人類牙周細胞培養於羊膜基質之研究

Study of human periodontal cells cultured on amniotic membrane matrix

程偉智(Wei-Jhih Cheng)

指導教授 ： 楊台鴻

國立臺灣大學/工學院/醫學工程學研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.10282

摘要

人類羊膜是一天然生物性材料，目前已有基礎及臨床研究報告顯示羊膜可被成功應用於一些醫療用途，例如作為皮膚燒燙傷之傷口貼附材料、眼角膜的重建等，至於利用羊膜做為口腔內組織再生的相關研究，迄今仍為數極少。本研究之目的是探討將人類羊膜作為細胞貼附基質時對於牙周細胞生長的影響，以評估羊膜應用於口腔內組織修復的可行性。
　　本研究第一部份是將兩種口腔細胞：牙周韌帶纖維細胞與牙齦纖維細胞分別在體外培養於羊膜基質層與經去細胞處理的基底膜層，並對其活性、形態與成骨分化潛力表現進行評估。第二部份則嘗試將幾丁聚醣與羊膜形成一複合材料，對其強度進行測試，並且進行動物實驗，將羊膜與此複合材料分別應用於口腔內頰側黏膜之傷口修復。
　　本實驗之結果發現: 相較於培養於組織培養皿上的生長，上述兩種口腔細胞培養在羊膜上時，都具有相當甚至更優的活性，並呈現類似纖維細胞的形態，此外，藉由聚合酶連鎖反應(RT-PCR)與鹼性磷酸酶(Alkaline phosphatase，ALP)活性測定發現，牙周韌帶纖維細胞在羊膜上仍具有顯著的ALP表現性，由於牙周韌帶細胞是一具有骨母細胞分化特性的纖維細胞，上述結果顯示羊膜能使牙周韌帶細胞保有其特殊功能性。
　　為加強羊膜此一天然生物材料的機械強度，本研究藉由覆蓋的方式使幾丁聚醣高分子溶液在經去細胞處理的羊膜基底膜上形成一層薄膜，藉由此方式形成的複合材料經單軸向拉伸測試發現其所能承載的拉力大小明顯上升。此外，本研究將羊膜與其複合材料貼附於米格魯犬頰側黏膜的傷口上，以評估其作為傷口敷材的效果，結果顯示: 無論是以羊膜或幾丁聚醣與羊膜複合材料覆蓋傷口，傷口在三週後都有完整且良好的癒合效果，尤其是羊膜複合材料覆蓋之頰側黏膜，傷口收縮程度最小，在組織學鏡檢下，可見表皮細胞遷移至傷口處，並生長形成多層的扁平麟狀表皮細胞，下方則充滿新生的膠原纖維組織。

關鍵字

人類羊膜 ； 牙周韌帶纖維細胞 ； 牙齦纖維細胞 ； 礦化誘導 ； 口腔黏膜

並列摘要

Human amniotic membrane is a nature biomaterial which has been used in several medical treatments such as cornea reconstruction and as a biological dressing for skin wounds and burns. The purpose of this study was to investigate the possibility of applying human amnions for oral tissue repair by evaluating the growth and　behavior of periodontal ligament fibroblast and gingival fibroblasts cultured on the denuded human amniotic membrane.
　　Periodontal ligament fibroblasts on the amnion stroma and gingival fibroblasts were cultured on the basement membrane of denuded amnion. Cell viabilities, morphologies and functions were evaluated. Moreover, we tried to combine the chitosan and the amnion to become a composite material. Then its mechanical properties and the performance being as a dressing material in the surgical defects of the Beagle dog’s oral mucosa were evaluated.
　　The results showed that gingival and periodontal ligament cells could grow very well when cultured on the basement membrane side and stromal side of denuded amnion, respectively. Furthermore, the periondontal ligament fibroblasts expressed significant ly elevated ALP activity when cultured on the stroma.
　　To increase the strength of the amnion, chitosan was coated onto the denuded amnion’s basement membrane. The result of tensile test reveals that this composite material indeed became stronger. In the animal experiment, dressing of amniotic membrane might facilitate the epithelialization of wound surface and neo-collagen formation underneath the wound surface. Our results suggest the potential of amniotic membrane being as the matrix scaffold for periodontal cell growth and that amniotic membrane transplantation might promote the wound healing of oral mucosa.

並列關鍵字

human amniotic membrane ； periodontal ligament fibroblasts ； gingival fibroblasts ； mineralization ； oral mucosa

參考文獻

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