 (c) and CMA3 (d).

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nanoparticles is very important as fine HA particles tend to agglomerate together and could be detrimental from a mechanical point of view. The SEM image of as prepared copolymer nanocomposite surface and the fracture morphology of the copolymer nanocomposites CNV3 and CMA3 are given in Fig. 4a and b and c and d respectively. Fig. 4a and b indicates that good dispersion of the HA in the polymer matrix has been observed. It further showed good interfacial bonding between the polymer and the HA nanoparticles with no cracks or voids observed in the interface of the two phases. The fracture morphology of the copolymer nanocomposites given in Fig. 4c and d reveals that the interphases of the copolymer nanocomposites undergo dimple fracture under low temperature stress. The debonding of the ceramic phase from the polymer matrix with the appearance of microcracks and nanopores leads to dimple fracture (Fig. 4d) at the interphase of the copolymer nanocomposites. The serrated structure of the copolymer composite (Fig. 4d) reflects the higher mechanical strength of the composite CMA3 [24]. The mechanical properties and contact angles of the copolymer nanocomposites are given in Table 1. The tensile strength and compressive strength of the copolymer nanocomposites increased with an increase for filler. Many factors contribute to the variation in mechanical properties of the copolymer nanocomposites, including size and shape of ceramic nanoparticles, ceramic/polymer phase composition, chemical interactions, and inherent properties of the polymer matrix [25,26]. The HA nanoparticles with a rod like shape were better suited to withstand the shear stress and to offer appreciable mechanical stability by forming a better mechanical interlock with the polymer. The data in Table 1 clearly demonstrate a progressive physical change from a weak material for CNV3 and CNV5 crosslinked with NVP to a stiffer material for CMA3 and CMA5 crosslinked with MMA. The higher mechanical properties observed with MMA based nanocomposites is attributed to the mobility and reorientation of needle like HA nanoparticles (30 nm) in one direction giving anisotropic nature to the composite during heat treatment at 140 °C. The mobility and reorientation of HA nanopaticles with MMA based nanocomposites is due to the lesser degree of crosslinking in comparison with that of NVP. The copolymer nanocomposites CMA3 and CMA5 demonstrated ultimate compressive strength similar to that of decellularized cancellous bone of femur head of porcine model [27]. To elucidate the surface properties of the copolymer nanocomposites contact angle measurements were carried out at neutral pH. Contact angle gives a clear picture of the hydrophilic/hydrophobic nature of the prepared materials. Optimal water contact angle required for materials used as bone regeneration biomaterials is about 50–70°. It has been reported that osteoblasts respond largely on optimum hydrophilicity [28]. The copolymer nanocomposites CMA3 and CMA5 exhibit optimum hydrophilicity, which is favorable to promote osteoblast growth and adhesion. The biodegradation of the copolymer nanocomposites in PBS and Ringer solution is depicted in Table 2. The degradation of the present nanocomposites is attributed to ester hydrolysis, with the generation of fragments to their respective monomeric carboxylic acids and diols. Aliphatic polyesters undergo bulk degradation, where material is lost from the entire polymer volume at the same time due to water penetrating the bulk. Therefore, the rate of degradation of these polymers depends on the extent of water accessibility to the matrix rather than

the intrinsic rate of ester cleavage [29]. The water accessibility to the matrix depends on several factors such as the polymer hydrophobicity and hydrophilicity, polymer crystallinity, and the dimensions of the sample [30]. In vitro demonstration of biodegradation requires experimental conditions mimicking the physiological characteristics of the living media. Thus, PBS (pH 7.4) and Ringer solution used as in vitro media, provide iso-osmolarity and aids in neutralizing the generated carboxyl groups. The degradation rate of the crosslinked copolymer nanocomposites were measured in terms of the weight loss over the period of exposure (Table 2). It was observed that the degradation and weight loss of CNV3 and CNV5 crosslinked with NVP in Ringer solution is lesser than that of MMA (CMA3 and CMA5) as reported elsewhere [31]. The lesser degree of degradation of CNV3 and CNV5 could also be due to the higher degree of crosslinking with NVP. Moreover, the degradation rate was found to be more in Ringer solution than in PBS, which has been explained by the synergistic buffering effect of the filler HA, and the PBS medium [32]. The higher weight loss observed in Ringer solution was due to the combined degradation of the composite and dissolution of HA from the composite at low pH since HA is not thermodynamically stable in acidic pH. It is also worth mentioning that the degradation decreases with an increase for filler. The filler HA helps in partially neutralizing the biodegraded acidic fragments of the polymer. These acid products can act as catalyst and accelerate the hydrolysis reaction. The hydroxyl groups in HA then reacts with the acids leading to inhibition of acid catalysis, which in turn reduces the biodegradation [32]. The result indicated that the mass loss of the nanocomposites was primarily due to the degradation of the polymers and that HA particles did not leach out from the network in noticeable amounts during degradation. The nanocomposites CMA3 and CMA5 maintained their dimensional integrity in spite of weight loss throughout the 12 weeks of immersion. It has also been reported that carbonated calcium phosphates are able to compensate acidity produced and buffer them within physiological pH range [33]. The pH change observed in the solutions after immersion could be a combination of acidic degradation byproducts resulting from the copolymers and any neutralization effects resulting from the HA filler. It has been reported that a drastic decrease in pH induces inflammation and subsequent necrosis due to the acid induced autocatalytic degradation [34]. However, in the present study the copolymer nanocomposites (Table 2) depict only a gradual decrease in pH drop, which can be effectively neutralized by the blood buffers in vivo. The tensile strength of CMA3 and CMA5 after immersion in PBS for 28 days is 11.3 ± 1.8 and 16.2 ± 2.9 MPa respectively. However, the values obtained are still within permissible levels to meet minimum demands during healing and restoration of the defect. The CMA3 and CMA5 copolymer nanocomposites with higher mechanical properties, optimal hydrophilicity, and degradation characteristics have been selected for further biological evaluation. The ability of bone bonding of the copolymer nanocomposites CMA3 and CMA5 has been evaluated through the development of an apatite layer in vitro, in SBF at physiological conditions. The formation of an apatite layer that has a Ca/P ratio higher than 1.4 promotes direct bone bonding [35]. The bioactivity of both the samples characterized by apatite formation in SBF in-vitro reveals that they were bioactive in nature (CMA3 copolymer nanocomposite as an example is represented in Fig. 5a–d). The

