Development Of Tissue Engineering Dermis Using Cell Sheets Technology In Vitro

OBJECTIVE:The large area defect of the skin is often difficult to completely heal due to the lack of autologous skin supply.The purpose of this study was to explore new strategies for constructing tissue-engineered dermis using cell sheets technology and to provide new ideas for the repair and healing of skin wounds.METHODS:(1)rBMSCs(rabbit Bone marrow mesenchymal stem cells rBMSCs)were obtained by whole bone marrow adherence method.HUVECs(HUVECs)and HDFs(Human dermal fibroblasts HDFs)were cultured by resuscitation and observe the morphology and growth state of seed cells under inverted phase contrast microscope.(2)P2 generation rBMSCs were inoculated in a 6-well plate at a density of 9×10 ~4cells/cm ~2;HDFs were cultured under the same conditions;both cells were cultured continuously for 14 days to harvest monolayers of cell sheets to 5×10 ~4 cells/cm~2 density of HUVECs were evenly seeded on the obtained undifferentiated rBMSCs sheets to form prevascularized cell sheets.At 1,3,7 and 14 days of culture,the microscopic morphology of the patch was observed under a microscope,HE staining and CD31 immunofluorescence staining were used to observe the distribution of cells and the formation of microvascular network;the fibroblasts under the microscope were observed under a microscope.The type I collagen and type III collagen immunohistochemical staining use to assessment of cell distribution and collagen expression.(3)Construction of three-dimensional prevascularized dermis-like tissue:A pre-vascularized cell sheet cultured for 7 days was sandwiched between two layers of fibroblast sheets to form a"sandwich"-like structure,which was then folded twice using tweezers and formed.A three-dimensional dermis-like tissue with a 12-layer membrane structure was obtained by sandwiching undifferentiated rBMSCs sheets between two layers of fibroblast sheets and folding them as a control group.Histological staining,immunofluorescence staining,and immunohistochemistry staining were performed in both groups after 24 hours.RESULTS:(1)The rBMSCs obtained by the whole bone marrow adherent method had good ability of proliferation and division,and they were irregular and polygonal under the microscope.The morphology of HUVECs cells is oval and paving stones.The morphology of HDFs cells is long spindle.All three types of cells grew in a swirling pattern on the bottom wall of the dish.(2)HUVECs were inoculated on the undifferentiated rBMSCs sheet.The vacuolar structure of HUVECs on the surface of the membrane was observed within 3 days.Type I collagen and type III collagen were positively expressed on the HDFs sheet.(3)The three-dimensional dermis-like tissue was constructed.In the experimental group,there was a"hive-like"network structure.There was no significant difference in the expression levels of type I collagen and type III collagen between the experimental group and the control group.CONCLUSIONS:(1)rBMSCs can be obtained by whole bone marrow adherence method and can be used in experiments after two passages of purification.The growth of HUVECs and HDFs is good.(2)HUVECs were uniformly inoculated onto undifferentiated rBMSCs sheets at a certain density to form prevascularized cell sheets in vitro;the constructed fibroblast sheets had ability of synthesize and secrete type I collagen and type III collagen in dermal tissue.(3)The matrix distribution of three-dimensional dermis-like tissues with HUVECs involvement was more orderly,the pre-vascularized dermis-like tissue constructed did not significantly affect collagen expression due to the formation of network blood vessels in a short period of time.