Cell and Tissue Journal

Abstract Introduction: Cartilage, a tissue without blood vessels and nerves, possesses inherently limited regenerative capacity following injury, often leading to progressive joint degeneration and conditions like osteoarthritis (OA) if left untreated. Current clinical interventions, such as surgical microfracture or autologous chondrocyte implantation (ACI), face significant challenges, including donor site morbidity, immune rejection, and the formation of fibrocartilage with inferior biomechanical properties. These limitations underscore the urgent need for novel therapeutic strategies that can effectively stimulate hyaline cartilage regeneration. In this context, mesenchymal stem cell-derived exosomes (MSC-Exos) have garnered attention as a cell-free regenerative tool, leveraging their cargo of bioactive molecules (e.g., miRNAs, cytokines, and growth factors) to modulate chondrogenesis, suppress inflammation, and enhance extracellular matrix (ECM) synthesis. Concurrently, glucosamine, a natural amino sugar and precursor for glycosaminoglycan (GAG) biosynthesis, has demonstrated dual functionality in joint health: not only does it serve as a building block for proteoglycans critical to cartilage integrity, but it also exhibits chondroprotective effects by mitigating ECM degradation and promoting stem cell chondrogenic differentiation. The potential synergy between MSC-Exos and glucosamine could thus address multiple facets of cartilage repair, combining anabolic stimulation (via exosomal signaling) with metabolic support (via glucosamine supplementation), offering a promising combinatorial approach to halt OA progression and restore functional cartilage.
Aims: This study aimed to investigate the combined effect of mouse bone marrow stem cell-derived exosomes and glucosamine on the expression of cartilage-specific genes, including Sox9, Acan, Col2a1, and Col10a1.
Materials and Methods: Bone marrow mesenchymal stem cells were prepared from NMRI mice. The mice were euthanized by cervical dislocation, the femoral heads were removed, and the bone marrow contents were transferred into a cell culture flask using a syringe containing culture medium. The bone marrow cells were cultured and were ready for use after 3 to 5 passages. The cell supernatant was separated, and exosomes were extracted from it by successive rounds of centrifugation followed by ultracentrifugation. Mesenchymal stem cell viability and determining the appropriate concentration of exosomes and glucosamine were performed using the MTT assay. The experiments were performed on mesenchymal stem cells in 4 groups: control, exosome, glucosamine, and exosome + glucosamine. The effects of exosomes and glucosamine on the expression of Sox9, Acan, Col2a1, and Col10a1 genes in mesenchymal stem cells were investigated in the presence of chondrogenic medium.
Results: According to the MTT assay results demonstrating the synergistic effect of exosomes and glucosamine, the combined concentrations of 15 μg/mL exosomes and 25 μg/mL glucosamine were chosen for subsequent applications. Real-time PCR results showed that the expression of Sox9, Acan, and Col2a1 genes in stem cells treated with exosomes and glucosamine significantly increased compared to the other groups after 14 days, while the expression of the Col10a1 gene significantly decreased compared to the other groups.
Discussion: The combined treatment of bone marrow–derived mesenchymal stem cell (BMSC) exosomes and glucosamine significantly upregulated the expression of key chondrogenic markers, including Sox9, Acan, and Col2a1, while downregulating the hypertrophic marker Col10a1. This gene expression profile suggests a dual beneficial effect: (1) promotion of chondrogenic differentiation and extracellular matrix (ECM) synthesis, and (2) suppression of hypertrophic differentiation, a critical factor in preventing cartilage calcification and osteoarthritis progression. These findings highlight the synergistic potential of BMSC exosomes and glucosamine as a combinatorial therapy for cartilage regeneration. By enhancing anabolic processes (Sox9-mediated chondrogenesis and aggrecan/collagen II deposition) and concurrently inhibiting catabolic pathways (Col10a1-associated hypertrophy), this strategy may offer a promising approach to delay or reverse early-stage cartilage degeneration in degenerative joint diseases
Conclusion: Our study reveals that combining bone marrow stem cell-derived exosomes with glucosamine synergistically enhances chondrogenesis by upregulating key cartilage markers (Sox9, Acan, Col2a1) while suppressing hypertrophy-related Col10a1. This dual action suggests that exosomes promote cartilage matrix synthesis through their bioactive cargo (e.g., miRNAs/growth factors), while glucosamine likely inhibits hypertrophic differentiation, potentially via modulation of the Wnt/β-catenin pathway. These findings support this combination as a promising strategy for improving cartilage repair and preventing OA progression, though further in vivo validation is needed.