The Effects Of Glutathione On The Differentiation Of MSCs With Steroid-associated Osteonecrosis Of Femoral Head

Part Ⅰ Isolation, Culture and Biological Characteristics Mesenchymal Stem Cells Dedrived from Human Bone MarrowObjective:Mesenchymal stem cells (MSCs) are proliferative and multipotent adult cells that have the ability to differentiate into multiple mesodermal lineages, such as osteoblasts, adipocytes, chondrocytes, neurocyte, vascular endothelial cells. Currently, many clinical trials are underway to treat many conditions with MSCs, including osteonecrosis of femoral head, spinal cord injury, immune diseases. The purpose of this study was to establish a method for effective isolation and caltivaion human bone marrow derived human MSCs and observed by morphology during osteogenic and adipogenic differentiation and analyze the dynamic expression of Runx2in the process of osteogenic differentiation and PPAR-yin the process of adiogenic differentiation..Methods:MSCs were isolated from human bone marrow by combination with gradient centrifugation and different adherent time. Morphology and growth characteristics were examined by phase contrast microscopy. The number of cells were counted by cells counting plate. surface markers CD90, CD34, and CD45were tested by flow cytometer. MSCs were induced in vitro to osteoblasts and adipocytes by osteogenic and adipogenic medium. Real time RT-PCR and Western blot were performed to detect the expression of the Runx2gene in the process of osteogenic differentiation and PPAR-ygene in the process of adiogenic differentiation.Results:Primary and passage cells were spindle-shaped and presented active proliferation in primary and passage cultures. The cell surface markers CD90were positive and CD34, and CD45were negative. Cells were successfully induced into osteoblasts and adipocytes with phenotypic characteristics. The expression of Runx2and PPAR-yof MSCs in osteogenic and adiogenic differentiation and was increased gradually.Conclusions:The method is successfully established to isolate and multiply MSCs from human bone marrow as seed cells and to verify by morphology and its osteogenic and adipogenic differentiation. Part ⅡThe study on cell number, function and differentiation of mesenchymal stem cells in patients with steroid-associated osteonecrosis of femoral headObjective:The dynamic balance between osteogenesis and adipogenesis of mesenchymal stem cells (MSCs) played a key role in metabolism homestasis of bone. The osteonecrosis of femoral head happened results from administration with ectogenic steroid with imbalance of osteogenesis and adipogenesis. The popurse of this study is to investigate the cell number, function and differentiation ability of MSCs in patients with steroid-associated osteonecrosis of femoral head and to investigate the effects of oxidative stress during differentiation.Methods:This study was approved by patients and the Ethics Committee of Wuhan Union Hospital. Between July2012and Desember2013,32patients (20men,12women; mean age50.3, ranging from38-66years) with GC-induced ONFH were selected at the authors’institution and21subjects with femoral neck fractures (12men and9women; mean age51.3, ranging from37-69years) were enrolled as controls. In the operating room,10ml of bone marrow was aspirated. MSCs were isolated and cultured and the cells in Passage3were used in this study. Cell colony formation ability was evaluated from all the patients. Treated with10μM Glutathione, we investigated reactive oxygen species(ROS), osteogenic or adipogenic ability of MSCs and the mark of osteogenic or adipogenic differentiation.Results:MSCs colony formation ability is less compared to the control group (20.3±7.7vs.27.3±5.8, P<0.01). The level of ROS in the GC-induced ONFH group is higher than the subjects group. Treated with10μM Glutathione, the level of ROS decreased(P<0.05). The ability of osteogenic differtiation is poor compared to control group, and better in adiogenic differentiation. The alter was restored by10μM Glutathione. Real-time PCR and western blot exhibted expression of Runx2and ALP level was down-regulated in the GC-induced ONFH group. The expression of PPAR-γ and C/EBP level was up-regulated in the GC-induced ONFH group. Moreover,10μM Glutathione could restore the dynamic balance between osteogenesis and adipogenesis by decreasing the level of ROS.Conclusion:The number and function MSCs were weakened in patients with steroid-associated osteonecrosis of femoral head. The osteogenesis ability decreased and adipogenesis ability increased and the level of ROS increased in MSC from patients with ONFH. Glutathione could restore the dynamic balance between osteogenesis and adipogenesis by decreasing the level of ROS Part ⅢGlutathione antagonizes adipogenesis and enhance osteogenesis in human MSCs by reducing dexamethasone-induced oxidative stressObjective:Increased oxidative stress in human mesenchymal stem cells(MSCs) is recently considered as a crucial cause of corticosteroid-induced necrosis of femoral head. The purpose of this study was to evaluate the effect of glutathione, a powerful antioxidant, on the level of oxidative stress, adipogenic and osteogenic differentiation markers of a human bone marrow derived MSCs. Mothed:The cells model of steroid induced necrosis of the femoral head was established upon treatment with10μM dexamethasone treated human MSCs. MSCs were treated with different5μM glutathione,10μM glutathione,50μM glutathione independently. Treatment of dexamethasone and antioxidant glutathione together at day7, adipogenic markers PPAR-yand C/EBP expression and osteogenic maker RUNX2and ALP expression were tested by quantitative real-time PCR and Western blot.Rezults:Upon treatment with dexamethasone at day21, MSCs containing lipid vesicles were distinguishable by Oil Red O staining.Treatment of dexamethasone at day7, adipogenic markers PPAR-γand C/EBP expression increased and osteogenic maker RUNX2and ALP expression decreased by quantitative real-time PCR and Western blot. Treatment of dexamethasone and antioxidant glutathione together at day7, adipogenic markers PPAR-yand C/EBP expression decreased and osteogenic maker RUNX2and ALP expression increased by quantitative real-time PCR and Western blot.10μM or50μM glutathione group had a greater effect than5μM glutathione group.Conclusion:Glutathione enhanced osteogenesis and simultaneously inhibited adipogenesis by human MSCs possibly through elimination of increased reactive oxygen species. The results indicated that glutathione can potentially be used for treatment or prevention of corticosteroid-induced osteonecrosis of femoral head.