Alleviation of oxidative stress damage in chondrocytes by a new Mn-containing bioceramic powder
BACKGROUND:Mn can participate in oxidation-reduction reactions in various organisms.For example,as a metal-assisted group in superoxide dismutase 2,Mn plays a role in helping to remove reactive oxygen species.Therefore,the development of novel anti-oxidative stress materials containing Mn has become a research focus in recent years.OBJECTIVE:To investigate the protective effect of Mn bioceramic powder material on oxidative stress damage to chondrocytes by reducing the reactive oxygen species pathway.METHODS:Bioceramic powders containing Mn were prepared by molten salt method.Primary mouse chondrocytes were isolated and cultured.Bioceramic powder containing 0,0.15,and 0.30 mg/mL of Mn was added into H2O2 solution.The H2O2 clearance rate was detected after incubation without light.The passage 2 to passage 4 chondrocytes were co-cultured with complete media containing Mn-containing bioceramic powder with different mass concentrations(0,0.15,and 0.30 mg/mL).Cell viability was detected by cell live/dead staining.The passage 2-4 chondrocytes(or cartilage tissue)were divided into four groups for intervention:Complete culture medium was added to the blank control group.The H2O2 group was added and cultured with complete medium containing H2O2.H2O2+low mass concentration Mn powder group was cultured by adding H2O2+0.15 mg/mL Mn-containing bioceramic powder.The complete medium containing H2O2+0.30 mg/mL Mn-containing bioceramic powder was added to the H2O2+high mass concentration Mn powder group.Viability of chondrocytes was detected by CCK-8 assay.Generation of reactive oxygen species of chondrocytes was detected by 2,7-dichlorofluorescein diacetate probe.Expression of chondrocyte-related factors was detected by qRT-PCR.The tissue structure and function of cartilage were detected by toluidine blue staining.RESULTS AND CONCLUSION:(1)Both doses of Mn-containing bioceramic powders could significantly remove H2O2 in vitro,and they were concentration dependent.The results of cell live/death staining showed that 0.15 mg/mL bioceramic powder containing Mn had chondrocyte safety,and 0.30 mg/mL bioceramic powder containing Mn had chondrocytotoxicity.(2)The results of CCK-8 assay showed that the two mass concentrations of Mn-containing bioceramic powders could significantly reduce the inhibitory effect of H2O2 on chondrocyte viability,and inhibit the generation of reactive oxygen species induced by H2O2 in chondrocytes in a mass concentration dependent manner.Both kinds of Mn-containing bioceramic powders could reverse the H2O2-induced increase of mRNA expression of a disintegrin and metalloproteinase with thrombospondin motifs-5 and decrease of proteoglycan mRNA expression in chondrocytes.(3)Toluidine blue staining results showed that both concentrations of Mn-containing bioceramic powder could protect the integrity of cartilage tissue structure under oxidative stress,and 0.30 mg/mL of Mn-containing bioceramic powder could also reduce the functional damage of cartilage tissue.(4)The results indicate that the Mn-containing bioceramic powder can protect chondrocytes under oxidative stress by clearing reactive oxygen species,maintaining the extracellular matrix homeostasis.However,0.30 mg/mL Mn-containing bioceramic powder has certain chondrocytotoxicity,so 0.15 mg/mL Mn-containing bioceramic powder is preferred for follow-up studies.
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维普
