32 Chapter CHAPTER Three-Dimensional Fibrous PLGA/HAp Composite Scaffolds for Bone Morphogenetic Protein-2 (BMP-2) Delivery † 3.1 Introduction Bone fracture is a common form of injuries that bring great inconvenience to victims. Bone grafting, as a common form of bone treatment, is done by transferring bone tissue from the donor site to the fractured site before adequate physiological treatment facilitates bone healing. For autologous bone treatment, the bone tissues are usually transferred from the iliac crest of the patient’s pelvis (Swan and Goodarce, 2006). While the advantages of autologous bone treatment may include easy access of donor site without requiring the reposition of the patient as well as very low risks of infectious diseases, post-operation complications have been reported to be as high as 15% (Swan and Goodarce, 2006; Mischkowski et al., 2006). Furthermore, the donor site may not be able to provide sufficient bone tissue to the injury site (Huang et al., 2005). Other implications of grafting including severe pain, persistent aching, scarring and infection have also been reported (Swan and Goodarce, 2006; Huang et al., 2005). Fortunately, there are less painful and risky alternatives to bone treatment. A group of proteins, known as bone morphogenetic proteins (BMPs), are known to facilitate bone † This chapter highlights the work published in H. Nie, B.W. Soh, Y.C. Fu and C.H. Wang. ThreeDimensional Fibrous PLGA/HAp Composite Scaffold for Bone Morphogenetic Protein-2 Delivery. Biotechnol. Bioeng. 99 (1), 223-234. 2008b. Chapter 33 healing without transferring bone tissues. By inducing marrow derived messenchymal stem cells (MSCs) to undergo chondroblastic and osteoblastic differentiation, BMPs can induce bone regeneration in vivo (Saito et al., 2005). Among this group of proteins, BMP2 has been shown to induce healing in segmental bone defects. Aebli et al. (Aebli et al., 2005) and Saito et al. (Saito et al., 2005) reported that BMPs improve bone regeneration in vivo. Over past years, many release dosage forms have been developed for drug or protein delivery, like nanoparticle and microsphere. However, one common problem is that the existence of a large burst over a narrow time period during the early stage of release. In view of this problem, a new type of scaffold is needed urgently, especially for bone regeneration to overcome this challenge, because nanoparticles and microspheres are not suitable due to the non-ideal release profile and their fluidity as well which hinders them from localizing themselves and giving new bone tissues enough support. Fiber is chosen in the present study as the release dosage form because of its more favourable release properties and morphology. Normally, a microsphere’s effectual release course can only sustain less than 30 days, which is far from enough for bone regeneration. Fiber has much lower release rate of drug or protein than microsphere because of its smaller surface/volume ratio (Wei et al., 2006). Moreover, compared with microsphere, compacted fibrous scaffold can give cell stable three-dimensional growth environment and provide newly generated bone good support. In this project, electrospinning (Kenawy et al., 2003) is employed to fabricate fibers due to its flexibility of operations and the Chapter 34 fiber diameter can be easily controlled by changing operation parameters such as voltages, polymer concentrations and organic/aqueous mixture composition. Hydroxylapatite (HAp), which is a major component of the bone, can be used as a subsidiary in the bone generation. HAp implants exhibit high mechanical strength and good biocompatibility. In addition, HAp has the added advantage of being able to bond directly to the bone since both of them have similar chemical structures. Despite the above qualities, HAp is usually not used alone as its brittle nature creates difficulties in fabricating the transplant block to the exact shape of the bone defect configuration (Rebecca and Wozney, 2001). A study by Takaoka et al. demonstrated that there is a lack of bone healing when HAp is used alone as a carrier for BMP-2 (Takaoka et al., 1988). The