Chondrocyte Phenotype Research Articles

Novel strategy of senescence elimination via toxicity-exempted kinome perturbations by nanoliposome-based thermosensitive hydrogel for osteoarthritis therapy

Cellular senescence and the senescence-associated secretory phenotype (SASP) have been implicated in osteoarthritis (OA). This study aims to determine whether multi-kinase inhibitor YKL-05-099 (Y099) has potential in senescence elimination and OA therapy and whether delivering Y099 by nanoliposmal hydrogel improves the performance of the kinase inhibitor. Y099 inhibited IL-1β-induced inflammation and catabolism and promoted anabolism of chondrocytes. To attenuate the inhibition of cell viability, nanoliposomal Y099-loaded thermosensitive hydrogel (Y099-Lip-Gel) was developed for sustained release and toxicity exemption. Notably, Y099-Lip-Gel exhibited a pronounced effect on promoting anabolism and suppressing catabolism and inflammation without causing the inhibition of chondrocyte viability. Moreover, Y099-Lip-Gel remarkably increased the master regulator of chondrocyte phenotype Sox9 expression. After four intra-articular injections of Y099-Lip-Gel in the OA murine model, the histological lesions of cartilage were attenuated by Y099-Lip-Gel with subchondral bone loss and osteoclast formation inhibited. Transcriptomic analysis and experimental validations revealed that Y099-Lip-Gel suppressed cellular senescence by inhibiting the expression of senescence inducers and SASP factors. Furthermore, the phosphoproteomic analysis showed that Y099-Lip-Gel exerted a significant influence on kinome phosphorylation, inhibiting the MAPK and NF-κB signaling activations. The protective effects of Y099-Lip-Gel were also validated in cultured human OA cartilage explants. In conclusion, nanoliposomal Y099-loaded thermosensitive hydrogel has considerable potential in OA therapy. Nanoliposome-based hydrogel system has strength in reducing kinase inhibition-induced cytotoxicity, enhancing cellular tolerance to kinome perturbation, and improving the performance of protein kinase inhibitors. Senescence elimination via toxicity-exempted kinome perturbations achieved by advanced nanotechnology is a promising strategy for OA.Graphical

Insufficiency of collagenases in establishment of primary chondrocyte culture from cartilage of elderly patients receiving total joint replacement.

Background Collagenases are frequently used in chondrocyte isolation from articular cartilage. However, the sufficiency of this enzyme in establishing primary human chondrocyte culture remains unknown. Methods Cartilage slices shaved from femoral head or tibial plateau of patients receiving total joint replacement surgery (16 hips, 8 knees) were subjected to 0.02% collagenase IA digestion for 16h with (N = 19) or without (N = 5) the pre-treatment of 0.4% pronase E for 1.5h. Chondrocyte yield and viability were compared between two groups. Chondrocyte phenotype was determined by the expression ratio of collagen type II to I. The morphology of cultured chondrocytes was monitored with a light microscope.Results Cartilage with pronase E pre-treatment yielded significantly higher chondrocytes than that without the pre-treatment (3,399 ± 1,637 cells/mg wet cartilage vs. 1,895 ± 688 cells/mg wet cartilage; P = 0.0067). Cell viability in the former group was also significantly higher than that in the latter (94% ± 2% vs. 86% ± 6%; P = 0.03). When cultured in monolayers, cells from cartilage with pronase E pre-treatment grew in a single plane showing rounded shape while cells from the other group grew in multi-planes and exhibited irregular shape. The mRNA expression ratio of collagen type II to I was 13.2 ± 7.5 in cells isolated from cartilage pre-treated with pronase E, indicating a typical chondrocyte phenotype. Conclusions Collagenase IA was not sufficient in establishing primary human chondrocyte culture. Cartilage must be treated with pronase E prior to collagenase IA application.

Effect of Collagen and GelMA on Preservation of the Costal Chondrocytes' Phenotype in a Scaffold in vivo.

