Matrix stiffening promotes chondrocyte senescence and the osteoarthritis development through downregulating HDAC3

Extracellular matrix(ECM)stiffening is a typical characteristic of cartilage aging,which is a quintessential feature of knee osteoarthritis(KOA).However,little is known about how ECM stiffening affects chondrocytes and other molecules downstream.This study mimicked the physiological and pathological stiffness of human cartilage using polydimethylsiloxane(PDMS)substrates.It demonstrated that epigenetic Parkin regulation by histone deacetylase 3(HDAC3)represents a new mechanosensitive mechanism by which the stiffness matrix affected chondrocyte physiology.We found that ECM stiffening accelerated cultured chondrocyte senescence in vitro,while the stiffness ECM downregulated HDAC3,prompting Parkin acetylation to activate excessive mitophagy and accelerating chondrocyte senescence and osteoarthritis(OA)in mice.Contrarily,intra-articular injection with an HDAC3-expressing adeno-associated virus restored the young phenotype of the aged chondrocytes stimulated by ECM stiffening and alleviated OA in mice.The findings indicated that changes in the mechanical ECM properties initiated pathogenic mechanotransduction signals,promoted the Parkin acetylation and hyperactivated mitophagy,and damaged chondrocyte health.These results may provide new insights into chondrocyte regulation by the mechanical properties of ECM,suggesting that the modification of the physical ECM properties may be a potential OA treatment strategy.

Mechanism of acupoint penetration acupuncture therapy regulating chondrocyte autophagy via the PI3K/Akt-mTOR pathway in KOA rats

Objective To investigate whether acupoint penetration acupuncture(APA)could regulate chondrocyte autophagy and apoptosis via the PI3K/Akt-mTOR signaling pathway to reduce cartilage degeneration in knee osteoarthritis(KOA)rats.Methods KOA was induced in rats via intra-articular injection of sodium iodoacetate resolution.Forty male Sprague-Dawley rats were randomly assigned to blank control,model,APA,electro-acupuncture(EA),and sham model groups(n=8)and those in the APA and EA groups received their respective therapies.Following completion of the treatment course,histological examinations of cartilage and muscle were conducted.Levels of apoptosis-and autophagy-related factors,including Bax,Bcl-2,mTOR,ULK-1,and Beclin-1 protein,and mRNAs were assessed.Additionally,β-endorphin(β-EP)concentrations in the brain and serum were measured.Results Histological analysis revealed that APA alleviated cartilage and muscle damage compared with the model group.APA inhibited cartilage degeneration by modulating the expression of apoptosis-and autophagy-related proteins and mRNA,thus preventing chondrocyte apoptosis.In the APA group,Bax and mTOR protein levels were significantly lower than those in the model group(both P=.024).Conversely,the Bcl-2 expression level was significantly higher than that in the EA group(P=.035).Additionally,ULK-1 expression was significantly lower than that in the EA group(P=.045).The mRNA level of Bax was significantly higher than that in the blank control group(P<.001).However,Beclin-1 levels were significantly higher than those in both the model and EA groups(both P<.001).ELISA results showed a significant decrease in the concentration ofβ-EP in the brains of the rats in the APA group compared with those in the model group(P=.032).Conclusions APA reduced osteoarthritis-related pain and alleviated cartilage damage by upregulating chondrocyte autophagy and down-regulating apoptosis via signaling pathways involving PI3K/Akt-mTOR in KOA rats.

AAV-mediated expression of p65shRNA and bone morphogenetic protein 4 synergistically enhances chondrocyte regeneration