objective of the research project is to conduct an in vitro study of recombinant BMP2 encapsulated in fibrous scaffolds by investigating the effects of HAp content and the different methods of protein loading on the biological and physical characteristics of the micro-fibers fabricated using the electrospinning method. The physical characteristics investigated are the surface morphology, thickness, crystallinity of HAp and residual DCM content. The biological characteristics investigated are the cell attachment and cytotoxicity of the fibrous scaffolds. Towards the end of this study, the protein encapsulation efficiency, the in vitro release profile of the protein, the probability of protein denaturation were also investigated. Chapter 35 3.2 Materials and methods 3.2.1 Materials Recombinant human bone morphogenetic protein-2 (rhBMP-2) (E. coli expressed, Cat. No. 355-BEC/CF) and its enzyme-linked immunosorbent assay (ELISA) kit were purchased from R&D Systems, Inc. (Minneapolis, US). Poly (D,L-lactide-co-glycolide) (PLGA) (Lot Number W3066-603 with L/G ratio 50:50, IV 0.57 and MW 51000) used in the experiment was manufactured by Alkermes Controlled Therapeutics II, (OH, US) and purchased from Lakeshore Biomaterials (AL, US). Dichloromethane (DCM) was manufactured in Tedia Company Inc. (Fairfield, Ohio, US). Hydroxylapatite (HAp) nanoparticles of 100nm were purchased from Berkeley Advanced Biomaterials Inc. (Berkeley, CA, US). Phosphate Buffer Saline (PBS) buffer used for in vitro release study was bought from Sigma Aldrich containing 0.1M sodium phosphate, 0.15M sodium chloride, pH 7.4. Dulbecco’s Modified Eagle Medium (DMEM), the cell culture medium in the experiment, was supplemented with 4mM-glutamine, 4.5g/L glucose, 25mM HEPES buffer, 10% fetal bovine serum (Gibco), 10U/mL penicillin G sodium, 10 mg/mL streptomycin, 25 mg/mL amphotericin B as Fungizone (Gibco) and 100mg/mL Lascorbic acid from Sigma-Aldrich, Oakville (Ontario, Canada) and the cells were extracted using trypsin-EDTA. Human marrow derived messenchymal stem cells (hMSCs) were purchased from Cambrex Bio Science Walkersville, Inc. (Newington, NH, US) and the PreMix WST-1 Cell Proliferatiom Assay System was purchased from Takara Bio Inc. (Otsu, Shiga, Japan). 36 Chapter 3.2.2 Preparation of fibrous scaffolds In all the experiments of the present work, the fibers were essentially fabricated from homogeneous emulsions formed from the sonication of organic and aqueous mixture. Table 3.1 summarises the composition of the emulsion of the four different experimental cases 1-4 and the fibrous scaffolds fabricated are named F1-F4 respectively. Table 3.1 Compositions of emulsions for preparing different scaffolds F1-F4 Experimental Scaffold Case Organic Phase Aqueous Phase DCM PLGA HAp BMP-2 F1 10mL 3g 0mg 5μg F2 10mL 3g 150mg 5μg F3 10mL 3g 300mg 5μg F4 10mL 3g 150mg 0* * For F1-F3, the BMP-2 solution was added directly into the aqueous fabrication solution for electrospinning. For F4, the BMP-2 solution was not added directly into the aqueous fabrication solution. Instead, the protein was added to each fibrous scaffold sample of F4 after scaffold was fabricated and dried for days using freeze dryer. Preparation of organic phase In each experimental case, a 30% wt/vol PLGA polymer solution using DCM as the solvent was prepared by dissolving 3g PLGA into 10 mL of DCM. The resultant mixture was agitated by applying vortex until a clear, homogeneous organic phase was formed. In order to compare the effect of polymer concentration on fiber morphology, fibers using 10% and 20% PLGA/DCM solutions were also prepared. Chapter 37 Preparation of aqueous phase In the experimental cases F1-F4, vials of BMP-2 of 10μg were dissolved in 90μL of 4mM hydrochloric acid (HCl) each and mixed well to produce a homogeneous BMP-2 solution. In each of the experiments for case 1- case 3, 50μL BMP-2 solution containing 5μg of the protein was dissolved in deionised water and mixed well with specified weight of HAp nanoparticles to give 800μL of homogeneous aqueous suspension. For experimental case 4, BMP-2 solution was not added directly into the aqueous