ELIXCYTE®, an Allogenic Adipose-Derived Stem Cell Product, Mitigates Osteoarthritis by Reducing Inflammation and Preventing Cartilage Degradation In Vitro.

Adipose-derived stem cells (ADSCs) comprise a promising therapy for osteoarthritis (OA). The therapeutic potential of ELIXCYTE®, an allogeneic human ADSC (hADSC) product, was demonstrated in a phase I/II OA clinical trial. However, the exact mechanism underlying such effects is not clear. Moreover, studies suggest that interleukin-11 (IL-11) has anti-inflammatory, tissue-regenerative, and immune-regulatory functions. Our aim was to unravel the mechanism associated with the therapeutic effects of ELIXCYTE® on OA and its relationship with IL-11. We cocultured ELIXCYTE® with normal human articular chondrocytes (NHACs) in synovial fluid obtained from individuals with OA (OA-SF) to investigate its effect on chondrocyte matrix synthesis and degradation and inflammation by assessing gene expression and cytokine levels. NHACs exposed to OA-SF exhibited increased MMP13 expression. However, coculturing ELIXCYTE® with chondrocytes in OA-SF reduced MMP13 expression in chondrocytes and downregulated PTGS2 and FGF2 expression in ELIXCYTE®. ELIXCYTE® treatment elevated anti-inflammatory cytokine (IL-1RA, IL-10, and IL-13) levels, and the reduction in MMP13 was positively correlated with IL-11 concentrations in OA-SF. These findings indicate that IL-11 in OA-SF might serve as a predictive biomarker for the ELIXCYTE® treatment response in OA, emphasizing the therapeutic potential of ELIXCYTE® to mitigate OA progression and provide insights into its immunomodulatory effects.

Insights into the Roles of Natural Killer Cells in Osteoarthritis.

Osteoarthritis (OA) is now widely acknowledged as a low-grade inflammatory condition, in which the intrinsic immune system plays a significant role in its pathogenesis. While the involvement of macrophages and T cells in the development of OA has been extensively reviewed, recent research has provided mounting evidence supporting the crucial contribution of NK cells in both the initiation and advancement of OA. Accumulated evidence has emerged in recent years indicating that NK cells play a critical role in OA development and progression. This review will outline the ongoing understanding of the utility of NK cells in the etiology of OA, focusing on how NK cells interact with chondrocytes, synoviocytes, osteoclasts, and other immune cells to influence the course of OA disease.

Exosomes Derived from Hypoxia-Cultured Human Adipose Stem Cells Alleviate Articular Chondrocyte Inflammaging and Post-Traumatic Osteoarthritis Progression.

Osteoarthritis (OA) is the most common age-related degenerative joint disease. Inflammaging, linking inflammation and aging, is found in senescent cells with the secretions of matrix-degrading proteins and proinflammatory cytokines. The senescence-associated secretory phenotype (SASP) plays a very important role in OA progression. However, there remains no effective way to suppress OA progression, especially by suppressing inflammaging and/or the chondrocyte SASP. Recent studies have shown that exosomes derived from hypoxia-cultured BMSCs can regenerate cartilage in OA animal models. Some reports have further indicated that exosomes secreted from MSCs contribute to the efficacy of MSC therapy in OA. However, whether hypoxia-cultured ADSC-secreted exosomes (hypoxia-ADSC-Exos) can alleviate the chondrocyte SASP or OA progression remains unclear. Accordingly, we hypothesized that hypoxia-ADSC-Exos have a beneficial effect on the normal functions of human articular chondrocytes (HACs), can attenuate the SASP of OA-like HACs in vitro, and further suppress OA progression in rats. Hypoxia-ADSC-Exos were derived from ADSCs cultured in 1% O2 and 10% de-Exo-FBS for 48 h. The molecular and cell biological effects of hypoxia-ADSC-Exos were tested on IL1-ß-induced HACs as OA-like HACs in vitro, and the efficacy of OA treatment was tested in ACLT-induced OA rats. The results showed that hypoxia-ADSC-Exos had the best effect on GAG formation in normal HACs rather than those cultured in normoxia or hypoxia plus 2% de-Exo-FBS. We further found that hypoxia-ADSC-Exos alleviated the harmful effect in OA-like HACs by decreasing markers of normal cartilage (GAG and type II collagen) and increasing markers of fibrous or degenerative cartilage (type I or X collagen), matrix degradation enzymes (MMP13 and ADAMT5), and inflammatory cytokines (TNFα and IL-6). More importantly, intra-articular treatment with hypoxia-ADSC-Exos suppressed OA progression, as evidenced by the weight-bearing function test and cartilage GAG quantification in ACLT rats. Moreover, through NGS and bioinformatic analysis, seven potential miRNAs were found in hypoxia-ADSC-Exos, which may contribute to regulating cellular oxidative stress and attenuating cell senescence. In summary, we demonstrated that hypoxia-ADSC-Exos, carrying potent miRNAs, not only improve normal HAC function but also alleviate HAC inflammaging and OA progression. The results suggest that hypoxia-ADSC-Exo treatment may offer another strategy for future OA therapy.