Chondrocytes were isolated from the costal cartilage of newborn rats using 0.15% collagenase solution in DMEM. The cells was characterized by glycosaminoglycan staining with alcian blue. Chondrocyte scaffolds were obtained from 4% type I porcine atelocollagen and 10% GelMA by micromolding and then implanted subcutaneously into the withers of two groups of Wistar rats. Histological and immunohistochemical studies were performed on days 12 and 26 after implantation. Tissue samples were stained with hematoxylin and eosin, alcian blue; type I and type II collagens were identified by the corresponding antibodies. The implanted scaffolds induced a moderate inflammatory response in both groups when implanted in animals. By day 26 after implantation, both collagen and GelMA had almost completely resorbed. Cartilage tissue formation was observed in both animal groups. The newly formed tissue was stained intensively with alcian blue, and the cells were positive for both types of collagen. Cartilage tissue was formed among muscle fibers. The ability of collagen type I and GelMA hydrogels to form hyaline cartilage in animals after subcutaneous implantation of scaffolds was studied. Both collagen and GelMA contributed to formation of hyaline-like cartilage tissue type in animals, but the chondrocyte phenotype is characterized as mixed. Additional detailed studies of possible mechanisms of chondrogenesis under the influence of each of the hydrogels are needed.

Hypoxia-induced Wnt/β-catenin signaling activation in subchondral bone osteoblasts leads to an osteoarthritis-like phenotype of chondrocytes in articular cartilage.

Background: Osteoarthritis (OA) is a whole-joint disease and characterized by alterations in the articular cartilage, subchondral bone, ligaments, and synovial membrane. The crosstalk between cartilage and subchondral bone plays a crucial role in the pathogenesis and progression of OA. Hypoxia has been reported to play an important role in cartilage degradation and subchondral bone remodeling in OA. In this study, we aimed to identify the involvement of hypoxia in modifying the osteoblast phenotypes and determine whether these alterations could influence the metabolism of chondrocytes. Methods: First, the levels of Hif-1α in subchondral bone of different compartments in patients with OA were assessed using immunohistochemistry (IHC). In in vitro, human primary osteoblasts were cultured under hypoxic and normoxic conditions, and the hypoxic or normoxic conditioned media (HCM and NCM) were used to culture human primary chondrocytes. Then, phenotypic changes in osteoblasts were assessed using reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and enzyme-linked immunosorbent assay (ELISA). Furthermore, the expression of type II collagen (COL2A1), aggrecan (ACAN), SRY-related high-mobility group-box gene 9 (SOX9), matrix metalloproteinase 13 (MMP13), and matrix metalloproteinase 3 (MMP3) in chondrocytes was measured using RT-PCR. Finally, the serum levels of Wnt-related proteins were determined using ELISA. Results: Hif-1α was significantly increased in severely sclerotic subchondral bone compared to less damaged subchondral bone. β-Catenin and SOST were identified as upregulated and downregulated in hypoxic osteoblasts, respectively. The hypoxia-induced results were confirmed by ELISA. Stimulating human primary chondrocytes with HCM significantly induced MMP13 and MMP3 and inhibited COL2A1, ACAN, and SOX9 mRNA expression. The serum levels of DKK-1 were significantly increased in human OA. Conclusion: Together, these findings revealed that hypoxia in subchondral bone is a key factor in the crosstalk between chondrocytes and osteoblasts and facilitates the shift of chondrocytes toward an OA-like phenotype probably by activating the Wnt/β-catenin signaling pathway in osteoblasts.

Differential Effects of Hypoxia versus Hyperoxia or Physoxia on Phenotype and Energy Metabolism in Human Chondrocytes from Osteoarthritic Compared to Macroscopically Normal Cartilage.

Chondrocyte phenotype and energy metabolism are altered in osteoarthritis (OA). However, most studies characterising the change in human chondrocyte behaviour in OA have been conducted in supraphysiological oxygen concentrations. The purpose of this study was to compare phenotype and energy metabolism in chondrocytes from macroscopically normal (MN) and OA cartilage maintained in 18.9% (standard tissue culture), 6% (equivalent to superficial zone of cartilage in vivo) or 1% oxygen (equivalent to deep zone of cartilage in vivo). MMP13 production was higher in chondrocytes from OA compared to MN cartilage in hyperoxia and physoxia but not hypoxia. Hypoxia promoted SOX9, COL2A1 and ACAN protein expression in chondrocytes from MN but not OA cartilage. OA chondrocytes used higher levels of glycolysis regardless of oxygen availability. These results show that differences in phenotype and energy metabolism between chondrocytes from OA and MN cartilage differ depending on oxygen availability. OA chondrocytes show elevated synthesis of cartilage-catabolising enzymes and chondrocytes from MN cartilage show reduced cartilage anabolism in oxygenated conditions. This is relevant as a recent study has shown that oxygen levels are elevated in OA cartilage in vivo. Our findings may indicate that this elevated cartilage oxygenation may promote cartilage loss in OA.