solution. Instead, the protein was loaded from a diluted BMP-2 solution (by 20 folds) using 50μL of the original BMP-2 solution with 750μL of deionised water. All the solution would then be evenly added to the blank (meaning no encapsulation of BMP-2) F4 scaffold prepared beforehand. To ensure that the viscosity of the emulsion is not affected by the organic-aqueous ratio, the volume of deionised water (HAp suspended without BMP-2) mixed with organic solution in experimental case is 800μL to keep the same ratio 10:0.8 as in case 1-3. Fabrication of fibrous scaffolds After adding the aqueous and organic phases together, the mixture was sonicated for 3040 seconds and the resultant emulsion was transferred to a 10 mL glass syringe (MICROMATE interchangeable 10cc hypodermic syringe, Popper & Sons, Inc., New Hyde Park, NY. US) fitted with a 29-g needle and set up in the elecontrospinning apparatus. The flow of polymer solution from the syringe into the spinneret (diameter 340 mm) was Chapter 38 controlled by a programmable syringe pump (KD Scientific, Holliston, MA, US). Scaffolds were electrospun at about a voltage difference 10 kV with a solution flow rate of 5mL /h. The spinneret (anode) was fixed at about 15 cm above the aluminium-covered rotating collection drum (cathode). The syringe was loaded into the syringe pump and aluminium foil was wrapped around the spinning motor to collect the fiber samples. 3.3 Characterization of scaffolds 3.3.1 Physical characterization Morphology of fibrous scaffolds Field emission scanning electron microscopy (FESEM, JSM-6700F, JEOL Technics Co. Ltd, Tokyo, Japan) was employed to study the surface morphology of the fibers produced in each experiment. Differential scanning calorimetry (DSC) Differential scanning calorimetry (DSC) can be employed to determine the amount of crystalline structure within the microfibers as well as the effects of HAp concentration on the glass transition temperature and the decomposition temperature of PLGA. The sample was heated from 30 °C to 400 °C at a constant temperature increment of 10 °C/minute and purged with nitrogen gas at 30 mL/min. X-ray diffractrometry (XRD) The HAp nanoparticles or fiber sample were placed in a sample holder and the surface of the sample was flattened. Next, the sample was placed in the XRD equipment 39 Chapter (SHIMADZU, Tokyo, Japan). A diffraction range of 10-35° (2θ) was selected and the XRD analysis was carried out at 2°/min. 3.3.2 Encapsulation efficiency (EE) determination The encapsulation efficiency (EE) of the BMP-2 in the scaffolds is defined as the percentage of the actual BMP-2 loading to the total (theoretical) amount of BMP-2 loading. In the EE analysis, about 5mg of each scaffold was dissolved in mL of DCM and mL of PBS (pH 7.4) was added subsequently. The mixture was vortexed for to extract BMP-2. Subsequently the system underwent centrifugation using a Hettich Zentrifugen system (Universal 32R, Andreas Hettich GmbH & Co KG, Tuttlingen, Germany) at 9000 rpm for 20 to separate the oil and water phases. At the same time, HAp nanoparticles settled to the bottom of tubes. The water phase was then carefully collected and kept frozen at -20 °C until it was analyzed for BMP-2 concentration using the ELISA BMP-2 Immunoassay kit mentioned above. The encapsulation efficiency of the BMP-2 in the fibers is the ratio of the actual amount of BMP-2 loaded into the fibers to the theoretical amount of BMP-2 loaded (Xie and Wang, 2005) by the following equation: EE = W BMP - + W PLGA + W HAp C BMP - × V water × × 100 % W sample W BMP - (3.1) where CBMP-2 is the BMP-2 concentration in the water phase of extraction; Vwater is the volume of water phase of extraction; Wsample is the weight of each scaffold sample used for EE analysis; WBMP-2, WPLGA and WHAp are the weights of BMP-2, PLGA and HAp used in the scaffolds fabrication process respectively. Chapter 40 3.3.3 In vitro release studies The in vitro release of BMP-2 was carried out over a period of 60 days and the cumulative release curve can be plotted. Approximately 25mg of microfiber samples made from each experiment were prepared and each of them was