【作者】 王华楠；

【导师】 李玉宝；

【作者基本信息】 四川大学 ， 生物医学工程， 2007， 硕士

【摘要】 骨组织工程的研究和发展为临床上骨缺损的修复和治疗提供了崭新的思路，并逐渐引起研究者的兴趣。其中，三维多孔支架材料是骨组织工程的重要基础，它不仅起支撑作用，保持原有组织的形状，而且还起到模板作用，为细胞提供赖以寄宿、生长、分化和增殖的场所，从而引导受损组织的再生和控制再生组织的结构。但目前骨组织工程支架的研究和应用仍面临着许多问题，如材料体内降解不可控，细胞繁殖效率低，降解产物引起炎性反应，细胞去分化等。尤其是在力学性能方面，单一的使用聚合物或陶瓷材料作为支架材料存在强度弱或脆性大的问题，都无法满足承力骨修复的要求。因此，为了解决目前骨组织工程支架存在的缺陷，满足支架的性能要求，我们使用具有良好生物学性能和与人体皮质骨相匹配力学性能的n-HA／PA66复合生物材料构建三维多孔支架，期望得到兼具良好的生物学性能和力学性能的支架材料。本研究采用相转移法(phase inversion)和相转移结合粒子沥滤法(particle leaching)的复合工艺制备了骨组织工程用多孔n-HA／PA66复合材料支架。用扫描电子显微镜(SEM)、X射线衍射(XRD)、红外光谱(IR)和力学性能测试等手段对多孔支架的理化性能进行表征。结果显示，使用相转移结合粒子沥滤工艺制备的n-HA／PA66复合材料支架具有较高且可控的孔隙率(90％)和孔隙贯通性；且该多孔支架表现出与人体松质骨相当的力学强度，足以满足骨组织工程支架的力学性能要求。骨髓间充质干细胞(Marrow-derived mesenchymal stem cells，MSCs)由于其易提取分离、易体外培养和在特定环境中定向分化的特点而广泛作为骨组织工程的种子细胞。本研究使用兔MSCs作为骨组织工程的种子细胞，并将其种植于n-HA／PA66支架上进行体外联合培养，以考察复合材料支架的体外生物相容性。同时，实验引入兔的下颌骨部位的极限骨缺损作为动物实验的模型，将种植有MSCs细胞的n-HA／PA66多孔支架和单纯n-HA／PA66支架植入缺损部分，通过考察支架材料的体内生物活性和成骨性能来研究n-HA／PA66复合材料骨组织工程支架修复骨缺损的可行性。我们使用光学显微镜、SEM、四唑盐比色法(MTT)检测、碱性磷酸酶(ALP)和Ⅰ型胶原(COLⅠ)免疫组化分析对MSCs在n-HA／PA66支架体外培养过程中细胞生长、繁殖和分化进行定性和定量的分析；并采用组织学切片、X光片分析和新骨计量学分析来定性和定量考察n-HA／PA66支架的体内生物活性和成骨性能。结果表明，n-HA／PA66复合材料骨组织工程支架对细胞的黏附、生长、繁殖和骨向分化均无不良影响，具有良好的体外生物相容性；与MSCs复合的n-HA／PA66支架具有较单纯支架更高的植入初期成骨效率，而长期体内实验显示两者的生物活性和成骨性能相当。这些结果都说明n-HA／PA66支架具有良好的生物相容性和体内成骨性能，是组织工程修复骨缺损的理想支架材料。同时n-HA／PA66多孔支架与MSCs的复合体系，在骨修复过程中表现出良好的骨重建能力，是一种有效的骨组织工程治疗骨缺损的方法。目前骨组织工程支架普遍存在的另一问题是新生骨组织往往在支架的外缘形成，这阻止了细胞渗透到支架内部，也妨碍了支架内部体液的相互交换，并最终导致支架内部长入组织的坏死。通常的支架材料是具有不规则、各向同性的孔隙结构以及相对较低的孔隙贯通性，这种结构也使得细胞和组织很难渗透到支架内部，而影响骨组织的重建。为了解决这一问题，制备出具有类似于自然骨在结构和性能上各向异性的特点的骨组织工程支架，促进细胞和组织向多孔支架内部生长，并提高养分和代谢产物交换的效率，我们设计并改进相转移工艺，来制备在孔隙结构和力学强度上都具有各向异性特点的n-HA／PA66骨组织工程支架。我们对该支架材料的制备工艺、理化性能、微观形貌进行评价，并对这种具有各向异性结构的支架进行体外细胞实验。结果表明，使用改进相转移法制备的n-HA／PA66多孔支架具有定向性多孔、各向异性的孔隙结构；其力学性能也表现出各向异性，支架平行于孔隙方向的抗压强度明显高于垂直于该方向的抗压强度；而细胞实验表明MSCs和成骨细胞更倾向于沿着孔隙方向生长、攀爬和增殖。更多还原

