【作者】 孙新君；

【导师】 王正国；

【作者基本信息】 第三军医大学 ， 外科学， 2004， 博士

【摘要】 临床上由于创伤、肿瘤切除、感染以及发育异常等原因而导致的骨缺损需要恢复骨骼的连续性、力学支撑以及再生修复。传统的骨缺损修复主要采用自体骨、同种异体骨移植、骨水泥、陶瓷及金属等。自体骨移植存在来源少,且常造成供骨部位并发症,会给病人带来一定的痛苦;而异体骨移植存在排斥反应问题,上述方法均存在不同程度的缺点。骨组织病损的修复可由周围健康的成骨前体细胞被趋化或植入缺损局部,通过再生最终实现。基于这种设想,组织工程,即细胞——基质(天然或人工合成材料)复合移植试图利用这一方法构建骨缺损修复的替代物。由于细胞必须依赖于细胞外基质的存在才能发挥其功能,基质材料必须具备三维结构,并有促进细胞粘附、分化的成分,而且还需要为组织再生提供一定的力学强度。因此,在骨组织工程中,细胞外基质替代物即种植基质材料的选择是一个重要方面。人工合成的高分子聚合材料因为具有适合成骨作用的前体细胞移动、增殖和分化的网状孔隙结构,常被用作促进骨组织再生的基质材料。但因生物力学特性差、代谢产物对周围组织的毒性作用、应力疲劳及对骨组织的应力遮挡等缺点,使其应用受到限制。其它材料中,天然材料衍生物,如各种方法处理的异体、异种脱钙骨等衍生材料,临床应用表明有促进骨缺损修复的作用。尽管动物实验及临床骨缺损应用表明脱钙骨基质(DBM)具有较好的生物相容性和吸收特性,但由于其力学特性差,而且有一定的抗原性,难以满足骨组织工程材料的要求。因此,研究和开发理想的骨组织工程基质材料有待进一步深入。研究显示脱细胞基质材料,如脱细胞真皮及血管等保留了良好的力学特性、生物相容性、生物降解特性,粗糙的表面和形状有利于细胞的粘附及生长,经处理后可避免疾病传播和免疫排斥反应。因此,我们有理由推测:经加工而成的脱细胞骨基质(ACBM)作为支架材料在骨组织工程中的应用前景广阔。本研究首先制备ACBM,并对其生物力学特性,生物相容性,骨诱导和骨传导作用进行评价。在此基础上,用纤维蛋白胶FBG,将具有骨诱导活性的重组人骨形态发生蛋白2(rhBMP-2)与ACBM复合在体外构建组织工程骨缓<WP=12>释载体,并观察材料的缓释作用。然后,将新西兰大耳白兔的骨髓间充质干细胞(MSC)与材料复合并观察对兔桡骨缺损的修复效果。主要结果和结论:本研究从构建组织工程骨的细胞外基质材料入手,将异种骨经系列处理后制备成具有适合管状骨修复的ACBM;体内外实验表明,ACBM具有低抗原性和良好的生物相容性。在此基础上,用人FBG将外源性rhBMP-2与ACBM复合;然后,分离、扩增MSC并在体外诱导分化;将细胞与上述材料复合后,体内植入修复兔桡骨骨缺损效果良好,得出以下主要结论。1.本实验采用理化联合生物化学的方法,实现了异种骨的脱细胞去抗原处理。体内外实验表明,对植入受体的细胞及体液免疫无明显影响,而且具有良好的生物安全性。2.优化了制作过程。分析表明,其主要成分胶原羟基磷灰石与正常骨组织相似。组织学观察表明,在制作中其形态结构无破坏,生物力学强度与新鲜骨组织无统计学差异。与现有的骨衍生材料比较,ACBM保留了骨组织的原有结构和生物力学强度,可作为骨缺损,尤其是管状骨缺损的修复材料。3.超微结构观察显示,ACBM为适合种子细胞生长的三维多孔支架材料,具有天然骨的网孔和孔隙率。体外种植发现,材料有利于种子细胞的粘附及活性表达;体内植入表明,ACBM具有骨传导和骨诱导双重作用。4.本研究结果表明,具有生物力学强度的块状ACBM材料,在修复骨缺损早期,其成骨量大于无力学强度的骨粒材料。适当的生物力学强度的材料有利于对骨缺损的修复。5.将FBG引入ACBM,用两步法将rhBMP-2复合进入ACBM,从而制备rhBMP-2的缓释载体。超微结构观察可见,复合材料的网孔结构及网孔率与复合前无明显差别。体外观察证实,缓释载体在4w内能够稳定而有效地释放rhBMP-2,达到缓释目的;体内植入修复骨缺损研究表明,复合rhBMP-2后,能够促进对局部组织中成骨前体细胞的趋化、诱导,加速对骨缺损的修复。6.本实验分离的MSC,体外具有成骨、成脂肪诱导分化的潜能;体外扩增及成骨诱导分化表明,无论其“质和量”均能够满足构建组织工程骨的需要;rhBMP-2的成骨诱导进一步表明细胞与材料复合的可行性。7.修复骨缺损的结果表明,4-8w,细胞-材料复合物对兔桡骨节段性骨缺损的修复效果优于无细胞的rhBMP-2/ACBM材料,而后者又较单纯ACBM材料修复为好。<WP=13>因此,材料-细胞复合物可以加速成骨,更利于骨缺损的修复。8w后,虽然成骨量在前二者无统计学差异,但在新骨的重建和塑型方面仍以细胞-材料复合移植效果为好。8.荧光小鼠MSC与材料复合移植后的示踪观察表明,12w时,表达绿色荧光蛋白的小鼠MSC不但可以成活,而且能够发挥成骨作用。更多还原

