| Objective To evaluate and verify whether the BMSCs transplanted to the defect of the femoral head can survive , live in situ , proliferate, differentiate into osteoblasts,increase bone formation,and repair the defect of the femoral head.Methods The study includes three parts.In part one,BMSCs (bone marrow stroma cell) were aquired from skeletally mature dogs via iliac crest aspiration and separated by adherent cell cytopheresis. BMSCs were cultured in vitro and collected from the second passage to experiment. Before reaching cell confluence,the purified MSCs were incubated with 5-bromodeoxyuridine (BrdU) at the concentrations of 10μmol/L BrdU for 48 h. After capsulotomy, a reformed trapdoor was created in the posterolateral surface of the femoral head and a 8-mm-diameter and 10-mm-depth subchondral area of bone was removed.The bone removed was devitalized by microwave. The devitalized bone was transplanted in situ ,in one lateral defect with the BrdU labelled BMSCs and in the contralateral defect without BMSCs. Samples were harvested at 5 week and embedded in paraform.In part two,BMSCs aquired via iliac crest aspiration were separated by density gradient centrifugation,and labelled with 5,6,2-carboxyfluorescein diacetate succinimidy ester (CFSE).The CFSE labelled BMSCs were transplanted into one lateral defect with gelatin sponges,and gelatin sponge were used into the contralateral defect without BMSCs as a control. Samples were harvested at 3 week.In part three,BMSCs from cultured in vitro were transplanted into one lateral defect with AECB(Antigen-extracted Cancellous Bone),and AECB were used into the contralateral defect without BMSCs as a control. Samples were harvested at 12 week. The precise location of CFSE-labeled BMSCs was observed under laser scanning confocalmicroscope (LSCM) and BrdU-labeled BMSCs by immunohistochemical staining. Roentgenogram,gross tissure observation, histochemical stain and image analysis were used. The osteoblasts specific differenciation markers were detected by immunohistochemical stain and fluoroimmunohistochemical stain.These markers include core binding factor al(Cbfa1,the master regulator of bone differentiation and also essential for the induction of hypertrophic chondrocytes), osteocalcin and osteopontin (the two noncollagenous extracellular matrix proteins,are also important in defining the progressive differentiation of osteoblasts), and type I collagen (the main collagenous extracellular matrix proteins,is very important in defining the progressive differentiation of osteoblasts),Results: Gross tissure observation demonstrated that the defect transplanted with BMSCs were healing better than without BMSCs. Microhistology also demonstrated that the BMSCs transplanted can increase bone formation and mineralization, improve osseous maturation, raise the number of fusiform shape mesenchymal cells, chondrocytes and osteoblasts. The osteoblasts specific differenciation markers are more intensive and abundant with BMSCs than without BMSCs.BrdU-labeled cells were found in the defect.The more bone formation,the more numer of the BrdU-labeled cells.Many fusiform shape mesenchymal cells, chondrocytes and osteoblasts are BrdU-labeled cells. Many vessel wall cells are also BrdU-labeled cells in new bone zones. Fluorescence microscopic study found that fluorescence signals from CFSE is also intense in the defect,and fluorescence signals from rhodamine-labeled antibody anti to Cbfa1, type I collagen osteocalcin and osteopontin is also intense in the same area,and yet the positive results will be interfered by high background.Conclution: This initial study demonstrated that the BMSCs transplanted to the defect of the femoral head can survive , live in situ and proliferate. BMSCs can differentiate into many types cells,including osteoblasts and vessel wall cells. BMSCs can increase bone and vessel formation,and repair the defect and osteonecrosis of the femoral head through differentiate into osteoblasts and vessel cells. |
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