Collagen和TGF-β1對於牙根尖幹細胞行為的影響

Abstract

實驗目的:牙齒再生組織工程中，細胞生長因子和細胞支架對細胞生長、分化、胞外間質形成以及牙本質生成扮演著重要角色。本實驗之目的在於探討β1型轉型生長因子（Transforming growth factor-β1, TGF-β1）和膠原蛋白(Collagen)對牙根尖細胞生長與分化所造成的影響。我們假設膠原蛋白可以影響根尖細胞的貼附、活性、分化、鹼性磷酸酶的表現。此外，在體外的環境將根尖細胞培養在細胞支架中，並加入轉型生長因子可以成功地刺激組織再生。 實驗方法：第一部分使用不同濃度的膠原蛋白培養人類牙根尖細胞 (Stem cells from apical papilla, SCAP)，檢測細胞的貼附能力並觀察細胞形態的改變，同時也以MTT測量細胞的活性並利用鹼性磷酸酶染色 （Alkaline phosphatase staining）觀察細胞分化的情形。第二部分使用不同濃度的TGF-β1和人類牙根尖細胞培養，以反轉錄聚合酶反應（RT-PCR）觀察結締組織生長因子(CTGF)和α1型組合蛋白(α1-integrin)的表現。最後將人類牙根尖細胞培養在兩種細胞支架(collagen/dentin scaffold)中並加入TGF-β1的刺激，以組織切片方式觀察細胞型態以及新生成的組織結構。

實驗結果：人類牙根尖細胞在覆有膠原蛋白的表面有助於細胞貼附的效果；在低濃度膠原蛋白表面生長會促進細胞的生長，而在高濃度膠原蛋白表面細胞活性會降低；鹼性磷酸酶染色也發現可看到根尖細胞在高濃度膠原蛋白的環境中鹼性磷酸酶的活性會略微降低。人類牙根尖細胞經過TGF-β1的刺激之後，CTGF和α1-integrin的表現量會隨著TGF-β1濃度升高而增加。將細胞培養在膠原蛋白細胞支架內再加入TGF-β1的刺激後初步結果可觀察到新生成的細胞間質堆積物；進一步將細胞放入牙本質細胞支架(dentin scaffold)中也可看到類似牙本質(dentin-like)的組織產生。 結論：TGF-β1和膠原蛋白對人類牙根尖細胞的附著、生長和分化都有調控的作用，在不同的條件配合下也可能誘導細胞產生不同的特性和分化的方向。在牙齒組織再生工程中，膠原蛋白和牙本質都是方便操作且具有潛力的細胞支架，若能合併適當的細胞和生長因子，未來可進一步應用在動物模型上甚至是臨床治療上，針對牙髓壞死的牙齒誘導牙本質和牙髓的再生。

Aim： In tissue regeneration, growth factors and scaffolds play important roles in cell proliferation, differentiation, extracellular matrix production and dentin formation. The purpose of this study is to investigate the effects of TGF-β1 and collagen on behaviors of stem cells from apical papilla (SCAP). We hypothesize that collagen can affect cell attachment, viability, and alkaline phosphatase (ALP) expression in SCAP. Besides, culturing SCAP in scaffold with combination of TGF-β1 can regenerate tissues successfully in vitro. Materials and Methods： SCAP were cultured on the surface coated with different densities of collagen. Then, SCAP were evaluated for cell attachment ability, cell viability by MTT test and cell differentiation by ALP activity. Besides, SCAP were treated with TGF-β1 with different concentrations. CTGF and α1-integrin expression were determined by reverse-transcription polymerase chain reaction（RT-PCR）. Finally, we cultured SCAP in collagen scaffolds and dentin disc scaffolds with combination of TGF-β1 to observe the morphological changes of cells and tissue regeneration under histological analysis.

Results： SCAP cultured on surfaces coated with collagen showed better attachment ability. SCAP viability elevated when cultured on the surface coated with lower densities of collagen. However, decreased cell viability and ALP activity were observed when cultured SCAP on the surfaces coated with high densities of collagen. TGF-β1 could promote CTGF and α1-integrin gene expression of SCAP in a dose-dependent manner. Furthermore, we cultured SCAP in collagen scaffold with TGF-β1 stimulation and observed the prominent cell proliferation and matrix-like tissue regeneration. When SCAP were cultured in dentin disc scaffold with TGF-β1 stimulation, regenerated dentin-like tissues containing dentinal tubules and calcified nodules were found. Conclusion： SCAP cultured on the collagen matrices with various concentrations showed different biological characteristics like cell attachment, proliferation, and differentiation. Besides, collagen and dentin disc scaffolds with combination of SCAP and TGF-β1 may be practical models in regeneration of dentin-pulp tissues. Further research may be engaged in applying the models in animal studies, and even in clinical practice to repair the teeth with pulp necrosis or immature root formation.