Search Results (187)

The aim of this preliminary study was to determine the effect of taurine on the expression of genes involved in glycolysis, oxidative phosphorylation, inflammation, autophagy, and regenerative activity in cultured peripheral blood mononuclear cells (PBMCs) and articular cartilage explants from patients with end-stage rheumatoid arthritis (RA). PBMCs and knee articular cartilage were obtained from 20 patients with RA (3 men and 17 women) aged 62.2 ± 10.9 years, with a mean disease duration of 17.5 years (range: 2–43), prior to arthroplasty. PBMCs and cartilage explants were cultured in the presence of 50 µM taurine. Gene expression was determined using real-time reverse transcriptase polymerase chain reaction (RT-PCR). Protein expression of the examined genes in PBMCs was quantified using ELISA. In the presence of 50 µM taurine PBMCs from patients with RA demonstrated a significant increase in the expression of genes encoding pyruvate kinase (PKM2), succinate dehydrogenase (SDHB), uncoupler of oxidation and phosphorylation (UCP2), ATP synthase (ATP5B), and unc-51-like kinase 1 (ULK1). At the same time a significant decrease in tumor necrosis factor (TNF)α and interleukin (IL)-1β expression was noted. In cartilage explants, taurine upregulated SDHB, UCP2, ULK1, and type 2 collagen gene (COL2A1), and decreased TNFα expression. We concluded that, under in vitro conditions, taurine can influence the expression of genes involved in glycolysis, oxidative phosphorylation, inflammation, autophagy, and regenerative processes in PBMCs and articular chondrocytes from patients with end-stage RA. Full article

The development of inks with suitable rheological, physicochemical, mechanical, and biological properties is crucial for the successful fabrication of functional scaffolds via extrusion-based 3D printing. In this study, collagen/chitosan hydrogels with varying polymer ratios were developed and characterized to evaluate their printability and suitability for cartilage tissue engineering. Rheological analyses revealed that all samples exhibited shear-thinning behavior and solid-like viscoelasticity, with the formulation of an 80:20 COL/CHI ratio (20CHI) demonstrating optimal filament formation and dimensional stability. Physicochemical analyses confirmed the preservation of the collagen triple helix and the formation of hydrogen bonding between chitosan and collagen. 20CHI scaffolds showed swelling capacity and high cohesiveness. In vitro studies confirmed the cytocompatibility of the scaffolds with murine fibroblasts and the ability of the scaffolds to promote adhesion, proliferation, and extracellular matrix production of both chondrocytes and adipogenic mesenchymal stem cells (aMSCs). Quantification of sulfated glycosaminoglycan (sGAG) indicated sustained matrix deposition over 28 days, particularly by chondrocytes. These findings demonstrate that 20CHI hydrogel is a promising candidate for 3D printing of biomimetic scaffolds for cartilage regeneration. Full article

Shoulder calcific tendinopathy is a common condition affecting adults and is has a higher incidence in women. This condition is due to a multifactorial process and is characterized by the deposition of hydroxyapatite crystals in the rotator cuff tendons. The disease shows a phenotypic transformation of tenocytes into chondrocyte-like cells, likely caused by metabolic and inflammatory changes and mechanical stress. Risk factors promoting this pathology include hyperlipidemia, advanced age, diabetes, female gender, and thyroid dysfunction. Recent studies highlight that metalloproteinases, oxidative stress, inflammatory mediators, bone morphogenetic proteins (BMPs), genetic and post-transcriptional alterations play a significant role in the pathogenesis of the disease. New therapeutic strategies are currently available that aim to modulate inflammation, osteogenic differentiation, and calcium homeostasis, showing promising results, especially in preclinical models. The aim of this review is to explore the different pathogenetic mechanisms and highlight future therapeutic developments for the treatment of shoulder calcification. Full article

Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) possess the potential for chondrogenic differentiation, offering a promising alternative source for cartilage regeneration. To address the limited availability and expansion capacity of autologous chondrocytes, we investigated the effect of co-overexpression of Sox9, TGFβ1, and type II collagen (Col II) on the chondrogenic differentiation of hUC-MSCs using both 2D and 3D pellet culture systems. Following transfection, the cells exhibited a chondrocyte-like morphology and a marked downregulation of the stemness marker Stro-1. After 21 days in a 3D pellet culture system, the cells formed cartilage-like tissue characterized by the strong expression of chondrocyte-specific genes (Sox9, TGFβ1, Col II, Aggrecan) along with the significant secretion of sulfated glycosaminoglycans (sGaGs). These effects were attributed to enhanced cell–cell contact and extracellular matrix interactions promoted by the 3D environment. Our findings suggest that genetically modified hUC-MSCs cultured in a 3D pellet system represent a robust in vitro model for cartilage regeneration, with potential applications in transplantation and drug toxicity screening. Full article

