Search

• FN is expressed early during mouse limb development at distinct regions within the growth plate and bone. • Cartilage-specific cFN and circulating pFN are distributed distinctly during embryonic and postnatal bone development. • Deletion of cFN and pFN leads to reduced bone growth and bone formation during early and adult postnatal development. • Absence of both FN isoforms in FN double knockout mice leads to altered chondrogenesis. • Ablation of both FN isoforms in developing long bones is associated with reduced TGFβ1 and phospho-AKT. Fibronectin (FN) is a ubiquitous extracellular matrix glycoprotein essential for the development of various tissues. Mutations in FN cause a unique form of spondylometaphyseal dysplasia, emphasizing its importance in cartilage and bone development. However, the relevance and functional role of FN during skeletal development has remained elusive. To address these aspects, we have generated conditional knockout mouse models targeting the cellular FN isoform in cartilage (cFNKO), the plasma FN isoform in hepatocytes (pFNKO), and both isoforms together in a double knockout (FNdKO). We used these mice to determine the relevance of the two principal FN isoforms in skeletal development from postnatal day one to the adult stage at two months. We identified a distinct topological FN deposition pattern in the mouse limb during different gestational and postnatal skeletal development phases, with prominent levels at the resting and hypertrophic chondrocyte zones and in the trabecular bone. Cartilage-specific cFN emerged as the predominant isoform in the growth plate, whereas circulating pFN remained excluded from the growth plate and confined to the primary and secondary ossification centers. Deleting either isoform independently (cFNKO or pFNKO) yielded only relatively subtle changes in the analyzed skeletal parameters. However, the double knockout of cFN in the growth plate and pFN in the circulation of the FNdKO mice significantly reduced postnatal body weight, body length, and bone length. Micro-CT analysis of the adult bone microarchitecture in FNdKO mice exposed substantial reductions in trabecular bone parameters and bone mineral density. The mice also showed elevated bone marrow adiposity. Analysis of chondrogenesis in FNdKO mice demonstrated changes in the resting, proliferating and hypertrophic growth plate zones, consistent alterations in chondrogenic markers such as collagen type II and X, decreased apoptosis of hypertrophic chondrocytes, and downregulation of bone formation markers. Transforming growth factor-β1 and downstream phospho-AKT levels were significantly lower in the FNdKO than in the control mice, revealing a crucial FN-mediated regulatory pathway in chondrogenesis and bone formation. In conclusion, the data demonstrate that FN is essential for chondrogenesis and bone development. Even though cFN and pFN act in different regions of the bone, both FN isoforms are required for the regulation of chondrogenesis, cartilage maturation, trabecular bone formation, and overall skeletal growth. [ABSTRACT FROM AUTHOR]

Osteoarthritis is a common and complex joint disorder that shows higher prevalence and greater disease severity in women. Here, we investigate genome-wide methylation profiles of primary chondrocytes from osteoarthritis patients. We compare genome-wide methylation profiles of macroscopically intact (low-grade) and degraded (high-grade) osteoarthritis cartilage samples matched from osteoarthritis patients undergoing knee replacement surgery. We perform an epigenome-wide association study for cartilage degeneration across 170 patients and separately in 96 women and 74 men. We reveal widespread epigenetic differences with enrichments of nervous system and apoptosis-related processes. We further identify substantial similarities between sexes, but also sex-specific markers and pathways. Together, we provide the largest genome-wide methylation profiles of primary cartilage to date with enhanced and sex-specific insights into epigenetic processes underlying osteoarthritis progression. [ABSTRACT FROM AUTHOR]

C-type natriuretic peptide (CNP), a member of the natriuretic peptide family, is a potent endochondral bone growth factor that exerts its biological effects via the guanylate cyclase B (GC-B) receptor. We previously demonstrated that CNP knockout (KO) mice exhibited midfacial hypoplasia along the sagittal plane; however, the effects of GC-B (the receptor for CNP) on endochondral ossification in the maxillofacial region remain unclear, and the mechanism of the CNP/GC-B system has not been elucidated. We investigated the physiological significance of GC-B in the cartilage of the craniofacial region through analysis of cartilage-specific GC-B KO mice. Morphological assessments were performed at 12 weeks old, with histological analyses performed at 2 weeks old. GC-B-KO mice exhibited sagittal midfacial hypoplasia, foramen magnum stenosis, and spinal canal stenosis. Histological examination revealed reduced thickness in the spheno-occipital synchondrosis (SOS), a critical growth center in cranio-maxillofacial skeletal development. The hypertrophic zone of the SOS exhibited reduced thickness, accompanied by a reduction in cell count in this area. This study highlights the essential role of GC-B receptors in craniofacial morphology contributing to our understanding of the mechanisms underlying facial morphological abnormalities, foramen magnum stenosis, and spinal canal stenosis. [ABSTRACT FROM AUTHOR]

AMP-activated protein kinase (AMPK) dysregulation is implicated in osteoarthritis (OA), but the mechanisms underlying this dysregulation remain unclear. We investigated the role of cereblon, a substrate-recognition protein within the E3-ligase ubiquitin complex, in AMPK dysregulation and OA pathogenesis. Cereblon expression was examined in human (n = 5) and mouse (n = 10) OA cartilage. The role of cereblon was investigated through its adenoviral overexpression (n = 10) or knockout (KO, n = 15) in the destabilization of the medial meniscus (DMM)-operated mice. The therapeutic potentials of the chemical cereblon degrader, TD-165, and the AMPK activator, metformin, were assessed through intra-articular (IA) injection to mice (n = 15). Immunostaining revealed that cereblon is upregulated in human and mouse OA cartilage. In DMM model mice, cartilage destruction was exacerbated by overexpression of cereblon in mouse joint tissues (OARSI grade; 1.11 [95% CI: 0.50 to 2.75]), but inhibited in global (−2.50 [95% CI: −3.00 to −1.17]) and chondrocyte-specific (−2.17 [95% CI: −3.14 to −1.06]) cereblon KO mice. The inhibitory effects were more pronounced in mice fed a high-fat diet compared to a regular diet. The degradation of cereblon through IA injection of TD-165 inhibited OA cartilage destruction (−2.47 [95% CI: −3.22 to −1.56]). Mechanistically, cereblon exerts its catabolic effects by negatively modulating AMPK activity within chondrocytes. Consistently, activation of AMPK by IA injection of metformin inhibited posttraumatic OA cartilage destruction (−1.20 ([95% CI: −1.89 to −0.45]). The cereblon-AMPK axis acts as a catabolic regulator of OA pathogenesis and seems to be a promising therapeutic target in animal models of OA. [ABSTRACT FROM AUTHOR]

During physical activities, chondrocytes experience coupled stimulation of hydrostatic pressure (HP) and a transient increase in temperature (T), with the latter varying within a physiological range from 32.5 °C to 38.7 °C. Previous short-term in vitro studies have demonstrated that the combined hydrostatic pressure-thermal (HP-T) stimuli more significantly enhance chondroinduction and chondroprotection of chondrocytes than isolated applications. Interestingly, this combined benefit is associated with a corresponding increase in HSP70 levels when HP and T are combined. The current study therefore explored the indispensable role of HSP70 in mediating the combined effects of HP-T stimuli on chondrocytes. In this mid-long-term study of in vitro engineered cartilage constructs, we assessed chondrocyte responses to HP-T stimuli using customized bioreactor in standard and HSP70-inhibited cultures. Surprisingly, under HSP70-inhibited conditions, the usually beneficial HP-T stimuli, especially its thermal component, exerted detrimental effects on chondrocyte homeostasis, showing a distinct and unfavorable shift in gene and protein expression patterns compared to non-HSP70-inhibited settings. Such effects were corroborated through mechanical testing and confirmed using a secondary cell source. A proteomic-based mechanistic analysis revealed a disruption in the balance between biosynthesis and fundamental cellular structural components in HSP70-inhibited conditions under HP-T stimuli. Our results highlight the critical role of sufficient HSP70 induction in mediating the beneficial effects of coupled HP-T stimulation on chondrocytes. These findings help pave the way for new therapeutic approaches to enhance physiotherapy outcomes and potentially shed light on the elusive mechanisms underlying the onset of cartilage degeneration, a long-standing enigma in orthopedics. [ABSTRACT FROM AUTHOR]

Chondrocytes, which typically rely on anaerobic metabolism, exhibit upregulated biosynthetic activity when subjected to conditions that elicit mixed aerobic-anaerobic metabolism. Previously, we observed that increasing media volume resulted in the transition from anaerobic to mixed aerobic-anaerobic metabolism. Maximal extracellular matrix (ECM) accumulation occurred at this transition as a result of changes in hypoxia-inducible factor 1α signaling and associated hypoxic gene expression. This study aimed to explore the effect of further increases in media availability on ECM synthesis and chondrocyte metabolism. Primary bovine chondrocytes were grown in 3D high-density tissue culture under varying levels of media availability (4–16 mL/106 cells). Changes in ECM accumulation and metabolism were determined through biochemical assays and 13C-metabolic flux analysis (13C-MFA). Increasing media volumes resulted in higher accumulation of cartilaginous ECM (collagen and proteoglycans) and cellularity. Extracellular metabolite measurements revealed that elevated media availability led to increased glucose and glutamine metabolism, along with increased anaerobic activity. 13C-MFA utilizing [U-13C] glucose demonstrated that increased media availability significantly impacted central carbon metabolism, upregulating all glucose-related metabolic pathways (glycolysis, lactate fermentation, the tricarboxylic acid (TCA) cycle, hexosamine biosynthetic pathway, and the malate-aspartate shuttle). Furthermore, 13C-MFA indicated that glutamine was donating carbons to the TCA cycle, and additional studies involving [U-13C] glutamine tracing supported this notion. Elevated media availability upregulates ECM synthesis and leads to significant changes in metabolic phenotype. Glutamine plays an important role in chondrocyte metabolism and increases in glutamine metabolism correlate with increases in ECM accumulation. [ABSTRACT FROM AUTHOR]

Neutralization of Interleukin (IL)-6-signaling by antibodies is considered a promising tool for the treatment of osteoarthritis (OA). To gain further insight into this potential treatment, this study investigated the effects of IL-6-signaling and IL-6 neutralization on chondrocyte metabolism and the release of IL-6-signaling-related mediators by human chondrocytes. Chondrocytes were collected from 49 patients with advanced knee/hip OA or femoral neck fracture. Isolated chondrocytes were stimulated with different mediators to analyze the release of IL-6, soluble IL-6 receptor (sIL-6R) and soluble gp130 (sgp130). The effect of IL-6 and IL-6/sIL-6R complex as well as neutralization of IL-6-signaling on the metabolism was analyzed. OA chondrocytes showed high basal IL-6 production and release, which was strongly negatively correlated with the production of cartilage-matrix-proteins. Chondrocytes produced and released sIL-6R and sgp130. The IL-6/sIL-6R complex significantly increased nitric oxide, prostaglandin E 2 and matrix metalloproteinase 1 production, decreased Pro-Collagen Type II and mitochondrial ATP production, and increased glycolysis in OA chondrocytes. Neutralization of IL-6-signaling by antibodies did not significantly affect the metabolism of OA chondrocytes, but blocking of glycoprotein 130 (gp130)-signaling by SC144 significantly reduced the basal IL-6 release. Although IL-6 trans-signaling induced by IL-6/sIL-6R complex negatively affects OA chondrocytes, antibodies against IL-6 or IL-6R did not affect chondrocyte metabolism. Since inhibition of gp130-signaling reduced the enhanced basal release of IL-6, interfering with gp130-signaling may ameliorate OA progression because high cellular release of IL-6 correlates with reduced production of cartilage-matrix-proteins. [ABSTRACT FROM AUTHOR]

