| Objective:To investigate rabbit bone marrow mesenchymal stem cells,osteoblast cells stained with human umbilical vein endothelial cells to construct three-dimensional micro-tissues in vitro and the osteogenic capacity of the micro-tissue in the subcutaneous transplanted model in nude mice.Method:1.Isolation and culture of rabbit bone marrow mesenchymal stem cells(BMSCs):rBMSCs were obtained by whole bone marrow adherence method and cultured to purify.They were passaged for morphological observation and growth record when adherent cells reached 90%.2.Induction of rabbit osteoblasts:The P2 generation of rBMSCs fused to 80%-90%were cultured with an osteogenic induction solution for osteogenic induction,and harvested14 days after continuous induction for subsequent experiments and detection of osteogenic characteristics.3.Resuscitation and culture of human umbilical vein endothelial cells:The human umbilical vein endothelial cells frozen in liquid nitrogen were taken out,and the primary cells were resuscitated in a water bath and sub-cultured for subsequent experiments.The morphology and growth status were observed under a microscope.4.Construction of three-dimensional micro-tissues in vitro:three-dimensional micro-tissues were divided into two groups,one group was osteoblasts and mesenchymal stem cells mixed in a ratio of one to one(OB group),and inoculated to a96-well low adhesion culture plate at a density of 5×10~4/cm~2,the medium replaced for7 days every other day;the other group was bone cells,mesenchymal stem cells and human umbilical vein endothelial cells mixed in a ratio of 1:1:1(co-culture group),and inoculated into a 96-well low-adhesion culture plate at a density of 5×10~4/cm~2,the medium replaced for 7 days every other day.5.transplantation of three-dimensional micro-tissues in nude mice:every 96 microspheres were injected into the subcutaneous site of a nude mouse and observed by general observation,HE staining,alizarin red staining,alkaline phosphatase staining and CD31 immunofluorescence staining at 2 and4 weeks after implantation to detect the osteogenesis and angiogenic ability of the implanted microspheres.Results:1.Rabbit bone marrow mesenchymal stem cells were successfully isolated and cultured,which were long fusiform and spirally growing.2.Rabbit bone marrow mesenchymal stem cells were successfully induced to differentiate into polygonal or cubic osteoblasts,with short protrusions on the surface,and orange-red calcium nodules were stained with alizarin red.3.Resuscitation of human umbilical vein endothelial cells:human umbilical vein endothelial cells are P1 generation after resuscitation,adherent growth,arranged in a"paving stone".The cells are flat and polygonal.4.Three-dimensional micro-tissues are successfully constructed:after cells were cultured in 96-well low-adhesion culture plates for 1 week,the cells were observed to be spheroidized by naked eyes.The alizarin red staining and alkaline phosphatase staining showed that both groups of microspheres had osteogenesis.The CD31 immunofluorescence showed an angiogenic potential in the co-culture group.5.After two weeks of implantation,tissue nodules were found in the OB group and the co-culture group.After 4 weeks,the tissue nodules at the implantation sites became smaller with clear boundary and the surface compression was hard.HE and alizarin red staining showed that the co-culture group formed more bone than OB.CD31 staining showed that the number of blood vessels formed in the co-culture group was much higher than that in the OB group.Conclusion:1.three-dimensional micro-tissue can be formed easily in 96-well low-adhesion culture plates,whose texture is relatively close.2.Three-dimensional micro-tissue constructed with bone marrow mesenchymal stem cells,osteoblasts and human umbilical vein endothelial cells in vitro has the capacity of osteogenesis and angiopoiesis. |
PDF Full Text Request