Osteoarthritis (OA) is a progressive joint disorder affecting over 250 million people globally and is characterized by chronic pain and disability. Among its key pathogenic mechanisms are mitochondrial dysfunction and elevated reactive oxygen species (ROS), often triggered by inflammatory mediators such as lipopolysaccharide (LPS). This study evaluates the protective effects of curcumin on mitochondrial function, autophagy, and apoptosis in an in vitro model of OA. Human bone marrow-derived mesenchymal stem cells (BMSCs) were differentiated into chondrocytes using MesenCult™-ACF medium. Differentiation was confirmed by histological staining for Type II Collagen, Alcian Blue, and Toluidine Blue. LPS was used to induce an OA-like inflammatory response. Mitochondrial membrane potential (ΔΨm) was assessed using Rhodamine 123 staining. Autophagy and apoptosis were evaluated using Acridine orange and propidium iodide staining, respectively. Western blotting was performed to analyze the expression of pro-caspase-3, Bcl-2, Beclin-1, LC3-I/II, and GAPDH. LPS significantly impaired mitochondrial function, limited autophagy, and enhanced apoptotic signaling (reduced pro-caspase-3). Curcumin (25 µM and 100 µM) restored ΔΨm, increased Beclin-1 and LC3-II, and maintained pro-caspase-3 expression, with Bcl-2 showing a non-monotonic response (higher at 25 µM than at 100 µM). Curcumin exerted cytoprotective effects in inflamed chondrocytes by stabilizing ΔΨm, promoting autophagy, and attenuating apoptotic activation, supporting its multi-target therapeutic potential in OA. Full article

Fibrin hydrogels are biocompatible but often lack instructive cues needed to sustain chondrocyte phenotype and cartilage-like matrix formation; therefore, we investigated whether a tricomposite fibrin hydrogel incorporating decellularized articular cartilage matrix (dACM) and decellularized amniotic membrane matrix (dAMM) enhances human articular chondrocyte performance in vitro. Human articular chondrocytes were encapsulated in tricomposite or fibrin-only hydrogels and cultured for 28 days, evaluating degradation kinetics, viability and cell density, histological remodeling (H&E, Masson’s trichrome, Safranin O), immunohistochemistry for type II collagen, aggrecan, and type I collagen, and qPCR of SOX9, COL2A1, ACAN, RUNX2, COL1A2, and COL10A1. The tricomposite remained cytocompatible (~99% viability), supported marked cell expansion (~250% by day 28), and degraded more slowly than fibrin controls. It increased chondrogenic gene expression (SOX9 >3-fold vs. control by day 28; sustained COL2A1 at 1.5–2-fold; early ACAN at 3–5-fold) while attenuating off-target transcriptional programs (RUNX2 ~50% of control, reduced COL1A2, and negligible COL10A1). Consistently, histology showed progressive lacuna-like morphology and proteoglycan-rich matrix accumulation, accompanied by strong type II collagen and aggrecan immunoreactivity and reduced type I collagen. Overall, adding dACM and dAMM to fibrin improved hydrogel biofunctionality and promoted hyaline-like extracellular matrix assembly, supporting further evaluation of this cell-instructive platform for focal articular cartilage repair. Full article

Microgravity (µg)-generated three-dimensional (3D) multicellular aggregates can serve as models of tissue and disease development. They are relevant in the fields of cancer and in vitro metastasis or regenerative medicine (tissue engineering). Driven by the 3R concept—replacement, reduction, and refinement of animal testing—µg-exposure of human cells represents a new alternative method that avoids animal experiments entirely. New Approach Methodologies (NAMs) are used in biomedical research, pharmacology, toxicology, cancer research, radiotherapy, and translational regenerative medicine. Various types of human cells grow as 3D spheroids or organoids when exposed to µg-conditions provided by µg simulating instruments on Earth. Examples for such µg-simulators are the Rotating Wall Vessel, the Random Positioning Machine, and the 2D or 3D clinostat. This review summarizes the most recent literature focusing on µg-engineered tissues. We are discussing all reports examining different tumor cell types from breast, lung, thyroid, prostate, and gastrointestinal cancers. Moreover, we are focusing on µg-generated spheroids and organoids derived from healthy cells like chondrocytes, stem cells, bone cells, endothelial cells, and cardiovascular cells. The obtained data from NAMs and µg-experiments clearly imply that they can support translational medicine on Earth. Full article