While chondrocytes have mitochondria, they receive little O 2 from the bloodstream. Sulfur respiration, an essential energy production system in mitochondria, uses supersulfides instead of O 2. Supersulfides are inorganic and organic sulfides with catenated sulfur atoms and are primarily produced by cysteinyl tRNA synthetase-2 (CARS2). Here, we investigated the role of supersulfides in chondrocyte proliferation and bone growth driven by growth plate chondrocyte proliferation. We examined the effects of NaHS, an HS−/H 2 S donor, and cystine, the cellular source of cysteine, on the proliferation of mouse primary chondrocytes and growth of embryonic mouse tibia in vitro. We also examined the effect of RNA interference acting on the Cars2 gene on chondrocyte proliferation in the presence of cystine. NaHS (30 μmol/L) enhanced tibia longitudinal growth in vitro with expansion of the proliferating zone of their growth plates. While NaHS (30 μmol/L) also promoted chondrocyte proliferation only under normoxic conditions (20 % O 2), cystine (0.5 mmol/L) promoted it under both normoxic and hypoxic (2 % O 2) conditions. Cars2 gene knockdown abrogated the ability of cystine (0.5 mmol/L) to promote chondrocyte proliferation under normoxic conditions, indicating that supersulfides produced by CARS2 were responsible for the cystine-dependent promotion of bone growth. The presented results indicate that supersulfides play a vital role in bone growth achieved by chondrocyte proliferation in the growth plates driven by sulfur respiration. [Display omitted] • NaHS, an HS−/H 2 S donor, promoted the growth of cultured bone under 20 % O 2. • NaHS enlarged the proliferating zone width of the growth plate under 20%O 2. • NaHS promoted chondrocyte proliferation under 20 % but not 2 % O 2. • Cystine promoted the proliferation of chondrocytes under both 20 % and 2 % O 2. • Cars2 knockdown canceled the enhancement of chondrocyte proliferation by cystine. [ABSTRACT FROM AUTHOR]

The purpose of this study was to investigate the effect of age and oxidative stress on regulation of nuclear factor erythroid-2-related factor 2 (Nrf2) in young, old, and osteoarthritic (OA) human articular chondrocytes. Levels of Nrf2 in primary human chondrocytes isolated from young, old, and OA donors were measured by immunoblotting, qPCR, and immunohistochemistry. Effects on levels of Nrf2, antioxidant proteins regulated by Nrf2, as well as p65, and the anabolic response to insulin-like growth factor-1 (IGF-1) were evaluated after induction of oxidative stress with menadione, Nrf2 knockdown with siRNA, and/or Nrf2 activation with RTA-408. Nrf2 protein levels were significantly lower in older adult chondrocytes (∼0.59 fold; p = 0.034) and OA chondrocytes (∼0.50 fold; p = 0.016) compared to younger cells. Menadione significantly increased Nrf2 protein levels in young chondrocytes by just under four-fold without changes in old chondrocytes. Nrf2 knockdown and activation differentially regulated levels of anti-oxidant proteins including sulfiredoxin and NAD(P)H quinone dehydrogenase 1. Nrf2 activation with RTA-408 also decreased basal p65 phosphorylation, increased aggrecan and type II collagen gene expression, and increased production of proteoglycans in OA chondrocytes treated with IGF-1. Targeted therapeutic strategies aimed at maintaining Nrf2 activity could be useful in maintaining chondrocyte homeostasis through maintenance of intracellular antioxidant function and redox balance. [ABSTRACT FROM AUTHOR]

Yes-associated protein (YAP) has been widely studied as a mechanotransducer in many cell types, but its function in cartilage is controversial. The aim of this study was to identify the effect of YAP phosphorylation and nuclear translocation on the chondrocyte response to stimuli relevant to osteoarthritis (OA). Cultured normal human articular chondrocytes from 81 donors were treated with increased osmolarity media as an in vitro model of mechanical stimulation, fibronectin fragments (FN-f) or IL-1β as catabolic stimuli, and IGF-1 as an anabolic stimulus. YAP function was assessed with gene knockdown and inhibition by verteporfin. Nuclear translocation of YAP and its transcriptional co-activator TAZ and site-specific YAP phosphorylation were determined by immunoblotting. Immunohistochemistry and immunofluorescence to detect YAP were performed on normal and OA human cartilage with different degrees of damage. Chondrocyte YAP/TAZ nuclear translocation increased under physiological osmolarity (400 mOsm) and IGF-1 stimulation, which was associated with YAP phosphorylation at Ser128. In contrast, catabolic stimulation decreased the levels of nuclear YAP/TAZ through YAP phosphorylation at Ser127. Following YAP inhibition, anabolic gene expression and transcriptional activity decreased. Additionally, YAP knockdown reduced proteoglycan staining and levels of type II collagen. Total YAP immunostaining was greater in OA cartilage, but YAP was sequestered in the cytosol in cartilage areas with more severe damage. YAP chondrocyte nuclear translocation is regulated by differential phosphorylation in response to anabolic and catabolic stimuli. Decreased nuclear YAP in OA chondrocytes may contribute to reduced anabolic activity and promotion of further cartilage loss. [ABSTRACT FROM AUTHOR]

Osteoarthritis is a disease that impacts millions around the world, leading to significant financial and medical burden for patients and the healthcare system. However, no effective biomarkers or disease modifying therapeutics exist for the early identification and management of the disease. Inflammation drives chondrocytes to express extracellular matrix (ECM) degrading enzymes and interruption of this pathway is a viable target to prevent degradation of cartilage. It has been demonstrated that inflammation can alter the intracellular metabolism of chondrocytes, a process known as metabolic reprogramming. This metabolic reprogramming is critical for cartilage breakdown by shifting chondrocytes to an ECM-catabolic state and likely as a potential therapeutic target for osteoarthritis. Metabolic modulators hold the potential to reduce chondrocyte inflammatory responses and protect cartilage. In this narrative review, we explore some of the existing examples of interactions between metabolism and inflammatory pathways in chondrocytes. We summarize the impact of inflammatory stimulation on various metabolic pathways and describe several examples by which targeting of metabolism is able to modulate ECM-degrading activity of chondrocytes to protect against cartilage damage. [ABSTRACT FROM AUTHOR]

Defects in autophagy contribute to joint aging and Osteoarthritis (OA). Identifying specific autophagy types could be useful for developing novel treatments for OA. An autophagy-related gene array was performed in blood from non-OA and knee OA subjects from the Prospective Cohort of A Coruña (PROCOAC). The differential expression of candidate genes was confirmed in blood and knee cartilage and a regression analysis was performed adjusting for age and BMI. HSP90A, a chaperone mediated autophagy (CMA) marker was validated in human knee joint tissues, as well as, in mice with aging-related and surgically-induced OA. The consequences of HSP90AA1 deficiency were evaluated on OA pathogenesis. Finally, the contribution of CMA to homeostasis was studied by assessing the capacity to restore proteostasis upon ATG5 -mediated macroautophagy deficiency and genetic HSP90AA1 overexpression. 16 autophagy-related genes were significantly down-regulated in blood from knee OA subjects. Validation studies showed that HSP90AA1 was down-regulated in blood and human OA cartilage and correlated with risk incidence of OA. Moreover, HSP90A was reduced in human OA joints tissues and with aging and OA in mice. HSP90AA1 knockdown was linked to defective macroautophagy, inflammation, oxidative stress, senescence and apoptosis. However, macroautophagy deficiency increased CMA, highlighting the CMA-macroautophagy crosstalk. Remarkably, CMA activation was sufficient to protect chondrocytes from damage. We show that HSP90A is a key chaperone for chondrocyte homeostasis, while defective CMA contributes to joint damage. We propose that CMA deficiency is a relevant disease mechanism and could represent a therapeutic target for OA. [ABSTRACT FROM AUTHOR]

• Tenacissoside G inhibited the mice knee cartilage loss induced by DMM surgery in vivo. • Tenacissoside G inhibited IL-1β-induced mice chondrocyte inflammatory response in vitro. • Tenacissoside G inhibited Inflammatory reaction via NF-κB /P65 pathway. Osteoarthritis (OA) is a degenerative joint disease characterized by the destruction of articular cartilage. Tenacissoside G is a flavonoid isolated from the dry roots of Marsdenia tenacissima (Roxb) and has been shown to have anti-inflammatory effects. However, there is no report on the protective effects of Tenacissoside G on OA. To identify the effects and mechanism of Tenacissoside G on OA. In vitro, primary mouse chondrocytes were induced with IL-1β to establish OA model. mRNA expression of MMP-13, MMP-3, TNF-α, IL-6 and iNOS, was detected by PCR. Protein expression of Collagen-II, MMP-13, p65, p-p65, and IκBα was detected by Western blot. Collagen-II in chondrocytes was also detected by immunofluorescence. In vivo, we established DMM OA mice model. The preventive effect of Tenacissoside G on OA was observed by micro-CT and histological analysis. In vitro , Tenacissoside G significantly inhibited the expression of iNOS, TNF-α, IL-6, MMP-3, MMP-13 and the degradation of collagen-II, Tenacissoside G also significantly suppressed NF-κB activation in chondrocytes by IL-1β-stimulated. In vivo , we demonstrated Tenacissoside G can decrease articular cartilage damage and reduce OARSI score. These results suggest that Tenacissoside G may serve as a potential drug for the prevention and treatment of OA. [Display omitted] [ABSTRACT FROM AUTHOR]

