超声结合碳纳米管应用于老龄牙周组织工程的研究

Periodontitis is the most common cause of tooth loss among adults and it is difficult to rebuild periodontal attachment through traditional treatments. Due to the decreasing amount and deteriorating function of stem cells, the treatment of periodontal defects has an even poorer prognosis among aged patients. Tissue engineering becomes an effective approach for periodontal regeneration. Researches indicate that low-intensity pulsed ultrasound can enhance osteogenic and cementogenic differentiation of mesenchymal stem cells. Carbon nanotubes can improve mechanical properties of collagen scaffolds and accelerate deposition of calcium, which makes up the shortage of the ultrasound in mineralization stage. And the signaling pathways triggered by them are tightly connected, so their combined application may have better effects in periodontal tissue engineering. In this project, the influence of ultrasound and carbon nanotubes to differentiation and mineralization of BMSCs will be studied, and their synergism and involved mechanism will be explored in an in vitro model. We will investigate their separate and combined application in periodontal tissue engineering based on exogenous BMSCs through an aged rat model. The differentiation and turnover of the implanted BMSCs will be studied and the key differentiation markers and proteins involved in signaling pathways will be verified in animal models. This research will provide theoretical and experimental basis for the application of ultrasound and carbon nanotubes in the periodontal and bone tissue regeneration.

牙周病是成年人牙齿缺失的主要原因，传统的方法难以建立新的附着。由于干细胞数量和功能的下降，老龄患者牙周缺损修复的效果更差。组织工程技术为牙周再生提供了有效途径。研究表明低强度脉冲超声可以促进间充质干细胞成骨向和成牙骨质向分化。碳纳米管可以改善胶原支架的性能并促进钙沉积，其可弥补超声在矿化期作用弱的缺点，且它们激活的信号途径联系紧密，两者结合应用于牙周组织工程可能会有良好的效果。本课题通过建立体外实验模型，研究超声和碳纳米管对老龄BMSCs分化和矿化的影响，分析两者是否具有协同作用，探索其作用的机制。建立老龄大鼠牙周缺损模型，考察两者分别以及结合使用对基于外源性BMSCs的牙周组织工程修复的影响，观察两者对植入的BMSCs分化和转归的作用，并进一步在动物模型中验证体外实验中获得的关键标志物和信号途径相关蛋白的变化情况。该研究可为超声结合碳纳米管在牙周以及骨组织工程中的应用提供实验和理论依据。

成牙骨质细胞在根面上形成牙骨质是牙周再生的一个很重要的步骤。实验中，我们研究了羧基功能化的多壁碳纳米管对成牙骨质细胞分化和矿化的促进作用，以及可能涉及的信号通路。在各种碳纳米管浓度下培养成牙骨质细胞，检测细胞活性以及在电镜下观察细胞的生长黏附情况，茜素红染色观察钙结节的形成。PCR和western blot检测成骨相关基因和蛋白的表达。结果显示5 µg/mL 的MWCNT-COOH没有明显细胞毒性且可明显促进细胞的分化。Alp, Ocn, Bsp, Opn, Col1 ，Runx2 ，Smad2 and Smad3基因的表达上调，smad7在第三天下调而第七天则变为上调。Smad2/3的变化在蛋白水平上得到验证。MWCNT-COOH通过TGF-β/Smad信号通路促进了成牙骨质细胞的分化和矿化。.阿司匹林对于干细胞以及在骨代谢过程的作用被认为近年用于治疗免疫相关疾病潜在机制。在这部分实验中，我们验证了阿司匹林可以增强骨髓间充质干细胞的成骨能力且提高细胞在动物牙周炎模型中的抗炎和免疫调节效果。在SD大鼠上建立牙周炎模型。与对照组相比，应用阿司匹林预处理的干细胞组炎症因子TNF-α, IL-17水平明显降低，然而IL-10水平上调。实验组中牙槽骨的吸收以及TRAP阳性的破细胞明显减少。免疫组化显示OPG的表达上调，RANKL的表达下调。结果显示，阿司匹林预处理的BMMSCs可以抑制牙槽骨的吸收，增加牙周炎骨缺损的修复，在牙周炎的治疗中有一定的应用前景。