使用多孔聚己內酯膜及人類牙齦纖維母細胞進行牙齦組織再生

Abstract

牙齦是一種在牙齒底下的組織，其組成的主要成分有：膠原蛋白（Collagen）、纖維母細胞（Fibroblast）、蛋白聚糖（Proteoglycans）。在健康的狀況下，牙齦組織呈現亮光澤粉色，若是牙齦開始變暗紅色且有化膿的現象，則要注意有可能是牙周病初期症狀，並可能造成牙齦萎縮的問題，目前臨床常用的治療方式為「游離牙齦移植手術」，但最大的缺點就是會帶給患者二次傷口。為了製造替代的人工移植體，本次實驗採用聚己內酯（Polycaprolactone, PCL）作為材料，並與目前臨床使用的Bio-Mend 膠原蛋白膜進行比較。在細胞生長的過程中有許多生長因子調控，其中Transforming growth factor β-1 (TGF-β1 )是非常重要的一個因子，其執行許多細胞功能，因此本研究藉由加入不同濃度的TGF-β1來探討纖維母細胞之膠原蛋白、纖連蛋白、蛋白聚糖蛋白質的表現量是否有影響，進而了解此人造膜對於未來應用於游離結締組織移植手術治療的可能性。 本研究先對材料進行基本性質鑑定（SEM、Porosity），接著為了解其體外降解程度，使用凝膠透滲層析儀（GPC）進行測試，接著利用CCK-8 kit測試細胞存活數，最後收集細胞蛋白進行西方墨點法了解其蛋白質表現。 結果顯示，聚己內酯膜在經過細胞培養7 ~ 21天後，分子量呈現趨勢性下降，培養液對聚己內酯膜的降解不具影響，而PCL膜的降解會隨細胞分泌出的lipase變多而變快，PCL膜在加入TGF-β1後細胞數並無大量上升，而Bio-mend的膠原蛋白膜在加入TGF-β1後細胞數雖有稍微上升，但發現PCL的組別在不加TGF-β1時就已能表現大量蛋白，而Bio-mend的膠原蛋白膜除Collagen外其餘蛋白表現量都偏低。 整體來說，膠原蛋白膜的整體表現都較聚己內酯膜差，或許聚己內酯膜在未來能成為替代膠原蛋白膜的一種材料。

Gingiva is a tissue located underneath the teeth, composed mainly of collagen, fibroblasts, and proteoglycans. In a healthy condition, the gingival tissue appears shiny pink. If the gingiva begins to turn dark red and purulent, it might be an early symptom of periodontal disease and may cause gum recession. Currently, the commonly used treatment method for this condition is "connective tissue graft”, but the major disadvantage is that it will bring secondary wounds to the patient. In order to create an alternative artificial graft, we used polycaprolactone (PCL) as the material in comparison with Bio-Mend Collagen membrane. Transforming growth factor β 1 (TGF-β1) is an important growth factor as it regulates many cellular functions. It is a secreted protein that performs many cellular function. To understand the future application of this artificial membrane for connective tissue graft, we examined the expression level of collagen, fibronectin, and proteoglycan proteins in fibroblasts induced by TGF-β1 addition. In this research, the surface morphology and porosity of PCL membrane were examined. Subsequently, gel permeation chromatography was used to investigate the degree of degradation in vitro. Cell viability was evaluated using the CCK-8 kit and cellular proteins were examined using Western blot analysis. The results showed that the PCL membrane with cell cultured gradually degraded within 7-21 days. The culture medium contributed negligible effect to the degradation of PCL membrane. By contrast, the degradation of PCL membrane increased with the increase of cell numbers because the lipase secreted by cells also increased. The addition of TGF-β1 to the PCL membrane did not cause significant cell proliferation. However, the cell number slightly increased after TGF-β1 addition in Bio-mend collagen membrane. Prominent protein expression was observed for PCL membrane without adding TGF-β1, but the protein expression levels were relatively low in the Bio-Mend collagen membrane except for collagen. Overall, the collagen membrane performance was inferior to PCL membrane regarding gingival regeneration. The PCL membrane might exhibit the potential to become an alternative material to replace collagen membranes in the future.