Objectives OA is a complex genetic disease with different risk factors contributing to its development. One of the genes, TNFRSF11B, previously identified with gain-of-function mutation in a family with early-onset OA with chondrocalcinosis, is among the highest upregulated genes in lesioned OA cartilage (RAAK-study). Here, we determined the role of TNFRSF11B overexpression in development of OA. Methods Human primary articular chondrocytes (9 donors RAAK study) were transduced using lentiviral particles with or without TNFRSF11B. Cells were cultured for 1 week in a 3 D in-vitro chondrogenic model. TNFRSF11B overexpression was confirmed by RT-qPCR, immunohistochemistry and ELISA. Effects of TNFRSF11B overexpression on cartilage matrix deposition, matrix mineralization, and genes highly correlated to TNFRSF11B in RNA-sequencing dataset (r >0.75) were determined by RT-qPCR. Additionally, glycosaminoglycans and collagen deposition were visualized with Alcian blue staining and immunohistochemistry (COL1 and COL2). Results Overexpression of TNFRSF11B resulted in strong upregulation of MMP13, COL2A1 and COL1A1. Likewise, mineralization and osteoblast characteristic markers RUNX2, ASPN and OGN showed a consistent increase. Among 30 genes highly correlated to TNFRSF11B, expression of only eight changed significantly, with BMP6 showing the highest increase (9-fold) while expression of RANK and RANKL remained unchanged indicating previously unknown downstream pathways of TNFRSF11B in cartilage. Conclusion Results of our 3D in vitro chondrogenesis model indicate that upregulation of TNFRSF11B in lesioned OA cartilage may act as a direct driving factor for chondrocyte to osteoblast transition observed in OA pathophysiology. This transition does not appear to act via the OPG/RANK/RANKL triad common in bone remodeling.

Zinc substituted hydroxyapatite (Zn-HAP) Ca5-xZnx(PO4)(3)OH (x = 0, 0.05, 0.2 and 1.0) was synthesized by hydrothermal method. Different weight ratio (30:70, 50:50 and 70:30) of polymer composite (parent HAP and Zn-HAP/PVA (poly vinyl alcohol)) were prepared by solvent casting method. The developed parent and Zn-HAP/PVA composites were characterized by XRD, FTIR and SEM-EDAX to examine the phase composition, functional groups and morphology. Porosity, swelling percentage, mechanical strength, antibacterial activity, hemocompatibility, bioactivity and cytotoxicity were studied. Increase in porosity (88.90 +/- 1.20-97.25 +/- 1.12 %) and mechanical strength (44.45-98.94 MPa) were observed with the increase in the wt% of Zn-HAP in the PVA network. Furthermore, In-vitro hemocompatibility assay showed that all composites were hemocompatible with less than 2% hemolytic ratio. In-vitro biomineralization of the optimized composite (Zn-HAP/PVA with 30:70 (w/w)) showed a new apatite formation on the surface of the composite after soaking in simulated body fluid (SBF) for 28 days. Further, the cytotoxicity of the optimized composite was evaluated against human osteoblast cells (HOS). Zn-HAP/PVA with 30:70 was found to have higher cell viability (p < 0.01) compared to the pure (HAP/PVA) composite.

Purpose: Founded by the hypothesis that the OA disease pathway of the previously identified high impact gain-of-function mutation in TNFRSF11B could be extrapolated to common, age-related, OA disease phenotypes, we set out to functionally study its effect. To this end, we established in vitro hiPS derived spherical chondrogenic and osteogenic pellets and compared TNFRSF11B mutated cells generated from affected family members to CRISPR/Cas9 repaired isogenic control cells. Given that the mutant OPG decoy receptor more efficiently antagonizes osteoclastogenesis, we expect that the mutation affects matrix mineralization hence inflicting the observed chondrocalcinosis phenotype. Methods: Using skin fibroblasts, hiPSCs of an affected family member were created (FOA) and differentiated to induced mesenchymal stromal stem cells (iMSCs). These were differentiated for 6 weeks to deposit neo-cartilaginous and neo-osseous pellets or neo-bone in parallel with a CRISPR-Cas9 repaired hiPSC line (FOA-R) serving as an isogenic control. Presence of glycosaminoglycans was determined by Alcian blue while mineralization was determined by Alizarin red staining. Gene expression analyses were performed to study anabolic markers and hypertrophy (ie, COL2A1, COL1A1, COL10A1) and matrix mineralization (ie, ALPL, MGP). Results: As demonstrated by the intensity of Alcian blue and Alizarin red staining of spherical neo-cartilaginous and neo-osseous pellets, respectively (Figure 1) the bi-directional differentiation via iPSC derived iMSCs appeared successful. Additionally, while only subtle differences in intensity were observed for the Alcian blue staining, the Alizarin red staining intensity in FOA mutated neo-osseous pellets was clearly higher as that of the FOA-repaired pellets, indicating increased matrix mineralization (Figure 1). Gene expression analyses of neo-cartilaginous pellets, showed, however, that in the presence of the FOA TNFRSF11B mutation, MGP levels were considerably decreased (FD=0.14 P=1.6x10-13), indicating cell signaling towards matrix mineralization also in the mutated neo-cartilaginous tissues. In line with this, neo-cartilaginous tissues from FOA mutated cells compared to FOA repaired cells expressed lower levels of COL2A1 (FD=-6.8, P-value=3.9x10-3) concomitant with higher levels of COL1A1 (FD=1.7, P-value=7.4x10-3). Additionally, in the FOA mutated neo-osseous pellets gene expression analysis showed a higher expression of RUNX2 (FD=2.6, P-value= 8.29x10-3) and a trend towards a higher expression of COL1A1 (FD=1.54, P-value= 0.079) and ALPL (FD=1.57, P-value=0.055) relative to FOA repaired neo-osseous pellets.supplements. Conclusions: Employing hiPS-cells from early-onset OA family members (FOA) and CRISPR/Cas9 repaired isogenic controls (FOA-R) in an established spherical 3D in vitro chondrogenic and osteogenic pellet model confirmed that the previously identified gain of function mutation in TNFRSF11B encoding OPG acts via increased matrix mineralization of cartilage and bone. In cartilage, likely, this is modulated via MGP expression as suggested by the significant lower levels in the mutant cells. This may raise the possibility to slow down OA progression in early-onset family members with the use of vitamin K.

Melanoma is the most aggressive and deadly form of skin cancer. The molecular variability involving microRNA (miRNA) expression plays a significant role in melanogenesis, which leads to poor prognostic effects in melanoma. Since there is a scarcity of comprehensive data on the prognostic role of miRNAs in melanoma patients, this study focuses on filling this knowledge gap through a systematic review and meta-analysis.