【作者】 陈铎；

【导师】 王学礼；

【作者基本信息】 河北医科大学 ， 人体解剖与组织胚胎学， 2003， 博士

【摘要】 由于疾病和外伤等原因所造成的组织或器官功能障碍或丧失，是人类健康面临的重要危害之一，也是人类疾病和死亡的最主要原因。近十几年来，随着细胞生物学、生物材料科学、化学及工程学突飞猛进的发展和各学科间的交叉渗透，组织工程学（tissue engineering）作为一门新兴边缘学科应运而生，并得到了迅猛发展，其目的是应用这些学科的原理与方法，研究和开发组织或器官替代物。其基本方法是制备具有三维空间结构的支架，将支架与活细胞复合，加以各种生长因子(grouth factors)，形成具有细胞生命特征、适应受体组织器官要求的复合体，为细胞及生物活性因子提供新陈代谢、生长发育和有效发挥活性的场所，也是形成新的具有形态和功能的组织、器官的物质基础。组织工程技术被认为是未来组织、器官缺损修复和再造的最有效方法之一。骨组织再造和骨缺损修复一直是骨科医生面临的难题之一，组织工程学的创立和发展为解决这一难题提供了新的思路和方法。骨组织工程(bone tissue engineering)是目前组织工程研究的热点问题之一，内容主要包括：①细胞种植基质材料的研究开发；②种子细胞性质的研究；③与促进骨再生有关的生长因子方面的研究。其中，细胞种植基质材料的研究是骨组织工程研究的重点内容，也是能否应用于临床的重要因素之一。本研究采用NaOH消蚀脱细胞技术制备出家兔长骨密质骨及松质骨骨细胞外基质（简称骨基质）材料作为支架，分别以新鲜骨髓及胚胎骨组织来源的成骨细胞作为种子细胞，以重组人骨形成蛋白（recombinant human bone morphogenetic protein，rhBMP2）作为骨形成的促进因子，对家兔进行了体内异位成骨及桡骨节段性骨缺损修复的实验研究。第一部分 采用NaOH消蚀技术制备骨组织工程支架材料及其生物相容性实验研究目的：研制一种新型的天然骨组织工程载体，观察其结构特征并<WP=5>探讨其在活体内的生物相容性及生物降解性。方法：取健康家兔桡骨骨干及髂嵴松质骨，经2%戊二醛/4%多聚甲醛混合液交联后，室温下6%NaOH水溶液消蚀脱细胞7天，蒸馏水超声震荡清洗，彻底清除细胞碎片及残存药液，直至清洗液清澈透明，pH值＜7，烘干。对制备好的两种骨基质材料行扫描电镜(scanning electric microscope,SEM)观察、生物力学测定、埋植于动物体内观察其生物相容性及可降解性，同时还对埋植前后的材料进行了X射线能谱化学元素分析，通过测定其化学元素的变化，了解其降解吸收的情况。结果：①骨基质材料的SEM特征 骨组织经NaOH消蚀及超声震荡清洗后，细胞成分被彻底消蚀掉。密质骨骨基质的主要成分胶原纤维和骨盐仍维持原有形态及结构特征，胶原纤维聚集、排列成规则的网状支架。骨盐沿胶原纤维排列。松质骨呈疏松的彼此沟通的多孔样结构，表面略粗糙，可见胶原纤维纹理。②生物力学测试结果 密质骨基质材料的三点抗弯曲强度结果为（0.0808±0.008）N，抗压缩强度为（0.2453±0.078）N，对照组结果分别为（0.1035±0.017）N和（0.4675±0.07）N，经NaOH消蚀后，力学强度低于正常骨组织(P＜0.05，P＜0.01）。③生物相容性实验结果 将埋植于家兔背部肌肉中的两种支架材料分别于术后1周、2周和4周取出。可见密质骨周围有纤维结缔组织包裹，少许淋巴细胞浸润和新生毛细血管；松质骨材料于埋植一周后即有结缔组织长入松质骨基质的孔隙内。随着埋植时间的延长密质骨支架结构逐渐疏松，松质骨支架逐渐减少，被降解吸收。④骨支架X射线能谱化学元素分析结果 经NaOH消蚀后，密质骨基质材料的主要化学元素仍为钙和磷。埋植后钙原子含量（%）为（68.18±6.13），明显低于埋植前水平（91.54±2.29），两者间有极显著差异（P＜0.001）。结论：经NaOH消蚀后的骨组织具有以下优点：①胶原纤维不受损伤且保持正常结构，骨盐成分羟基磷灰石未被破坏，使该支架仍具有一定的生物力学强度，适宜修复较大的节段性骨缺损；②NaOH去除了骨组织中的细胞、脂类及杂蛋白，降低了支架的免疫原性，组织相容性好；③在体内易于降解吸收；④制作工艺简便，可根据缺损形状而塑形。因而可将其作为种子细胞和生长因子的载体用于骨组织工程。<WP=6>第二部分 骨基质支架复合rhBMP2及自体红骨髓修复兔桡骨节段性骨缺损的实验研究目的：探讨骨基质支架复合rhBMP2及自体红骨髓修复兔桡骨节段性骨缺损的成骨效果。为该支架作为种子细胞和生长因子载体可行性提供进一步的实验依据。方法：将两种支架与自体红骨髓（autologous red marrow ，ARM）和rhBMP2复合后，植入保留有骨膜的兔桡骨15 mm节段性骨缺损动物模型中。分为A、B两个实验组，A组移植密质骨基质支架（compact bone matrix frame, CBMF），又分为A1组、A2组、A3组分别移植CBMF+ARM+ rhBMP2、CBMF+ARM及CBMF+rhBMP2；B组移植松质骨基质支架（pongy bone matrix frame,SBMF），又分为B1组、B2组、B3组，组合方式同A组；对照组分为D1组（单纯CBMF）和D2组（单纯SBMF）。ARM自髂嵴处抽取，每侧植入约0.5ml，注入密质骨管腔或松质骨孔隙内，再将复合有0.8mg rhBMP2的胶原膜贴附于支架表面，骨膜原位缝合。分别于术后4周、8周、12周观察X?更多还原