Systemic inhibition or global deletion of CaMKK2 protects against post-traumatic osteoarthritis.

OBJECTIVE: To investigate the role of Ca2+/calmodulin-dependent protein kinase 2 (CaMKK2) in post-traumatic osteoarthritis (PTOA). METHODS: Destabilization of the medial meniscus (DMM) or sham surgeries were performed on 10-week-old male wild-type (WT) and Camkk2-/- mice. Half of the DMM-WT mice and all other cohorts (n = 6/group) received tri-weekly intraperitoneal (i.p.) injections of saline whereas the remaining DMM-WT mice (n = 6/group) received i.p. injections of the CaMKK2 inhibitor STO-609 (0.033 mg/kg body weight) thrice a week. Study was terminated at 8- or 12-weeks post-surgery, and knee joints processed for microcomputed tomography imaging followed by histology and immunohistochemistry. Primary articular chondrocytes were isolated from knee joints of 4-6-day-old WT and Camkk2-/- mice, and treated with 10 ng/ml interleukin-1ß (IL)-1ß for 24 or 48 h to investigate gene and protein expression. RESULTS: CaMKK2 levels and activity became elevated in articular chondrocytes following IL-1ß treatment or DMM surgery. Inhibition or absence of CaMKK2 protected against DMM-associated destruction of the cartilage, subchondral bone alterations and synovial inflammation. When challenged with IL-1ß, chondrocytes lacking CaMKK2 displayed attenuated inflammation, cartilage catabolism, and resistance to suppression of matrix synthesis. IL-1ß-treated CaMKK2-null chondrocytes displayed decreased IL-6 production, activation of signal transducer and activator of transcription 3 (Stat3) and matrix metalloproteinase 13 (MMP13), indicating a potential mechanism for the regulation of inflammatory responses in chondrocytes by CaMKK2. CONCLUSIONS: Our findings reveal a novel function for CaMKK2 in chondrocytes and highlight the potential for its inhibition as an innovative therapeutic strategy in the prevention of PTOA.

Dynamics of Connexin 43 Down Modulation in Human Articular Chondrocytes Stimulated by Tumor Necrosis Factor Alpha.

Connexin 43 (Cx43) exerts pivotal functions in articular chondrocytes (CH). It is involved in the communication among cells and between cells and the extracellular environment, and it contributes to the maintenance of the correct cell phenotype. The pro-inflammatory cytokine TNFα induces a reduction in Cx43 expression in CH. Here, we studied the dynamics of this decrease in expression. We evaluated Cx43 protein and gene expression and the involvement of C-terminal domain (CTD) cleavage and proteasomal degradation. Treatments able to counteract TNFα action were also examined, together with Gap Junction (GJ) functionality and Cx43 localization. TNFα induced a significant reduction in Cx43 expression already at day 1, and the down modulation reached a peak at day 3 (-46%). The decrease was linked to neither gene expression modulation nor CTD cleavage. Differently, the proteasome inhibitor MG132 reverted TNFα effect, indicating the involvement of proteasomal degradation in Cx43 reduction. In addition, the co-treatment with the anabolic factor TGF-ß1 restored Cx43 levels. Cx43 decrease occurred both at the membrane level, where it partially influenced GJ communication, and in the nucleus. In conclusion, TNFα induced a rapid and lasting reduction in Cx43 expression mostly via the proteasome. The down modulation could be reverted by cartilage-protective factors such as MG132 and TGF-ß1. These findings suggest a possible involvement of Cx43 perturbation during joint inflammation.

Unsulfated biotechnological chondroitin by itself as well as in combination with high molecular weight hyaluronan improves the inflammation profile in osteoarthritis in vitro model.

