两种牙周组织工程支架体内异位成骨能力的实验研究

发布时间：2018-03-25 08:18

  本文选题：牙周组织工程　切入点：骨髓基质细胞　出处：《山西医科大学》2013年硕士论文

【摘要】：目的 本实验通过对壳聚糖-丝素蛋白（chitosan-silk fibroinCS-SF）支架与壳聚糖-丝素蛋白-磷酸三钙（chitosan-silk fibroin-tricalcium CS-SF-TCP）支架的基本性能、力学性能及异位成骨能力进行研究，寻求更适合牙周组织工程的支架。 方法 采用二次冻干技术制备两种支架，，液体替代法、冰冻切片、INSTRON5544拉伸实验机对其一般性能及力学性能进行检测、全髓贴壁法培养骨髓基质细胞（BMSCs）。将裸鼠24只、雄性、体重20～25g，采用完全随机法分为4组，每组6只，CS-SF组、CS-SF+BMSCs组、CS-SF-TCP组、CS-SF-TCP+BMSCs组，所有的植入物均植入裸鼠背部皮下。每只裸鼠背部植入3块。每组分别于术后4、8周各处死动物3只。通过大体观察、组织学与Ⅰ型胶原免疫组织化学染色观察两种支架的成骨能力。 结果 1、支架的一般性能及力学性能的观察 CS-SF与CS-SF-TCP支架大体外观呈白色疏松多孔海绵样物质，有一定的弹性与脆性。两种支架的孔隙率约为70%-80%，孔径值为100-200μm。置于无水乙醇中有溶胀现象但未发生溶解。冰冻切片观察支架的内部结构，可见支架孔径较均匀且彼此相连。力学性能的结果显示：CS-SF支架的压缩强度为0.44±0.15MPa，CS-SF-TCP支架的压缩强度为1.44±0.96MPa。 2、大体及组织学结果 所有的植入物均被一层致密的纤维薄膜包绕，并发生肉眼可见的吸收现象，触之稍硬。组织学染色显示：4周时，CS-SF组与CS-SF-TCP组仅见支架的边缘有纤维膜包绕，支架发生吸收断裂，支架的中心并未见细胞的长入及胶原样物质生成。CS-SF+BMSCs组与CS-SF-TCP+BMSCs组支架的边缘有纤维膜包绕，支架的中心可见细胞及胶原样物质生成。8周时，CS-SF组与CS-SF-TCP组支架的吸收更明显，仍未见细胞长入。CS-SF+BMSCs组与CS-SF-TCP+BMSCs组可见细胞样物质增多,胶原样物质增多增粗。所有组均未见有明显的骨及类骨质样物质生成。 3、Ⅰ型胶原免疫组织化学染色 所有组4、8周时均可见Ⅰ型胶原阳性表达，CS-SF组与CS-SF-TCP组阳性表达主要在支架的边缘，CS-SF+BMSCs组与CS-SF-TCP+BMSCs组阳性表达主要在支架的孔隙表面及中央。4、8周时CS-SF-TCP+BMSCs的平均光密度值均高于CS-SF+BMSCs组，差别有统计学意义（P0.05）。CS-SF组的平均光密度值最低，与其他组差别有统计学意义（P0.05）。 结论 CS-SF-TCP支架能更好的诱导骨髓基质细胞向成骨细胞分化的能力，故更适合于牙周组织工程。
[Abstract]:Purpose. In this experiment, the basic properties, mechanical properties and ectopic osteogenesis of chitosan-silk fibroin CS-SF scaffolds and chitosan-silk fibroin-tricalcium CS-SF-TCP-based scaffolds were studied in order to find a more suitable scaffold for periodontal tissue engineering. Method. Two kinds of scaffolds were prepared by secondary freeze-drying technique, liquid substitution method, frozen section INSTRON5544 tensile test machine to test their general and mechanical properties, bone marrow stromal cells were cultured by whole pulp adherent method, 24 nude mice were male, The body weight was 20 ~ 25 g, and was randomly divided into 4 groups with 6 rats in each group, CS-SF BMSCs group, CS-SF-TCP BMSCs group, CS-SF-TCP group, CS-SF-TCP group, CS-SF-TCP group, CS-SF-TCP BMSCs group, and CS-SF-TCP BMSCs group. All the implants were implanted subcutaneously into the back of the nude mice. The osteogenic ability of the two scaffolds was observed by histological and immunohistochemical staining. Results. 1. General and mechanical properties of the scaffold. The appearance of CS-SF and CS-SF-TCP scaffolds was white and porous sponge. There is some elasticity and brittleness. The porosity of the two kinds of scaffolds is about 70-80 and the pore size is 100-200 渭 m. There is swelling phenomenon in anhydrous ethanol, but no dissolution. Frozen sections are used to observe the internal structure of the scaffolds. The results of mechanical properties show that the compression strength of the W CS-SF stent is 0.44 卤0.15MPA and the compression strength of CS-SF-TCP scaffold is 1.44 卤0.96MPa. 2. Gross and histological results. All the implants were wrapped in a dense fibrous film, and the absorption phenomenon was visible to the naked eye, and it was slightly hard to be touched. Histological staining showed that in the CS-SF group and the CS-SF-TCP group at 4 weeks, only the fibrous membrane was wrapped around the edge of the scaffold, and the scaffold was absorbed and broken. There was no cell-growth and collagen-like substance formation in the center of the scaffold. The scaffolds in CS-SF BMSCs group and CS-SF-TCP BMSCs group had fibrous membrane wrapping around the edge, and the absorption of scaffold in CS-SF group and CS-SF-TCP group was more obvious at 8 weeks after the formation of collagen like substance and cells in the center of the scaffold. In the CS-SF BMSCs group and CS-SF-TCP BMSCs group, the cell like substance increased and the collagen-like substance increased and thickened. No bone and bone-like substances were found in all the groups. Immunohistochemical staining of type 3, type I collagen. Positive expression of type I collagen was found in all groups at 48 weeks. The positive expression of type 鈪

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