Postmortem chondrocyte viability in porcine articular cartilage: Influence of time, temperature, and burial under winter conditions.

The aim of the present study was to investigate the effects of time, temperature, and burial in a natural environment on the viability of chondrocytes in porcine femoral condyles using confocal laser scanning microscopy. Hind trotters from 10 pigs were buried or left unburied. Samples were collected daily and stained with a combination of vital dyes (calcein-AM and ethidium homodimer-1). The chondrocytes showed an intense staining corresponding to their vitality. In the first 3 days, viability decreased slowly and showed no statistical difference between buried and unburied samples. After the first 3 days, it decreased rapidly, with the viability of the buried samples being 66% on day 4, decreasing to 25% on day 8 and to 16% on day 10, while in the unburied samples it decreased to 43% on day 4, 13% on day 8 and 5% on day 10. Our results indicate a time, temperature, and burial dependent decrease in chondrocyte viability and suggest the use of chondrocyte viability as a marker for estimating PMI in both the natural environment and in animals, as well as its potential use in humans.

Gulp1 regulates chondrocyte growth arrest and differentiation via the TGF-ß/SMAD2/3 pathway.

Chondrocyte differentiation is crucial for cartilage formation. However, the complex processes and mechanisms coordinating chondrocyte proliferation and differentiation remain incompletely understood. Here, we report a novel function of the adaptor protein Gulp1 in chondrocyte differentiation. Gulp1 expression is upregulated during chondrogenic differentiation. Gulp1 knockdown in chondrogenic ATDC5 cells reduces the expression of chondrogenic and hypertrophic marker genes during differentiation. Furthermore, Gulp1 knockdown impairs cell growth arrest during chondrocyte differentiation and reduces the expression of the cyclin-dependent kinase inhibitor p21. The activation of the TGF-ß/SMAD2/3 pathway, which is associated with p21 expression in chondrocytes, is impaired in Gulp1 knockdown cells. Collectively, these results demonstrate that Gulp1 contributes to cell growth arrest and chondrocyte differentiation by modulating the TGF-ß/SMAD2/3 pathway.

NOTCH2 sensitizes the chondrocyte to the inflammatory response of tumor necrosis factor α.

Notch regulates the immune and inflammatory response and has been associated with the pathogenesis of osteoarthritis in humans and preclinical models of the disease. Notch2tm1.1Ecan mice harbor a NOTCH2 gain-of-function and are sensitized to osteoarthritis, but the mechanisms have not been explored. We examined the effects of tumor necrosis factor α (TNFα) in chondrocytes from Notch2tm1.1Ecan mice and found that NOTCH2 enhanced the effect of TNFα on Il6 and Il1b expression. Similar results were obtained in cells from a conditional model of NOTCH2 gain-of-function, Notch22.1Ecan mice, and following the expression of the NOTCH2 intracellular domain in vitro. Recombination signal-binding protein for immunoglobulin Kappa J region partners with the NOTCH2 intracellular domain to activate transcription; in the absence of Notch signaling it inhibits transcription, and Rbpj inactivation in chondrocytes resulted in Il6 induction. Although TNFα induced IL6 to a greater extent in the context of NOTCH2 activation, there was a concomitant inhibition of Notch target genes Hes1, Hey1, Hey2, and Heyl. Electrophoretic mobility shift assay demonstrated displacement of recombination signal-binding protein for immunoglobulin Kappa J region from DNA binding sites by TNFα explaining the increased Il6 expression and the concomitant decrease in Notch target genes. NOTCH2 enhanced the effect of TNFα on NF-κB signaling, and RNA-Seq revealed increased expression of pathways associated with inflammation and the phagosome in NOTCH2 overexpressing cells in the absence and presence of TNFα. Collectively, NOTCH2 has important interactions with TNFα resulting in the enhanced expression of Il6 and inflammatory pathways in chondrocytes.

Hes1 marks peri-condensation mesenchymal cells that generate both chondrocytes and perichondrial cells in early bone development.

