Chondrocyte Phenotype Research Articles

ER stress-induced YAP upregulation leads to chondrocyte phenotype loss in age-related osteoarthritis

BackgroundOsteoarthritis (OA) is a common degenerative joint disease, leading to pain and restricted mobility. Age-related endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of OA, but the underlying mechanisms remain unclear. This study aims to explore the relationship between age-related ER stress, YAP overexpression, and chondrocyte phenotype loss in the development of OA.MethodsCartilage samples were collected from patients undergoing amputation, and age-related ER stress markers and YAP expression were assessed using immunohistochemical staining and qPCR. Transgenic mice with cartilage-specific YAP overexpression (YAPOE) were created, and Pamrevlumab was administered to evaluate its therapeutic effects.ResultsHigher expression of ER stress markers and YAP were showed in aged tissues compared to younger tissues. YAP overexpression led to decreased levels of cartilage phenotype markers and increased osteogenesis-related proteins. In vivo, YAPOE mice exhibited OA-like cartilage degeneration, which was mitigated by Pamrevlumab treatment.ConclusionAge-related ER stress induces YAP overexpression, contributing to OA pathogenesis. Pamrevlumab effectively prevents this phenotype loss in YAPOE mice, suggesting its potential as a therapeutic agent for OA. These findings provide new insights into the molecular mechanisms of OA and highlight the importance of targeting the ER stress-YAP-CTGF signaling pathway in OA treatment and prevention.

TRPM2-mediated feed-forward loop promotes chondrocyte damage in osteoarthritis via calcium-cGAS-STING-NF-κB pathway

IntroductionsOsteoarthritis (OA) is a significant contributor to disability in the elderly population. However, current therapeutic options are limited. The transient receptor potential melastatin 2 (TRPM2) is involved in a range of disease processes, yet its role in OA remains unclear. ObjectivesTo investigate the role of TRPM2 in OA. MethodsCartilage samples were collected from patients with osteoarthritis (OA) and mice with OA to examine TRPM2 expression levels. To investigate the effects of TRPM2 modulation on the destabilization of the medial meniscus (DMM) induced knee OA in mice, we utilized TRPM2 knockout mice and employed adenovirus-mediated overexpression of TRPM2. Furthermore, siRNA-mediated TRPM2 knockdown or plasmid-mediated TRPM2 overexpression was conducted to explore the role of TRPM2 in IL-1β-induced chondrocytes. The regulatory mechanism of IL-1β on TRPM2 expression was screened by signaling pathway inhibitors, and the transcription factors and binding sites of TRPM2 were predicted using the database. The binding of RELA (NF-κB-p65) to the Trpm2 promoter was verified by chip-PCR and ChIP-qPCR. The therapeutic potential of Ca2+ chelation with BAPTA-AM for the treatment of osteoarthritis (OA) was investigated. ResultsAn increased expression of TRPM2 was observed in the cartilage of OA patients and OA mice. Furthermore, mice deficient in Trpm2 exhibited a protective effect against DMM-induced OA progression. In contrast, TRPM2 overexpression resulted in exacerbation of DMM-induced OA and thepromotion of an OA-like phenotype of chondrocytes. TRPM2 was upregulated by IL-1β in an NF-κB-p65-dependent manner. Subsequently, the TRPM2-Ca2+-mtDNA-cGAS-STING-NF-κB axis in the progression of OA was validated. Furthermore, inhibition of the TRPM2-Ca2+ axis with BAPTA-AM effectively attenuated established OA. ConclusionsOur data collectively revealed a pathological feedback loop involving TRPM2, Ca2+, mtDNA, cGAS, STING, and NF-κB in OA chondrocytes. This suggests that disrupting this loop could be a viable therapeutic approach for OA.