To elucidate the mechanism underlying the inhibitory effect of acupotomy on chondrocyte apoptosis in rabbits with knee osteoarthritis (KOA) through screening, analysis, and verification of apoptosis-related differentially expressed proteins via proteomics. Thirty New Zealand white rabbits were randomly divided into the normal, model, and acupotomy groups, with 10 rabbits in each group. The Vidman method was used to prepare KOA models in the model and acupotomy groups. In the acupotomy group, acupotomy was performed at the midpoints of the patellar ligament and medial and lateral collateral ligaments on the left knees once a week for three weeks. Rabbit articular cartilage protein was quantified in each group, and the proteins with differential expression were profiled via proteomics. The apoptosis-related proteins were further screened, and differentially expressed proteins common among the normal, model, and acupotomy groups were collected for cluster analysis. Pathological changes in chondrocytes were observed via HE staining, and chondrocyte apoptosis was observed under an electron microscope. Quantitative real-time PCR (RT-PCR) and western blot were performed to determine the mRNA and protein expression levels, respectively, of the common apoptosis-related proteins in the three groups. Western blot and RT-PCR showed that the protein and mRNA expression levels of TXN, CAP1 and FABP3 were lower (all P < 0.05), whereas that of LTF, SCIN and IL-10 were higher (all P < 0.01) in the acupotomy group than that in the model group. The antiapoptotic effect of acupotomy on chondrocytes in KOA rabbits may be achieved by modulating the protein and mRNA expression of differentially expressed apoptosis-related proteins (TXN, CAP1, FABP3, LTS, SCIN, and IL-10). [ABSTRACT FROM AUTHOR]

• Increasing tool speeds while drilling into the osteochondral area of bone leads to an increase in temperature. • Increasing drill bit sizes while drilling into the osteochondral area of bone leads to an increase in temperature. • Drilling in bone can lead to an increase in temperature that can cause osteonecrosis and cell damage. Drilling is a common technique used in orthopedic surgery procedures but causes increases in temperature that can lead to cell damage and death. The extent of this depends largely on the magnitude of the increase in temperature. The commonly accepted limit to prevent osteonecrosis is less than 47 °C for 60 s. There is controversy when it comes to the optimal drilling parameters that limit temperature increases and cell death. In addition to this, less research has been done on the drilling effects in the osteochondral area of joints. Osteochondral tissue damage can interfere with the daily lives of patients and if severe enough will need to be treated. We hypothesize that increasing tool speed and drill bit size will increase temperature that could be above the osteonecrosis limit. Ex-vivo experiments were conducted on porcine shoulder joints that tested the thermal effects of different tool speeds and drill bit sizes. A thermal camera was used to record and measure real time temperature changes while drilling. Three drill bit sizes and five tool speeds were used. Statistical analyses includes Welch's ANOVA with Games-Howell Post Hoc analyses, multivariate linear regression, and surface response regression were used to explore the association of tool speeds and drill bit size on temperature. All the tool speed and drill bit size combinations lead to an increase in temperature that were under the commonly accepted limit. The highest temperature reached was 44 °C with a tool speed of 1150 RPM and 3070 RPM and drill bit size 5.159 mm. It was found that increasing the tool speed increased the temperature change and increasing the drill bit size increased the temperature change. [ABSTRACT FROM AUTHOR]

Osteoarthritis-related cartilage extracellular matrix remodeling is dependent on changes in chondrocyte protein expression. Yet, the role of ribosomes in chondrocyte translation regulation is unknown. In this exploratory study, we investigated ribosomal RNA (rRNA) epitranscriptomic-based ribosome heterogeneity in human articular chondrocytes and its relevance for osteoarthritis. Sequencing-based rRNA 2′-O-methylation profiling analysis (RiboMethSeq) was performed on non-OA primary human articular chondrocytes (n = 5) exposed for 14 days to osteoarthritic synovial fluid (14 donors, pooled, 20% v/v). The SW1353 SNORD71 KO cell pool was generated using LentiCRISPRv2/Cas9. The mode of translation initiation and fidelity were determined by dual-luciferase reporters. The cellular proteome was analyzed by LC–MS/MS and collagen type I protein expression was evaluated by immunoblotting. Loading of COL1A1 mRNA into polysomes was determined by sucrose gradient ultracentrifugation and fractionation. We discovered that osteoarthritic synovial fluid instigates site-specific changes in the rRNA 2′-O-me profile of primary human articular chondrocytes. We identified five sites with differential 2′-O-me levels. The 2′-O-me status of 5.8S-U14 (one of identified differential 2′-O-me sites; decreased by 7.7%, 95% CI [0.9–14.5%]) was targeted by depleting the level of its guide snoRNA SNORD71 (50% decrease, 95% CI [33–64%]). This resulted in an altered ribosome translation modus (e.g., CrPV IRES, FC 3, 95% CI [2.2–4.1]) and promoted translation of COL1A1 mRNA which led to increased levels of COL1A1 protein (FC 1.7, 95% CI [1.3–2.0]). Our data identify a novel concept suggesting that articular chondrocytes employ rRNA epitranscriptomic mechanisms in osteoarthritis development. [ABSTRACT FROM AUTHOR]

The incidence of osteoarthritis (OA) in menopausal women is significantly higher than in same-aged men. Investigating the role of subchondral osteoblasts in estrogen deficiency-induced OA may help elucidate the pathological mechanism, providing new insights for the diagnosis and treatment of menopausal OA. A classical ovariectomy-induced OA (OVX-OA) rat model was utilized to isolate primary articular chondrocytes and subchondral osteoblasts, which were identified and then cocultured in Transwell. The expression of chondrocyte anabolic and catabolic indicators was evaluated. The differentially expressed proteins in the conditioned medium (CM) of osteoblasts were identified by Liquid Chromatograph-Mass Spectrometer (LC–MS/MS). Normal chondrocytes were treated with osteoblast CM, and then RNA sequencing was performed on the treated chondrocytes. KEGG was used to identify significant enrichment of signaling pathways, and Simple Western was used to verify the expression of related proteins in the signaling pathways. Coculture of OVX-OA subchondral osteoblasts with chondrocytes significantly downregulated the expression of the anabolic indicators and upregulated the expression of the catabolic indicators in chondrocytes. 1,601 proteins were identified in both normal and OVX osteoblast culture supernatants. Protein–protein interaction network analysis revealed that Sparc was one of the hub proteins. The AMPK/Foxo3a signaling pathway of chondrocytes was downregulated by OVX-OA osteoblasts CM. AICAR, the AMPK agonist, partially reversed the catabolic effect of OVX-OA osteoblasts on chondrocytes. Sparc secreted by OVX-OA subchondral osteoblasts can downregulate the AMPK/Foxo3a signaling pathway of chondrocytes, thereby promoting chondrocyte degeneration. [ABSTRACT FROM AUTHOR]

The field of osteoarthritis (OA) biology is rapidly evolving and brilliant progress has been made this year as well. Landmark studies of OA biology published in 2021 and early 2022 were selected through PubMed search by personal opinion. These papers were classified by their molecular mechanisms, and it was largely divided into the intracellular signaling mechanisms and the inter-compartment interaction in chondrocyte homeostasis and OA progression. The intracellular signaling mechanisms involving OA progression included (1) Piezo1/transient receptor potential channels of the vanilloid subtype (TRPV) 4-mediated calcium signaling, (2) mechanical load-F-box and WD repeat domain containing 7 (FBXW7) in chondrocyte senescence, (3) mechanical loading-primary cilia-hedgehog signaling, (4) low grade inflammation by toll-like receptor (TLR)-CD14-lipopolysaccharide-binding protein (LBP) complex and inhibitor of NF-κB kinase (IKK) β-nuclear factor kappa B (NF-κB) signaling, (5) selenium pathway and reactive oxygen species (ROS) production, (6) G protein-coupled receptor (GPCR) and cyclic adenosine monophosphate (cAMP) signaling, (7) peroxisome proliferator-activated receptor α (PPARα)-acyl-CoA thioesterase 12 (ACOT12)-mediated de novo lipogenesis and (8) hypoxia-disruptor of telomeric silencing 1-like (DOT1L)-H3-lysine 79 (H3K79) methylation pathway. The studies on inter-compartment or intercellular interaction in OA progression included the following subjects; (1) the anabolic role of lubricin, glycoprotein from superficial zone cells, (2) osteoclast-chondrocyte interaction via exosomal miRNA and sphingosine 1-phosphate (S1P), (3) senescent fibroblast-like synoviocyte and chondrocyte interaction, (4) synovial macrophage and chondrocyte interaction through Flightless I, (5) αV integrin-mediated transforming growth factor beta (TGFβ) activation by mechanical loading, and (6) osteocytic TGFβ in subchondral bone thickening. Despite the disastrous Covid-19 pandemic, many outstanding studies have expanded the boundary of OA biology. They provide both critical insight into the pathophysiology as well as clues for the treatment of OA. [ABSTRACT FROM AUTHOR]

• iBMSC-EVs attenuates osteoarthritis progression in vitro and in vivo. • iBMSC-EVs facilitates synovial macrophage M1/M2 polarization during OA. • Rpl14 is responsible for iBMSC-EVs mediated synovial macrophage polarization and OA progression. Bone marrow mesenchymal stem cells (BMSCs) and extracellular vesicles (EVs) were considered promising methods for the treatment of diverse diseases, including osteoarthritis (OA). However, the role of inflammatory-stimulated BMSCs driven EVs (iBMSC-EVs) in OA remains unclear. This study aims to investigate the potential function of iBMSC-EVs in ameliorating OA and its underlying molecular mechanism. In this study, EVs were isolated from inflammatory-stimulated BMSCs and destabilization of the medial meniscus (DMM) surgery was performed to induce knee OA model in mice. Micro-CT, H&E Staining, Safranin O-Fast Green Staining and immunofluorescence were used to examine the in vivo therapeutic effect of iBMSC-EVs on OA. Public single-cell RNA-seq data was downloaded and bulk RNA-seq data was acquired to identify critical genes promoted by iBMSC-EVs in macrophage polarization during OA. Real-time quantitative polymerase chain reaction (RT-qPCR), western blot, enzyme-linked immunosorbent assay (ELISA), cell counting kit-8 (CCK-8), transwell and scratch assays were used to further confirm the in vitro effect of iBMSC-EVs on macrophage polarization and chondrocyte proliferation, migration, as well as ECM anabolism and catabolism. In vivo experiments demonstrated that iBMSC-EVs could alleviate OA in mice and promote the reprogramming of M1 macrophages towards an M2 phenotype. RNA-Seq and scRNA-Seq analyses indicated that iBMSC-EVs altered the expression profiles of OA-related genes, with Rpl14 identified as a critical gene regulating macrophage reprogramming in OA. In vivo and in vitro experiments further confirmed that iBMSC-EVs, by downregulating Rpl14 expression, promoted M1 macrophage reprogramming to M2, facilitating chondrocyte proliferation and migration, inhibiting ECM degradation and ultimately alleviating OA progression. In conclusion, our data revealed the substantive role of iBMSC-EVs in ameliorating OA progression via Rpl14 mediated synovial macrophage polarization, presenting a promising therapeutic method for the treatment of OA. [ABSTRACT FROM AUTHOR]