Bone development starts with condensations of undifferentiated mesenchymal cells that set a framework for future bones within the primordium. In the endochondral pathway, mesenchymal cells inside the condensation differentiate into chondrocytes and perichondrial cells in a SOX9-dependent mechanism. However, the identity of mesenchymal cells outside the condensation and how they participate in developing bones remain undefined. Here we show that mesenchymal cells surrounding the condensation contribute to both cartilage and perichondrium, robustly generating chondrocytes, osteoblasts, and marrow stromal cells in developing bones. Single-cell RNA-seq analysis of Prrx1-cre-marked limb bud mesenchymal cells at E11.5 reveals that Notch effector Hes1 is expressed in a mutually exclusive manner with Sox9 that is expressed in pre-cartilaginous condensations. Analysis of a Notch signaling reporter CBF1:H2B-Venus reveals that peri-condensation mesenchymal cells are active for Notch signaling. In vivo lineage-tracing analysis using Hes1-creER identifies that Hes1+ early mesenchymal cells surrounding the SOX9+ condensation at E10.5 contribute to both cartilage and perichondrium at E13.5, subsequently becoming growth plate chondrocytes, osteoblasts of trabecular and cortical bones, and marrow stromal cells in postnatal bones. In contrast, Hes1+ cells in the perichondrium at E12.5 or E14.5 do not generate chondrocytes within cartilage, contributing to osteoblasts and marrow stromal cells only through the perichondrial route. Therefore, Hes1+ peri-condensation mesenchymal cells give rise to cells of the skeletal lineage through cartilage-dependent and independent pathways, supporting the theory that early mesenchymal cells outside the condensation also play important roles in early bone development.

Analysis of N-glycosylation protein of Kashin-Beck disease chondrocytes derived from induced pluripotent stem cells based on label-free strategies with LC-MS/MS.

We aimed to compare N-glycosylation proteins in Kashin-Beck disease (KBD) chondrocytes and normal chondrocytes derived from induced pluripotent stem cells (iPSCs). KBD and normal iPSCs were reprogrammed from human KBD and normal dermal fibroblasts, respectively. Subsequently, chondrocytes were differentiated from KBD and normal iPSCs separately. Immunofluorescence was utilized to assay the protein markers of iPSCs and chondrocytes. Differential N-glycosylation proteins were screened using label-free strategies with LC-MS/MS. Bioinformatics analyses were utilized to interpret the functions of differential N-glycosylation proteins. Immunofluorescence staining revealed that both KBD-iPSCs and normal-iPSCs strongly expressed pluripotency markers OCT4 and NANOG. Meanwhile, chondrocyte markers collagen II and SOX9 are presented in KBD-iPSC-chondrocytes and normal-iPSC-chondrocytes. We obtained 87 differential N-glycosylation sites which corresponded to 68 differential proteins, which were constructed into 1 cluster. We obtained collagen type I trimer and 9 other biological processes; polysaccharide binding and 9 other molecular functions; regulation of transcription by RNA polymerase II and 9 other cellular components from GO; the Pl3K-Akt signaling pathway and 9 other KEGG pathways; peroxisome and 7 other subcellular locations; and integrin alpha chain, C-terminal cytoplasmic region, conserved site and 9 other classifications of domain annotations, and 2 networks. FGFR3 and LRP1 are expressed at higher levels in KBD-iPSC-chondrocytes, while the expressions of COL2A1, TIMP1, UNC5B, NOG, LEPR, and ITGA1 were down-regulated in KBD-iPSC-chondrocytes. The differential expressions of these N-glycosylation proteins may lead to the abnormal function of KBD chondrocytes.

Enhancement of mesenchymal stem cells' chondrogenic potential by type II collagen-based bioscaffolds.

