矽酸鈣陶瓷微環境變化影響牙髓細胞生理機制探討

本研究主要探討不同微環境對於矽酸鈣(Calcium silicate，CS)相關材料影響 細胞生理機制的探討。首先口腔環境中的複雜的離子環境會影響磷灰石沉澱， 推測將影響矽酸鈣材料中矽離子的釋放。因此，本研究將著重於是否磷灰石的 沉積，將會調節參與人類牙髓細胞(Human dental pulp cells，hDPCs)的相關生 理行為。實驗中，將 hDPCs 培養於 CS 上並搭配正常的細胞培養基(DMEM) 或是不含磷酸的培養基(P-free DMEM)，除了觀察材料表面型態以外，更進 一步分析細胞型態以及後續成骨分化的表現。分析了 CS 水泥浸泡在 DMEM 和 無磷 DMEM 中的微觀結構和組成。結果顯示 CS 與 DMEM 接觸後，材料表面 上將會被球狀的磷灰石層覆蓋，但是在 P-free DMEM 中將不會出現這樣的狀 況。但是 P-free DMEM 組別則會釋放更多的 Si 離子，並且刺激 hDPCs 分泌更 多的細胞外基質，如 collagen I(COL)和 fibronectin(FN)等。這些細胞外基 質將幫助 hDPCs 在 CS 表面的貼附及生長。本研究另外也將模擬根管充填時的 實際發炎環境，將 hDPCs 先以脂多醣(LPS)誘導成發炎狀態後，在持續培養 於三氧礦化合物(MTA)或 CS 材料，並持續觀察該細胞的相關反應。結果顯 示，誘導發炎後的 hDPCs 培養一天後，能夠觀察到 CS 組別中的細胞數量明顯 大於 MTA。此外在培養兩天後，MTA 組別的培養基中分析到較高的 Si 濃度， 推測是引起 hDPCs 進入細胞凋亡的原因。以上兩個研究能夠證實，由實驗室自 行開發的矽酸鈣陶瓷，對於正常細胞及發炎細胞皆具有良好的生物相容性，並 且能夠持續地提供細胞良好生長及分化的環境，未來在臨床上將是一具有潛力 的材料。

This study unraveling the mechanisms behind the processes of cell attachment and the enhanced proliferation that occurs as a response to the presence of calcium silicate-based materials needs to be better understood so as to expand the applications of silicate-based materials. Ions in the environment may influence apatite precipitation and affect silicate ion release from silicate-based materials. Thus, the involvement of apatite precipitate in the regulation of cell behavior of human dental pulp cells (hDPCs) is also investigated in the present study, along with an investigation of the specific role of cell morphology and osteocalcin protein expression cultured on calcium silicate (CS) with different Dulbecco’s modified Eagle’s medium (DMEM). The results demonstrate that CS is completely covered by the apatite layer with tiny spherical shapes on the surface in the DMEM, but not in the P-free DMEM. The CS immersed in P-free DMEM group was released more Si ion increased COL and FN secretion, which promotes cell attachment more effectively than DMEM. In addition, we also compared the biological behaviors of lipopolysaccharide (LPS)-treated hDPCs cells directly cultured on mineral trioxide aggregate (MTA) and CS cements. The results indicated the absorbance values of LPS-treated cells cultured on CS cement were higher than a tissue culture plate. A significant difference (P < 0.05) in cell viability was observed between cells on CS and MTA cements 24 h after seeding. At 48 h, a high concentration of Si (5 mM) was released from MTA, which induced LPS-treated DP cell apoptosis. The present study demonstrates that CS cement is biocompatible with cultured LPS-treated hDPCs cells. MTA stimulates inflammation in LPS-treated hDPCs cells, which leads to greater IL- 1β expression and apoptosis. The above two studies can confirm that CS materials developed by our lab have good biocompatibility for primary cells and inflammatory cells and can continue to provide a good environment for cell growth and differentiation. Therefore, we believe CS will be a potential biomaterial in the future clinically.