Objective To investigate the effects of exosomes derived from hypoxia-treated mesenchymal stem cells on chondrocyte injury.Methods After mesenchymal stem cells were subjected to hypoxic treatment,the secreted exosomes were collected and co-cultured with IL-1β-stimulated chondrocytes.Cell viability was assessed using the CCK-8 assay,while apoptosis was evaluated by flow cytometry and the measurement of Caspase-3 and PARP activities.Intracellular levels of ROS,Fe2+,and MDA were quantified using commercial assay kits.The expression of GPX4,SLC7A11,and ACSL4 was analyzed at both mRNA and protein levels via qRT-PCR and Western blot,respectively.Additionally,the secretion of COL2A1,MMP13,ADAMTS5,TNF-α,IL-6,and PGE2 was determined by ELISA.Results Chondrocyte viability was significantly enhanced following the uptake of exosomes derived from hypoxia-treated mesenchymal stem cells(H-Exo)(P<0.05).IL-1β treatment reduced chondrocyte viability,increased Caspase-3 and PARP activities,and promoted apoptosis(P<0.05);however,H-Exo effectively reversed IL-1 β-induced apoptotic effects.Furthermore,IL-1 β markedly down-regulated the expression of GPX4 and SLC7A11,up-regulated ACSL4 expression,and elevated intracellular levels of ROS,Fe2+,and MDA(P<0.05),indicating the induction of ferroptosis.Both the ferroptosis inhibitor and H-Exo significantly attenuated IL-1β-triggered ferroptosis,and H-Exo counteracted the detrimental effects of IL-1β as well as those induced by a ferroptosis inducer.Additionally,IL-1β suppressed the expression of the chondrogenic marker COL2A1,up-regulated the catabolic enzymes MMP13 and ADAMTS5,and enhanced the secretion of pro-inflammatory cyto-kines TNF-α,IL-6,and PGE2(P<0.05).Notably,H-Exo alleviated IL-1β-mediated inflammation and restored the balance between chondrogenic anabolism and catabolism.Conclusions Exosomes secreted by mesenchymal stem cells under hypoxic conditions can effectively inhibit chondrocyte apoptosis and ferroptosis,thereby alleviating cellular injury.These findings suggest that such exosomes exert a protective effect on chondrocytes and hold prom-ise as a novel therapeutic strategy for cartilage repair.

Objective To investigate the effects of exosomes derived from hypoxia-treated mesenchymal stem cells on chondrocyte injury.Methods After mesenchymal stem cells were subjected to hypoxic treatment,the secreted exosomes were collected and co-cultured with IL-1β-stimulated chondrocytes.Cell viability was assessed using the CCK-8 assay,while apoptosis was evaluated by flow cytometry and the measurement of Caspase-3 and PARP activities.Intracellular levels of ROS,Fe2+,and MDA were quantified using commercial assay kits.The expression of GPX4,SLC7A11,and ACSL4 was analyzed at both mRNA and protein levels via qRT-PCR and Western blot,respectively.Additionally,the secretion of COL2A1,MMP13,ADAMTS5,TNF-α,IL-6,and PGE2 was determined by ELISA.Results Chondrocyte viability was significantly enhanced following the uptake of exosomes derived from hypoxia-treated mesenchymal stem cells(H-Exo)(P<0.05).IL-1β treatment reduced chondrocyte viability,increased Caspase-3 and PARP activities,and promoted apoptosis(P<0.05);however,H-Exo effectively reversed IL-1 β-induced apoptotic effects.Furthermore,IL-1 β markedly down-regulated the expression of GPX4 and SLC7A11,up-regulated ACSL4 expression,and elevated intracellular levels of ROS,Fe2+,and MDA(P<0.05),indicating the induction of ferroptosis.Both the ferroptosis inhibitor and H-Exo significantly attenuated IL-1β-triggered ferroptosis,and H-Exo counteracted the detrimental effects of IL-1β as well as those induced by a ferroptosis inducer.Additionally,IL-1β suppressed the expression of the chondrogenic marker COL2A1,up-regulated the catabolic enzymes MMP13 and ADAMTS5,and enhanced the secretion of pro-inflammatory cyto-kines TNF-α,IL-6,and PGE2(P<0.05).Notably,H-Exo alleviated IL-1β-mediated inflammation and restored the balance between chondrogenic anabolism and catabolism.Conclusions Exosomes secreted by mesenchymal stem cells under hypoxic conditions can effectively inhibit chondrocyte apoptosis and ferroptosis,thereby alleviating cellular injury.These findings suggest that such exosomes exert a protective effect on chondrocytes and hold prom-ise as a novel therapeutic strategy for cartilage repair.

Objective In order to understand the chondrogenesis differentiation of mesenchymal stem cells in either hydrogel or pellet culture,we applied the two methods and reveal the possible mechanism and for further investigation.Methods In C3H10T1/2 chondrogenic differentiation,we apply extracellular matrix hydrogel mixed the cell suspensions of freshly prepared (including scaling chondroitin sulfate,sodium hyaluronate synthesis and cross-linking agent) co-culture system and high cell density pellet formed by centrifugation.Chondrogenic differentiation of C3H10T1/2 was induced by treatment with TGF-β3 (10 ng/ml),dexamethasone (100 nmol/L),ascorbic acid (50 ug/ml),1 ∶ 100 dilution ITS+Premix and high glucose-DMEM medium with 0.2 volume fraction fetal bovine serum.And high glucose-DMEM medium with 0.2 volume fraction fetal bovine serum is for control group.Histochemistry staining was utilized to identify extracellular proteoglycan and real-time PCR was performed to assess gene expression of SOX9,collagen Ⅱa1/Ⅹa1 and aggrecan for the 1st,2nd and 3rd week respectively.Results In the hydrogel model for 3 weeks chondrogenic differentiation,the expression of master transcription factor SOX9 was upregulated in both culture models.While the marker genes of collagen Ⅱa1 and collagen Ⅹa1 were all promoted in hydrogel culture,the aggrecan gene expression was peaked in pellet culture.In addition,immunocytochemistry analysis of the hydrogel and pellet for 3 week illustrated the expression of extracellular matrix and more obviously in the hydrogel model.Conclusion In compared with pellet culture,the MSCs in the hydrogel were more likely promoted chondrogenesis leading to the eventual expression of marker genes.And the hydrogel would be applied in regeneration of cartilage injury.

Objective To analyze the treatment effects of cervical disc herniation treated by ozone combined with radiofrequency ther-mocoagulation. Methods Ninety cases of cervical intervertebral disc herniation were collected from our hospital in July 2009 to December 2013,who were treated by ozone combined with radiofrequency thermocoagulation. The patients were followed up for at least 3~6 months and the improvement rate was calculated according to the Macnab improved standard. Results All the patients were followed up for at least 3~6 months,according to the Macnab standard improved,50 cases were excellent,good in 25 cases,in 10 cases and poor in 5 cases,the improve-ment rate was 94. 4%. Conclusion The treatment of cervical disc herniation by ozone combined with radiofrequency thermocoagulation is one of the interventional therapy methods,which is minimally invasive and relatively safe. The treatment method has the following advantages, such as,satisfactory effect,accurate operation safety,less complications and without destroying the stability of the spinal structure.