Activity Of Chondrocytes Research Articles

Mechanical overloading induces GPX4-regulated chondrocyte ferroptosis in osteoarthritis via Piezo1 channel facilitated calcium influx

IntroductionsExcessive 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. ObjectivesTo identify whether excessive mechanical stress can cause ferroptosis. To explore the role of mechanical overloading in chondrocyte ferroptosis. MethodsChondrocytes 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. ResultsHuman 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. ConclusionsOur 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.

Apoptosis Activation and Autophagy Inhibition of Chondrocytes by Leptin by the Upregulation of LOXL3 in Osteoarthritis Pathogenesis.

Background Osteoarthritis is one of the usual chronic musculoskeletal dysfunctions. It is one of the primary leading causes which leads to limitation of movement and absenteeism in the working adult population. Chondrocytes are the singlecellular-based component found in the cartilage which has an important role in the degradation of the cartilage. In recent studies, autophagy is observed to protect the human chondrocytes from stress.Leptin an adipokine managing food consumption and energy outlay. Chondrocytes indicate prolonged isoform of the leptin receptor where inside these cells theleptin signals individually or combine with the remaining molecules and promptthe indication of the pro-inflammatory molecules and cartilage disintegration enzymes. Materials and Methods mRNA expressions of Lysyl oxidase-like 3 in tissues of cartilage and concentration of leptin from synovial fluidwere measured from all samples from disease-induced groups, sham group, and RAPA-treated groups via RT-PCR and immunoassays. Histopathological analysis was also performed post-induction of the rat osteoarthritis model by the anterior cruciate ligament transection method. Western blot analysis was done, and expressions were analyzed by autophagy and apoptosis regulatory markers. Cell apoptosis and cell survival were evaluated with the help of flow cytometry, respectively, in all groups. Result mRNA of LOXL3 was increased in osteoarthritis models which were directly related to leptin concentration in SF. ACLT surgery caused an increase in cleaved caspase 3 protein levels, while a significant reduction in Bcl-2, Beclin1, and LC3 I was noted (figure 4,5). When LOXL3 was silenced in the ACLT group and leptin-treated group apoptosis was inhibited and autophagy, cell proliferation was promoted in primary chondrocytes. A significant increase in LOXL3 caused inhibition of autophagy in chondrocytes. Conclusion LOXL3 has stimulated apoptosis while inhibited autophagy in chondrocytes; hence LOXL3 is a prominent target for treating osteoarthritis. Keywords:chondrocytes, LOXL3, Leptin, osteoarthritis, qRT-PCR, ACLT, mRNA.

Ear reconstruction research using animal models: The effect of fat grafting on costal cartilage stents

ObjectFor congenital microtia patients with a depressed mastoid area, it is unclear whether autologous fat grafting to fill the depressed area of the cheek will affect the survival of the subsequent grafted costal cartilage stent. An animal model was used for in vivo research to provide guidance for clinical applications. MethodsAutologous costal cartilage was implanted in nude mice. Fat samples were collected at different time points and histological examination performed to analyze the activity of chondrocytes and the deposition of the chondrocyte matrix. ResultsThis nude mouse fat transplantation model study showed that there were statistical differences in chondrocyte viability between the fat filling group and the control group, but there was no statistical difference in the effect on collagen content. ConclusionTransplanting fat reduces the viability of chondrocytes, but has little effect on collagen matrix deposition.

Role of Exosomal Non-Coding RNAs in Bone-Related Diseases.

Bone-related diseases seriously affect the lives of patients and carry a heavy economic burden on society. Treatment methods cannot meet the diverse clinical needs of affected patients. Exosomes participate in the occurrence and development of many diseases through intercellular communication, including bone-related diseases. Studies have shown that exosomes can take-up and “package” non-coding RNAs and “deliver” them to recipient cells, thereby regulating the function of recipient cells. The exosomal non-coding RNAs secreted by osteoblasts, osteoclasts, chondrocytes, and other cells are involved in the regulation of bone-related diseases by inhibiting osteoclasts, enhancing chondrocyte activity and promoting angiogenesis. Here, we summarize the role and therapeutic potential of exosomal non-coding RNAs in the bone-related diseases osteoporosis, osteoarthritis, and bone-fracture healing, and discuss the clinical application of exosomes in patients with bone-related diseases.

