BACKGROUND:Bone marrow mesenchymal stem cells play a crucial role in treatment of diseases,such as femoral head necrosis,and the therapeutic efficacy is closely related to the quality of the cells.The empowerment of cells has become a research focus.OBJECTIVE:To investigate the effects of hypoxia mimetic dimethylglyoxal glycine pretreatment on mitophagy and differentiation capacity of human bone marrow mesenchymal stem cells.METHODS:Bone marrow mesenchymal stem cells were extracted from the bone marrow of patients' iliac crest and cultured in vitro to the third passage.The cells were treated with dimethylglyoxal glycine at 0,10,50,and 100 μmol/Lfor 24 hours,followed by the replacement with an osteogenic induction differentiation medium,which constituted the pretreatment group.The continuous treatment group was cultured directly in osteogenic induction medium containing 0,10,50,and 100 μmol/L dimethylglyoxal glycine after cell adhesion.After 7 days of induction,alkaline phosphatase staining was performed to select the most favorable conditions for osteogenic differentiation for subsequent experiments,with normally cultured bone marrow mesenchymal stem cells serving as the control group.Alkaline phosphatase staining,alkaline phosphatase activity,oil red O staining,and related RT-qPCR were used to evaluate the osteogenic and adipogenic differentiation differences of bone marrow mesenchymal stem cells between the two groups.MitoSox staining was used to detect mitochondrial reactive oxygen species levels.Mito-tracker and Lyso-tracker staining were used to detect the co-localization of mitochondria and lysosomes.The fluorescent probe JC-1 was used to measure mitochondrial membrane potential.RESULTS AND CONCLUSION:Alkaline phosphatase staining indicated that the most beneficial treatment for bone marrow mesenchymal stem cell osteogenesis was pretreatment with 10 μmol/L dimethylglyoxal glycine for 24 hours.Compared with the control group,the experimental group showed enhanced alkaline phosphatase staining expression,increased alkaline phosphatase activity and osteogenic gene expression,reduced lipid droplet formation and adipogenic gene expression as indicated by oil red O staining,decreased mitochondrial reactive oxygen species production,increased co-localization of mitochondria and lysosomes,and elevated mitochondrial membrane potential.The results suggest that 10 μmol/L dimethylglyoxal glycine pretreatment can promote osteogenic differentiation of bone marrow mesenchymal stem cells,inhibit adipogenic differentiation,and enhance mitophagy.

BACKGROUND:Bone marrow mesenchymal stem cells play a crucial role in treatment of diseases,such as femoral head necrosis,and the therapeutic efficacy is closely related to the quality of the cells.The empowerment of cells has become a research focus.OBJECTIVE:To investigate the effects of hypoxia mimetic dimethylglyoxal glycine pretreatment on mitophagy and differentiation capacity of human bone marrow mesenchymal stem cells.METHODS:Bone marrow mesenchymal stem cells were extracted from the bone marrow of patients' iliac crest and cultured in vitro to the third passage.The cells were treated with dimethylglyoxal glycine at 0,10,50,and 100 μmol/Lfor 24 hours,followed by the replacement with an osteogenic induction differentiation medium,which constituted the pretreatment group.The continuous treatment group was cultured directly in osteogenic induction medium containing 0,10,50,and 100 μmol/L dimethylglyoxal glycine after cell adhesion.After 7 days of induction,alkaline phosphatase staining was performed to select the most favorable conditions for osteogenic differentiation for subsequent experiments,with normally cultured bone marrow mesenchymal stem cells serving as the control group.Alkaline phosphatase staining,alkaline phosphatase activity,oil red O staining,and related RT-qPCR were used to evaluate the osteogenic and adipogenic differentiation differences of bone marrow mesenchymal stem cells between the two groups.MitoSox staining was used to detect mitochondrial reactive oxygen species levels.Mito-tracker and Lyso-tracker staining were used to detect the co-localization of mitochondria and lysosomes.The fluorescent probe JC-1 was used to measure mitochondrial membrane potential.RESULTS AND CONCLUSION:Alkaline phosphatase staining indicated that the most beneficial treatment for bone marrow mesenchymal stem cell osteogenesis was pretreatment with 10 μmol/L dimethylglyoxal glycine for 24 hours.Compared with the control group,the experimental group showed enhanced alkaline phosphatase staining expression,increased alkaline phosphatase activity and osteogenic gene expression,reduced lipid droplet formation and adipogenic gene expression as indicated by oil red O staining,decreased mitochondrial reactive oxygen species production,increased co-localization of mitochondria and lysosomes,and elevated mitochondrial membrane potential.The results suggest that 10 μmol/L dimethylglyoxal glycine pretreatment can promote osteogenic differentiation of bone marrow mesenchymal stem cells,inhibit adipogenic differentiation,and enhance mitophagy.

Objective To investigate the changes of microarchitecture and gene expression of subchondral bone in the initial stage of traumatic arthritis ,to explore the characteristics of subchondral bone remodeling and its role in the articular cartilage de‐generation .Methods The medial meniscal tear (MMT) was performed on the right knees of 13 SD rats to simulate the traumatic osteoarthritis ,while sham operation on the control group .Three weeks later ,all the rats were executed and dissected ,with proximal tibiae being kept and distributed into the two groups ,10 respectively .Micro‐computed tomography (micro‐CT) was adopted to re‐construct and analyze the subchondral bone .After being fixed by 4% paraformaldehyde ,all the samples were decalcified until six weeks passed ,followed by paraffin‐sectioning ,safranin O and fast green staining ,and examining and photographing under an ordina‐ry optical microscope .The RNA of another 3 SD rats′subchondral bone was extracted ,and a real‐time PCR test was carried out to illuminate the expression variation of bone‐formation marker genes (ALP ,RUNX2 ,and OCN) ,and bone‐resorption marker genes (TRAP ,CTSK and MMP9) ,between the two groups .Results Three weeks after MMT surgery ,subchondral bone disorders were observed among the experimental samples through micro‐CT scanning .There was lesser BV/TV ,Conn .D and Tb .Th(P<0 .05) and more Tb .Sp(P<0 .05) in the experimental group compared with the control group .In the pathological section ,arthritic degen‐eration was not spotted in both groups ,but trabeculae of the experimental group were found to be sparse .Compared with control group ,the level of mRNA expression of the bone‐formation marker genes of the experimental group was decreased(P<0 .05) ,while bone‐resorption related genes increased(P<0 .05) .Conclusion The model of initial traumatic osteoarthritis induced by MMT in rats′knees showed an active bone remodeling ,more bone absorbing than bone formation ,lowered bone volume ,and microarchitec‐ture changing of the subchondral bone .