Table. 1 Mechanical and physical properties of the as prepared nanocomposites. Sample

Hardness (shore D)

Compressive strength (MPa)

Tensile strength (MPa)

Young's modulus (tensile) (MPa)

Contact angle (theta) Advancing

Receding

CNV3 CMA3 CNV5 CMA5

36 44 39 46

8.73 22.43 15.46 34.99

9.86 20.3 14.3 26.8

149.7 525.3 247.6 691.3

43 60 49 65

52 55 54 55

± ± ± ±

2 1.8 3.1 2.3

± ± ± ±

0.73 2.27 0.98 2.21

± ± ± ±

1.24 1.42 2.56 2.98

± ± ± ±

78.6 83.5 153.4 115.2

± ± ± ±

8.2 7.9 9.4 8.1

± ± ± ±

10.3 6.7 7.6 7.9

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157

Table. 2 Gravimetric dry weight and pH changes after immersion of nanocomposites in different media. Medium PBS

Ringer

PBS

Ringer

Immersion period (days)

CNV3

CMA3

7 28 50 7 28 50

Weight loss (%) 6.2 ± 0.3 12.5 ± 0.3 16.3 ± 0.3 8.6 ± 0.9 10.0 ± 0.7 19.0 ± 0.2

8.3 14.4 22.5 10.8 12.9 24.6

± ± ± ± ± ±

0.1 0.3 0.5 0.1 0.8 0.1

4.4 10.2 14.3 6.1 9.7 17.2

± ± ± ± ± ±

0.2 0.3 0.2 0.1 0.9 0.2

3.6 9.2 12.1 8.23 11.5 19.6

± ± ± ± ± ±

0.2 0.4 0.01 0.1 0.5 0.4

7 28 50 7 28 50

pH 7.3 7.2 6.9 5.9 4.9 4.3

7.4 7.5 7.3 6.2 6.1 5.5

± ± ± ± ± ±

0.1 0.5 0.1 0.2 0.1 0.2

7.2 7.3 7.0 6.4 5.5 4.9

± ± ± ± ± ±

0.2 0.1 0.2 0.1 0.5 0.1

7.3 7.1 7.2 6.5 6.3 5.8

± ± ± ± ± ±

0.2 0.1 0.2 0.2 0.1 0.2

± ± ± ± ± ±

0.3 0.3 0.3 0.1 0.9 0.2

morphology and composition of the CMA3 nanocomposites were analyzed over time after immersion in SBF. The AFM micrographs (Fig.5a and c) depict the surface of the CMA3 nanocomposite after immersion in SBF. Fig. 5a and c represents the nanocomposite immersed for 2 h and two weeks respectively. It has been observed that globular crystals called spherulites appeared on the surface of the CMA3 sample after a two day immersion in SBF. These apatite spherulites increased in both number and size with an increase in immersion time as indicated in Fig. 5c which corresponds to immersion for 2 weeks [36]. The morphological evaluation of the apatite layer grows from a rough dune like surface to the smooth hillocks indicating a polynuclear growth process, during which the new nuclei start rapidly before the

CNV5

CMA5

proceeding layer growth is completed [36]. The EDAX pattern of the CMA3 copolymer nanocomposites soaked in SBF after 2 h and two weeks is shown in Fig. 5b and d respectively. The EDAX spectra indicated that the globules were mainly composed of calcium and phosphorous which were distinctive components of the apatite phase. There has been a relative increase in intensity of Ca and P peaks for the 2 week sample when compared to the two day sample. The apatite layer formed in SBF has been found to be stable and it enhances fast bone bonding at the interface. Bone tissue may accept it more easily without a foreign body response because of its similarity to bone apatite [37]. It has also been established that a bone-like apatite layer plays an important role in establishing the bone-bonding interface between biomaterials and living tissue [38].

