Induction Of Human Gingival Fibroblasts Into Human Vascular Endothelium-like Cells

Objectives:Four types of tissues have been known in human body, such as epithelialtissue, connective tissue, muscle tissue and nerve tissue. As a monolayer ofsquamous epithelium, consisting of vascular endothelial cells, vascularendothelium belongs to epithelial tissue. Vascular endothelial cells not onlymaintain the integrity of vessel walls and the smoothness of internal surface,but also keep the permeability of vessel walls and normal hemokinesis.Furthermore, vascular endothelial cells can secrete considerable endotheliumbioactive substances. Thus, vascular endothelial cells tend to be the seed cellsof vascular tissue engineering and the cores of construction of blood vessels.In oral maxillofacial regions, gingiva is mainly constructed by connectivetissue, and is one kind of periodental supporting tissue mainly containingfibroblasts. More over, gingiva tissue is easy to regenerate, repair, and obtain.Many researches indicated that gingival tissue could become the source ofmultipotential cells.In this study, gingival fibroblasts were cultured and induced into vascularendothelium-like cells in vitro, in order to reveal the possibility if humangingival fibroblasts could differentiate into vascular endothelial cells. Thisstudy provided a new source of the seed cells for tissue engineering vascularendothelium.Methods:1The cell culture of human gingival fibroblastsNormal human gingiva was obtained from a patient with an extraction ofimpacted teeth in Department of Oral Maxillofacial Surgery, Hospital ofStomatology, Hebei Medical University. Primary culture of gingival tissueblock was adopted, and the fibroblasts of the human gingiva were cultured in DMEM-LOW GLUCOSE containing15%fetal bovine serum. The cells weredigested by0.25%trypsin.2The identification of human gingival fibroblastsThe second generation of human gingival fibroblasts was identified byimmunocytochemical and immunofluorescent stain against vimentin andcollagen III respectively.3The induction into vascular endothelium-like cellsThe second generation of human gingival fibroblasts was cultured inserum-free DMEM-LOW GLUCOSE containing8ng/ml VEGF,100U/mlpenicillin and100U/ml streptomycin for50days.4Identification of the vascular endothelium-like cellsAfter induction, the cells were identified by immunocytochemical andimmunofluorescent stain against CD34and CD31respectively.5The observation of vascular endothelium-like cells by transmissionelectron microscope (TEM)After being cultured with8ng/ml VEGF for50days, the vascularendothelium-like cells were collected and fixed in2.5%glutaraldehyde. Thesamples were examined through transmission electron microscopy (TEM).Results:1The cell culture of human gingival fibroblastsAfter5~7days of primary culture of human gingiva, spindle-shapedfibroblast cells with clear outline and large, spherical or elliptic nucleiemigrated from the tissues.2The identification of human gingival fibroblastsImmunocytochemical and immunofluorescent stain showed vimentin andcollagen III positive in the cytoplasms of the fibroblasts.3The induction into vascular endothelium-like cellsAfter being cultured with8ng/ml VEGF for50days, spindle-shaped cellsturned into polygon or cube epithelial-like cells by inverted microscope. Somecells joined together gradually and formed cobblestone-like or slabstone-likegrowth pattern. Some vascular endothelium-like cells arranged in vascular lumen-like structures.4Identification of the vascular endothelium-like cellsImmunocytochemical and immunofluorescent stain showed CD34andCD31positive in the cytoplasms of vascular endothelium-like cells arrangedin vascular lumen-like structures.5The observation of vascular endothelium-like cells by transmissionelectron microscope (TEM)Microvillus and lysosomes can be observed, and Weibel-Palade body likestructure was found in the cytoplasms of endothelium-like cells.Conclusion:Human gingival fibroblasts have the potentialities to differentiate intovascular endothelium-like cells.