Evaluation of Polyether sulfone/nanohydroxyapatite nanofiber
Transcription
Evaluation of Polyether sulfone/nanohydroxyapatite nanofiber
Dharmalingam Sangeetha J.Biosci Tech,Vol 6(1),2015,607-619 ISSN: 0976-0172 Journal of Bioscience And Technology www.jbstonline.com Evaluation of Polyether sulfone/nanohydroxyapatite nanofiber composite as bone graft materials Kalambettu Aravind and Dharmalingam Sangeetha* Department of Mechanical Engineering, Anna University, Sardar Patel Road, Chennai 600 025, Tamil Nadu, India. *E-mail [email protected], ABSTRACT Nanofiber mats of polyether sulfone/nano hydroxyapatite (PES/nHA) were prepared by electrospinning technique. The fabricated composites were characterized using FTIR, XRD and SEM. The composite nanofiber mats were subjected to in vitro biological studies namely bioactivity, haemocompatibility and cytocompatibility. The XRD analysis showed an increase in the amorphous nature of the composite with the addition of nHA (filler) suggesting an interaction between the polymer matrix and the filler. The bioactivity studies performed using Simulated Body Fluid (SBF) showed that the bioactivity was observed to be higher in the composites containing nHA. It was observed that the amount of protein (Bovine Serum Albumin) adsorption as well as the blood coagulation decreased with the addition of the filler content which improved in turn the cell adhesion and proliferation of osteoblast (MG-63) cells. The in vivo studies, performed on a pilot scale by implantation of the PES/nHA in the tibia of rabbits, suggested an intense inflammatory host response. Hence from the study, it was concluded that although PES/nHA showed promising in vitro properties, their unfavuorable in vivo response meant that these composites need to be analysed further to determine the reason behind that inflammatory response and the means of overcoming it. 1. INTRODUCTION Total hip arthroplasty (THA) is a commonplace procedure, though for younger patients there is a need to develop new materials that can extend the life of the artificial joint beyond 20 years. Dramatic advances in the field of cell and molecular biology, genetics, tissue engineering and material science have given rise to the remarkable new cross disciplinary field of tissue engineering which uses synthetic or naturally derived, engineered biomaterials to replace damaged or defective tissues such as bone, skin, and even organs. A potential material for use as a scaffold in tissue engineering must fulfill a number of necessities including biocompatibility, cytocompatibility and biodegradation to nontoxic products within the time frame required for the application, processability to complicated shapes with appropriate porosity, ability to support cell growth and KEY WORDS: Nano fiber composite; Haemocompatibility; Osteoblast; Bioactivity; In vivo. proliferation, along with appropriate mechanical properties, as well as maintaining mechanical strength during most part of the tissue regeneration process [1]. When developing new biomaterials for bone regeneration, surface properties must be modulated, ideally in order to mimic the tissue to be replaced. Furthermore, a strong bonding between the host bone and the osteoconductive surface of the implant is required [2]. Among the several methods available for designing materials for tissue engineering, electrospun fibers have garnered special interest over the recent years in different areas of research and more particularly, in the field of tissue engineering as suitable materials for wound dressings, tissue scaffolds and drug-delivery systems [3-6]. Nanofiber composite, obtained by the electrospinning process, offers a large surface area to weight ratio which would 607 Dharmalingam Sangeetha J.Biosci Tech,Vol 6(1),2015,607-619 help in the migration of cells as well as aid cell growth. The polymer nanofiber composites could be made from biocompatible and biodegradable polymers which could have potential applications in the replacement of structurally or physiologically deficient tissues and organs in humans. The use of nanofibers in tissue restoration is expected to result in an efficient and rapid recovery process of the organ owing to the large surface area offered by nanofibers made from polymer. Polymeric nanofibers have been successfully used for the epithelialization of implants and the construction of biocompatible prostheses, cosmetics, face