added to a tube with 5mL PBS, the release medium in the experiment. The resultant mixture was placed in an orbital shaker bath (GFL® 1092) at 37 °C, 120rpm. mL of sample mixture was extracted at specific intervals (16h, days 1, 2, 3, 5, 7, 10, 12, 14, 16, 19, 23, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57 and 60) from each test tube. mL of PBS solution was then added to each mixture to make up mL again and all the mixtures were incubated in the orbital shaker bath again before the next set of sample mixtures were extracted. The ELISA kit was used to test the concentrations of BMP-2 inside the PBS solutions The optical density of each well was determined using the micro plate reader (Tecan Trading AG, Switzerland), while setting the wavelength to 450nm with correction wavelength of 570nm. 3.3.4 Protein integrity and secondary structure check Continuous Native-polyacrylamide gel electrophoresis (continuous Native-PAGE) To evaluate the effects of fabrication process on the molecular integrity and biological activity of BMP-2, in vitro release sample was centrifuged and the supernatant was analyzed by Native-PAGE to determine the integrity and conformation of BMP-2. In order to avoid stacking-induced aggregation, a continuous buffer system was used. The electrophoresis buffer with pH 7.4 was prepared according to the MaLellan method (43 mM Histidine + 35mM HEPES). Each sample or native BMP-2 was diluted in sample Chapter 41 buffer [1.0 mL electrophoresis buffer, 3.0 mL glycerol, 0.2 mL 0.5% bromophenol blue, and 5.8 mL deionised water in each 10 mL sample buffer] before 10μl sample or native BMP-2 was loaded into each well of a 6% polyacrylamide gel and electrophoresis was conducted using a Bio-Rad Mini-PROTEAN electrophoresis system (Cat No: 165-3301 and 165-3302) at a constant voltage difference (100V). Protein bands were detected by Coomassie G-250 staining using GelCode Blue Stain Reagent (24590, Pierce Biotechnology Inc., Rockford, IL, US). A Bioimaging system, Gene Genius (Syngene, Synoptics Ltd, Cambridge, United Kingdom) was used to image the gels. Fourier transform infrared Spectroscopy (FTIR) FTIR spectroscopy, conducted using a Bio- Rad FTS3500 (Bio-Rad Laboratories, Cambridge, MA) was employed to explore the secondary structure of proteins in PBS solution. A total of 32 scans at a resolution of cm-1 were averaged for each sample. To determine the secondary structure of protein, all spectra were analyzed by second derivatization in the amide I region (1700-1600cm-1) for their component composition and BMP-2 secondary structure quantified by Gaussian curve fitting after Fourier selfdeconvolution of the corrected spectra by Peakfit 4.0 (SPSS Science). The area of each peak in the amide І region was calculated and used to determine the secondary structure of the protein using procedures reported by Nahar and Tajmir-Riahi (Nahar and TajmirRiahi, 1996). Chapter 44 a large surface area for the release of BMP-2 as well as promoting cell interaction and growth (Lazzeri et al., 2005). Figure 3.1 Comparison of the fibers formed from emulsions with different PLGA concentrations. 1A-1B: 10% PLGA, 2A-2B: 20% PLGA, 3A-3B: 30% PLGA, where A and B have different amplifications. Chapter 45 Figure 3.2 SEM micrographs of fibrous scaffolds F1-F4 fabricated in experimental cases 1-4, respectively. Differential scanning calorimetry was performed to determine the physical state of HAp nanoparticles within the overall structure of the fabricated scaffolds. In the DSC thermogram, as shown in Figures 3.3 and 3.4, all the fibrous samples including the pure PLGA fiber sample showed exothermic peaks at approximately 50 °C, which is the glass transition temperature of PLGA. The glass transition temperature of PLGA obtained in this experiment is approximately consistent with the glass transition temperature for PLGA microspheres of about 50 °C obtained by Xie et al. and about 40 ± °C obtained by Calis et al. (Xie and Wang, 2006; Calis et al., 2002). Chapter 46 Figure 3.3 A comparison of the DSC thermogram for all fibrous samples F1-F4, pure PLGA fiber and HAp nanoparticles from 30-400 °C. F1-BMP-2 loaded, no