BACKGROUND: Osteoarthritis (OA) is a common degenerative chronic disease accounting for physical pain, tissue stiffness and mobility restriction. Current therapeutic approaches fail to prevent the progression of the disease considering the limited knowledge on OA pathobiology. During OA progression, the extracellular matrix (ECM) of the cartilage is aberrantly remodeled by chondrocytes. Chondrocytes, being the main cell population of the cartilage, participate in cartilage regeneration process. To this end, modern tissue engineering strategies involve the recruitment of mesenchymal stem cells (MSCs) due to their regenerative capacity as to promote chondrocyte self-regeneration. METHODS AND RESULTS: In the present study, we evaluated the role of type II collagen, as the main matrix macromolecule in the cartilage matrix, to promote chondrogenic differentiation in two MSC in vitro culture systems. The chondrogenic differentiation of human Wharton's jelly- and dental pulp-derived MSCs was investigated over a 24-day culture period on type II collagen coating to improve the binding affinity of MSCs. Functional assays, demonstrated that type II collagen promoted chondrogenic differentiation in both MSCs tested, which was confirmed through gene and protein analysis of major chondrogenic markers. CONCLUSIONS: Our data support that type II collagen contributes as a natural bioscaffold enhancing chondrogenesis in both MSC models, thus enhancing the commitment of MSC-based therapeutic approaches in regenerative medicine to target OA and bring therapy closer to the clinical use.

The Effect of CaV1.2 Inhibitor Nifedipine on Chondrogenic Differentiation of Human Bone Marrow or Menstrual Blood-Derived Mesenchymal Stem Cells and Chondrocytes.

Cartilage is an avascular tissue and sensitive to mechanical trauma and/or age-related degenerative processes leading to the development of osteoarthritis (OA). Therefore, it is important to investigate the mesenchymal cell-based chondrogenic regenerating mechanisms and possible their regulation. The aim of this study was to investigate the role of intracellular calcium (iCa2+) and its regulation through voltage-operated calcium channels (VOCC) on chondrogenic differentiation of mesenchymal stem/stromal cells derived from human bone marrow (BMMSCs) and menstrual blood (MenSCs) in comparison to OA chondrocytes. The level of iCa2+ was highest in chondrocytes, whereas iCa2+ store capacity was biggest in MenSCs and they proliferated better as compared to other cells. The level of CaV1.2 channels was also highest in OA chondrocytes than in other cells. CaV1.2 antagonist nifedipine slightly suppressed iCa2+, Cav1.2 and the proliferation of all cells and affected iCa2+ stores, particularly in BMMSCs. The expression of the CaV1.2 gene during 21 days of chondrogenic differentiation was highest in MenSCs, showing the weakest chondrogenic differentiation, which was stimulated by the nifedipine. The best chondrogenic differentiation potential showed BMMSCs (SOX9 and COL2A1 expression); however, purposeful iCa2+ and VOCC regulation by blockers can stimulate a chondrogenic response at least in MenSCs.

NFATc1 marks articular cartilage progenitors and negatively determines articular chondrocyte differentiation.

The origin and differentiation mechanism of articular chondrocytes remain poorly understood. Broadly, the difference in developmental mechanisms of articular and growth-plate cartilage is still less elucidated. Here, we identified that the nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) is a crucial regulator of articular, but not growth-plate, chondrocyte differentiation during development. At the early stage of mouse knee development (embryonic day 13.5), NFATc1-expressing cells were mainly located in the flanking region of the joint interzone. With development, NFATc1-expressing cells generated almost all articular chondrocytes but not chondrocytes in limb growth-plate primordium. NFATc1-expressing cells displayed prominent capacities for colony formation and multipotent differentiation. Transcriptome analyses revealed a set of characteristic genes in NFATc1-enriched articular cartilage progenitors. Strikingly, the expression of NFATc1 was diminished with articular chondrocyte differentiation, and suppressing NFATc1 expression in articular cartilage progenitors was sufficient to induce spontaneous chondrogenesis while overexpressing NFATc1 suppresses chondrogenesis. Mechanistically, NFATc1 negatively regulated the transcriptional activity of the Col2a1 gene. Thus, our results reveal that NFATc1 characterizes articular, but not growth-plate, cartilage progenitors during development and negatively determines articular chondrocyte differentiation at least partly through regulating COL2A1 gene transcription.