Moderate-intensity exercise alleviates pyroptosis by promoting autophagy in osteoarthritis via the P2X7/AMPK/mTOR axis

Instability and excessive use of the knee joint can cause osteoarthritis (OA). Reasonable exercise can enhance the stability of the knee joint and prevent and relieve the occurrence and development of OA. As a key switch for inflammation, P2X purinoceptor 7 (P2X7) has attracted much attention in studies of OA. Exercise can regulate P2X7 expression and activation. However, the role of P2X7 in exercise-based prevention and treatment of OA is unknown. We previously showed that moderate-intensity exercise can significantly alleviate OA symptoms. Accordingly, in this study, we evaluated the effects of exercise on P2X7 expression and activation in chondrocytes. Micro-computed tomography, hematoxylin, and eosin staining, Toluidine Blue O staining, immunohistochemistry, and terminal deoxynucleotidyl transferase dUTP nick-end labeling experiments showed that P2X7 expression was lower in the moderate-intensity exercise group than in the inflammation and low- and high-intensity exercise groups. Additionally, chondrocyte death, cartilage destruction, and the degree and severity of pyroptosis were significantly reduced, whereas autophagy levels were significantly increased in the moderate-intensity exercise group. Cell Counting Kit-8 assay, lactate dehydrogenase release, flow cytometry, enzyme-linked immunosorbent assay, cell fluorescence, western blot, reverse transcription-quantitative polymerase chain reaction, and transmission electron microscopy experiments showed that moderate activation of P2X7 promoted autophagy through the AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway and promoted autolysosome targeting for degradation of the inflammasome component NLRP3, thereby inhibiting pyroptosis. Additionally, the use of AMPK and mTOR activators and inhibitors indicated that the AMPK-mTOR signaling pathway, as the downstream of P2X7, played a key role in delaying the occurrence and development of OA. We propose that moderate-intensity exercise promoted chondrocyte autophagy through the P2X7/AMPK/mTOR signal axis to alleviate pyroptosis. Our findings provide novel insights into the positive and preventative effects of exercise on OA.

CDDO-Im ameliorates osteoarthritis and inhibits chondrocyte apoptosis in mice via enhancing Nrf2-dependent autophagy.

Osteoarthritis (OA) is the most prevalent chronic degenerative joint disease with few treatment options. The pathogenesis of OA is characterized by sustained inflammation, oxidative stress and chondrocyte apoptosis that eventually lead to cartilage degradation and joint dysfunction. In the present study, we identified a synthetic triterpenoid CDDO-Im(1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl] imidazole) as an activator of Nrf2 (nuclear factor erythroid 2-related factor 2) that displayed strong anti-OA effects. We showed that CDDO-Im (20 nM) significantly alleviated TNF-α-induced apoptosis of primary human chondrocytes and extracellular matrix degradation. In a mouse OA model incurred by DMM (destabilization of medial meniscus), administration of CDDO-Im (2.5 mg/kg, ip, every other day for 8 weeks) effectively reduced knee joint cartilage erosion and serum levels of inflammatory cytokines IL-1β and IL-6. We revealed that CDDO-Im (20 nM) significantly enhanced autophagy activities in chondrocytes, whereas the autophagy inhibition by chloroquine (CQ, 50 μM) or 3-methyladenine (3-MA, 5 mM) abrogated the anti-apoptosis and chondroprotective effects of CDDO-Im in TNF-α-treated chondrocytes. Moreover, we confirmed that CDDO-Im (1-20 nM) dose-dependently activated Nrf2 pathway in TNF-α-treated chondrocytes, and its chondroprotective and autophagy-enhancing effects were significantly diminished when Nrf2 signaling was blocked by Nrf2 inhibitor ML385 (20 μM) or siRNA-mediated Nrf2 knockdown. Together, our results demonstrate that CDDO-Im exhibits prominent chondroprotective and anti-OA activities owing to its Nrf2 activation and autophagy-enhancing properties, which might provide new insights into the strategies of OA clinical prevention and treatment.

