低濃度樹脂單體10-MDP與HEMA對於HEPM細胞凋亡及壞死機制之探討

Abstract

牙科黏著劑是現代復形治療不可或缺的材料，尤其在以複合樹脂為主的修復臨床中，黏著劑的選擇與應用對治療成敗有著關鍵性的影響。牙科黏著系統歷經多代演進，從早期多瓶、分步驟的設計，發展到近年強調「一瓶到底」與多功能應用的單瓶式系統。這些單瓶式黏著劑大多整合了酸蝕劑、底劑與黏結樹脂，並以10-MDP（10-Methacryloyloxydecyl dihydrogen phosphate）及HEMA（2-Hydroxyethyl methacrylate）等功能性單體為主要成分，以提升其物理性質與長期穩定性。然而，這些功能性單體除了賦予材料良好的黏著強度與臨床操作便利性外，其生物相容性問題也逐漸受到重視。 在本研究中，針對不同濃度的10-MDP與HEMA長期暴露於人類胚胎上顎間質細胞（HEPM）所造成的生物學影響進行評估。細胞實驗包括Alamar Blue assay 觀察細胞存活率、免疫螢光試驗觀察細胞形態、流式細胞儀檢測細壞死及凋亡情況，粒線體膜電位盒量測膜電位改變。 結果發現，在初期（1至7天）暴露下，不論單體濃度高低，細胞的代謝活性與對照組相近，顯示短期內細胞粒線體功能與能量代謝尚能維持正常。然而，隨著暴露時間延長至14至28天，特別是在高濃度組（如400 μM HEMA + 200 μM 10-MDP），細胞的代謝活性明顯下降，細胞存活率大幅減少，凋亡及壞死細胞比例則顯著上升，呈現濃度與時間依賴性的毒性反應。進一步以粒線體膜電位檢測發現，初期高濃度單體組合會出現粒線體膜電位上升，反應細胞為了應對化學壓力而暫時增強能量代謝，試圖維持存活。但隨著暴露時間拉長，膜電位波動加劇，最終在高濃度組出現顯著下降，顯示粒線體功能受損，與細胞凋亡訊號啟動密切相關。細胞形態學觀察亦顯示，高濃度單體暴露下，細胞密度下降、骨架結構拉長且排列異常，細胞核出現濃縮與不規則形態，貼附與存活能力明顯降低。 這些現象與國內外文獻一致，均指出10-MDP與HEMA的細胞毒性具濃度與時間依賴性，且累積性損傷效果明顯。雖然10-MDP與HEMA提升了黏著劑的臨床性能，但其潛在細胞毒性不容忽視，特別是在深層修復或大量暴露情境下。以本實驗結果來看，HEMA在200 μM而10-MDP在100 μM到200 μM的範圍應較不會造成明顯細胞毒性。 未來材料設計應兼顧黏著強度與生物安全性，改良單體組成、降低未聚合單體釋放。總結來說，高濃度及長期暴露於10-MDP與HEMA會對HEPM細胞造成明顯的細胞毒性，表現為細胞活性下降、凋亡壞死比例上升與粒線體功能障礙。這些現象提醒臨床應謹慎選擇與應用含有這些單體的黏著劑，以確保修復治療的長期安全與成功。

Dental adhesives are indispensable materials in contemporary restorative dentistry, particularly in clinical procedures that utilize composite resins as the primary restorative material. Over the years, dental adhesive systems have undergone multiple generations of evolution, progressing from early multi-bottle, multi-step designs to the recent development of single-bottle systems that emphasize streamlined application and multifunctionality. These single-bottle adhesives typically integrate etchants, primers, and bonding resins, with functional monomers such as 10-MDP and HEMA serving as major components to enhance physical properties and long-term stability. However, alongside the improved bond strength and clinical convenience provided by these functional monomers, increasing attention has been paid to their biocompatibility. In this study, the biological effects of prolonged exposure to various concentrations of 10-MDP and HEMA on human embryonic palatal mesenchymal （HEPM） cells were evaluated. Cellular experiments included the Alamar Blue assay to assess cell viability, immunofluorescence staining to observe cell morphology, flow cytometry to detect cell necrosis and apoptosis, and measurement of mitochondrial membrane potential to monitor changes in mitochondrial function. The results demonstrated that during the initial exposure period （days 1–7）. Regardless of monomer concentration, cellular metabolic activity remained comparable to the control group, indicating that mitochondrial function and energy metabolism were largely unaffected in the short term. However, as the exposure period extended to 14–28 days, a marked decrease in metabolic activity was observed, accompanied by a substantial reduction in cell viability and a significant increase in the proportion of apoptotic and necrotic cells. These findings revealed a concentration- and time-dependent cytotoxic response. Further analysis of mitochondrial membrane potential showed that, in the early phase, the combinations of high-concentration monomer combinations led to an increase in membrane potential, reflecting a compensatory enhancement of energy metabolism in response to chemical stress. However, with prolonged exposure, fluctuations in membrane potential intensified, ultimately leading to a significant decline in the high-concentration groups. This indicates mitochondrial dysfunction closely associated with the activation of apoptotic signaling pathways. The results of morphological observations also revealed that high-concentration monomer exposure resulted in decreased cell density, elongated and abnormally arranged cytoskeletal structures, and condensed or irregularly shaped nuclei, all indicative of compromised cell adhesion and viability. These phenomena are consistent with findings in the literature, which collectively indicate that the cytotoxicity of 10-MDP and HEMA is both concentration- and time-dependent, with pronounced cumulative damage. Although 10-MDP and HEMA improve the clinical performance of dental adhesives, their potential cytotoxicity cannot be overlooked, particularly in deep restorations or scenarios involving extensive exposure. The design of future materials should be committed to balancing adhesive strength and biological safety by optimizing monomer composition and reducing the release of unpolymerized monomers. In summary, high concentrations and prolonged exposure to 10-MDP and HEMA result in significant cytotoxic effects on HEPM cells, manifested as decreased cell activity, increased rates of apoptosis and necrosis, and mitochondrial dysfunction. These findings underscore the importance of prudent clinical selection and application of adhesives containing these monomers to ensure the long-term safety and success of restorative treatments.