Several studies suggest that inflammation has a pivotal role during the progression of osteoarthritis (OA) and cytokines have been identified as the main process mediators. This study aimed to explore the ability to modulate the main OA pro-inflammatory biomarkers of novel gels (H-HA/BC) based on high molecular weight hyaluronan (H-HA) and unsulfated biotechnological chondroitin (BC). For the first time, BC was tested also in combination with H-HA on human primary cells isolated from pathological knee joints. Specifically, the experiments were performed using an OA in vitro model based on human chondrocytes and synoviocytes. To evaluate the anti-inflammatory effects of H-HA/BC in comparison with H-HA and BC single gels, NF-kB, COMP-2, MyD88, MMP-13 and a wide range of cytokines, known to be specific biomarkers in OA (e.g., IL-6, IL-8, and TNF-α), were evaluated. In addition, cell morphology and proliferation occurring in the presence of either H-HA/BC or single components were assessed using time-lapse video microscopy. It was shown that synovial fluids and cells isolated from OA suffering patients, presented a cytokine pattern respondent to an ongoing inflammation status. H-HA and BC significantly reduced the levels of 23 biomarkers associated with cartilage damage. However, H-HA/BC decreased significantly 24 biological mediators and downregulated 19 of them more efficiently than the single components. In synoviocytes cultures, cytokine analyses proved that H-HA/BC gels re-established an extracellular environment more similar to a healthy condition reducing considerably the concentration of 11 analytes. Instead, H-HA and BC significantly modulated 7 (5 only with a longer treatment) and 8 biological cytokines, respectively. Our results suggest that H-HA/BC beyond the viscosupplementation effect typical for HA-based gels, can improve the inflammation status in joints and thus could be introduced as a valid protective and anti-inflammatory intraarticular device in the field of Class III medical devices for OA treatments.

Contribution of neural crest-derived stem cells and nasal chondrocytes to articular cartilage regeneration.

Due to poor self-regenerative potential of articular cartilage, stem cell-based regeneration becomes a hopeful approach for the treatment of articular cartilage defects. Recent studies indicate that neural crest-derived cells (NCDCs) have the potential for repairing articular cartilage with even greater chondrogenic capacity than mesoderm-derived cells (MDCs): a conventional stem cell source for cartilage regeneration. Given that NCDCs originate from a different germ layer in the early embryo compared with MDCs that give rise to articular cartilage, a mystery remains regarding their capacity for articular cartilage regeneration. In this review, we summarize the similarities and differences between MDCs and NCDCs including articular and nasal chondrocytes in cell origin, anatomy, and chondrogenic differentiation and propose that NCDCs might be promising cell origins for articular cartilage regeneration.

Red panda: a novel method for detecting variants in single-cell RNA sequencing.

BACKGROUND: Single-cell sequencing enables us to better understand genetic diseases, such as cancer or autoimmune disorders, which are often affected by changes in rare cells. Currently, no existing software is aimed at identifying single nucleotide variations or micro (1-50 bp) insertions and deletions in single-cell RNA sequencing (scRNA-seq) data. Generating high-quality variant data is vital to the study of the aforementioned diseases, among others. RESULTS: In this study, we report the design and implementation of Red Panda, a novel method to accurately identify variants in scRNA-seq data. Variants were called on scRNA-seq data from human articular chondrocytes, mouse embryonic fibroblasts (MEFs), and simulated data stemming from the MEF alignments. Red Panda had the highest Positive Predictive Value at 45.0%, while other tools-FreeBayes, GATK HaplotypeCaller, GATK UnifiedGenotyper, Monovar, and Platypus-ranged from 5.8-41.53%. From the simulated data, Red Panda had the highest sensitivity at 72.44%. CONCLUSIONS: We show that our method provides a novel and improved mechanism to identify variants in scRNA-seq as compared to currently existing software. However, methods for identification of genomic variants using scRNA-seq data can be still improved.

Acid-sensing ion channel 1a mediates acid-induced pyroptosis through calpain-2/calcineurin pathway in rat articular chondrocytes.

