| 摘要: | 緒論
直接覆髓治療和斷髓治療皆是保護外露牙髓組織活性的保守治療方式。在修復的過程中，活化的牙髓幹細胞會增生，接著遷徙到受損處並分化成類牙本質母細胞以分泌鈣化物質形成修復性牙本質作為牙髓組織及外界的屏障。過去的研究指出靜磁場可以以生物力學的形式刺激不同細胞的生長，遷徙及分化。然而，至目前還沒有相關的研究指出靜磁場對於修復性牙本質生成是否也具有相同的正面效果。因此，本研究的主要目的就是探討靜磁場是否可促進牙髓幹細胞的增生，遷徙及牙本質分化，以及其可能的機制。
實驗材料與方法
為了探討細胞的生長，本實驗使用MTT方法分析細胞的生長速率以及利用流式細胞儀分析細胞週期。在細胞遷徙的實驗中則是採用刮痕方法比較對照組和實驗組的差異。檢測細胞牙本質母細胞的分化則是利用即時聚合酶鏈反應分析DSPP和DMP-1的表現及使用Alizarin Red S染劑染色最終鈣離子的沉積。為了探討靜磁場對牙髓幹細胞行為改變的可能機制，細胞膜的流動性，胞內鈣離子的動態平衡，細胞骨架的結構塑形和MAPK訊息傳遞路徑也一一進行分析。本實驗利用螢光染劑TMA-DPH的螢光偏極光特性分析細胞膜的異位性。胞內鈣離子的動態平衡和細胞骨架則是分別利用Fura-2AM螢光染劑及肌動蛋白/肌球蛋白染劑標定並於螢光顯微鏡下觀察比較。而MAPK訊息傳遞在靜磁場的作用下之探討則是分別加入三種不同的抑制劑以抑制ERK，JNK和p38 MAPK路徑。最後，為了轉移體外的實驗結果到臨床上的應用，本實驗也進行實體牙髓外露的牙齒模型及體內動物實驗，嘗試印證靜磁場應用於覆髓治療的可能性。
結果
MTT實驗結果顯示暴露靜磁場的細胞比對照組細胞有更高的生長速率，但其細胞週期並沒有顯著的改變。在刮痕實驗中也可發現暴露靜磁場的細胞在細胞遷徙的速度上也比較快，而且即時聚合酶鏈反應和鈣離子沉積的結果都顯示暴露靜磁場的細胞，有顯著較高的表現。此外，暴露靜磁場細胞其細胞膜各向異性（r值）比對照組的高，胞內鈣離子的活性也增加及細胞骨架呈現高度活躍的塑形狀態。而且，細胞活性實驗和即時聚合酶鏈反應的結果顯示牙髓幹細胞在0.4 T靜磁場的暴露下其p38 MAPK信息傳遞路徑會活化。後續的實驗結果也發現細胞在加入p38 MAPK信息傳遞路徑的抑制劑SB203580時，原本靜磁場促進細胞遷徙和牙本質分化的生物效應也會一併被抑制。最後，實體牙髓外露的牙齒模型及體內動物實驗的結果都呈現靜磁場刺激的組別有更活躍的細胞生長及修復性牙本質的生成。
結論
依據本研究的實驗結果顯示0.4 T靜磁場可以促進牙髓幹細胞的生長，遷徙和牙本質分化。這些細胞行為的改變主要是細胞在受到靜磁場的機械外力後，藉由細胞膜及細胞骨架的結構改變來完成直接性機械外力傳遞。另外，鈣離子的動態平衡變化和p38 MAPK訊息傳遞路徑的活化則是以間接性機械外力傳遞方式把靜磁場的刺激傳遞至細胞核以影響細胞行為的表現。另外，體內動物實驗的結果也印證了靜磁場在臨床上可作為覆髓治療的輔助工具。
Introduction:
Direct pulp capping and partial pulpotomy are conservative treatments for preserving the vitality of exposed dental pulp tissue. In the reparation process, the proliferated dental pulp stem cells (DPSCs) migrate to the trauma site and differentiate into odontoblast-like cells which replace the necrotic odontoblasts. As the cellular biomechanical concept, the static magnetic fields (SMFs) enhance cell proliferation, migration, and differentiation in difference cell types. However, whether SMFs have a similar positive effect on the reparative formation of DPSCs remain unknown. Therefore, the purpose of the present study was investigating the effects of SMFs on the proliferation, migration, and odontogenesis of DPSCs and its putative mechanism.
Materials and methods:
For the cell proliferation analysis, MTT and cell cycle experiments were performed. In vitro scratch assay was performed to evaluate the effect of 0.4 T SMF on DPSC migration. The odontogenic evaluation was performed by analyzing the expressions of odontogenic genes using a quantitative real-time polymerase chain reaction (qRT-PCR) process and Alizarin Red S staining. To evaluate the putative mechanism of SMFs to DPSCs, cell membrane fluidity, calcium ion homeostasis, cytoskeleton reorganization and the mitogen-activated protein kinase (MAPK) signaling pathways were analyzed. The cell membrane anisotropy was tested using a fluorescence polarization-depolarization assay. The intracellular calcium ions of the SMF-treated cells were analyzed using Fura-2 acetoxymethyl ester labelling. The cytoskeletons of exposed and unexposed control cells were labelled with actin fluorescence dyes. Cell viability, cell migration and dentinogenesis were checked when the tested cells were cultured with inhibitors of ERK, JNK and p38 to discern the possible signaling cascade involved in the proliferative effect of the SMF on the DPSCs. To translating research into practice, a traumatic pulp exposure model and in vivo animal study were also performed.
Results:
The MTT results showed the proliferation of SMF-treated DPSCs was higher than the sham-exposed group, while the cell cycles were no significant different. The scratch assay results showed that the application of 0.4 T SMF enhanced cell migration toward the scratch wound. For the SMF-treated group, the DMP-1 and DSPP genes exhibited a higher expression than the sham-exposed DPSCs. The distribution of calcified nodules was also found to be denser in the SMF-treated group. Furthermore, SMF-treated cells demonstrated a higher anisotropy value. The intracellular calcium ions were also activated by SMFs. In addition, fluorescence microscopy images demonstrated that SMF-treated cells exhibit higher fluorescence intensity of the actin cytoskeletal structure. Cell viability and qRT-PCT suggested that the p38 signaling cascade was activated when the DPSCs were exposed to a 0.4 T SMF. The cell migration and dentinogenesis were also reduced by SB203580 incorporated. The traumatic pulp exposure model and in vivo animal study showed the SMF-treated groups have better cell activation and reparative dentin formation.
Conclusion:
The present results suggest that 0.4 T SMF enhances DPSC proliferation, migration and dentinogenesis through the change of cellular membrane and cytoskeleton architecture as direct mechanotransduction. Also, it stimulates the intracellular well-known second messenger calcium ion activity and activation of the p38 MAPK related pathway as indirect mechanotransduction. The in vivo study was also showed the possibility use of SMF as an adjuvant for pulp regenerative therapy. |