Fig. 5. AFM micrograph (a) and EDAX pattern (b) of CMA3 after immersion in SBF for 2 h. AFM micrograph (c) and EDAX pattern (d) of CMA3 after immersion in SBF for 2 weeks respectively.

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Blood cell aggregation is a highly undesirable phenomenon that can induce serious circulatory disorders, even lethal toxicity. The blood compatibility of the copolymer nanocomposites CMA3 and CMA5 was confirmed in vitro by the results of cell aggregation and hemolytic activity. Negligible aggregation was detected in RBC incubated with the copolymer nanocomposites signifying the nontoxic nature of the prepared nanocomposites on normal RBCs. The micrographs of RBCs upon interaction with the nanocomposites along with normal saline and PEI as controls are shown in Fig. 6. It was observed that the nanocomposites did not promote aggregation like the positive control PEI and the images obtained were comparable to normal saline. Several images were taken for each of the samples and there was no evidence of rouleaux formation. Similarly, the nanocomposites gave no significant hemolysis. The hemolysis (%) values of CMA3 and CMA5 copolymer nanocomposites are 0.7 ± 0.4 and 0.98 ± 0.14 respectively. The results indicate that when implanted in vivo they may display blood compatibility. Toxicity is the major issue that limits the practical clinical applicability of various materials and biocompatibility studies are critical for analyzing the actual toxicity of the engineered materials. Considering this, we tested the toxicity of the CMA3 and CMA5 copolymer nanocomposites on fibroblast cell line (L929) using the MTT [39] and NR assays. The results obtained in the assay reveal that the copolymer nanocomposites offer good viability to the proliferating L929 fibroblasts. The CMA3 and CMA5 copolymer nanocomposites exhibit viability of over 80% after incubation for 72 h with MTT, which are comparable with NR assay. The negative and positive control cells had 100 and 0% viabilities respectively. Upon contact with the copolymer nanocomposites there is no significant change in the morphology of cells observed after growing the L929 cells beneath the CMA3 copolymer composite (Fig. 7b) with respect to the control (Fig.7a). Similar results were obtained for the CMA5 copolymer nanocomposites. These studies thus proved that the degradation products and the particles leaching out from the copolymer

nanocomposites CMA3 and CMA5 are nontoxic to cell viability indicating compatibility for in vivo applications. The live dead staining images of the L929 cells seeded onto the CMA3 and CMA5 copolymer nanocomposites indicated the presence of abundant healthy cells, which was revealed by the green fluorescence of acridine orange. Almost all the fibroblasts seeded on the copolymer nanocomposite CMA3 (Fig. 7d) displayed a normal green fluorescence similar to the control (Fig. 7c) showing that these cells were healthy and viable with no evidence of apoptotic nuclei. Different approaches are under way in the fast evolving panorama of bone tissue engineering and several attempts have been reported to overcome shortcomings and to design composite materials with optimum properties. The data obtained in the present study depict that the copolymer nanocomposites CMA3 and CMA5 possess many desirable attributes like optimum hydrophilicity, gradual degradation, good mechanical properties, low cytotoxicity and good blood compatibility. In addition, they further demonstrated efficient bioactivity. Overall the results obtained indicate that the copolymer nanocomposites CMA3 and CMA5 may suit the requirement for bone applications. 5. Conclusions In the present study, in situ polymerized biodegradable copolyesters with hydroxyapatite (HA) and its nanocomposites were prepared to demonstrate their feasibility for bone applications. The structure of the nanocomposites was characterized by Fourier transform infrared spectroscopy and confirmed by Raman techniques. Raman spectral imaging clearly reveals a uniform homogenous distribution of the individual molecular constituents in the copolymer nanocomposites. Bright field TEM images presented distinct, homogenous, high aspect ratio needle like hydroxyapatite particles. The serrated morphology of the copolymer nanocomposites showed good dispersion of the HA nanoparticles in the polymer matrix. It has been observed that a stiffer and compact

Fig. 6. Micrographs of RBC cells with positive control (a) and negative control (b) and after incubation with CMA3 (c) and CMA5 (d) respectively.

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Fig. 7. Cell adhesion of control (a), test sample (b), live/dead assay of control (c), and test sample (d) of L929 cells seeded on CMA3 nanocomposites respectively after 5 days. The images were taken under 20× magnification.

thermoset was formed when crosslinked with MMA when compared to NVP. This difference in morphology reflects in the difference in the observed mechanical properties. It was observed that the in-vitro degradation of the copolymer crosslinked with NVP is slower than that of MMA and the degradation rate decreases with an increase in the amount of the HA filler. The copolymer nanocomposites exhibited that good compatibility, bioactivity, and the cytotoxicity evaluation by MTT assay revealed no apparent toxicity. The results led us to comprehend that these designed copolymer nanocomposites could eventually be applicable for intramedullary rod like bone applications.

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