【Abstract】 Critical size bone defects arising from trauma, tumor resection, infection and skeletal abnormalities require assistance to provide skeletal continuity, mechanical support and eventual regeneration. Traditionally, materials strategies to address bone defects include the use of autogeneous grafts and flaps, allograft bone, non-degradable bone cement, metals and ceramics. All of these options have their associated problems such as the finite amount of tissue that can be harvested for autografts, the difficulty to shape grafts into the desired form, and the risk of immune rejection or disease transfer with allografts.The regeneration of fractured bone is based on the hypothesis that healthy progenitor cells, either recruited or delivered to an injured site, can eventually replace the lost or damaged bone tissue. Based on this suggestion, tissue engineering approaches attempt to create tissue replacements by culturing cells onto nature or synthetic three-dimensional matrixes. It is difficult to repair tissues by using cultured cells alone. In fact, a biomaterial with appropriate composition and three-dimensional structure to promote cellular adhesion, differentiation and the mechanic strength to provide as a scaffold for tissue regeneration is needed. So, it is very important for materials to be used in bone engineering.Three-dimensional porous polymeric matrixes are seen as one approach to enhance bone regeneration by creating and maintaining channels that facilitate progenitor cell migration, proliferation, and differentiation. However, their application is limited because of some shortcomings such as poor mechanical properties, toxicity of degradation product to cells and stress shielding to the surrounding bone or fatigue failure of the implants. For other materials to be considered, among which are natural bone derived biomaterials. DBM for example, has been shown to accelerate wound healing, and be used as a biocompatible and absorbable material in both animal and human bone defects. However, these materials can not meet the requirements of bone tissue engineering because of their poor mechanical properties and allergic reactions. Therefore, the research of improving ideal materials in <WP=9>bone engineering should be continuously carried out.Acellular matrixes, such as dermis and blood vessels had been shown to have good mechanical properties, biocompatibility, biodegradability. Also, their gross architecture and shape facilitate cells adhesion and proliferation. At the same time, allergic reactions and disease transmission may be avoided after treatment.Therefore, it is reasonable to predict that ACBM can be used as a prospective candidate to form a structural framework for bone engineering. In this study, ACBM material was prepared and evaluated for mechanical properties, general biocompatibility, effect of osteoinductive and osteoconductive. Subsequently, it was combined with rhBMP-2 by fibrin gelatin to constitute the slowly releasing carrier of tissue engineering bone. The scaffold was seeded with mesenchymal stem cells harvested from bone marrow of NZW rabbits to evaluate their performance as potential bone substitutes in vitro and in vivo.The main results and conclusions are as follows: 1. United with physical, chemical and biochemical methods, we successfully realized deantigenic treatment of xenogeneic bone in the formulations of ACBM. In vivo and in vitro evaluation of cellular and humoral immunity in recipients proved that ACBM has lower or no antigen with a good reliability. 2. Not only the components and structure of bone are being preserved in ACBM, but also the mechanical properties are similar to that of fresh bone. Compared with natural derived bone matrixs existing now, ACBM reserves the original structure and biomechanics. So, it is a suitable material for the repair of bone defects, especially for segmental bone defects in tubular bone.3. Scanning with SEM, it proved that the three-dimensional porous ACBM maintain channels that facilitate progenitor cell migration, proliferati更多还原