应用牙龈角质细胞构建组织工程化牙龈的实验研究

Experimental in vitro construction of tissue engineered gingiva with human gingival keratinocytes

目的:建立体外连续培养的人牙龈角质细胞系,探讨构建人工复合牙龈的方法,为种植体周围软组织改善提供帮助。方法:体外无血清培养人牙龈角质细胞,探索细胞体外生长的动力学规律。将角质细胞与脱细胞真皮基质复合,构建组织工程化牙龈。结果:体外连续传代培养人牙龈角质细胞,可传4～6代,存活30～50d;培养细胞具有角质细胞的典型形态和染色特征。第1、2代细胞群体倍增时间最短,贴壁率、克隆形成率最高。角质细胞可以接种于脱细胞真皮基质上,形成组织工程化牙龈。结论:体外培养扩增人的牙龈角质细胞,第2、3代较适合用于组织工程化牙龈的构建。脱细胞真皮基质可作为组织工程化牙龈的支架材料。

PURPOSE： To establish a model of culturing normal human gingival keratinocytes, and develop a composite human gingiva equivalent for peri-implant soft tissue management. METHODS： Gingival keratinecytes were cultured in a chemically defined serum free medium and identified with morphological study and immunohistochemical staining. The growth kinetics of keratinecytes were studied with recording cell growth curve, attached ratio, colony forming efficiency and PDT. Keratinocytes were seeded on Renov,a kind of acellular dermal matrix,to form a tissue-engineered gingiva. RESULTS： Gingival keratinocytes could be maintained in culture up to 4-6 passages and 30-50 days. The cells showed typical morphology of keratinecytes and positive staining for cytokeratin antibody. The PDT of passage 1 and passage 2 was the shortest, with the highest attached ratio and colony forming efficiency. Composite gingiva was developed by seeding keratinocytes on ADM. CONCLUSIONS： Considering the factors such as cell proliferation function, cell quantity, the second and third passages of keratinecytes cultured in vitro is the optimal seeding cells for construction of tissue- engineered gingiva. ADM could be used as scaffold of composite gingiva.