masks, bone substitutes, artificial blood vessels, valves and drug delivery applications [7]. Most commonly used devices have a bearing surface of ultrahigh molecular weight polyethylene (UHMWPE) which has an unsolved problem such as a lack of cytocompatibility properties for promoting bone cell growth [8]. Sulfur containing aromatic polymers have good chemical stability and have been used in medicine for various applications like hemodialysis membranes, scaffolds for bio mineralization, load bearing implants for fixation in bone, etc [9-12]. The presence of sulfur creates a stable bond between the polymer and nano hydroxyapatite [9, 10]. This holds many advantages over conventional UHMWPE/hydroxyapatite composites where leaching of the apatite and polymer particle are a matter of concern. Contact of blood with artificial surfaces during extracorporeal circulation procedures is associated with activation of blood cells as well as plasma proteolytic enzyme systems, such as the complement, coagulation, fibrinolytic and FXII–kallikrein–kinin cascades. Numerous attempts have been made to solve these problems by modifying the surface chemistry of blood contacting materials in order to make them more ISSN: 0976-0172 Journal of Bioscience And Technology www.jbstonline.com thrombo resistant. High haemocompatibility is essential for medical devices such as catheters and cardiovascular implants that are in contact with blood in clinical use. At present, one of the most serious challenges is surface-induced thrombus formation immediately after implantation of such a device within the living system. Much research has been focused on this problem using various polymer materials or surface modification methods [13]. Cell adhesion is the most important aspect of cell interaction with a biomaterial because it is the prerequisite for further cellular activity such as spreading, proliferation and differentiation. Initial osteoblast material interactions may be conveniently characterized by four stages: (i) protein adsorption to the surface, (ii) contact of rounded cells, (iii) attachment of cells to the substrate, and (iv) spreading of cells. Initial cell attachment is generally influenced by the original surface characteristics of the materials [7]. Hydrophobic properties of the polymer are not favorable to direct cellular adhesion and further population of cells [1415]. The introduction of bioceramics such as nHA within the PES nanofiber is considered to improve the hydrophilicity and cellular affinity and thus to better allow its use as tissue regenerative matrices and to promote adhesions and proliferation of osteoblast (bone-forming) cells. Hydroxyapatite (HA), has been extensively investigated due to its excellent biocompatibility, bioactivity and osteoconductivity as well as its similarities to the main mineral component of bone. However, the poor compressive strength and fatigue failure limits its applicability to the low or non-load bearing sites in human body [16-17]. Amongst the various polymers employed for medical applications, polyethersulfone (PES) has been used for separation and filtration purposes. However, one of the drawbacks of 608 Dharmalingam Sangeetha J.Biosci Tech,Vol 6(1),2015,607-619 the PES membrane is its low blood compatibility which results in the adsorption of proteins in the blood onto the PES membrane surface and thus forming a protein layer [18]. Prihandana et al [18] demonstrated the improved the antithrobigenicity of PES by coating it with fluoridated diamond like carbon films. Ardeshirylajimi et al [19] fabricated PES nanofiber scaffolds and studied the differentiation of pluripotent stem cells into osteoblastic lineage. The present study i s focused on the preparation of nanofibers of sulfur containing aromatic polymer, PES and its composite using electrospinning having a bioceramic, nHA as the filler. The prepared nanofiber composites were characterized using X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). The nanofiber composites were then subjected to in vitro studies to evaluate their bioactivity, protein adsorption, haemocompatibility and cytocompatibility properties inorder to test its sustability for tissue engineering application. Subsequently there in vivo performance was evaluated by implanting them in the tibia of rabbits. 