【作者】 彭浩；

【导师】 罗成群；

【作者基本信息】 中南大学 ， 烧伤外科， 2009， 博士

【摘要】 背景运用组织工程骨是修复因创伤(烧伤)、炎症、肿瘤导致的各种骨缺损的主要发展方向,而其中培养具有正常骨的基本形态和基本功能的功能骨是研究工作的主要目标。近年来,骨组织工程的研究在种子细胞,支架材料,培养手段等方面取得了很大进展,干细胞培养和纳米材料技术在骨组织工程中得到运用。目前组织工程骨的构建大多仍沿袭直接将单个种子细胞种植于支架在体内成骨这一基本模式,将组织工程骨广泛运用于临床仍需克服大量技术问题,主要包括:种子细胞的体外规模化扩增方法;支架材料的选择;种子细胞与支架材料有效均匀的结合;大块组织工程骨的中心供养;组织工程骨的体外最佳培养时间及方法;组织工程骨中支架材料的降解残留问题;体内组织工程骨的血管化;不同形态组织工程骨的构建等。构建具有正常骨的基本形态和基本功能的功能性组织工程骨是目前骨组织工程研究的热点问题,也是组织工程骨广泛运用的基础。目的采用2周-8周兔的骨髓间充质干细胞(Bone marrow cellsBMSCs)作为种子细胞,电纺纳米仿生材料聚已内酯-Ⅰ型小牛胶原(polycaproactone collagenⅠPCL-CO)作为支架材料,运用细胞成膜技术将兔BMSCs膜与PCL-CO在旋转式组织反应器中培养形成管状骨后埋植于裸鼠皮下,进而组织工程出血管化的功能骨。通过力学测试、组织切片、免疫组化及现代分子生物学等方法分析组织工程管状功能骨的机械强度,组织结构及细胞功能,此组织工程方法为进一步在临床上运用组织工程骨修复骨缺损奠定实验基础。方法1.采用密度梯度离心及贴壁培养相结合的方法分离兔的BMSCs,进行纯化扩增,对其细胞生物学特性进行观察;使用传代2-3期细胞进行三相诱导成为成骨细胞,脂肪细胞及软骨细胞。2.电纺合成电纺纳米纤维PCL-CO采用细胞活力检测,电镜检测,激光共聚焦显微镜检测电纺纳米纤维PCL-CO的基本结构,生物相容性。3.将经成骨诱导后两周的兔BMSCs细胞膜片(cell-sheet)与电纺纳米纤维PCL-CO膜复合,在旋转式组织反应器中继续诱导分化动态培养4周及8周后,进行组织切片HE、茜素红、番红-快绿染色(safaranin-O/fast green)和免疫组化检测Ⅰ型胶原、OCN、OPN、Ⅱ型胶原表达及组织生物力学测试。4.将经过在旋转式组织反应器中诱导分化动态培养8周形成的管状功能骨植入裸鼠皮下,观察局部组织反应情况,分别于植入后4周、8周取出,进行组织切片和免疫组化检测Ⅰ型胶原、OCN表达及生物力学测试。结果1.采用密度梯度离心及贴壁培养相结合的方法可分离得到纯化2周-8周兔的BMSCs细胞在合适的诱导条件下,体现出三相分化能力,在相应的诱导中,细胞表现出特异的生理生化反应以及特异蛋白和组织学特征。2.电纺纳米纤维PCL-CO有良好的物理特性及细胞相容性。3.BMSCs细胞膜片与电纺纳米纤维PCL-CO膜复合以后,在成骨诱导下及生物反应器中,可以形成有机械强度的和骨特异蛋白表达的组织。4.体外培养的人工组织可以在体内形成高度血管化的管状骨,机械强度可超过200MPa,组织中含有软骨及松质骨结构,大部新生骨组织为软骨内成骨。结论1.采用密度梯度离心及贴壁培养相结合的方法分离的2周-8周龄的兔BMSCs在合适的培养条件下,具有细胞性状稳定,纯化扩增较容易,在特定的诱导条件下,能向成骨细胞、软骨细胞、脂肪细胞等不同细胞分化,是理想的组织工程种子细胞。2.采用电纺技术合成的纳米纤维PCL-CO对兔BMSCs细胞的增殖活性无影响,对BMSCs的吸附性较好,是一种较理想的组织工程材料。3.运用Cell-sheet技术在生物反应器的动态培养下,兔BMSCs细胞膜片结合纳米可降解电纺纤维支架技术培养出不断生长的组织工程骨,该方法及技术系在骨组织工程的首次尝试。4.兔BMSCs细胞膜片/纳米PCL-CO组织工程骨埋入裸鼠皮下后能够以软骨成骨的方式形成高度血管化的管状骨。5.将兔BMSCs细胞膜片/纳米PCL-CO组织工程骨埋入裸鼠皮下成骨模型,可以为研究预构复合组织骨瓣(皮瓣复合组织工程骨骨瓣)提供试验基础。更多还原