BACKGROUND:Adeno-associated virus(AAV)gene therapy has been proven to be reliable and safe for the treatment of osteoarthritis in recent years.However,given the complexity of osteoarthritis pathogenesis,single gene manipulation for the treatment of osteoarthritis may not produce satisfactory results.Previous studies have shown that nuclear factorκB could promote the inflammatory pathway in osteoarthritic chondrocytes,and bone morphogenetic protein 4(BMP4)could promote cartilage regeneration.OBJECTIVE:To test whether combined application of AAV-p65shRNA and AAV-BMP4 will yield the synergistic effect on chondrocytes regeneration and osteoarthritis treatment.METHODS:Viral particles containing AAV-p65-shRNA and AAV-BMP4 were prepared.Their efficacy in inhibiting inflammation in chondrocytes and promoting chondrogenesis was assessed in vitro and in vivo by transfecting AAV-p65-shRNA or AAV-BMP4 into cells.The experiments were divided into five groups:PBS group;osteoarthritis group;AAV-BMP4 group;AAV-p65shRNA group;and BMP4-p65shRNA 1:1 group.Samples were collected at 4,12,and 24 weeks postoperatively.Tissue staining,including safranin O and Alcian blue,was applied after collecting articular tissue.Then,the optimal ratio between the two types of transfected viral particles was further investigated to improve the chondrogenic potential of mixed cells in vivo.RESULTS AND CONCLUSION:The combined application of AAV-p65shRNA and AAV-BMP4 together showed a synergistic effect on cartilage regeneration and osteoarthritis treatment.Mixed cells transfected with AAV-p65shRNA and AAV-BMP4 at a 1:1 ratio produced the most extracellular matrix synthesis(P<0.05).In vivo results also revealed that the combination of the two viruses had the highest regenerative potential for osteoarthritic cartilage(P<0.05).In the present study,we also discovered that the combined therapy had the maximum effect when the two viruses were administered in equal proportions.Decreasing either p65shRNA or BMP4 transfected cells resulted in less collagen II synthesis.This implies that inhibiting inflammation by p65shRNA and promoting regeneration by BMP4 are equally important for osteoarthritis treatment.These findings provide a new strategy for the treatment of early osteoarthritis by simultaneously inhibiting cartilage inflammation and promoting cartilage repair.

Exploring the effect of Bushen Bitong recipe-containing serum on IL-1β-induced chondrocyte apoptosis based on SOX9/NF-κB/MMP-13 signaling pathway

Objective:To observe the effect and possible mechanism of action of Bushen Bitong recipe(BSBT)containing serum on IL-1β-induced chondrocyte apoptosis.Methods:Generation 3 rat chondrocytes were randomized into Control,IL-1β,IL-1β+BSBT(L),IL-1β+BSBT(M),and IL-1β+BSBT(H)groups(5%,10%and 15%BSBT-containing serum),and then 24h after intervention respectively,the cell proliferation and Apoptosis rate;Western blot detected the expression levels of Bcl-2,BAX,Caspase-3,SOX9,NF-κB p65,MMP-13 proteins in chondrocytes.ELISA detected the levels of TNF-α,IL-6,and bFGF in the supernatants of chondrocyte culture.Results:Compared with Control group,cell proliferation activity decreased,apoptosis rate increased,NF-κB p65,MMP-13 protein level and TNF-α,IL-6 level increased,and SOX9 protein level and bFGF level decreased in IL-1βgroup;compared with IL-1βgroup,different concentrations of BSBT-containing serum group,cell proliferation activity increased,and apoptosis rate decreased.NF-κB p65,MMP-13 protein level and TNF-α,IL-6 level decreased,SOX9 protein level and bFGF level increased;compared with IL-1β+BSBT(L)group,cell proliferation activity increased,apoptosis rate decreased in IL-1β+BSBT(M)and IL-1β+BSBT(H)groups,and NF-κB p65,MMP-13 protein level and TNF-αlevel decreased.13 protein levels and TNF-αand IL-6 levels decreased,and SOX9 protein levels and bFGF levels increased.Conclusion:BSBT-containing serum may promote IL-1β-induced proliferation of chondrocytes,reduce apoptosis,improve the microenvironment of chondrocytes,and promote cartilage repair through the SOX9/NF-κB/MMP-13 signaling pathway.

LOXL3 Inhibits Autophagy of Chondrocytes by Activating Rheb in Osteoarthritis

Objective This study aimed to investigate the potential mechanisms by which lysyl oxidase like 3(LOXL3)affects the autophagy in chondrocytes in osteoarthritis(OA),specifically through the activation of mammalian target of rapamycin complex 1(mTORC1).Methods To establish an OA model,rats underwent anterior cruciate ligament transection(ACLT).Chondrocytes were isolated from cartilage tissues and cultured.Western blotting was performed to assess the expression of LOXL3,Rheb,phosphorylation of p70S6K(p-p70S6K,a downstream marker of mTORC1),and autophagy markers.The autophagy of chondrocytes was observed using an immunofluorescence assay.Results The expression levels of both LOXL3 and Rheb proteins were upregulated in chondrocytes isolated from the OA model cartilage,in comparison to those from the normal cartilage.The silencing of LOXL3 resulted in a decrease in the protein levels of Rheb and p-p70S6K,as well as an increase in the expression of autophagy-related proteins.Additionally,the effect of LOXL3 could be reversed through the silencing of Rheb.The results of the immunofluorescence assay confirmed the impact of LOXL3 and Rheb on chondrocyte autophagy.Conclusion LOXL3 inhibits chondrocyte autophagy by activating the Rheb and mTORC1 signaling pathways.