HAp; F2BMP-2 loaded, 5% HAp; F3-BMP-2 loaded, 10% HAp; F4-BMP-2 loaded outside, 5% HAp. A closer examination at the larger exothermic peaks of the fibrous samples F1-F4 in Figure 3.4 showed that the temperature in which the peaks occurred generally increased with the content of HAp nanoparticles. For F1, where no HAp is added, the exothermic peak occurred at around 350 °C, while for F3, where the weight/weight ratio of HAp to PLGA is 10%, the peak occurred at around 375 °C. This is because like PLGA, HAp nanoparticles will also absorb heat during the heating process. Therefore, more time is required for the PLGA in the fibers to absorb enough heat to reach the decomposition point. Recalling that the temperature varies linearly with time, it is logical that as HAp content in the fibers increases, the exothermic peak of the fibers will occur at a higher temperature. Overall, the DSC plots show that HAp nanoparticles were incorporated into the fibers. Otherwise, all the exothermic peaks would have occurred at around the same temperature. Chapter 47 Figure 3.4 A comparison of the DSC thermogram for all fibrous samples, pure PLGA fiber and HAp nanoparticles from 30-200 °C. F1-BMP-2 loaded, no HAp; F2-BMP-2 loaded, 5% HAp; F3-BMP-2 loaded, 10% HAp; F4-BMP-2 loaded outside, 5% HAp. Figure 3.4 gives a clearer comparison of the DSC curves of the fibrous scaffolds F1-F4 together with pure HAp nanoparticles. An exothermic peak at approximately 115-120 °C reveals the glass transition temperature of HAp nanoparticles, but the exothermic peak is absent in all the DSC curves of fibrous samples. The lack of the exothermic peak suggests the lack of HAp clusters in all the fibrous samples and this shows that the HAp nanoparticles were well distributed and poorly crystallized in all samples. This is consistent with the result from Kim and colleagues (Kim et al., 2005), who find that HAp particles are poorly crystallized as long as HAp concentration (to polymer) is below 15%. Figure 3.5 shows the XRD results of the protein loaded fibrous scaffolds F1-F4 as well as the XRD results of pure PLGA fibers and pure HAp nanoparticles. The purpose of the XRD characterization is to verify the hypothesis from the DSC thermogram that the HAp nanoparticles encapsulated are evenly dispersed on scaffold surfaces. Overall, the shapes of the XRD curves obtained from all fibrous scaffolds were similar to the PLGA fiber Chapter 48 curve, and all revealed small broad peaks at approximately 22-23° (2θ). However, the XRD curves of all the fibrous scaffolds did not reveal any peaks at around 28° or 32° (2θ), which are the characteristic peaks in the HAp nanoparticle curve as shown in XRD results. The absence of 28° and 32° (2θ) peaks shows that the HAp nanoparticles were well dispersed within the fibrous scaffolds, not just appearing on the surface. Figure 3.5 A comparison of the XRD patterns of the protein loaded scaffolds F1-F4. From the results tabulated in Table 3.2, the encapsulation efficiency of F4 is the highest because the protein is directly loaded into the scaffold after its fabrication; therefore it can be considered that full amount of protein is adsorbed and the encapsulation efficiency (EE) is recorded as 100%. As for scaffolds F1-F3, where the protein is loaded into the emulsion before the fabrication of fibers, the encapsulation efficiency in all scaffolds tested lie in moderate values between 40-70%. Such values of encapsulation efficiency is reasonable considering the fact that the emulsion was made up of organic and aqueous components that are barely miscible, and it is difficult to obtain a high encapsulation efficiency without adding a surfactant to stabilise the emulsion. 49 Chapter Table 3.2 BMP-2 encapsulation efficiency in the groups of fibrous scaffolds (F1-F4) F1 F2 F3 F4 49.39±5.70 44.03±5.90 65.89±7.43 100* Encapsulation Efficiency (%) *For the EE of F4, the BMP-2 was added into scaffolds directly after the fabrication of scaffold, so its EE can be considered to be 100%. 