Effects Of Different Proportions Of Fibroblasts On The Construction Of Tissue-Engineered Dermal Membrane Of Bone Marrow Mesenchymal Stem Cells,Endothelial Cells,and Fibroblasts

Large areas of burns,soft tissue trauma,and skin necrotizing diseases can cause dermal defects that severely affect the quality of life of patients.Tissue engineering dermis offers the possibility to repair defective dermis.The dermis contains a large amount of bone marrow mesenchymal stem cells,umbilical vein endothelial cells,and dermal fibroblasts.This experiment aims to explore the proportion of bone marrow mesenchymal stem cells,umbilical vein endothelial cells and dermal fibroblasts co cultured to form tissue engineering dermis,so as to provide a reference for future tissue engineering dermis construction.Methods:1)Resuscitation,cultured and morphological observation of primary human dermal fibroblasts and umbilical vein endothelial cells:10mg/ml polylysine coated flask,recovery of the primary cells,observed the cell morphology and proliferation under inverted phase contrast microscope,took cells with better growth activity for experiments.2)Extraction,isolation,in vitro culture and morphological observation of rabbit bone marrow mesenchymal stem cells:Two-week-old New Zealand white rabbits were harvested from the hind limb bone marrow under aseptic conditions.Mesenchymal stem cells were isolated and purified using whole bone marrow adherence method.Observation of cell morphology and proliferation under inverted phase contrast microscope,the third generation of cells is used for experiments.3)Co-cultured of rabbit bone marrow mesenchymal stem cells,human umbilical vein endothelial cells,and human dermal fibroblasts and construction of cell sheets.Rabbit bone marrow mesenchymal stem cells were seeded in a six-well plate at 9×10 4cells/cm2,after 14 days,Human umbilical vein endothelial cells were seeded on the stem cell membrane at 5×10 4 cells/cm2,at the same time,human dermal fibroblasts were seeded on the stem cell sheets at a ratio of 0%,40%,60%,and 80%,and cultured under the same conditions.4)Observed cell sheets growth and morphology,At 1,3,7,and 14 days,the growth of cell sheets was observed under an inverted phase contrast microscope and recorded.5)surveyed the thickness of the sheets :took 1,3,7 and 14 days of cell sheets to make frozen sections,HE staining,and surveyed the thickness of the sheets the scale repeatedly.6)CD31 immunofluorescence staining and observation:took 1,3,7 and 14 days of cell sheets to make immunofluorescence staining of CD31.observed the formation of endothelial vascular network under Inverted fluorescence microscope and recorded.7)Immunohistochemical staining: took 1,3,7,14 days of the cell sheets of each group to maka immunohistochemical staining for collagen type I and type III collagen,and observed and photographed under inverted microscope.IPP6.0 calculated the content of type I collagen and type III collagen.8)Identification of rabbit bone marrowmesenchymal stem cells by flow cytometry:after 7 days,the cell sheets of each group were digested with trypsin and CD90 mark,performed flow cytometry.9)Single factor analysis of variance and T tests were performed on the results using spss23 statistical software.Result:1)Primary umbilical vein endothelial cells and dermal fibroblasts showed high activity and morphology.2)The bone marrow mesenchymal stem cells were long spindle or polygonal in shape and grew in monoclonal way.3)Surveyed of the thickness of cell sheets:at 1,3,7 and 14 days,the thickness of cell sheets increased with time and the proportion of fibroblasts.The cells were tightly packed,cluttered and there are no spaces between cells.At the 7days,the 60% and 80% groups of fibroblasts,at the 14 th day,the 40% and the 60% group were the most typical.4)CD31 immunofluorescent staining results:with the increased of culture time and the proportion of fibroblasts,at 1,3,7 and 14 days,the vascular network formed by the endothelial cells in the cell sheets became dense,At the 7th and 14 th days,60% of the fibroblasts and 80% of the fibroblasts were most typical.5)Immunohistochemical staining of type I collagen:With the increased of culture time and the proportion of fibroblasts,the content of type I collagen in cell membranes showed a trend of first increased and then reduced.At the 7th day,in the 60% and 80%of fibroblasts,the content of human dermal type I collagen was closest.Similarly,with the increase of culture time and proportion of fibroblasts,the content of collagen III increased first and then decreased.On the first day and the seventh day,the 60% and 80% groups of fibroblasts were closest to the content of type III collagen of human dermis.7)Flow cytometry results:CD90-marked stem cells appeared in cell sheets in 7 days.Conclusion:Rabbit bone marrow mesenchymal stem cells,human umbilical vein endothelial cells,and human dermal fibroblasts were co-cultured at a ratio of 9:5:21 could form the cell sheets with partial properties of the dermis at the 7 day.