D-氨基酸通过c-di-GMP信号通路对种植体表面生物膜的破坏作用

Peri-implant mucositis together with its subsequent peri-implantitis are the major causes of implant failure. The formation of biofilm is an initiating factor for the failure. In clinical practice, no established medications are aimed to remove the biofilm yet. Recently, D-amino acids have been found to destroy the biofilm formed by in vitro cultured bacteria. The finding suggests that the D-amino acids could be used to prevent the biofilm formation and /or destroy the formed biofilm for maintaining the implants in vivo. In addition, cyclic di-guanosine monophosphate (c-di-GMP), a vitally crucial second messenger for bacteria, has been identified to regulate the biofilm formation, with the evidence that L-amino acids promote the biofilm formation by acting on the c-di-GMP signaling pathway. Thus, we presume that D-amino acids could destroy the biofilms by targeting the c-di-GMP signaling pathway because the D-amino acids could function appositively to their enantiomeric L-amino acids.. In this project, we are planning to test whether the D-amino acids can destroy the biofilm formed with porphyromonas gingivalis, one of the major microbes causing the implant failure, by acting on c-di-GMP signaling pathway. The D-amino acids will be prepared in temperature-sensitive slow-releasing gels and tested in peri-implantitis dog model, being observed for their efficacies to remove the biofilm on implant and prevent peri-implantitis. The results of this study will provide a theoretical basis for developing a novel medication for the long term maintenance of the implants.

种植体周围黏膜炎及继发的种植体周围炎是导致种植失败的主要原因，生物膜的形成是其始动因素。然而目前临床上并没有清除生物膜的药物。近期发现D-氨基酸能够破坏体外细菌形成的生物膜，提示D-氨基酸在体内可能通过阻止生物膜的形成或破坏已形成的生物膜来维护种植体。环二鸟苷酸（c-di-GMP），作为细菌至关重要的第二信使调控生物膜形成。L-氨基酸通过c-di-GMP信号通路可促进生物膜的形成。由于D-氨基酸起到其对映异构L-氨基酸相反的作用，推测D-氨基酸可能通过c-di-GMP信号通路破坏生物膜。. 本研究拟检测D-氨基酸是否通过c-di-GMP信号通路破坏牙龈卟啉单胞菌（导致植体失败最重要的细菌）形成的生物膜。构建负载D-氨基酸的温敏缓释凝胶并用于种植体周围炎模型，观察D-氨基酸对种植体表面生物膜的清除及种植体周围炎的防治效果。研究结果可能为临床上维持种植体长期稳定的新药研制提供理论依据。

主要研究内容：P.gingivalis ATCC 33277的培养和鉴定，D-氨基酸对 P.gingivalis ATCC 33277生长及其生物膜形成和分散的影响；评价 D-氨基酸的细胞毒性，初步筛选出无毒性作用的浓度；探讨 D-氨基酸在 L-氨基酸作用下对细菌生物膜形成作用的影响；通过研究有效 D-氨基酸对 P.gingivalis ATCC 33277细胞内 c-di-GMP浓度的影响，探讨有效 D-氨基酸对 P.gingivalis ATCC 33277生物膜分散作用机制；负载 D-氨基酸水凝胶的构建及表征；探讨负载 D氨基酸水凝胶在体内对种植体周围炎的预防和治疗作用。.重要结果：D-氨基酸可有效抑制 P.gingivalis ATCC 33277细菌活性，并通过影响细胞内 c-di-GMP浓度抑制其生物膜形成和分散成熟的生物膜；D-氨基酸具有良好的生物安全性，对 L-929细胞无毒性；在 L-氨基酸存在的情况下，不同种类的 D-氨基酸可表现为协同作用、抑制作用和无相关性，即不同种类的氨基酸其左旋右旋结构对其作用影响不同；项目组成功构建了生物安全的负载D-氨基酸水凝胶；并在体内实验证实了负载 D-氨基酸水凝胶对种植体周围炎的预防及治疗作用。.科学意义：负载 D-氨基酸水凝胶可作为治疗菌斑性种植体周围炎的潜在药物。