狗牙周膜细胞与壳聚糖-磷酸三钙三维培养的实验研究  

A experimental study of three dimension on the chitosan-tricalcium phosphate combined with cultured dog periodontal ligament cells

作　　者：林志勇[1] 单秀莉[2] 王青[3] 

机构地区：[1]山东省立医院口腔内科,山东济南250021 [2]济南市第五人民医院口腔科,山东济南250022 [3]山东大学口腔医院牙体牙髓科,山东济南250012

出　　处：《山东大学学报（医学版）》2006年第7期666-670,共5页Journal of Shandong University:Health Sciences

基　　金：山东省科技发展计划资助课题(032050110)

摘　　要：目的:建立狗牙周膜细胞(PDLCs)体外三维立体培养模型,探讨以狗PDLCs为种子细胞,以壳聚糖-磷酸三钙复合体为支架材料应用于牙周组织工程的可行性。方法:冷冻干燥法制备壳聚糖-磷酸三钙多孔复合支架材料,组织块法培养狗牙周膜细胞,取第4代细胞传代扩增后接种到三维支架上,体外继续培养。免疫组化方法鉴定细胞起源;MTT法检测支架对狗PDLCs增殖的影响;取培养3、6 d的细胞-支架复合物的标本,用倒置相差显微镜和扫描电镜观察细胞的形态及在支架上的粘附、生长情况。结果:免疫组化显示,狗PDLCs抗波形丝蛋白阳性,抗角蛋白阴性,证实为中胚层来源。MTT法检测结果显示,支架材料对狗PDLCs的生长、增殖与空白对照组相比差异均无统计学意义(P>0.05)。扫描电镜示壳聚糖-磷酸三钙复合体具有良好的多孔网状结构,狗PDLCs在支架上贴附牢固,伸展充分,增殖旺盛并分泌大量的细胞外基质。结论:壳聚糖-磷酸三钙具有良好的粘附性和细胞相容性,狗PDLCs与之复合培养生长良好,表明以自体PDLCs为种子细胞、以壳聚糖-磷酸三钙为支架材料进行的牙周组织工程是可行的。Objective： To establish a three-dimensional cultured model of dog periodontal ligament cells （PDLC） , and therefore to evaluate the feasibility of chitosan/tricalcium phosphate（CTCP）as the scaffolds of periodontal tissue engineering. Methods： The CTCP was prepared by freeze-drying, and then the dog periodontal ligament cells cultured in vitro were collected and seeded on CTCP three-dimensional scaffold. The PDLCs were identified by immunohistochemistry method. Cell morphology and growth pattern were observed under reverse microscope, and cell proliferation was assayed using MTT method. The cells growth on the scaffolds was observed by scanning electron microscope（SEM）. Results： The PDLCs were identified by positive staining for vimentin and negative for keratin. Growth and proliferation of dog PDLCs of CTCP group was not significantly different from that of the control group. SEM showed the porous structure of the CTCP and the dog PDLCs could adhere well, proliferate adequately and extend excessively on the three-dimensional scaffold, secreting lots of extracellular atrix. Conclusion： CTCP showed excellent adhesion and good biocompatibility with the dog PDLCs in vitro. It is feasible to use CTCP as scaffold and PDLCs as seed cell in periodantal tissue engineering.

关 键 词：牙周膜细胞 壳聚糖 磷酸三钙 三维培养 组织工程 

分 类 号：R781.4[医药卫生—口腔医学] R318.08[医药卫生—临床医学]