Low deacetylation degree chitosan oligosaccharide protects against IL-1β induced inflammation and enhances autophagy activity in human chondrocytes

Osteoarthritis (OA) is a degenerative joint disease, which can lead to joint pain, stiffness, deformity and dysfunction, that seriously affects the quality of life in patients. At present, the treatments of OA mainly include early pharmacological treatment and late joint replacement. However, current pharmacological treatment has limited efficacy and undesired side effects. Chitosan oligosaccharide (COS) is a kind of nontoxic and biodegradable oligo-saccharide, which is composed of 2–20 glucosamine or N-acetylglucosamine linked by β-1,4 glycosidic bond. Studies have shown that COS has significant biological properties like antimicrobial, anti-inflammatory, antioxidant, and anti-tumor, as well as immunoregulation ability. However, the effects of COS on OA have not been clarified. In this study, we explored the protective effects of COS with different degrees of deacetylation on chondrocytes stimulated by interleukin 1β (IL-1β) in vitro. The results showed that IL-1β inhibited cell proliferation and promoted cell apoptosis. Besides that, IL-1β increased the expression of the major chondro-degrading genes MMP13 and ADAMTS-5, while decreased the expression of COL2A and ACAN. COS with different degrees of deacetylation (HDACOS, MDACOS, LDACOS) had different effects on IL-1β induced inflammation. LDACOS had the most obvious anti-inflammatory effects to inhibit the expression of MMP13 and ADAMTS-5 while promoted the expression of COL2A and ACAN. In addition, we found that the expression of autophagy-related gene Beclin-1 was up-regulated, and the ratio of LC3-II/LC3-I was increased in the LDACOS group. Furthermore, transmission electron microscopy (TEM) analysis showed that the number of intracellular autophagosomes increased significantly with the treatment of LDACOS. Based on our research, we suggested that LDACOS could inhibit chondrocytes inflammation and promote cell autophagy, and might be a protective drug for the treatment of OA.

Protective Effects of Annatto Tocotrienol and Palm Tocotrienol-Rich Fraction on Chondrocytes Exposed to Monosodium Iodoacetate

Background: This study aimed to compare the chondroprotective efficacy and mechanism of annatto tocotrienol (AnTT) and palm tocotrienol-rich fraction (PT3) using SW1353 chondrocytes treated with monosodium iodoacetate (MIA). Methods: The chondrocytes were incubated with AnTT or PT3 in advance or concurrently with MIA for 24 h. The viability of the cells was tested with an MTT assay. The 8-isoprostane F2-α, extracellular matrix proteins, metalloproteinase and sex-determining region Y box protein 9 (SOX9) levels were determined using immunoassays. Results: AnTT and PT3 reversed an MIA-induced decrease in chondrocyte viability when incubated together with MIA (p < 0.05). Prior incubation with both mixtures did not produce the same effects. AnTT and PT3 cotreatment could suppress 8-isoprostane F2-α level in chondrocytes exposed to MIA (p < 0.01). Co-exposure to tocotrienols and MIA increased the type II collagen/type I collagen ratio in chondrocytes (p < 0.01). In addition, the co-exposure of AnTT and MIA for 24 h significantly upregulated SOX9, type II collagen and aggrecan levels (p < 0.05), which was not observed with co-exposure of PT3 and MIA, AnTT or PT3 exposure alone. Conclusion: AnTT and PT3 could prevent a reduction in chondrocyte viability following MIA exposure by reducing oxidative stress. In addition, AnTT might induce self-repair and anabolic activities in chondrocytes challenged with MIA.

SETD7 regulates chondrocyte differentiation and glycolysis via the Hippo signaling pathway and HIF‑1α.

Chondrocytes are well adapted to hypoxia and produce more functional extracellular matrix in low oxygen environments in vitro. In our previous study, methyltransferase SET domain containing (SETD)7 regulated chondrocyte activity in hypoxic conditions. However, the precise association between SETD7 and chondrocyte differentiation under low oxygen partial pressure remains unclear. The association between SETD7 and chondrocyte differentiation was studied by silencing SETD7 in chondrocytes in vitro. The results showed that the silencing of SETD7 in ATDC5 cells inhibited the Hippo signaling pathway, decreased Yes-associated protein (YAP) phosphorylation and increased the levels of YAP and hypoxia inducible factor-1α (HIF-1α) in the nucleus. YAP combined with HIF-1α to form a complex that promoted the expression of genes involved in chondrogenic differentiation and the glycolytic pathway. Thus, SETD7 inhibited chondrocyte differentiation and glycolysis via the Hippo signaling pathway. The present study demonstrated that SETD7 was a potential molecular target that maintained the chondrocyte phenotype during cartilage tissue engineering and cartilage-associated disease.

Phenotype and synthesis activity of joint chondrocytes extracted from newborn rats with prenatal ethanol exposure.

