Chondrocyte Phenotype Research Articles

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.

Dendrobine Alleviates Cellular Senescence and Osteoarthritis via the ROS/NF-κB Axis.

Osteoarthritis (OA) is a degenerative joint disease characterized by low-grade inflammation and cartilage degradation. Dendrobine (DEN) is reported to inhibit inflammation and oxidative stress in some diseases, but its role in chondrocyte senescence and OA progress has not yet been elucidated. Our study aimed to explore the protective effects of DEN on OA both in vitro and in vivo. We found that DEN inhibited extracellular matrix (ECM) degradation and promoted ECM synthesis. Meanwhile, DEN inhibited senescence-associated secretory phenotype (SASP) factors expression and senescence phenotype in IL-1β-treated chondrocytes. Furthermore, DEN improved mitochondrial function and reduced the production of intracellular reactive oxygen species (ROS). Also, DEN suppressed IL-1β-induced activation of the NF-κB pathway. Further, using NAC (ROS inhibitor), we found that DEN might inhibit NF-κB cascades by reducing ROS. Additionally, X-ray, micro-CT, and histological analyses in vivo demonstrated that DEN significantly alleviated cartilage inflammation, ECM degradation, and subchondral alterations in OA progression. In conclusion, DEN inhibits SASP factors expression and senescence phenotype in chondrocytes and alleviated the progression of OA via the ROS/NF-κB axis, which provides innovative strategies for the treatment of OA.

Perspective in Achieving Stratified Articular Cartilage Repair Using Zonal Chondrocytes.

Articular cartilage is composed of superficial, medial, and deep zones, which endow the tissue with biphasic mechanical properties to withstand shearing force and compressional loading. The tissue has very limited self-healing capacity once it is damaged due to its avascular nature. To prevent the early onset of osteoarthritis, surgical intervention is often needed to repair the injured cartilage. Current noncell-based and cell-based treatments focus on the regeneration of homogeneous cartilage to achieve bulk compressional properties without recapitulating the zonal matrix and mechanical properties, and often oversight in aiding cartilage integration between host and repair cartilage. It is hypothesized that achieving zonal architecture in articular cartilage tissue repair could improve the structural and mechanical integrity and thus the life span of the regenerated tissue. Engineering stratified cartilage constructs using zonal chondrocytes have been hypothesized to improve the functionality and life span of the regenerated tissues. However, stratified articular cartilage repair has yet to be realized to date due to the lack of an efficient zonal chondrocyte isolation method and an expansion platform that would allow both cell propagation and phenotype maintenance. Various attempts and challenges in achieving stratified articular cartilage repair in a clinical setting are evaluated. In this review, different perspectives on achieving stratified articular cartilage repair using zonal chondrocytes are described. The effectiveness of different zonal chondrocyte isolation and zonal chondrocyte phenotype maintenance methodologies during expansion are compared, with the focus on recent advancements in zonal chondrocyte isolation and expansion that could present a possible strategy to overcome the limitation of applying zonal chondrocytes to facilitate zonal architecture development in articular cartilage regeneration. Impact Statement The zonal properties of articular cartilage contribute to the biphasic mechanical properties of the tissues. Recapitulation of the zonal architecture in regenerated articular cartilage has been hypothesized to improve the mechanical integrity and life span of the regenerated tissue. This review provides a comprehensive discussion on the current state of research relevant to achieving stratified articular cartilage repair using zonal chondrocytes from different perspectives. This review further elaborates on a zonal chondrocyte production pipeline that can potentially overcome the current clinical challenges and future work needed to realize stratified zonal chondrocyte implantation in a clinical setting.

The epigenetic players and the chromatin marks involved in the articular cartilage during osteoarthritis.