2. EXPERIMENTAL 2.1 MATERIALS AND METHODS PES (Ultrason E6020P) and HA nano powder (CAS 12167-74-7) were procured from BASF, Germany and Sigma Aldrich, USA respectively. Dimethyl formamide (DMF) was used as a solvent, and was obtained from SRL Pvt. Ltd, India. The composition of the dope solution used for this study was 2/0.5/7.5 of PES/nHA/DMF. The dope solution was dissolving PES in DMF and was incorporated into it quantities. The whole content prepared by the filler nHA in calculated was kept under ISSN: 0976-0172 Journal of Bioscience And Technology www.jbstonline.com magnetic stirring overnight and then subjected to ultrasonication for 30 min prior to the start of electrospinning process in order to disperse the nHA uniformly in the solution. In a typical electrospinning process, t h e p olymer solution with a specific concentration of nHA was loaded onto a 2 ml syringe which was linked to a high voltage power supply with a capacity to generate high voltage of up to 50 kV. The flow rate of the syringe pump was regulated using the PICO Espin 2.0 version software. The electrospinning was performed with an electric voltage supplied at 20 kV at a needle tip to collector distance of 15 cm. The flow rate was adjusted to 0.3 ml/h and the collecting drum was regulated to rotate at a speed of 2000 rpm. From amongst the prepared PES/nHA nanofiber composite with different concentrations of nHA, the PES nanofiber composite with 5 wt% of nHA showed the best fiber formation. Hence PES/nHA nanofiber composite mat with 5 wt% of nHA alone was considered for further studies. 2.2 Characterization Studies 2.2.1 XRD The phase analysis of the nanofiber composite samples were done by XRD using 35 mA, and 40 kV current, with a monochromatic CuKα radiation (λ=1.5405˚A) with a step size of 0.04 º 2θ, a scan rate of 0.02º 2θ/s, and a scan range from 2θ = 10 to 60°. 2.2.2 FTIR The functional groups present in the polymer and the interaction between the polymer/nHA nanofiber composites were analyzed by using Alpha T Bruker Optics FTIR spectrophotometer. The spectra obtained were recorded in transmission mode within the scanning range of 4000–500cm-1. 609 Dharmalingam Sangeetha J.Biosci Tech,Vol 6(1),2015,607-619 ISSN: 0976-0172 Journal of Bioscience And Technology www.jbstonline.com 2.2.3 SEM 2.5 Blood coagulation The morphology and dispersion of particles in the polymer matrix were observed using HITACHI S-3400 model SEM. The surface of the materials was sputter coated with gold before being subjected to SEM in order to make them electro conductive. Blood coagulation tests were carried out with PES and its nanofiber composite individually using blood samples collected from healthy individuals. Buffered citrate was added to the blood samples which functioned as an anticoagulant agent. The samples were separately immersed in the blood contained in the test tubes and left undisturbed for 2 h. The morphology of the cells before and after the treatment with the samples was analyzed using Light microscope (Labomed, LX-400). 2.3 Bioactivity Simulated body fluid (SBF) was prepared in laboratory according to procedure developed by Kokubo [2, 9]. In vitro tests were carried out to assess the bioactivity of the prepared composite samples. The samples were immersed in SBF solution for 30 days and maintained at 37 ºC. The samples were retrieved from SBF solution, dried and then their surface was analyzed by SEM to observe the growth of hydroxyapatite. 2.4 Protein adsorption The protein adsorption experiments were made with Bovine Serum Albumin (BSA) solutions. The known concentration of BSA was prepared in phosphate buffered saline (PBS, pH=7.4). The nanofiber composite with an area of 1 cm2 was incubated in distilled water for 24 h, washed 3 times with PBS solution, and then immersed in the protein solution for 2 h. After protein adsorption, the membranes were carefully rinsed 3 times with PBS solution and then rinsed with distilled water. The adsorbed proteins were quantitatively eluted with 1.0 ml of 2% SDS solution for 6 h. The amount of protein in the sodium dodecyl sulfate (SDS) solution was quantified by protein analysis (Micro BCA protein assay reagent kit) [20]. 