【Abstract】 BackgroundBone tissue engineering research hold great potential for treating bone defect caused by trauma(burns),inflammation and tumor.The main objective of the study was to tissue engineer the functional bones which have the mechanical force and function of normal bones.In recent years,the vast progress of the cell seeding technology and scaffold materials research have made functional bone tissue engineering possible.Currently,the construction of bone tissue engineering still follows the basic model that single cell suspension were seeded into scafolds directly differentiate into bone in vivo.Moreover,the application of engineered bone in clinical encountered numerous technical problems.These problems consisted of the large-scale amplification of seed cells in vitro,the selcetion of the scaffold materials, the effective and uniform combination of the seeded cells and the scaffold materials,the central support large piece of tissue engineering bone,the optimal cultivation techniques,degradation of the scaffold, vascularization of the tissue engineering bones in vivo,the construction of the tissue engineered bones of different forms,etc.The reconstitution of functional bones with suitable mechanical force in vitro is the key points of the bone tissue enginerring research.ObjectiveThe rabbit(from 2 weeks to 8weeks) BMSCs(bone marrow stromal cells) were used as seeded cells in this study,and the electric nano-biomimetic material PCL-CO(polycaprolactoone-collagenⅠ) was used as scaffold materials.The cell sheet technique will transformed the BMSCs membrane and PCL-CO to tubular functional bones in the rotary bio-reactor.The tubular functional bones were implanted subcutaneously in the nude mice.Diffrent analysis from the mechanical testing,histological section,immunohistochemistry to molecular biology were applied in study.The research results would play an important role in bone defects repair and functional tissue engineering area. Methods1.Rabbit BMSCs were purified and amplified with the density gradient centrifugation and adherent culture method.Cells were well characterized by cell growth,cell morphology and multi-induction potential and so on.Passage of 2-3 cells were directly differneitated into osteoblasts,adipocytes and chondrocytes.2.Characterization of the electric spinning nanofiber PCL-CO’s basic structure,biocompatibility through cell viability testing,electron microscopy,laser scanning confocal microscope,etc.3.The compound of the cell sheet of the rabbit BMSCs and the electrospin PCL-CO nanofibers were contructed,differentiated and dynamically cultivated in rotary tissue reactor for 4 and 8 weeks. Utilization of histological section,HE stain,alizarin stain, safranine-O/fast green stain and immunohistochemistry to detect the expression of collagen typeⅠ、collagen typeⅡ、OCN and OPN. Samples were underwent biomechanics analysis as well.4.Implantation the in vitro engineered tubular bones subcutaneously in the nude mice and observation of regional tissue reaction.After 4 and 8 weeks implantation,samples were analysised by histological section and immunohistochemistry as well as biomechanics test.Results1.Protocol of the combination of the density gradient centrifugation and adherent culture method could be applied to isolate and purify rabbit BMSCs,which presented the ability of three-stage differentiation under appropriate induction conditions.Cells showed specific physiological, biochemical reactions as well as the specific protein and histological characteristics.2.Electric spinning nanofiber PCL-CO possed satisfactory physical characteristic and cell compatibility.3.The compound of the cell sheet of BMSCs and PCL-CO membrane formed the tissue with suitable mechanical strength and the expression of bone-specific proteins under the osteogenesis induction in bioreactor.4.The artificial tissue grew into vascularized tubular bones with the mechanical strength more than 200MPa in nude mice.The tissue contained cancellous bone structure and chondrocytes,indicating endochondral ossification.Conclusions1.The rabbit BMSCs isolated by the method of the combination of the density gradient centrifugation and adherent culture method posseed stable cell trait,and it was easy to be purified and amplified.It should be the ideal seed cells since it could be differentiated into osteoblast, cartilage cells,fat cells.2.PCL-CO had no negative effects on the proliferative activity of rabbit BMSCs and it presented good biocompatility.It may be one of ideal tissue engineering materials for bone.3.Under dynamic culture in a rotary bioreactor through cell-sheet technique,the co-constructed complex of BMSCs sheet and PCL-CO membrane formed growing tubular bone-like tissue.4.The enginerred bones implanted subcutaneously in the nude mice formed highly vascularized tubular bones by the way of cartilaginous osteogenesis,containing both cartilage and cancellous bone structure.5.The model of function bone engineering with the rabbit BMSs cell sheet and nano-PCL-CO subcutaneous tissue engineering bone provided an experimental basis for clinical bone defeat repair and prefabricating composite flaps(skin flap/tissue engineering bone flaps).更多还原