【作者】 刘玲蓉；

【导师】 张其清；

【作者基本信息】 中国协和医科大学 ， 生物医学工程， 2007， 博士

【摘要】 骨肿瘤和外伤等引起骨缺损发病率的逐年增加已对人类健康造成极大的威胁。目前,治疗骨缺损一般采用自体骨和异体骨移植的方法,但由于自体骨和异体骨移植存在供体有限、免疫反应和传染病的危险等。因此,急需开发人工骨修复材料用于修复骨组织的缺损。目前,用于骨组织再生修复生物材料的研究和临床应用已从第一代生物惰性材料、第二代生物活性材料及生物可降解材料发展到基于细胞和分子水平的第三代生物材料。同时,组织工程、原位组织再生等新理论和新方法的出现为骨缺损的治疗带来了新技术,生物活性与生物可降解性结合的第三代生物材料的研究使应用组织工程和原位组织再生方法修复病缺损组织成为可能。因此,研究开发新型骨诱导生物活性和生物降解性结合的骨修复材料以满足医学科学发展和临床需要是生物医学工程学领域研究的重要方向之一。本论文模拟天然骨组织的组成和结构特征,构建胶原—羟基磷灰石(HA)复合材料并引入细胞因子骨形态发生蛋白(BMP),研制用于原位骨组织再生修复和骨组织工程的骨诱导生物活性和生物降解性结合的生物材料。主要研究结果如下:1.胶原—HA复合骨修复材料的制备及其理化、生物安全性表征选用生物相容性好的天然胶原、HA为主原料,通过共混、化学交联等过程制备胶原—HA复合人工骨修复材料,并对材料的结构和理化性能进行了表征,研究了材料与血浆接触后材料对血浆蛋白吸附及对血浆中钙磷影响,参照GB/T16886医疗器械生物学评价系列标准等对材料进行了系统的生物安全性评价。研究结果显示,胶原—HA复合人工骨修复材料为非致密多孔状,可见HA间的胶原纤维,HA均匀分散在胶原纤维中;复合材料的理化性能和生物安全性符合体内植入物相关标准的要求,具有良好的生物相容性;复合材料与血浆接触后,血浆溶液中血浆总蛋白(TP),纤维蛋白原(FiB)及纤维粘连蛋白(FNC)的降低表明了材料表面有各种蛋白质的吸附,材料吸附某些特异性非胶原蛋白,将益于骨组织形成和矿化;血浆溶液中Ca、P值均有增加,与材料表面离子释放有关,可参与体内代谢,对骨诱导将会起到促进作用。2.胶原—HA/BMP复合材料异位成骨效应的研究将BMP与胶原—HA复合材料通过物理吸附的方法复合,利用鼠股部肌袋模型,通过组织学观察、碱性磷酸酶(ALP)染色以及组织中ALP定量检测等手段研究了复合BMP后材料的异位诱导成骨效应。实验结果显示复合BMP的胶原—HA复合材料和HA材料均有异位诱导成骨作用,而未复合BMP的材料均没有表现异位诱导成骨作用。3.胶原—HA/BMP复合材料修复节段性骨缺损及成骨效应的研究为进一步研究和评价研制的骨修复材料对骨缺损的修复能力,将复合BMP的胶原—HA复合材料植入兔桡骨10mm骨缺损,通过血生化检测、生物力学测定、扫描电镜及组织学染色分析等手段,观察了新骨形成及材料的降解规律。实验结果显示胶原HA复合材料对骨缺损有较强的修复能力,具有良好的可降解吸收性;胶原—HA复合材料/BMP骨缺损修复能力大于胶原—HA复合材料,BMP的加入可有效地促进新骨的形成。结合异位成骨实验结果说明,胶原—HA复合材料可与BMP构成优良的承载系统,保护BMP的生物活性,使BMP能在组织局部聚集成较高的浓度,诱导成骨。4.胶原—HA复合骨组织工程支架材料及复合蛋白药物的释放特性的研究采用冷冻干燥法制备多孔胶原-HA复合支架材料用于骨组织工程,对材料的结构形态、性能进行了表征;以牛血清白蛋白(BSA)为模型药物,应用离子交联法制备载BSA壳聚糖胶体颗粒,并与支架材料复合,对胶原-HA骨组织工程支架材料作为生物活性因子释放载体的体内外释放特性进行了研究。现究结果显示,制备的胶原-HA骨组织工程支架材料为多孔三维结构,支架孔径平均50—150μm;胶原与HA复合后支架材料的强度得到提高;甲醛交联和碳化二亚胺交联使支架材料的降解速率明显降低,力学强度增加;支架材料具有良好的细胞相容性。应用离子交联法制备的壳聚糖胶体颗粒粒径为200-300nm,先制备载BSA壳聚糖胶体颗粒,再和支架材料复合,可明显增强缓释作用,延长药物在植入局部存留的时间,体外释放的趋势与体内释放的基本一致。更多还原

