【作者】 谢恩；

【导师】 胡蕴玉；

【作者基本信息】 第四军医大学 ， 外科学， 2008， 博士

【摘要】 当前各种原因造成的难治性骨缺损是临床上面临的一大难题,为了避免传统的自体骨及同种异体骨移植所带来的问题,人们开发了许多种类的人工骨修复材料,时至今日,这一领域已成为创伤骨科和骨组织工程学研究的一个热点。在这些材料中,58s生物玻璃以其良好的生物相容性引起人们的广泛重视。为了研制一种理想的仿生型骨修复材料,本课题在综合分析大量文献的基础上,选用化学组成与生物体的自然骨骼相似、容易与周围的骨骼形成紧密牢固的化学键合、经生物降解形成新的骨骼成分、具有良好生物相容性和骨传导性的生物活性玻璃(Bioactive glass,BG)作为复合支架的基体,与天然骨基质主要成分中的I型胶原(Collagen,COL)、透明质酸(Hyaluronicacid,HYA)、磷酸丝氨酸(Phosphatidylserine,PS)交联,并与骨形态发生蛋白(Bone morphogenetic protein.BMP)复合,利用冷冻干燥技术,制备BMP-BG-COL-HYA-PS复合支架,通过体内外系列实验研究观察其理化性能、生物相容性及其诱导、传导成骨能力。实验分四个部分进行观察研究:1.仿生型BMP-BG-COL-HYA-PS复合支架材料的制备及性能表征研究。58s生物活性玻璃(58s BG)采用了溶胶凝胶技术制备工艺。BG-COL-HYA-PS(nano-bioactive glass(BG),collagen(COL),hyaluronic acid(HYA) and phosphatidylserine(PS),BG-COL-HYA-PS)与BMP根据不同的比率,采用了交联和冷冻干燥进行复合。SEM显微形貌显示BMP-BG-COL-HYA-PS复合支架均具有相互交错、连续的不规则多孔结构。孔径大小在100微米到400um间分布。冷冻干燥后孔隙率为80%。BMP-BG-COL-HYA-PS网络支架具有紧密的结构,生物玻璃颗粒包裹在胶原蛋白-透明质酸-磷酸丝氨酸复合聚合物中,颗粒间通过纤维状的有机大分子相连。采用三点弯曲试验法分析BMP-BG-COL-HYA-PS复合支架的机械性能,测试结果显示BMP-BG-COL-HYA-PS复合支架的断裂强度(Strength at break)为1.406±0.019MPa;弹性模量(Elastic modulus)为35.812±0.016kPa。说明BG-COL-HYA-PS复合支架具有较高的韧性。2.仿生型BMP-BG-COL-HYA-PS复合支架材料的生物相容性检测倒置显微镜、扫描电镜显示骨髓基质干细胞在BMP-BG-COL-HYA-PS材料上能良好粘附、增殖,并向支架孔内生长,用倒置相差显微镜观察骨髓基质干细胞与支架联合培养时的动力学生长情况可以观察到细胞具有明显向材料聚集的生长趋势。BMP-BG-COL-HYA-PS材料浸提液注入动物体内的急性毒性反应试验结果依据规定的判定标准判定为无毒性。制备材料体外浸泡第1天、第7天的浸提液,将材料及其浸提液分别与人骨髓基质干细胞(MSCs)体外共培养,通过SEM观察、MTT测定,探讨复合材料的细胞粘附能力及其浸提液对于人MSCs增殖的影响。SEM结果显示:复合材料与MSCs细胞可良好粘附、增殖,细胞形态正常。体外培养时复合材料不同时间的浸提液对MSCs细胞的增殖具有促进作用。说明仿生复合材料BMP-BG-COL-HYA-PS具有良好的骨细胞相容性和生物安全性。3.BMP-BG-COL-HYA-PS复合人工骨的异位成骨活性研究将制备的BMP-BG-COL-HYA-PS,BG-COL-HYA-PS块分别植入小鼠两侧股部肌袋内,术后2周、4周取材,进行大体观察、X线、组织学检测,观察成骨状况和组织学反应,以评价载体材料的组织相容性及异位成骨能力。X线检查示材料植入4周在复合材料周围有高密度影出现,组织学观察两种植入物在植入初期周围均有轻度非特异性炎症反应,材料周围有软骨细胞出现。4周后炎症消失,材料周围出现新骨,BMP-BG-COL-HYA-PS组成骨量明显。表明复合材料具有较强的异位诱导成骨能力。4.仿生型BMP-BG-COL-HYA-PS复合支架修复兔挠骨缺损的实验研究将52只兔子随机分成3组,BMP-BG-COL-HYA-PS组、BG-COL-HYA-PS组和空白对照组。左右两侧桡骨制造15mm骨缺损,分别植入相应材料。②在植入材料后2,4,8,12周观察动物饮食、活动及伤口愈合等大体情况,以及缺损部位X射线变化,并取材分别进行组织形态学、扫描电镜、以及骨密度的检测。结果分析:①兔大体观察结果:术后所有动物伤口愈合良好,未发生骨折,活动、进食情况和精神状态基本正常。②兔桡骨缺损区X射线检查结果:术后2周,BMP-BG-COL-HYA-PS组两截骨端有明显致密影,外层有少量骨痂形成;术后8周骨皮质连接完整;12周缺损完全修复,髓腔基本再通。③兔桡骨缺损区组织形态学观察结果:术后2周BMP-BG-COL-HYA-PS组即可见成骨细胞沿支架材料爬行并分泌骨基质;4周可见大量新生骨小梁向缺损中心生长;12周缺损区内基本看不到支架材料,完全由新生骨组织替代,髓腔已再通。④各组兔桡骨缺损区骨密度测定结果:BMP-BG-COL-HYA-PS组术后4,12周骨密度分别是BG-COL-HYA-PS组的4.71倍和1.675倍,析因设计方差分析不同实验组F=1262.398,P＜0.01;LSD-t两两比较差异均有显著性意义,P＜0.01。从以上试验结果表明:BMP-BG-COL-HYA-PS复合支架具有与天然骨组织及细胞外基质相似的组成成分、良好的生物相容性和可降解性,在诱导成骨和促进矿化方面性能优越,作为骨缺损修复替代材料应用前景广阔。更多还原

【Abstract】 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.更多还原