Within the body are about 300 joints connecting bones together. Many factors ­ including trauma, inflammation, aging, and genetic changes ­ can affect the cushion tissue covering the end of the bones in these joints known as articular cartilage. This can lead to diseases such as osteoarthritis which cause chronic pain, and in some cases disability. To treat such conditions, it is essential to know how cells in the articular cartilage are formed during development. In the embryo, most cells come from groups of progenitor cells that are programmed to produce specific types of tissue. But which progenitor cells are responsible for producing the main cells in articular cartilage, chondrocytes, and the mechanisms that govern this transformation are poorly understood. In 2016, a group of researchers found that the gene for the protein NFATc1, which is important for building bone, is also expressed in a group of progenitor cells at the site where ligaments insert into bone in mice. Inactivation of NFATc1 in these progenitor cells has also been shown to cause abnormal cartilage to form, a condition termed osteochondromas. Building on this work, Zhang, Wang et al. ­ including some of the researchers involved in the 2016 study ­ set out to find whether NFATc1 is also involved in the normal development of articular chondrocytes. To investigate, the team used genetically modified mice in which any cells with NFATc1 also had a green fluorescent protein, and tracked these cells and their progeny over the course of joint development. This led them to discover a group of NFATc1-containing progenitor cells that gave rise to almost all articular chondrocytes in the knee joint. Further experiments revealed that when NFATc1 was removed, this made the progenitors become articular chondrocytes very quickly. In contrast, when the cells had excess amounts of the protein, the formation of articular chondrocytes was significantly reduced. This suggests that the level of NFATc1 governs when progenitors develop into articular chondrocytes. These findings have provided a way to track the progenitors of articular chondrocytes throughout development and study how articular cartilage is formed. In the future, this work could help researchers develop treatment strategies for osteoarthritis and other cartilage-based diseases. However, before this can happen, further work is needed to confirm that the effects observed in this study also relate to humans.

The Expression of αvß3 and Osteopontin in Osteoarthritic Knee Cartilage and Their Correlations With Disease Severity and Chondrocyte Senescence.

Osteoarthritis (OA) is the main joint disease associated with aging. Previous studies have confirmed that both osteopontin (OPN) and αvß3 integrin are involved in the progression of knee OA. The purpose of this study was to determine the expression of OPN and αvß3 integrin and chondrocyte senescence levels in OA. Forty-six cartilage tissues from normal and knee OA patients were divided into 4 groups of normal, minor, moderate, and severe lesions based on the Mankin score. Immunohistochemistry and western blotting were used to determine the expression of αvß3, OPN, and senescent-associated-ß-galactosidase (SAß-gal) in articular cartilage. Then, Spearman's correlation was used to analyze the correlations between the Mankin scores and αvß3, OPN and SAß-gal. Pearson correlation analysis was used to analyze the correlations among αvß3, OPN, and SAß-gal. The expression of OPN, αvß3, and SAß-gal in articular cartilage was explored. αvß3, OPN, and SAß-gal proteins were all elevated in OA cartilage, and the correlation coefficient between the Mankin score and the average optical density value of αvß3, OPN, SAß-gal were r =0.60, r =0.75, and r =0.87, respectively, all P <0.001; the correlation between the average optical density value of αvß3 and OPN was r =0.3191, P <0.05; the correlation between αvß3 and SAß-gal was r =0.4955, P <0.001; and the correlation between OPN and SAß-gal was r =0.7821, P <0.001. The correlations among αvß3, OPN, and SAß-gal expression in articular cartilage might be important in OA progression and pathogenesis. Nonetheless, more research is needed to elucidate the exact contribution of αvß3, OPN, and SAß-gal to the degenerative process of OA.

Epstein-Barr virus-induced gene 3 commits human mesenchymal stem cells to differentiate into chondrocytes via endoplasmic reticulum stress sensor.