2.6 MTT Assay Cytotoxicity studies using the nanofiber composite were analyzed in 96 well plates using normal and osteoblast cell lines (MG63) by MTT assay. MG-63 cell lines were cultured using Dulbecco’s modified Eagle’s medium (Himedia) supplemented with 5% fetal bovine serum (FBS) and 1% penicillinstreptomycin and then seeded into the 96 well plate. The wells were sterilized with 70% ethanol followed by UV treatment for 4 h and were neutralized with phosphate buffer (pH 7). The wells without the polymer samples were the control groups for the experiment. The MG-63 cell lines were seeded at a density of 6-7x103 cells per well and incubated at 37 °C in a humidified atmosphere containing 5% CO2. In all culture conditions, the medium was renewed every 24 h. After 3 days of incubation, the supernatant of each well was removed and washed with PBS. MTT, diluted in serumfree medium, was added to each well and the plates incubated at 37 °C for 3 h. After aspirating the MTT solution, acidified isopropanol (0.04N HCl in isopropanol) was added to each well and pipetted up and down to dissolve the dark blue formazan crystals and then left at room temperature for a few minutes to ensure the dissolution 610 Dharmalingam Sangeetha J.Biosci Tech,Vol 6(1),2015,607-619 of all crystals. Finally, the absorbance was measured at 570 nm using an ELISA reader. Each experiment was performed at least three times for reproducibility. 2.7 In vivo studies PES/nHA (5 wt%) were implanted in the tibia of rabbits and the type of host response was studied histologically. Institutional Animal Ethical Committee (IAEC) approval (Approval number: IAEC/ XXX/SRU/ 225/2012) was obtained at Sri Ramachandra University, Chennai, prior to performing the in vivo experiments. Three healthy male New Zealand white rabbits, aged about 16 weeks and weighing between 1.8 and 2 kg were selected for the study. The PES/nHA composite samples were autoclaved prior to implantation. 2.7.1 Preparation of the animals The medial side of rabbit tibial proximal epiphysis was chosen as the site of implantation since it had the least muscle attachments and was considered to be anatomically favourable. For this purpose, the fur around the proposed site of surgery was removed and the site cleaned using povidoneiodine solution. 2.7.2 Anaesthesia protocol The three rabbits were anaesthetised using a combination of diazepam (5 mg/kg) and ketamine (60 mg/kg) i.m. for induction and maintained using Isoflurane until the end of the surgical procedure. 2.7.3 Surgical procedure Once the rabbits were sufficiently anaesthetised, the surgical site was exposed by placing an incision on the skin at the medial side of the tibia and deflecting the underlying fascia. A carbide fissure bur fixed to a dental handpiece was used to drill two holes in the bone with each hole measuring ISSN: 0976-0172 Journal of Bioscience And Technology www.jbstonline.com about 5 mm wide and depth until the marrow was reached. The two holes were placed in a straight line and 1 cm apart. The PES/nHA composite samples were cut into tiny pieces (approximately square shaped of about 1 mm2) using a pair of sterile scissors. The graft material was then slightly wet with saline and then implanted into the first defect in as much quantity so as to slightly overfill the first defect. In the second defect of all the three rabbits, a biphasic calcium phosphate commercial bone graft material (DM bone, MetaBiomed, Korea) was filled. Subsequently, the soft tissues overlying the bone were sutured using catgut sutures and the skin was sutured using silk suture threads. The surgical site was then protected by a bandage. The animals were then revived and kept under observation. Analgesia was ensured by administering Ketorolac (Ketorol, Dr Reddy Laboratories, India) injections i.m. for 10 days. Antibiotic coverage was provided for 7 days post operatively through cefalexin (15 mg/kg SC). The animals were then maintained for 8 weeks and then euthanised by giving an overdose of ketamine i.v. Subsequently, the right tibia of each animal was harvested and fixed in 10% neutral buffered formalin, decalcified in 10% formic acid, dehydrated in series of graded alcohol and embedded in paraffin. From these samples, 3 – 4 µm thick vertical serial slices were prepared using a microtome and surface staining was performed with haematoxylin and eosin (H&E). Some of the samples were not H&E stained and used for SEM analysis to study the cell spreading. The H & E stained bone sections were examined under Optika Trinocular fluorescence microscope and evaluated for the following parameters 1. Detection of inflammatory cells 2. Detection of type of healing 3. Detection of osteoblasts/ osteoclasts both around as well as on the surface of implant. 