Thirteen female Wistar rats were divided into two groups: one treated with ethanol and the other of untreated. Four newborns from each mother were selected and weighed, measured, and evaluated for physical characteristics. From these neonates, chondrocytes were extracted from the articular cartilages of the femur and tibia, and cultivated in a chondrogenic medium at 37oC and 5% CO2. At 7, 14, and 21days of cultivation, alkaline phosphatase activity tests, MTT conversion to formazan, and percentage area covered by cells per field were performed. At 21days, the percentage of PAS+ areas in 3D cultures was performed, as well as the evaluation of gene transcript expression for aggrecan, SOX-9, collagen type II, collagen X, Runx-2, and VEGF by real-time RT-PCR. The means were compared by Student's t-test. The weight of the ethanol group neonates was significantly lower than that of the controls. Chondrocyte cultures from the ethanol group showed significantly higher AP activity, MTT conversion, and cell percentage. There was higher expression of collagen type II and lower expression of SOX-9 in the ethanol group. There was no difference in the percentage of PAS+ areas in pellets and in expression of aggrecan, collagen X, Runx-2, or VEGF between groups. In conclusion, prenatal exposure to ethanol alters the phenotype and activity of offspring chondrocytes, which may be mechanisms by which endochondral bone formation is compromised by maternal ethanol consumption.

The Interaction between microRNAs and the Wnt/β-Catenin Signaling Pathway in Osteoarthritis.

Osteoarthritis (OA) is a chronic disease affecting the whole joint, which still lacks a disease-modifying treatment. This suggests an incomplete understanding of underlying molecular mechanisms. The Wnt/β-catenin pathway is involved in different pathophysiological processes of OA. Interestingly, both excessive stimulation and suppression of this pathway can contribute to the pathogenesis of OA. microRNAs have been shown to regulate different cellular processes in different diseases, including the metabolic activity of chondrocytes and osteocytes. To bridge these findings, here we attempt to give a conclusive overview of microRNA regulation of the Wnt/β-catenin pathway in bone and cartilage, which may provide insights to advance the development of miRNA-based therapeutics for OA treatment.

Randomised, double-blind comparison of a fixed co-formulation of intra-articular polynucleotides and hyaluronic acid versus hyaluronic acid alone in the treatment of knee osteoarthritis: two-year follow-up

BackgroundA first-year interim analysis of this two-year study suggested that intra-articular injections of highly purified, natural-origin polynucleotides and hyaluronic acid (HA) as a fixed combination (PNHA) might improve knee function and joint pain more effectively than HA alone in patients with knee osteoarthritis (OA). The purpose of the second-year analysis herein described was to verify whether the first-year interim outcomes persist over the whole two-year period.MethodsRandomised, double-blind, HA-controlled clinical trial in 100 knee OA patients (98 randomised, 79 completing the study) in a high-specialisation tertiary care setting. The hypothesised difference of efficacy between PNHA and HA for the original sample size estimate is 20%. Treatment cycle: three intra-articular knee injections of either PNHA or HA, at baseline and weekly for two weeks. Evaluations: Western Ontario and McMaster Universities (WOMAC) score and Knee Society Score (KSS) as, respectively, primary and secondary endpoints, evaluated at baseline and after 2, 6, 12, and 24 months; synovial fluid levels of mediators (at baseline and the end of the treatment cycle). Adverse effects investigated at each control visit. Statistical analysis: Kruskal-Wallis test for independent samples (nonparametric one-way analysis of variance) after correction of means for age, Body Mass Index and Kellgren-Lawrence grade. If significant, pairwise post-hoc Sidak multiple comparisons.ResultsKSS total score and KSS pain item: significant improvement in both groups, with significantly more pain improvement in patients treated with PNHA (2-point reduction) than HA (1-point reduction). Both groups experienced significant long-term reductions in WOMAC total scores: significantly stronger in PNHA-treated patients after 24 months with a steady difference of 16% favouring PNHA in WOMAC pain subscore. No clinically significant adverse events in either group.ConclusionsThe outcomes of the 2-year study confirmed that a short cycle of intra-articular treatment (3 weekly double-blind injections) with polynucleotides (long-acting viscosupplementation properties, chondrocyte activation, pain-relieving properties) in fixed combination with high molecular weight hyaluronic acid is more effective in improving knee function and pain in knee OA patients than HA alone. PNHA may be elective for viscosupplementation in knee OA patients with fastidious and resistant pain and worsening disease.Trial registrationNCT02417610.Registration, 15/04/2015.ClinicalTrials.gov database link:

The Gasotransmitter Hydrogen Sulfide (H2S) Prevents Pathologic Calcification (PC) in Cartilage.

