【作者】 沈健坚；

【导师】 丁焕文；

【作者基本信息】 广州中医药大学 ， 中西医结合临床， 2011， 硕士

【摘要】 目的：将组织工程学(TE)、计算机辅助设计(CAD)、快速成型(RP)、逆向工程(RE)、有限元分析(FEA)等技术相结合；通过对比格犬骨髓间充质干细胞(BMSCs)进行分离、培养及定向诱导分化为成骨细胞,为骨缺损修复的种子细胞；计算机辅助设计、快速成型技术制作个性化骨缺损修复支架,在体外联合构建成骨细胞和个性化骨缺损修复支架的组织工程骨支架；采用有限元法对不同内固定方式+个性化骨缺损修复体修复重建骨缺损、计算机辅助设计个性化骨缺损体修复肿瘤型骨缺损进行静力结构分析,为个性化骨缺损修复提供力学依据。方法：1.采用密度梯度离心法和贴壁细胞分离法相结合的方法,对比格犬骨髓间充质干细胞进行分离、培养、扩增、以及骨髓间充质干细胞的生物学特性的鉴定。2.采用成骨细胞诱导液对BMSCs进行定向诱导分化,通过对诱导后的细胞进行形态学、组织化学和免疫组化染色等鉴定。3.计算机辅助设计骨组织工程支架内部结构的基本结构单元；有限元法分析不同基本结构单元的的应力和整体变形情况,筛选获取合适的基本结构单元；计算机辅助设计孔径、孔隙率和连通性可控的骨组织工程支架。4.通过立体光固化成型制作骨组织工程支架的负型模具和外套的光敏树脂模型；在骨组织工程支架负型模具中灌注β-TCP浆体,通过原位凝固和高温烧结,形成孔径、孔隙率和连通性可控的骨组织工程支架。5.将诱导后的成骨细胞复合多孔β-TCP陶瓷支架构建组织工程骨支架,观察成骨细胞在多孔β-TCP陶瓷支架上的粘附和增殖情况。6.计算机辅助建立胫骨骨缺损的三维模型,计算机辅助设计与骨缺损外形相匹配的个性化骨修复体的三维模型；计算机辅助建立Golf钢板和髓内钉固定修复骨缺损的三维模型；采用有限元法分析不同固定方式重建骨缺损后的应力情况。7.计算机辅助建立胫骨近端骨肿瘤的三维模型、测量肿瘤病灶范围、设计肿瘤病灶切除截骨平面；计算机辅助设计个性化骨修复体、建立Golf钢板固定修复骨缺损的三维装配体模型。采用有限元法分析Golf钢板+个性化同种异体骨修复体修复重建膝关节周围肿瘤切除后大段骨缺损的应力、应变分布情况。结果：1.经密度梯度离心法和贴壁细胞发分离获取的骨髓间充质干细胞原代培养14天细胞达90%以上融合,细胞呈长梭形、并沿一定方向放射状排列生长,连续传代培育3代,细胞形态、增殖特性未发生改变；P2代BMSCS的G1期为74.38%、G2期为0.4%、S期为25.23%；BMSCS组织化学染色结果：糖原PAS呈阳性,油红O染色为阴性,碱性磷酸酶染色为阴性。2.采用含有β-甘油磷酸钠、抗坏血酸、地塞米松和10%FBS/DMEM的成骨细胞诱导液对BMSCs诱导培养14天后的细胞呈多边形或多角形；组织化学染色显示：碱性磷酸酶染色阳性、茜素红染色阳性、银染色阳性；I型胶原免疫反应阳性；证实诱导后的细胞为成骨细胞。3.成功实现计算机辅助设计骨组织工程支架不同的基本结构单元；有限元分析证实：开口立方体结构单元力学性能最强、立方体-圆柱结构单元次之、立方体-球体结构单元最差；在计算机辅助设计软件中,可以设计出孔径、孔隙率和连通性可控的骨组织工程支架,以及与支架阴阳互补的负型模具。4.采用快速成型技术,可以制作出孔径、孔隙率和连通性可控的骨组织工程支架负型模具；采用原位凝固成型法结合快速原型技术可以制备孔径、孔隙率和连通性可控的β-TCP陶瓷支架。5.成骨细胞与多孔β-TCP陶瓷支架体外培养7天后,见成骨细胞在支架中粘附、增殖情况良好,HE染色显示：多孔β-TCP陶瓷支架的孔壁均有成骨细胞的粘附生长。6.计算机辅助建立Golf钢板固定和髓内钉联合个性化骨缺损修复体修复骨缺损的三维模型；建立‘’Golf钢板+个性化骨缺损修复体”、“髓内钉+个性化骨缺损修复体”的有限元模型；Golf冈板固定修复骨缺损的最大等效应力为24.37Mpa,髓内钉固定修复骨缺损的最大等效应力13.51Mpa；对于胫骨骨干处的大段骨缺损修复,髓内钉固定较钢板固定合适。7.借助于逆向工程软件和计算机辅助设计软件,成功设计与肿瘤切除后骨缺损病灶外形匹配的个性化骨缺损修复体；有限元分析显示：轴向加压试验中,Golf钢板固定修复胫骨近端肿瘤型骨缺损的最大等效应力为18.26Mpa；扭转试验时最大等效应力为64.466Mpa; Golf钢板+个性化骨缺损修复体修复胫骨近端肿瘤型骨缺损能够提供足够的稳定性。结论：1.骨髓间充质干细胞是骨组织工程研究理想的种子细胞。2.采用计算机辅助设计技术,可以设计出不同内部结构的骨组织工程支架。3.计算机辅助设计-快速成型制作相结合,可以制备孔径、孔隙率和连通性可控的β-TCP陶瓷支架。4.β-TCP具有良好的生物相容性。5.逆向工程-计算机辅助设计相结合,可以设计出与骨缺损病灶外形相匹配的个性化骨缺损修复体。6.有限元分析可以为骨缺损修复重建提供力学依据。更多还原

【Abstract】 Objective:To combine with new technologies, such as tissue engineering(TE), computer aided design (CAD),rapidprototyping(RP), reverse engineering(RE), finite element analysis(FEA), to design and manufacture individual bone defect scaffold. The purpose of this research is for separating and culturing the bone marrow stromal cells from Beagle dog, and induce differentiate to into the osteoblast. Individual bone defect scaffolds are designed and manufactured by using computer aided design and rapid prototyping. To constructe the tissue engineering bone scaffold in vitro with osteoblast and individual bone defectβ-tricalcium phosphate,β-TCP) ceramic scaffold. Using the finite element method to analysy the stress value in difference ways of fixationg with individual bone defect scaffold for reconstructing bone defect, and computer-aided design individual bone defect scaffold to repair tumor bone defect,to provid mechanics parameter in bone defect reconstruction.Method:1. The Beagle dogs’bone marrow stromal