611 Dharmalin ngam Sangeethaa J.Biosci Tecch,Vol 6(1),201 15,607-619 ISSN:: 0976-0172 Jourrnal of Biosciennce And Technnology www.jbbstonline.com 612 Dharmalin ngam Sangeethaa J.Biosci Tecch,Vol 6(1),201 15,607-619 ISSN:: 0976-0172 Jourrnal of Biosciennce And Technnology www.jbbstonline.com 613 Dharmalin ngam Sangeethaa J.Biosci Tecch,Vol 6(1),201 15,607-619 ISSN:: 0976-0172 Jourrnal of Biosciennce And Technnology www.jbbstonline.com 614 Dharmalin ngam Sangeethaa J.Biosci Tecch,Vol 6(1),201 15,607-619 ISSN:: 0976-0172 Jourrnal of Biosciennce And Technnology www.jbbstonline.com 3. RESU ULTS AND DISCUSSIION 3.1 XRD D The XRD D is a verssatile techniique to stud dy the grow wth of any crystalline c phase on oth her amorphous or crysttalline matrrix. Hence in the preseent study, the t capabilitty of PES to facilitate the grow wth of HA on it was w X The XRD patteern investigaated using XRD. of electrrospun PES S nanofiber is shown in Figure 1a, 1 which co onfirmed th he amorphou us nature of PES. The XRD D pattern of electrosp pun PES placced in SBF for 30 days is shown in n Figure 1b b. From the Figure, th he evidence for the gro owth of HA was noted by b the appeearance of a peak closee to 32° (2θθ). Hence th he capability y of PES to permit p grow wth of HA on o its surfaace was verrified. As th he intensity of the refllection due to HA was w found to be very low w, the grow wth of HA on o PES/nHA A nanofibeer compositte was also studied using the same SBF. The XR RD patterns of PES/nH HA nanofib ber composiite and of th he same mateerial placed in SBF for 30 3 days are represeented in Figgure 1c annd 1d respecctively. Preesence of H HA phase was evidennt in PES/nnHA compossite nanofibeer by the chharacteristic reflections at 26°, 32°,, 40°, 47°, 550°, 53° andd 64° (2θ). IIn Figure 1dd, the intenssity of thee reflectionns due to HA increaased, thus prroving the ggrowth of H HA on the pprevious nnHA already presentt in PES/nnHA compoosite nanofi fiber. Hencee the study confirmed tthat the inheerent properrty of F was nHA ttiny crystalss to grow furrther in SBF not aaffected byy PES. T Though PES S is hydropphobic, thee growth oof nHA att the interfaace was not suppresssed. This sstudy confirrmed that either PES S or PES//nHA nanofi fiber compossite could bee used as a m matrix for thee growth of nHA in SBF F. (Fig. 1) 3.2 FT TIR The fformation oof apatite layer over the nanofi fiber was cconfirmed by ATR-F FTIR spectrroscopy. A ATR-FTIR sspectra of PES nanofi fiber and its nanofiberr compositee are shownn in Figure 2. All thhe spectra sshow 615 Dharmalingam Sangeetha J.Biosci Tech,Vol 6(1),2015,607-619 their strongest band in the spectrum region below 1800 cm-1. The peaks observed at 1125 and 1242 cm-1 correspond to diaryl sulfone (Ar-SO2-Ar) and aryl ether (Ar-OAr) groups respectively, which is the back bone of PES [21]. The vibration of aromatic C=C group that occur at 1586 cm-1 belong to benzene ring. The broad band at about 2800 cm-1 correspond to the absorbed hydrate and the sharp medium and short peaks at 632 and 3570 cm-1 corresponds to stretching vibrations of lattice OH- ions of hydroxyapatite. And also the peaks at 632 and 3570 cm-1 are the characteristic bands for stoichiometric nHA. The symmetric P-O (PO4-3 ion) stretching mode for nHA occurs at 995 cm-1 which indicates typical nHA structure (Figure 2d). From the investigation, it is proved that the nanofiber composite soaked in SBF after nHA addition are able to induce apatite nucleation. (Fig. 2) 3.3 SEM The morphology of PES and its nanofibers composite are shown in the SEM image in Figure 3 (a, b). The average diameter of the obtained fiber between 150-480 nm offers more surface area to weight ratio that helps the deposition and growth of apatite and provides huge surface area for the proliferation of bone forming cells (Osteoblast) and also improves the angiogenesis for blood vessels to penetrate through the nanofiber [22]. Figure 3b shows the PES nanofiber with adhereded nHA that acted as stimuli for the growth of apatite when immersed in SBF. (Fig. 3) 3.4 Bioactivity The bioactivity of the nanofiber mats of polymer and the composite was observed from the SEM images (Figure 4), for evidence of apatite formation after 30 days ISSN: 0976-0172 Journal of Bioscience And Technology www.jbstonline.com of immersion in a metastable calcium phosphate solution (SBF). From the earlier studies [23] it was