The Role of Cartilage Tissue Engineering in Osteoarthritis Treatment: The Bench to Bedside Translation

ABSTRACTCartilage tissue engineering holds huge promise for joint defects and osteoarthritis (OA) conditions which otherwise have limited treatment options due to cartilage's inherent inability to self‐repair. Chemical cues play a pivotal role in regulating chondrocyte behavior and matrix synthesis. Strategies utilizing growth factors, small molecules, and biomaterial‐based delivery systems aim to modulate chondrogenic differentiation, proliferation, and matrix deposition, while recent insights emphasize the significance of mimicking native tissue gradients for optimal regeneration outcomes. Mechanical stimuli profoundly influence chondrocyte phenotype and function, necessitating precise control of the mechanical microenvironment in tissue engineering strategies. Advances in biomaterial design, scaffold fabrication, and bioreactor systems facilitate the tailored modulation of mechanical cues, including substrate stiffness, topography, and dynamic loading regimes. This review showcases the latest advancements in engineering both the chemical and mechanical microenvironment to enhance chondrocyte regeneration. Furthermore, exploring the synergistic effects of combining chemical and mechanical cues underscores the importance of multifaceted approaches in promoting robust chondrocyte regeneration. The review also addresses challenges and future directions in the field, such as achieving spatially organized tissue architecture and integrating patient‐specific factors, to propel advancements in cartilage tissue engineering. We also conducted a comprehensive enlistment for the clinical trials and tissue engineering‐based marketed products for OA therapy.

Hydrogel composition and mechanical stiffness of 3D bioprinted cell-loaded scaffolds promote cartilage regeneration

ObjectiveTo investigate the impact of different component ratios and mechanical stiffness of the gelatin-sodium alginate composite hydrogel scaffold, fabricated through 3D bioprinting, on the viability and functionality of chondrocytes.MethodsThree different concentrations of hydrogel, designated as low, medium, and high, were prepared. The rheological properties of the hydrogel were characterized to optimize printing parameters. Subsequently, the printability and shape fidelity of the cell-loaded hydrogel scaffolds were statistically evaluated, and the chondrocyte viability was observed. Dynamic mechanical analysis was conducted to measure the modulus, thereby assessing the scaffold’s stiffness. Following a 21-day culture period, RT-PCR, histological staining, and immunostaining were employed to assess chondrocyte activity, chondrosphere aggregates formation, and cartilage matrix production.ResultsBased on rheological analysis, optimal printing temperatures for each group were determined as 27.8°C, 28.5°C, and 30°C. The optimized printing parameters could ensure the molding effect of the scaffolds on the day of printing, with the actual grid area of the scaffolds was close to the theoretical grid area. And the scaffolds exhibited good cell viability (93.24% ± 0.99%, 92.04% ± 1.49%, and 88.46% ± 1.53%). After 7 days of culture, the medium and high concentration groups showed no significant change in grid area compared to the day of printing (p > 0.05), indicating good morphological fidelity. As the hydrogel’s bicomponent ratio increased, both the storage modulus and loss modulus increased, while the loss factor remained relatively constant. The highest number of chondrocytes-formed chondrosphere aggregates in the medium concentration group was observed by light microscopy. RT-PCR results indicated significantly higher expression levels of chondrogenic genes SOX9, Agg, and Col-II in the low and medium concentration groups compared to the high concentration group (p < 0.05). Histological staining results showed that the middle concentration group formed the highest number of typical cartilage lacunae.ConclusionThe aforementioned results indicate that in 3D bioprinted cell-loaded GA-SA composite hydrogel scaffolds, the scaffolds with the composition ratio (10:3) and mechanical stiffness (∼155 kPa) exhibit sustained morphological fidelity, effectively preserve the hyaline phenotype of chondrocytes, and are more conducive to cartilage regeneration.

The Therapeutic Potential of Adipose-Derived Mesenchymal Stem Cell Secretome in Osteoarthritis: A Comprehensive Study

