牙齦纖維母細胞在去細胞化之豬牙齦基質之生長與貼附

Abstract

牙齦組織覆蓋在牙根、齒槽骨、直至齒頸部。由於牙齒周圍的牙齦組織皆為角化牙齦，因此可以保護牙根及其周圍組織。諸多因素都有可能會造成牙齦組織的萎縮與缺損，進而影響牙齦與齒槽骨的高度。在臨床上，對於大面積牙齦組織的萎縮與缺損，會應用口腔黏膜組織再生工程的技術來做填補與修復。 目的 目前臨床上用來修補口腔黏膜缺損的材料中，無論是化合物組成的再生膜、或是自體組織移植的方式，都會有無法避免的困難。然而，豬與人類在形態學與免疫學上相近。因此將去細胞後的豬牙齦組織製作成牙齦基底後，若可成功培養細胞，將可成為施行牙根覆蓋術的理想選擇材料之一。因此，本研究之目的是探討纖維母細胞在去細胞化之豬牙齦組織上的生長性，以做為未來發展與利用去細胞之豬牙齦組織做為口腔黏膜再生工程之支架材料。 材料及方法 本實驗使用1周大的ICR ( Institute of Cancer Research ) 品種小鼠來培養牙齦纖維母細胞。豬牙齦去細胞的處理方法則是參考Tasaki Y.處理去細胞皮膚基底的方式。之後，將牙齦纖維母細胞培養於豬牙齦組織塊以觀察生長情況。 另一方面，以紫木蘇與伊紅染色法對細胞核與細胞質染色，以便觀察和比較豬牙齦組織在未經去細胞、去細胞後豬牙齦組織、以及去細胞後豬牙齦基底烘乾後細胞殘存的情形。 將繼代培養的牙齦纖維母細胞分別培養至去細胞豬牙齦基底上，在4、8、16、24、與32小時的時間點，分別觀察細胞的生長型態。利用MTT ( 3-(4, 5-cimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide ) 來測量細胞在細胞外基質上的存活率與活性。 另外，以電子顯微鏡觀察未經處理的豬牙齦組織、經去細胞後處理的豬牙齦基底組織、以及牙齦纖維母細胞等標本在不同時間的型態。並且用梅生三色染色劑來區別在周圍結締組織的細胞類型及組成。 結果與結論 本實驗利用Trypsin和EDTA加上Triton X-100對全皮層的豬牙齦組織進行去細胞的方式雖然可以明顯去除上皮細胞，但對於殘存的細胞殘骸仍須藉由烘乾並以PBS沖洗後才能完整的去除。在去細胞的豬牙齦基底上培養的牙齦纖維母細胞，也能維持好的活性與細胞的型態，並利用纖維母細胞可促進完整基底膜生成的特性，以增加上皮細胞的生長。另外，在去細胞處理後可以觀察到牙齦基底因絲狀的不規則排列所產生的孔洞，其孔洞大小可利於細胞直接穿入內部。豬牙齦組織適合培養上皮細胞的生長，並且適用於需要大面積修補的牙齦缺損，對於應用於臨床牙周再生的材料中提供了另一種選擇。

The extent of the coverage of gingival tissue is over the root of tooth, alveolar bone, and the cervical portion of the tooth. Because all of the periodontal gingival tissue is keratinized gingiva, therefore, they can protect the root of tooth and the peripheral tissue. Many factors would be likely to cause the recession and defect of the gingival tissue, and this will affect the height of the gingiva and alveolar bone. Clinically, with regard to large area of the recession and defect of the gingival tissue, the tissue engineering of oral mucosa will be applied for the filling and repair. Aim The present materials, whether regeneration membrane composed of chemical compound or autologous graft, for the repair of the defect of oral mucosa both have unavoidable difficulties. However, the pig are similar with human beings in morphology and immunology. Hence, if the culture of the cells could be successful in the gingival matrix made from acellular porcine gingival tissue, it could be one ideal choice of material for the root coverage. Therefore, the aim of this study is to discuss the growth of the fibroblast in acellular porcine gingival tissue, and the development and application of the acellular porcine gingival tissue as scaffold and material for the tissue engineering of oral mucosa in the future. Materials and Methods The one-week-old ICR ( Institute of Cancer Research ) mice was used for the culture of the gingival fibroblasts, and the method of acellularization of the porcine gingiva was referred to the management of acellularization of the dermal matrix by Tasaki Y.. Thereafter, the growth of the gingival fibroblasts cultured in porcine gingival sheets were observed. On the other hand, the Hematoxylin & Eosin stain applied to the nucleus and cytoplasm was used for observation and comparison of the residual cells in the porcine gingival tissue with the condition of pre-acellularization, post-acellularization, and dried post-acellularized matrix. The subcultured gingival fibroblasts was cultured in the acellular porcine gingival matrix, and the growth and state were observed at the time after 4, 8, 16, 24, and 32 hours. The MTT ( 3-(4, 5-cimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide ) was applied for the detection of the survival rate and activity of the cells in the extracellular matrix. In addition, the morphology of the pre-acellular porcine gingival tissue, post-acellular porcine gingival matrix, and gingival fibroblast were observed by the scanning electronic microscope. The Masson’s Trichrome Staining was used for the differentiation of the cellular types and composition in peripheral connective tissue. Results and Conclusion This study used trypsin and EDTA with Triton X-100 for acellularization of the full-thickness porcine gingival tissue, although this method definitely clear the epidermal cells, the residual cellular debris was still needed drying as well as washing with PBS for the purpose of complete clearance. Fibroblasts cultured in the acellular porcine gingival tissue maintained a good activities and cellular morphology, and the character of promoting complete development of the basement membrane of these fibroblasts can be used for increasing growth of the epithelial cells. After acellularization, filamentous and irregular arrangement of the gingival matrix would show some holes, which facilitated cells entering the inner site directly. Porcine gingival tissue is suitable for the growth of the epithelial cells, and it can be applied to the big-size gingival recession. Therefore, it provides an alternative choice of matrix of the clinical periodontal tissue engineering.