Background: Articular cartilage has limited self-repair capacity. While thermoresponsive poly N-isopropyl acrylamide (pNIPAAm)-based Cell Sheet Engineering (CSE) is a promising scaffold-free strategy, its inherent material properties pose limitations. This study developed and validated a novel, non-thermoresponsive CSE platform for functional cartilage regeneration. Methods: A culture platform was fabricated by grafting the biocompatible polymer poly gamma-glutamic acid (γ-PGA) and a disulfide-containing amino acid onto porous PET membranes. This design enables intact cell sheet detachment with its native extracellular matrix (ECM) via specific cleavage of the disulfide bonds by a mild reducing agent. Results: The hydrated substrate exhibited a biomimetic stiffness (~16.2 MPa) that closely mimics native cartilage. The platform showed superior biocompatibility and supported the cultivation of multi-layered rabbit chondrocyte sheets rich in Collagen II and Glycosaminoglycans. Critically, in a rabbit full-thickness defect model, transplanted autologous cell sheets successfully regenerated integrated, hyaline-like cartilage at 12 weeks, as confirmed by MRI, CT, and histological analyses. Conclusions: This novel CSE platform, featuring highly biomimetic stiffness and a gentle, chemically specific detachment mechanism, represents a highly promising clinical strategy for repairing articular cartilage defects. Full article

Background and Objectives: Heterotopic ossification (HO) involves abnormal bone growth in soft tissues. Current treatments are ineffective and prone to adverse effects, suggesting the need for new HO therapies. Intramembranous bone growth is led by osteoblasts. Since osteoblastogenesis and adipogenesis are opposed and mutually controlled processes, this study aims to identify a new repurposed therapeutic tool to inhibit osteoblastogenesis through adipogenesis promotion. Methods: We performed docking experiments between peroxisome proliferator-activated receptor-γ and bone metabolism-affecting drugs, namely, thiazolidinediones (rosiglitazone, pioglitazone), indomethacin, and dexamethasone, to test tritherapy antiosteoblastogenic effect. Mouse mesenchymal stem cells (C3H10T1/2), human osteoblast-like cells (SaOS2 and primary preosteoblasts), and mouse chondrocytes (ATDC5) were differentiated in the presence of these compounds. The effects on osteoblastogenesis, adipogenesis, and endochondral ossification were analysed through marker gene expression via RT–qPCR. Additionally, primary human HO cells and a congenital HO patient were treated with the selected drug combination (P-tritherapy). Results: Tritherapy significantly and synergistically promoted the expression of an adipogenic marker (fatty acid-binding protein 4) and decreased the expression of an osteoblastogenic marker (osteopontin). In an endochondral ossification model, it reduced ossification markers (collagen-2α1) expression, and in HO cells, it increased adipogenesis markers’ expression. Clinically, P-tritherapy administration prompted bone resorption in a patient with progressive osseous heteroplasia. Conclusions: Tritherapy induced adipogenesis while inhibiting osteoblastogenesis and endochondral ossification, demonstrating its potential as a new therapeutic tool to prevent abnormal bone growth. These results were consistent with bone turnover modification observed in a congenital HO patient. This concordance underscores tritherapy potential for rapid and safe translation to prevent HO. Full article