α-parvin controls chondrocyte column formation and regulates long bone development

Endochondral ossification requires proper control of chondrocyte proliferation,differentiation,survival,and organization.Here we show that knockout ofα-parvin,an integrin-associated focal adhesion protein,from murine limbs causes defects in endochondral ossification and dwarfism.The mutant long bones were shorter but wider,and the growth plates became disorganized,especially in the proliferative zone.With two-photon time-lapse imaging of bone explant culture,we provide direct evidence showing thatα-parvin regulates chondrocyte rotation,a process essential for chondrocytes to form columnar structure.Furthermore,loss ofα-parvin increased binucleation,elevated cell death,and caused dilation of the resting zones of mature growth plates.Single-cell RNA-seq analyses revealed alterations of transcriptome in all three zones(i.e.,resting,proliferative,and hypertrophic zones)of the growth plates.Our results demonstrate a crucial role ofα-parvin in long bone development and shed light on the cellular mechanism through whichα-parvin regulates the longitudinal growth of long bones.

Regulatory role of NFAT1 signaling in articular chondrocyteactivities and osteoarthritis pathogenesis

Osteoarthritis (OA), the most common form of joint disease, is characterized clinically by joint pain, stiffness,and deformity. OA is now considered a whole joint disease;however, the breakdown of the articular cartilage remains themajor hallmark of the disease. Current treatments targeting OA symptoms have a limited impact on impeding orreversing the OA progression. Understanding the molecular and cellular mechanisms underlying OA development isa critical barrier to progress in OA therapy. Recent studies by the current authors’ group and others have revealedthat the nuclear factor of activated T cell 1 (NFAT1), a member of the NFAT family of transcription factors, regulatesthe expression of many anabolic and catabolic genes in articular chondrocytes of adult mice. Mice lacking NFAT1exhibit normal skeletal development but display OA in both appendicular and spinal facet joints as adults. Thisreview mainly focuses on the recent advances in the regulatory role of NFAT1 transcription factor in the activities ofarticular chondrocytes and its implication in the pathogenesis of OA.

The Effect of Nivalenol on Metabolism of Cultured Chondrocytes and the Protection of Selenium

Objective: To investigate the possible effect of nivalenol on metabolism ofthe cultured chondrocytes and the protection of selenium. Methods: The quantitative analyses ofmetabolism in single- layer cultured chondrocytes were performed by biocliemical means and theimpairment of DNA was observed by both of the single cell microgel electrophoresis assay and theagarose gel electrophoresis assay. Results: In the media containing different concentrations ofnivalenol (0. 000 5-0. 020 0 mg/L), the amounts of DNA and proteoglycan in matrix of thechondrocytes were decreased. The syn-thesis of protein was reduced and the impairment of DNAdeteriorated with the increase of the concentrations of nivalenol in tlte given dose. When seleniumwas added into the media, the impairment by nivalenol was decreased. In the media containingdifferent concentrations of nivalenol, however, the lipid peroxidation of the chondrocytes was notaffected by nivalenol, yet the amount of lipid peroxides significantly declined. Conclusion:Nivalenol may evidently cause impairment of the chondrocytes when its concentrations are in thepresent experimental range. Selenium can protect cultured cliondrocytes, but cannot prevent theirDNA from being impaired.