Pathologic calcification (PC) is a painful and disabling condition whereby calcium-containing crystals deposit in tissues that do not physiologically calcify: cartilage, tendons, muscle, vessels and skin. In cartilage, compression and inflammation triggered by PC leads to cartilage degradation typical of osteoarthritis (OA). The PC process is poorly understood and treatments able to target the underlying mechanisms of the disease are lacking. Here we show a crucial role of the gasotransmitter hydrogen sulfide (H2S) and, in particular, of the H2S-producing enzyme cystathionine γ-lyase (CSE), in regulating PC in cartilage. Cse deficiency (Cse KO mice) exacerbated calcification in both surgically-induced (menisectomy) and spontaneous (aging) murine models of cartilage PC, and augmented PC was closely associated with cartilage degradation (OA). On the contrary, Cse overexpression (Cse tg mice) protected from these features. In vitro, Cse KO chondrocytes showed increased calcification, potentially via enhanced alkaline phosphatase (Alpl) expression and activity and increased IL-6 production. The opposite results were obtained in Cse tg chondrocytes. In cartilage samples from patients with OA, CSE expression inversely correlated with the degree of tissue calcification and disease severity. Increased cartilage degradation in murine and human tissues lacking or expressing low CSE levels may be accounted for by dysregulated catabolism. We found higher levels of matrix-degrading metalloproteases Mmp-3 and -13 in Cse KO chondrocytes, whereas the opposite results were obtained in Cse tg cells. Finally, by high-throughput screening, we identified a novel small molecule CSE positive allosteric modulator (PAM), and demonstrated that it was able to increase cellular H2S production, and decrease murine and human chondrocyte calcification and IL-6 secretion. Together, these data implicate impaired CSE-dependent H2S production by chondrocytes in the etiology of cartilage PC and worsening of secondary outcomes (OA). In this context, enhancing CSE expression and/or activity in chondrocytes could represent a potential strategy to inhibit PC.

Sex-specific effects of 17β-estradiol and dihydrotestosterone (DHT) on growth plate chondrocytes are dependent on both ERα and ERβ and require palmitoylation to translocate the receptors to the plasma membrane

Rat costochondral cartilage growth plate chondrocytes exhibit cell sex-specific responses to 17β-estradiol (E2), testosterone, and dihydrotestosterone (DHT). Mechanistically, E2 and DHT stimulate proliferation and extracellular matrix synthesis in chondrocytes from female and male rats, respectively, by signaling through protein kinase C (PKC) and phospholipase C (PLC). Estrogen receptors (ERα; ERβ) and androgen receptors (ARs) are present in both male and female cells, but it is not known whether they interact to elicit sex-specific signaling. We used specific agonists and antagonists of these receptors to examine the relative contributions of ERs and ARs in membrane-mediated E2 signaling in female chondrocytes and DHT signaling in male chondrocytes. PKC activity in female chondrocytes was stimulated by agonists of ERα and ERβ and required intact caveolae; PKC activity was inhibited by the E2 enantiomer and by an inhibitor of ERβ. Western blots of cell lysates co-immunoprecipitated for ERα suggested the formation of a complex containing both ERα and ERß with E2 treatment. DHT and DHT agonists activated PKC in male cells, while AR inhibition blocked the stimulatory effect of DHT on PKC. Inhibition of ERα and ERβ also blocked PKC activation by DHT. Western blots of whole-cell lysates, plasma membranes, and caveolae indicated the translocation of AR to the plasma membrane and specifically to caveolae with DHT treatment. These results suggest that E2 and DHT promote chondrocyte differentiation via the ability of ARs and ERs to form a complex. The results also indicate that intact caveolae and palmitoylation of the membrane receptor(s) or membrane receptor complex containing ERα and ERβ is required for E2 and DHT membrane-associated PKC activity in costochondral cartilage cells.