The pyroptosis is a causative agent of rheumatoid arthritis, a systemic autoimmune disease merged with degenerative articular cartilage. Nevertheless, the precise mechanism of extracellular acidosis on chondrocyte pyroptosis is largely unclear. Acid-sensing ion channels (ASICs) belong to an extracellular H+ -activated cation channel family. Accumulating evidence has highlighted activation of ASICs induced by extracellular acidosis upregulate calpain and calcineurin expression in arthritis. In the present study, to investigate the expression and the role of acid-sensing ion channel 1a (ASIC1a), calpain, calcineurin, and NLRP3 inflammasome proteins in regulating acid-induced articular chondrocyte pyroptosis, primary rat articular chondrocytes were subjected to different pH, different time, and different treatments with or without ASIC1a, calpain-2, and calcineurin, respectively. Initially, the research results showed that extracellular acidosis-induced the protein expression of ASIC1a in a pH- and time-dependent manner, and the messenger RNA and protein expressions of calpain, calcineurin, NLRP3, apoptosis-associated speck-like protein, and caspase-1 were significantly increased in a time-dependent manner. Furthermore, the inhibition of ASIC1a, calpain-2, or calcineurin, respectively, could decrease the cell death accompanied with the decreased interleukin-1ß level, and the decreased expression of ASIC1a, calpain-2, calcineurin, and NLRP3 inflammasome proteins. Taken together, these results indicated the activation of ASIC1a induced by extracellular acidosis could trigger pyroptosis of rat articular chondrocytes, the mechanism of which might partly be involved with the activation of calpain-2/calcineurin pathway.

The Lysine Specific Demethylase-1 Negatively Regulates the COL9A1 Gene in Human Articular Chondrocytes.

Osteoarthritis (OA) is a degenerative disease of the joints which is associated with an impaired production of the cartilage matrix by the chondrocytes. Here, we investigated the role of Lysine-Specific Demethylase-1 (LSD1), a chromatin remodeling enzyme whose role in articular chondrocytes was previously associated with a catabolic activity and which is potentially involved during OA. Following a loss of function strategy and RNA sequencing analysis, we detail the genes which are targeted by LSD1 in human articular chondrocytes and identify COL9A1, a gene encoding the α1 chain of the cartilage-specific type IX collagen, as negatively regulated by LSD1. We show that LSD1 interacts with the transcription factor SOX9 and is recruited to the promoter of COL9A1. Interestingly, we observe that OA cartilage displays stronger LSD1 immunostaining compared with normal, and we demonstrate that the depletion of LSD1 in OA chondrocytes prevents the decrease in COL9A1 following Il-1ß treatment. These results suggest LSD1 is a new regulator of the anabolic activity of articular chondrocytes potentially destabilizing the cartilage matrix, since it negatively regulates COL9A1, a gene encoding a crucial anchoring collagen molecule. This newly identified role played by LSD1 may thus participate in the alteration of the cartilage matrix during OA.

Regulation of articular chondrocyte catabolic genes by growth factor interaction.

Osteoarthritis is characterized by a loss of articular cartilage homeostasis in which degradation exceeds formation. Several growth factors have been shown to promote cartilage formation by augmenting articular chondrocyte anabolic activity. This study tests the hypothesis that such growth factors also play an anticatabolic role. We transferred individual or combinations of the genes encoding insulin-like growth factor-I, bone morphogenetic protein-2, bone morphogenetic protein-7, transforming growth factor-ß1, and fibroblast growth factor-2, into adult bovine articular chondrocytes and measured the expression of catabolic marker genes encoding A disintegrin and metalloproteinase with thrombospondin motifs-4 and -5, matrix metalloproteinases-3 and -13, and interleukin-6. When delivered individually, or in combination, these growth factor transgenes differentially regulated the direction, magnitude, and time course of expression of the catabolic marker genes. In concert, the growth factor transgenes regulated the marker genes in an interactive fashion that ranged from synergistic inhibition to synergistic stimulation. Synergistic stimulation prevailed over synergistic inhibition, reaching maxima of 15.2- and 2.7-fold, respectively. Neither the magnitude nor the time course of the effect of the transgene combinations could be predicted on the basis of the individual transgene effects. With few exceptions, the data contradict our hypothesis. The results demonstrate that growth factors that are traditionally viewed as chondrogenic tend also to promote catabolic gene expression. The competing actions of these potential therapeutic agents add an additional level of complexity to the selection of regulatory factors for restoring articular cartilage homeostasis or promoting repair.

Downregulation of microRNA-23b protects against ischemia-reperfusion injury via p53 signaling pathway by upregulating MDM4 in rats.