Osteoarthritis (OA) is a degenerative joint disease characterized by cartilage degradation and inflammation. This study investigates the therapeutic potential of secretome derived from adipose tissue mesenchymal stem cells (ASCs) in mitigating inflammation and promoting cartilage repair in an in vitro model of OA. Our in vitro model comprised chondrocytes inflamed with TNF. To assess the therapeutic potential of secretome, inflamed chondrocytes were treated with it and concentrations of pro-inflammatory cytokines, metalloproteinases (MMPs) and extracellular matrix markers were measured. In addition, secretome-treated chondrocytes were subject to a microarray analysis to determine which genes were upregulated and which were downregulated. Treating TNF-inflamed chondrocytes with secretome in vitro inhibits the NF-κB pathway, thereby mediating anti-inflammatory and anti-catabolic effects. Additional protective effects of secretome on cartilage are revealed in the inhibition of hypertrophy markers such as RUNX2 and COL10A1, increased production of COL2A1 and ACAN and upregulation of SOX9. These findings suggest that ASC-derived secretome can effectively reduce inflammation, promote cartilage repair, and maintain chondrocyte phenotype. This study highlights the potential of ASC-derived secretome as a novel, non-cell-based therapeutic approach for OA, offering a promising alternative to current treatments by targeting inflammation and cartilage repair mechanisms.

Senolytic therapy combining Dasatinib and Quercetin restores the chondrogenic phenotype of human osteoarthritic chondrocytes by the release of pro-anabolic mediators.

Cellular senescence is associated with various age-related disorders and is assumed to play a major role in the pathogenesis of osteoarthritis (OA). Based on this, we tested a senolytic combination therapy using Dasatinib (D) and Quercetin (Q) on aged isolated human articular chondrocytes (hACs), as well as in OA-affected cartilage tissue (OARSI grade 1-2). Stimulation with D + Q selectively eliminated senescent cells in both, cartilage explants and isolated hAC. Furthermore, the therapy significantly promoted chondroanabolism, as demonstrated by increased gene expression levels of COL2A1, ACAN, and SOX9, as well as elevated collagen type II and glycosaminoglycan biosynthesis. Additionally, D + Q treatment significantly reduced the release of SASP factors (IL6, CXCL1). RNA sequencing analysis revealed an upregulation of the anabolic factors, inter alia, FGF18, IGF1, and TGFB2, as well as inhibitory effects on cytokines and the YAP-1 signaling pathway, explaining the underlying mechanism of the chondroanabolic promotion upon senolytic treatment. Accordingly, stimulation of untreated hAC with conditioned medium of D + Q-treated cells similarly induced the expression of chondrogenic markers. Detailed analyses demonstrated that chondroanabolic effects could be mainly attributed to Dasatinib, while monotherapeutical application of Quercetin or Navitoclax did not promote the chondroanabolism. Overall, D + Q therapy restored the chondrogenic phenotype in OA hAC most likely by creating a pro-chondroanabolic environment through the reduction of SASP factors and upregulation of growth factors. This senolytic approach could therefore be a promising candidate for further testing as a disease-modifying osteoarthritis drug.

Protein folding dependence on selenoprotein M contributes to steady cartilage extracellular matrix repressing ferroptosis via PERK/ATF4/CHAC1 axis

ObjectiveInitiation of endoplasmic reticulum (ER) stress is pivotal to the advancement of osteoarthritis (OA). We aimed to explore the function of ER-resident selenoprotein M (SELM) in cartilage-forming chondrocytes, investigating how SELM participates in cartilage extracellular matrix (ECM) metabolism and ER stress modulation. MethodsArticular cartilage samples with knee OA undergoing total knee arthroplasty were categorized into OA-smooth and OA-damaged groups, with primary chondrocytes extracted from smooth areas. Destabilization of the medial meniscus was induced in male C57BL6/J mice, with sham operations on the left knee as controls. After 8 weeks, knee joint tissues were collected for analysis. Histology and immunohistochemistry examined cartilage damage. Molecular biology techniques investigated how SELM affects ECM metabolism and ER stress regulation. RNA sequencing revealed the pathway changes after SELM intervention. AlphaFold demonstrated how SELM interacts with other molecules. Cultured cartilage explants helped determine the effects of SELM supplementation. ResultsSELM expression was reduced in the damaged cartilage. Increasing SELM levels positively impacted ECM equilibrium. Decreasing SELM expression activated genes linked to degenerative ailments and impaired the cellular response to misfolded proteins, initiating the PERK/P-EIF2A/ATF4 pathway and exacerbating GSH/GSSG imbalance via the ATF4/CHAC1 axis. SELM likely participated in protein folding and modification by leveraging its thioredoxin domains. In vitro SELM supplementation mitigated IL-1β effects on damaged cartilage explants and suppressed beneficial chondrocyte phenotypes. ConclusionsOur results confirm the involvement of SELM in ER stress-induced cartilage damage as well as protein folding, pointing to new directions in molecular therapy for degenerative diseases.