The art of building bone: emerging role of chondrocyte-to-osteoblast transdifferentiation in endochondral ossification

There is a worldwide epidemic of skeletal diseases causing not only a public health issue but also accounting for a sizable portion of healthcare expenditures. The vertebrate skeleton is known to be formed by mesenchymal cells condensing into tissue elements(patterning phase) followed by their differentiation into cartilage(chondrocytes) or bone(osteoblasts) cells within the condensations. During the growth and remodeling phase, bone is formed directly via intramembranous ossification or through a cartilage to bone conversion via endochondral ossification routes. The canonical pathway of the endochondral bone formation process involves apoptosis of hypertrophic chondrocytes followed by vascular invasion that brings in osteoclast precursors to remove cartilage and osteoblast precursors to form bone. However, there is now an emerging role for chondrocyte-to-osteoblast transdifferentiation in the endochondral ossification process. Although the concept of "transdifferentiation" per se is not recent,new data using a variety of techniques to follow the fate of chondrocytes in different bones during embryonic and post-natal growth as well as during fracture repair in adults have identified three different models for chondrocyte-to-osteoblast transdifferentiation(direct transdifferentiation, dedifferentiation to redifferentiation, and chondrocyte to osteogenic precursor). This review focuses on the emerging models of chondrocyte-to-osteoblast transdifferentiation and their implications for the treatment of skeletal diseases as well as the possible signaling pathways that contribute to chondrocyte-to-osteoblast transdifferentiation processes.

Altered Gene Expression in Articular Chondrocytes of Smad3^(ex8/ex8) Mice, Revealed by Gene Profiling Using Microarrays

It has been previously reported that small mother against decapentaplegic 3 （Smad3） gene knockout （Smad3^ex8/ex8） mice displays phenotypes similar to human osteoarthritis, as characterized by abnormal hypertrophic differentiation of articular chondrocytes. To further clarify the crucial target genes that mediate transformation growth factor-β （TGF-β）/Smad3 signals on articular chondrocytes differentiation and investigate the underlying molecular mechanism of osteoarthritis, microarrays were used to perform comparative transcriptional profiling in the articular cartilage between Smad3^ex8/ex8and wild-type mice on day five after birth. The gene profding results showed that the activity of bone morphogenetic protein （BMP） and TGF-β/cell division cycle 42 （Cdc42） signaling pathways were enhanced in Smad3^ex8/ex8 chondrocytes. Moreover, there was altered gene expression in growth hormone/insulin-like growth factor 1 （Igfl） axis and fibroblast growth factor （Fgf） signaling pathway. Notably, protein synthesis related genes and electron transport chain related genes were upregulated in Smad3^ex8/ex8 chondrocytes, implying that accelerated protein synthesis and enhanced cellular respiration might contribute to hypertrophic differentiation of articular chondrocytes and the pathogenesis of osteoarthritis.

T-2 toxin-induced apoptosis involving Fas,p53,Bcl-xL,Bcl-2,Bax and caspase-3 signaling pathways in human chondrocytes

Objective:To investigate the effects of T-2 toxin on expressions of Fas,p53,Bcl-xL,Bcl-2,Bax and caspase-3 on human chondrocytes.Methods:Human chondrocytes were treated with T-2 toxin(1～20 ng/ml)for 5 d.Fas,p53 and other apoptosis-related proteins such as Bax,Bcl-2,Bcl-xL,caspase-3 were determined by Western blot analysis and their mRNA expressions were determined by reverse transcriptase-polymerase chain reaction(RT-PCR).Results:Increases in Fas,p53 and the pro-apoptotic factor Bax protein and mRNA expressions and a decrease of the anti-apoptotic factor Bcl-xL were observed in a dose-dependent manner after exposures to 1～20 ng/ml T-2 toxin,while the expression of the anti-apoptotic factor Bcl-2 was unchanged.Meanwhile,T-2 toxin could also up-regulate the expressions of both pro-caspase-3 and caspase-3 in a dose-dependent manner.Conclusion:These data suggest a possible underlying molecular mechanism for T-2 toxin to induce the apoptosis sig- naling pathway in human chondrocytes by regulation of apoptosis-related proteins.