3.4.2 In vitro release results The shape of the release curve (Figure 3.6) obtained from this experiment is consistent with the in vitro release profiles of many studies of bone engineering (Jansen et al., 2005; Ruhe et al., 2004; Hedberg et al., 2002; Luginbuehl et al., 2005), but some differences should be emphasized after introducing HAp. Generally, there is a higher percentage of BMP-2 released when more HAp nanoparticles were added. The percentage of BMP-2 released in F1 was slightly less than 25% after more than 360 hours (15 days) of in vitro release study, and the percentages were much higher, of about 30% and 45% for F2 and F3 respectively, but the percentage is more than 96% for F4. The profile for F4 is consistent with which has been observed in the BMP-2 release from microspheres (Woo et al., 2001). A logical reason could be the morphology change rate during the release course due to adding of HAp nanoparticles. What is the most important is that adding the hydrophilic HAp increased the hydrophilicity of the scaffolds; therefore F2, F3 and F4 are much easier to disassemble than F1. In this process of degradation, BMP-2 in F2, F3 and F4 will diffuse out much faster than in F1. Chapter 50 Figure 3.6 The cumulative in vitro release curves of the scaffolds over a period of 60 days. The plot was presented in terms of the percentage mass released over the original mass of protein present. The percentage release rate of BMP-2 is the highest for scaffold F4 at the early stage, with more than 96% of the protein released after 15 days of in vitro release study. With the protein loaded after the fibrous scaffolds were fabricated, the protein molecules were essentially located outside the fibers and stayed in the interstitial spaces within the 3D network. Hence, it is easier for the protein molecules to diffuse into the release medium without requiring the fibers to undergo biodegradation before they can be released. Another reason maybe that BMP-2 was added into F4 as aqueous solution, which means the hydrophilicity of F4 was increased slightly although most of the water evaporated already before F4 was used in release test. Chapter 51 Figure 3.7 A comparison of morphology of F1-F4 after 30 days of in vitro release. The morphology change was also checked after 30 days of release, which is shown in Figure 3.7. The result shows clearly the effects of HAp content and protein encapsulation method on the morphology. After 30 days, many HAp particles appeared on the surface of F2 and compared with F1, the surface of F2 are much weaker with fractures and many holes on its surface. It is interesting that F3 and F4 can not maintain their complete morphologies after 30 days and broke into an amorphous mass. Although F2 and F4 have the same content of HAp nanoparticles, they showed different morphologies after 30 days of release. F4 seems less integrated and this finding indirectly explains why BMP-2 in F4 was released much faster than all other fibers. Chapter 52 Controlled and sustained release of protein is crucial for any protein drug delivery system, so early burst and short-term release course should both be avoided. Furthermore, a very low concentration of release is not wanted if it is below the effective (therapeutic) concentration level. For F4, its release curve shows a large release at a very early stage for bone regeneration because 15 days is far from the desired target. Normally, at least 30 days of sustained release is required. As for F1, its release maybe too slow if its concentration on site is below the effective concentration. Results from Figure 3.7 illustrate the control of BMP-2 release rate from PLGA fibrous scaffolds by altering the HAp content. From the trend shown, increasing HAp content can accelerate the release rate of BMP-2 from fibrous scaffold. 