Activating Mitochondrial Sirtuin 3 in Chondrocytes Alleviates Aging-Induced Fibrocartilage Layer Degeneration and Promotes Healing of Degenerative Rotator Cuff Injury

The present study aimed to examine the impact of mitochondrial sirtuin 3 (SIRT3) on the degenerative rotator cuff injury, which is a prevalent issue among the elderly population primarily due to aging-related tissue degradation. The study hypothesized that SIRT3, as a major deacetylase in mitochondria, is a significant factor in controlling the quality of mitochondria and the deterioration of fibrocartilage, a crucial component of the rotator cuff. Results showed that the aging process led to weakened biomechanical properties and degeneration of the fibrocartilage layer in mice, accompanied by a decrease in SIRT3 expression. SIRT3 activation ameliorated the aging-related disruption of chondrocyte phenotype and fibrocartilage degradation. SIRT3 activator honokiol improved the phenotype of senescent chondrocytes and promoted rotator cuff healing in aged mice through SIRT3 activation. In conclusion, the findings suggested that the decline in SIRT3 levels with age contributes to rotator cuff degeneration and chondrocyte senescence, and that SIRT3 activation through the use of honokiol is an effective approach for promoting rotator cuff healing in the elderly population.

Microenvironmental mechanoactivation through Yap/Taz suppresses chondrogenic gene expression.

Chondrocyte phenotype is preserved when cells are round and the actin cytoskeleton is cortical. Conversely, these cells rapidly dedifferentiate in vitro with increased mechanoactive Rho signaling, which increases cell size and causes large actin stress fiber to form. While the effects of Rho on chondrocyte phenotype are well established, the molecular mechanism is not yet fully elucidated. Yap, a transcriptional coregulator, is regulated by Rho in a mechanotransductive manner and can suppress chondrogenesis in vivo. Here, we sought to elucidate the relationship between mechanoactive Rho and Yap on chondrogenic gene expression. We first show that decreasing mechanoactive state through Rho inhibition results in a broad increase in chondrogenic gene expression. Next, we show that Yap and its coregulator Taz are negative regulators of chondrogenic gene expression, and removal of these factors promotes chondrogenesis even in environments that promote cell spreading. Finally, we establish that Yap/Taz is essential for translating Rho-mediated signals to negatively regulate chondrogenic gene expression, and that its removal negates the effects of increased Rho signaling. Together, these data indicate that Rho is a mechanoregulator of chondrogenic differentiation, and that its impact on chondrogenic expression is exerted principally through mechanically induced translocation and activity of Yap and Taz.

CELL MORPHOLOGY AS A BIOLOGICAL FINGERPRINT FOR DESCRIBING CHONDROCYTE PHENOTYPE UNDER INFLAMMATORY ATTACK

While cell morphology has been recognized as a fundamental regulator of cell behavior, few studies have measured the complex cell morphological changes of chondrocytes using quantitative cell morphometry descriptors in relation to inflammation and phenotypic outcome.Acute vs. persistent exposure to IL-1β and how IL-1β modulated dynamic changes in cell morphology in relation to the phenotype, donor and OA grade in healthy and osteoarthritis (OA) chondrocytes was investigated. A panel of quantitative cell morphometry descriptors was measured using an automated high-throughput method. Absolute quantification of gene expression was measured by ddPCR followed by correlation analyses.In OA chondrocytes, chronic IL-1β significantly decreased COL2A1, SOX9, and ACAN, increased IL-6 and IL-8 levels and caused chondrocytes to become less wide, smaller, longer, slimmer, less round and more circular, consistent with a de-differentiated phenotype. In healthy chondrocytes, 3 days after acute (72 h) IL-1β exposure, COL1A2 and IL-6 significantly increased but had minor effects on cell morphology. However, in healthy chondrocytes, persistent IL-1β led to more profound effects in all cell morphology descriptors and chondrocytes expressed significantly less COL2A1 and more IL-6 and IL-8 vs. controls and acutely-stimulated chondrocytes. In both OA and healthy chronically-stimulated chondrocytes, area, width and circularity were sensitive to the persistent presence of the IL-1β cytokine. Moreover, there were many significant and strong correlations among the measured parameters, with several indications of an IL-1β-mediated mechanism.Cell morphology combined with gene expression analysis could guide researchers interested in understanding inflammatory effects in the complex domain of cartilage/chondrocyte biology. Use of quantitative cell morphometry could complement classical approaches by providing numerical data on a large number of cells, thereby providing a biological fingerprint for describing chondrocyte phenotype, which could help to understand how changes in cell morphology lead to disease progression.

