納米纖維絲素蛋白與脫細胞基質(zhì)真皮在口腔黏膜修復重建中的動物實驗研究
發(fā)布時間：2018-11-21 12:32
【摘要】：研究目的： 利用納米纖維絲素蛋白支架與脫細胞基質(zhì)（牛皮，人皮）真皮材料，在實驗動物Wister大鼠體內(nèi)建立口腔黏膜修復重建的動物模型，針對重建后的口腔黏膜進行組織病理學評價，通過對比，評估納米纖維絲素蛋白作為組織工程口腔黏膜修復材料的可行性，為絲素蛋白材料修復重建口腔黏膜提供理論依據(jù)。 實驗方法： 1、動物實驗：清潔級雄性Wister大鼠80只，隨機分成四組，造成口腔黏膜缺損模型，A組為納米纖維絲素蛋白修復口腔頰黏膜圓形缺損（直徑10mm），n=20；B組為牛皮脫細胞基質(zhì)修復口腔頰黏膜圓形缺損（直徑10mm），n=20；C組為人皮脫細胞基質(zhì)組修復口腔頰黏膜圓形缺損（直徑10mm），n=20；D組為空白對照組，凡士林油紗覆蓋口腔頰黏膜圓形缺損（直徑10mm），n=20，分別于術后一周、兩周、三周、四周后行大體觀察，組織學及免疫組織化學觀察。 2、口腔黏膜重建模型組織學觀察： 1）口腔黏膜重建術后一周、兩周、三周、四周分別進行全層口腔黏膜取材。 2）取材后進行大體觀察，HE染色和免疫組化染色，從組織學水平動態(tài)評價新生口腔黏膜變化并用免疫組化技術對各組新生黏膜的血管化及上皮角化程度進行鑒定，對新生黏膜炎性細胞及成纖維細胞進行對比。 結果： 1、大鼠口腔黏膜重建手術模型成功，手術切口全部如期愈合，未見感染、血腫、排斥等并發(fā)癥。預期時間段取材，未見口腔黏膜攣縮，塌陷或修復材料脫出等，再生口腔黏膜組織生長良好。 2、納米纖維絲素蛋白與脫細胞基質(zhì)材料進行口腔黏膜缺損修復效果基本一致，但初始上皮化的時間略晚。術后一周絲素蛋白材料排列較整齊，材料周圍炎性反應明顯，絲素蛋白材料中較多炎性細胞，成纖維細胞及少量毛細血管長入，尚未見到上皮結構。絲素蛋白纖維上可見成纖維細胞。術后二周術區(qū)創(chuàng)緣可見新生上皮細胞，新生上皮層較薄，層數(shù)為3-4層，平坦，上皮細胞排列不規(guī)則，疏松，上皮細胞生長活躍，固有層可見散在的小血管，毛細血管，固有層膠原排列較疏松。炎性反應較前明顯減輕，炎性細胞數(shù)量減少，，成纖維細胞及毛細血管數(shù)量增多，絲素蛋白材料部分吸收降解，絲素材料短而稀疏。術后三周，新生上皮較厚，呈現(xiàn)多層結構，上皮腳及釘突出現(xiàn)較多，上皮多層結構極向恢復，排列較緊密，固有層內(nèi)炎性細胞較前明顯減少，固有層內(nèi)生成較多清晰血管，固有層內(nèi)膠原排列較前緊密，可見散在的平滑肌。絲素蛋白支架降解成碎片狀。四周新生上皮較厚，呈現(xiàn)多層結構，上皮腳釘突明顯，結構與正常大鼠口腔黏膜無明顯區(qū)別，固有層內(nèi)膠原排列整齊，可見部分肌束形成。上皮廣譜角蛋白抗體免疫組化染色證明新生口腔黏膜有上皮組織存在，且從重建第二周開始出現(xiàn)陽性，其結果與HE染色觀察結果一致。 結論： 1、進一步完善實驗動物大鼠構建口腔黏膜缺損的模型制備，得到新生口腔黏膜組織學證據(jù)，模型構建穩(wěn)定可靠，證明實驗用Wister大鼠是進行口腔缺損性疾病研究的理想實驗動物。 2、從實驗動物圍手術期和術后短期觀察結果以及新生口腔黏膜的大體和組織學觀察，證明利用納米纖維絲素蛋白材料來修口腔黏膜，可以取得與脫細胞基質(zhì)真皮材料類似的效果，可以作為口腔黏膜重建的修復材料，且因其制作材料為天然絲素，來源廣泛， 價格低廉，具有廣闊的應用前景。3、納米纖維絲素蛋白材料能夠促進口腔黏膜上皮增生，加速創(chuàng)面愈合，抑制口腔黏膜創(chuàng)面的收縮，減輕瘢痕攣縮。
[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
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R783
本文編號：2346973
[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
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R783
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