3.4.3 Protein integrity and secondary structure testing results In order to examine whether there is a loss in protein integrity through the loss of amino acid groups or structural damage caused in the process of fabrication, SDS-PAGE was carried out to check for losses in the molecular weight of the protein. In addition, FTIR was executed to check for structural changes within the protein. Figure 3.8 shows the Native-PAGE electrophoretic patterns of the BMP-2 released in vitro from scaffolds F1-F4 after days together with native BMP-2. The results demonstrate that the released BMP-2 has retained its structural integrity and structural conformation, as evident by the bands present on the gel. The BMP-2 in all the five wells has about the same molecular weight of 28kDa, which is accorded with the specification from the manufacturer. In other words, the released BMP-2 has survived potentially harsh Chapter 53 processes (for instance emulsification and electrospinning as well as post-processing conditions like scaffold handling and incubation) that may result in partial removal of amino acid groups and conformation change in the protein. Figure 3.8 Native PAGE results of BMP-2 released from four kinds of fibrous scaffolds F1-F4 suspended in PBS after day. Lane 1: Native BMP-2; Lane 2: BMP-2 released from scaffold F1; Lane 3: BMP-2 released from scaffold F2; Lane 4: BMP-2 released from scaffold F3; Lane 5: BMP-2 released from scaffold F4. Chapter 54 Figure 3.9 A comparison of α-helix proportion of native BMP-2 ( ) and BMP-2 released from released from F1 ( ), F2 ( ), F3 ( ) and F4 ( ). Values represent mean ± S.D., n=3. From literature (Nahar and Tajmir-Riahi, 1996; Woo et al., 2001), the peak of α-helix structure should lie within the range 1647-1660cm-1. For β-sheet, the peak should lie within the range 1615-1636cm-1. For antiparallel β-sheet, the range should be within 1681-1692cm-1. As for turn and random structure, the range should lie within 1660-1680 and 1637-1647cm-1 respectively. Finally, the proportion of each type of protein structure was determined by evaluating the area of the peak under the curve. The result of the deconvolution of the absorption curves are summarised at Table 3.3. Generally, the proportion of all the protein conformations are well maintained for the BMP-2 loaded in all the scaffolds, with the proportion of the α-helix structure at the Amid I band of the protein most intact at around 12%. The wavenumber in which the peaks of the various structures occurred also agrees well with the values obtained from literature. Overall, the FTIR characterization gives a satisfactory result because the overall small changes in the proportion of the protein structures in each scaffold showed that only a small proportion Chapter 55 of the loaded protein was denatured, especially for the BMP-2 released from F2, F3 and F4. This claim is supported by the data because the BMP-2 from these three scaffolds showed very similar percentages of α-helix and β-sheet, while the BMP-2 protein from F1 shows a much bigger error bar in terms of the percentage of α-helix and β-sheet, hence it has demonstrated that the extent of BMP-2 denaturation maybe higher when the HAp content is lower. This result shows that the BMP-2 in F1 was perhaps damaged in the electrospinning process, which can be explained by the long time of contact of BMP2 with the hydrophobic organic solvent DCM. The BMP-2 in F2 and F3 escaped the harsh environment because of the presence of HAp nanoparticles due to their similar hydrophilicity. BMP-2 tried their best to attach to HAp nanoparticles to elude the contact with DCM. For F4, the BMP-2 was dripped and coated after fiber fabrication, so the BMP-2 did not experience the harsh process of electrospinning and kept its native structure wonderfully, as shown in Table 3.3 and Figure 3.10. Figure 3.10 A comparison of the relative cell attachment ability of F1 ( ), F2 ( ), F3 ( ) and F4 ( ) compared with TCPS well control ( ). Values represent means ± S.D., n=3 (*p[...]