One of the major processes occurring during the healing of a fractured long bone is chondrogenesis, leading to the formation of the soft callus, which subsequently undergoes endochondral ossification and ultimately bridges the fracture site. Thus, understanding the molecular mechanisms of chondrogenesis can enhance our knowledge of the fracture repair process. One such molecular process is calciun (Ca++) signaling, which is known to play a critical role in the development and regeneration of multiple tissues, including bone, in response to external stimuli. Despite the existence of various mouse models for studying Ca++ signaling, none of them were designed to specifically examine the skeletal system or the various musculoskeletal cell types. As such, we generated a genetically engineered mouse model that is specific to cartilage (crossed with Col2a1 Cre mice) to study chondrocytes. Herein, we report on the characterization of this transgenic mouse line using conditional expression of GCaMP6f, a Ca++-indicator protein. Specifically, this mouse line exhibits increased GCaMP6f fluorescence following Ca++ binding in chondrocytes. Using this model, we show real-time Ca++ signaling in embryos, newborn and adult mice, as well as in fracture calluses. Further, robust expression of GCaMP6f in chondrocytes can be easily detected in embryos, neonates, adults, and fracture callus tissue sections. Finally, we also report on Ca++ signaling pathway gene expression, as well as real-time Ca++ transient measurements in fracture callus chondrocytes. Taken together, these mice provide a new experimental tool to study chondrocyte-specific Ca++ signaling during skeletal development and regeneration, as well as various in vitro perturbations. • Generated transgenic mouse line using chondrocyte specific expression of GCaMP6f • Demonstrated live imaging of embryonic and postnatal Ca++ signaling • Detected in vivo Ca++ signaling during fracture repair • Quantitatively measure Ca++ signaling in fracture callus slices • First demonstration of real time Ca++ signaling in fracture callus chondrocytes [ABSTRACT FROM AUTHOR]

• Myrislignan prevent ECM degradation induced by IL-1β and stimulate ECM production in chondrocytes. • Myrislignan ameliorates IL-1β-Induced chondrocyte inflammation and osteoarthritis. • Myrislignan inhibits NF-κB and modulates the IL-1β-activated Nrf2/HO-1/JNK signaling pathway in chondrocytes. Osteoarthritis (OA) is a prevalent disease of the musculoskeletal system that causes functional deterioration and diminished quality of life. Myrislignan (MRL) has a wide range of pharmacological characteristics, including an anti-inflammatory ability. Although inflammation is a major cause of OA, the role of MRL in OA treatment is still not well-understood. In this study, we analyze the anti-inflammatory and anti-ECM degradation effects of MRL both in vivo and in vitro. Rat primary chondrocytes were treated with interleukin-1β (IL-1β) to simulate inflammatory environmental conditions and OA in vitro. The in vivo OA rat model was established by anterior cruciate ligament transection (ACLT) on rat. Our investigation discovered that MRL lowers the IL-1β-activated tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX2) and inducible nitric-oxide synthase (iNOS) expression in chondrocytes. Moreover, MRL effectively alleviates IL-1β-induced extracellular matrix (ECM) degradation and promotes ECM synthesis in chondrocytes by upregulating the mRNA level expression of collagen-II and aggrecan (ACAN), downregulating the expression of matrix metalloproteinases-3,-13 (MMP-3, MMP-13), and a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5). Gene expression profiles of different groups identified DEGs that were mainly enriched in functions associated with NF-κB signaling pathway, and other highly enriched in functions related to TNF, IL-17, Rheumatoid arthritis and cytokine-cytokine receptor signaling pathways. Venn interaction of DEGs from the abovementioned five pathways showed that Nfkbia, Il1b, Il6, Nfkb1, Ccl2, Mmp3 were highly enriched DEGs. In addition, our research revealed that MRL suppresses NF-κB and modulates the Nrf2/HO-1/JNK signaling pathway activated by IL-1β in chondrocytes. In vivo research shows that MRL slows the progression of OA in rats. Our findings imply that MRL might be a viable OA therapeutic choice. [ABSTRACT FROM AUTHOR]

• ASA VI Preserves Mitochondrial Homeostasis by Activating Sirtuin 3. • ASA VI Ameliorates IL-1β-Induced Mitochondrial Dysfunction in Chondrocytes. • ASA VI Suppresses Chondrocyte Degeneration by Maintaining Mitochondrial Homeostasis through Sirtuin 3 Activation. The aim of this study was to investigate whether ASA VI controls osteoarthritis (OA) by regulating mitochondrial function. Primary chondrocytes were isolated and cultured from rat knee joints. The chondrocytes were treated with ASA VI and interleukin-1β (IL-1β) to simulate the inflammatory environment of OA. Cell viability, apoptosis, inflammatory cytokine levels, and extracellular matrix (ECM) component levels were assessed. Mitochondrial function, including ATP levels, mitochondrial membrane potential, reactive oxygen species (ROS) levels, and mitochondrial DNA content, was evaluated. The expression of Sirtuin 3 (Sirt3), a key regulator of mitochondrial homeostasis, was examined. Additionally, a rat OA model was established by destabilizing the medial meniscus, and the effects of ASA VI on cartilage degeneration were assessed. ASA VI treatment improved cell viability, reduced apoptosis, and decreased IL-6 and TNF-α levels in IL-1β-induced chondrocytes. ASA VI also upregulated Collagen II and Aggrecan expression, while downregulating ADAMTS5 and MMP-13 expression. Furthermore, ASA VI mitigated IL-1β-induced mitochondrial dysfunction by increasing ATP levels, restoring mitochondrial membrane potential, reducing ROS production, and preserving mitochondrial DNA content. These effects were accompanied by the activation of Sirt3. In the rat OA model, ASA VI treatment increased Sirt3 expression and alleviated cartilage degeneration. ASA VI exerts chondroprotective and anti-inflammatory effects on IL-1β-induced chondrocytes by improving mitochondrial function through Sirt3 activation. ASA VI also attenuates cartilage degeneration in a rat OA model. These findings suggest that ASA VI may be a potential therapeutic agent for the treatment of osteoarthritis by targeting mitochondrial dysfunction. [ABSTRACT FROM AUTHOR]

Facet joint osteoarthritis (FJOA) is a prevalent condition contributing to low back pain, particularly in the elderly population. This study aimed to investigate the potential role of Cytokine Receptor-like Factor 1 (CRLF1) in FJOA pathogenesis and its therapeutic implications. Bioinformatics analysis was utilized to identify CRLF1 as the target gene, followed by quantification of CRLF1 expression levels and joint degeneration degree using immunohistochemistry (IHC). In primary chondrocytes, the inhibition of CRLF1 expression by siRNA was performed, and Western blot analysis was conducted to evaluate the involvement of the extracellular matrix and MAPK/ERK signaling pathway. Flow cytometry was employed to assess the apoptosis rate of chondrocytes, while immunofluorescence (IF) was utilized to evaluate the localization of CRLF1, cleaved-caspase3, MMP13, COL2A1, and ERK. The expression of CRLF1 was found to be significantly elevated in FJOA tissues compared to normal tissues. Through the use of loss-of-function assays, it was determined that CRLF1 not only enhanced the rate of apoptosis in chondrocytes, but also facilitated the degradation of the extracellular matrix in vitro. Furthermore, CRLF1 was found to activate the ERK1/2 pathways. The pro-arthritic effects elicited by CRLF1 were mitigated by treatment with the MEK inhibitor U0126 in chondrocytes. These results suggest that CRLF1 enhances chondrocytes apoptosis and extracellular matrix degration in FJOA and thus may therefore be a potential therapeutic target for FJOA. • The study identifies CRLF1 as a crucial role in FJOA pathogenesis. • The CRLF1/MAPK/ERK signaling axis exacerbates FJOA by promoting extracellular matrix degradation and chondrocyte apoptosis. • CRLF1-siRNA shows promise as a therapeutic strategy to target the CRLF1/MAPK/ERK axis for mitigating FJOA progression. [ABSTRACT FROM AUTHOR]

Temporomandibular joint osteoarthritis (TMJOA) is considered to be a low-grade inflammatory disease involving multiple joint tissues. The crosstalk between synovium and cartilage plays an important role in TMJOA. Synovial cells are a group of heterogeneous cells and synovial microenvironment is mainly composed of synovial fibroblasts (SF) and synovial macrophages. In TMJOA, SF and synovial macrophages release a large number of inflammatory cytokines and extracellular vesicles and promote cartilage destruction. Cartilage wear particles stimulate SF proliferation and macrophages activation and exacerbate synovitis. In TMJOA, chondrocytes and synovial cells exhibit increased glycolytic activity and lactate secretion, leading to impaired chondrocyte matrix synthesis. Additionally, the synovium contains mesenchymal stem cells, which are the seed cells for cartilage repair in TMJOA. Co-culture of chondrocytes and synovial mesenchymal stem cells enhances the chondrogenic differentiation of stem cells. This review discusses the pathological changes of synovium in TMJOA, the means of crosstalk between synovium and cartilage, and their influence on each other. Based on the crosstalk between synovium and cartilage in TMJOA, we illustrate the treatment strategies for improving synovial microenvironment, including reducing cell adhesion, utilizing extracellular vesicles to deliver biomolecules, regulating cellular metabolism and targeting inflammatory cytokines. [Display omitted] • Temporomandibular joint osteoarthritis (TMJOA) involves all inter-compartments of joints, including cartilage, subchondral bone, synovium, and adipose tissue. • There is complex crosstalk in each compartment of TMJOA. • Adhesion, exosomes, metabolite and ligand-receptor binding are cellular crosstalk pathways between synovium and cartilage. • Through its various actions, regulating crosstalk between synovium and cartilage is a promising option in TMJOA management. [ABSTRACT FROM AUTHOR]

Temporomandibular disorder can cause degenerative pathological changes by aseptic inflammation in the temporomandibular joint (TMJ). Vitamin D (VD) is known for maintaining calcium homeostasis, and recent studies indicated that VD and the vitamin D receptor (VDR) are important in inflammatory responses. In this study, we explored the anti-inflammatory effect of VD-VDR signaling axis in TMJ pathological degeneration. Mice ablated for Vdr (Vdr −/−res) were fed with a rescue diet to avoid hypocalcemia. With abnormal mechanical stimulation, unilateral anterior crossbite (UAC) induced temporomandibular disorders in mice. Histological staining, immunohistochemistry staining, and micro-CT analysis were performed to evaluate TMJ pathological changes. To identify the mechanisms in the aseptic inflammatory process, in vitro experiments were conducted on wild-type (WT) and Vdr −/− chondrocytes with compressive mechanical stress loading, and the related inflammatory markers were examined. Vdr −/−res mice did not develop rickets with a high calcium rescue diet. The TMJ cartilage thickness in Vdr −/−res mice was significantly decreased with mechanical stress stimulation compared to WT mice. UAC-induced bone resorption was obvious, and the number of osteoclasts significantly increased in Vdr −/−res mice. The proliferation was inhibited and the gene expression of Il1b , Mmp3 , and Mmp13 was significantly increased in Vdr −/− chondrocytes. However, WT chondrocytes showed significantly increased Tnfa gene expression as a response to mechanical stress but not in Vdr −/− chondrocytes. VD-VDR is crucial in TMJ pathological changes under abnormal mechanical stimulation. Deletion of Vdr exacerbated inflammatory response excluding TNFα, inhibited chondrocyte proliferation, and promoted bone resorption in TMJ. [Display omitted] • Mice ablated for Vdr show temporomandibular joint (TMJ) malformations but can be partially treated with a rescue diet rich in calcium and phosphorus • Even though calcium metabolic homeostasis was maintained, abnormal mechanical stress induced more severe TMJ pathological degeneration in Vdr -/- mice than in WT mice. • In vitro compressive mechanical stress upregulated the gene expression of pro- inflammatory cytokines, excluding Tnfa , in Vdr -/- chondrocytes [ABSTRACT FROM AUTHOR]