Matrix Viscoelasticity Tunes the Mechanobiological Behavior of Chondrocytes.

In articular cartilage, the pericellular matrix acting as a specialized mechanical microenvironment modulates environmental signals to chondrocytes through mechanotransduction. Matrix viscoelastic alterations during cartilage development and osteoarthritis (OA) degeneration play an important role in regulating chondrocyte fate and cartilage matrix homeostasis. In recent years, scientists are gradually realizing the importance of matrix viscoelasticity in regulating chondrocyte function and phenotype. Notably, this is an emerging field, and this review summarizes the existing literatures to the best of our knowledge. This review provides an overview of the viscoelastic properties of hydrogels and the role of matrix viscoelasticity in directing chondrocyte behavior. In this review, we elaborated the mechanotransuction mechanisms by which cells sense and respond to the viscoelastic environment and also discussed the underlying signaling pathways. Moreover, emerging insights into the role of matrix viscoelasticity in regulating chondrocyte function and cartilage formation shed light into designing cell-instructive biomaterial. We also describe the potential use of viscoelastic biomaterials in cartilage tissue engineering and regenerative medicine. Future perspectives on mechanobiological comprehension of the viscoelastic behaviors involved in tissue homeostasis, cellular responses, and biomaterial design are highlighted. Finally, this review also highlights recent strategies utilizing viscoelastic hydrogels for designing cartilage-on-a-chip.

Macrophage and chondrocyte phenotypes in inflammation.

Inflammation is a complex biological process protecting the body from diverse external threats. Effectively performing this task requires an intricate, well-regulated interplay of different cells and tissues. Furthermore, several cells participating in inflammation can assume diverse phenotypes. A classic and relatively well-studied example of phenotypic diversity in inflammation is macrophage polarization. Based on the TH1/TH2 phenotypes of T helper cells, this scheme has proinflammatory "classical/M1" activation contrasted with the anti-inflammatory and healing-promoting "alternative/M2" phenotype. Some authors have extended the concept into an M17 phenotype induced by the classic TH17 cytokine IL-17. Phenotypic changes in chondrocytes have also been studied especially in the context of osteoarthritis (OA), and there are indications that these cells can also assume polarized phenotypes at least partly analogous to those of TH cells and macrophages. The therapeutic success of biological agents targeting TH1/TH2/TH17 inductor and/or effector cytokines displays the utility of the concept of polarization. The aim of this focused review is to survey the internal and external factors affecting macrophage and chondrocyte phenotypes (such as inflammatory cytokines, widely used medications and natural products) and to explore the possibility of ameliorating pathological states by modulating these phenotypes.

Chemically programmed metabolism drives a superior cell fitness for cartilage regeneration.

The rapid advancement of cell therapies underscores the importance of understanding fundamental cellular attributes. Among these, cell fitness-how transplanted cells adapt to new microenvironments and maintain functional stability in vivo-is crucial. This study identifies a chemical compound, FPH2, that enhances the fitness of human chondrocytes and the repair of articular cartilage, which is typically nonregenerative. Through drug screening, FPH2 was shown to broadly improve cell performance, especially in maintaining chondrocyte phenotype and enhancing migration. Single-cell transcriptomics indicated that FPH2 induced a super-fit cell state. The mechanism primarily involves the inhibition of carnitine palmitoyl transferase I and the optimization of metabolic homeostasis. In animal models, FPH2-treated human chondrocytes substantially improved cartilage regeneration, demonstrating well-integrated tissue interfaces in rats. In addition, an acellular FPH2-loaded hydrogel proved effective in preventing the onset of osteoarthritis. This research provides a viable and safe method to enhance chondrocyte fitness, offering insights into the self-regulatory mechanisms of cell fitness.

