牙周膜干细胞结合内皮化碳纳米管复合支架材料修复牙周组织缺损的应用基础研究

Periodontal tissue defects cause loosing, shifting and loss of the teeth, which severely affect one's appearance and function of stomatognathic system, and bring difficulties to orthodontic tooth movement in adults. Periodontal tissue restoration is the problem urgently to be solved for periodontal therapy and adult orthodontics. Rising of tissue engineering provides new idea for resolving the problem. However, the distinctiveness of periodontal tissue leads to the complication of periodontal tissue engineering. Vascularization and functionalization are critical for success of the therapy. The characteristics of arrangement orientation and sustained release of carbon nanotube draw our attention. Based on the latest research progress of the field and our previous foundation, the project plans to: (i) study the effect of mechanical stress on periodontal ligament stem cells and blood vessels/vascular endothelial cells in vitro and in vivo, and provide theoretic basis for application of biomechanics on periodontal engineering; (ii) prepare oriented composite of carbon nanotubes and chitosan/collagen as scaffolds, co-seed periodontal ligament stem cells and vascular endothelial cells to establish engineered periodontal tissue, and explore the impact of mechanical force on cell-scaffolds composite; (iii) establish the animal model of periodontal tissue defects, repair them with the engineered periodontal tissue constructed in our study, apply mechanical loading and assess the functionalization of regenerated periodontal tissue. The successful implement of the project will make important breakthroughs on the two crucial issues of vascularization and functionalization in periodontal tissue engineering, and the research findings will bring favorable social and economic effects.

牙周组织缺损引起牙齿松动、移位、脱落，严重影响颜貌美观及口颌功能，牙周组织缺损修复是牙周炎治疗及成人正畸亟待解决的难题。组织工程的兴起为解决该难题提供了新的思路。然而，牙周组织结构的特殊性决定了牙周组织工程的复杂性，血管化和功能化是其关键。碳纳米管因其具有取向性和缓释功能，为我们所关注。基于该领域最新研究进展和本课题组的前期基础，本项目拟：（1）研究应力刺激对体内外牙周膜干细胞和血管/血管内皮细胞的影响，为生物力学应用于牙周组织工程提供依据；（2）制备取向性碳纳米管复合壳聚糖/胶原支架材料，共接种牙周膜干细胞和血管内皮细胞，构建工程化牙周组织，并探讨应力刺激对其的作用；（3）建立牙周组织缺损动物模型，用本研究构建的工程化牙周组织修复，加载应力评估再生牙周组织的功能情况。本项目的成功实施，有望在牙周组织工程中功能性及血管化两个关键问题上实现重要突破，其研究成果将具有良好的社会和经济效益。

牙周组织缺损引起牙齿松动、移位、脱落，严重影响颜貌美观及口颌功能，牙周组织缺损修复是牙周炎治疗及成人正畸亟待解决的难题。组织工程的兴起为解决该难题提供了新的思路。基于该领域最新研究进展和本课题组的前期基础，我们做了四个方面的研究：.（1）应力刺激对血管/血管内皮细胞的影响.我们建立了Beagle犬牙周膜体内应力加载模型，探究应力对血管的影响。结果表明：在应力刺激下牙周膜微血管E2F1及ANG2表达明显上调，新生血管数目增加。.（2）缺氧条件下牙周膜干细胞的成骨分化.我们建立了缺氧条件下PDLSCs与ECs单独培养及间接共培养模型，研究表明缺氧能促进PDLSCs的增殖活性、成骨分化、矿化形成及旁分泌，并证明MAPK/ERK及p38 MAPK信号通路参与了此过程。另一方面证明了缺氧能提高COX-2/PEG2/VEGF通路介导的ECs的迁移能力和PGE2、 VEGF的分泌水平，共培养的ECs能通过COX-2/PEG/VEGF通路进一步增强PDLSCs的成骨分化。.（3）三维有序纳米电纺纤维支架在牙周组织方向性再生中的作用 .我们合成了壳聚糖粘合的多层有序电纺聚己内酯-聚乙二醇三维支架来引导牙周组织定向再生。结果表明：与无序支架及多孔支架相比，有序支架可以显著促进牙周干细胞的增殖及向材料内部的渗透迁移，引导其方向性排列，增加牙周韧带相关基因的表达，促进干细胞向牙周韧带组织分化。且三维有序支架组中新生的类牙周膜组织有明显方向性，牙周韧带的成熟和牙支持组织矿化显著。证明了有序支架适用于定向的牙周组织再生，并可能可更广泛的应用于人类其他组织的定向再生。.（4）复合改性的碳纳米管/胶原/羟基磷灰石多孔支架在骨组织再生中的作用.我们合成了胶原羟基磷灰石（Col-HA）及复合改性的碳纳米管的胶原羟基磷灰石（Col/HA /CNT）泡沫状多孔支架，检测到CNT的加入能显著增强支架材料的机械性能，以0.5wt%CNT组材料硬度最佳。通过体内体外实验证明含有CNT的胶原/羟磷灰石支架材料能够显著的促进细胞增殖及成骨向分化，促进新生骨的形成，且FAK、RhoA、YAP、TAZ可能参与此过程。