• circSLTM, miR-515-5p, and VAPB form a novel regulatory axis in osteoarthritis (OA) pathogenesis. • circSLTM is upregulated in OA patients and LPS-treated chondrocytes. • Silencing circSLTM enhances proliferation and reduces apoptosis of LPS-treated chondrocytes. • circSLTM interacts with miR-515-5p to upregulate VAPB in OA progression. • circSLTM silencing alleviates cartilage damage in ACLT-induced OA mouse model. Osteoarthritis (OA) is a prevalent joint disorder characterized by cartilage degeneration. Circular RNAs (circRNAs) have emerged as pivotal players in OA progression, orchestrating various biological processes such as proliferation, apoptosis, inflammation, and extracellular matrix (ECM) reorganization. Among these circRNAs, circSLTM exhibits aberrant expression in OA, yet its precise regulatory mechanism remains elusive. This study aimed to elucidate the regulatory mechanisms of circSLTM in OA pathogenesis, with a focus on its role as a competing endogenous RNA (ceRNA). Human cartilage tissues were procured from both OA patients and non-OA individuals, while human chondrocyte cells were subjected to lipopolysaccharide (LPS) treatment to mimic OA-like conditions. Our findings revealed upregulation of circSLTM in OA patients and LPS-treated chondrocytes. Loss-of-function assays were conducted, demonstrating that silencing circSLTM via shRNAs mitigated LPS-induced effects on chondrocytes, as evidenced by enhanced proliferation, reduced apoptosis, and inflammatory factors, and altered expression of extracellular matrix proteins. Further exploration into the regulatory mechanism of circSLTM unveiled its interaction with microRNA-515-5p (miR-515-5p) to modulate vesicle-associated membrane protein (VAPB) expression in chondrocytes. VAPB, also upregulated in OA, was positively regulated by circSLTM. Rescue assays corroborated that VAPB overexpression reinstated the protective effects of circSLTM knockdown on LPS-treated chondrocytes. Moreover, concurrent knockdown of both circSLTM and VAPB demonstrated synergistic protection against LPS-induced chondrocyte injury. Additionally, we delineated that LPS triggered the activation of the NF-κB pathway in chondrocytes, which was counteracted by circSLTM silencing. To assess the effects of circSLTM on OA in vivo, anterior cruciate ligament transection (ACLT) mouse models were established, revealing that circSLTM deficiency ameliorated cartilage defects in vivo. In conclusion, circSLTM exacerbates osteoarthritis progression by orchestrating the miR-515-5p/VAPB axis and activating the NF-κB pathway, providing novel insights for targeted therapy in OA management. [ABSTRACT FROM AUTHOR]

Chondrocytes respond to mechanical stimuli by increasing their intracellular calcium concentration. The response depends on the cellular environment. Previous studies have investigated chondrocytes under slow strain rates or cells embedded in hydrogels, but the response of chondrocytes in their native environment under physiologically relevant cyclic loads and dynamic hydrostatic pressure has not been studied. This study investigated the calcium signaling response of in-situ chondrocytes under physiological cyclic compressive loads and hydrostatic pressure with varying frequency and load rates. Bovine cartilage explants were stained with a fluorescent calcium indicator dye and subjected to physiologically relevant cyclic loads using a custom-built loading device secured on a confocal/multiphoton microscope. Calcium fluorescence intensities of the cells were tracked and analyzed. Loading groups were compared using one-way ANOVA followed by a post-hoc test with Tukey correction (α = 0.05). The percentage of cells signaling increased in all compressive loading conditions compared to the no-load baseline. The percentage of cells responding under 1 Hz load was significantly greater than the slow ramp and 0.1 Hz group (p < 0.05). The number of compression cycles had no effect on the calcium signaling response (p > 0.05). The width and time between consecutive peaks were not different between different loading conditions (p > 0.05). Calcium signaling of in-situ chondrocytes did not increase under dynamic hydrostatic pressure of magnitudes up to 0.2 MPa at frequencies of 0.5 Hz and 0.05 Hz (p > 0.05). In conclusion, in-situ chondrocytes respond to physiological compressive loads in a strain rate-dependent manner with an increased number of responsive cells and unaltered temporal characteristics. [ABSTRACT FROM AUTHOR]

Otolaryngology is an important specialty in the field of surgery that deals with the diagnosis and treatment of the ear, nose, throat, trachea, as well as related anatomical structures. Various otolaryngological disorders are difficult to treat using established pharmacological and surgical approaches. The advent of molecular and cellular therapies led to further progress in this respect. This article reviews the therapeutic strategies of using stem cells, immune cells, and chondrocytes in otorhinolaryngology. As the most widely recognized cell derivatives, exosomes were also systematically reviewed for their therapeutic potential in head and neck cancer, otitis media, and allergic rhinitis. Finally, we summarize the limitations of stem cells, chondrocytes, and exosomes, as well as possible solutions, and provide an outlook on the future direction of cell- and derivative-based therapies in otorhinolaryngology, to offer a theoretical foundation for the clinical translation of this therapeutic modality. Common ENT diseases include hearing loss, sinusitis, bronchial defects, head and neck cancer, etc. Cells and their derivatives used in the study and treatment of these diseases mainly include stem cells, chondrocytes, and exosomes, with systemic and local drug administration, in vitro tissue culture, and genetic engineering being the common interventions. The main logic of the research consists of two parts: one is to use the differentiation ability of stem cells to culture them into target organs or target tissues, so as to replace their functions; the other is to use the transport properties of exosomes to deliver drugs or use them as biomarkers to predict disease progression. [Display omitted] [ABSTRACT FROM AUTHOR]

[Display omitted] • Our study proved that mechanical overloading induces ferroptosis of chondrocyte, which might be a potential therapeutic target for mechanical damage of chondrocyte and OA. • Our study demonstrated Piezo1 facilitated calcium influx leads to reduction of GSH, decrease of Gpx4 and activation of oxidative stress in chondrocyte under high strain mechanical stimulation. • Mechanical signals were converted into ferroptosis-associated signals through Piezo1 channel induced calcium influx, which might shed light on therapeutic interventions for treatment of OA and other diseases associated with ferroptosis. Excessive mechanical stress is closely associated with cell death in various conditions. Exposure of chondrocytes to excessive mechanical loading leads to a catabolic response as well as exaggerated cell death. Ferroptosis is a recently identified form of cell death during cell aging and degeneration. However, it's potential association with mechanical stress remains to be illustrated. To identify whether excessive mechanical stress can cause ferroptosis. To explore the role of mechanical overloading in chondrocyte ferroptosis. Chondrocytes were collected from loading and unloading zones of cartilage in patients with osteoarthritis (OA), and the ferroptosis phenotype was analyzed through transmission electron microscope and microarray. Moreover, the relationship between ferroptosis and OA was analyzed by GPX4-conditional knockout (Col2a1-CreERT: GPX4flox/flox) mice OA model and chondrocytes cultured with high strain mechanical stress. Furthermore, the role of Piezo1 ion channel in chondrocyte ferroptosis and OA development was explored by using its inhibitor (GsMTx4) and agonist (Yoda1). Additionally, chondrocyte was cultured in calcium-free medium with mechanical stress, and ferroptosis phenotype was tested. Human cartilage and mouse chondrocyte experiments revealed that mechanical overloading can induce GPX4-associated ferroptosis. Conditional knockout of GPX4 in cartilage aggravated experimental OA process, while additional treatment with ferroptosis suppressor protein (FSP-1) and coenzyme Q10 (CoQ10) abated OA development in GPX4-CKO mice. In mouse OA model and chondrocyte experiments, inhibition of Piezo1 channel activity increased GPX4 expression, attenuated ferroptosis phenotype and reduced the severity of osteoarthritis. Additionally, high strain mechanical stress induced ferroptosis damage in chondrocyte was largely abolished by blocking calcium influx through calcium-free medium. Our findings show that mechanical overloading induces ferroptosis through Piezo1 activation and subsequent calcium influx in chondrocytes, which might provide a potential target for OA treatment. [ABSTRACT FROM AUTHOR]

Objective: This study was performed to characterize selected rhodanine derivatives as potential preclinical disease-modifying drugs for experimental osteoarthritis (OA) in mice.Methods: Three rhodanine derivatives, designated rhodanine (R)-501, R-502, and R-503, were selected as candidate OA disease-modifying drugs. Their effects were evaluated by intra-articular (IA) injection in OA mouse models induced by DMM (destabilization of the medial meniscus) or adenoviral overexpression in joint tissues of hypoxia-inducible factor (HIF)-2α or zinc importer ZIP8. The regulatory mechanisms impacted by the rhodanine derivatives were examined in primary-culture chondrocytes and fibroblast-like synoviocytes (FLS).Results: All three rhodanine derivatives inhibited OA development caused by DMM or overexpression of HIF-2α or ZIP8. Compared to vehicle-treated group, for example, IA injection of R-501 in DMM-operated mice reduced median OARSI grade from 3.78 (IQR 3.00-5.00) to 1.89 (IQR 0.94-2.00, P = 0.0001). R-502 and R-503 also reduced from 3.67 (IQR 2.11-4.56) to 2.00 (IQR 1.00-2.00, P = 0.0030) and 2.00 (IQR 1.83-2.67, P = 0.0378), respectively. Mechanistically, the rhodanine derivatives inhibited the nuclear localization and transcriptional activity of HIF-2α in chondrocytes and FLS. They did not bind to Zn2+ or modulate Zn2+ homeostasis in chondrocytes or FLS; instead, they inhibited the nuclear localization and transcriptional activity of the Zn2+-dependent transcription factor, MTF1. HIF-2α, ZIP8, and interleukin-1β could upregulate matrix-degrading enzymes in chondrocytes and FLS, and the rhodanine derivatives inhibited these effects.Conclusion: IA administration of rhodanine derivatives significantly reduced OA pathogenesis in various mouse models, demonstrating that these derivatives have disease-modifying therapeutic potential against OA pathogenesis. [ABSTRACT FROM AUTHOR]