Osteoarthritis (OA) is a degenerative joint disease characterized by progressive cartilage breakdown, synovial inflammation, and subchondral bone remodeling. Previous studies have shown that cellular communication network factor 3 (CCN3) expression increases with age in cartilage, and its overexpression promotes OA-like changes by inducing senescence-associated secretory phenotypes. This study aimed to investigate the effect of Ccn3 knockout (KO) on OA development using a murine OA model. Destabilization of the medial meniscus (DMM) surgery was performed in wild-type (WT) and Ccn3-KO mice. Histological scoring and staining were used to assess cartilage degeneration and proteoglycan loss. Gene and protein expressions of catabolic enzyme (Mmp9), hypertrophic chondrocyte marker (Col10a1), senescence marker, and cyclin-dependent kinase inhibitor 1A (Cdkn1a) were evaluated. Single-cell RNA sequencing (scRNA-seq) data from WT and Sox9-deficient cartilage were reanalyzed to identify Ccn3⁺ progenitor populations. Immunofluorescence staining assessed CD44 and Ki67 expression in articular cartilage. The effects of Ccn3 knockdown on IL-1β-induced Mmp13 and Adamts5 expression in chondrocytes were examined in vitro. Ccn3 KO mice exhibited reduced cartilage degradation and catabolic gene expression compared with WT mice post-DMM. scRNA-seq revealed enriched Ccn3-Cd44 double-positive cells in osteoblast progenitor, synovial mesenchymal stem cell, and mesenchymal stem cell clusters. Immunofluorescence showed increased CCN3⁺/CD44⁺ cells in femoral and tibial cartilage and meniscus. Ki67⁺ cells were significantly increased in DMM-treated Ccn3 KO cartilage, mostly CD44⁺. In vitro Ccn3 knockdown attenuated IL-1β-induced Mmp13 and Adamts5 expressions in chondrocytes. Ccn3 contributes to OA pathogenesis by promoting matrix degradation, inducing hypertrophic changes, and restricting progenitor cell proliferation, highlighting Ccn3 as a potential therapeutic target for OA. Full article

Articular cartilage regeneration remains a major challenge due to its limited self-repair capacity. Bone marrow-derived skeletal stem cells (SSCs) and mesenchymal stem cells (MSCs) are promising candidates for cartilage engineering, although they differ in their chondrogenic potential. This study explored whether co-culturing SSCs and MSCs in three-dimensional (3D) organoid systems under cartilage physioxia (5% O₂) and chondrogenic induction could improve cartilage tissue formation. SSCs, MSCs, and SSC–MSC co-cultures were characterized for morphology, phenotype, and differentiation capacity. Organoids were generated and cultured for 10 days, followed by analysis of morphology, viability, gene expression (SOX9, RUNX2, ACAN, COL2A1, COL10A1, PRG4, and PDPN), chondrocyte-associated antigens (CD44, CD105, CD146, and PDPN), and cartilage ECM proteins (aggrecan, collagen types I, II, and X, and PRG4). SSCs showed robust chondrogenic and osteogenic potential, while MSCs exhibited a balanced multipotency. Co-culture-derived organoids enhanced chondrogenesis and reduced adipogenesis, with higher expression of cartilage-specific ECM and lower hypertrophic marker levels. These findings highlight the functional synergy between SSCs and MSCs in co-culture, promoting the formation of stable, cartilage-like structures under physioxia. The approach offers a promising strategy for generating preclinical models and advancing regenerative therapies for hyaline cartilage repair. Full article

Endometrial mesenchymal stem cells (eMSCs) are a novel and biologically potent source of multipotent stromal cells with potential beyond reproductive medicine. This study explored their phenotypic profile, trilineage differentiation, and the cytoprotective effects of their conditioned media (eMSCCM) on oxidatively stressed neonatal and adult chondrocytes. Canine eMSCs displayed typical fibroblast-like morphology and expressed high levels of mesenchymal surface markers CD29 and CD44, low hematopoietic markers CD34/CD45, and variable CD90, confirming a mesenchymal identity. Differentiation assays revealed osteogenic and chondrogenic differentiation, whereas adipogenic activity was limited. Using eMSCCM at 25% and 50% concentrations, chondrocyte viability was assessed after exposure to 200 µM H₂O₂. eMSCCM significantly enhanced the viability of H₂O₂-stressed chondrocytes in a dose-dependent manner, particularly at 50%, with marked effects at 24 and 48 h. Although metabolic activity declined at 72 h, the treated cells remained more metabolically active than untreated controls. These findings suggest that eMSCCM offers promising cytoprotective effects for cartilage-related oxidative stress conditions. Full article

