Experimental Study On The Novel Biomimetic Scaffold Composite Of BMP-BG-COL-HYA-PS

Nowadays complex bone defects from various injuries have become a great challenge to orthopedists.To deal with complications associated with autogenous and allogenous bone grafting,people developed various kinds of artificial bone repairing materials(bone substitute),which has become a focus of intensive research in traumaology and tissue engineering.Of all materials available,58s BG has received considerable attention.To develop an optimal bio-mimetic material for bone repair,this study,based on thorough analysis of literature data,selected bioactive glass(BG) as the principal constituent of the scaffold composite, considering that its chemical components are similar to natural bones and it readily integrates with surrounding bone tissues through tight chemical bond formation.It induces bone formation through biodegradation and has good biocompatibility and osteoconductivity.The composite scaffold BG-COL-HYA-PS was fabricated by crosslinking BG with the major components of natural matrix TypeⅠcollagen(COL),hyaluronic acid(HYA) and phosphatidylserine(PS) and then combined with bone morphogenetic protein(BMP),using the freeze-drying technology.The composite scaffold was evaluated by a series of in vitro and in vivo experiments,for its physical-chemical properties,biocompatibility, osteoinductivity and osteoconductivity.The experiments were divided into four parts:1.Fabrication and characterization of the biomimetic BMP-BG-COL-HYA-PS composite material.The 58S bioactive glass(58SBG) was prepared by solvent-casting process.The BMP-BG-COL-HYA-PS scaffold composites were prepared by mixing nano powders of the bioactive glass,TypeⅠcollagen, hyaluronic acid(HYA),BMP and phosphatidylserine according to different ratios,using a technology of cross-linking followed by freeze-drying.The morphology,porous size and porosity of the composite scaffolds were examined by scanning electron microscopy(SEM). Mechanical strength and characteristics were evaluated by three-point bending tests.Inflation dynamics was examined using phosphate-buffered saline(PBS) as the immersion media.Strength at break is 1.406士0.019MPa;Elastic modulus is 35.812士0.016kPa.2.Biocompatibility evaluation of the biomimetic BMP-BG-COL-HYA-PS composite material. Using in vitro cell culture technique,the MSCs were seeded onto composites BMP-BG-COL-HYA-PS.Cell growth and reciprocity were monitored using MTT assay,inversion phase contrast microscope and SEM.Biocompatibility studies were performed on healthy Kunming mice selected according to the evaluation criteria for medical implant materials and the recommended biological and animal tests in GB/T16886.5-1997-ISO 10993-5:1992.These results were used for the evaluation of biological compatibility and safety of the composite materials.3.Ectopic osteogenic activity of BMP-BG-COL-HYA-PS composite.To investigate the ectopic osteoinduction and biocompatibility of BMP-BG-COL-HYA-PS composite and BG-COL-HYA-PS composite,they were synthesized and implanted into the muscle pouches in the right and left thigh of mice,respectively.At 2 and 4 week after implantation,the rats were killed,the samples were harvested,and the ectopic new bone formation was detected by X-ray analysis and histologic examination.X-ray analysis revealed that more callus-like shadow appeared adjacent to the implant of BMP-BG-COL-HYA-PS after 4 weeks.Histologically,there was slight acute inflammation in the surrounding tissue of both types of implants at the early stage.With new chondrocytes being evident at all BMP-BG-COL-HYA-PS implanted sites within 2 weeks after implantation.In 4 weeks,all of the implants with BMP-BG-COL-HYA-PS showed new bone formation with characteristics hematopoietic marrow and osseous trabeculae,and the amount of new bone as a result of the use of BMP-BG-COL-HYA-PS was significantly higher than that obtained with BG-COL-HYA-PS.The biocompatibility of composite samples was assessed by cell adhesion,proliferation,and differentiation assays using MSCs cells.MSCs were cultured with the 24h extraction medium of 1st,7th day of BMP-BG-COL-HYA-PS and BG-COL-HYA-PS samples.Cell-material interaction on the surface of the composites was observed by SEM.Cell proliferation and differentiation were measured by MTT.The effects of BMP-BG-COL-HYA-PS on the behavior of MSCs ceils were evaluated in comparison with those of BG-COL-HYA-PS.Morphology investigation of adhered cells by SEM indicated that MSCs cells could adhere,proliferate on the surface of the composite presenting normal morphology.MSCs cells,cultured in the presence of BMP-BG-COL-HYA-PS composite extract samples had a higher proliferation rate as compared to the control sample after 7 days of incubation.All these results suggest that the composite is biocompatible, nontoxic,and in some cases shows an increase in the proliferation rate of the cells as compared to the control.These results indicate that BMP-BG-COL-HYA-PS is more effective in inducing ectopic osteogenesis,that would be useful for repair bone defects.4.Biomimetic scaffold composite BMP-BG-COL-HYA-PS in the repair of rabbit radius defectIn vivo studies of the biomimetic BMP-BG-COL-HYA-PS composite scaffold for the repair of rabbit radius defects.52 Healthy adult New Zealand rabbits were used as model to create radius defects (center-section belt periosteum 15mm bone damage)on both sides.The defects were implanted with various grafts:BG-COL-HYA-PS and BMP-BG-COL-HYA-PS composites,no implant as the control group. X-Ray radiophotography was taken at weeks 2,4,8 and 12 after operation,and tissue samples were taken for histomorphological and SEM examination.Bone density,mineral apposition rate and bone formation rate were determined at weeks 4 and 12 after operation to evaluate the effectiveness of BMP-BG-COL-HYA-PS composite scaffold for repair of the bone defects.RESULTS:①Gross observation results: The wounds healed by first intention after operation,no fracture occurred. The activity,eating and mental state of the animals were normal basically.②Results of X-ray examination in the defect region:two weeks after the operation,there was significant dense shadow and a degree of porosity at ends of the oseotomy in the BMP-BG-COL-PS-HYA group.At week 8 after operation,cortical bone was fully connected.At week 12 defects were fully repaired and medullary cavity was recanalized basically.③Findings of histomorphologic observation in defect region of rabbits:In the BMP-BG-COL-PS-HYA group,two weeks after operation,osteoblasts were found along the scaffold material and excreted matrix.At weeks 4 it was found that a mass of new bone trabecula grew towards defect center. At week 12 there was no scaffold material in the defect region,which was replaced by new bone tissue fully,and the medullary cavity was recanalized.④Determination results of BMD in defect region of each group:The BMD in the BMP-BG-COL-PS-HYA group was 4.71 times and 1.675 times that in the BG-COL-PS-HYA group at weeks 4 and 12 after operation.F value was equal to 1262.398,P＜0.01 in different experimental groups detected with factorial design and variance analysis. There was significant difference in the LSD-t pair wise comparison P＜0.01.On the basis of these observations.We can conclude that the biomimetic BMP-BG-COL-PS-HYA composites scaffold has similar composition and structure to the natural bone and extracellular matrix,as well as good biocompatibility and biodegradability.It holds promise in induction of bone formation and mineralization,and should have broad applications as a bone substitute material.