CHANGES TO THE F-ACTIN DISTRIBUTION OF THE CHONDROCYTE CYTOSKELETON IN EARLY OSTEOARTHRITIS

Cartilage degeneration and loss are key events in the initiation and progression of osteoarthritis (OA). Changes to chondrocyte volume and morphology (in the form of cytoplasmic processes) and thus cell phenotype are implicated, as they lead to the production of a mechanically-weakened extracellular matrix. The chondrocyte cytoskeleton is intimately linked to cell volume and morphology and hence we have investigated alterations to levels and distribution of chondrocyte F-actin that occur during early OA.The femoral heads (FH) from hip joints (N=16) were obtained with ethical permission and patient consent following femoral neck fracture. Cartilage was assessed as grade 0 (non-degenerate) and grade 1 (superficial fibrillation) using OARSI criteria. In situ chondrocyte volume and F-actin distribution were assessed using the fluorescent indicators (5-chloromethyl fluorescein diacetate (CMFDA)) and phalloidin, and imaged and quantified by confocal microscopy, ImarisTM and ImageJ software.There were no differences between the volume or total F-actin levels of in situ chondrocytes within the superficial zone of grade 0 (n=164 cells) compared to grade 1 (n=145) cartilage (P>0.05). However, a more detailed analysis of phalloidin labelling was performed, which demonstrated significant increases in both intense punctuate (IP) or intense areas (IA) (P<0.0001; P=0.0175 respectively). A preliminary analysis of IP and IA F-actin labelling suggested that while the former did not appear to be associated with changes to chondrocyte morphology, most of the cytoplasmic processes were associated with the presence of IA at the starting point of the protrusion.These results demonstrate marked changes to F-actin distribution in chondrocytes in the very early stages of cartilage degeneration as occurs in OA. These subtle changes are probably an early indication of a change to the chondrocyte phenotype and thus worthy of further study as they may lead to deleterious alterations to matrix metabolism and ultimately cartilage weakening.

Tropoelastin-Pretreated Exosomes from Adipose-Derived Stem Cells Improve the Synthesis of Cartilage Matrix and Alleviate Osteoarthritis.

Mesenchymal stem cells (MSCs) have recently been widely used to treat osteoarthritis (OA). Our prior research shows that tropoelastin (TE) increases MSC activity and protects knee cartilage from OA-related degradation. The underlying mechanism might be that TE regulates the paracrine of MSCs. Exosomes (Exos), the paracrine secretion of MSCs, have been found to protect chondrocytes, reduce inflammation, and preserve the cartilage matrix. In this study, we used Exos derived from TE-pretreated adipose-derived stem cells (ADSCs) (TE-ExoADSCs) as an injection medium, and compared it with Exos derived from unpretreated ADSCs (ExoADSCs). We found that TE-ExoADSCs could effectively enhance the matrix synthesis of chondrocytes in vitro. Moreover, TE pretreatment increased the ability of ADSCs to secrete Exos. In addition, compared with ExoADSCs, TE-ExoADSCs exhibited therapeutic effects in the anterior cruciate ligament transection (ACLT)-induced OA model. Further, we observed that TE altered the microRNA expression in ExoADSCs and identified one differentially upregulated microRNA: miR-451-5p. In conclusion, TE-ExoADSCs helped maintain the chondrocyte phenotype in vitro, and promoted cartilage repair in vivo. These therapeutic effects might be related with the altered expression of miR-451-5p in the ExoADSCs. Thus, the intra-articular delivery of Exos derived from ADSCs with TE pretreatment could be a new approach to treat OA.