Epigenetics defines the modifications of the genome that do not involve a change in the nucleotide sequence of DNA. These modifications constitute a mechanism of gene regulation poorly explored in the context of cartilage physiology. They are now intensively studied by the scientific community working on articular cartilage and its related pathology such as osteoarthritis. Indeed, epigenetic regulations can control the expression of crucial gene in the chondrocytes, the only resident cells of cartilage. Some epigenetic changes are considered as a possible cause of the abnormal gene expression and the subsequent alteration of the chondrocyte phenotype (hypertrophy, proliferation, senescence…) as observed in osteoarthritic cartilage. Osteoarthritis is a joint pathology, which results in impaired extracellular matrix homeostasis and leads ultimately to the progressive destruction of cartilage. To date, there is no pharmacological treatment and the exact causes have yet to be defined. Given that the epigenetic modifying enzymes can be controlled by pharmacological inhibitors, it is thus crucial to describe the epigenetic marks that enable the normal expression of extracellular matrix encoding genes, and those associated with the abnormal gene expression such as degradative enzyme or inflammatory cytokines encoding genes. In this review, only the DNA methylation and histone modifications will be detailed with regard to normal and osteoarthritic cartilage. Although frequently referred as epigenetic mechanisms, the regulatory mechanisms involving microRNAs will not be discussed. Altogether, this review will show how this nascent field influences our understanding of the pathogenesis of OA in terms of diagnosis and how controlling the epigenetic marks can help defining epigenetic therapies.

Sustained intra-articular reactive oxygen species scavenging and alleviation of osteoarthritis by biocompatible amino-modified tantalum nanoparticles.

Recent studies highlight the vital role of oxidative stress and reactive oxygen species (ROS) during progression of osteoarthritis (OA). Attenuating oxidative stress and reducing reactive oxygen species generation in joints represent reasonable strategies for the treatment of osteoarthritis. To address the potential question for clinical translation, and improve the biocompatibility and long-term performance of current antioxidants, the present study provided high biocompatible small positively charged tantalum nanoparticles (Ta-NH2 NPs) with sustained intra-articular catalase activity and first applied to osteoarthritis intervention. Our in vitro results showed that Ta-NH2 NPs were stable with good biocompatibility, and protected viability and hyaline-like phenotype in H2O2-challenged chondrocytes. In addition, the in vivo biodistribution data demonstrated a sustained retention of Ta-NH2 NPs in the joint cavity, particularly in articular cartilage without organ toxicity and abnormality in hemogram or blood biochemistry indexes. Finally, compared with catalase (CAT), Ta-NH2 NPs exhibited long-term therapeutic effect in monosodium iodoacetate (MIA) induced osteoarthritis model. This study preliminarily explored the potential of simply modified metal nanoparticles as effective reactive oxygen species scavenging agent for osteoarthritis intervention, and offered a novel strategy to achieve sustained reactive oxygen species suppression using biocompatible Ta-based nano-medicine in oxidative stress related diseases.

Synergistic prevention and reparative effects of sesquiterpene farnesol in a rabbit model of surgical resection-induced osteoarthritis.

Articular cartilage may regenerate poorly after injury or during aging. In vitro, farnesol can modulate extracellular matrix synthesis and restore chondrocyte phenotypes by increasing type II collagen (COL II) and glycosaminoglycan (GAG) production. Here, we evaluated farnesol's preventive and reparative effects against osteoarthritis (OA) in vivo. We induced OA in rabbits through resection of the lateral collateral ligament and meniscus. After 2 weeks, the affected limb was treated with 0.5 ml of 0.4 mM farnesol, hyaluronan (HA) nanoparticle-encapsulated 0.8 mM farnesol (Farn/HA), or HA nanoparticles intra-articularly. After 2 and 6 treatment weeks, synovial inflammatory cytokine levels were analyzed. We also removed the entire joint cartilage from lateral femoral condyles for histological investigation. The half-maximum inhibitory concentration of farnesol was 0.5 mM. Farn/HA had relatively low cytotoxicity showing cells remained viable after being treated with 1 mM a concentration Farn/HA. Untreated lateral condyle exhibited extensive wear. By contrast, 0.4 mM farnesol or 0.8 mM Farn/HA led to a relatively transparent and bright appearance. After 2 and 6 treatment weeks, farnesol, particularly 0.8 mM Farn/HA, reduced matrix metalloproteinase 1 and 13 levels considerably. Therefore, 0.8 mM Farn/HA, which enabled slow drug release, demonstrated the highest anti-inflammatory and OA preventive effects. After 6 treatment weeks, farnesol also promoted COL II and GAG synthesis and, thus, aided healing.

In vitro study to identify ligand-independent function of estrogen receptor-α in suppressing DNA damage-induced chondrocyte senescence.