Mesenchymal stem cells (MSC) can differentiate into chondrocytes. Epstein-Barr virus-induced gene 3 (EBI3) is differentially expressed during chondrogenic differentiation and can be produced by MSC. EBI3 is also a subunit of interleukin (IL)-27 and IL-35, and it accumulates in the endoplasmic reticulum (ER) when its partners, such as IL-27 p28 and IL-35 p35, are insufficient. ER stress induced by protein accumulation is responsible for chondrogenic differentiation. However, the role of EBI3 and its relevance to the ER stress in chondrogenic differentiation of MSC have never been addressed. Here, we demonstrate that EBI3 protein is expressed in the early stage of chondrogenic differentiation of MSC. Additionally, knockdown, overexpression, or induction of EBI3 through IL-1ß inhibits chondrogenesis. We show that EBI3 localizes and accumulates in the ER of MSC after overexpression or induction by IL-1ß and TNF-α, whereas ER stress inhibitor 4-phenylbutyric acid decreases its accumulation in MSC. Moreover, EBI3 modulates ER stress sensor inositol-requiring enzyme 1 α (IRE1α) after induced by IL-1ß, and MSC-like cells coexpress EBI3 and IRE1α in rheumatoid arthritis (RA) synovial tissue. Altogether, these data demonstrate that intracellular EBI3 commits to chondrogenic differentiation by regulating ER stress sensor IRE1α.

Acid-sensitive ion channel 1a mediates osteoarthritis chondrocyte senescence by promoting Lamin B1 degradation.

Osteoarthritis (OA) is a common and debilitating chronic joint disease, which is characterized by degeneration of articular cartilage and the aging of chondrocytes. Acid-sensitive ion channel 1a (ASIC1a) is a proton-activated cationic channel abundant in chondrocytes, which senses and regulates joint cavity pH. Our previous study demonstrated that ASIC1a was involved in acid-induced rat articular chondrocyte senescence, but the mechanistic basis remained unclear. In this study, we explored the mechanism of ASIC1a in chondrocyte senescence and OA. The results showed that senescence-related-ß-galactosidase, senescence-related markers (p53 and p21) and the autophagy-related protein Beclin-1 were found to be increased, but Lamin B1 was found to be reduced with acid (pH 6.0) treatment. These effects were inhibited by ASIC1a-specific blocker psalmotoxin-1 or ASIC1a-short hairpin RNA respectively in chondrocytes. Moreover, Silencing of Lamin B1 enhanced ASIC1a-mediated chondrocyte senescence, this effect was reversed by overexpression of Lamin B1, indicating that Lamin B1 was involved in ASIC1a-mediated chondrocyte senescence. Further, blockade of ASIC1a inhibits acid-induced autophagosomes and Beclin-1 protein expression, suggesting that ASIC1a is involved in acid-induced chondrocyte autophagy. Blocking autophagy with chloroquine inhibited Beclin-1 and increased Lamin B1 in acid-induced chondrocyte senescence. We further demonstrated that ASIC1a-mediated reduction of Lamin B1 expression was caused by autophagy pathway-dependent protein degradation. Finally, blocking ASIC1a protected cartilage tissue, restored Lamin B1 levels and inhibited chondrocyte senescence in a rat OA model. In summary, these findings suggest that ASIC1a may promote Lamin B1 degradation to mediate osteoarthritis chondrocyte senescence through the autophagy pathway.

GDF5+ chondroprogenitors derived from human pluripotent stem cells preferentially form permanent chondrocytes.

It has been established in the mouse model that during embryogenesis joint cartilage is generated from a specialized progenitor cell type, distinct from that responsible for the formation of growth plate cartilage. We recently found that mesodermal progeny of human pluripotent stem cells gave rise to two types of chondrogenic mesenchymal cells in culture: SOX9+ and GDF5+ cells. The fast-growing SOX9+ cells formed in vitro cartilage that expressed chondrocyte hypertrophy markers and readily underwent mineralization after ectopic transplantation. In contrast, the slowly growing GDF5+ cells derived from SOX9+ cells formed cartilage that tended to express low to undetectable levels of chondrocyte hypertrophy markers, but expressed PRG4, a marker of embryonic articular chondrocytes. The GDF5+-derived cartilage remained largely unmineralized in vivo. Interestingly, chondrocytes derived from the GDF5+ cells seemed to elicit these activities via non-cell-autonomous mechanisms. Genome-wide transcriptomic analyses suggested that GDF5+ cells might contain a teno/ligamento-genic potential, whereas SOX9+ cells resembled neural crest-like progeny-derived chondroprogenitors. Thus, human pluripotent stem cell-derived GDF5+ cells specified to generate permanent-like cartilage seem to emerge coincidentally with the commitment of the SOX9+ progeny to the tendon/ligament lineage.