Increase of TNFα-stimulated Osteoarthritic Chondrocytes Apoptosis and Decrease of Matrix Metalloproteinases 9 by NF-κB Inhibition

Objective To investigate the in vitro effect of caffeic acid phenethyl ester （CAPE）, a NF-KB inhibitor, on the apoptosis of osteoarthritic （OA） chondrocytes and on the regulation of the gelatinases matrix metalloproteinase 2 （MMP-2） and matrix metalloproteinase 9 （MMP-9）. Methods Annexin V-FITC/propidium iodide （PI） labeling and western blotting were used to observe and determine the apoptosis in TNFa-stimulated primary cultured osteoarthritic chondrocytes. Also, gelatin zymography was applied to examine MMP-2 and MMP-9 activities in supernatants. Results it was confirmed by both flow cytometry and western blotting that chondrocytes from OA patients have an apoptotic background. Use of CAPE in combination with 10 ng/mL of TNFa for 24 h facilitated the apoptosis. MMP-9 in the supernatant could be autoactivated （from proMMP-9 to active MMP-9）, and the physiologic calcium concentration （2.5 mmol/L） could delay the autoactivation of MMP-9. The activities of MMP-2 and MMP-9 in the fresh supernatant increased significantly in response to stimulation by 10 ng/mL of TNFa for 24 h. The stimulatory effect of TNFa just on proMMP-9 was counteracted significantly by CAPE. Conclusion NF-KB could prevent chondrocytes apoptosis though its activation was attributed to the increase of proMMP-9 activity induced by TNFa （a pro-apoptotic factor）. Therefore, therapeutic NF-KB inhibitor was a ＇double-edged swords＇ to the apoptosis of chondrocytes and the secretion of MMP-9.

Sustained Akt signaling in articular chondrocytes causes osteoarthritis via oxidative stress-induced senescence in mice

Osteoarthritis(OA) is an age-related disorder that is strongly associated with chondrocyte senescence. The causal link between disruptive PTEN/Akt signaling and chondrocyte senescence and the underlying mechanism are unclear. In this study, we found activated Akt signaling in human OA cartilage as well as in a mouse OA model with surgical destabilization of the medial meniscus.Genetic mouse models mimicking sustained Akt signaling in articular chondrocytes via PTEN deficiency driven by either Col2a1-Cre or Col2a1-Cre^(ERT2) developed OA, whereas restriction of Akt signaling reversed the OA phenotypes in PTEN-deficient mice.Mechanistically, prolonged activation of Akt signaling caused an accumulation of reactive oxygen species and triggered chondrocyte senescence as well as a senescence-associated secretory phenotype, whereas chronic administration of the antioxidant N-acetylcysteine suppressed chondrocyte senescence and mitigated OA progression in PTEN-deficient mice. Therefore,inhibition of Akt signaling by PTEN is required for the maintenance of articular cartilage. Disrupted Akt signaling in articular chondrocytes triggers oxidative stress-induced chondrocyte senescence and causes OA.

Expression of miRNA-140 in Chondrocytes and Synovial Fluid of Knee Joints in Patients with Osteoarthritis

Objective To investigate the expression of miRNA-140 in chondrocytes and synovial fluid of osteoarthritis(OA) patients, and explore the relationship between the miRNA-140 expression and OA severity. Methods This study enrolled 30 OA patients who underwent total knee arthroplasty for chondrocytes sampling and 30 OA patients who underwent intra-articular injection for synovial fluid sampling. All OA patients were grouped into mild [Kellgren and Lawrence(KL) grade 1-2], moderate(KL grade 3) and severe(KL grade 4), with 10 in each subgroups for each sampling purposes. 7 non-OA patients and 10 patients with knee injury were collected for cartilage and synovial fluid sampling respectively as control groups. Chondrocytes were isolated from the cartilage tissue and cultured in vitro. Quantitative real time PCR for miRNA-140 in chondrocytes and synovial fluid were performed, and the U6 sn RNA was used as internal control. The expression difference of miRNA-140 among groups and correlation between the expression and the KL grade of OA were analysed using one-way ANOVA and Spearman test respectively. Results The expression of miRNA-140 in chondrocytes of knees in OA patients was reduced than that in normal knees, and the between-group difference was statistically significant(F=305.464, P<0.001). miRNA-140 could be detected in synovial fluid of both normal knees and OA knees, its relative expression level was reduced in synovial fluid of OA group compared with normal group, and the between-group difference was statistically significant as well(F=314.245, P<0.001). The relative expression level of miRNA-140 in both chondrocytes and synovial fluid were negatively correlated with the KL grade of OA(r=-0.969, P<0.001; r=-0.970, P<0.001). Conclusion miRNA-140 could be detected in chondrocytes and synovial fluid of OA patients, and its expression was negatively correlated with the severity of OA.