In osteoarthritis (OA), chondrocytes undergo many pathological alternations that are linked with cellular senescence. However, the exact pathways that lead to the generation of a senescence-like phenotype in OA chondrocytes are not clear. Previously, we found that loss of estrogen receptor-α (ERα) was associated with an increased senescence level in human chondrocytes. Since DNA damage is a common cause of cellular senescence, we aimed to study the relationship among ERα levels, DNA damage, and senescence in chondrocytes. We first examined the levels of ERα, representative markers of DNA damage and senescence in normal and OA cartilage harvested from male and female human donors, as well as from male mice. The influence of DNA damage on ERα levels was studied by treating human chondrocytes with doxorubicin (DOX), which is an often-used DNA-damaging agent. Next, we tested the potential of overexpressing ERα in reducing DNA damage and senescence levels. Lastly, we explored the interaction between ERα and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway. Results indicated that the OA chondrocytes contained DNA damage and displayed senescence features, which were accompanied by significantly reduced ERα levels. Overexpression of ERα reduced the levels of DNA damage and senescence in DOX-treated normal chondrocytes and OA chondrocytes. Moreover, DOX-induced the activation of NF-κB pathway, which was partially reversed by overexpressing ERα. Taken together, our results demonstrated the critical role of ERα in maintaining the health of chondrocytes by inhibiting DNA damage and senescence. This study also suggests that maintaining the ERα level may represent a new avenue to prevent and treat OA.

Effect of increasing hyaluronic acid content in collagen scaffolds on the maintenance of chondrogenic phenotype in chondrocytes and mesenchymal stem cells

IntroductionIn articular cartilage, hyaluronic acid (HA) has been shown to increase adhesion, differentiation, and proliferation of cartilage cells, and its addition to three-dimensional (3D) scaffolds has been shown to improve chondrocyte phenotype in vitro. ObjectivesThe purpose of this study was to determine (1) whether increasing the HA levels in collagen scaffolds leads to better maintenance of the chondrogenic phenotype in chondrocytes in a dose-dependent manner and (2) whether collagen scaffolds infused with HA promote the differentiation of mesenchymal stem cells (MSCs) into chondrocytes within 3D collagen/HA scaffolds. MethodsBovine chondrocytes and rat MSCs were seeded onto 2% or 6% collagen/HA scaffolds, or no scaffold for controls. Cells were harvested at days 6, 12 and 18. Real-time polymerase chain reaction was used to measure expression of Type I and Type II collagen, SOX-9, and aggrecan. ResultsExpression of Type I and II collagen was increased at day 18 in the chondrocytes of both groups. For the MSCs, at day 18 there was increased expression of Type I collagen and increased expression of aggrecan in both the 2 and 6% scaffold groups, plus increased SOX-9 expression in the 2% HA group. ConclusionsThough there was no significant dose-dependent change between the HA groups in their effects on Type II collagen production, both HA scaffold groups showed significantly increased expression of Type II collagen as compared to monolayer controls at 18 days. Future experiments should extend timepoints to elucidate these effects and human chondrocytes and MSCs should be used to determine biological applicability.

Pentosan polysulfate sodium promotes redifferentiation to the original phenotype in micromass-cultured canine articular chondrocytes and exerts molecular weight-dependent effects.

Pentosan polysulfate sodium (PPS) is a heparin-like polysaccharide that is applied as a therapeutic treatment for osteoarthritis (OA) in animals. This study investigated the efficacy of different molecular weights PPS (1,500-7,000 Da) on the phenotype regulatory and chondrogenic properties of canine articular chondrocytes. The cytotoxicity of PPS on chondrocytes was assessed using flow cytometry and 3-(4,5-dimehylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay. After 72 hr of exposure, PPS did not induce chondrocyte apoptosis, regardless of molecular weight. In addition, chondrogenic properties were determined according to the mRNA and protein levels in micromass-cultured chondrocytes. Quantitative polymerase chain reaction analysis confirmed that PPS promotes a chondrogenic phenotype in chondrocytes in a molecular weight-dependent manner, with significant upregulation of collagen type II alpha 1 chain, aggrecan, and SRY-box transcription factor 9 (SOX9) mRNA levels relative to those in the control. However, the collagen type I alpha 2 chain mRNA level simultaneously increased after 7,000 Da PPS treatment. PPS exposure also increased collagen type II and SOX9 protein production in a molecular weight-dependent manner and inhibited Akt phosphorylation in chondrocytes. Alcian blue staining indicated that PPS treatment enhanced proteoglycan deposition in micromass cultures, with stronger effects observed in 5,000 and 7,000 Da groups. Overall, these results indicate that PPS exerts protective effects on the chondrocyte phenotype and may represent a potential therapeutic target for OA treatment. Increasing the molecular weight of PPS could enhance these anabolic effects.