【Abstract】 Bone defects caused by disease, trauma, or surgery, increase with years, and threaten the human health. The traditional treatment for bone defects include autografts and allografts, though there are some restrictions, such donor site morbidity and donor shortage for autograft, immunologic response and risk of infecting disease for allografts. Artificial bone substitute materials are also used for bone repair. The biomedical materials used for bone repair and regeneration have developed from the first generation-bioinert materials, the second generation-bioactive materials and biodegradable materials, to the third generation-the biomaterials based on celuller and molecular level. At the same time, the new theories and methods of tissue engineering and in situ tissue regeneration bring the new technology for the treatment of bone defects. The research of the third generation biomedical materials, which combine the bioactivity with biodegradation, is the base for applying the methods of tissue engineering and in situ tissue regeneration for repair of bone defects. So, research and develop osteoinductive and biodegradable bone substitute materials for the need of clinic is the major aspect in the area of biomedical engineering.In this study, simulating the composition and structure of nature bone, we prepared the collagen-hydroxyapaptite(HA) compsite, and incorporated the BMP into the material to develop the biomedical material for bone tissue engineering and in situ tissue regeneration which combine the osteoinduction with biodegradation. The paper includes four parts as following:1.Preparation and characterization of the collagen-HA compositeUsing collagen and HA as main raw material, the collagen-HA composite material was prepared by the process of blending, chemical crosslinking and so on, the structure, physical and chemical properties of the material were characterized, the biocompatibility of the material was evaluated according to GB/T16886 series standard. After the material was contacted with plasma, the capability of the material to absorb plasma protein and effect on calcium(Ca), phosphorus(P) were investigated. SEM photogragh of collagen-HA composite show the material has porous microstructure. It possesses excellent biocompatibility for its chemical, physical and biological properties accord with standards about the biomedical materials. After the material is contacted with plasma, the TP, FiB and FNC in plasma decreased. It indicate the absorption of protein on the surface of the materials, which may benefit for the formation and mineralization of bone. The increasing of Ca and P in plasma is related with the releasing of ions from the material, it may participate in metabolization and accelerate the osteoinduction.2. The ectopic osteoinduction of collagen-HA/BMP compositeThe collagen-HA composite was combined with BMP by absorption, and implanted into the muscle of rat. The ectopic osteoinduction of the material was estimated by histological observation, alkaline phosphatase(ALP) stain and measurement. The results show that Collagen-HA composite and HA which is combined with BMP have obvious ectopic osteoinduction, and the materials which is not combined with BMP have no ectopic osteoinduction.3. New bone formation and segmental bone defects repair with collagen-HA/BMP composite in vivoCollagen-HA/BMP composite was implanted into bone defect about 10mm in the shaft of the radius of rabbits. In order to observe the new bone formation and the degradation process of the material, a series of examination were carried out including blood biochemical test, SEM, biomechanical test and histological observation. The results show that the collagen-HA composite have the capability to repair bone defect and can be biodegraded, incorporation of BMP can accelerate the formation of new bone. The material can be used as the carrier for BMP to sustain its releasing, protect its bioactivity, improve the osteoinduction.4.The research of collagen-HA composite scaffold for bone tissue engineering and the release properties of the scaffold incorporated proteinThe porous collagen-HA scaffolds were prepared by lyophilization. Microstructure and properties of the scaffold was characterized. Chitosan colloid particle carrying model drug BSA was prepared by the method of ionic gelatin, and incorporated into the scaffold. BSA release from the system was studied in vitro and in vivo. The collagen-HA scaffolds used for bone tissue engineering are porous with three-dimension interconnected fiber microstructure, and pore sizes are 50-150μm. Compared with pure collagen, the mechanical property of collagen-HA composite improves significantly. Crosslinking by formaldehyde and EDC both decrease the degradation rate of the scaffold and increase the mechanical property. The scaffold has good biocompatibility and suitable for used as bone tissue engineering.The size of chitosan particle prepared by ion crosslink is 200-300nm. In vivo release studies show that encapsulating BSA by chitosan particle, then incorporating the chitosan particle into the scaffold, would apparently enhance the controlled release effect to prolong the local retention of the protein after implantation. The results of in vitro release studies show good pertinency with respective in vivo results.更多还原