Targeting Fascin1 maintains chondrocytes phenotype and attenuates osteoarthritis development

Osteoarthritis (OA) is the most common form of arthritic disease, and phenotypic modification of chondrocytes is an important mechanism that contributes to the loss of cartilage homeostasis. This study identified that Fascin actin-bundling protein 1 (FSCN1) plays a pivotal role in regulating chondrocytes phenotype and maintaining cartilage homeostasis. Proteome-wide screening revealed markedly upregulated FSCN1 protein expression in human OA cartilage. FSCN1 accumulation was confirmed in the superficial layer of OA cartilage from humans and mice, primarily in dedifferentiated-like chondrocytes, associated with enhanced actin stress fiber formation and upregulated type I and III collagens. FSCN1-inducible knockout mice exhibited delayed cartilage degeneration following experimental OA surgery. Mechanistically, FSCN1 promoted actin polymerization and disrupted the inhibition of Decorin on TGF-β1, leading to excessive TGF-β1 production and ALK1/Smad1/5 signaling activation, thus, accelerated chondrocyte dedifferentiation. Intra-articular injection of FSCN1-overexpressing adeno-associated virus exacerbated OA progression in mice, which was mitigated by an ALK1 inhibitor. Moreover, FSCN1 inhibitor NP-G2-044 effectively reduced extracellular matrix degradation in OA mice, cultured human OA chondrocytes, and cartilage explants by suppressing ALK1/Smad1/5 signaling. These findings suggest that targeting FSCN1 represents a promising therapeutic approach for OA.

Activating UCHL1 through the CRISPR activation system promotes cartilage differentiation mediated by HIF-1α/SOX9.

Developing strategies to enhance cartilage differentiation in mesenchymal stem cells and preserve the extracellular matrix is crucial for successful cartilage tissue reconstruction. Hypoxia-inducible factor-1α (HIF-1α) plays a pivotal role in maintaining the extracellular matrix and chondrocyte phenotype, thus serving as a key regulator in chondral tissue engineering strategies. Recent studies have shown that Ubiquitin C-terminal hydrolase L1 (UCHL1) is involved in the deubiquitylation of HIF-1α. However, the regulatory role of UCHL1 in chondrogenic differentiation has not been investigated. In the present study, we initially validated the promotive effect of UCHL1 expression on chondrogenesis in adipose-derived stem cells (ADSCs). Subsequently, a hybrid baculovirus system was designed and employed to utilize three CRISPR activation (CRISPRa) systems, employing dead Cas9 (dCas9) from three distinct bacterial sources to target UCHL1. Then UCHL1 and HIF-1α inhibitor and siRNA targeting SRY-box transcription factor 9 (SOX9) were used to block UCHL1, HIF-1α and SOX9, respectively. Cartilage differentiation and chondrogenesis were measured by qRT-PCR, immunofluorescence and histological staining. We observed that the CRISPRa system derived from Staphylococcus aureus exhibited superior efficiency in activating UCHL1 compared to the commonly used the CRISPRa system derived from Streptococcus pyogenes. Furthermore, the duration of activation was extended by utilizing the Cre/loxP-based hybrid baculovirus. Moreover, our findings show that UCHL1 enhances SOX9 expression by regulating the stability and localization of HIF-1α, which promotes cartilage production in ADSCs. These findings suggest that activating UCHL1 using the CRISPRa system holds significant potential for applications in cartilage regeneration.

Interleukin-4-Loaded Gelatin Methacryloyl Hydrogel Promotes Subcutaneous Chondrogenesis of Engineered Auricular Cartilage in a Rabbit Model.