Previous research has shown that T-2 toxin can damage cartilage, resulting in a disease phenotype similar to osteoarthritis. The precise molecular mechanism by which T-2 toxin causes chondrocyte injury, however, is unknown. The purpose of this study was to look into the role of YAP in T-2 toxin-induced rat chondrocyte injury. Based on research results, T-2 toxin decreased the levels of collagen II and PCNA while increasing the expression of matrix metalloproteinase MMP13. These findings supported the T-2 toxin's detrimental effect on chondrocytes. YAP's role in T-2 toxin-induced chondrocyte injury was also investigated. Total YAP and related nuclear proteins were found to decrease as the concentration of T-2 toxin increased. While PYAP expression was not significantly altered in response to T-2 toxin, the PYAP/YAP ratio decreased as the T-2 toxin concentration increased, implying that the HIPPO signaling pathway was activated. Furthermore, the YAP-specific inhibitor Verteporfin was used to investigate the role of YAP in T-2 toxin-induced chondrocyte injury. YAP inhibition increased MMP13 expression while decreasing COL2 and PCNA levels. In summary, the current study found that T-2 toxin decreased the levels of COL2 and PCNA while increasing the expression of MMP13 in chondrocytes after inhibiting YAP, providing a new insight into the mechanism of T-2 toxin-induced cartilage damage. Schematic diagram for the molecular mechanism of YAP in T-2 toxin-induced chondrocyte injury. T-2 toxin phosphorylates YAP in the cytoplasm and inhibits the entry of YAP into the nucleus for binding to transcription factors, resulting in the upregulation of MMP13, the downregulation of Collagen II, the reduction of PCNA, and ultimately chondrocyte damage. [Display omitted] • Yes-related protein is the most critical effector of HIPPO signaling pathway to regulate proliferation and apoptosis. • Nuclear expression of Yes-related proteins was decreased when chondrocytes were treated with T-2 toxin. • T-2 toxin stimulates the expression of MMP 13 and degradation of collagen type II through Yes-related protein. • T-2 toxin inhibits chondrocyte proliferation, perhaps by suppressing the expression of Yes-related protein. [ABSTRACT FROM AUTHOR]

Aseptic loosening after total hip replacement brings adverse health outcomes and increased risk for complications. The resorptive activity of inflammatory cells activated by the presence of wear-generated debris plays a critical role in debris-induced osteolysis. Previous studies indicate that the abnormally expressed LINC01534 plays a critical role in inflammatory responses. In this study, we aimed to elucidate the functional role and underlying mechanism of LINC01534 in debris-induced osteolysis. We first confirmed that LINC01534 was highly expressed in hip cartilage tissues from aseptic loosening patients. By using an IL-1β-induced inflammation model mimicking debris-induced osteolysis, we demonstrated that LINC01534 promoted IL-1β-induced inflammatory response in hip chondrocytes. Knockdown of LINC01534 inhibited the expression of inflammatory IL-6, IL-8, and TNF-α in hip chondrocytes. Our results showed that LINC01534 functioned as a competing endogenous RNA (ceRNA) by sponging miR-135b-5p in hip chondrocytes. Moreover, bioinformatics analysis and luciferase reporter assay demonstrated that CCHC-Type Zinc Finger Nucleic Acid Binding Protein (PTPRT) is a downstream target of miR-135b-5p. Knockdown of PTPRT attenuated the IL-1β-induced inflammatory responses in hip chondrocytes. In addition, we revealed that inhibition of miR-135b-5p or overexpression of PTPRT could antagonize the effects of LINC01534 knockdown on inflammation attenuation in hip chondrocytes. Mechanistically, we demonstrated that LINC01534/miR-135b-5p/PTPRT axis regulated the NF-κB signaling pathway in hip chondrocytes. Taken together, our findings suggest that LINC01534/miR-135b-5p/PTPRT axis might be a valuable therapeutic target for the treatment of debris-induced osteolysis. [ABSTRACT FROM AUTHOR]

Knee osteoarthritis (KOA) is a prevalent and disabling clinical condition affecting joint structures worldwide. The Yiqi Yangxue formula (YQYXF) is frequently prescribed in clinical settings for the treatment of KOA. Existing research has primarily focused on alterations in drug metabolism, with limited investigation into the epigenetic effects of YQYXF, particularly in relation to non-coding RNA. Exploring the effects of YQYXF on critical factors of long chain non-coding RNA UFC1/miR-34a/matrix metalloproteinase-13 (MMP-13) axis and their interrelationships. UHPLC-QE-MS technology was used to identify the YQYXF ingredients in rat serum. KEGG and GO analysis were performed on the targets of blood components acting on KOA using a database. Simultaneously, a protein interaction network was constructed using target proteins and metabolites to identify the core components and key pathways of YQYXF. The KOA rat model was established using an improved Hulth method. SPF SD rats were randomly divided into normal group, sham surgery group, model group, celecoxib capsules group (18 mg/kg), YQYXF low, medium and high dose groups (4.6 g/kg, 9.2 g/kg, 18.4 g/kg). Observe the synovial and cartilage tissues of rats using pathological methods. RT-PCR was used to detect the levels of UFC1, miR-34a, and MMP-13 in cartilage. Immunohistochemistry was used to detect the levels of MMP-13 and ADAMTS-5 in cartilage. ELISA method was used to detect the levels of MMP-13 and ADAMTS-5 in serum. In addition, we further validated the regulation of crucial factor expression levels of UFC1/miR-34a/MMP-13 axis in rat chondrocytes and degenerative chondrocytes of KOA patients by YQYXF, providing a basis for its treatment of KOA. The compounds that YQYXF enters the bloodstream mainly contain flavonoids and phenylpropanoid compounds. The core components that act on OA include quercetin, fisetin, and demethylweldelolactone. The main target pathways are the IL-17 signaling pathway, lipid and atherosclerosis, cellular sensitivity, inflammatory mediator regulation of TRP channels, TNF signaling pathway, relaxin signaling pathway and C-type lectin receptor signaling pathway. YQYXF inhibited the expression of miR-34a and MMP-13 mRNA, and reduced the protein levels of MMP-13 and ADAMTS-5. In vitro studies have confirmed that 20% YQYXF serum promoted UFC1 and reduce miR-34a levels. In addition, miR-34a in sh-UFC1+10% YQYXF serum and sh-UFC1+20% YQYXF serum groups significantly decreased compared to the sh-UFC1 group. The anti-KOA cartilage degeneration effect of YQYXF might be related to inhibiting cell apoptosis and promoting cell proliferation, which regulated the lncRNA-UFC1/miR-34a/MMP-13 axis. [Display omitted] • The compounds that YQYXF enters the bloodstream mainly contain flavonoids and phenylpropanoid compounds. • The core components that act on OA include quercetin, fisetin, and demethylweldelolactone. • LncRNA-UFC1/miR-34a/MMP-13 axis regulates KOA cartilage injury. • YQYXF alleviates KOA through lncRNA-UFC1/miR-34a/MMP-13 axis. [ABSTRACT FROM AUTHOR]

• Ruscogenin has demonstrated therapeutic promise in orthopedic and joint disorders. • New Perspectives on Macrophage Reprogramming for Osteoarthritis. • Oxidative stress regulates macrophage reprogramming. Synovial inflammation, Cartilage erosion, and subchondral osteosclerosis, which are collectively referred to as the triad of pathogenesis, contribute to osteoarthritis (OA) progression. Specifically, the M1 macrophage in the synovium worsens the development of the illness and is a significant factor in the deterioration and functioning of cartilage. To investigate whether Ruscogenin attenuates progressive degeneration of articular cartilage in rats with anterior cruciate ligament transection (ACLT)-induced osteoarthritis (OA) by modulating macrophage reprogramming and to explore its specific mechanism of action. In vitro, SW1353 cells and RAW264.7 cells were applied to elucidate the mechanisms by which Ruscogenin protects articular cartilage. Specifically, the expression levels of molecules related to cartilage ECM synthesis and degradation enzymes and macrophages were analysed. In vivo, a rat osteoarthritis model was established using ACLT. The protective effect of Ruscogenin on articular cartilage was observed. Ruscogenin significantly reversed LPS-induced macrophage inflammatory response and promoted cartilage regeneration-related factors. In addition, Ruscogenin had a significant protective effect on the knee joint of ACLT rats, effectively preventing cartilage degeneration. These positive therapeutic effects were achieved on the one hand by Ruscogenin regulating macrophage reprogramming by targeting Sirt3, and on the other hand Ruscogenin could attenuate the ROS level of chondrocytes thereby inhibiting chondrocyte ferroptosis. Ruscogenin exerts chondroprotective effects by regulating macrophage reprogramming and inhibiting chondrocyte ferroptosis. [ABSTRACT FROM AUTHOR]

• Nuciferine(Nuc) exerts anti-inflammatory and ameliorate the degradation of extracellular matrix in IL-1β-induced chondrocytes. • Nuciferine(Nuc) mitigates the progression of osteoarthritis in a mouse model. • Nuciferine(Nuc) exerts a protective effect on chondrocytes by activating the PTEN/AKT/NF-κB signaling pathway. Osteoarthritis (OA) is the most common musculoskeletal disease and a leading cause of pain and disability. A key hallmark of OA is cartilage degradation, which occurs due to an imbalance between the synthesis and degradation of the extracellular matrix (ECM). Interleukin-1β(IL-1β) has been reported to regulate ECM metabolism. Nuciferine (Nuc), a natural peptide extracted from the lotus leaf, possesses several significant pharmacological properties. However, the anti-inflammation of Nuc in OA has not been reported. In this study, ELISA and Western blot analyses were used to measure the production of inflammatory mediators in IL-1β-Induced mouse chondrocytes. Additionally, mice with or without surgical destabilization of the medial meniscus (DMM) were treated with intra-articular injection of Nuc. We found that Nuc significantly reduces the level of iNOS, PEG2, and IL-6 in IL-1β-induced chondrocytes. Furthermore, Nuc can ameliorate the development of OA in mice. Mechanistically, we found that the chondrocyte-protective effects of Nuc occur via the PTEN/NF-κB pathway. These findings suggest that Nuc could be a potential therapeutic agent for improving OA development. [ABSTRACT FROM AUTHOR]

Synovitis and cartilage destruction are crucial characteristics of osteoarthritis (OA). Inflammatory cytokines, such as IL-1β, are secreted by synovial macrophages, leading to cartilage destruction. Pyroptosis is a lytic form of programmed cell death, which could be triggered by the NLRP3 inflammasome of macrophages. Pyroptosis promotes the secretion of IL-1β and is supposed as a potential biomarker for OA. However, the function of Pyroptosis and NLRP3 inflammasome and its regulatory mechanism for activation is unclear in OA. In this study, we found that Degrasyn could alleviate the GSDMD-mediated pyroptosis of macrophages and the release of IL-1β, caspase-1, and LDH. Furthermore, it selectively impedes the form of ASC oligomer and speckle to effectively suppress the NLRP3 inflammasome during its assembly phase. Notably, Degrasyn exhibited potential chondroprotective effects in a co-culture system. Additionally, these results also indicate that Degrasyn mitigates synovitis and cartilage damage in a murine model of destabilization of the medial meniscus (DMM)-induced OA. In summary, Degrasyn emerges as a promising pharmaceutical agent for synovitis, paving the way for innovative therapeutic approaches to OA. Our findings underscore the potential of Degrasyn as a viable candidate for OA therapeutics, demonstrating its ability to regulate pyroptosis and NLRP3 inflammasome activation. • Degrasyn alleviate NLRP3 inflammasome activation and the Pyroptosis of macrophages induced by DAMPs in the joint cavity. • Degrasyn specifically blocked ASC oligomerization and play a widespread inhibition function on inflammasomes. • Degrasyn could possess chondroprotective effects in a modified co-culture system and DMM-OA mice. [ABSTRACT FROM AUTHOR]

