| Objective To promote the osteogenesis and angiogenesis by transplanting endothelial progenitor cells(EPCs) and bone marrow stromal cells(BMSCs) into the femoral head necrotic zone.Methods 1 BMSCs isolated from rabbit bone marrow by density gradient centrifugation, were cultured and identification. 2 using the same method to obtain the rabbit EPCs and identification. 3 ALP activity was assayed for the determination of their best ratio. 4 The expressions of cells osteogenesis and angiogenesis related genes were detected by the Real-Time PCR. 5 Rabbit models of femoral head ischemia necrosis were established by hormone method. 6 Cells were divided into four groups:blank group(group A);BMSCs transplantation group(group B);EPCs transplantation group(group C);BMSCs and EPCs transplantation group(group D). 7 Cells preparation for transplanting. 8 The cultured cells transplanted into the rabbit model of necrotic femoral head. 9 Detection of avascular necrosis of the femoral head region.(1) HE staining of histological section(2) BMP-2 expression was detected by immunohistochemistry.(3) Immunohistochemical detection of neovascularizationResults 1 Identification of rabbit BMSCs(1) Inverted phase microscopy showing characteristics of BMSC colony on day 6 after culture and confluent growth on day 9 after culture.(2) H & E staining showing thriving BMSCs growth on day 14 after culture(3) Alizarin red staining showing calcium nodules.(4) The positive rates of BMSCs surface markers determined by the flow cytometry were CD44(97.85±1.56)%,CD90(96.83±1.83)%, CD34(5.63±2.21)%.2 Detection of rabbit EPCs(1) Microscopy showing typical crazy-paving morphology of EPCs on day 15 after culture.(2) Immunofluorescence showing binding of EPCs to FITC-UEA-1 and Dil-Ac- LDL and dual staining was EPCs(3) The positive rates of EPCs surface markers determined by the flow cytometry were CD34(81.35±3.52)% and CD133(85.64±2.44)%.3 The best ratio of co-cultured EPCs and BMSCs was 1:14 Analysis of osteoblast marker genes and angiogenic marker genes expression detected by Real-Time PCR The expression of osteoblast marker genes in co-culture of BMSCs and EPCs group was higher than that in group of BMSCs at day 7(P<0.05)and angiogenic marker genes expression in co-culture group was also higher than that in group of EPCs at day 7(P<0.05).5 X-ray showing necrotic femoral head at week 6, H & E staining showing less osteoblasts in medullary canal, lipid change in bone cells, and increased bone lacuna.6 Detection of avascular necrosis of the femoral head region(1) Histology showed no obvious bone-like tissue and vascular structure in group A, bone-like tissue in group B,vascular structure in group C with less bone-like tissue, and vascular structure and bone-like tissue in group D.(2) The BMP-2 protein expression level was higher in group C,group B,group D than that in group A(P<0.05);the expression in group B and group D was higher than that in group C(P<0.05);group D was higher than group B(P<0.05).(3) New blood vessels were formed in all four groups,the number of vessels in group C and group D was higher than in group A and B(P<0.05);those in group D were higher than in group C(P<0.05).Conclusions 1 EPCs co-cultured with BMSCs can promote osteogenesis and angiogenesis 2 Transplantation of EPCs and BMSCs into femoral head necrotic zone at the ratio of 1:1 can effectively promote the osteogenesis and angiogenesis. |
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