The Osteogenic And Tracking Study Of Bone Marrow Stromal Stem Cells

Background and objectives:Recent years,cell therapy and gene therapy are under investigation as potential treatments for many kinds of diseases.To objectively evaluate curative effect after cells transplantation,most traditional techniques for the study of stem cell transplantation in animal models require histological analysis to determine the fate and migration of cells.It is difficult to exactly observe the cell differentiation and make a objective evaluation to the curative effect of stem cell transplantation,which is tremendously interfere the development of clinical therapy of cellular transplantation. With the development of molecular biology and the concept of molecular imaging was proposed,mechanism of diseases has been explained from cellular and molecular level. For example we can use Magnetic resonance image(MR) to monitor transplanted cells, the good quality of MRI is its high tissue and spatial resolution.Magnetic resonance image(MR)permit multi-sequences and multi-angles imaging and easily showing anatomic structure of soft tissue.The greatest advantages of using MR is MRI can perform multi-times repeative detection noninvasively.To specifically imaging transplanted cells and target cells in MR,the recent investigation generally using superparamagnetic iron oxide(SPIO) to label MSCs,which should enable serial tracking and quantification of MSC transplantation.Since iron is an paramagnetic material,which can generate significantly greater T2 effects,thus enable to obviously lower the signal intensity of T2WI and T2~*WI in target area and thereby generate negativity contrast effect.With this characteristic of feridex,SPIO labeled transplanted cells able to be imaging with low signal by MR,which thereby indirectly represent the distribution and localization of transplanted cells.The purpose of this study was to evaluate and verify whether the MSCs 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 by detecting and tracking magnetically labeled MSCs transplanted into a dog model of femoral head osteonecrosis.We hope to make a groundwork for tracking cell transplantation in vivo in the future and get some theoretical evidences for clinical using MSCs to treat osteonecrosis.Methods The study includes two parts.Part one,the SPIO labeling of MSCs(bone marrow stromal cell) in vitro:MSCs were aquired from skeletally mature dogs via iliac crest aspiration and separated by adherent cell cytopheresis.MSCs were cultured in vitro and collected to experiment.Before reaching cell confluence,the purified MSCs were incubated with SPIO at different concentrations SPIO for 18 h.We need to evaluate labeling efficiency of MSCs with different labeling concentrations of SPIO nanoparticles as well as detection of characteristics and signal attenuation rules by MRI at 1.5T in vitro. Part two,transplantation and tracking of labeled MSCs in dog model of femoral head necrosis:After cell labelling,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 and the devitalized bone was transplanted in situ.4 weeks later,we remove the necrosis bone and transplant the SPIO labelled MSCs in one lateral defect with and in the contralateral defect without BMSCs.Samples were harvested at different week and embedded in paraform.Samples were harvested at different week.The precise location of SPIO-labeled MSCs was observed by immunohistochemical staining.Roentgenogram,gross tissue observation,histochemical stain and image analysis were used.The osteoblasts specific differenciation markers were detected by immunohistochemical stain and fluoroimmunohistochemical stain.These markers include AKP and typeⅠcollagen.Results:MSCs were efficiently labeled by SPIO in vitro and SPIO can keep in cells for long time.9μg Fe per ml medium was an appropriate concentration liminal value.MSCs loaded with SPIO has no alterations to viability and proliferation profiles at this labeling concentration.They can be detected by MRI at certainly cell concentration in vitro.Gross tissure observation demonstrated that the defect transplanted with MSCs were healing better than without MSCs.SPIO-labeled cells were found in the defect.The more bone formation,the more number of the SPIO-labeled cells.Many vessel wall cells are also SPIO-labeled cells in new bone zones.Microhistology demonstrated that the MSCs transplanted can increase bone formation and mineralization,improve osseous maturation, raise the number of fusiform shape mesenchymal cells,chondrocytes and osteoblasts.The osteoblasts specific differentiation markers are more intensive and abundant with induced MSCs than non-induced MSCs.Fluorescence signals from typeⅠcollagen and AKP is also more intensive than the contralateral.Conclution:This initial study demonstrated that MSCs can be easily and efficiently labeled by SPIO without restriction of cells viability and proliferation and can be detected by MRI in vitro,,the MSCs can survive after transplantation to the necrosis area of the femoral head,live in situ and proliferate.MSCs can efficiently increase bone and vessel formation through differentiate into osteoblasts and vessel cells.