加载rhBMP2纳米短肽水凝胶RADA16-I促进hUC-MSCs牙向分化再生牙髓的实验研究

Dental pulp regeneration has been a research focus in the field of restorative dentistry. Cell, scaffold and growth factors are the three key materials for tissue engineering. As the sources of seed cells for pulp tissue engineering, dental stem cells often are insufficient due to lack of resources. In comparison, human umbilical cord mesenchymal stem cells(hUC-MSCs) may be the ideal seeding cells for pulp tissue engineering because of their abundant availability and multi-lineage differentiation potential. Our previous study indicated that dental pulp cells (DPCs) can induce hUC-MSCs differentiation into odontoblast in the presence of BMP-2. BMP-2 play a important role on the differentiation of odontoblast. Self-assembly peptide nanofiber hydrogel scaffold materials as a new member of scaffold materials because of its excellent biological compatibility and biological activity, has been widely used in tissue engineering and regenerative medicine. RADA16-I (AcN-RADARADARADARADA-CNH) is a new type of injection self assembling peptide nanofiber hydrogel scaffold materials. Researches indicated that subcutaneous implantation of the RADA16-I containing dental stem cells showed the formation of dental pulp-like tissue. It is not clear whether DPCs can induce hUC-MSCs differentiating into odontoblast in RADA16-I containing rhBMP-2. In this study, for the first time, we encapsulate these two cells in RADA16-I supplement with BMP-2 and investigate the feasibility of its applications in pulp tissue regeneration in vitro and in nude mice, and further explore its molecular mechanisms, which may offer the experimental basis for treatment of pulp injury.

牙髓再生是牙齿修复研究的热点。种子细胞、支架材料和细胞因子是实现牙髓生物再生的三个关键因素。牙源性干细胞做为种子细胞来源匮乏，而脐带间充质干细胞(hUC-MSCs)来源广泛，多向分化能力优良。前期研究表明牙髓细胞在骨形态发生蛋白2 ( BMP-2) 作用下，促进hUC-MSCs分化为成牙本质细胞。RADA16-I 是一种新型自组装多肽纳米纤维水凝胶支架材料。研究表明牙源性干细胞与RADA16-I混合植入体内可形成牙髓样组织。目前不清楚hUC-MSCs在加载BMP-2的RADA16-I中与牙髓细胞三维共培养能否分化为成牙本质细胞，在体内能否形成牙髓样组织。本实验首次将上述两种细胞混合接种于加载BMP-2的RADA16-I，初步探索三维共培养策略构建牙髓组织的可行性，并移植裸鼠体内，检验其在动物体内的长期稳定性，探讨其相关分子机制，为进一步研究hUC-MSCs治疗牙髓损伤提供实验依据。

牙髓再生是牙齿修复研究的热点。种子细胞、支架材料和细胞因子是实现牙髓生物再生的三个关键因素。牙源性干细胞做为种子细胞来源匮乏，而脐带间充质干细胞(hUC-MSCs)来源广泛，多向分化能力优良。前期研究表明牙髓细胞在骨形态发生蛋白2(BMP-2)作用下，促进hUC-MSCs分化为成牙本质细胞。RADA16-I是一种新型自组装多肽纳米纤维水凝胶支架材料。研究表明牙源性干细胞与RADA16-I混合植入体内可形成牙髓样组织。目前不清楚hUC-MSCs在加载BMP-2的RADA16-I中与牙髓细胞三维共培养能否分化为成牙本质细胞，在体内能否形成牙髓样组织。本实验首次将上述两种细胞混合接种于加载BMP-2的RADA16-I，初步探索三维共培养策略构建牙髓组织的可行性，并移植裸鼠体内，检验其在动物体内的长期稳定性，探讨其相关分子机制，为进一步研究hUC-MSCs治疗牙髓损伤提供实验依据。本课题研究结果表明：1.二维培养体系中hUC-MSCs与hDPCs按照1:1的比例共培养可促进hUC-MSCs牙向分化并增强细胞的促血管形成因子的表达。该实验结论说明hUCMSCs有分化为牙髓组织相关细胞的能力，为丰富hUCMSCs的多向分化能力以及牙髓再生的种子细胞来源提供了依据。2.适宜人脐带间充质干细胞生长的水凝胶支架最佳浓度为0.25%。所得的结论为进一步研究脐带间充质干细胞与支架材料相互作用提供理论依据和实验参数。3.LPS浓度为10μg/mL时可促进DPCs增殖、且引起细胞因子IL-6,IL-10,HGF表达发生显著变化。4.共培养体系可促进细胞增殖、抑制促炎因子IL-6表达、促进抑炎因子IL-10和HGF的表达，并降低IL-6/IL-10值与IL-6/HGF值。5.将BMP-2诱导的hUCMSCs和hDPCs在水凝胶中混合共培养，建立一种更接近细胞生长微环境的三维共培养新模式，这种水凝胶共培养方式能促进细胞的增殖和向牙源性分化。其作用机制尚需进一步研究，目前的研究结果为后续的相关研究奠定了基础。