Tissue engineering technology offers a promising solution for ear reconstruction; however, it faces the challenge of foreign body reaction and neocartilage malformation. This study investigates the impact of interleukin-4 (IL-4), an anti-inflammatory factor, on cartilage regeneration of hydrogel encapsulating autologous auricular chondrocytes in a rabbit subcutaneous environment. Initially, we assessed the influence of IL-4 on chondrocyte proliferation and determined the appropriate concentration using the CCK-8 test in vitro. Subsequently, we loaded IL-4 into gelatin methacryloyl (GelMA) hydrogel containing chondrocytes and measured its release profile through ELISA. The constructs were then implanted autologously into rabbits' subcutis, and after 3, 7, 14, and 28 days, cartilage matrix formation was evaluated by histological examinations, and gene expression levels were detected by qRT-PCR. Results demonstrated that IL-4 promotes chondrocyte proliferation in vitro, and maximum release from constructs occurred during the first week. In the rabbit subcutaneous implantation model, IL-4-loaded constructs (20 ng/mL) maintained a superior chondrocytic phenotype compared to controls with increased expression of anti-inflammatory factors. These findings highlight IL-4 as a potential strategy for promoting chondrogenesis in a subcutaneous environment and improving ear reconstruction.

A tetrahedral framework nucleic acids-based gene therapeutic nanococktail alleviates cartilage damage and protects against osteoarthritis progression

Gene therapy based on therapeutic microRNAs (miRNAs) shows great potential in Osteoarthritis (OA) treatment. However, individual miRNAs application is limited by the single target, the instability, and the low efficiency of cell internalization. Here, we identified two potential candidate miRNAs, miR-140-5p and miR-455-3p, exhibited combined enhancing effects against OA progression. Furthermore, a nanococktail therapeutic platform, T-(140 + 455) was constructed by employing tetrahedral framework nucleic acids (tFNAs) to codeliver miR-140-5p/miR-455-3p and achieve enhanced gene therapy for OA. Compared with the delivery of individual miRNAs, T-(140 + 455) can effectively reverse the cell phenotype and metabolism of OA chondrocytes. Moreover, the nanococktail T-(140 + 455) reprogrammed macrophage polarization by modulating the crosstalk between chondrocytes and macrophages, combatting the damage caused by inflammation. The OA rat model treated with nanococktail T-(140 + 455) exhibited favorable performance in halting cartilage damage and regulating extracellular matrix (ECM) metabolic homeostasis. Thus, tFNA-based nanococktails enhance the delivery and efficacy of miRNAs, offering a promising strategy for OA gene therapy.

Repair of Mechanical Cartilage Damage Using Exosomes Derived from Deer Antler Stem Cells.

Articular cartilage has limited self-repair capacity, and current clinical treatment options for cartilage defects are inadequate. However, deer antler cartilage possesses unique regenerative properties, with the ability to rapidly repair itself. This rapid self-repair process is closely linked to the paracrine factors released by deer antler stem cells. These findings present potential for the development of cell-free therapies for cartilage defects in clinical settings. The aim of this study was to investigate a novel method for repairing cartilage. A rat model with articular cartilage defects was established through surgery. Hydrogels loaded with exosomes (Exos) derived from antler stem cells (ASC-Exos) were implanted into the rat cartilage defects. The extent of cartilage damage repair was assessed using histological methods. The effects of ASC-Exos on chondrocytes and rat bone marrow mesenchymal stem cells (BMSCs) were evaluated using cell viability assays, proliferation assays, and scratch assays. Additionally, the maintenance of the chondrocyte phenotype by ASC-Exos was assessed using real-time fluorescence quantitative PCR (qPCR) and western blot analysis. The protein components contained of the Exos were identified using data-independent acquisition (DIA) mass spectrometry. ASC-Exos significantly promoted the repair of cartilage tissue damage. The level of cartilage repair in the experimental group (ASC-Exos) was higher than that in the positive control (human adipose-derived stem cells, hADSC-Exos) and negative control (dulbecco's modified eagle medium) groups (p < 0.05). In vitro experiments demonstrated that ASC-Exos significantly enhanced the proliferation abilities of chondrocytes and the proliferation abilities and the migration abilities of BMSCs (p < 0.05). ASC-Exos up-regulated the expression levels of Aggrecan, Collagen II (COLII), and Sox9 mRNA and proteins in chondrocytes. Analysis of ASC-Exos protein components revealed the presence of active components such as Serotransferrin (TF), S100A4, and Insulin-like growth factor-binding protein 1 (IGF1). ASC-Exos have a significant effect on cartilage damage repair, which may be attributed to their promotion of chondrocyte and BMSCs proliferation and migration, as well as the maintenance of chondrocyte phenotype. This effect may be mediated by the presence of TF, S100A4, and IGF1.