proved that the formation of apatite layer on polymer/nHA composite was due to the presence of nHA which was used as a stimulus and has the ability to induce the formation of a bone-like apatite layer over the nanofiber. Hence in the present study also, nHA was used to induce bioactivity. Figure 4b showed that the nHA, present in the nanofiber composite, acted as a nucleation site resulting in increased apatite formation over a period of time and is expected to not only promote the tissue growth adjacent to the implant site but also facilitate a strong bond between the tissue and the implant. It was observed that the PES nanofiber, after 30 days of immersion in SBF, showed evidence of apatite formation and from which it was inferred that, the minerals present in the SBF encouraged apatite formation albeit at very low level as shown in Figure 4a. Our results were in agreement with previous reports where nHA was used as a stimulus for apatite formation [24]. (Fig. 4) 3.5 Protein adsorption Protein adsorption on the material surface is a common phenomenon during thrombus formation upon contact of the biomaterial with blood. Thus, the amount of protein adsorbed on the PES nanofiber is considered to be one of the important factors in evaluating the haemocompatibility [20, 21]. Figure 5 shows adsorption of BSA on the nanofiber and its composite. From these observations it was found that the amount of protein adsorbed on the surface of apatite forming nanofiber composites was lower (Figure 5b and 5d) than that of the plain nanofiber (Figure 5a and 5c). Hydrophobicity of the polymer favors the adsorption of protein on the surface and may 616 Dharmalingam Sangeetha J.Biosci Tech,Vol 6(1),2015,607-619 lead to undesirable results, such as platelet adhesion, aggregation and coagulation [25]. The incorporation of nHA increased the hydrophilicity of the nanofiber mat thereby decreasing the protein adsorption, which was expected to improve its biocompatibility. (Fig.5) 3.6 Blood coagulation From the microscopic image in 6a, the floating of blood cells with spherical shape was observed which was due to the presence of anti-thrombogenic agent (buffered citrate) and is seen prior to contact with the nanofiber. The morphology of blood cells changed along with the formation of a clot when the polymer nanofiber c a m e in contact with blood. The microscopic image in figure 6b was taken after 2 h contact with PES nanofiber. This observation underlined the thrombogenic nature of bare PES nanofiber. Interestingly diminishing of blood coagulation with oval shaped blood cells were observed on apatite formed nanofiber composite (Figure 6c). The observed decrease in thrombogenicity could be attributed to the enhanced hydrophilicity of the nanofiber [26], which in turn resulted in decreasing adsorption of fibrinogen, which eventually lowered the clot formation [27]. The results revealed the fact that the formation of blood clot constantly decreased with addition of nHA in the feed mixture. The results may be explained on the basis of the fact that nHA increases hydrophilicity of the polymer and therefore, are not expected to induce any damage to blood cells or any change in the structure of the plasma proteins [28]. Hence, based on the above studies, it was demonstrated that the antithrombogenic property of the PES nanofiber was achieved with apatite formation and improved the haemocompatibility of the nanofiber. (Fig. 6) ISSN: 0976-0172 Journal of Bioscience And Technology www.jbstonline.com 3.7 Cell Morphology The morphology of osteoblast cells (MG-63) on the nanofiber composite was observed in vitro by SEM images after 5 and 10 days of seeding. It was observed that the bare nanofiber mat remained unchanged after seeding with bone forming cells as seen from the SEM images shown in figures 7a and 7b. SEM images in figures 7c and 7d shows the migration of osteoblast cell lines over the PES/nHA nanofiber composites after incubation in SBF. It was noted that the addition of nHA increased the hydrophilicity of the scaffold and thus eventually helping the adhered osteoblastic cells to spread and migrate on the surface of the nanofiber composite. It has been reported that the osteoblast-like cells prefer more hydrophilic surfaces [29]. The SEM examination of the osteoblast cell (MG-63) growth on the surface of the apatite formed nanofiber composite showed that almost the whole surface of the