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Background: Osteoarthritis (OA), the most common joint disorder, is characterized by a progressive degradation of articular cartilage. Increasing evidence suggests that OA is closely associated with cartilage pathologies including chondrocyte hypertrophy and fibrosis. Methods: In this study, we showed that asiatic acid (AA) treatment reduced chondrocyte hypertrophy and fibrosis. First, the cytotoxicity of AA (0, 5, 10, and 20 μM) to chondrocytes was evaluated, and 5 μM was selected for subsequent experiments. Then, we detected the gene and protein level of chondrocyte hypertrophic markers including type X collagen (COL-X), matrix metalloproteinase-13 (MMP-13), alkaline phosphatase (ALP), and runtrelated transcription factor 2 (Runx2); chondrocyte fibrosis markers including type I collagen (COL-Ι) and alphasmooth muscle actin (α-SMA); and chondrogenic markers including SRY-related HMG box 9 (SOX9), type II collagen (COL-II), and aggrecan (ACAN). Further, we tested the mechanism of AA on inhibiting chondrocyte hypertrophy and fibrosis. Finally, we verified the results in an anterior cruciate ligament transection (ACLT) rat OA model. Results: We found that AA treatment inhibited the hypertrophic and fibrotic phenotype of chondrocytes, without affecting the chondrogenic phenotype. Moreover, we found that AA treatment activated AMP-activated protein kinase (AMPK) and inhibited phosphoinositide-3 kinase/protein kinase B (PI3K/AKT) signaling pathway in vitro. The results in an ACLT rat OA model also indicated that AA significantly attenuated chondrocyte hypertrophy and fibrosis. Conclusion: AA treatment could reduce hypertrophic and fibrotic differentiation and maintain the chondrogenic phenotype of articular chondrocytes by targeting the AMPK/PI3K/AKT signaling pathway. Our study suggested that AA might be a prospective drug component that targets hypertrophic and fibrotic chondrocytes for OA treatment.

Ca2+ has been recognized as a key molecule for chondrocytes, however, the role and mechanism of spontaneous [Ca 2+] i signaling in cartilaginous extracellular matrix (ECM) metabolism regulation are unclear. Here we found that spontaneous Ca 2+ signal of in‐situ porcine chondrocytes was [Ca 2+] o dependent, and mediated by [Ca 2+] i store release. T‐type voltage‐dependent calcium channel (T‐VDCC) mediated [Ca 2+] o influx was associated with decreased cell viability and expression levels of ECM deposition genes. Further analysis revealed that chondrocytes expressed both inositol 1,4,5‐trisphosphate receptor (InsP3R) and Orai isoforms. Inhibition of endoplasmic reticulum (ER) Ca 2+ release and store‐operated calcium entry significantly abolished spontaneous [Ca 2+] i signaling of in‐situ chondrocytes. Moreover, blocking ER Ca 2+ release with InsP3R inhibitors significantly upregulated ECM degradation enzymes production, and was accompanied by decreased proteoglycan and collagen type II intensity. Taken together, our data provided evidence that spontaneous [Ca 2+] i signaling of in‐situ porcine chondrocytes was tightly regulated by [Ca 2+] o influx, InsP3Rs mediated [Ca 2+] i store release, and Orais mediated calcium release‐activated calcium channels activation. Both T‐VDCC mediated [Ca 2+] o influx and InsP3Rs mediated ER Ca 2+ release were found crucial to cartilaginous ECM metabolism through distinct regulatory mechanisms.

Intra-articular injection of mesenchymal stem cells (MSCs) is a promising strategy for osteoarthritis (OA) treatment. However, more and more studies reveal that the injected MSCs have poor adhesion, migration, and survival in the joint cavity. A recent study shows that tropoelastin (TE) regulates adhesion, proliferation and phenotypic maintenance of MSCs as a soluble additive, indicating that TE could promote MSCs-homing in regenerative medicine. In this study, we used TE as injection medium, and compared it with classic media in MSCs intra-articular injection such as normal saline (NS), hyaluronic acid (HA), and platelet-rich plasma (PRP). We found that TE could effectively improve adhesion, migration, chondrogenic differentiation of infrapatellar fat pad MSCs (IPFP-MSCs) and enhance matrix synthesis of osteoarthritic chondrocytes (OACs) in indirect-coculture system. Moreover, TE could significantly enhance IPFP-MSCs adhesion via activation of integrin β1, ERK1/2 and vinculin (VCL) in vitro . In addition, intra-articular injection of TE-IPFP MSCs suspension resulted in a short-term increase in survival rate of IPFP-MSCs and better histology scores of rat joint tissues. Inhibition of integrin β1 or ERK1/2 attenuated the protective effect of TE-IPFP MSCs suspension in vivo . In conclusion, TE promotes performance of IPFP-MSCs and protects knee cartilage from damage in OA through enhancement of cell adhesion and activation of integrin β1/ERK/VCL pathway. Our findings may provide new insights in MSCs intra-articular injection for OA treatment.