| The treatment of large bone defect caused by comminuted fracture, tumor excision, or osteomyelitis still remains a challenge in clinic. As the gold standard for bone defects’ treatment, autogenous bone grafts bring problems such as donor site pain and limited supply. Allogeneic bone are alternatives but the issue is high cost, risk of virus transmission and adverse host immune reaction which restrain their application. Osteoinductive biomaterials are a promising method to solve this problem and provide an alternative to autogenous bone or allografts. Some growth factors for promoting bone healing, such as bone morphogenetic protein-2(BMP-2) and bone morphogenetic protein-7(BMP-7), have been combined with biomaterials as carriers and used in clinic for fracture healing or spinal fusion. However, the doses of BMP used in clinical situation was far above physiological levels which causes many side effects such as local soft tissue edema, spinal radiculitis, ectopic ossification, bone resorption around implant and cancer risk. So, the ideal synthetic bone graft substitutes which can be widely used in clinical situation needs further research.In the natural process of bone healing after fracture a variety of growth factors, such as BMPs, vascular endothelial growth factor (VEGF), and insulin-like growth factor (IGF) can regulate cellular activities and induce mesenchymal stem cells (MCS) to osteogenic differentiation and promote bone formation. BMP-2and BMP-7have been proved to have strong osteoinductive activity. Previous study have indicated that BMP-2reaches the peak on the first day after the fracture, and BMP-7emerge after2weeks in the natural healing process of fracture. Recently some studies have shown that the combined delivery of BMP-2and BMP-7enhances bone regeneration and healing of fracture compared with single factor delivery. So if scaffolds can release these growth factors sequentially and slowly in a biomimetic manner, it would optimize the effect of bone defect repair within the safe concentration range.Microcapsule technology just uses natural or synthetic polymer material as the capsule wall material to encapsulate the solid or liquid drugs for microcapsule. By diffusion and permeation mode wrapped drug is released in an appropriate rate in the setted position to be more effective. So far, more than200kinds of drugs have been encapsulated as microcapsule form. In recent years microcapsule technology have been successfully used in biological macromolecules, including proteins, polypeptides, genes, growth factors and antigens, which can protect of the structure and activity of biological macromolecules and achieve its control release style to make it more controllable, effective and safer. Recently, many scholars have tried to encapsulate BMPs with different polymer to prepare microcapsules for sustained release or fabrication of artificial bone scaffold. The in vitro and in vivo experiments got satisfactory results, so how to optimize the BMPs delivery system to avoid side effects caused by high doses and keep its osteoinductive activity as soon as possible remain the trend of present research.For a long time, microcapsule has been only used as one controlled release component of scaffolds or hydrogel for tissue engineering. In2007, Ana Jaklenec et al invented a simple novel method to fabricate scaffolds fused only by protein-loaded microspheres using dichloromethane vapor for control release. In the following year, he continued to prepare a scaffold fused by IGF-I and TGF-β1loaded microcapsules for cartilage tissue engineering. The final scaffold could succeed to deliver bioactive growth factors in a sequential manner and have shown its promising applications in cartilage or bone tissue engineering field. However no further relative studies were reported in this area. The advantage of this microcapsule-based scaffold is its simple progress of fabrication. The one-step preparation process can avoid adding various organic solvents, chemical stabilizers or cross-linking agent, and do not require freeze-dried, high temperature, high pressure and other physical processing, so it can protect the structure and bioactivity of proteins. Meanwhile, the synthetic viscoelastic character is helpful to repair bone defect with different forms, and the interlinked pores are suitable for nutrient solution infiltration.In the present study, the rhBMP-2loaded microcapsules were prepared using double emulsion solvent evaporation method firstly. PLA-PEG-PLA triblock copolymer (PELA) was used as wall material of microcapsule. We wanted to find optimal entrapping conditions for high encapsulation efficiency by response surface methodology. Then we employed and modified the Ana Jaklenec et al. method to prepare the scaffolds for bone healing. The scaffolds fused directly by BMPs loaded