cells are separated in the method of the density gradient centrifuge process and pastes wall cell separation law unifies. And The bone marrow stromal cells was cultured, amplificated, and biological characteristics of bone marrow stromal cells was also appraised.2. The bone marrow stromal cells are induced differentiate to into the osteoblast using ascorbic acid,β-sodium glycerophosphate, dexamethasone and 10%FBS/DMEM. The evaluation of morphology, histochemistry, immunity dyeing of the cells after inducing are carried on.3. The bone tissue engineering scaffolds with different basic internal architecture units are designed using computer-aided design. And the stress and total deformity in the basic architecture units are tested in the ways of finite element analysis. After that the bone tissue engineering scaffolds with precise pore size, porosity, connectivity are designed using computer aided design.4. The photoallergy resin of minor and exterior die of bone tissue engineering scaffolds are manufactured using stereo lithography apparatus. After that theβ-TCP ceramic slurry perfuse into the minor die of bone tissue engineering scaffolds, The bone tissue engineering scaffolds with precise pore size, porosity, connectivity,are manufactured using in-situ consolidation and high sintering.5. To construct bone tissue engineering scaffolds with with osteoblast and individual bone defectβ-TCP ceramic scaffold in vitro, surveyed the capability adhere and proliferation of osteoblast after cultured in 7 days.6. The tibial bone defet module is built by computer-aided technologies, and the module of individual bone defect scaffolds matching with the profile of bone defect are designed by computer aided design. The module of bone defect reconstruction with golf plate and intramedullary pin fixation are installed by computer aided design. The value of the stress in different fixation of bone defect reconstruction are measured by finite element analysis.7. The proximal tibia bone tumor module is built by computer-aided technologies,and measure the range of the tumor bouncary, design the plane of tumor resection. The computer-aided design technologies are used to design individual bone defect scaffold,build the 3D module of golf plate fix bone defect. The value of the stress and strain in proximal tibia bone tumor reconstruction with golf plate and individual bone defect scaffold are measured by finite element analysis.Results:1. The bone marrow stromal cells harvest in the method of the density gradient centrifuge process and pastes wall cell separation law unifies. The BMSCs showed long fusiform shape and spread orderly along a direction, had a 90% fusion after cultured 14 days, The form and multiplication characteristic of the BMSCs has not changed after transfer of culture 3 times. The P2 BMSCs has 74.38% in Gl stage,0.4% was in G2 stage, and 25.23% was in S stage. The characterization of BMSCs histochemistry dyeing results showed that the glucogen PAS(+),oil red "0" (－).AKP(－).2. The cells inducted and cultured from BMSCs in 14 days, shows polygon and turn bigger and