组织工程牙周膜的体外构建

Construction of tissue engineered periodontal ligament in vitro

背景:利用牙周膜干细胞的多向分化潜能修复牙周缺损是近来研究的热点课题。目的:探讨利用可吸收生物材料冻干胶原膜和犬牙周膜干细胞在体外构建组织工程牙周膜的可行性。设计、时间及地点:单一样本观察,于2003-11/2004-10在解放军第四军医大学口腔医院组织工程实验中心完成。材料:生物膜为冻干胶原膜由解放军第四军医大学口腔医院组织工程实验中心提供。从4只1周岁犬的下颌前牙中分离培养牙周膜干细胞。方法:取第3代犬牙周膜干细胞扩增至1×107数量级,种植在冻干胶原膜的表面,形成细胞-生物材料复合物,每日更换培养液,倒置相差显微镜动态观察细胞形态和黏附生长状况,体外培养1周左右,组织做扫描电镜、透射电镜和苏木精-伊红染色,观察细胞在冻干胶原膜材料中的生长、增殖以及基质分泌情况。主要观察指标:①光镜观察细胞接种后生长状况。②电镜和组织学观察细胞支架复合结果。结果:培养6d后,犬牙周膜干细胞分泌的基质与支架材料形成网状结构。复合物体外培养1周,细胞黏附在生物支架上,细胞增殖和生长良好,分泌大量基质并形成纤维状结构,细胞复层生长,伸入膜的内部,增殖的细胞和分泌的基质与膜交接成一整体,复合物成为纤维网状组织,基本具备牙周膜的纤维状结构。结论:利用冻干胶原膜为支架,犬牙周膜干细胞为种子细胞,可在体外成功构建组织工程化牙周膜。

BACKGROUND： Repairing periodontal tissue defects with periodontal ligament stem cells, which have the multiple differentiation potential, have become the hot topic in recent studies. OBJECTIVE： To investigate the feasibility of construction of tissue engineered periodontal ligament in vitro using expanded dog periodontal ligament stem cells and tyophilization collagen membrane. DESIGN, TIME AND SETTING： A single sample observation was performed at the Experimental Center of Tissue Engineering, Stomatological College of Fourth Military Medical University of Chinese PLA from November 2003 to October 2004. MATERIALS： The lyophilization collagen membrane was supplied by Experimental Center of Tissue Engineering, Stomatological College of Fourth Military Medical University of Chinese PLA. Periodontal ligament stem cells were obtained from mandibular anterior teeth of 4 dogs with 1 week old. METHODS： PDLSCs （107/mL） of 3rd passage were expanded and seeded onto lyophilization collagen membrane to form a cell-scaffold complex. The complexes were cultured in DMEM. The cell-scaffold complexes were observed continuously by inverted microscopy. The cultured complexes were harvested at 1 week for electromicroscope, histological analysis. MAIN OUTCOME MEASURES： The growth of cells after seeded was observed by light microscope. The result of binding between cell and scaffold was measured by electron microscope and histological observation. RESULTS： At day 6 after culture, the matrix secreting by periodontal ligament stern cells combined scaffold materials in reticular formation. At 1 week after in vitro culture, cells adhered on scaffold with well proliferation and growth. Fibrous structure was formed There were multiple layers of the cells extended into the inner part of membrane, the proliferative cells and excretive matrix formed collagen fibers-like tissues, which showed fibrous structure as peridental membrane. CONCLUSION： Periodontal ligament tissue is possible to be constructed in vitro using periodontal ligament stem cells and collagen membrane.