microscapsules was fabricated to promote impaired bone healing. The microcapsules encapsulating BMP-7were covered by BMP-2. The scaffold fused by these microcapsules could deliver BMP-2and BMP-7sequentially. Further, we investigated whether the BMPs release style of the scaffold can mimic the natural bone regeneration cascade. The release kinetics of growth factors and degradation character were studied. The effect on bone regeneration was investigated in vitro using rat’s MSCs and in vivo through rat’s femoral bone defect models.Object:To fabricate the microcapsule-based scaffold (BMP-2/PELA/BMP-7) which can be prepared simply and release BMP-2and BMP-7sequently. We will investigate its viscoelasticity, morphology, biocompatibility, release kenitics of growth factors, osteogenic induction ability and the bioactivity of released BMPs. Further we will employ rat’s bone defect model to evaluate the scaffold osteogenic capability in vivo and we hope to provide a new approach for future therapy of bone defects in clinic.Method:1Preparation of rhBMP-2loaded PELA microcapsules and parameters optimization for high encapsulation efficiency (EE) The rhBMP-2loaded PELA microcapsules were prepared using modified double emulsion solvent evaporation method. This work was conducted to optimize entrapping conditions of rhBMP-2loaded PELA copolymer. The effect of different molecular weight of PEG in copolymer, PELA amount, rhBMP-2amount, span-20concentration, polyvinyl alcohol (PVA) concentration and stirring time on EE tested. On the basis of single-factor experiments, the optimum parameters for highest EE were achieved by response surface methodology (RSM). The effects of three independent variables at three levels were investigated using Box-Behnken experimental design with software DPS13.0and software STATISTICA7.0was employed to draw the response surface plot for EE of rhBMP-2. The statistical significance of the model was determined by the application of F-test and P<0.05was considered statistically significant difference. Morphology of these microcapsules is observed by scanning electron microscope (SEM).2Preparation of BMPs/PELA microcapsule-based scaffold and its release keniticsIn this study, we employed and modified the Ana Jaklenec et al. method to prepare the microcapsules scaffolds. PLA-PEG-PLA triblock copolymer (PELA) was used as wall material of microcapsule. The microcapsules encapsulating BMP-7were covered by BMP-2. The scaffold was fused directly with these microscapsules by dichloromethane vapor (BMP-2/PELA/BMP-7, group A). Meanwhile PELA/BMP-7(group B), BMP-2/PELA (group C) and PELA (group D) scaffolds were fused as control group. In this chapter preparation method of scaffold was studied and the morphology of was imaged by SEM. Scaffold’s swelling and degradation tests, were performed in10ml PBS (pH7.4) at37℃. The weight loss and swelling ratio were recorded within46days. The BMPs’ release kenitics of the scaffold were studied in the same condition. Each sample was run in six copies to minimize the error.3Effect of BMPs/PELA scaffold on rat’s mesenchymal stem cells’(MSCs) osteogenic differentiation in vitroRat’s MSCs were employed to investigate osteogenic ability and cytotoxicity of BMPs/PELA scaffold. First bone marrow MSCs were isolated from male wistar rats and identified through the observation of cell morphology and detection of cell surface markers with flow cytometry. MTT assay was used to evaluate the activity of MSCs seeded on scaffold at day3,7and14. To estimate the bioactivity of released BMPs from scaffolds and its ability to induce osteogenic differentiation of MSCs, cells’alkline phosphatase (AKP) activity in different group were test. Six copies for each sample were carried out to reduce the error. All data are presented as mean+standard deviation. One-way ANOVA followed by SNK test was performed to compare multiple groups of data with SPSS19.0software. The difference in groups was considered significant if P<0.05.4BMPs/PELA scaffold repair the rat’s bone defect in vivoWe investigated the in vivo effect of BMPs loaded PELA scaffolds in bone regeneration through rat’s femoral bone defect models. Twenty-four wistar rats were randomly assigned to four groups. Under general anesthesia a5mm×5mm unicortical hole in condyle was filled with50mg biomaterials. At4and8weeks after implantation, the rats were sacrificed and the femurs removed. The general situation of femur and soft tissue around was observed. Quantitative3D analysis of bone ingrowth in the scaffolds was performed by micro-computed tomographic (mCT). The newly formed bone in each group was analyzed with the parameters the ratio of bone volume and total defected volume (BV/TV), bone mineral density (BMD) and trabecular number (Tb.N). To obtain more detailed information about new bone tissue and degradation