nanofiber composite was covered with the cells in contrast to the case with the bare PES nanofiber. This implied that the addition of nHA promoted better cell adherence and proliferation. (Fig. 7) 3.8 Cell viability The viability of cells in terms of percentage, after 40 and 80 h of seeding on the nanofiber composites are shown in Figure 8. From the figure, it was evident that after 80 h, 65-90% of cells could survive on these nanofibers. Not surprisingly, the number of cells on the apatite formed polymer nanofiber composite was noted to be always higher than that on the plain polymer nanofiber during the culture period and was likely due to the higher cell adhesion on the apatite formed nanofiber [30]. It was also evident that, the viability of cells on the bare PES nanofiber decreased with culturing time. From the observed result, it was deciphered 617 Dharmalingam Sangeetha J.Biosci Tech,Vol 6(1),2015,607-619 that the apatite formation on the PES/nHA apparently increases the cytocompatibility. (Fig. 8) 3.9 In vivo study The results of the in vivo studies using PES/nHA composites were disappointing. All the three samples showed negligible evidence for new bone formation as visualized in figure 9. The histology pictures showed a strong presence of inflammatory cells and multinucleated giant cells. There was also evidence for local bone necrosis surrounding the implant site. This unfavourable host response was observed despite the favourable in vitro response with MG 63 cell line. This contrary observations could be explained by considering that while in in vitro studies, the scaffolds are exposed to only one cell line (MG 63 in the present case), in the in vivo experiments, the entire host immune system (including macrophages, inflammatory cells and interleukins) comes into play.( Fig. 9) 4. CONCLUSION The characterization of the electrospun PES/nHA nanofiber composite using XRD and FTIR discovered the apatite formation. Hydrophilicity of the composite enhanced by the nHA was identified with low adsorption of protein which leads to improved cell viability of the biomaterial. The PES/nHA composite nanofiber showed excellent haemocompatibility compared with bare PES nanofiber due to low protein adsorption rendered by improved hydrophilicity of the polymer. In vitro study combined with SEM characterization of apatite formed nanofiber revealed substantial biomineralization. The exhibited hydrophilicity of the nanofiber composite appeared to have enhanced cell adhesion and proliferation rates of osteoblast (MG-63) cell ISSN: 0976-0172 Journal of Bioscience And Technology www.jbstonline.com lines which were further confirmed by SEM. In addition, the in vitro cell viability of the osteoblast also increased the life span of the biomaterial. Surprisingly, the success achieved in the in vitro studies was not reproducible in the in vivo studies. But considering the favourable in vitro cell response of MG 63 cells, it would be justified to attempt either PEGylation or other chemical modifications (such as grafting/Layer By Layer technique) of PES and then evaluate the in vivo response. However, the best method would be to find out first the reason behind the adverse in vivo response and later attempt at overcoming the limitation. It is widely recognized that by increasing the hydrophilicity of PES surface better haemocompatibility can be achieved. At the same time, the roughness of the PES scaffold is also known to determine the cell response. One method of modifying the surface roughness is through CO2 pulsed layer irradiation [31]. Acknowledgements: The authors would like to thank Indian Council of Medical Research (ICMR), New Delhi, India for funding the study (Vide letter No. 5/20/5(Bio)/09-NCD letter dated 26.02.2010) and All India Council for Technical Education (AICTE), New Delhi, India for the Doctoral fellowship awarded to Aravind (vide their letter no. vide their letter no. 110/RID/NDFPG (5)/2009-10). The help rendered by SRMC, Chennai in carrying out the study is also gratefully acknowledged. 5. REFERENCES Journals: [1] Meredith, P. A., Elliott, H. L., Clin. Pharmacokinet. 1992, 22, 22 – 31. [2] Yamamoto, K., Hagino, M., Kotaki, H., Iga, T., J. Chromatogr. B 1998, 720, 251 – 255. 618 Dharmalingam Sangeetha J.Biosci Tech,Vol 6(1),2015,607-619 Books: [1]. [2]. [3]. [4]. [5]. [6]. [7]. [8]. [9]. [10]. [11]. [12]. [13]. 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