【Abstract】 The development of bone tissue engineering brings forward a promisingalternative for the clinic treatment on bone defects, and attracts gradually increasinginterest from the scientific researchers. In bone tissue engineering, scaffold served asthe matrices of tissue formation plays a pivotal role, and has to fulfill a few basicrequirements, that is, high porosity and proper pore size, required surface propertiespermitting cell adhesion, differentiation and proliferation, desirable mechanicalintegrity to maintain the predesigned tissue structure, non-cytotoxicity andosteoconductivity.The selection of the most appreciate material to produce a scaffold to be used inbone tissue engineering application is a critically important step towards theconstruction of a tissue engineered product. Hydroxyapatite (HA) has beenconsidered to be the ideal material to build bone tissue engineering scaffold due toits osteoconductivity and osteoinductivity. But its brittleness and poor performanceof mechanical stability limit its use for the regeneration of load-bearing bone defects.On the other hand, biocompatible polymers have also been regarded as the propercandidates for tissue-engineered scaffolds. However, a number of tough practicalproblems still persist, that is, difficulty in controlling the in vivo degradation ofbioresorbable polymers, low efficiency of cell seeding, cytotoxicity of thebreakdown products produced during scaffold degradation, and more commonlyhappened, poor mechanical properties incomparable with natural hard tissues.To overcome these shortages presented by bioceramics and biopolymers,ceramic/polymer composite scaffolds, and prepare three-dimensionally porous structure with adequate mechanical properties, we fabricated a novel compositetissue-engineered scaffold using n-HA/PA66 composite biomaterials which hasalready been proven to possess both good bioactivity and comparative mechanicalstrength to that of cortical bone. In this study, we employed phase inversion andphase inversion in combination with particle leaching processing methods to prepareporous scaffold. Then the morphologies and properties of the scaffolds werecharacterized by scanning electronic microscope (SEM), X-ray diffraction (XRD),infrared spectroscopy (IR) and mechanical testing. The results indicated thatn-HA/PA66 composite scaffold possessed ideal pore size (100～500μm) andinterconnectivity suitable for new bone ingrowth, and the porosity can be controlledby changing the parameters of the processing method. Moreover, the scaffoldexhibited good mechanical strength that was similar to the cancellous bone(2～10MPa). Because of its non-biodegradability, this composite scaffold is able toprovide a long-term stable support for the regeneration of new bone.On the other hand, the other important aspect of bone tissue engineering is theintroduction of bioactive cells into the three-dimensionally porous scaffold.Mesenchymal stem cells (MSCs) are present in many human tissues and can bedirectly derived from marrow, easily isolated, easily cultured and rapidlyproliferated in the laboratory setting. More importantly, MSCs serve as a readilyavailable source of undifferentiated cells that are capable to give rise to diversetissues, including bone, cartilage, tendon, muscle and so on. The advantages ofMSCs encourage us to introduce these cells into n-HA/PA composite scaffolds forbone tissue engineering application. Therefore, in this study, we investigate thebiocompatibility and cytotoxicity of the n-HA/PA composite scaffolds in directcontact with MSCs by in vitro tests, and study the in vivo biocompatibility andosteogenesis of the MSCs hybridized n-HA/PA scaffold in the animal experiments.The MSCs/scaffold constructs were cultured for up to 7 days and the adhesion,proliferation and differentiation of MSCs into osteoblastic phenotype weredetermined using MTT assay, alkaline phosphatase (ALP) activity and collagen typeⅠ(COLⅠ) immunohistochemical staining and scanning electronic microscopy (SEM). The results confirm that n-HA/PA scaffolds are biocompatible and have no negativeeffects on tile MSCs in vitro. To investigate the in vivo biocompatibility andosteogenesis of the composite scaffolds, both pure n-HA/PA scaffolds andMSCs/scaffold constructs were implanted in rabbit mandibles and studiedhistologically and microradiographically. The results show that n-HA/PA compositescaffolds exhibit good biocompatibility and extensive osteoconductivity with hostbone. Moreover, the introduction of MSCs to the scaffolds dramatically enhancesthe efficiency of new bone formation, especially at the initial stage afterimplantation. In long term (more than 12 weeks implantation), however, the purescaffolds show as good biocompatibility and osteogenesis as the hybrid ones.Nowadays, another common problem encountered when using such scaffolds fortissue engineering is the rapid formation of tissue on the outer edge, which leads tothe development of a necrotic core due to the limitations of cell penetration andnutrient exchange. Most tissue-engineered scaffolds with irregular and isotropicporous architecture normally possess relatively low interconnectivity, which makesit difficult for the cells and newly formed tissue to penetrate into the center ofscaffold and influences bone reconstruction. To solve this problem and prepare ascaffold with similar anisotropic property in both structure and physical propertieslike bone, which improve the ingrowth of cells and tissues into the interior part ofthe scaffold and enhance the nutrient exchange, we design and prepare n-HA/PA66scaffold with anisotropic structure and mechanical strength using a modified phaseinversion processing method. Then the processing method, physical and chemicalproperties, macro-morphology and mechanical property of the anisotropic scaffoldwere investigated, and the biocompatibility was evaluated using in vitro cells culture.Consequently, this n-HA/PA66 scaffold presents anisotropy in both morphology andmechanical activity, that is, the compressive strength of the scaffold in the directionparallel to the pore direction is much larger than that perpendicular to the poredirection. Additionally, the result of cells culture indicates that both MSCs andosteoblasts have the tendency to grow, migrate and proliferate along the orientationparallel to the pore direction. After all, these results indicate that n-HA/PA66 composite scaffolds fulfill thebasic requirements of bone tissue engineering scaffold, and have the potential to beapplied in orthopedic, reconstructive and maxillofacial surgery.更多还原