circler with osteoblast induction nutrient solution,which contain ascorbic acid,β-sodium glycerophosphate, dexamethasone and 10% FBS, DMEM. The characterization of cells histochemistry dyeing results showed that AKP(+), alizarin Bordeaux(+), VON KOSSA(+),I style collagen immunity dyeing(+), The results prove the cells is osteoblast.3. We finished successful the different basic internal architecture units which designed using computer-aided design technologies. The results of the FEA showed that the value of the stress and total deformity has the lowest in open cube unit, cube-cast unit is the second, and cube-spheroplast unit is the worst. We can design the the bone tissue engineering scaffolds with precise pore size, porosity, connectivity and the minor module of the scaffold using computer aided design software.4. The photoallergy resin of minor die of bone tissue engineering scaffolds can be manufacture using rapid prototyping technologies. Theβ-TCP ceramic scaffold with pore size, porosity, connectivity,can be manufactured precisely using in-situ consolidation and rapid prototyping technologies.5. The osteoblast proliferate and adhere very well in the porosityβ-TCP ceramic scaffold after 7 days culture in vitro. The HE dyeing result showed that all of the scaffold conduit wall have osteoblast proliferation and adherent.6. The 3D module of bone defect reconstruction with golf plate and individual bone defect scaffold, intramedullary pin and individual bone defect scaffold were built using computer-aided design technologies. And the finite element module was also built in the FEA software. The value of stress in module of bone defect reconstruction with golf plate and individual bone defect scaffold is 24.37 Mpa, intramedullary pin and individual bone defect scaffold is 13.51Mpa. The intramedullary pin fixation is a good choice for bone defect reconstruction in shaft of tibia.7. With the help of computer aided design and reverse engineering software, We designed successful individual bone defect scaffold matching with the profile of bone defect. In the finite element analysis axial direction pressurize test, the maximum value of stress is 18.26Mpa. In torsion test, the maximum value of stress is 64.466Mpa. The ways of golf plate and individual bone defect scaffold to reconstruct the proximal tibia bone tumor bone defect can provide a enough mechanics stabilize. Conclusion:1. Bone marrow stromal cells is an ideal seed cell for bone tissue engineering.2. The bone tissue engineering scaffolds with different internal architecture are designed using computer-aided design technologies.3. Theβ-TCP ceramic scaffold with precise pore size, porosity, connectivity, can be manufactured using computer aided design and rapid prototyping technologies.4. Theβ-TCP ceramic scaffold is of well biocompatibility.5. Individual bone defect scaffold matching with the profile of bone defect can be designed by computer aided design and reverse engineering technologies.6. Finite element analysis is a good method for providing mechanics parameter in bone defect reconstruction.更多还原