Thermosensitive injectable hydrogel loaded with hypoxia-induced exosomes maintains chondrocyte phenotype through NDRG3-mediated hypoxic response

Current clinical treatments cannot effectively delay the progression of osteoarthritis (OA). Consequently, joint replacement surgery is required for late-stage OA when patients cannot tolerate pain and joint dysfunction. Therefore, the prevention of OA progression in the early and middle stages is an urgent clinical problem. In a previous study, we demonstrated that NDRG3-mediated hypoxic response might be closely related to the development and progression of OA. In this study, an injectable thermosensitive hydrogel was established by cross-linking Pluronic F-127 and hyaluronic acid (HA) for the sustained release of hypoxia-induced exosomes (HExos) derived from adipose-derived mesenchymal stem cells. We demonstrated that for OA at the early and middle stages, the HExos-loaded HP hydrogel could maintain the chondrocyte phenotype by enhancing chondrocyte autophagy, reducing chondrocyte apoptosis, and promoting chondrocyte activity and proliferation through the NDRG3-mediated hypoxic response. This novel composite hydrogel, which could activate the NDRG3-mediated hypoxic response, may provide new ideas and a theoretical basis for the treatment of early- and mid-stage OA.

Chondrocyte Hypertrophy in Osteoarthritis: Mechanistic Studies and Models for the Identification of New Therapeutic Strategies.

Articular cartilage shows limited self-healing ability owing to its low cellularity and avascularity. Untreated cartilage defects display an increased propensity to degenerate, leading to osteoarthritis (OA). During OA progression, articular chondrocytes are subjected to significant alterations in gene expression and phenotype, including a shift towards a hypertrophic-like state (with the expression of collagen type X, matrix metalloproteinases-13, and alkaline phosphatase) analogous to what eventuates during endochondral ossification. Present OA management strategies focus, however, exclusively on cartilage inflammation and degradation. A better understanding of the hypertrophic chondrocyte phenotype in OA might give new insights into its pathogenesis, suggesting potential disease-modifying therapeutic approaches. Recent developments in the field of cellular/molecular biology and tissue engineering proceeded in the direction of contrasting the onset of this hypertrophic phenotype, but knowledge gaps in the cause-effect of these processes are still present. In this review we will highlight the possible advantages and drawbacks of using this approach as a therapeutic strategy while focusing on the experimental models necessary for a better understanding of the phenomenon. Specifically, we will discuss in brief the cellular signaling pathways associated with the onset of a hypertrophic phenotype in chondrocytes during the progression of OA and will analyze in depth the advantages and disadvantages of various models that have been used to mimic it. Afterwards, we will present the strategies developed and proposed to impede chondrocyte hypertrophy and cartilage matrix mineralization/calcification. Finally, we will examine the future perspectives of OA therapeutic strategies.

Chondrocyte De-Differentiation: Biophysical Cues to Nuclear Alterations.

Autologous chondrocyte implantation (ACI) is a cell therapy to repair cartilage defects. In ACI a biopsy is taken from a non-load bearing area of the knee and expanded in-vitro. The expansion process provides the benefit of generating a large number of cells required for implantation; however, during the expansion these cells de-differentiate and lose their chondrocyte phenotype. In this review we focus on examining the de-differentiation phenotype from a mechanobiology and biophysical perspective, highlighting some of the nuclear mechanics and chromatin changes in chondrocytes seen during the expansion process and how this relates to the gene expression profile. We propose that manipulating chondrocyte nuclear architecture and chromatin organization will highlight mechanisms that will help to preserve the chondrocyte phenotype.

Aging Affects the Efficacy of Platelet-Rich Plasma Treatment for Osteoarthritis.