The Effect of Crosslinking Density on Nasal Chondrocytes' Redifferentiation.

Hydrogels appear to be an attractive class of biomaterial for cartilage tissue engineering due to their high water content, excellent biocompatibility, tunable stiffness, etc. The crosslinking density of the hydrogel can affect their viscoelastic property, and therefore potentially impact the chondrogenic phenotype of re-differentiated chondrocytes in a 3D microenvironment through physical cues. To understand the effect of crosslinking densities on chondrocytes phenotype and cellular interaction with the hydrogel, this study utilized a clinical grade thiolate hyaluronic acid and thiolate gelatin (HA-Gel) hydrogel, crosslinked with poly(ethylene glycol) diacrylate to create various crosslinking densities. The HA-Gel hydrogels were then mixed with human nasal chondrocytes to generate neocartilage in vitro. The influence of the hydrogel crosslinking density and the viscoelastic property on the cell behaviours on the gene and matrix levels were evaluated using biochemistry assays, histology, quantitative polymerase chain reaction (qPCR) and next-generation sequencing (RNA seq). In general, the differences in the storage modulus of the HA-Gel hydrogel are not enough to alter the cartilaginous gene expression of chondrocytes. However, a positively correlated trend of PPAR-γ gene expression to the crosslinking density was measured by qPCR. The RNA-seq results have shown that 178 genes are significantly negatively correlated and 225 genes are positively correlated to the crosslinking density, which is worth investigating in the future studies.

Inhibition of TRADD ameliorates chondrocyte necroptosis and osteoarthritis by blocking RIPK1-TAK1 pathway and restoring autophagy

Osteoarthritis (OA) is an age-related disease characterized by cartilage degeneration. TNFR1-associated death domain protein (TRADD) is a key upstream molecule of TNF-α signals but its role in OA pathogenesis is unknown. This study aimed to verify that whether inhibition of TRADD could protect against chondrocyte necroptosis and OA, and further elucidate the underlying mechanism. We demonstrated that TNF-α-related OA-like phenotypes including inflammation response, extracellular matrix degradation, apoptosis, and necroptosis in chondrocytes were inhibited by TRADD deficiency. Furthermore, TRADD interacted with TRAF2 and knockdown of TRADD suppressed the activation of RIPK1-TAK1-NF-κB signals and restored impaired autophagy. ICCB-19, the selective inhibitor of TRADD, also attenuated necroptosis in chondrocytes. Mechanismly, ICCB-19 blocked the phosphorylation of TAK1-NF-κB signals and restored impaired autophagy, whereas inhibiting autophagic process with 3-Methyladenine compromised these effects of ICCB-19. The in vivo study showed that the intra-articular injection of ICCB-19 rescued the expression of collagen alpha-1(II) chain and LC3, and mitigated the cartilage degeneration of OA mice. This study demonstrates that TRADD mediates TNF-α-induced necroptosis and OA-like phenotypes of chondrocytes and suggests that ICCB-19 suppresses chondrocyte damage and cartilage degeneration by inhibiting TNF-α-TRADD-mediated signals and dysregulation of autophagy in chondrocytes. ICCB-19 may serve as an important option for OA therapy.

Daurisoline attenuates H2O2-induced chondrocyte autophagy by activating the PI3K/Akt/mTOR signaling pathway