The lncRNA MIAT/miR-181a-5p axis regulates osteopontin (OPN)-mediated proliferation and apoptosis of human chondrocytes in osteoarthritis.

Osteoarthritis (OA) is a slow-progressing degenerative joint disease mainly characterized by progressive cartilage loss and subchondral bone remodeling. Osteopontin (OPN) is a matrix extracellular glyco-phosphoprotein capable of regulating the expression levels of multiple factors linked with OA pathogenesis. This study explores the upstream regulatory molecular mechanism of OPN on proliferation and apoptosis of human chondrocytes in OA. Chondrocytes were isolated from OA cartilage and identified by toluidine blue staining and immunofluorescent staining of type II collagen. An MTT assay was used for cell viability, and a BrdU assay was applied for DNA synthesis. Cell apoptosis was detected by a flow cytometry assay. A lncRNA MIAT/miR-181a-5p/OPN axis regulating OA chondrocyte proliferation and apoptosis were identified. miR-181a-5p directly targeted OPN and inhibited OPN expression in OA chondrocytes. miR-181a-5p overexpression inhibited OA chondrocyte viability, suppressed DNA synthesis, and promoted apoptosis. OPN overexpression exerted opposite effects on OA chondrocytes and significantly attenuated the roles of miR-181a-5p overexpression in OA chondrocytes. A total of six long non-coding RNAs (lncRNAs) were predicted to target miR-181a-5p, and MIAT was the most up-regulated in OA cartilage tissues among the six lncRNAs. Through direct targeting, MIAT inhibited miR-181a-5p expression. MIAT silencing inhibited cell viability, suppressed DNA synthesis, and promoted cell apoptosis. Moreover, miR-181a-5p inhibition partially reversed the effects of MIAT silencing on OA chondrocytes. The lncRNA MIAT/miR-181a-5p/OPN axis could modulate OA chondrocyte proliferation and apoptosis. The comprehensive function of this axis on OA requires further in vivo and clinical investigations.

Pin1-mediated regulation of articular cartilage stem/progenitor cell aging.

Cartilage stem/progenitor cells (CSPCs) was recently isolated and identified from the cartilage tissue. CSPCs is essential for repair and regeneration of cartilage in osteoarthritis (OA). Aging is a primary risk factor for cartilage damage and joint OA. Although studies have confirmed the link between cell aging and OA, the underlying molecular mechanisms regulating CSPCs aging are not fully understood. In this study, we investigated the role of Pin1 in the aging of rat knee joint CSPCs. We isolated CSPCs from rat knee joints and demonstrated that, in long-term in vitro culture, Pin1 protein levels are significantly reduced. At the same time, expression of the senescence-related ß-galactosidase and the senescence marker p16INK4A were markedly elevated. In addition, Pin1 overexpression reversed the progression of cellular senescence, as evidenced by the down-regulation of senescence-related ß-galactosidase, increased EdU positive cells and diminished levels of p16INK4A. In contrast, Pin1 siRNA incorporation promoted CSPCs senescence. In addition, we also observed the distribution of cell cycles through flow cytometry and revealed that Pin1 deficiency results in cell cycle arrest in the G1 phase, suggesting severe lack of proliferation ability, a sign of cellular senescence. Collectively, these results validated that Pin1 is an essential regulator of CSPCs aging.

PRDM paralogs antagonistically balance Wnt/ß-catenin activity during craniofacial chondrocyte differentiation.

