牙本质浸提液促进脐带间充质干细胞定向分化协同SCF诱导造血祖细胞归巢重建牙髓组织的实验研究

After removal of the pulp because of pulpitis and periapical diseases, the tooth becomes more fragile for the lack of blood supply and immune defense. Pulp regeneration by tissue engineering method is one of the ideal ways for maintaining the living pulp and function of the tooth. However, the current strategies for pulp tissue engineering encounter three obstacles: Clinical difficulties for obtaining autogenous odontogenic stem cells, lack of clinically easy-handling intracanal scaffold and lack of extensive, sufficient blood supply for the regenerated pulp. Our research has confirmed that Stem Cell Factor (SCF) has the function of inducing stem cells, especially hematopoietic progenitor cells migrating and homing, and promoting microvessels formation. Therefore, in the current research, we are going to optimize the conditions for dental pulp regeneration by: .1. Usage of umbilical cord mesenchymal stem cells (UCMSCs) as the stem cell for dental pulp regeneration. UCMSCs have the multi-lineage differentiation ability and exhibit much more stemness than other adult stem cells. The notable superiority of UCMSCs is the easily acquisition clinically. The liquid extract of dentin matrix is going to be extracted and added into the medium of UCMSCs to provide a stem cell niche for UCMSCs differentiation into dental pulp fibroblasts and odontoblasts. .2. Injectable Self-assembled polypeptide hydrogel has the advantages of easy-handling, and it can also accelerate cells adhesion and proliferation. Therefore, Self-assembled polypeptide hydrogel is going to be used as the scaffold for pulp regeneration in the present study..3. The animal model of mini-pig is going to be established for pulp regeneration. After removal of the pulp and cleaning of the root canals of the tooth, SCF and UCMSCs after detinal matrix extracted liquid treatment are going to be added into the scaffold in the root canals. Homing effect of SCF on hematopoietic progenitor cells and the formation of the microvessels will be observed after 14, 28 and 90 days. .The present study will optimize the conditions for pulp regeneration and promote the clinical translation process of pulp regeneration.

牙髓的保存对于牙齿的生理机能具有重要作用，去除牙髓后，患牙缺少血液供应及防御能力降低表现为抗性下降、脆弱、易折。因此，利用组织工程的方法实现牙髓再生是牙髓炎及根尖周炎理想的治疗方案。目前牙髓再生的主要问题在于：自体牙源性干细胞的获取难题；缺乏适合于根管内注射的理想的支架材料；难以实现快速、充分的血液供应。我们的研究已表明，脐带间充质干细胞（UCMSCs）有向成牙本质细分化的可能性，SCF具有诱导干细胞归巢，促进血管生成的作用。因此，本研究拟采用具有多项分化潜能、“干性”更强、临床易于获得的UCMSCs作为种子细胞，利用牙本质浸提液为UCMSCs提供分化微环境；选择适合于临床操作、可注射的自组装多肽水凝胶为支架材料，以小型猪为牙髓重建的动物模型，利用SCF诱导造血祖细胞归巢至根管支架材料内，促进血管生成，实现牙髓再生。本研究对牙髓再生的临床转化及牙髓炎、根尖周炎的治疗具有重要的科学意义。

牙髓的保存对于牙齿的生理机能具有重要作用，去除牙髓后，患牙缺少血液供应及防御能力降低表现为抗性下降、脆弱、易折。因此，利用组织工程的方法实现牙髓再生是牙髓炎及根尖周炎理想的治疗方案。目前牙髓再生的主要问题在于：自体牙源性干细胞的获取难题；缺乏适合于根管内注射的理想的支架材料；难以实现快速、充分的血液供应。因此，本研究拟采用具有多项分化潜能、“干性”更强、临床易于获得的UCMSCs作为种子细胞，利用牙本质浸提液为UCMSCs提供向成牙本质细胞分化微的环境；利用VEGF诱导UCMSCs向内皮样细胞分化，并将二者构建共培养体系，接种于SCF-RADA16-1自组装多肽水凝胶上，利用体外牙根模型中植入裸鼠皮下，探讨在体内进行牙髓再生的可能性。.实验结果表明：①实验所分离培养的人脐带间充质干细胞（UCMSCs）表达高水平的间充质干细胞标记物（CD90和CD73）及CD105,且可向成骨及成脂方向分化；②牙本质浸提液（LE-TDM）可增强UCMSCs细胞迁移和黏附能力，并可促进其向成牙本质细胞方向分化。③构建载SCF自组装多肽水凝胶支架并进行生物学评价，结果表明该支架材料既保持RADA16-1作为生物支架的优势，还可保持SCF活性，在促进细胞的增殖的同时还可实现对牙髓干细胞和血管内皮细胞的趋化性，为干细胞提供了增殖、粘附、分化的三维微环境，是一种较为理想的牙髓再生支架材料。④利用VEGF诱导UCMSCs向内皮细胞方向分化，结果表明将UCMSCs与经VEGF诱导的UCMSCs（V-UCMSCs）共培养后，HIF-1信号通路被激活，血管形成能力增强。⑤将上述细胞接种于SCF-RADA16-A支架材料上，利用移植牙根进行动物体内牙髓再生的实验研究发现，发现仅有支架材料的牙根内没有牙髓样组织生成，根管内所有移植细胞组（单独培养组和共培养组）都有牙髓样组织生成，与单独培养组相比，共培养组细胞外基质形成较多，胶原沉积量高有血管生成的趋势。以上结果为牙髓再生的临床转化提供了实验依据，提示合适的种子细胞和支架材料对牙髓再生具有重要作用。