丁香油酚之牙本質滲透性對於健康人類牙髓細胞的影響

Abstract

實驗目的：過去的研究顯示丁香油酚可經由牙本質緩慢的滲透進入發炎的牙髓腔中，達到抗發炎、止痛的效果；不過丁香油酚藉由牙本質滲透進入牙髓腔的濃度，各研究存在差異。另外，過去的研究也較少著墨於丁香油酚對於健康人類牙髓細胞之影響。牙科臨床上利用丁香油酚抗發炎與止痛特性的同時，應同時瞭解這個材料對健康細胞的影響。本研究欲探討丁香油酚對於牙本質之滲透濃度、及對於健康人類牙髓細胞的細胞毒性和發炎反應的影響，並嘗試了解相關的細胞訊息傳導路徑。 實驗方法：第一部分先藉由Transwell Dentin Disc Model體外實驗觀察並以質譜儀（Mass Spectrometers）檢測分析不同濃度丁香油酚於12小時、24小時滲透0.5mm牙本質的能力。第二部分自拔除的牙齒培養人類牙髓細胞，參考第一部分滲透後丁香油酚濃度刺激細胞，以光學顯微鏡觀察細胞形態的變化，再以MTT細胞存活率分析檢測細胞活性。第三部分將健康人類牙髓細胞以丁香油酚處理不同時間後，以西方墨點法（Western blot）及免疫螢光染色法（Immunofluorescence）觀察丁香油酚對於促發炎路徑及細胞膜內訊息分子的影響。接著使用U0126（MEK/ERK的抑制劑）預先處理人類牙髓細胞，來反向驗證丁香油酚對於訊息傳導路徑的影響。實驗中分析的訊息傳導路徑的蛋白包括ERK 1/2、p38 mitogen-activated protein kinases（p38）、nuclear factor-kappa B（NF-κB）、Cyclooxygenase-2（COX-2）。 實驗結果：IRM®和10mM丁香油酚滲透0.5mm牙本質12小時後丁香油酚之滲透濃度分別約為82.6µM、61.3µM；24小時後丁香油酚之滲透濃度分別約為242.3µM、387µM。小於500µM丁香油酚的濃度對人類牙髓細胞的細胞活性及細胞形態的影響較小，濃度高於500µM開始會使細胞活性及細胞形態改變，而濃度大於1.5mM在12小時即觀察到細胞死亡。在健康人類牙髓細胞加入500µM或1000µM的丁香油酚處理約三小時後，觀察到ERK 1/2、p38及NF-κB磷酸化表現達到高峰； COX-2在丁香油酚濃度500μM或1000µM處理下，隨著時間的增加呈現上升趨勢。抑制劑預處理的結果顯示，U0126對於丁香油酚所引發之COX-2表現量的上升，具有抑制效果。 結論(1)IRM®和丁香油酚可以透過很薄的牙本質滲透進入牙髓腔。不論是使用IRM®或是含有10mM的丁香油酚溶液，在透過牙本質試片模型進行滲透實驗時，24小時的滲透濃度都低於500µM。這樣的滲透濃度不會對人類牙髓細胞產生細胞毒性，對人類牙髓細胞的影響也很小。(2)小於500µM濃度的丁香油酚對人類牙髓細胞的細胞活性及細胞形態的影響較小，高於500µM開始會使細胞活性及細胞形態改變。濃度大於1.5mM在12小時即觀察到細胞死亡，顯現高濃度的丁香油酚的細胞毒性高。(3)丁香油酚加入健康人類牙髓細胞後，可能透過MEK/ERK訊息傳導路徑誘導COX-2的表現。 目前丁香油酚廣泛應用於窩洞填補和間接覆髓，並具有抗菌、抗發炎、止痛和鎮靜神經的效果。善用這些特性或許能減少未來根管治療的需求。不過高濃度的丁香油酚具有細胞毒性，應避免直接接觸牙髓細胞。研究丁香油酚對牙髓細胞的影響對牙科臨床治療很重要，未來需深入探究其對COX-2表現相關訊息傳遞路徑及調控蛋白的影響，以優化臨床應用。

Aim：Past studies have indicated that eugenol can slowly permeate into the inflamed dental pulp chamber through dentin, resulting in anti-inflammatory and analgesic effects. However, there are variations in the eugenol concentration that penetrates through dentin in different research studies. Moreover, there has been limited research on the impact of eugenol on healthy human dental pulp cells（hDPCs）. While utilizing eugenol for its anti-inflammatory and analgesic properties in dental clinical practice, understanding its effects on healthy cells is crucial. This study aims to explore the eugenol permeation through dentin, its cytotoxicity, and its impact on inflammatory response in healthy hDPCs, as well as investigating relevant cellular signaling pathways. Materials and methods： The study consists of three main parts. The first part involves an in vitro experiment using the transwell dentin disc model with mass spectrometers to detect and analyze the permeation ability of varying concentrations of eugenol through 0.5mm dentin over 12 and 24 hours. In the second part, hDPCs obtained from extracted teeth are cultured and exposed to eugenol at concentrations determined in the previous part. Cell morphology are observed using an optical microscope, and cell viability is analyzed using the MTT assay. The third part involves treating healthy hDPCs with eugenol for different time durations. The effects of eugenol on pro-inflammatory pathways and intracellular signaling molecules are examined using Western blot and Immunofluorescence techniques. Additionally, pre-treatment with U0126 （MEK/ERK inhibitor） was conducted in healthy hDPCs to investigate eugenol's influence on signaling pathways. Results： After 12 hours of permeation through 0.5mm dentin, the eugenol concentrations of IRM® and 10mM were approximately 82.6µM and 61.3µM, respectively. After 24 hours, the eugenol concentrations were approximately 242.3µM and 387µM, respectively. Eugenol concentrations below 500µM had minimal impact on the cell viability and morphology of human dental pulp cells. However, concentrations higher than 500µM started to induce changes in cell viability and morphology. Cell death observed at concentrations greater than 1.5mM within 12 hours. In healthy hDPCs exposed to 500µM or 1000µM eugenol for approximately three hours, we observed the highest levels of phosphorylation in ERK 1/2, p38, and NF-κB. Concurrently, the expression of COX-2 exhibited an upward trend over time following treatment with 500μM or 1000µM eugenol. U0126 inhibited the increase in COX-2 levels in healthy hDPCs induced by eugenol. Conclusion：(1)Both IRM® and eugenol can penetrate through dentin. Whether using IRM® or a 10mM eugenol solution, the permeation concentrations were below 500µM in the dentin disc model. Such permeation concentrations do not exhibit cytotoxicity to human dental pulp cells and have minimal impact on their behavior.(2)Eugenol concentrations below 500µM have limited effects on the cell viability and morphology of human dental pulp cells, while concentrations above 500µM start to induce changes in cell viability and morphology. At concentrations higher than 1.5mM, cell death was observed within 12 hours, indicating high cytotoxicity of eugenol at high concentrations.(3)U0126 inhibited the increase in COX-2 levels in healthy hDPCs induced by eugenol. Currently, eugenol is widely used in cavity filling and indirect pulp capping procedures due to its antibacterial, anti-inflammatory, analgesic, and nerve-calming effects. Utilizing these properties may reduce root canal treatments in the future. However, high concentrations of eugenol can be cytotoxic to cells, and direct contact with dental pulp cells should be avoided. Research on the effects of eugenol on dental pulp cells is crucial for dental clinical treatment, and further investigations should explore its impact on COX-2 expression and related signaling pathways to optimize its clinical applications.