引導骨組織再生膜之細胞模式研究

In Vitro Study of Guided Bone Tissue Regeneration Membrane

林郁敏(Yu-Min Lin)

指導教授 ： 鍾仁傑

國立臺北科技大學/工程學院/生物科技研究所/碩士(2012年)

摘要

本研究旨在進行引導骨組織再生膜之細胞模式研究。以原花青素與第一型膠原蛋白進行交聯反應，並製備出含 10 % 原花青素之組織再生薄膜 (簡稱為P-C film)，實驗以人類類骨母細胞 (MG63) 與人類皮膚纖維母細胞 (WS1) 細胞株進行研究。評估類骨母細胞對 P-C film 之細胞毒性、貼附、增生、分化的影響。並利用孔徑分析儀分析材料孔徑率，以及將 MG63 與 WS1 進行共培養實驗來探討組織再生膜是否能阻止細胞移行穿透，並達到阻隔的作用。
研究結果顯示，在 MTT assay 與細胞週期細胞活性測試方面，P-C film 不影響 MG63 類骨母細胞之生長。在 SEM 及 H33258 螢光染色細胞貼附測試方面，隨著 P-C film 與 MG63 cell 貼附的時間增長，細胞貼附於組織再生膜上之數目隨之增加。在細胞增生與分化測試方面，P-C film 能刺激 MG63 cell 的增生，提早骨母細胞於第 7 天開始進行骨分化，並於第 14 天觀察到鈣沉積的生成。而於組織再生膜之細胞穿透性探討方面，P-C film 孔洞分佈範圍在 3.3 ~ 101.6 μm，其中孔洞平均直徑範圍落在 7 ~ 9 μm，得知組織再生膜孔徑非常小。於細胞穿透性測試方面，經由 MG63 cell 與 WS1 cell 單面培養，細胞貼附於材料表面情形良好，且材料下方並無細胞核被染出，可知我們的組織再生膜確實具有阻隔細胞穿透之功能。
綜合以上結果，此組織再生膜具有良好的生物相容性，不僅可刺激骨母細胞的增生及分化，還具有阻隔細胞移行穿透的功能，故此研究製備的 P-C film 在引導骨組織再生膜的應用上有很大的潛力。

關鍵字

第一型膠原蛋白 ； 低聚合原花青素 ； 引導組織再生膜 ； 細胞實驗

並列摘要

The purpose of this research was to carry out the detailed in vitro study of guided bone tissue regeneration membrane. The fabricated membrane, P-C film, was made of type I collagen cross-linked using oligomeric proanthocyanidins (OPCs) as the cross-linking agent. The human osteoblast cell line (MG63) and human skin fibroblast cell line (WS1) were cultured for in vitro studies. The effects of P-C film on MG63 cell viability, adhesion, proliferation, alkaline phosphatase activity and differentiation were evaluated. In order to investigate the barrier ability of the tissue regeneration membrane, capillary flow porometer was used to analyze the pore size; separated co-culture experiments using MG63 and WS1 were carried out to observe the cell migration behavior.
The results showed that P-C film did not affect cell growth and had good biocompatibility. The cells attached to the P-C film surface increased with time. We also found that MG63 cells grew faster on P-C film, compared to the culture flask. On the other hand, P-C film promoted osteoblast to enter the differentiation stage, indicated by higher alkaline phosphatase activity and calcium deposition. In addition, the mean pore size of P-C film ranged at 7~9 μm, which was small enough to block the migration of cells through the membrane.
In conclusions, P-C film does not affect cell growth, and is able to promote the differentiation of osteoblast. Results of in vitro experiments indicate that P-C film has great potential in the applications of guided bone regeneration and bone tissue engineering.

並列關鍵字

Type I Collagen ； Oligomeric Proanthocyanidins ； Guided Tissue Regeneration Membrane ； In Vitro Study

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