Cell volume and shape changes play a pivotal role in cellular mechanotransduction, governing cellular responses to external loading. Understanding the dynamics of cell behavior under loading conditions is essential to elucidate cell adaptation mechanisms in physiological and pathological contexts. In this study, we investigated the effects of dynamic cyclic compression loading on cell volume and shape changes, comparing them with static conditions. Using a custom-designed platform which allowed for simultaneous loading and imaging of cartilage tissue, tissues were subjected to 100 cycles of mechanical loading while measuring cell volume and shape alterations during the unloading phase at specific time points. The findings revealed a transient decrease in cell volume (13%) during the early cycles, followed by a gradual recovery to baseline levels after approximately 20 cycles, despite the cartilage tissue not being fully recovered at the unloading phase. This observed pattern indicates a temporal cell volume response that may be associated with cellular adaptation to the mechanical stimulus through mechanisms related to active cell volume regulation. Additionally, this study demonstrated that cell volume and shape responses during dynamic loading were significantly distinct from those observed under static conditions. Such findings suggest that cells in their natural tissue environment perceive and respond differently to dynamic compared to static mechanical cues, highlighting the significance of considering dynamic loading environments in studies related to cellular mechanics. Overall, this research contributes to the broader understanding of cellular behavior under mechanical stimuli, providing valuable insights into their ability to adapt to dynamic mechanical loading. [ABSTRACT FROM AUTHOR]

Actin is a central mediator of the chondrocyte phenotype. Monolayer expansion of articular chondrocytes on tissue culture polystyrene, for cell-based repair therapies, leads to chondrocyte dedifferentiation. During dedifferentiation, chondrocytes spread and filamentous (F-)actin reorganizes from a cortical to a stress fiber arrangement causing a reduction in cartilage matrix expression and an increase in fibroblastic matrix and contractile molecule expression. While the downstream mechanisms regulating chondrocyte molecular expression by alterations in F-actin organization have become elucidated, the critical upstream regulators of F-actin networks in chondrocytes are not completely known. Tropomyosin (TPM) and the RhoGTPases are known regulators of F-actin networks. The main purpose of this study is to elucidate the regulation of passaged chondrocyte F-actin stress fiber networks and cell phenotype by the specific TPM, TPM3.1, and the RhoGTPase, CDC42. Our results demonstrated that TPM3.1 associates with cortical F-actin and stress fiber F-actin in primary and passaged chondrocytes, respectively. In passaged cells, we found that pharmacological TPM3.1 inhibition or siRNA knockdown causes F-actin reorganization from stress fibers back to cortical F-actin and causes an increase in G/F-actin. CDC42 inhibition also causes formation of cortical F-actin. However, pharmacological CDC42 inhibition, but not TPM3.1 inhibition, leads to the re-association of TPM3.1 with cortical F-actin. Both TPM3.1 and CDC42 inhibition, as well as TPM3.1 knockdown, reduces nuclear localization of myocardin related transcription factor, which suppresses dedifferentiated molecule expression. We confirmed that TPM3.1 or CDC42 inhibition partially redifferentiates passaged cells by reducing fibroblast matrix and contractile expression, and increasing chondrogenic SOX9 expression. A further understanding on the regulation of F-actin in passaged cells may lead into new insights to stimulate cartilage matrix expression in cells for regenerative therapies. • During chondrocyte dedifferentiation filamentous actin reorganizes from a cortical to a stress fiber organization. • Tropomyosin 3.1 associates with F-actin in both primary and passaged chondrocytes. • Tropomyosin 3.1 inhibition in passaged cells reduces stress fibers, promotes cortical actin and suppresses the dedifferentiated phenotype. • CDC42 inhibition enables the formation of cortical F-actin with Tropomyosin 3.1 association, and suppresses the dedifferentiated phenotype. • Inhibition of Tropomyosin 3.1 or CDC42 suppresses dedifferentiation via myocardin related transcription factor nuclear export. [ABSTRACT FROM AUTHOR]

Cellular therapy based on chondrocytes implantation is the most widely used procedure for inducing cartilage regeneration. However, the dedifferentiation process that these cells suffer and their limited capacity of proliferation, when they are cultured in vitro, restrict their use in cellular therapy protocols. To investigate the capacity of mesenchymal stromal cells (MSCs) to promote chondrogenesis from chondrocytes or chondrons in 2D and 3D coculture systems. Murine chondrocytes and chondrons were cocultured with MSCs at different cell ratios (100/0, 50/50, 70/30, 0/100) in two-dimensional (2D) and three-dimensional (3D) culture systems. High proliferation of cells with chondrocyte morphology, enhanced GAG production and expression of cartilage genes (aggrecan, type II collagen, and SOX-9) were observed in chondrocytes/MSCs cocultures. In contrast, fibroblastoid cells, down-regulation of cartilage gene expression and reduction of GAG production were observed in chondrons/MSCs cocultures. Chondrocytes within cartilage lacunae and surrounded by extracellular matrix were observed in chondrocytes/MSC pellets. MSCs promote the proliferation of functional chondrocytes in 2D and 3D culture systems. Transplantation of chondrogenic construct based on MSCs and chondrocytes may constitute a potential treatment for inducing cartilage repair. [ABSTRACT FROM AUTHOR]

Objective: In knee cartilage from patients with osteoarthritis (OA), both preserved cartilage and damaged cartilage are observed. In this study, we aim to compare preserved with damaged cartilage to identify the molecule(s) that may be responsible for the mechanical loading-induced differences within cartilage degradation.Methods: Preserved and damaged cartilage were harvested from the same OA knee joint. RNA Sequencing was performed to examine the transcriptomic differences between preserved and damaged cartilage cells. Estrogen receptor-α (ERα) was identified, and its function of was tested through gene knockin and knockout. The role of ERα in mediating chondrocyte response to mechanical loading was examined via compression of chondrocyte-laded hydrogel in a strain-controlled manner. Findings from the studies on human samples were verified in animal models.Results: Level of estrogen receptor α (ERα) was significantly reduced in damaged cartilage compared to preserved cartilage, which were observed in both human and mice samples. Knockdown of ESR1, the gene encoding ERα, resulted in an upregulation of senescence- and OA-relevant markers in chondrocytes. Conversely, knockin of ESR1 partially reversed the osteoarthritic and senescent phenotype of OA chondrocytes. Using a three-dimensional (3D) culture model, we demonstrated that mechanical overload significantly suppressed ERα level in chondrocytes with concomitant upregulation of osteoarthritic phenotype. When ESR1 expression was suppressed, mechanical loading enhanced hypertrophic and osteogenic transition.Conclusion: Our study demonstrates a new estrogen-independent role of ERα in mediating chondrocyte phenotype and its response to mechanical loading, and suggests that enhancing ERα level may represent a new method to treat osteoarthritis. [ABSTRACT FROM AUTHOR]

The ECM of cartilage is composed of proteoglycans (PG) that contain glycosaminoglycan (GAG), aggrecan, hyaluronic acid (HA) and other molecular components which play an important role in regulating chondrocyte functions via cell-matrix interactions, integrin-mediated signalling etc. Implantation of chondrocytes encapsulated in scaffolds that mimic the micro-architecture of proteoglycan, is expected to enhance cartilage repair. With an aim to create a hydrogel having macromolecular structure that resembles the cartilage-specific ECM, we constructed a hierarchal structure that mimic the PG. The bottle brush structure of the aggrecan was obtained using chondroitin sulphate and carboxymethyl cellulose which served as GAG and core protein mimic respectively. A proteoglycan-like structure was obtained by cross-linking it with modified chitosan that served as a HA substitute. The physico-chemical characteristics of the above cross-linked injectable hydrogel supported long term human articular chondrocyte subsistence and excellent post-injection viability. The chondrocytes encapsulated in the PMH expressed significant levels of articular cartilage specific markers like collagen II, aggrecan, GAGs etc. , indicating the ability of the hydrogel to support chondrocyte differentiation. The biocompatibility and biodegradability of the hydrogels was confirmed using suitable in vivo studies. The results revealed that the PG-mimetic hydrogel could serve as a promising scaffold for chondrocyte implantation. [Display omitted] [ABSTRACT FROM AUTHOR]

The mammalian skeleton develops through two distinct modes of ossification: intramembranous ossification and endochondral ossification. During the process of skeletal development, SRY-box containing gene 9 (Sox9), runt-related transcription factor 2 (Runx2), and Sp7 work as master transcription factors (TFs) or transcriptional regulators, underlying cell fate specification of the two distinct populations: bone-forming osteoblasts and cartilage-forming chondrocytes. In the past two decades, core transcriptional circuits underlying skeletal development have been identified mainly through mouse genetics and biochemical approaches. Recently emerging next-generation sequencer (NGS)-based studies have provided genome-scale views on the gene regulatory landscape programmed by the master TFs/transcriptional regulators. With particular focus on Sox9, Runx2, and Sp7, this review aims to discuss the gene regulatory landscape in skeletal development, which has been identified by genome-scale data, and provide future perspectives in this field. [ABSTRACT FROM AUTHOR]

Multiple human tissue engineered cartilage constructs are showing promise in advanced clinical trials but identifying important measures of manufacturing reproducibility remains a challenge. FDA guidance suggests measuring multiple mechanical properties prior to implantation, because these properties could affect the long term success of the implant. Additionally, these engineered cartilage mechanics could be sensitive to the autologous chondrocyte source, an inherently irregular manufacturing starting material. If any mechanical properties are sensitive to changes in the autologous chondrocyte source, these properties may need to be measured prior to implantation to ensure manufacturing reproducibility and quality. Therefore, this study identified variability in the compressive, friction, and shear properties of a human tissue engineered cartilage constructs due to the chondrocyte source. Over 200 constructs were created from 7 different chondrocyte sources and tested using 3 distinct mechanical experiments. Under confined compression, the compressive properties (aggregate modulus and hydraulic permeability) varied by orders of magnitude due to the chondrocyte source. The friction coefficient changed by a factor of 5 due to the chondrocyte source and high intrapatient variability was noted. In contrast, the shear modulus was not affected by changes in the chondrocyte source. Finally, measurements on the local compressive and shear mechanics revealed variability in the depth dependent strain fields based on chondrocyte source. Since the chondrocyte source causes large amounts of variability in the compression and local mechanical properties of engineered cartilage, these mechanical properties may be important measures of manufacturing reproducibility. Although the FDA recommends measuring mechanical properties of human tissue engineered cartilage constructs during manufacturing, the effect of manufacturing variability on construct mechanics is unknown. As one of the first studies to measure multiple mechanical properties on hundreds of human tissue engineered cartilage constructs, we found the compressive properties are most sensitive to changes in the autologous chondrocyte source, an inherently irregular manufacturing variable. This sensitivity to the autologous chondrocyte source reveals the compressive properties should be measured prior to implantation to assess manufacturing reproducibility. [Display omitted] [ABSTRACT FROM AUTHOR]