Despite the increased use of platelet-rich plasma in the treatment of osteoarthritis, whether and how age of the platelet-rich plasma donor affects therapeutic efficacy is unclear. In vitro, male osteoarthritic human chondrocytes were treated with platelet-rich plasma from young (18-35 yrs) or old (≥65 yrs) donors, and the chondrogenic profile was evaluated using immunofluorescent staining for two markers of chondrogenicity, type II collagen and SOX-9. In vivo, we used a within-subjects design to compare Osteoarthritis Research Society International scores in aged mouse knee joints injected with platelet-rich plasma from young or old individuals. In vitro experiments revealed that platelet-rich plasma from young donors induced a more youthful chondrocyte phenotype, as evidenced by increased type II collagen ( P = 0.033) and SOX-9 expression ( P = 0.022). This benefit, however, was significantly blunted when cells were cultured with platelet-rich plasma from aged donors. Accordingly, in vivo studies revealed that animals treated with platelet-rich plasma from young donors displayed a significantly improved cartilage integrity when compared with knees injected with platelet-rich plasma from aged donors ( P = 0.019). Injection of platelet-rich plasma from a young individual induced a regenerative effect in aged cells and mice, whereas platelet-rich plasma from aged individuals showed no improvement in chondrocyte health or cartilage integrity.

Self-Assembly Culture Model for Engineering Musculoskeletal Tissues.

The goal of a self-assembly tissue engineering is to create functional tissue following a natural cell-driven process that mirrors natural development. This approach to tissue engineering has tremendous potential for the development of reparative strategies to treat musculoskeletal injuries and diseases, especially for articular cartilage which has poor regenerative capacity. Additionally, many bioengineering and culture methods fail to maintain the chondrocyte phenotype and contain the correct matrix composition in the long term. Existing cartilage-engineering approaches have been developed, but many approaches involve complicated culture techniques and require foreign substances and biomaterials as scaffolds. While these scaffold-based approaches have numerous advantages, such as an instant or rapid creation of biomechanical properties, they frequently result in dedifferentiation of cells in part, due to the adherence to foreign scaffold materials. In this chapter, we describe a novel approach of developing a scaffold-less cartilage-like biomaterial, using the simple principle that cells at high density bear a capacity to coalesce when they cannot attach to any culture substrate. We refer to the biomaterial formed as a cartilage tissue equivalent or CTA and have published to describe their characteristics and utility in high-throughput drug screening. The method is described to generate reproducible cartilage analogs using a specialized high-density suspension culture technique using a hydrogel poly-2-hydroxyethyl methacrylate (polyHEMA) coating of a culture dish. We have demonstrated that this approach can rapidly form biomass of chondrocytes that over time becomes very synthetically active producing a cartilage-like extracellular matrix that closely mimics the biochemical and biomechanical characteristics of native articular cartilage. The culture approach can also be used to form CTA from other than articular cartilage-derived chondrocytes as well as mesenchymal stem cells (MSCs) (while differentiating MSCs into chondrocytes). Some of the advantages are phenotype stability, reproducible CTA size, and biomechanical and biochemical characteristics similar to natural cartilage.

Loss of RAP2A Aggravates Cartilage Degradation in TMJOA via YAP Signaling

Abnormal stress loading has been considered a major contributor to the initiation of temporomandibular joint osteoarthritis (TMJOA), but studies to date have not identified a functional molecule that transforms physical stress into biological or biochemical signaling in chondrocytes in response to excessive mechanical stress. Ras-related protein Rap-2a (RAP2A) is reportedly a molecular switch that relays extracellular matrix rigidity signals via the Hippo/Yes-associated protein (YAP) pathway. In the present study, RAP2A diminished with cartilage degradation in unilateral anterior crossbite-induced TMJOA mice, as well as severe cartilage matrix degeneration and TMJOA formation in Cre-loxP–mediated conditional RAP2A knockout mice. RAP2A in chondrocytes regulated the Hippo/YAP pathway directly in response to matrix stiffness, and RAP2A/Hippo/YAP was critical for a chondrocyte phenotype switch and matrix synthesis function. Loss of RAP2A impaired cartilage homeostasis and altered chondrocyte phenotype via Hippo/YAP/SRY-box transcription factor 9 signaling. It may be possible to generate therapeutic strategies using RAP2A or YAP to attenuate the TMJOA pathological process at an early stage. This is the first study to reveal the molecular function of RAP2A in TMJOA progression as a mechanotransduction molecule in condylar chondrocytes.

