永生化牙髓干细胞外泌体基质诱导内源性细胞归巢实现牙髓再生的研究

Dental pulp necrosis, as the most common outcome of dental pulp inflammation, has a high incidence in dental clinic. It is widely accepted that realizing dental pulp regeneration is the best choice for treating teeth with dental pulp necrosis. Compared with exogenous cells transplantation, it is more suitable for clinical application to realize dental pulp regeneration via endogenous cell homing. For realizing dental pulp regeneration based on cell-homing strategy, it is necessary to establish a matrix micro-environment suitable for dental pulp repairing in root canals. Exosome, as a vital media of cell-cell communication, can affect surrounding cells by transporting RNAs and proteins. We demonstrated dental pulp stem cells(DPSC) can secrete a series of factors involving cell migration, differentiation and tissue regeneration. Dental pulp specific extracellular matrix(ECM) is the micro-environment for dental pulp cells growth and maintenance, and our pilot experiment proved that dental pulp ECM can promote the expressions of associated proteins. We have made a matrix based on DPSC exosomes and dental pulp ECM for establishing micro-environment in root canals, and will evaluate its application potential by investigating the capacity of inducing neighboring stem cells to migrate into canal space and differentiate to form dental pulp and dentine. Our team have established immortalized dental pulp stem cells (iDPSC) to avoid repeated isolation of primary DPSC. We have also demonstrated iDPSC had the similar paracrine profile as primary DPSC. Besides, exosomes derived from iDPSC (iDPSC-Exo) were proved to enhance stem cell migration. This present project is to acquire exosomes derived from iDPSC and investigate 1) profile of biological molecules in iDPSC-Exo, 2) effect of iDPSC-Exo on cell migration and odontogenic differentiation in vitro, 3) effect of iDPSC-Exo on inducing dental pulp regeneration in situ in animal model. Then the feasibility of iDPSC-Exo on dental pulp regeneration is evaluated.

牙髓坏死是牙髓感染最常见的结局，发病率高。再生牙髓是最佳治疗方式，诱导内源性细胞归巢相比外源性细胞植入更适合临床应用。即在根管内构建适合牙髓修复的微环境，诱导内源性细胞归巢实现牙髓再生。外泌体是细胞间交流的媒介，通过传递蛋白和RNA作用于其它细胞。我们研究发现牙髓干细胞(DPSC)能分泌多种促细胞迁移分化及组织再生的因子，牙髓特异性胞外基质(ECM)是维持牙髓细胞特性的微环境，促进细胞表达相关蛋白。我们用DPSC外泌体和ECM在根管内构建了基质微环境，评估其诱导周围干细胞迁移至根管并分化形成牙髓牙本质的能力论证其应用价值。为避免反复提取原代细胞，我们构建永生化DPSC，发现其分泌蛋白同原代细胞相似，其外泌体(iDPSC-Exo)能促进干细胞迁移。本课题拟研究iDPSC-Exo①生物活性成分②体外对细胞迁移分化的作用③原位促牙髓再生的作用,以评估基于iDPSC-Exo的牙髓再生模式的可行性。

牙髓坏死是牙髓感染最常见的结局，发病率高。再生牙髓是最佳治疗方式，诱导内源性细胞归巢相比外源性细胞植入更适合临床应用。即在根管内构建适合牙髓修复的微环境，诱导内源性细胞归巢实现牙髓再生。前期研究发现牙髓干细胞(DPSC)能分泌多种促细胞迁移分化及组织再生的因子，牙髓特异性胞外基质(ECM)是维持牙髓细胞特性的微环境，促进细胞表达相关蛋白。本课题旨在用DPSC外泌体（DPSC-Exo）和ECM在根管内构建了基质微环境，评估其诱导周围干细胞迁移至根管并分化形成牙髓牙本质的能力论证其应用价值。首先成功分离DPSC-Exo，用TEM、WB和粒径分析进行鉴定，采用质谱法和miRNA测序分析外泌体的成分，获取DPSC-Exo生物活性成分的数据，证明通过低氧诱导和3D培养可调节优化外泌体的成分。DPSC-Exo体外功能实验证明了DPSC-Exo具有促进细胞增殖，迁移和体外管状结构形成。研究结果证明外泌体miRNA可以逆转LPS引起的细胞炎症，并通过基因测序筛选了发挥作用的信号通路。制备了人牙髓ECM，基于牙髓特异性ECM制备了ECM水凝胶搭载DPSC-Exo，负载外泌体的ECM水凝胶具有良好的生物相容性和再生功效，此外制备了适合牙髓组织再生的明胶-纤维蛋白支架作为备选。我们采用多种细胞及牙髓ECM成功构建了3D人牙髓organoids模型，可用于快速筛查盖髓材料的毒性。基于这一模型验证了DPSC-Exo可提高成牙相关蛋白表达。进行了异位和原位的牙髓再生动物实验，发现负载外泌体的水凝胶构建局部基质微环境，能促进根管内新生类似牙髓样组织，且具有血管结构和DSP阳性细胞。本课题证明了DPSC-Exo和牙髓ECM在牙髓再生中的应用潜能。开发出一种可负载细胞外泌体的水凝胶载体，用于牙髓组织工程研究。本课题提供了一种牙髓再生性治疗新策略，有望今后临床转化。发表文章3篇，专利授权2项，申请1项。