基于骨微环境设计的纤维支架促骨再生研究

发布时间：2019-06-24 22:47

【摘要】：炎症、先天畸形和手术治疗等原因引起的口腔颌面部骨缺损是口腔医学科学领域常见的临床表现，这就使骨缺损修复成为该领域面临的重要问题。目前采用自体骨移植、异体骨移植、生物材料以及牵张成骨技术治疗骨缺损取得了一定的进步，，但由于存在继发损伤、塑形性差、免疫排斥反应、技术要求高、价格昂贵以及成骨能力有限等问题而限制了其应用。随着细胞生物学和生物材料学的发展，应用组织工程学技术构建的人工骨治疗骨缺损成为目前研究的热点。骨生物化学微环境是骨组织工程的生物学基础，也是骨组织工程支架材料的设计及生物活性因子的选择的依据。对骨生物化学微环境的认识和理解将极大促进骨组织工程的发展。理想的骨组织工程支架材料应具备良好的骨传导性和骨诱导活性。骨组织工程设计目标是实现骨细胞外基质微环境体系和骨生物化学信号分子体系重建与再生，以快速实现骨组织再生。骨细胞外基质中两大主要成分是胶原和羟基磷灰石，因此骨组织工程支架材料理论上应包含这两种成分；而通过选择合适的生物活性因子重建骨生物化学信号分子可显著提高骨修复效率。基于上述理论，我们采用静电纺丝方法制备了明胶和羟基磷灰石改性的聚乳酸-羟基乙酸共聚物纤维，通过SEM和AFM对支架材料的表面形貌进行表征；将MC3T3-E1细胞和骨髓基质细胞接种于支架表面，通过过激光共聚焦显微镜、MTT和实时定量PCR等实验方法检测改性后纤维支架对成骨细胞粘附、增殖、分化的影响。通过体内模型检测纳米纤维的体内宿主反应。通过上述实验我们证实了改性后的纳米纤维具有很好的成骨诱导活性和生物安全性，因此明胶/纳米羟基磷灰石改性PLGA纳米支架可作为骨组织支架材料用于骨再生领域；明胶/纳米羟基磷灰石可提高骨组织工程支架材料骨诱导和骨引导活性。我们还制备了仿生Gelatin/HA静电纺丝纤维支架材料，并将成骨诱导条件培养基中的常用成分β甘油磷酸钠（beta-glycerol phosphate disodium salt，β-GP）和抗坏血酸（Ascorbic acid，AA）直接纺入Gelatin/HA纤维中。通过扫描电子纤维镜观察制备的交联前后的纤维支架材料；通过建立大鼠颅骨极量骨缺损模型检测载药纳米纤维体内促骨再生能力。通过MICRO CT和组织学评价其修复效果。结果显示载药纳米纤维能够快速修复骨缺损，能够作为模板引导骨组织再生并快速诱导矿化。因此载β-GP和AA的仿生Gelatin/HA纤维支架是优良的自体骨替代材料，可用于临床用于骨缺损治疗。另外，通过冻干的方法，我们还将骨生化微环境中的激素类信号分子EPO蛋白直接与Gelatin/HA纤维支架复合，利用静电纺丝支架很高的比表面积和孔隙率负载EPO蛋白用于修复大鼠颅骨缺损。通过体内建立5mm的大鼠颅骨极量骨缺损模型并植入载EPO蛋白纤维支架材料。通过MICRO CT和组织学评价其修复效果。结果显示载EPO纤维支架材料能够显著促进骨修复，EPO能够刺激成骨细胞增殖并大量分泌骨基质。因此载EPO的Gelatin/HA的纤维支架材料是优秀的骨组织工程替代骨。
[Abstract]:Oral and maxillofacial bone defect caused by inflammation, congenital malformation and surgical treatment is a common clinical manifestation in the field of stomatology and science, which makes the repair of bone defect an important problem in the field. The present invention has made some progress in the treatment of bone defects by using autologous bone graft, allogenic bone grafting, biological material and distraction osteogenesis technology, but because of secondary injury, poor shaping property, immune rejection reaction and high technical requirement, And the application thereof is limited by the problems of high price and limited osteogenic capacity. With the development of cell biology and biological materials, the artificial bone-treated bone defect constructed by the tissue engineering technology has become the hot spot of the present research. The micro-environment of bone biochemistry is the biological basis of bone tissue engineering, and is the basis for the design of bone tissue engineering scaffold material and the selection of biological activity factors. The understanding and understanding of the micro-environment of bone biochemistry will greatly promote the development of bone tissue engineering. The ideal bone tissue engineering scaffold material should have good bone conductivity and bone-induced activity. The aim of the bone tissue engineering is to realize the reconstruction and regeneration of the micro-environment system of the extracellular matrix and the molecular system of the bone biochemical signal, so as to rapidly realize the regeneration of the bone tissue. The two main components of the extracellular matrix are the collagen and the hydroxyapatite, so the material of the bone tissue engineering scaffold material should contain the two components; and the bone biochemical signal molecules can be reconstructed by selecting the appropriate biological active factor to significantly improve the bone repair efficiency. Based on the above theory, we used the electrostatic spinning method to prepare the polylactic acid-glycolic acid copolymer fiber modified by gelatin and hydroxyapatite. The surface morphology of the stent material was characterized by SEM and AFM. The MC3T3-E1 cells and the bone marrow stromal cells were inoculated into the scaffold table. The adhesion, proliferation and differentiation of osteoblasts were detected by laser confocal microscope, MTT and real-time quantitative PCR. in response to that in-vivo host inversion of the nanofiber by in vivo model It is proved that the modified nano-fiber has good osteoinductive activity and biological safety through the above experiment, so the gelatin/ nano-hydroxyapatite-modified PLGA nano-scaffold can be used as a bone tissue scaffold material for the bone regeneration collar. Domain; Gelatin/ nano-hydroxyapatite can improve bone induction and bone-guided activity of bone tissue engineering scaffold Sex. We also prepared the bionic Gelatin/ HA electrostatic spinning fiber support material, and spun the common components of the osteogenic induction condition culture medium into the Gelatin/ HA by directly spinning the sodium glycerophosphate (beta-glycinol phosphate salt, HCO3-GP) and ascorbic acid (AA) into the Gelatin/ HA. The fiber support material before and after the cross-linking preparation is observed by scanning an electron-fiber mirror; and the bone growth-promoting effect of the drug-loaded nano-fiber body is detected through the establishment of a rat skull polar-volume bone defect model, Students' ability to be tested by MICCRO CT and Histology. The result shows that the drug-loaded nano-fiber can be used as a template to guide the regeneration of bone tissue and induce the rapid induction of bone defect. The biomimetic Gelatin/ HA fiber scaffold of the carrier-GP and AA is an excellent substitute for autogenous bone, which can be used in the clinical application of bone defect. In addition, through the freeze-drying method, the EPO protein in the bone biochemical micro-environment is directly compounded with the Gelatin/ HA fiber support, and the high specific surface area and the porosity of the electrostatic spinning support are used for repairing the large amount of the EPO protein Rat skull defect. A 5 mm rat skull bone defect model was established in vivo and an EPO protein fiber was implanted. Dimensional stent material. Through the MICS and histological evaluation of the MICS The results show that the EPO fiber support material can significantly promote bone repair, and the EPO can stimulate the proliferation of the osteoblast and a large amount of the EPO. The bone matrix is secreted. Therefore, the fiber scaffold material of the Gelastin/ HA carrying the EPO is an excellent bone tissue.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R318.08

【共引文献】