Total knee arthroplasty is a commonly performed safe procedure and typically executed in severe knee arthritis, but it also triggers ischemia-reperfusion injury (IRI). More recently, microRNAs (miRs) have been reported to play a contributory role in IRI through the key signaling pathway. Hence, the current study aimed to investigate the effect and specific mechanism of microRNA-23b (miR-23b), murine double minute 4 (MDM4), and the p53 signaling pathway in IRI rat models. First, the IRI model was established, and the expression pattern of miR-23b, MDM4, and the p53 signaling pathway-related genes was characterized in cartilaginous tissues. Then, miR-23b mimics or inhibitors were applied for the elevation or the depletion of the miR-23b expression and siRNA-MDM4 for the depletion of the MDM4 expression in the articular chondrocytes. By means of immunohistochemistry, quantitative real-time polymerase chain reaction, and Western blot analysis, IRI rats exhibited increased miR-23b expression, activated p53 signaling pathway, and decreased MDM4 expression. MDM4 was verified as a target gene of miR-23b through. Downregulated miR-23b increased the expression of MDM4, AKT, and Bcl-2, but decreased the expression of p53, p21, and Bax. In addition, a series of cell experiments demonstrated that downregulated miR-23b promoted articular chondrocyte proliferation and cell cycle entry, but inhibited articular chondrocyte apoptosis. The absence of the effects of miR-23b was observed after MDM4 knocked down. Our results indicate that silencing miR-23b could act to attenuate IRI and reduce the apoptosis of articular chondrocytes through inactivation of the p53 signaling pathway by upregulating MDM4, which provide basic therapeutic considerations for a novel target against IRI.

IL-10 Could Play a Role in the Interrelation between Diabetes Mellitus and Osteoarthritis.

The association between osteoarthritis (OA), obesity and metabolic syndrome suggests an interrelation between OA and diabetes mellitus (DM). Little is known about the role of anti-inflammatory cytokine interleukin (IL)-10 in the interrelation between OA and DM. Hence, the effects of IL-10 under hyperglycemia (HG) and hyperinsulinemia (HI) in human articular chondrocytes (hAC) and chondrosarcoma cell line Okayama University Medical School (OUMS)-27 were examined. HAC and OUMS-27, cultured in normoglycemic (NG) and HG conditions were stimulated with insulin and/or IL-10. Cell survival, metabolic activity, proliferation and extracellular matrix (ECM) synthesis were immunocytochemically examined. No significant differences in vitality of hAC neither in pure NG (NGw/o) nor HG (HGw/o) conditions were found. Applying HI and/or IL-10 in both conditions reduced significantly the vitality of hAC but not of OUMS-27. HG impaired significantly hAC metabolism. When combined with HI + IL-10 or IL-10 alone it decreased also significantly hAC proliferation compared to NGw/o. In OUMS-27 it induced only a trend of impaired proliferation compared to NGw/o. hAC but not OUMS-27 reduced significantly their collagen type (col) I, SOX9 and proteoglycan (PG) synthesis in HG combined with HI +/- IL-10 compared to NGw/o. IL-10 could not moderate HI and HG effects. In contrast to hAC OUMS-27 showed limited sensitivity as DM model.

High glycine concentration increases collagen synthesis by articular chondrocytes in vitro: acute glycine deficiency could be an important cause of osteoarthritis.

Collagen synthesis is severely diminished in osteoarthritis; thus, enhancing it may help the regeneration of cartilage. This requires large amounts of glycine, proline and lysine. Previous works of our group have shown that glycine is an essential amino acid, which must be present in the diet in large amounts to satisfy the demands for collagen synthesis. Other authors have shown that proline is conditionally essential. In this work we studied the effect of these amino acids on type II collagen synthesis. Bovine articular chondrocytes were cultured under a wide range of different concentrations of glycine, proline and lysine. Chondrocytes were characterized by type II collagen immunocytochemistry of confluence monolayer cultures. Cell growth and viability were assayed by trypan blue dye exclusion method. Type II collagen was measured in the monolayer, every 48 h for 15 days by ELISA. Increase in concentrations of proline and lysine in the culture medium enhances the synthesis of type II collagen at low concentrations, but these effects decay before 1.0 mM. Increase of glycine as of 1.0 mM exceeds these effects and this increase continues more persistently by 60-75%. Since the large effects produced by proline and lysine are within the physiological range, while the effect of glycine corresponds to a much higher range, these results demonstrated a severe glycine deficiency for collagen synthesis. Thus, increasing glycine in the diet may well be a strategy for helping cartilage regeneration by enhancing collagen synthesis, which could contribute to the treatment and prevention of osteoarthritis.

