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Human synovium-derived stem cells (hSSCs) are an attractive source of cells for cartilage repair. At present, the quality of tissue and techniques used for cartilage regeneration have scope for improvement. A small compound, TD-198946, was reported to enhance chondrogenic induction from hSSCs; however, other applications of TD-198946, such as priming the cell potential of hSSCs, remain unknown. Our study aimed to examine the effect of TD-198946 pretreatment on hSSCs. HSSCs were cultured with or without TD-198946 for 7 days during expansion culture and then converted into a three-dimensional pellet culture supplemented with bone morphogenetic protein-2 (BMP2) and/or transforming growth factor beta-3 (TGFβ3). Chondrogenesis in cultures was assessed based on the GAG content, histology, and expression levels of chondrogenic marker genes. Cell pellets derived from TD-198946-pretreated hSSCs showed enhanced chondrogenic potential when chondrogenesis was induced by both BMP2 and TGFβ3. Moreover, cartilaginous tissue was efficiently generated from TD-198946-pretreated hSSCs using a combination of BMP2 and TGFβ3. Microarray analysis revealed that NOTCH pathway-related genes and their target genes were significantly upregulated in TD-198946-treated hSSCs, although TD-198946 alone did not upregulate chondrogenesis related markers. The administration of the NOTCH signal inhibitor diminished the effect of TD-198946. Thus, TD-198946 enhances the chondrogenic potential of hSSCs via the NOTCH3 signaling pathway. This study is the first to demonstrate the gradual activation of NOTCH3 signaling during chondrogenesis in hSSCs. The priming of NOTCH3 using TD-198946 provides a novel insight regarding the regulation of the differentiation of hSSCs into chondrocytes.

There is no therapy currently available for fully repairing articular cartilage lesions. Our laboratory has recently developed a visible light-activatable methacrylated gelatin (mGL) hydrogel, with the potential for cartilage regeneration. In this study, we further optimized mGL scaffolds by supplementing methacrylated hyaluronic acid (mHA), which has been shown to stimulate chondrogenesis via activation of critical cellular signalling pathways. We hypothesized that the introduction of an optimal ratio of mHA would enhance the biological properties of mGL scaffolds and augment chondrogenesis of human bone marrow-derived mesenchymal stem cells (hBMSCs). To test this hypothesis, hybrid scaffolds consisting of mGL and mHA at different weight ratios were fabricated with hBMSCs encapsulated at 20 × 106 cells/ml and maintained in a chondrogenesis-promoting medium. The chondrogenenic differentiation of hBMSCs, within different scaffolds, was estimated after 8 weeks of culture. Our results showed that mGL/mHA at a 9:1 (%, w/v) ratio resulted in the lowest hBMSC hypertrophy and highest glycosaminoglycan production, with a slightly increased volume of the entire construct. The applicability of this optimally designed mGL/mHA hybrid scaffold for cartilage repair was then examined in vivo. A full-thickness cylindrical osteochondral defect was surgically created in the rabbit femoral condyle, and a three-dimensional cell-biomaterial construct was fabricated by in situ photocrosslinking to fully fill the lesion site. The results showed that implantation of the mGL/mHA (9:1) construct resulted in both cartilage and subchondral bone regeneration after 12 weeks, supporting its use as a promising scaffold for repair and resurfacing of articular cartilage defects, in the clinical setting.

Mesenchymal stem cells (MSCs) derived from induced pluripotent stem cells (iPSCs) are a promising cell source for the repair of skeletal disorders. Recently, neural crest cells (NCCs) were reported to be effective for inducing mesenchymal progenitors, which have potential to differentiate into osteochondral lineages. Our aim was to investigate the feasibility of MSC-like cells originated from iPSCs via NCCs for osteochondral repair. Initially, MSC-like cells derived from iPSC-NCCs (iNCCs) were generated and characterized in vitro. These iNCC-derived MSC-like cells (iNCMSCs) exhibited a homogenous population and potential for osteochondral differentiation. No upregulation of pluripotent markers was detected during culture. Second, we implanted iNCMSC-derived tissue-engineered constructs into rat osteochondral defects without any preinduction for specific differentiation lineages. The implanted cells remained alive at the implanted site, whereas they failed to repair the defects, with only scarce development of osteochondral tissue in vivo. With regard to tumorigenesis, the implanted cells gradually disappeared and no malignant cells were detected throughout the 2-month follow-up. While this study did not show that iNCMSCs have efficacy for repair of osteochondral defects when implanted under undifferentiated conditions, iNCMSCs exhibited good chondrogenic potential in vitro under appropriate conditions. With further optimization, iNCMSCs may be a new source for tissue engineering of cartilage.