HMGB2 promotes chondrocyte proliferation under negative pressure through the phosphorylation of AKT

Cells in articular cartilage are zonal arranged. Cells in superficial zone cartilage are generally small and proliferative. Appropriate negative pressure stimulation is beneficial to cell survival and tissue repair. Whether negative pressure has promotive impact on the proliferation activity of the superficial zone chondrocytes is of interest. In this study, we isolated superficial chondrocytes from the mandibular condylar cartilage of rats. After negative pressure treatment, the cells were collected for RNA-sequencing, quantitative real-time PCR and western blotting assays, aiming to detect the proliferative responses of chondrocytes to negative pressure and explore the potential molecular mechanisms. Data from RNA-sequencing analysis indicated that the superficial chondrocytes responded to the 4 h −10 kPa treatment by a significant increase in proliferation. In addition, the expression of high-mobility group box 2 (HMGB2) and the phosphorylation of AKT were obviously promoted. Knockdown of HMGB2 decreased AKT phosphorylation and diminished the negative pressure-induced proliferation of chondrocytes, as shown by decreased expression of Ki67 and cyclin-dependent kinase 6 (CDK6). In contrast, overexpression of HMGB2 enhanced AKT phosphorylation and further promoted proliferative activity. Moreover, LY294002, an AKT inhibitor, suppressed the proliferative activity of chondrocytes under negative pressure, while SC79, an activator of AKT phosphorylation, enhanced the proliferation of chondrocytes. Our data demonstrated that HMGB2 exhibits a promotion impact on chondrocyte proliferation under negative pressure via the phosphorylation of AKT. These results provide a new perspective for superficial zone chondrocytes proliferation under negative pressure, which should be benefit for cartilage regeneration.

Osteoarthritis-Related Inflammation Blocks TGF-β's Protective Effect on Chondrocyte Hypertrophy via (de)Phosphorylation of the SMAD2/3 Linker Region.

Osteoarthritis (OA) is a degenerative joint disease characterized by irreversible cartilage damage, inflammation and altered chondrocyte phenotype. Transforming growth factor-β (TGF-β) signaling via SMAD2/3 is crucial for blocking hypertrophy. The post-translational modifications of these SMAD proteins in the linker domain regulate their function and these can be triggered by inflammation through the activation of kinases or phosphatases. Therefore, we investigated if OA-related inflammation affects TGF-β signaling via SMAD2/3 linker-modifications in chondrocytes. We found that both Interleukin (IL)-1β and OA-synovium conditioned medium negated SMAD2/3 transcriptional activity in chondrocytes. This inhibition of TGF-β signaling was enhanced if SMAD3 could not be phosphorylated on Ser213 in the linker region and the inhibition by IL-1β was less if the SMAD3 linker could not be phosphorylated at Ser204. Our study shows evidence that inflammation inhibits SMAD2/3 signaling in chondrocytes via SMAD linker (de)-phosphorylation. The involvement of linker region modifications may represent a new therapeutic target for OA.

Cultured Horse Articular Chondrocytes in 3D-Printed Chitosan Scaffold With Hyaluronic Acid and Platelet Lysate.

Three-dimensional (3D) printing has gained popularity in tissue engineering and in the field of cartilage regeneration. This is due to its potential to generate scaffolds with spatial variation of cell distribution or mechanical properties, built with a variety of materials that can mimic complex tissue architecture. In the present study, horse articular chondrocytes were cultured for 2 and 4 weeks in 3D-printed chitosan (CH)-based scaffolds prepared with or without hyaluronic acid and in the presence of fetal bovine serum (FBS) or platelet lysate (PL). These 3D culture systems were analyzed in terms of their capability to maintain chondrocyte differentiation in vitro. This was achieved by evaluating cell morphology, immunohistochemistry (IHC), gene expression of relevant cartilage markers (collagen type II, aggrecan, and Sox9), and specific markers of dedifferentiated phenotype (collagen type I, Runx2). The morphological, histochemical, immunohistochemical, and molecular results demonstrated that the 3D CH scaffold is sufficiently porous to be colonized by primary chondrocytes. Thereby, it provides an optimal environment for the colonization and synthetic activity of chondrocytes during a long culture period where a higher rate of dedifferentiation can be generally observed. Enrichment with hyaluronic acid provides an optimal microenvironment for a more stable maintenance of the chondrocyte phenotype. The use of 3D CH scaffolds causes a further increase in the gene expression of most relevant ECM components when PL is added as a substitute for FBS in the medium. This indicates that the latter system enables a better maintenance of the chondrocyte phenotype, thereby highlighting a fair balance between proliferation and differentiation.

