纳米纤维丝素蛋白与脱细胞基质真皮在口腔黏膜修复重建中的动物实验研究

发布时间：2018-11-21 12:32

【摘要】：研究目的：利用纳米纤维丝素蛋白支架与脱细胞基质（牛皮，人皮）真皮材料，在实验动物Wister大鼠体内建立口腔黏膜修复重建的动物模型，针对重建后的口腔黏膜进行组织病理学评价，通过对比，评估纳米纤维丝素蛋白作为组织工程口腔黏膜修复材料的可行性，为丝素蛋白材料修复重建口腔黏膜提供理论依据。实验方法： 1、动物实验：清洁级雄性Wister大鼠80只，随机分成四组，造成口腔黏膜缺损模型，A组为纳米纤维丝素蛋白修复口腔颊黏膜圆形缺损（直径10mm），n=20；B组为牛皮脱细胞基质修复口腔颊黏膜圆形缺损（直径10mm），n=20；C组为人皮脱细胞基质组修复口腔颊黏膜圆形缺损（直径10mm），n=20；D组为空白对照组，凡士林油纱覆盖口腔颊黏膜圆形缺损（直径10mm），n=20，分别于术后一周、两周、三周、四周后行大体观察，组织学及免疫组织化学观察。 2、口腔黏膜重建模型组织学观察： 1）口腔黏膜重建术后一周、两周、三周、四周分别进行全层口腔黏膜取材。 2）取材后进行大体观察，HE染色和免疫组化染色，从组织学水平动态评价新生口腔黏膜变化并用免疫组化技术对各组新生黏膜的血管化及上皮角化程度进行鉴定，对新生黏膜炎性细胞及成纤维细胞进行对比。结果： 1、大鼠口腔黏膜重建手术模型成功，手术切口全部如期愈合，未见感染、血肿、排斥等并发症。预期时间段取材，未见口腔黏膜挛缩，塌陷或修复材料脱出等，再生口腔黏膜组织生长良好。 2、纳米纤维丝素蛋白与脱细胞基质材料进行口腔黏膜缺损修复效果基本一致，但初始上皮化的时间略晚。术后一周丝素蛋白材料排列较整齐，材料周围炎性反应明显，丝素蛋白材料中较多炎性细胞，成纤维细胞及少量毛细血管长入，尚未见到上皮结构。丝素蛋白纤维上可见成纤维细胞。术后二周术区创缘可见新生上皮细胞，新生上皮层较薄，层数为3-4层，平坦，上皮细胞排列不规则，疏松，上皮细胞生长活跃，固有层可见散在的小血管，毛细血管，固有层胶原排列较疏松。炎性反应较前明显减轻，炎性细胞数量减少，，成纤维细胞及毛细血管数量增多，丝素蛋白材料部分吸收降解，丝素材料短而稀疏。术后三周，新生上皮较厚，呈现多层结构，上皮脚及钉突出现较多，上皮多层结构极向恢复，排列较紧密，固有层内炎性细胞较前明显减少，固有层内生成较多清晰血管，固有层内胶原排列较前紧密，可见散在的平滑肌。丝素蛋白支架降解成碎片状。四周新生上皮较厚，呈现多层结构，上皮脚钉突明显，结构与正常大鼠口腔黏膜无明显区别，固有层内胶原排列整齐，可见部分肌束形成。上皮广谱角蛋白抗体免疫组化染色证明新生口腔黏膜有上皮组织存在，且从重建第二周开始出现阳性，其结果与HE染色观察结果一致。结论： 1、进一步完善实验动物大鼠构建口腔黏膜缺损的模型制备，得到新生口腔黏膜组织学证据，模型构建稳定可靠，证明实验用Wister大鼠是进行口腔缺损性疾病研究的理想实验动物。 2、从实验动物围手术期和术后短期观察结果以及新生口腔黏膜的大体和组织学观察，证明利用纳米纤维丝素蛋白材料来修口腔黏膜，可以取得与脱细胞基质真皮材料类似的效果，可以作为口腔黏膜重建的修复材料，且因其制作材料为天然丝素，来源广泛，价格低廉，具有广阔的应用前景。3、纳米纤维丝素蛋白材料能够促进口腔黏膜上皮增生，加速创面愈合，抑制口腔黏膜创面的收缩，减轻瘢痕挛缩。
[Abstract]:The purpose of the study: An animal model of oral mucosa repair and reconstruction is established in a Wister rat of an experimental animal by using a nano-fiber silk fibroin scaffold and a acellular matrix (cowhide, human skin) dermal material, and the tissue pathology evaluation is carried out on the reconstructed oral mucosa, To compare and evaluate the feasibility of nano-fiber silk fibroin as a repair material for oral mucosa of tissue engineering, to provide a theory for the repair and reconstruction of oral mucosa of silk fibroin material. On the basis of. Experimental method: 1. Animal experiment: 80 rats in the clean-grade male Wister rats were randomly divided into four groups, resulting in the oral mucosa defect model. The group A was a nano-fiber silk fibroin to repair the circular defect of the buccal mucosa (diameter 10 (mm), n = 20; group B was a cowhide acellular matrix to repair the oral buccal mucosa circular defect (diameter: 10mm), n = 20; Group C was a human skin decell matrix group to repair the oral buccal mucosa circular defect (diameter: 10mm), n = 20; and the D group was the blank control group, and the vaseline oil gauze covers the circular defect of the buccal mucosa of the oral cavity. (diameter: 10 mm), n = 20, followed by general observation, histology, and post-operative week, two-week, three-week, four-week follow-up. immunohistochemical study. histological observation of the mucosal reconstruction model: 1) one week, two weeks and three weeks after the oral mucosa reconstruction, The whole-layer oral mucosa was obtained from all four weeks. After the materials were obtained, the general observation, HE staining and immunohistochemical staining were carried out. The changes of the new oral mucosa were dynamically evaluated from the level of the histological level. the vascularization and the degree of the epithelial keratosis of the mucosa are identified, submucous membrane Comparison of inflammatory cells and fibroblasts. Results: 1. The model of the rat oral