BMP-2/EMPs控释纳米涂层修饰的仿牙周复合体支架构建及其诱导组织再生的研究

Orthodontic treatment is closely related with periodontal remodeling, and the healthy bone-periodontal ligament-cementum complex is important for physiological periodontal remodeling and tooth movement. Preliminary results have been achieved in the regeneration of the periodontal tissue. However, few reports have been delivered on the successful regeneration of complete periodontal complex containing three layer structure (alveolar bone, periodontal membrane and cementum) in vivo. Some Parameters of the scaffolds, such as raw materials ratio, delamiantion structure, porosity and degradation properties, are yet to be improved. Firstly, the three phase-integrated bionic scaffolds, which are similar to the normal human periodontal complex, are prepared via electrospinning techniques according to biomimetic raw materal ratio, in which the middle phase contains CTGF. Then, the two surfaces of each bionic scaffold are modified via layer-by-layer self-assembled technology, in order to establish a stable controlled released system of BMP-2 and EMPs. The periodontal ligament stem cells may differentiate into osteoblast, fibroblast and cementoblast induced by the released BMP-2, CTGF and EMPs, which may be verified by various molecular biological methods. After the periodontal defect models of Beagle dogs are established, PDLSCs-seeded multiphase scaffolds are implanted. The efficiency of the materials for the periodontal complex regeneration may be investigated via morphological and histochemical analysis.

正畸牙移动伴随牙周改建，健康的牙槽骨-牙周膜-牙骨质复合体是牙周改建及牙移动的基础。各种原因引起的牙周组织丧失严重影响正畸进展及疗效。近年来通过组织工程实现牙周再生的研究中，少有大型动物体内实验成功构建完整牙周复合体的报道。支架材料在原料、配比、层次结构、孔隙率及降解性能等方面尚有待改进。本课题拟从仿生学角度，模拟牙周复合体分层结构和主要成分，通过静电纺丝技术，按不同原料配比和层次厚度连续喷涂构建三相一体化仿牙周复合体薄膜支架，并在中层结构层包埋成纤维诱导因子，然后运用层层自组装技术分别在薄膜两侧建立稳定的成骨/成牙骨质因子控释系统，诱导牙周膜干细胞成骨、成纤维、成牙骨质的定向分化，并通过分子生物学方法加以验证。通过建立犬牙周组织缺损动物模型，将复合牙周膜干细胞的仿牙周复合体薄膜支架材料植入缺损处，采用形态学和组织化学手段分析评估牙周复合体再生效果。

正畸牙齿移动伴随牙周组织改建，健康的牙槽骨-牙周膜-牙骨质复合体是正常牙周改建及牙齿移动的基础。各种原因引起的牙周支持组织丧失严重影响矫治进展及疗效。课题组通过构建EMPs/BMP-2纳米控释涂层修饰的仿牙周复合体支架以期望该材料能够诱导牙周组织再生。课题组严格按照原研究计划要求，完成了以下工作：1）制备不同COL-I和HA含量及构成比的静电纺丝膜支架材料（PCL＋5％COL膜和PCL＋5％COL＋1％HA膜），证实牙周膜细胞在PCL＋5％COL＋1％HA 膜上的细胞增殖和活性显著优于其他两种膜片。2）验证培养得到的PDLSCs为间充质干细胞。通过三维细胞培养板得到牙周膜干细胞球，证明三维培养条件下牙周膜干细胞活性基本能维持7天左右，成骨分化能力较二维培养细胞明显增强。3）采用层层自组装技术构建{[(BMP-2/PAH)/PSS]/[COL/ALG]}20控释涂层修饰PCL纺丝支架，证实该支架材料能缓释BMP-2，并且可诱导PDLSCs分化成为具有较强矿化能力的细胞。4）采用层层自组装技术构建{[(PAH+EMPs)/PSS]/[COL/ALG]}20控释涂层修饰PCL纺丝支架，证实该支架材料能缓释EMPs，并促进PDLSCs的黏附和增殖，显著提高细胞成牙骨质/成骨分化能力。5）构建EMPs/BMP-2控释纳米涂层修饰PCL纺丝膜，体外研究证实EMPs/BMP-2涂层修饰PCL纺丝膜能够促进 PDLSCs增殖和细胞成牙骨质/成骨分化。6）构建仿牙周复合体支架，即EMPs/BMP-2涂层修饰PCL膜-细胞膜片复合体，复合体内部连接紧密，结构完整且稳定。7）裸鼠皮下体内异位移植证实EMPs/BMP-2控释纳米涂层修饰PCL膜-细胞膜片复合体在体内可形成少量新生牙骨质样结构。8）已成功构建Beagle犬牙周组织缺损模型，并采用原位自体移植方法将EMPs/BMP-2涂层修饰PCL膜-PDLSC细胞膜片复合体植入犬牙周膜缺损组织处，样本的后续组织染色研究需等待脱钙完成后继续跟进。本项目构建了EMPs/BMP-2控释纳米涂层修饰PCL膜-细胞膜片复合体三维支架材料，证实其能促进细胞成骨/成牙骨质分化，体内可形成牙骨质样结构，为进一步改良支架材料并将其应用于牙周组织缺损修复提供了重要的实验依据。