BackgroundOsteoarthritis (OA) is a chronic degenerative joint disease characterized by cartilage degeneration and intra-articular inflammation. Daurisoline (DAS) is an isoquinoline alkaloid isolated from Rhizoma Menispermi, whose antitumor and anti-inflammatory pharmacological effects have been demonstrated, but the effects of DAS on OA have rarely been researched. In this study, we aimed to explore the potential role of DAS in OA and its partial mechanism.Materials and methodsThe cytotoxicity of H2O2 and DAS toward chondrocytes was detected by the Cell Counting Kit-8 assay. Safranin O staining was used to detect chondrocyte phenotype changes. Cell apoptosis was measured by both flow cytometry and quantitative analysis of the protein levels of the apoptosis-related factors Bax, Bcl-2 and cleaved caspase-3 by western blot. Western blotting and immunofluorescence were used to assess the expression of the autophagy-related proteins LC3, Beclin-1 and p62. In addition, key signal pathway targets and matrix-degrading indicators were measured by western blot.ResultsOur results indicated that H2O2 induced human chondrocyte apoptosis and activated autophagy in a dose-dependent manner. DAS treatment dose-dependently reversed the expression of apoptosis-related proteins (Bax, Bcl-2 and cleaved caspase3) and the apoptosis rate induced by H2O2. Western blot and immunofluorescence analyses showed that DAS decreased the H2O2-induced upregulation of the autophagy marker Beclin-1 and the LC3 II/LC3 I ratio and upregulated the p62 protein level. Mechanistically, DAS inhibited autophagy through the activation of the classical PI3K/AKT/mTOR signaling pathway and protected chondrocytes from apoptosis. In addition, DAS alleviated the H2O2-induced degradation of type II collagen and the high expression of matrix metalloproteinase 3 (MMP3) and MMP13.ConclusionOur research demonstrated that DAS alleviated chondrocyte autophagy caused by H2O2 through activation of the PI3K/AKT/mTOR signaling pathway and protected chondrocytes from apoptosis and matrix degradation. In conclusion, these findings suggest that DAS may serve as a promising therapeutic strategy for OA.

WNT16 Regulation of the Articular Chondrocyte Phenotype in Mice.

The anabolic effects of WNT16 on osteoblasts are well established, however, little is known regarding the role of WNT16 in chondrocytes. In this study, we evaluated Wnt16 expression and its biological effects on mouse articular chondrocytes (ACs), since these cells are key to the development of osteoarthritis. While ACs derived from the long bone epiphysis of 7-day old C57BL/6J mice express multiple Wnts, Wnt5b and Wnt16 represent the two most highly expressed Wnts (expressed at several-fold higher levels than other Wnts). Treatment of serum-free AC cultures, with 100 ng/mL of recombinant human (rh) WNT16 for 24 h (hrs), increased proliferation (20%, p < 0.05) and expression levels of makers (Sox9 and Col2) of immature chondrocytes at both 24 h and 72 h, while Acan increased at 72 h. Expression of Mmp9, a marker of mature chondrocytes was decreased at 24 h. Additionally, WNT16 treatment regulated expression levels of Wnt ligands in a biphasic manner, inhibiting its expression at 24 h, while stimulating expression at 72 h. To determine whether WNT16 exerted anabolic effects on the AC phenotype, ex vivo cultures of tibial epiphyses were treated with rhWNT16 or vehicle for 9 days, and the articular cartilage phenotype was evaluated by safranin O cartilage staining and expression of articular cartilage marker genes. Both articular cartilage area and expression levels of AC markers were increased after rhWNT16 treatment. Our data suggest that Wnt16 expressed in ACs may play a role in regulating joint cartilage homeostasis via its direct effect, as well as through modulating the expression of other Wnt ligands.

Hypoxia Promotes Cartilage Regeneration in Cell-Seeded 3D-Printed Bioscaffolds Cultured with a Bespoke 3D Culture Device

In this study, we investigated the effect of oxygen tension on the expansion of ADMSCs and on their differentiation toward their chondrocytic phenotype, regenerating a lab-based cartilaginous tissue with superior characteristics. Controversial results with reference to MSCs that were cultured under different hypoxic levels, mainly in 2D culturing settings combined with or without other biochemical stimulus factors, prompted our team to study the role of hypoxia on MSCs chondrogenic differentiation within an absolute 3D environment. Specifically, we used 3D-printed honeycomb-like PCL matrices seeded with ADMSCs in the presence or absence of TGF and cultured with a prototype 3D cell culture device, which was previously shown to favor nutrient/oxygen supply, cell adhesion, and infiltration within scaffolds. These conditions resulted in high-quality hyaline cartilage that was distributed uniformly within scaffolds. The presence of the TGF medium was necessary to successfully produce cartilaginous tissues with superior molecular and increased biomechanical properties. Despite hypoxia's beneficial effect, it was overall not enough to fully differentiate ADMSCs or even promote cell expansion within 3D scaffolds alone.