... proportions of native BMP -2 Amide I BMP -2 BMP -2 BMP -2 BMP -2 released released released released from conformations BMP -2 from from from F1 components native F2 F3 F4 (cm-1) 1647-1660 α-Helix 12 1 9 2 11 2 11 2 12 1 1615-1636 β- sheet 26 ±4 19±7 24 ±6 26 2 25 2 1637-1646 random 10±1 10±3 10±1 10 2 11±1 1660-1680 Turn 26 2 38±18 30 2 29±1 26 2 1681-16 92 β- Antiparallel 26 ±3 24 ±4 25 2 24±1 26 2 α-helix percentage... F3 and F4 can not maintain their complete morphologies after 30 days and broke into an amorphous mass Although F2 and F4 have the same content of HAp nanoparticles, they showed different morphologies after 30 days of release F4 seems less integrated and this finding indirectly explains why BMP -2 in F4 was released much faster than all other fibers Chapter 3 52 Controlled and sustained release of protein... consistent with the in vitro release profiles of many studies of bone engineering (Jansen et al., 20 05; Ruhe et al., 20 04; Hedberg et al., 20 02; Luginbuehl et al., 20 05), but some differences should be emphasized after introducing HAp Generally, there is a higher percentage of BMP -2 released when more HAp nanoparticles were added The percentage of BMP -2 released in F1 was slightly less than 25 % after more... released BMP -2 has survived potentially harsh Chapter 3 53 processes (for instance emulsification and electrospinning as well as post-processing conditions like scaffold handling and incubation) that may result in partial removal of amino acid groups and conformation change in the protein Figure 3.8 Native PAGE results of BMP -2 released from four kinds of fibrous scaffolds F1-F4 suspended in PBS after... 1: Native BMP -2; Lane 2: BMP -2 released from scaffold F1; Lane 3: BMP -2 released from scaffold F2; Lane 4: BMP -2 released from scaffold F3; Lane 5: BMP -2 released from scaffold F4 Chapter 3 54 Figure 3.9 A comparison of α-helix proportion of native BMP -2 ( ) and BMP -2 released from released from F1 ( ), F2 ( ), F3 ( ) and F4 ( ) Values represent mean ± S.D., n=3 From literature (Nahar and Tajmir-Riahi,... diameter of 6mm, sterilized using gamma radiation and placed in the wells of 96-well plates About 20 0μL of hMSC suspension (2. 5 x 105 cells/mL) were added into each well and the well plates were incubated in a humid atmosphere at 37 °C and 5% CO2 For cell attachment test, after incubation for 4 hours, all scaffolds were rinsed and removed from wells and the cell number inside wells was assessed and compared... percentages of α-helix and β-sheet, while the BMP -2 protein from F1 shows a much bigger error bar in terms of the percentage of α-helix and β-sheet, hence it has demonstrated that the extent of BMP -2 denaturation maybe higher when the HAp content is lower This result shows that the BMP -2 in F1 was perhaps damaged in the electrospinning process, which can be explained by the long time of contact of BMP2 with... Ontario, Canada) and incubated at 37 °C and 5% CO2 humid atmosphere in 75cm2 cell culture flasks The cells were extracted with PBS solution containing 0 .25 wt% trypsin and 0.02wt% ethylenediaminetetraacetic (EDTA) acid The cells were normally sub-cultured at a density of 2 x 104 cells/cm2 Cell attachment and cytotoxicity test of scaffolds Before cell testing, all scaffolds were punched into round sections... attachment and cytotoxicity tests show that the incorporation of HAp nanoparticles can enhance cell attachment and cell viability Furthermore, PLGA/HAp composite scaffolds are promising for BMP -2 delivery and bone regeneration Chapter 3 59 3.5 Conclusions This study investigated two methods to load BMP -2 into three dimensional fibrous scaffolds using an electrospinning method, including encapsulating into... that adding the hydrophilic HAp increased the hydrophilicity of the scaffolds; therefore F2, F3 and F4 are much easier to disassemble than F1 In this process of degradation, BMP -2 in F2, F3 and F4 will diffuse out much faster than in F1 Chapter 3 50 Figure 3.6 The cumulative in vitro release curves of the scaffolds over a period of 60 days The plot was presented in terms of the percentage mass released . grafting including severe pain, persistent aching, scarring and infection have also been reported (Swan and Goodarce, 20 06; Huang et al., 20 05). Fortunately, there are less painful and risky. activity of BMP -2, in vitro release sample was centrifuged and the supernatant was analyzed by Native-PAGE to determine the integrity and conformation of BMP -2. In order to avoid stacking-induced. Canada) and incubated at 37 ° C and 5% CO 2 humid atmosphere in 75cm 2 cell culture flasks. The cells were extracted with PBS solution containing 0 .25 wt% trypsin and 0.02wt% ethylenediaminetetraacetic