Novel amidrazone derivatives: Design, synthesis and activity evaluation.

A series of new 6-styryl-naphthalene-2-amidrazone derivatives were synthesized and evaluated as potential ASIC1a inhibitors. Among them, compound 5e showed the most activity to inhibit [Ca2+]i. elevation in acid-induced articular chondrocytes. Together with the important role of ASIC1a in the pathogenesis of tissue acidification diseases including rheumatoid arthritis, these results might provide a meaningful hint or inspiration in developing drugs targeting at tissue acidification diseases.

Measuring clinically relevant endpoints in a serum-free, three-dimensional, primary cell culture system of human osteoarthritic articular chondrocytes.

Osteoarthritis (OA) is characterized by degeneration of articular cartilage within the joint, inflammation and pain. The purpose of this study was to develop a primary, serum free cell culture system of human osteoarthritic articular chondrocytes (HOACs) with which to study manifestations of the disease process. Joint tissues were obtained from OA patients undergoing total knee arthroplasty (TKA). HOACs isolated from the femoral condyles and tibial plateau of the same side were combined, plated in three-dimensional, alginate beads and cultured for five days in serum, hormone and protein free medium. More living cells were obtained from the femoral condyles than the tibial plateau. The optimal plating density was 2.5 × 106 cells/ml of alginate. The amounts of DNA, RNA, proteoglycans and total collagen were similar in cultures prepared from the sides of least and greatest pathology. More type 1 than type 2 collagen was detected in the medium on days 2 and 5. A greater percentage of type 1 than type 2 collagen was degraded. The inflammatory cytokine interleukin-1 beta was present in the medium and alginate associated matrix. Although variation in the metabolic profiles between subjects was observed, HOACs from all patients continued to reflect the OA phenotype for five days in culture. This serum free, three-dimensional primary culture system of HOACs provides a platform with which to measure clinically relevant endpoints of OA and screen potential disease modifying OA therapeutics.

Acetazolamide protects rat articular chondrocytes from IL-1ß-induced apoptosis by inhibiting the activation of NF-κB signal pathway.

Because the excessive apoptosis of articular chondrocytes contributes to extracellular matrix (ECM) loss and cartilage damage in rheumatoid arthritis (RA), inhibiting chondrocyte apoptosis might be a promising strategy for RA. Aquaporin1 (AQP1) is overexpressed in RA cartilage and synovial tissues, and play a vital pathogenic role in RA development. Particularly, we previously reported that acetazolamide (AZ) as an AQP1 inhibitor suppressed secondary inflammation and promoted ECM production in cartilage of adjuvant-induced arthritis rats. Here, we investigated the antiapoptotic effect of AZ on interleukin-1ß (IL-1ß)-induced apoptosis, a classic in vitro model of chondrocyte apoptosis. AZ treatment could inhibit IL-1ß-induced apoptosis, evidenced by increasing cell viability, relieving apoptotic nuclear morphology, decreasing apoptosis rates, and restoring mitochondrial membrane potential. Additionally, AZ reversed IL-1ß-induced decrease of Bcl-2 protein and reduced IL-1ß-induced increases of Bax and caspase 3 protein, accompanied by inhibiting IκBα degradation and phosphorylation in cytoplasm, reducing NF-κB p65 protein level in nucleus and preventing NF-κB p65 translocation from cytoplasm to nucleus. In conclusion, our findings indicated that AZ could effectively attenuate IL-1ß-induced chondrocyte apoptosis mediated by regulating the protein levels of apoptosis-related genes and inhibiting the activation of NF-κB signal pathway, suggesting that AZ might be of potential clinical interest in RA treatment.

Influence of Mechanical Unloading on Articular Chondrocyte Dedifferentiation.

