The Study On The Treatment Of Steroid-associated Osteonecrosis Of Femoral Head Using Epigenetically Reprogramming MSCs

Objective:The maintenance of bone metabolism homestasis relies on dynamic balance between osteogenesis and adipogenesis of MSCs. Administration with ectogenic steroid could make osteogenesis and adipogenesis imbalanced, then the osteonecrosis of femoral head developed. This study is to investigate the cell number, function and the Wnt signaling pathway of mesenchymal stem cells in patients with steroid-associated osteonecrosis of femoral head.Methods:This study was approved by the Ethics Committee of Wuhan Union Hospital. Between July2011and June2012,22patients (10men,12women; mean age49.3, ranging from39-65years) with GC-induced ONFH were selected at the authors’ institution (Union Hospital, Wuhan, China) and25subjects with femoral neck fractures (15men and10women; mean age52.3, ranging from36-68years) were enrolled as controls. In the operating room,5ml of bone marrow was aspirated Human mesenchymal stem cells were isolated, cultured and identified, then the cells in Passage3were used in this study. Cell colony formation ability was evaluated from all the patients. Then we investigated the cellular viability, cell cycle, mitochondrial membrane potential, reactive oxygen species, osteogenic or adipogenic potential of MSCs and the Wnt signaling pathway activation from the three groups.Results:MSCs colony formation ability is poor compared to the control group (19.6±7.7vs.262±6.2, P<0.001). Treated with25mmol/L LiCL, the cellular viability in the GC-induced ONFH group increased while the expected increased ROS showed an evident decrease and the polarization state of the mitochondria was restored. Moreover, LiCL could make the dynamic balance between osteogenesis and adipogenesis restore by the activation of the Wnt signaling pathway. immunofluorescence, real-time PCR and western blot exhibted expression of Wnt-related genes, Runx2and PPAR-y both in transcriptive level and translational level was down-regulated and LiCL can up-regulate it.Conclusion:The MSCs’ number, function and the Wnt signaling pathway of mesenchymal stem cells in patients with steroid-associated osteonecrosis of femoral head were weakened. Steroids could make MSCs dysfunctional and adipogenic by affecting the Wnt signaling pathway. Finally, the femoral heads were necrotic and collapsed. Objective:The Epigenetic Regulation plays an important role in differentiation of stem cells. In each step from pluripotency to terminal differentiation, it is necessary for stem cells to keep a stable epigenetic state. The most widely studied epigenetic modification in humans is the cytosine methylation of DNA within the dinucleotide CpG This study is to observe DNA hypermethylation in mesenchymal stem cells of patients with steroid-associated osteonecrosis and its impact on MSCs dysfunction and explore the underlying mechanism.Methods:This study was approved by the Ethics Committee of Wuhan Union Hospital. Between July2012and May.2013,20patients (10men,10women; mean age53.5, ranging from39-67years) with GC-associated ONFH were selected at the authors’ institution (Union Hospital, Wuhan, China) and22subjects with femoral neck fractures (12men and10women; mean age56.2, ranging from40-70years) were enrolled as controls. Human mesenchymal stem cells were isolated and cultured, then the cells from Passage3were used in this study. LUMA was used to test the genome-wide methylation rate of MSCs from the three groups. Then Bisulfite sequencing detected CpG islands hypermethylation of ABCB1, Oct4and Nanog. Real-time PCR and western blot detected expression of Oct4, Nanog, ABCB1, Runx2, PPAR-y and osteocalcin both in transcriptive level and translational level. We also investigated the viability, mitochondrial membrane potential, reactive oxygen species and osteogenic or adipogenic potential of MSCs from the three groups.Results:The result of LUMA showed that the frequencies.of methylation for the MSCs from ONFH group was obviously higher than the control group. The treatment with15μM5’-Aza-dC can lower methylation rate. Bisulfite sequencing detected the promoters of ABCB1, Oct4and Nanog were hypermethylated. Treated with15μM5’-Aza-dC, the cellular viability in the GC-induced ONFH group increased while the expected increased ROS showed an evident decrease and the polarization state of the mitochondria was restored. Moreover,5’-Aza-dC could make the dynamic balance between osteogenesis and adipogenesis restore. Real-time PCR and western blot exhibted expression of Oct4, Nanog, ABCB1, Runx2, PPAR-y and osteocalcin both in transcriptive level and translational level was down-regulated and5’-Aza-dC can up-regulate it.Conclusion:The genome-wide DNA was hypermethylated in mesenchymal stem cells of patients with steroid-associated osteonecrosis. Hypermethylation of ABCB1, Oct4and Nanog led to the loss of pluripotency and defence capability, then MSCs became dysfunctional and adipogenic. Finally, the femoral heads were necrotic and collapsed. Objective:To explore the feasibility of the treatment of of steroid-associated osteonecrosis of femoral head at early stage using reprogramming MSCs and the related mechanism.Methods:Forty SD rats were randomly assigned to four groups:blank control group, experimental control group, reprogramming MSCs group and MSCs group,10rats in each group. We established rat models of ONFH by intramuscular injection of25mg/kg prednisolone acetate twice a week, for4weeks while intramuscular injection of physiological saline in the blank control group. Then we cut along the inner side of the patella and turned the patellar outside of the both knees of the experimental control group, reprogramming MSCs group and MSCs group, and drilled from the intercondylar until the femoral neck, finally MSCs suspension was dropped into. Gelatin sponge closed the pore.8weeks post-operation, the femoral heads were pathologically examinationed.Results:Rats MSCs were isolated and identified. They highly expressed CD44, CD90and lowly expressed CD45. The MSCs in experimental control group had poor activity compared to other groups.5μM5’-Aza-dC can promote the proliferation of the MSCs in experimental control group while up-regulate the transcripts of OCT4, NANOG and ABCB1. H&E staining showed that the treatment with the reprogramming MSCs lowered the rate of empty lacuna. TUNEL and DKK1staining showed that the treatment with the reprogramming MSCs lowered the rate of positive area while PCNA and β-catenin staining showed that the treatment with the reprogramming MSCs enhanced the rate of positive area.Conclusion:The transplantation of reprogramming MSCs can prevent the steroid-associated osteonecrosis of femoral head at early stage by activating Wnt signaling pathway.