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The ubiquitin protease pathway plays important role in human bone marrow-derived mesenchymal stem cell (hBMSC) differentiation, including osteogenesis. However, the function of deubiquitinating enzymes in osteogenic differentiation of hBMSCs remains poorly understood. In this study, we aimed to investigate the role of ubiquitin-specific protease 53 (USP53) in the osteogenic differentiation of hBMSCs. Based on re-analysis of the Gene Expression Omnibus database, USP53 was selected as a positive regulator of osteogenic differentiation in hBMSCs. Overexpression of USP53 by lentivirus enhanced osteogenesis in hBMSCs, whereas knockdown of USP53 by lentivirus inhibited osteogenesis in hBMSCs. In addition, USP53 overexpression increased the level of active β-catenin and enhanced the osteogenic differentiation of hBMSCs. This effect was reversed by the Wnt/β-catenin inhibitor DKK1. Mass spectrometry showed that USP53 interacted with F-box only protein 31 (FBXO31) to promote proteasomal degradation of β-catenin. Inhibition of the osteogenic differentiation of hBMSCs by FBXO31 was partially rescued by USP53 overexpression. Animal studies showed that hBMSCs with USP53 overexpression significantly promoted bone regeneration in mice with calvarial defects. These results suggested that USP53 may be a target for gene therapy for bone regeneration.

Traumatic and degenerative lesions of articular cartilage usually progress to osteoarthritis (OA), a leading cause of disability in humans. MicroRNAs (miRNAs) can regulate the differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) and play important roles in the expression of genes related to OA. However, their functional roles in OA remain poorly understood. Here, we have examined miR-449a, which targets sirtuin 1 (SIRT1) and lymphoid enhancer-binding factor-1 (LEF-1), and observed its effects on damaged cartilage. The levels of chondrogenic markers and miR-449a target genes increased during chondrogenesis in anti-miR-449a-transfected hBMSCs. A locked nucleic acid (LNA)-anti-miR-449a increased cartilage regeneration and expression of type II collagen and aggrecan on the regenerated cartilage surface in acute defect and OA models. Furthermore, intra-articular injection of LNA-anti-miR-449a prevented disease progression in the OA model. Our study indicates that miR-449a may be a novel potential therapeutic target for age-related joint diseases like OA.

To investigate the functional effects of resveratrol (RSV) on mesenchymal stem cells (MSCs), we treated MSCs with RSV continuously during ex vivo expansion. MSCs were continuously treated with RSV from passage (P) 0 to P5. A proliferative capacity of RSV-treated MSCs was higher than that of non-treated MSCs and similar with P1-MSCs. Continuous treatment of RSV on MSCs increased the stemness and inhibited the senescence. During chondrogenic differentiation in vitro, RSV-treated MSCs had higher differentiation potential and reduced hypertrophic maturation, which are limitations for hyaline cartilage formation. The histological analysis of micromass demonstrated increased chondrogenic differentiation potential. We further explored the therapeutic effectiveness of this method in a rabbit osteochondral defect model. A rabbit osteochondral defect model was established to investigate the hyaline cartilage regeneration potential of RSV-treated MSCs. Moreover, the cartilage regeneration potential of RSV-treated MSCs was greater than that of untreated MSCs. The expression levels of chondrogenic markers increased and those of hypertrophic markers decreased in RSV-treated MSCs compared with untreated MSCs. Sustained treatment of RSV on MSCs during ex vivo expansion resulted in the maintenance of stemness and enhanced chondrogenic differentiation potential. Consequentially, highly efficient MSCs promoted superior hyaline cartilage regeneration in vivo. This novel treatment method provides a basis for cell-based tissue engineering.

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