Cartilage damage, particularly in the knee joint, presents a significant challenge in regenerative medicine due to its limited capacity for self-repair. Conventional treatments like microfracture surgery, autologous chondrocyte implantation (ACI), and osteochondral allografts often fall short, particularly in cases of larger defects or degenerative conditions. This has led to a growing interest in tissue engineering approaches that utilize biomaterial scaffolds to support cartilage regeneration. Among the many materials explored, chitosan—a naturally derived polysaccharide—has gained attention for its biocompatibility, biodegradability, and structural resemblance to the extracellular matrix (ECM) of cartilage. Recent advances in scaffold design have focused on modifying chitosan to improve its mechanical properties and enhance its biological performance. These modifications include chemical crosslinking, the incorporation of bioactive molecules, and the development of composite formulations. Such enhancements have allowed chitosan-based scaffolds to better support mesenchymal stem cell (MSC) differentiation into chondrocytes, paving the way for improved regenerative strategies. This review explores the latest progress in chitosan scaffold fabrication, preclinical findings, and the transition toward clinical applications. It also discusses the challenges that need to be addressed, such as mechanical stability, degradation rates, and the successful translation of research into viable therapeutic solutions. Full article

Osteoarthritis (OA) is a prevalent and debilitating joint disease in older adults with a complex etiology. We investigated the role of SUMOylation, a post-translational modification, in OA pathogenesis, focusing on the mitochondrial chaperone Prohibitin (PHB1) and the cartilage homeostasis transcription factor PITX1. We hypothesized that oxidative stress-induced SUMOylation promotes PHB1 nuclear accumulation, leading to PITX1 downregulation and contributing to OA development. Analysis of cartilage specimens from 27 OA patients and 4 healthy controls revealed an increased nuclear accumulation of PHB1 in OA chondrocytes, accompanied by elevated levels of SUMO-1 and SUMO-2/3. Mechanistically, nuclear PHB1 interacted indirectly with SUMO-1 through a SUMO-interacting motif (SIM), and the deletion of this SIM prevented PHB1 nuclear trapping in OA cells. Furthermore, the SUMO-conjugating enzyme E2 (UBC9) encoded by the UBE2I gene was upregulated in knee OA cartilage, and its overexpression in vitro enhanced PHB1 nuclear accumulation. Consistently, transgenic mice overexpressing the Ube2i gene exhibited increased UBC9 in their knee cartilage, resulting in Pitx1 downregulation and the emergence of an early OA-like phenotype in articular chondrocytes. Our findings uncover a novel role for UBC9-mediated SUMOylation in primary knee and hip OA. This pathway enhances PHB1 nuclear accumulation, contributing to PITX1 repression and subsequent OA development. These results underscore the importance of SUMOylation in OA pathogenesis and suggest potential molecular targets for early diagnosis and therapeutic intervention. Full article

Background/Objectives: Lumbar disc herniation (LDH) is the most common condition associated with low back pain, and it adversely impacts individuals’ health. The interplay between energy metabolism and apoptosis is critical, as the loss of viable cells in the intervertebral disc (IVD) can lead to a cascade of degenerative changes. Efferocytosis is a key biological process that maintains homeostasis by removing apoptotic cells, resolving inflammation, and promoting tissue repair. Therefore, enhancing mitochondrial energy metabolism and efferocytosis function in IVD cells holds great promise as a potential therapeutic approach for LDH. Methods: In this study, energy metabolism and efferocytosis-related differentially expressed genes (EMERDEGs) were identified from the transcriptomic datasets of LDH. Machine learning approaches were used to identify key genes. Functional enrichment analyses were performed to elucidate the biological roles of these genes. The functions of the hub genes were validated by RT-qPCR. The CIBERSORT algorithm was used to compare immune infiltration between LDH and Control groups. Additionally, we used single-cell RNA sequencing dataset to analyze cell-specific expression of the hub genes. Results: By using bioinformatics methods, we identified six EMERDEGs hub genes (IL6R, TNF, MAPK13, ELANE, PLAUR, ABCA1) and verified them using RT-qPCR. Functional enrichment analysis revealed that these genes were primarily associated with inflammatory response, chemokine production, and cellular energy metabolism. Further, we identified candidate drugs as potential treatments for LDH. Additionally, in immune infiltration analysis, the abundance of activated dendritic cells, neutrophils, and gamma delta T cells varied significantly between the LDH group and Control group. The scRNA-seq analysis showed that these hub genes were mainly expressed in chondrocyte-like cells. Conclusions: The identified EMERDEG hub genes and pathways offer novel insights into the molecular mechanisms underlying LDH and suggest potential therapeutic targets. Full article