【Abstract】 The loss or failure of an organ or tissue is one of the most severe human health problems. It is also the main reason of human diseases and death. During the last two decades, because of the enormous develepment and intersection between cytobiology and biomaterial engineering science, tissue engineering has forged ahead rapidly as a novel frontier science. Its purpose is to research and invent the ideal tissue and orgen substitues based on principles and technique of those scientific disciplines. For this purpose, a complex should be preparaed by using a three-dimensional frame, a number of active seed cells expanded in vitro and some kinds of growth factors. The complex can provide an excellent environment of metabolism, proliferation and effective performing activity for seed cells and biological active growth factors. It is also the material base for forming the new tissues and organs which possess normal structure and function. Tissue engineering technology has been universally acknowledged as one of the most effective therapies for defective repairing and restructuring of some tissues and organs.The bone tissue reconstruction and reparation are one of the difficulties facing by orthopedist. Tissue engineering provides a new thought and way for solving these problems. Bone tissue engineering is a hot spot among the reseaches nowadays. Its main content includes three aspects of study, ①scaffolding materials which cells can be seeded on; ②seed cells characteristics; ③growth factors related with accelerating bone reconstruction. Of which, the first one is the core for the tissue engineering and the key if it can be for the clinical application. In this study, we took some long compact bones and spongy bones from rabbits to prepare the extracellular matrix material by applying NaOH cell-maceration method<WP=12>for the carrier of seed cells, autologous red marrow and osteoblast came from rabbit embryo’s bone tissue and rhBMP2. We carried out a series of experimental studies to repair the segmental radius defects and allotopic osteoanagenesis in rabbits.Part One: Experimental Studies of Preparation of the Bone Tissue Engineering Frame Material by NaOH-Maceration Method and its BiocompatibilityObjective： To prepare a new natural bone tissue engineering carrier, observe its structure and research its biocompatibility and biodegradation in vivo.Methods：Radial diaphyses and iliac crests taken from the healthy rabbits were cross-linked with 2%glutaradehyde/4%paraformaldehyde mixed solution and immersed in 6%aqaeous solution of NaOH for 7days at room temperature, and then rinsed in distilled water for a couple of days, using ultrasonic wave (USW) cleaning to clear cellular pieces and removed reagent untill the distilled water was clear and pH＜7. Then putting it in the drying oven to make them dry. A series of examination about the materials were carried out including scanning electron microscope (SEM), biomechanics, experiment of biocompatibility and biodegradation in vivo and chemical composition analysis before and after implanted.Results：①SEM characteristic of the bone extracellular matrix material: Maceration of the bone tissues with a NaOH solution followed thorough washing by distilled water and USW effectively and sheerly removed the cellular elements. The main composition of extracellular matrix of compact bone, collagen fibrils and minerals, were preserved in the natural position and structure characteristic. The collagen fibrils arrangement formed networks and the minerals adhering to the surfaces of the collagen fibrils. The spongy bone treated by NaOH present with numerous orifices interlinked. Its surface looked rough and collagen fibrils’ streaks could be seen on the surface. ②Results of the biomechanics: The<WP=13>results of resistant bending strength at three points and the resistant compressed strength of compact bone extracellular matrix frame(CBMF) were （0.0808±0.008）N, and（0.2453±0.078）N.The control group were（0.1035±0.017）N and（0.4675±0.07）. The mechanical更多还原