progress of scaffold in vivo, histological analysis was performed with hematoxylin and eosin (HE) and Masson Trichrome methods. The results were analysed by One-way ANOVA method with software SPSS19.0, followed by SNK test for the determination of the significance of difference among groups. Statistically difference was defined as P<0.05.Result:1Preparation of rhBMP-2loaded PELA microcapsules and parameter optimization for high EEIn this study, it was successful to employ response surface methodology to optimize the preparing parameters of double emulsion solvent evaporation technique for high EE of rhBMP-2loaded PELA microcapsules. In preparing process, many factors such as the molecular weight of PEG block in polymer, amount of raw material (PELA and rhBMP-2), stirring time of double emulsion, concentration of emulsifier in oil phase and PVA concentration in outer aqueous phase could influence the final EE of rhBMP-2apparently. The result showed that the highest EE of BMP-2was achieved at PEG molecule weight4000Da, PELA330mg, BMP3μg, span-20concentration0.5%, PVA concentration0.5%, stirring time30min in800rpm/min.Then the effects of three independent variables X1(PELA), X2(PVA concentration) and X3(rhBMP-2) at three levels on EE were investigated using Box-Behnken experimental design. The average EE was taken as the response Y. An second order polynomial model be found and the R square was98.46%. Y=27.33602+0.29086X1+86.69629X2-30.05764X3-0.00055X12-48.92593X22+2.79563X32+0.02088X1X3-7.36111X2X3. The3D response surface plot showed that rhBMP-2amount had significant interaction with amount of PELA and PVA concentration on EE. The optimal preparing parameters could be determined from this model:the amount of PELA282.3mg, rhBMP-21μg and PVA concentration0.79%. Under these conditions, the actual EE value (75%) was in agreement with the predicted highest EE (76.5%). The data proved that the developed model could adequately represent the real relationship between the chosen parameters and EE. Most of microcapsules were rounded and the mean diameter of microcapsule was40μn by SEM.2Preparation of BMPs/PELA microcapsule-based scaffold and its release keniticsMicrocapsules encapsulating BMP-7and covered by BMP-2were prepared using the improved double emulsion solvent evaporation technique, as our previous method. Porous scaffolds (BMP-2/PELA/BMP-7) were fused by the dichloromethane vapor. Even after fusion the spheres’ underlying structure was still visible with SEM. Its inherent pores could be found with the size ranged from50μm to200μm. Scaffolds’ swelling and degradation tests showed that in the first two weeks, the swelling ratio increased obviously and attained a peak in day14in PBS solution. There was no significant mass loss until day12, then the weight began to decease quickly. In vitro BMPs release study showed that the BMP-2/PELA/BMP-7porous scaffolds could control release rhBMP-2and rhBMP-7sequently. The release profile of two growth factors were different. RhBMP-2had a burst release on day2, followed by a long sustained release until the end of experiment. To rhBMP-7the initial burst release was relatively low. After a period of slow release an accelerated release was found at2weeks later. Controlled-release interval was more than42days which can partially mimic the BMPs’ release profile in natural fracture healing process.3Effect of BMPs/PELA scaffold on rat’s MSCs’ osteogenic differentiation in vitroMTT assay was used to evaluate the rat’s MSCs activity which cultured with BMPs/PELA scaffold. After statistical analysis cells’ activity had no significant different between PELA scaffolds and positive control at day3(F=2.08, P=0.135), day7(F=0.12, P=0.954) and day14(F=43.98, P=0.732). The result indicates the BMPs loaded PELA scaffold has no negative impact on cell activity in the short term.Then MSCs were used to cultured with the releasates of4groups of scaffolds and the AKP activity was test on day7and14. Results showed that the difference at day7was statistically significant (F=28.57, P=0.000). The releasates from group A, B and C could markedly enhance cells’AKP activity at day7compared to the PELA group (P=0.000) and there was no statistical difference between these three groups. At day14the difference of AKP activity was also statistically significant (F=43.98, P=0.000). The group A was significantly higher than the group B(P=0.007) and group C (P=0.014). There was no statistical difference between group B and C. The PELA group’s AKP activity was lower than B (P=0.000) and C group (P=0.007). Thus our observations suggest that:BMPs/PELA scaffold has not been found with obvious cytotoxicity in a short term; the BMPs released from BMPs/PELA scaffold can keep their bioactivity and incorporation of BMP-2and BMP-7has the synergistic effect on promotion to rat’s MSCs’osteogenic