IL-40 is up-regulated in the synovial fluid and cartilage of osteoarthritis patients and contributes to the alteration of chondrocytes phenotype in vitro

IntroductionIL-40 is a novel cytokine associated with autoimmune connective tissue disorders such as rheumatoid arthritis (RA) or Sjögren syndrome. We have previously shown an accumulation of IL-40 in the RA joint and its expression by immune cells and fibroblasts. Therefore, we aimed to assess the role of IL-40 in association with hyaline cartilage and chondrocyte activity.MethodsImmunohistochemistry was employed to detect IL-40 in paired samples of loaded and unloaded regions of osteoarthritis (OA) cartilage (n=5). Synovial fluid IL-40 was analysed by ELISA in OA (n=31) and control individuals after knee injury (n=34). The impact of IL-40 on chondrocytes was tested in vitro.ResultsIL-40 was found in chondrocytes of the superficial zone of the OA cartilage, both in loaded and unloaded explants. Additionally, only biopsies from loaded explants showed significant IL-40 positivity in transitional zone chondrocytes. Levels of IL-40 were significantly elevated in the synovial fluid from OA patients compared to controls (p<0.0009) and correlated with synovial fluid leukocyte counts in OA (r=0.444, p=0.014). Chondrocytes exposed to IL-40 dose dependently increased in the secretion of pro-inflammatory cytokines IL-6 (p<0.0001) and IL-8 (p=0.004). Moreover, a dose dependent up-regulation of matrix degrading metalloproteinases MMP-1 (p=0.004), MMP-3 (p=0.031) and MMP-13 (p=0.0002) upon IL-40 treatment was observed in contrast to untreated chondrocytes.ConclusionThis study is the first to demonstrate the accumulation of IL-40 in OA cartilage and its up-regulation in the synovial fluid of OA patients compared to controls. In addition, extracellular IL-40 appears to play a role in promoting inflammation and cartilage destruction by driving chondrocyte behaviour towards a more aggressive phenotype.

Plasma Membrane-Derived Biomimetic Apoptotic Nanovesicles Targeting Inflammation and Cartilage Degeneration for Osteoarthritis.

Osteoarthritis (OA) is a degenerative whole-joint disease in which the synovium and joint cartilage become inflamed and damaged. The essential role of inflammation in the development of OA has been recognized recently. Accordingly, simultaneous regulation of local inflammation and tissue degeneration is proposed as a promising therapeutic strategy. Herein, multifunctional biomimetic apoptotic nanovesicles (Apo-NVs) are constructed with plasma membrane derived from apoptotic T cells. The anti-inflammatory microRNA-124 is further encapsulated into Apo-NVs in the hope of achieving an enhanced immunomodulatory effect. It is found that apoptotic nanovesicles, including Apo-NVs and Apo-NVs-miR-124, both efficiently promote the M2 repolarization of M1 macrophages and inhibit the degenerative phenotype of chondrocytes. Further in vivo studies show that Apo-NVs and Apo-NVs-miR-124 alleviate synovial inflammation and protect cartilage tissue from degeneration in OA mice. The study highlights the potential of Apo-NVs in treating OA and other inflammation-related diseases.

Influence of storage conditions on the viability of spheroids from human chondrocytes