Knee osteoarthritis (KOA) is a chronic joint disorder involving the articular cartilage and tissues around the synovial joint. The key objective of this study was to determine the effect of miR-186-5p administration on the expression of pathogenic signalling in the chondrocytes using a surgical destabilization of the medial meniscus (DMM) model of KOA, and to testify the mechanism of P2X7-mediated regulation of RUNX2/ADAMTS5 axis by miR-186 in the KOA rats. After eight weeks of intra-articular injection of the miR-186-5p and negative control lentivirus samples, the knee cartilage tissues were subjected to histopathological analysis Safranin-O/Fast green staining. Further, the articular chondrocytes were separated and analysed for various proteins including P2X7, cathepsin-K, RUNX2 and ADAMTS5 using Western blotting method. We observed that the protein expressions of P2X7, cathepsin-K/RUNX2/ADAMTS5, and also MMP-13 were upmodulated in the KOA rats, while intra-articular miR-186-5p lentivirus administration prevented these aberrations. Hence, the study concludes that miR-186 orchestrates P2X7 expression and the P2X7-mediated cathepsin-K/RUNX2/ADAMTS5 axis and regulates the pathogenesis of KOA. In light of this evidence, we propose that molecular therapeutic interventions targeting miR-186 activation might attenuate osteoarthritic cartilage degeneration. [ABSTRACT FROM AUTHOR]

Osteoarthritis occurs when protective cartilage of bones worn out. Similarlty, cartilage damage occurs mainly in the pannus cartilage in rheumatoid arthritis. It is a potentially debilitating condition, affecting women two to three times more often than men. The cause and prognosis of rheumatoid and osteoarthritis are still poorly known. However, advances in the study of disease pathogenesis have encouraged the creation of new therapeutics with improved outcomes. The purpose of this study is to investigate the differentially expressed genes potentially involved in dysregulated rheumatoid arthritis (RA) and their association to other types of arthritis, including osteoarthritis (OA). Complete RNAs were isolated for RNA expression profiling using next-generation sequencing from human primary cultured normal and RA chondrocytes. From RNA sequencing results 250 differentially expressed genes were identified using bioinformatics analysis, of which 32 were found to be significantly playing role in RA pathogenesis and its associated diseases. Molecular ontologies of the identified genes showed they are connected to Innate immune response, Protein phosphorylation, Transcription initiation from RNA polymerase II promoter, Immune response, Neoplasms of bones, as well as osteorthritis, and Rheumatoid arthritis. Among the identified genes, TRAF1, TRAF2, BAMP, STX11, MEOX2, AES, REL, FHL3, PNMA1, SGTA, LZTS2, SIAH2, PNMA1, and TFCP2 were found to be highly enriched in the protein-protein interaction network. The significant cross talks were found in Hypertrophic cardiomyopathy, Small cell lung cancer, Proteasome, p53 signaling pathway, Arrhythmogenic right ventricular cardiomyopathy, Small cell lung cancer, SNARE interactions in vesicular transport, RIG-I-like receptor signaling pathway, and Hypertrophic cardiomyopathy pathways. The results offer new opportunities for target gene control in RA and OA cartilage destruction. [ABSTRACT FROM AUTHOR]

Introduction& Aims: Non Steroidal Anti-Inflammatory drugs (NSAIDs) are potent inhibitors of post-traumatic pain. Several studies have highlighted that NSAIDs could exert a negative effect on bone healing process possibly by down-regulating chondrogenesis and endochondral ossification. The aim of the study is to explore the potential mechanism though which NSAIDs can affect chondrogenesis. M&M: Trabecular bone from the fracture site was isolated from 10 patients suffering from long bone fractures. Mesenchymal Stem Cells (MSCs) were isolated following collagenase digestion and functional assays to assess the effect of diclofenac sodium on chondrogenesis were performed. Gene expression analysis of 84 key molecules was performed.Results: Diclofenac sodium inhibits chondrogenic differentiation and induces a strong inhibition of prostaglandin E-2 (PGE-2) production during chondrogenic differentiation. Replenishment of PGE-2 did not reverse this negative effect. Chondrogenic inhibition is similar in cells treated only for the first week of chondrogenic differentiation or continuously for 3 weeks. Gene analysis shows a strong downregulation of TGF-β3 and FGF-1 while TNF was upregulated.Conclusion: NSAIDs seem to affect the transition phase of mesenchymal stem cells towards functional chondrocytes. This effect is unrelated to the endogenous production of PGE-2. The downregulation of the expression of key molecules like TGF-β3 seem to be the underlying mechanism. [ABSTRACT FROM AUTHOR]

Objective: Due to the small size of the murine knee joint, extracting the chondrocyte transcriptome from articular cartilage (AC) is a major technical challenge. In this study, we demonstrate a new pragmatic approach of combining bulk RNA-sequencing (RNA-seq) and single cell (sc)RNA-seq to address this problem.Design: We propose a new cutting strategy for the murine femur which produces three segments with a predictable mixed cell population, where one segment contains AC and growth plate (GP) chondrocytes, another GP chondrocytes, and the last segment only bone and bone marrow. We analysed the bulk RNA-seq of the different segments to find distinct genes between the segments. The segment containing AC chondrocytes was digested and analysed via scRNA-seq.Results: Differential expression analysis using bulk RNA-seq identified 350 candidate chondrocyte gene in the AC segment. Gene set enrichment analysis of these genes revealed biological processes related- and non-related to chondrocytes, including, cartilage development (adj. P-value: 3.45E-17) and endochondral bone growth (adj. P-value 1.22E-4), respectively. ScRNA-seq of the AC segment found a cluster of 131 cells containing mainly chondrocytes. This cluster had 759 differentially expressed genes which enriched for extracellular matrix organisation (adj. P-value 7.76E-40) and other joint development processes. The intersection of the gene sets of bulk- and scRNA-seq contained 75 genes.Conclusions: Based on our results, we conclude that the combination of the two RNA-seq methods is necessary to precisely delineate the chondrocyte transcriptome and to study the disease phenotypes of chondrocytes in murine OA models in the future. [ABSTRACT FROM AUTHOR]

[Display omitted] • Chondrocyte ferroptosis promotes OA progression. • ICA inhibits inflammatory mediator release and reduces cartilage extracellular matrix degradation. • ICA inhibits chondrocyte ferroptosis by enhancing the SLC7A11/GPX4 signaling. • ICA attenuates the degradation of articular cartilage tissue and delays the progression of OA. Chondrocyte ferroptosis plays a critical role in the pathogenesis of osteoarthritis (OA), regulated by the SLC7A11/GPX4 signaling pathway. Icariin (ICA), a flavonoid glycoside, exhibits strong anti-inflammatory and antioxidant activities. This study investigated whether ICA could modulate the SLC7A11/GPX4 signaling to inhibit chondrocyte ferroptosis and alleviate OA. The objective was to explore the impact of ICA on chondrocyte ferroptosis in OA and its modulation of the SLC7A11/GPX4 signaling pathway. The anti-ferroptosis effects of ICA were evaluated in an interleukin-1β (IL-1β)-treated SW1353 cell model, using Ferrostatin-1 (Fer-1) and Erastin (Era) as ferroptosis inhibitor and inducer, respectively, along with GPX4 knockdown via lentivirus-based shRNA. Additionally, the therapeutic efficacy of ICA on OA-related articular cartilage damage was assessed in rats through histopathology and immunohistochemistry (IHC). IL-1β treatment upregulated the expression of OA-associated matrix metalloproteinases (MMP3 and MMP1), a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS-5), and increased intracellular ROS, lipid ROS, and MDA levels while downregulating collagen II and SOX9 expression in SW1353 cells. ICA treatment countered the IL-1β-induced upregulation of MMPs and ADAMTS-5, restored collagen II and SOX9 expression, and reduced intracellular ROS, lipid ROS, and MDA levels. Furthermore, IL-1β upregulated P53 but downregulated SLC7A11 and GPX4 expression in SW1353 cells, effects that were mitigated by ICA or Fer-1 treatment. Significantly, ICA also alleviated Era-induced ferroptosis, whereas it had no effect on GPX4-silenced SW1353 cells. In vivo , ICA treatment reduced articular cartilage damage in OA rats by partially restoring collagen II and GPX4 expression, inhibiting cartilage extracellular matrix (ECM) degradation and chondrocyte ferroptosis. ICA treatment mitigated chondrocyte ferroptosis and articular cartilage damage by enhancing the SLC7A11/GPX4 signaling, suggesting its potential as a therapeutic agent for OA interventions. [ABSTRACT FROM AUTHOR]

The transcription factor, signal transducer, and stimulator of transcription 3 (STAT3) is a potential target in osteoarthritis (OA) treatment. Although xanthatin (XA), a biologically active substance derived from Xanthium strumarium L, specifically inhibits STAT3 phosphorylation at Tyr705, the mechanism underlying its inhibitory effect on OA progression remains unclear. In this study, our objective was to explore the therapeutic effects exerted by XA on OA and the underlying molecular mechanisms. The effects of XA treatment on mouse OA models subjected to destabilization of the medial meniscus using medial collateral ligament transection, as well as on interleukin-1β (IL-1β)-induced mouse chondrocytes, were examined. Histological changes in cartilage and subchondral bone (SCB), as well as changes in the expression levels of osteophytes, cartilage degeneration- and osteoclast differentiation-related factors, and the role of XA-related signaling pathways in human cartilage tissue, were studied using different techniques. XA inhibited STAT3 phosphorylation at Tyr705 and further attenuated the activity of nuclear factor-κB (NF-κB) in chondrocytes and osteoclasts. In vitro , XA administration alleviated pro-inflammatory cytokine release, extracellular matrix catabolism, and RANKL-mediated osteoclast differentiation. In vivo , intraperitoneal injection of XA exerted a protective effect on cartilage degeneration and SCB loss. Similarly, XA exerted a protective effect on human cartilage tissue by inhibiting the STAT3/NF-κB signaling pathway. Overall, our study elucidated the therapeutic potential of XA as a small-molecule inhibitor of STAT3-driven OA progression. This discovery may help enhance innovative clinical interventions against OA. [Display omitted] • XA was identified as a specific STAT3 (Tyr705) inhibitor for OA treatment. • XA protects AC and SCB via STAT3/NFκB signaling. • XA selectively targets Tyr705 activation in STAT3. • XA prevents early SCB osteoporosis and cartilage decomposition. [ABSTRACT FROM AUTHOR]