The effect of allyl isothiocyanate on chondrocyte phenotype is matrix stiffness-dependent: Possible involvement of TRPA1 activation.

Osteoarthritis (OA) is a chronic joint disease with increasing prevalence. Chondrocytes (CHs) are highly differentiated end-stage cells with a secretory phenotype that keeps the extracellular matrix (ECM) balanced and the cartilage environment stable. Osteoarthritis dedifferentiation causes cartilage matrix breakdown, accounting for one of the key pathogenesis of osteoarthritis. Recently, the activation of transient receptor potential ankyrin 1 (TRPA1) was claimed to be a risk factor in osteoarthritis by causing inflammation and extracellular matrix degradation. However, the underlying mechanism is still unknown. Due to its mechanosensitive property, we speculated that the role of TRPA1 activation during osteoarthritis is matrix stiffness-dependent. In this study, we cultured the chondrocytes from patients with osteoarthritis on stiff vs. soft substrates, treated them with allyl isothiocyanate (AITC), a transient receptor potential ankyrin 1 agonist, and compared the chondrogenic phenotype, containing cell shape, F-actin cytoskeleton, vinculin, synthesized collagen profiles and their transcriptional regulatory factor, and inflammation-related interleukins. The data suggest that allyl isothiocyanate treatment activates transient receptor potential ankyrin 1 and results in both positive and harmful effects on chondrocytes. In addition, a softer matrix could help enhance the positive effects and alleviate the harmful ones. Thus, the effect of allyl isothiocyanate on chondrocytes is conditionally controllable, which could be associated with transient receptor potential ankyrin 1 activation, and is a promising strategy for osteoarthritis treatment.

Effect of the uronic acid composition of alginate in alginate/collagen hybrid hydrogel on chondrocyte behavior.

Introduction: Developing a culture system that can effectively maintain chondrocyte phenotype and functionalization is a promising strategy for cartilage repair. Methods: An alginate/collagen (ALG/COL) hybrid hydrogel using different guluronate/mannuronate acid ratio (G/M ratio) of alginates (a G/M ratio of 64/36 and a G/M ratio of 34/66) with collagen was developed. The effects of G/M ratios on the properties of hydrogels and their effects on the chondrocytes behaviors were evaluated. Results: The results showed that the mechanical stiffness of the hydrogel was significantly affected by the G/M ratios of alginate. Chondrocytes cultured on Mid-G/M hydrogels exhibited better viability and phenotype preservation. Moreover, RT-qPCR analysis showed that the expression of cartilage-specific genes, including SOX9, COL2, and aggrecan was increased while the expression of RAC and ROCK1 was decreased in chondrocytes cultured on Mid-G/M hydrogels. Conclusion: These findings demonstrated that Mid-G/M hydrogels provided suitable matrix conditions for cultivating chondrocytes and may be useful in cartilage tissue engineering. More importantly, the results indicated the importance of taking alginate G/M ratios into account when designing alginate-based composite materials for cartilage tissue engineering.

Mechanical memory stored through epigenetic remodeling reduces cell therapeutic potential

Understanding how cells remember previous mechanical environments to influence their fate, or mechanical memory, informs the design of biomaterials and therapies in medicine. Current regeneration therapies, such as cartilage regeneration procedures, require 2D cell expansion processes to achieve large cell populations critical for the repair of damaged tissues. However, the limit of mechanical priming for cartilage regeneration procedures before inducing long-term mechanical memory following expansion processes is unknown, and mechanisms defining how physical environments influence the therapeutic potential of cells remain poorly understood. Here, we identify a threshold to mechanical priming separating reversible and irreversible effects of mechanical memory. After 16 population doublings in 2D culture, expression levels of tissue-identifying genes in primary cartilage cells (chondrocytes) are not recovered when transferred to 3D hydrogels, while expression levels of these genes were recovered for cells only expanded for eight population doublings. Additionally, we show that the loss and recovery of the chondrocyte phenotype correlates with a change in chromatin architecture, as shown by structural remodeling of the trimethylation of H3K9. Efforts to disrupt the chromatin architecture by suppressing or increasing levels of H3K9me3 reveal that only with increased levels of H3K9me3 did the chromatin architecture of the native chondrocyte phenotype partially return, along with increased levels of chondrogenic gene expression. These results further support the connection between the chondrocyte phenotype and chromatin architecture, and also reveal the therapeutic potential of inhibitors of epigenetic modifiers as disruptors of mechanical memory when large numbers of phenotypically suitable cells are required for regeneration procedures.