Cranial neural crest cell (NCC)-derived chondrocyte precursors undergo a dynamic differentiation and maturation process to establish a scaffold for subsequent bone formation, alterations in which contribute to congenital birth defects. Here, we demonstrate that transcription factor and histone methyltransferase proteins Prdm3 and Prdm16 control the differentiation switch of cranial NCCs to craniofacial cartilage. Loss of either paralog results in hypoplastic and disorganized chondrocytes due to impaired cellular orientation and polarity. We show that these proteins regulate cartilage differentiation by controlling the timing of Wnt/ß-catenin activity in strikingly different ways: Prdm3 represses whereas Prdm16 activates global gene expression, although both act by regulating Wnt enhanceosome activity and chromatin accessibility. Finally, we show that manipulating Wnt/ß-catenin signaling pharmacologically or generating prdm3-/-;prdm16-/- double mutants rescues craniofacial cartilage defects. Our findings reveal upstream regulatory roles for Prdm3 and Prdm16 in cranial NCCs to control Wnt/ß-catenin transcriptional activity during chondrocyte differentiation to ensure proper development of the craniofacial skeleton.

Four-Octyl Itaconate Protects Chondrocytes against H2O2-Induced Oxidative Injury and Attenuates Osteoarthritis Progression by Activating Nrf2 Signaling.

Nrf2 is a critical regulator of the antioxidant defense systems in cellular protection. Emerging evidence has shown that four-octyl itaconate (OI) activates Nrf2 cascade. In this study, the chondroprotective effects of OI on H2O2-stimulated chondrocytes and DMM-induced osteoarthritis (OA) progression were investigated. In primary murine chondrocytes, OI interrupted the binding of Keap1 and Nrf2, leading to accumulation and nuclear translocation of Nrf2 protein, as well as transcription and expression of Nrf2-dependent genes, such as HO-1, NQO1, and GCLC. Furthermore, OI inhibited cell death and apoptosis, as well as H2O2-stimulated ROS generation, lipid peroxidation, superoxide accumulation, and mitochondrial depolarization in chondrocytes, which were abolished by the silence or depletion of Nrf2. In addition, in vivo experiments revealed the therapeutic effects of OI on OA progression in a DMM mouse model. Collectively, these results suggested that OI might serve as a potential treatment for OA progression.

Silence of TANK-binding kinase 1 (TBK1) regulates extracellular matrix degradation of chondrocyte in osteoarthritis by janus kinase (JAK)-signal transducer of activators of transcription (STAT) signaling.

TANK-binding kinase 1 (TBK1) was previously reported to be critical for the regulation of osteoclast differentiation. However, its function in osteoarthritis (OA) has not yet been determined. This study aims to reveal the role of TBK1 in the extracellular matrix (ECM) degradation in OA. C57BL/6 J mice were subjected to anterior cruciate ligament transection (ACLT) surgery to establish an OA animal model. ATDC5 cells were treated with IL-1ß to construct a cell model of OA. Changes in the expression of TBK1 were analyzed by qRT-PCR, Western blotting, and immunohistochemistry. Safranin O-fast green staining, ELISA, and Western blotting were performed to evaluate the ECM degradation. By searching GSE75181 and GSE6119 datasets, TBK1 was found to be highly expressed in the OA model. Its upregulation was also confirmed in ACLT mice and in a cell model of OA. Silencing of TBK1 reduced cartilage degradation, OARSI score, and serum levels of CTX-II and COMP. Silencing of TBK1 attenuated ECM degradation, as ADAMTS-4, MMP3, and MMP13 were downregulated, whilst SOX9, collagen II, and aggrecan were upregulated. Furthermore, TBK1 activates the JAK/STAT signaling pathway. Transfection of cells with the STAT3 overexpression plasmid blocked the beneficial effects of TBK1 silencing. In conclusion, TBK1 is highly expressed in OA. Silencing of TBK1 inhibited ECM degradation.

Identification of thrombin as a key regulator of chondrocyte catabolic activity through RNA-Seq and experimental verification.