Notch signaling controls chondrocyte hypertrophy via indirect regulation of Sox9

RBPjk-dependent Notch signaling regulates both the onset of chondrocyte hypertrophy and the progression to terminal chondrocyte maturation during endochondral ossification. It has been suggested that Notch signaling can regulate Sox9 transcription, although how this occurs at the molecular level in chondrocytes and whether this transcriptional regulation mediates Notch control of chondrocyte hypertrophy and cartilage development is unknown or controversial. Here we have provided conclusive genetic evidence linking RBPjk-dependent Notch signaling to the regulation of Sox9 expression and chondrocyte hypertrophy by examining tissuespecific Rbpjk mutant（Prx1Cre;Rbpjkf/f）, Rbpjk mutant/Sox9 haploinsufficient（Prx1Cre;Rbpjkf/f;Sox9f/1）,and control embryos for alterations in SOX9 expression and chondrocyte hypertrophy during cartilage development. These studies demonstrate that Notch signaling regulates the onset of chondrocyte maturation in a SOX9-dependent manner, while Notch-mediated regulation of terminal chondrocyte maturation likely functions independently of SOX9. Furthermore, our in vitro molecular analyses of the Sox9 promoter and Notch-mediated regulation of Sox9 gene expression in chondrogenic cells identified the ability of Notch to induce Sox9 expression directly in the acute setting, but suppresses Sox9 transcription with prolonged Notch signaling that requires protein synthesis of secondary effectors.

The effects of interleukin-1β in modulating osteoclast-conditioned medium's influence on gelatinases in chondrocytes through mitogen-activated protein kinases

Osteoarthritis is recognised to be an interactive pathological process involving the cartilage, subchondral bone and synovium. The signals from the synovium play an important role in cartilage metabolism, but little is known regarding the influence of the signalling from bone. Additionally, the collagenases and stromelysin-1 are involved in cartilage catabolism through mitogen-activated protein kinase （MAPK） signalling, but the role of the gelatinases has not been elucidated. Here, we studied the influence of osteoclastic signals on chondrocytes by characterising the expression of interleukin-1β （IL-1β）-induced gelatinases through MAPK signalling. We found that osteoclast-conditioned media attenuated the gelatinase activity in chondrocytes. However, IL-1β induced increased levels of gelatinase activity in the conditioned media group relative to the mono-cultured chondrocyte group. More specifically, IL-1β restored high levels of gelatinase activity in c-Jun N-terminal kinase inhibitor-pretreated chondrocytes in the conditioned media group and led to lower levels of gelatinase activity in extracellular signal-regulated kinase or p38 inhibitor-pretreated chondrocytes. Gene expression generally correlated with protein expression. Taken together, these results show for the first time that signals from osteoclasts can influence gelatinase activity in chondrocytes. Furthermore, these data show that IL-11~ restores gelatinase activity through MAPK inhibitors; this information can help to increase the understanding of the gelatinase modulation in articular cartilage.

A high-resolution route map reveals distinct stages of chondrocyte dedifferentiation for cartilage regeneration

Articular cartilage damage is a universal health problem.Despite recent progress,chondrocyte dedifferentiation has severely compromised the clinical outcomes of cell-based cartilage regeneration.Loss-of-function changes are frequently observed in chondrocyte expansion and other pathological conditions,but the characteristics and intermediate molecular mechanisms remain unclear.In this study,we demonstrate a time-lapse atlas of chondrocyte dedifferentiation to provide molecular details and informative biomarkers associated with clinical chondrocyte evaluation.We performed various assays,such as single-cell RNA sequencing(scRNA-seq),live-cell metabolic assays,and assays for transposase-accessible chromatin with high-throughput sequencing(ATAC-seq),to develop a biphasic dedifferentiation model consisting of early and late dedifferentiation stages.Early-stage chondrocytes exhibited a glycolytic phenotype with increased expression of genes involved in metabolism and antioxidation,whereas late-stage chondrocytes exhibited ultrastructural changes involving mitochondrial damage and stress-associated chromatin remodeling.Using the chemical inhibitor BTB06584,we revealed that early and late dedifferentiated chondrocytes possessed distinct recovery potentials from functional phenotype loss.Notably,this two-stage transition was also validated in human chondrocytes.An image-based approach was established for clinical use to efficiently predict chondrocyte plasticity using stage-specific biomarkers.Overall,this study lays a foundation to improve the quality of chondrocytes in clinical use and provides deep insights into chondrocyte dedifferentiation.

