下顎智齒拔除術後神經損傷修復之研究：電刺激促進齒源性幹細胞神經分化

Abstract

研究背景 智齒手術為常見的牙科手術，手術併發症中的下顎齒槽神經損傷，其伴隨的症狀常對患者之後的生活品質造成影響。對於神經損傷，傳統治療包含藥物治療、轉介之復健和心理治療等。然而，藥物使用雖有一定的幫助，尚仍缺乏長期或可預期性的結果。近年來，組織工程於醫學領域的應用，對於周邊神經損傷之患者，組織工程輔助療法似乎成為一道曙光。其中，幹細胞療法及生物支架已證實對於脊髓神經損傷的患者有正向的療效。本研究之主要目標係以細胞實驗模式針對牙髓幹細胞輔以不同電壓之電刺激，評估其誘導分化成神經細胞的能力。進一步輔以文獻回顧及臨床資料庫之建立，期能初步建立提供未來影響醫源性三叉神經損傷於臨床治療之處置流程之基礎研究。 實驗方法 針對細胞研究，係收集人類牙髓幹細胞，進行分離、純化及驗證牙髓幹細胞以完成細胞之製備，其後進行兩次的誘導分化，於第二次誘導分化時分組以不同電壓(0.5V、1.0V、1.5V)進行短期電刺激，再以細胞型態、免疫螢光染色及流式細胞儀確認細胞分化及生長結果。 實驗結果 本研究已由文獻回顧確認下顎智齒手術術後之神經損傷之臨床重要性，並以細胞實驗證實牙髓幹細胞於適當的神經分化液驅導之下，有分化為神經細胞的潛能；同時設計出一電刺激裝置，確認若在細胞分化成熟過程中，輔以1.0V的電刺激，可達成最多的神經細胞生長及分化。並經由細胞型態、免疫螢光染色及流式細胞儀確認。 結論 本實驗已順利開發出電刺激裝置，用於評估齒源性幹細胞誘導分化成神經細胞的研究模式。經由此一模式，也證實適當的電刺激有驅導牙髓幹細胞分化之潛能，並可得出一較佳電壓強度。雖針對神經損傷之臨床處置，目前仍有極大發展空間，本研究已初步建立神經損傷於臨床治療之處置流程之基礎研究。

Background Mandibular third molar surgery was one of the most common dentoalveolar surgery. However, the potential complication of inferior alveolar nerve injury after the surgery played a profound role in the patients’ quality of life. The conventional management of nerve injury included medication, physical therapy, psychological consultation, and also surgical repair, but there was still lack of predictable long-term outcome and required further investigation. Recently, tissue engineering was a thriving topic in the field of medical care. The optimistic outcome of application of stem cell therapy and biomechanical scaffold in treatment of patients with spinal cord injury became a shining light of hope for many other patients suffered from nerve injury. Therefore, our specific aim was to investigate the effect of electric stimuli on odontogenic stem cells on nerve differentiation in vitro. Accompanied with literature review and establishing a clinical dataset, a proper clinical management protocol of iatrogenic trigeminal nerve injury may be developed. Material and method In the in vitro study, we collected extracted human teeth, and performed isolation, purification, culture and identification of human dental pulp stem cells. After neural differentiation on dopaminergic neural induction medium, short term electric stimuli were indicated on the stem cells. Last but not least, cell morphology, immunocytochemistry and flow cytometry were done to confirm the result of neural differentiation. Results Iatrogenic trigeminal nerve injury significantly affected the quality of life of patients. In in vitro study, we confirmed that dental pulp stem cells(DPSCs) owned the potential of neural differentiation after proper induction. Meanwhile, we designed an appliance to perform electric stimuli on DPSCs. The result showed better neural differentiation after short term electric stimuli with 1.0 voltage, and was verified by cell morphology, immunocytochemistry and flow cytometry. Conclusion Our result supported that electric stimuli with specific voltage had the potential of positive effect on neural differentiation of DPSCs. An appliance was also designed and manufactured to provide electric stimuli. However, lack of solid evidence was still a major challenge to the clinical protocol of nerve repair. We have initially established a clinical treatment scheme for iatrogenic trigeminal nerve injury based on the in vitro study.