三维智能成形脱细胞牙髓水凝胶支架诱导内源性干细胞再生功能性牙髓的机制研究

Pulp regeneration by tissue engineering is a new concept for the treatment of pulp periapical diseases, but there is no effective biological scaffold at present. Decellularized dental pulp can promote the differentiation of dental pulp tissue. However, it is difficult to provide three-dimensional growth environment for cells because of its lack of morphological adaptability. Therefore, the construction of in situ shaping decellularized dental pulp tissue engineering scaffold is the key to achieve pulp regeneration. In our previous study, we successfully prepared decellularized dental pulp hydrogel scaffolds which can be formed automatically at body temperature. At the same time, we found that decellularized dental pulp hydrogel scaffolds can promote the survival of cells under oxidative stress by improving the antioxidant capacity of stem cells. In view of the importance of stem cell migration and survival in damaged microenvironment for regeneration, we believe that decellularized dental pulp hydrogel scaffolds can promote functional pulp regeneration by improving the migration and survival of endogenous stem cells, but the specific mechanism is not clear. This study is to establish animal models of pulp regeneration in nude mice and beagle dogs to verify the effect of decellularized dental pulp hydrogel scaffolds on pulp regeneration in vivo. Proteomics and RNA-seq techniques were used to analyze the specific mechanism of the scaffolds promoting stem cell migration and influencing the antioxidant signaling pathway of stem cells, so as to provide theoretical basis for the improving of the biological function of decellularized dental pulp hydrogel scaffolds and their clinical transformation.

利用组织工程方法实现牙髓再生是治疗牙髓根尖周病的新理念，但目前缺乏有效的生物支架。脱细胞牙髓可促进牙髓组织形成，然而，因其不具备形态可适应性难以为细胞提供三维生长环境。因此，构建原位塑形的脱细胞牙髓组织工程支架是实现牙髓再生的关键。课题组前期成功制备了体温下自动成形的脱细胞牙髓水凝胶支架，证实其可三维填充根管系统，诱导牙髓干细胞迁移，同时可通过提高干细胞抗氧化能力促进细胞的存活。鉴于干细胞迁移及其在损伤微环境中的存活对再生至关重要，我们认为脱细胞牙髓水凝胶支架能够通过提高内源性干细胞迁移能力及存活率促进功能性牙髓再生，但体内效果及具体机制未明。本研究拟建立裸鼠、比格犬牙髓再生模型，验证脱细胞牙髓水凝胶支架体内再生牙髓的效果，采用蛋白组学、RNA-seq等技术，分析该支架促进干细胞迁移、影响干细胞抗氧化信号通路的具体机制，为提高脱细胞牙髓水凝胶支架的生物功能及促进其临床转化应用提供理论依据。

脱细胞牙髓基质在促进牙髓再生方面具有巨大潜能，基于其在牙髓再生中的应用存在缺陷与不足的现状，本项目创新性地制备了一种更适用于复杂多变根管系统的脱细胞牙髓基质水凝胶。本课题通过脱细胞等一系列处理制备脱细胞牙髓基质水凝胶，该支架可实现原位注射，能在体温下启动凝胶化转变，在此基础上检测其理化性能，研究脱细胞牙髓基质水凝胶对牙髓干细胞粘附、迁移及增殖的影响，并探究脱细胞牙髓基质水凝胶对牙髓干细胞成牙本质、成神经及血管化三向分化的影响和对人脐静脉内皮细胞成管的影响。进一步通过体内实验验证脱细胞牙髓基质水凝胶诱导牙髓再生的潜能。本项目发现脱细胞处理后水凝胶仍存在纳米级纤维结构，提示脱细胞牙髓基质水凝胶依然保存细胞外基质成分及空间结构；蛋白组学分析检测出多种与细胞生物学行为相关的蛋白，其中包括细胞迁移、粘附、增殖以及多向分化。免疫荧光染色和细胞迁移实验提示实验组的脱细胞牙髓基质水凝胶促进牙髓干细胞的粘附和迁移，效果均优于其他组；细胞增殖实验显示实验组促进牙髓干细胞增殖效果优于其他组。以上结果表明脱细胞牙髓基质水凝胶能促进牙髓干细胞的粘附、迁移以及增殖。通过免疫荧光染色、Western Blot和qRT-PCR检测发现，牙髓干细胞成牙本质向分化相关蛋白（DSPP）含量和基因（DSPP, DMP-1）在脱细胞牙髓基质水凝胶组的表达上调且高于其他组；成神经向相关蛋白（Tuj-1）和基因（GFAP, Nestin）在脱细胞牙髓基质水凝胶组的表达上调且高于其他组；在人脐静脉内皮细胞成管实验中，脱细胞牙髓基质水凝胶组相对结节数和管长高于其他组，实验组的血管化相关蛋白（KDR, CD31）和基因（VEGF, CD31）表达增强且高于其他组。注射有脱细胞牙髓基质水凝胶的牙本质环于裸鼠皮下植入4周和8周，HE染色可见有类成牙本质细胞及血管形成。结果表明脱细胞牙髓基质水凝胶能在体内促进牙髓样组织的形成。本项目为牙髓再生组织工程支架的选择提供新思路，为三维成形的脱细胞牙髓基质水凝胶应用于牙髓再生提供理论依据。