BACKGROUND: The use of tissue-engineered products for treating hyaline cartilage injuries is currently a promising direction for regenerative medicine. The study of storage and transportation conditions of such preparations is essential in product development because it determines the viability and functional activity of the cellular component of the product. In addition, such work should be performed following the requirements of Russian legislation before using the developed drug product in clinical trials. AIM: To characterize the storage conditions for tissue-engineered preparations such as chondrospheres. METHODS: Chondrospheres were formed from human chondrocytes obtained from the biopsy material of the knee joint cartilage of patients diagnosed according to ICD-10 codes M15–M19. After the formation of chondrospheres, cell viability was examined using the metabolic dye PrestoBlue, and the expression of chondrogenic markers (SOX9, aggrecan, collagen type 1 and type 2) was preserved using real-time polymerase chain reaction under various transportation conditions, i.e., at different storage times, medium composition, and temperature. RESULTS: The analysis of the expression of chondrocyte markers confirmed that cells in chondrospheres retain a chondrocyte phenotype. In the cell viability study of tissue-engineered products under various conditions (temperature, solution, and time), the most favorable environment for storing the product was phosphate buffer saline and 0.9% sodium chloride solution (р 0.05). The metabolic activity of the cells that make up the tissue-engineered product was maintained when stored at +4 °C for up to 3 days (р 0.05). CONCLUSION: Isotonic solution and temperature regime +4 °C for not more than 2 days showed the preferable medium and conditions for storing and transporting the prototype cartilage implant as chondrospheres.

Coaxial electrohydrodynamic printing of core–shell microfibrous scaffolds with layer-specific growth factors release for enthesis regeneration

The rotator cuff tear has emerged as a significant global health concern. However, existing therapies fail to fully restore the intricate bone-to-tendon gradients, resulting in compromised biomechanical functionalities of the reconstructed enthesis tissues. Herein, a tri-layered core–shell microfibrous scaffold with layer-specific growth factors (GFs) release is developed using coaxial electrohydrodynamic (EHD) printing for in situ cell recruitment and differentiation to facilitate gradient enthesis tissue repair. Stromal cell-derived factor-1 (SDF-1) is loaded in the shell, while basic fibroblast GF, transforming GF-beta, and bone morphogenetic protein-2 are loaded in the core of the EHD-printed microfibrous scaffolds in a layer-specific manner. Correspondingly, the tri-layered microfibrous scaffolds have a core–shell fiber size of (25.7 ± 5.1) μm, with a pore size sequentially increasing from (81.5 ± 4.6) μm to (173.3 ± 6.9) μm, and to (388.9 ± 6.9 μm) for the tenogenic, chondrogenic, and osteogenic instructive layers. A rapid release of embedded GFs is observed within the first 2 d, followed by a faster release of SDF-1 and a slightly slower release of differentiation GFs for approximately four weeks. The coaxial EHD-printed microfibrous scaffolds significantly promote stem cell recruitment and direct their differentiation toward tenocyte, chondrocyte, and osteocyte phenotypes in vitro. When implanted in vivo, the tri-layered core–shell microfibrous scaffolds rapidly restored the biomechanical functions and promoted enthesis tissue regeneration with native-like bone-to-tendon gradients. Our findings suggest that the microfibrous scaffolds with layer-specific GFs release may offer a promising clinical solution for enthesis regeneration.

CKIP-1-Loaded Cartilage-Affinitive Nanoliposomes Reverse Osteoarthritis by Restoring Chondrocyte Homeostasis.

Osteoarthritis (OA) is a chronic joint disease characterized by cartilage imbalance and disruption of cartilage extracellular matrix secretion. Identifying key genes that regulate cartilage differentiation and developing effective therapeutic strategies to restore their expression is crucial. In a previous study, we observed a significant correlation between the expression of the gene encoding casein kinase-2 interacting protein-1 (CKIP-1) in the cartilage of OA patients and OA severity scores, suggesting its potential involvement in OA development. To test this hypothesis, we synthesized a chondrocyte affinity plasmid, liposomes CKIP-1, to enhance CKIP-1 expression in chondrocytes. Our results demonstrated that injection of CAP-Lipos-CKIP-1 plasmid significantly improved OA joint destruction and restored joint motor function by enhancing cartilage extracellular matrix (ECM) secretion. Histological and cytological analyses confirmed that CKIP-1 maintains altered the phosphorylation of the signal transduction molecule SMAD2/3 of the transforming growth factor-β (TGF-β) pathway by promoting the phosphorylation of the 8T, 416S sit. Taken together, this work highlights a novel approach for the precise modulation of chondrocyte phenotype from an inflammatory to a noninflammatory state for the treatment of OA and may be broadly applicable to patients suffering from other arthritic diseases.