The present study was designed to explore the relationship between thrombin and catabolic activity in chondrocytes. Primary rat chondrocytes were cultured for 24 h with rat serum (RS), rat plasma (RP), or rat plasma supplemented with thrombin (RPT). RNA-sequencing was then performed. Cell proliferation was analyzed by EdU uptake, CCK-8 assays and protein-protein interaction (PPI) network of proliferation-related genes. Heatmaps were used to visualize differences in gene expression. Gene Ontology (GO) enrichment analyses of up- and down-regulated differentially expressed genes were conducted. Molecular probes were used to label the endoplasmic reticulum in chondrocytes from three treatment groups. Immunofluorescence and Safranin O staining were used to assess type II collagen (Col2a1) expression and proteoglycan synthesis, whereas Lox expression was assessed by immunocytochemistry. The expression of enzymes involved in the synthesis and maturation of extracellular matrix (ECM) components and chemokines were measured by qPCR while matrix metalloproteinases (MMPs) levels were evaluated by Western blotting. Relevant nodules were selected through further PPI network analyses. A total of 727 and 1162 genes were up- and down-regulated based on the Venn diagrams comparison among groups. Thrombin was thus able to promote chondrocyte proliferation and a shift towards fibrotic morphology, while upregulating MMPs and chemokines linked to ECM degradation. In addition, thrombin decreased the enzyme expression involved in the synthesis and maturation of ECM.

The functionality and translatability of neocartilage constructs are improved with the combination of fluid-induced shear stress and bioactive factors.

Neocartilage tissue engineering aims to address the shortcomings of current clinical treatments for articular cartilage indications. However, advancement is required toward neocartilage functionality (mechanical and biochemical properties) and translatability (construct size, gross morphology, passage number, cell source, and cell type). Using fluid-induced shear (FIS) stress, a potent mechanical stimulus, over four phases, this work investigates FIS stress' efficacy toward creating large neocartilage derived from highly passaged minipig costal chondrocytes, a species relevant to the preclinical regulatory process. In Phase I, FIS stress application timing was investigated in bovine articular chondrocytes and found to improve the aggregate modulus of neocartilage by 151% over unstimulated controls when stimulated during the maturation stage. In Phase II, FIS stress stimulation was translated from bovine articular chondrocytes to expanded minipig costal chondrocytes, yielding a 46% improvement in aggregate modulus over nonstimulated controls. In Phase III, bioactive factors were combined with FIS stress to improve the shear modulus by 115% over bioactive factor-only controls. The translatability of neocartilage was improved in Phase IV by utilizing highly passaged cells to form constructs more than 9-times larger in the area (11 × 17 mm), yielding an improved aggregate modulus by 134% and a flat morphology compared to free-floating, bioactive factor-only controls. Overall, this study represents a significant step toward generating mechanically robust, large constructs necessary for animal studies, and eventually, human clinical studies.

Effect of passage number of genetically modified TGF-ß3 expressing primary chondrocytes on the chondrogenesis of ATDC5 cells in a 3D coculture system.

Due to the lack of blood vessels, nerves and lymphatics, articular cartilage is difficult to repair once damaged. Tissue engineering is considered to be a potential strategy for cartilage regeneration. Successful tissue engineering strategies depend on the effective combination of biomaterials, seed cells and biological factors. In our previous study, a genetically modified coculture system with chondrocytes and ATDC5 cells in an alginate hydrogel has exhibited a superior ability to enhance chondrogenesis. In this study, we further evaluated the influence of chondrocytes at various passages on chondrogenesis in the coculture system. The results demonstrated that transfection efficiency was hardly influenced by the passage number of chondrocytes. The coculture system with passage 5 (P5) chondrocytes had a better effect on chondrogenesis of ATDC5 cells, while chondrocytes in this coculture system presented higher levels of dedifferentiation than other groups with P1 or P3 chondrocytes. Therefore, P5 chondrocytes were shown to be more suitable for the coculture system, as they accumulated in sufficient cell numbers with more passages and had a higher level of dedifferentiation, which was prone to form a favorable niche for chondrogenesis of ATDC5 cells. This study may provide fresh insights for future cartilage tissue engineering strategies with a combination of a coculture system and advanced biomaterials.