IL-6, IL-1β and TNF-α regulation of the chondrocyte phenotype: a possible mechanism of haemophilic cartilage destruction

ABSTRACTObjective:Proinflammatory cytokines are considered to be one of the key causes of haemophilic cartilage destruction by inducing chondrocyte apoptosis and extracellular matrix degradation. However, few studies have focused on how proinflammatory cytokines regulate the phenotypic changes of chondrocytes, which may be an important factor in haemophilic cartilage degradation pathogenesis. More understanding is needed about the effect of proinflammatory cytokines on phenotypic changes of the chondrocyte. The objective of this study was to examine how IL-6, TNF-α and IL-1β regulate the chondrocyte phenotype, which may be an important factor in haemophilic cartilage degradation pathogenesis.Methods:HUM-iCell-s018 chondrocytes were treated with increasing concentrations of TNF-α, IL-6 or IL-1β (0, 1, 5, 10 ng/ml) for 24 h, then FGF23 and SOX9 expression was determined by qRT-PCR and WB, respectively.Results:We found that TNF-α, IL-6 and IL-1β induced FGF23 and suppressed SOX9 expression in chondrocytes in a dose-dependent manner. IL-1β had a stronger regulatory effect on FGF23, while TNF-α and IL-6 had stronger regulatory effects on SOX9.Conclusions:These findings suggest that IL-6, IL-1β and TNF-α may be involved in haemophilic cartilage destruction pathogenesis by altering the chondrocyte phenotype through modulation of FGF23 and SOX9 gene expression.

Cell morphology as a biological fingerprint of chondrocyte phenotype in control and inflammatory conditions.

Little is known how inflammatory processes quantitatively affect chondrocyte morphology and how single cell morphometric data could be used as a biological fingerprint of phenotype. We investigated whether trainable high-throughput quantitative single cell morphology profiling combined with population-based gene expression analysis can be used to identify biological fingerprints that are discriminatory of control vs. inflammatory phenotypes. The shape of a large number of chondrocytes isolated from bovine healthy and human osteoarthritic (OA) cartilages was quantified under control and inflammatory (IL-1β) conditions using a trainable image analysis technique measuring a panel of cell shape descriptors (area, length, width, circularity, aspect ratio, roundness, solidity). The expression profiles of phenotypically relevant markers were quantified by ddPCR. Statistical analysis, multivariate data exploration, and projection-based modelling were used for identifying specific morphological fingerprints indicative of phenotype. Cell morphology was sensitive to both cell density and IL-1β. In both cell types, all shape descriptors correlated with expression of extracellular matrix (ECM)- and inflammatory-regulating genes. A hierarchical clustered image map revealed that individual samples sometimes responded differently in control or IL-1β conditions than the overall population. Despite these variances, discriminative projection-based modeling revealed distinct morphological fingerprints that discriminated between control and inflammatory chondrocyte phenotypes: the most essential morphological characteristics attributable to non-treated control cells was a higher cell aspect ratio in healthy bovine chondrocytes and roundness in OA human chondrocytes. In contrast, a higher circularity and width in healthy bovine chondrocytes and length and area in OA human chondrocytes indicated an inflammatory (IL-1β) phenotype. When comparing the two species/health conditions, bovine healthy and human OA chondrocytes exhibited comparable IL-1β-induced morphologies in roundness, a widely recognized marker of chondrocyte phenotype, and aspect ratio. Overall, cell morphology can be used as a biological fingerprint for describing chondrocyte phenotype. Quantitative single cell morphometry in conjunction with advanced methods for multivariate data analysis allows identifying morphological fingerprints that can discriminate between control and inflammatory chondrocyte phenotypes. This approach could be used to assess how culture conditions, inflammatory mediators, and therapeutic modulators regulate cell phenotype and function.