- 學位論文
以組織工程技術於牙本質再生之應用
The Application of Tissue Engineering on Dentin Regeneration
摘要
牙齒在攝取營養、外觀及與人溝通方面，都是不可或缺的。齲齒、牙髓炎、牙周疾病及外傷等因素，將導致牙齒缺失，進而影響正常咀嚼、發音等生理功能，並造成外型上的困擾。若是能發展出生物性的替代牙齒便能取代缺損的牙齒，將是臨床治療的一大突破；而幹細胞可遷移至器官受損處進行修復，並具有可自我更新及分化的潛力，若能運用幹細胞組織工程技術來達到此目標，將是一大進展。 為了評估這個目標，我們取出大鼠臼齒的牙胚細胞與大鼠骨髓間葉幹細胞分離成單細胞方式共養後，評估有無共養之細胞活性及對牙生成有幫助的相關基因表現之比較。共養之細胞與牙胚細胞分別接種在具生物分解性的雙層支架中並植入免疫缺失鼠背部皮下4、8、12週後，以X光放射線攝影、組織化學染色及免疫組織化學染色進行檢測。 實驗結果顯示，大鼠骨髓間葉幹細胞經鑑定後確定其幹細胞之特性，而經共養之後的大鼠骨髓間葉幹細胞與大鼠牙胚細胞在細胞活性及基因表現量上都有提升。第12週共養之細胞與牙胚細胞由X光放射線攝影圖中看到硬組織的產生、組織化學染色中有表現鈣鹽，推測已有礦化的產生。在第4、8、12週時，共養之細胞與牙胚細胞在免疫組織化學染色都有表現Dentin Matrix Protein-1 ( DMP-1)蛋白，確定已有類牙本質的產生，而從染色結果得知，共養之細胞在DMP-1蛋白表現量遠高於牙胚細胞。可確定的是共養之細胞與牙胚細胞在具生物分解性的雙層支架中，大鼠骨髓間葉幹細胞與牙胚細胞可以進行重組而形成礦化與類牙本質之結構。
並列摘要
The human dentition is indispensable for nutrition, communication and physiology. Caries, pulpitis, apical periodontitis and trauma may lead to loss of tooth, causing the problem of pronunciation, mastication, and appearance difficulties. A biological tooth substitute that could replace lost teeth would provide a vital alternative to currently available clinical treatment. Stem cells are multipotent cells which are capable of self-renewing and differentiating into multi- cell lineages. In this study, tissue engineering approach will be addressed to evaluate this possibility to reconstruct tooth. To achieve this goal, we separately dissociated Wistar rat Tooth Bud Cells and Mesenchymal Stem Cells ( MSC ). Then two kinds of cells were seeded either in monoculture ( Tooth Bud Cells only ) or co-culture ( MSC + Tooth Bud Cells ) condition to evaluate cell viability and gene expression in vitro. Co-culture Cells and Tooth Bud Cells were then respectively seeded onto biodegradable bilayered scaffold and implanted in subcutaneous layers in SCID mice. After implantation for 12 weeks, the results were studied by X ray, histology and immunohistochemistry. From the results, the cell phenotype of rat msenchymal stem cells was identified by flow cytometry. Cells viability and gene expression were elevated in co-culture group. At 12 weeks, the mineralization in Co-culture Cells group and Tooth Bud Cells group was visualized in X ray, Von Kossa and Alizarin Red S. At 4, 8, 12 weeks, the dentin matrix protein-1 ( DMP-1) were deposited and stained in Co-culture Cells group and Tooth Bud Cells group by immunohistochemistry, representing dentin-like structure formation. In addition, Co-culture Cells group expressed stronger than Tooth Bud Cells group. It seems like that rat Mesenchymal Stem Cells and Tooth Bud Cells could reorganize themselves on biodegradable bilayered scaffold into arrangements that favor formation of mineralization / dentin-like tissue.