Gene expression profile of hypertrophic chondrocytes treated with H2O2:A Preliminary investigation

Objective To identify the osteogenesis genes whose expression is altered in hypertrophic chondrocytes treated with H2 O2.Methods Murine chondrogenitor cells(ATDC5) were differentiated into hypertrophic chondrocytes by InsulinTransferrin-Selenium(ITS) treatment, and then treated with H2 O2. Suitable conditions(concentration, time) were determined by using the MTT assay. After total RNA isolation and cD NA synthesis, the levels of 84 genes were determined using the PCR array, whereas quantitative RT-PCR was carried out to validate the PCR array data. Results We identified 9 up-regulated genes and 12 down-regulated genes, encoding proteins with various functions, such as collagen proteins, transcription factors, proteins involved in skeletal development and bone mineral metabolism, as well as cell adhesion molecules. Quantitative RT-PCR confirmed the altered expression of 5 down-regulated genes(Smad2, Smad4, transforming growth factor β receptor 1, transforming growth factor β receptor 3, and matrix metalloproteinase 10). Conclusions H2 O2 significantly changed the expression of several genes involved in a variety of biological functions. Because of the link between oxidative damage and Kashin-Beck disease, these genes may also be involved in the deep-zone necrosis of the cartilage observed in Kashin-Beck disease.

Protective Effect of Pyrroloquinoline Quinone on TNF-α-induced Mitochondrial Injury in Chondrocytes

Osteoarthritis(OA)is a degenerative disease characterized by matrix degradation and cell death leading to a gradual loss of articular cartilage integrity.As a bacterial synthesis of quinine,pyrroloquinoline quinone(PQQ)is a strong redox cofactor with a variety of biological benefits,including antioxidant,anti-inflammation-induced mitochondrial metabolism regulation.This study was designed to investigate the effect of PQQ on TNF-α-induced mitochondrial damage in chondrocytes.Chondrocytes isolated from C57BL/6 mice were exposed to TNF-α50 ng/mL,TNF-α50 ng/mL+PQQ 10µmol/L for 24 h.Then,morphological study,functional study and mechanism study were taken.The results revealed TNF-α-induced chondrocyte mitochondrion damage could be reduced by application of PQQ,evidenced by elevated number of mitochondria,well-kept mtDNA integrity,preserved ATP level,reestablished mitochondrial membrane potential,and prevented mitochondrial function.The present work strongly suggests that the mitochondrion is an important target for OA chondrocyte damage induced by TNF-αand the PQQ protection from this damage ameliorates mitochondrial dysfunction induced by TNF-α.PQQ might be a potential chemical for OA intervention.

Dietary fat-associated osteoarthritic chondrocytes gain resistance to lipotoxicity through PKCK2/STAMP2/FSP27

Free fatty acids(FFAs), which are elevated with metabolic syndrome, are considered the principal offender exerting lipotoxicity. Few previous studies have reported a causal relationship between FFAs and osteoarthritis pathogenesis. However, the molecular mechanism by which FFAs exert lipotoxicity and induce osteoarthritis remains largely unknown. We here observed that oleate at the usual clinical range does not exert lipotoxicity while oleate at high pathological ranges exerted lipotoxicity through apoptosis in articular chondrocytes. By investigating the differential effect of oleate at toxic and nontoxic concentrations, we revealed that lipid droplet(LD) accumulation confers articular chondrocytes, the resistance to lipotoxicity. Using high fat diet-induced osteoarthritis models and articular chondrocytes treated with oleate alone or oleate plus palmitate, we demonstrated that articular chondrocytes gain resistance to lipotoxicity through protein kinase casein kinase 2(PKCK2)—six-transmembrane protein of prostate 2(STAMP2)—and fat-specific protein 27(FSP27)-mediated LD accumulation. We further observed that the exertion of FFAs-induced lipotoxicity was correlated with the increased concentration of cellular FFAs freed from LDs, whether FFAs are saturated or not. In conclusion, PKCK2/STAMP2/FSP27-mediated sequestration of FFAs in LD rescues osteoarthritic chondrocytes. PKCK2/STAMP2/FSP27 should be considered for interventions against metabolic OA.