【作者】 李雪盛；

【导师】 孙建军； 孔维佳；

【作者基本信息】 华中科技大学 ， 耳鼻咽喉科学， 2007， 博士

【摘要】 [目的]研究原位骨组织工程技术及其在特殊含气空腔部位修复骨缺损的可能行性,为此技术应用于耳鼻咽喉头颈外科领域的骨缺损修复重建提供实验依据。[方法]1.原位骨组织工程相关基础研究:制备脱细胞松质骨,观察其结构及生物相容性;取兔骨髓基质干细胞,体外培养,复合于含BMP-2的脱细胞松质骨,于皮下移植,观察BMP-2原位诱导骨髓基质干细胞原位成骨分化能力;将BMP-2与脱细胞松质骨复合,异位移植,观察BMP-2原位诱导间充质干细胞成骨分化及促进新生骨形成的能力。2.原位骨组织工程相关应用研究:选择健康兔,制作颅骨标本,观察听泡大体形态及位置,取活体兔听泡,使用耳内镜及病理学方法观察其结构特点。选择耳后手术径路,观察活体兔此径路术中手术标志;采用耳后进路,于兔听泡后外侧骨壁钻孔,清除听泡内部分粘膜组织,植入复合BMP-2听骨赝复物,单纯脱细胞骨小柱植入作为对照。术后3个月,通过大体及病理切片观察听骨赝复物成骨情况;制备复合BMP-2的片状脱细胞松质骨,单侧以生物蛋白胶固定薄层胶原海绵作为修复材料,未加BMP-2材料用做对照。手术去除兔上颌窦外侧骨壁及附着粘膜,胶原海绵侧面向窦腔,原位修复缺损,术后3个月进行大体标本及病理切片观察,了解其修复效果及成骨情况。[结果]1、松质骨经脱细胞处理后,保持海绵状多孔结构,孔径约在200-600μm左右,机械性能无明显变化,植入体内后无明显免疫排斥发生,具有良好生物相容性;体内植入实验术后3个月材料形状无明显变化,周围组织有少量血管增生,无明显免疫排斥现象,脱细胞松质骨/BMP-2/骨髓基质细胞组、脱细胞松质骨/BMP-2组病理学检查发现有新骨形成,对照组仅有纤维结缔组织充填。2、兔听泡体积较大,听骨及内耳结构主要集中于听泡前方,取其耳后切口,沿骨性外耳道后壁,寻找茎突样结构,经听泡后壁可顺利进入听泡,不会损伤重要结构;BMP-2/脱细胞松质骨小柱植入兔听泡后3个月,大体观察材料与听泡骨壁接触部位结合紧密,表面被再生黏膜覆盖,形态无明显变化。病理学检查发现有新骨形成,对照组仅有黏膜覆盖,无新骨形成;BMP-2/脱细胞松质骨材料可很好地修复上颌窦外侧骨壁缺损,窦腔内侧面有可形成新生粘膜覆盖,材料孔内有新生骨形成,对照组仅有纤维结缔组织长入。[结论]1、脱细胞松质骨可保持多孔结构,无明显免疫原性,适用于原位骨组织工程支架材料。2、BMP-2可原位诱导间充质干细胞成骨分化,并可于异位形成新生骨组织。3、耳后切口,经听泡后壁可进入兔听泡,不损伤重要结构,此径路可应用于基于兔听泡的相关实验研究。4、复合BMP-2的脱细胞松质骨可于听泡内形成新生自体骨组织,形态无明显变化,有可能成为听骨链缺损重建材料。5、BMP-2具有诱导成骨能力,胶原海绵可用作粘膜再生支架,脱细胞松质骨/BMP-2/胶原海绵,可用于修复含气窦腔局部骨壁伴粘膜缺损。[意义]本实验为原位骨组织工程技术应用于特殊部位骨缺损修复提供了理论和实验依据。更多还原