mucosa reconstruction was successful and the operation was successful. The incision was completed as scheduled, no complications such as infection, hematoma, rejection, etc. were not seen. The expected time period was not found in the oral mucosa contracture. the tissue growth of the regenerative oral mucosa is good; and 2, the nano-fiber silk fibroin and the acellular matrix material are carried out The repair effect of the oral mucosa defect is basically the same, but the time of the initial epithelialization is slightly late. The material of the silk fibroin material in the week is more orderly, the inflammatory reaction around the material is obvious, and the more inflammatory property in the silk fibroin material Cells, fibroblasts, and a small number of capillaries The epithelial structure was not seen on the silk fibroin fiber. The newly-born epithelial cells were found in the wound margin of the two-week operation area. The newly-born epithelial cells were thin, the number of layers was 3-4, the flat, the epithelial cells were irregular, loose, and the epithelial cells were active and solid. There are small vessels, capillaries, and lamina propria, which are scattered in the layer. The inflammatory response is significantly reduced, the number of inflammatory cells is reduced, and the fibroblasts and capillaries The amount of silk fibroin was partially absorbed and degraded, and the material of silk fibroin was short and sparse. Three weeks after the operation, the newly-born epithelium was thicker, the multi-layer structure was present, the epithelial and nail processes were more and more, the multi-layer structure of the epithelial layer was more closely aligned, and the inflammatory cells in the intrinsic layer were significantly reduced. and more in the intrinsic layer. Clear blood vessels, the collagen in the lamina propria The smooth muscle of the smooth muscle and the silk fibroin scaffold are in the form of fragments. The new epithelium of the periphery is thicker, the multi-layer structure is present, the epithelial-foot nail is obvious, and the structure is in contact with the normal rat's oral cavity. There was no obvious difference in the membrane, the collagen in the intrinsic layer was arranged in order, and the visible part of the muscle bundle was formed. The immunohistochemical staining of the epithelial-wide-spectrum keratin antibody demonstrated the presence of epithelial tissue in the oral mucosa of the newborn and from the reconstruction The results were consistent with the results of HE staining. Conclusion: 1. The model preparation of the oral mucosa defect was further improved, and the histological evidence of the new oral mucosa was obtained. The model was stable and reliable. The experiment was proved to be the ideal experimental animal for the study of oral defect in Wister rats. 2. The results of the perioperative and postoperative short-term observation of the experimental animals and the gross and histological observation of the oral mucosa of the newborn were proved. The rice fiber silk fibroin material can be used for repairing the oral mucosa, and the effect of similar to the acellular matrix dermal material can be obtained. The fruit can be used as a repair material for the reconstruction of the oral mucosa, broad application prospect. 3. Nanofiber silk fibroin material
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R783

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