| Summary: | 碩士 === 國立臺灣大學 === 臨床牙醫學研究所 === 100 === 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.
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