Experimental Study Of RBX Filled With Mesenchymal Stem Cells Transfected By VEGF Gene On Repairing Nontraumatic Osteonecrosis Of Femoral Head In Rabbit

OBJECTIVE:1.To explore the approach to isolate and culture mesenchymal stem cells(MSCs)in vitro and identify it.2.To study the expression of VEGF gene transfected MSCs.3.To investigate the possible therapeutic effect of RBX filled with mesenchymal stem cells transfected by VEGF gene in treatment of nontraumatic osteonecrosis of femoral head(NONFH).METHODS:1.MSCs derived from Qingzilan rabbits aged 4 to 6 months and weighted 2.5-3.5kg with the methods of adherent culture screening method and density gradient centrifugation were cultured in vitro.Morphoginic characteristics were identified using mouse anti-rabbit antibody CD44,CD34 and CD90.2.Lipofectin method was used to introduce hVEGF-165 gene into mesenchymal stem cells.The transcription and transient and stable expression of VEGF gene were investigate by using RT-PCR,Western Blot assay and immuno-histochemistry S-P method respectively.3.Animal model of rabbits was introduced through frozen by liquid nitrogen to make a model of osteonecrosis of femoral head.Twenty-two rabbits(48 hips)were divided into two equal-sized groups:a treatment group and a control group.two groups were treatment with two different interventional options:the treatment group is applied with RBX Filled with Mesenchymal Stem Cells transfected by VEGF gene,the other only with RBX.X-ray examination,histopathological evaluation and comparative bone density analysis were conducted.Dataed in SPSS13.0 software to proceed statistical analysis to find out if the differences between each group had statistical significance.RESULTS:1.The cultured cells positively reacted to antigen CD44 and CD90,but negative to antigen CD34 by immuocytochemistry,morphocytology obviously.2.the cell proliferation of the transfected MSCs were similar to those of the control group. The transcription and expression of VEGF gene were found distinctly in gene transfected MSCs after 72h of transfection and 4 weeks of culture.The control group were not found the transcription and expression.3.Roentgenographically,two week, little difference between the two groups;six weeks in treatment of bone defects more bone callus formation,the control group,only a small amount of bone callus formation. The bone density of the treatment group was higher than that of the control group at the 6_thweek;density of the treatment group was similar with that of the normal animals at the 12^thweek,while that of the control group was still much lower.In the bone density and mineral quality content,after 2 week,there were no significant differences between the two groups,after 6 and 12 weeks,significant differences were found between the two groups(P<0.01).Histopathologically,osteocytes,chondrocytes and bone marrow cells were completely necrotic but bone trabecula remained intact after 2 weeks;6 weeks after,osteocytes,capillaries were increased at the sites of implantation of RBX filled with mesenchymal stem cells transferred by VEGF gene compared with the control group;after 12 weeks,nascent osseous tissue reached to cartilage in the treatment group,pieces of implanted bone remained in the control group.CONCLUSION:1,It is a simple,effective and practical method to separate and to obtain higher purity and activity of MSCs from the bone marrow of rabbits by gradient centrifugation and adherent culture screening method.MSCs can grow quickly when cultured.2.Transfected by VEGF gene,the MSCs can transcript and express the VEGF protein,the transfection is succeeding and effective by RT-PCR,Western Blot and immuno-histochemistry S-P method analysis at the level of transcription and translation.3.RBX Filled with Mesenchymal Stem Cells transfected by VEGF gene can not only enhance the formation of new microscopic blood vessels but also the activity of osteoinduction.therefore it has apparent promotion effect on the repairing process of NONFH..