Automated quantification of live articular chondrocyte fluorescent staining using a custom image analysis framework.

The goal of this study was to develop, validate, and implement an image analysis framework to automatically analyze chondrocytes in 3D image stacks of cartilage acquired using a fluorescent confocal microscope. Source specimens consist of viable osteochondral tissue co-stained with multiple live-cell dyes. Our framework utilizes a seeded watershed-based algorithm to automatically segment individual chondrocytes in each 2D slice of the confocal image stack. The resulting cell segmentations are colocalized in 3D to eliminate duplicate segmentation of the same cell resulting from the visibility of fluorescence signal in multiple imaging planes, and the 3D cell distribution is used to automatically define the cartilage tissue volume. The algorithm then provides chondrocyte density data, and the associated segmentation can be used as a mask to extract and quantify per cell intensity of a secondary, functional dye co-staining the chondrocytes. The accuracy of the automated chondrocyte segmentation was validated against manual segmentations (average IOU = 0.79). When applied to a cartilage surrogate, this analysis framework estimated chondrocyte density within 10% of the true density and demonstrated a good agreement between framework's counts and manual counts (R2 = 0.99). In a real application, the framework was able to detect the increased dye signal of monochlorobimane (MCB) in chondrocytes treated with N-acetylcysteine (NAC) after mechanical injury, quantifying intracellular biochemical changes in living cells. This new framework allows for fast and accurate quantification of intracellular activities of chondrocytes, and it can be adapted for broader application in many imaging and treatment modalities, including therapeutic OA research.

Growth factor-loaded microspheres in mPEG-polypeptide hydrogel system for articular cartilage repair.

We developed an injectable hydrogel system with a sustained release of TGF-β3 through growth factor-loaded microsphere to mimic the cartilage-like microenvironment. Poly(lactic-co-glycolic acid) (PLGA) microspheres incorporated in three dimensional (3D) scaffolds were chosen because of its regulatory approval, good biodegradability, and acting as carriers with sustained release behavior. We evaluated sustained release of TGF-β3 by PLGA microspheres encapsulated in methoxy poly(ethylene glycol)-poly(alanine) (mPA) hydrogels and the resulting enhanced chondrogenic effects. We reported here the effect of the proposed system for sustained release of growth factors on chondrogenesis in cartilage regeneration. PLGA microspheres were used in our thermosensitive mPA hydrogel system with bovine serum albumin as a stabilizing and protecting agent for the emulsion and TGF-β3 enabling sustained release. Gelation, structural properties, and in-vitro release of this composite, that is, microspheres in hydrogel, system were investigated. Using PLGA microspheres to carry growth factors could complement the mPA hydrogel's ability to provide an excellent 3D microenvironment for the promotion of chondrogenic phenotype as compared the systems using mPA hydrogel or microspheres alone. Our study demonstrated that this synthesized composite hydrogel system is capable of modulating the biosynthetic and differentiation activities of chondrocytes. The sustained release of TGF-β3 in this novel hydrogel system could improve biomedical applicability of mPEG-polypeptide scaffolds. The distinctive local growth factor delivery system successfully combined the use of both polymers to be a suitable candidate for prolonged articular cartilage regeneration.

ATF6 aggravates angiogenesis-osteogenesis coupling during ankylosing spondylitis by mediating FGF2 expression in chondrocytes

SummaryAlthough angiogenesis-osteogenesis coupling is important in ankylosing spondylitis (AS), therapeutic agents targeting the vasculature remain elusive. Here, we identified activating transcription factor 6 (ATF6) as an important regulator of angiogenesis in the pathogenesis of AS. First, we found that ATF6 and fibroblast growth factor 2 (FGF2) levels were higher in SKG mice and in cartilage of pateints with AS1. The proangiogenic activity of human chondrocytes was enhanced by the activation of the ATF6-FGF2 axis following 7 days of stimulation with inflammatory factors, e.g., tumor necrosis factor alpha (TNF-α), interferon-γ (IFN-γ) or interleukin-17 (IL-17). Mechanistically, ATF6 interacted with the FGF2 promotor and promoted its transcription. Treatment with the ATF6 inhibitor Ceapin-A7 inhibited angiogenesis in vitro and angiogenesis-osteogenesis coupling in vivo. ATF6 may aggravate angiogenesis-osteogenesis coupling during AS by mediating FGF2 transcription in chondrocytes, implying that ATF6 represents a promising therapeutic target for AS.