【Abstract】 [Objective]To study the in situ bone tissue engineering technique and the ability of this technique to reconstruct the bone defects in special parts contained air, and to provid experimental evidence for reconstructing the bone defects in the aera of otolaryngology–head-neck.[Methods]1. Fundamental research for in situ bone tissue engineering: To prepare the acellcular cancellous bone, observe the structure and the biocompatibility of the materials; To culture the rabbit’s bone marrow stem cells on the acellcular cancellous bone/BMP-2 in vitro, and then transplant the compound into partes subcutanea. We observed the osteogenesis ability of bone marrow stem cell induced by BMP-2 in situ; To add the BMP-2 into the acellcular cancellous bone, and trandplant into allotopia parts, then observe the osteogenesis ability of mesenchymal stem cell induced by BMP-2 in situ .2. Application study for in situ bone tissue engineering: To choose healthy rabbit to make skull bone specimen, observe the shape and position of the bullae. take fresh bullae to observe the detailed structure through endoscope and pathology technique. and then observe the landmarks of the retroauricular approach in a real surgery; Then from retroauricular approach, to make a hole in the posterolateral bone wall of the acoustic vesicle, and the prepared materials were implanted. After 3 month, we observed the osteogenesis of the prothesis with macroscopical anatomy and pathology; To prepare the acellular cancellous bone combined with BMP-2, and collagen sponges lamella were fixed on the material unilaterally by protein glue. The same material without BMP-2 were used in the control group. To remove part of the maxillary sinus lateral bone wall with mucosa, repair it with the prepared material, and the aspects of collagen sponge were put medially in situ. After 3 month, we observed the osteogenesis and reparation of the defects with macroscopical anatomy and pathology.[Results] 1. The acellular cancellous bone maintained porous structure just like sponge, the diameter of the pores were about 200-600μm, and the intension of the materials weren’t significant change than the cancellous bone. No obvious immunological reaction occoured after the materials were transplanted in vivo; In the osteogenesis test, the implanted acellular cancellous bones maintained the shape after they were transplanted 3 month. There were some angiogenesis around the materials, no obvious immune reaction occured. In the acellcular cancellous bone/BMP-2/bone marrow stem cells and the acellcular cancellous bone/BMP-2 groups, osteogenesis were observed. In control groups, no osteogenesis occurred in simple acellular cancellous bone.2. There is a large cavity in the bullae. The rabbit’s ossicle and inner ear are in the anterior of the bullae. From the retroauricular incision, following the posterior bone wall of the external auditory canal, we can find the processus styloideus-like structure. Trough the posterior wall , we can open the bullae with the important structure protected well; After 3 month, the implanted acellular cancellous bones were banded tightly with the bone of the acoustic vesicle bone wall. The surface of all materials were covered with mucosa. No obvious transformation of the materials were observe, and in the material with BMP-2, osteogenesis occurred; In the maxillary sinus wall defects repair test, the rabbits recovered soon after surgery, the implanted acellular cancellous bones were banded tightly with the bone around the defects, the medial aspects of the material were covered with mucosa., and new bone were formed after 3 month.[Conclusion]1. The acellcular cancellous bone can maintain its porous structure, no obvious antigenicity, and it is a suitable material for bone tissue engineering.2. BMP-2 can induce the mesenchymal stem cells differentiate into osteobalsts, and induce osteogenesis in allotopia.3. Through the retroauricular incision and surgical approach, we can enter the rabbit’s bullae easily, and this technique can be used in the experiments based on rabbit’s bullae. 4. The acellular cancellous bone with BMP-2 could induce osteogenesis in acoustic vesicle with primary shape, this material might be use to reconstruct the defects of ossicular chain.5. The BMP-2 could induce osteogenesis, the collagen sponges could support the mucosal regeneration, collagen sponge and acellular cancellous bone combined with BMP-2 could be used to repair the defects of the maxillary sinus bone wall and mucosa.[Significance]This study can provide the theoretic and experimental basis for the bone defects in special parts using in situ bone tissue engineering trchnique.更多还原