Due to the limited self-repair capacity of articular cartilage, the surgical restoration of defective cartilage remains a major clinical challenge. The cell-based approach, which is known as autologous chondrocyte transplantation (ACT), has limited success, presumably because the chondrocytes acquire a fibroblast-like phenotype in monolayer culture. This unwanted dedifferentiation process is typically addressed by using three-dimensional scaffolds, pellet culture, and/or the application of exogenous factors. Alternative mechanical unloading approaches are suggested to be beneficial in preserving the chondrocyte phenotype. In this study, we examined if the random positioning machine (RPM) could be used to expand chondrocytes in vitro such that they maintain their phenotype. Bovine chondrocytes were exposed to (a) eight days in static monolayer culture; (b) two days in static monolayer culture, followed by six days of RPM exposure; and, (c) eight days of RPM exposure. Furthermore, the experiment was also conducted with the application of 20 mM gadolinium, which is a nonspecific ion-channel blocker. The results revealed that the chondrocyte phenotype is preserved when chondrocytes go into suspension and aggregate to cell clusters. Exposure to RPM rotation alone does not preserve the chondrocyte phenotype. Interestingly, the gene expression (mRNA) of the mechanosensitive ion channel TRPV4 decreased with progressing dedifferentiation. In contrast, the gene expression (mRNA) of the mechanosensitive ion channel TRPC1 was reduced around fivefold to 10-fold in all of the conditions. The application of gadolinium had only a minor influence on the results. This and previous studies suggest that the chondrocyte phenotype is preserved if cells maintain a round morphology and that the ion channel TRPV4 could play a key role in the dedifferentiation process.

Co-culture of infrapatellar fat pad-derived mesenchymal stromal cells and articular chondrocytes in plasma clot for cartilage tissue engineering.

BACKGROUND: Cell source plays a deterministic role in defining the outcome of a cell-based cartilage regenerative therapy and its clinical translational ability. Recent efforts in the direction of co-culture of two or more cell types attempt to combine the advantages of constituent cell types and negate their demerits. METHODS: We examined the potential of co-culture of infrapatellar fat pad-derived mesenchymal stromal cells (IFP MSCs) and articular chondrocytes (ACs) in plasma clots in terms of their ratios and culture formats for cartilage tissue engineering. RESULTS AND DISCUSSION: It was observed that IFP MSCs and ACs interact positively to produce a better quality hyaline cartilage-like matrix. While a supra-additive deposition of sulfated Glycosaminoglycans (sGAG), collagen type II, aggrecan and link protein was observed, deposition of collagen type I and X was sub-additive. (Immuno)-histologically similar cartilage was generated in vitro in IFP MSC:AC ratio of 50:50 and pure AC groups thus yielding a hyaline cartilage with 50% reduced requirement of ACs. Subsequently, we investigated if this response could be improved further by enabling better cell-cell interactions using scaffold-free systems such as self-assembled cartilage or by encapsulating cellular micro-aggregates in plasma clot. However, it was inferred that while self-assembly may have enabled better cell-cell interaction, poor cell survival negated its overall beneficial role, whereas the micro-aggregate group demonstrated highly heterogeneous matrix deposition within the construct, thus diminishing its translational utility. Overall, it was concluded that co-culture of IFP MSCs and ACs at a ratio of 50:50 within plasma clots demonstrated potential for cell-based cartilage regenerative therapy.

ASIC1a Promotes Acid-Induced Autophagy in Rat Articular Chondrocytes through the AMPK/FoxO3a Pathway.

Acid-sensing ion channel 1a (ASIC1a) is a member of the extracellular H⁺-activated cation channels family. Our previous studies suggested that ASIC1a contributed to acid-induced rat articular chondrocytes autophagy. However, its potential mechanisms remain unclear. The present study demonstrated the effect of ASIC1a on rat articular chondrocytes autophagy and explored the underlying molecular mechanisms. The results demonstrated that ASIC1a contributed to acid-induced autophagy in rat articular chondrocytes, and which was associated with an increase in (Ca2+)i, as indicated that acid-induced increases in mRNA and protein expression of LC3B-II and other autophagy-related markers were inhibited by ASIC1a-specific blocker, PcTx1 and calcium chelating agent, BAPTA-AM. Furthermore, the results showed that extracellular acid increased level of Forkhead box O (FoxO) 3a, but was reversed by inhibition of ASIC1a and Ca2+ influx. Moreover, gene ablation of FoxO3a prevented acid-induced increases in mRNA and protein expression of LC3B-II, Beclin1 and the formation of autophagosome. Finally, it also showed that ASIC1a activated adenine nucleotide (AMP)-activated protein kinase (AMPK). In addition, suppression of AMPK by Compound C and its small interfering RNA (siRNA) prevented acid-induced upregulation of total and nuclear FoxO3a and increases in mRNA and protein expression of LC3B-II, Beclin1, and ATG5. Taken together, these findings suggested that AMPK/FoxO3a axis plays an important role in ASIC1a-mediated autophagy in rat articular chondrocytes, which may provide novel mechanistic insight into ASIC1a effects on autophagy.