differentiation.4BMPs/PELA scaffold repair the rat’s bone defect in vivoBMPs/PELA scaffold could repair the rat’s femoral defect so well. The femur’s mCT analysis showed that at week4after implantation the group A enhanced more new bone ingrowth in femoral defect than other groups, and new bone formation in the group B and C were more than the group PELA, which implied BMP-2and BMP-7released from scaffolds could keep bioactivity and promote new bone formation compared to control group. At week8all groups showed more new bone formation than week4. The femoral hole filled with the group A was covered completely by new bone tissue and full cortical bone could be found. There was more new bone formation in the group B and C than group D, the group C seemed to have stronger ability to promote bone healing than the group B. And for the group D, there was partial new bone could be found in the defect site until8weeks.The regenerated bone mineral density (BMD), the volume of new bone formation per total scaffold volume (BV/TV) and the trabecular number of defect area (Tb.N) were calculated for more accurately analysis. All parameters of group A were higher than other groups at week4and week8. The BMD’s difference of each group was statistically significant at week4(F=58.86, P=58.86). The group A was higher than other groups significantly (P=0.000). The difference between group B and C was not significant (P=0.54), but both were higher than group D (P=0.004; P=0.001). At week8the BMD’s difference was also statistically significant (F=22.57, P=22.57). The group A was still higher than group B (P=0.000), group C (P=0.039) and group D (P=0.000). The mean BMD of group C was high than group C (P=0.013).The difference of BV/TV value between four groups was statistically significant at week4(F=171.34, P=0.000). The group A was higher than other groups significantly (P=0.000); The difference between group B and C was not significant (P=0.134), but both were higher than group D (P=0.044; P=0.001). At week8the difference of all groups was also statistically significant (F=67.95, P=0.000). The group A was still higher than other groups(P=0.000). The group C had higher BV/TV value than group B (P=0.002). The Tb.N value of all groups showed similar trend. At week4, the difference of Tb.N between four groups was statistically significant (F=20.35, P=0.000). The group A was higher than other groups significantly (P=0.000); Both group B and C were higher than group D (P=0.005; P=0.010). At week8the difference of all groups was also statistically significant (F=12.01, P=0.000). The group A was still higher than group B (P=0.000) and group D (P=0.000). The group C was high than group B (P=0.018). These results indicated in vivo BMP-2/PELA/BMP-7material have the stronger osteogenetic ability than other groups; The long term osteoinductive ability of BMP-2/PELA scaffold was stronger than PELA/BMP-7scaffold; All BMPs combined PELA scaffold material shows better osteogenetic effect than simple PELA polymer.Histological analysis of rats’ femur showed that in the group A new bone was observed to surround the residual materials and plenty of blood cells were around at4weeks. The copolymers seemed to degrade along with the new bone formation. At week8, much more and thicker new bone was observed and tended to become lamellar bone, Some osteon with vessels can be seen in the bone matrix. In both the group B and C, limited new woven bone combined with materials was observed in the bone matrix at week4, and material’s degradation product was found to be surrounded by new bone tissue. At week8, more new bone was observed, but no matured lamellar bone was found in both groups. In the group PELA, no new bone formation was found at week4. Until week8new bone began to appear and replace the biomaterials. The results of histological observation consisted with the image of mCT.Conclusion:The rhBMP-2loaded PELA microcapsules have been successfully prepared by double emulsion solvent evaporation technique and the optimum parameters for high EE (75.0%) were achieved by response surface methodology. Then the microcapsules encapsulating rhBMP-7and covered by rhBMP-2were prepared and the porous scaffolds (BMP-2/PELA/BMP-7) were fused with these microcapsules by the dichloromethane vapor. The scaffold could control release rhBMP-2and rhBMP-7for more than42days which can partially mimic the BMPs’release profile in natural fracture healing process. Except superior biocompatibility and biodegradation, the scaffold could enhance rat’s MSCs osteogenic differentiation in vitro and repair the rat’s bone defect in vivo so well. Together, the results indicate promising potential for the use of these microcapsule-based PELA scaffolds in the treatment of bone defect in the future. |
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