An integrated in silico-in vitro approach for identifying therapeutic targets against osteoarthritis

BackgroundWithout the availability of disease-modifying drugs, there is an unmet therapeutic need for osteoarthritic patients. During osteoarthritis, the homeostasis of articular chondrocytes is dysregulated and a phenotypical transition called hypertrophy occurs, leading to cartilage degeneration. Targeting this phenotypic transition has emerged as a potential therapeutic strategy. Chondrocyte phenotype maintenance and switch are controlled by an intricate network of intracellular factors, each influenced by a myriad of feedback mechanisms, making it challenging to intuitively predict treatment outcomes, while in silico modeling can help unravel that complexity. In this study, we aim to develop a virtual articular chondrocyte to guide experiments in order to rationalize the identification of potential drug targets via screening of combination therapies through computational modeling and simulations.ResultsWe developed a signal transduction network model using knowledge-based and data-driven (machine learning) modeling technologies. The in silico high-throughput screening of (pairwise) perturbations operated with that network model highlighted conditions potentially affecting the hypertrophic switch. A selection of promising combinations was further tested in a murine cell line and primary human chondrocytes, which notably highlighted a previously unreported synergistic effect between the protein kinase A and the fibroblast growth factor receptor 1.ConclusionsHere, we provide a virtual articular chondrocyte in the form of a signal transduction interactive knowledge base and of an executable computational model. Our in silico-in vitro strategy opens new routes for developing osteoarthritis targeting therapies by refining the early stages of drug target discovery.Graphical

Effects of Bushen Zhuangjin Decoction drug serum on SOX9 expression and Chondrocyte phenotype in vitro.

Introduction Bushen Zhuangjin Decoction (BZD), a well-known formulation in Traditional Chinese Medicine, has been widely used for the treatment of osteoarthritis (OA). Due to the poor intrinsic repair capacity of chondrocytes, promoting the proliferation of chondrocytes is an efficient treatment to delay the progression of cartilage degradation.Hypothesis/Gap Statement Therefore, to explore the regulatory mechanism of Bushen Zhuangjin Decoction in chondrocytes will contribute to the repair of chondrocyte injury in OA, and may serve as a potential therapy for OA diseases.Aim To investigate the expression and distribution of SOX9 mediated by serum containing Bushen Zhuangjin Decoction (BZD) and its therapeutic effect on chondrocyte injury in rats.Methodology. The subcultured second-generation rat chondrocytes were randomly divided into four groups, and they were intervened with medium containing different serums, including: blank serum group, low-concentration BZD group, medium-concentration BZD group, and high-concentration BZD group. The viability, proliferation and apoptosis of chondrocytes were detected by MTT assay and flow cytometry. The gene and protein levels of SOX9, aggrecan and type II collagen genes were analysed by qRT-PCR and Western blot analysis. Immunofluorescence staining was used to analyse the expression and distribution of SOX9. Inflammatory factors in different culture mediums of chondrocytes were detected by ELISA.Results Compared with the control group, the activity of chondrocytes in the BZD drug-containing serum group was significantly enhanced, and the degree of apoptosis was significantly decreased. The gene and protein levels of SOX9, proteoglycan aggrecan and collagen II in chondrocytes increased significantly. The inflammatory factors in the culture medium also decreased significantly. And in the above experiments, the medium concentration group BZD drug-containing serum had the best effect.Conclusion Our research results show that BZD medicated serum can up-regulate the expression of SOX9, reduce the release of inflammatory factors, and promote changes in the phenotype of chondrocytes, which protects chondrocytes from damage.

Formulation and evaluation of a bioink composed of alginate, gelatin, and nanocellulose for meniscal tissue engineering.

1The necessity to preserve meniscal function prompts the research and development of novel treatment options, like three-dimensional (3D) bioprinting. However, bioinks for meniscal 3D bioprinting have not been extensively explored. Therefore, in this study, a bioink composed of alginate, gelatin, and carboxymethylated cellulose nanocrystal (CCNC) was formulated and evaluated. Firstly, bioinks with varying concentrations of the aforementioned components were subjected to rheological analysis (amplitude sweep test, temperature sweep test, and rotation). The optimal bioink formulation of 4.0% gelatin, 0.75% alginate, and 1.4% CCNC dissolved in 4.6% D-mannitol was further used for printing accuracy analysis, followed by 3D bioprinting with normal human knee articular chondrocytes (NHAC-kn). The encapsulated cells' viability was > 98%, and collagen II expression was stimulated by the bioink. The formulated bioink is printable, stable under cell culture conditions, biocompatible, and able to maintain the native phenotype of chondrocytes. Aside from meniscal tissue bioprinting, it is believed that this bioink could serve as a basis for the development of bioinks for various tissues.