Osteoarthritis is a global problem and the treatment of early disease is a clear area of unmet clinical need. Treatment strategies include cell therapies utilising chondrocytes e.g. autologous chondrocyte implantation and mesenchymal stem/stromal cells (MSCs) e.g. microfracture. The result of repair is often considered suboptimal as the goal of treatment is a more accurate regeneration of the tissue, hyaline cartilage, which requires a more detailed understanding of relevant biological signalling pathways. In this study, we describe a modulator of regulatory pathways common to both chondrocytes and MSCs. The chondrocytes thought to be cartilage progenitors are reported to reside in the superficial zone of articular cartilage and are considered to have the same developmental origin as MSCs present in the synovium. They are relevant to cartilage homeostasis and, like MSCs, are increasingly identified as candidates for joint repair and regenerative cell therapy. Both chondrocytes and MSCs can be regulated by the Wnt and TGFβ pathways. Dishevelled Binding Antagonist of Beta-Catenin (Dact) family of proteins is an important modulator of Wnt and TGFβ pathways. These pathways are key to MSC and chondrocyte function but, to our knowledge, the role of DACT protein has not been studied in these cells. DACT1 and DACT2 were localised by immunohistochemistry in the developing joints of mouse embryos and in adult human cartilage obtained from knee replacement. RNAi of DACT1 and DACT2 was performed on isolated chondrocytes and MSCs from human bone marrow. Knockdown efficiency and cell morphology was confirmed by qPCR and immunofluorescence. To understand which pathways are affected by DACT1, we performed next-generation sequencing gene expression analysis (RNAseq) on cells where DACT1 had been reduced by RNAi. Top statistically significant (p < 0 .05) 200 up and downregulated genes were analysed with Ingenuity® Pathway Analysis software. We observed DACT1 and DACT2 in chondrocytes throughout the osteoarthritic tissue, including in chondrocytes forming cell clusters. On the non-weight bearing and visually undamaged cartilage, DACT1 and DACT2 was localised to the articular surface. Furthermore, in mouse embryos (E.15.5), we observed DACT2 at the interzones, sites of developing synovial joints, suggesting that DACT2 has a role in cartilage progenitor cells. We subsequently analysed the expression of DACT1 and DACT2 in MSCs and found that both are expressed in synovial and bone marrow-derived MSCs. We then performed an RNAi knockdown experiment. DACT1 knockdown in both chondrocyte and MSCs caused the cells to undergo apoptosis within 24 hours. The RNA-seq study of DACT1 silenced bone marrow-derived MSCs, from 4 different human subjects, showed that loss of DACT1 has an effect on the expression of genes involved in both TGFβ and Wnt pathways and putative link to relevant cell regulatory pathways. In summary, we describe for the first time, the presence and biological relevance of DACT1 and DACT2 in chondrocytes and MSCs. Loss of DACT1 induced cell death in both chondrocytes and MSCs, with RNA-seq analysis revealing a direct impact on transcript levels of genes involved in the Wnt and TFGβ signalling, key regulatory pathways in skeletal development and repair

Introduction. Due to its remarkable stoichiometric flexibility and surface chemistry, hydroxyapatite (HAp) is the fundamental structural material in all vertebrates. Natural HAp's properties inspired an investigation into silicon nitride (Si. 3. N. 4. ) to see if similar functionality could be engineered into this bioceramic. Biological and in situ spectroscopic analyses were used to monitor the response of osteosarcoma cells (SaOS-2) to surface-modulated Si. 3. N. 4. and a titanium alloy after long-term in vitro exposure. Materials and Methods. Four groups of Si. 3. N. 4. discs, Ø12.7×1.0mm, (Amedica Corporation, Salt Lake City, UT USA) were subjected to surface treatments: (i) “As-fired;” (ii) HF-etched (5% HF solution for 45 s); (iii) Oxidized (1070°C for 7 h); and (iv) Nitrogen-annealed (1400°C for 30 min, 1.1 bar N. 2. gas). 1. Titanium alloy discs (Ti6Al4V, ASTM F136) were used as a control group. SaOS-2 cells cultured for 24 h at 37. °. C were deposited (5×10. 5. cells/ml) and incubated on the UV sterilized discs in an osteogenic medium for 7 days at 37°C. Cell proliferation was monitored using scanning electron and laser microscopy. The Receptor Activator of NF-kB Ligand (sRANKL) and the insulin growth factor 1 (IGF-1) were used to evaluate osteoclast formation and cell proliferation efficiency, respectively. In situ Raman spectroscopy was employed to monitor metabolic cell activity. Statistics (n≥3) were analyzed using the Student's t-test or one-way Analysis of Variance with p<0.05 considered significant. Results. Results are presented in Figure 1(a)∼(c) for HAp formation, free sRANKL, and IGF-1, respectively. These data indicate that the N. 2. -annealed Si. 3. N. 4. samples had the highest amount of HAp formation followed by the as-fired, oxidized, and HF samples. The Ti-alloy showed moderate HAp formation; but it had a higher amount of free sRANKL as compared to all Si. 3. N. 4. samples. These data suggest that the Si. 3. N. 4. represented a friendlier environment for SaOS-2 cell differentiation. The IGF-1 concentration did not differ among the Si. 3. N. 4. samples, but they were all higher than the Ti-alloy. Higher IGF-1 stimulates cells to proliferate and differentiate. 2. In Figure 2, in situ collected Raman spectra confirmed enhanced formation of HAp on the Si. 3. N. 4. samples, especially the N. 2. -annealed material. Discussion. Enhanced apatite formation was found to originate from a high density of positively charged surface groups, including nitrogen vacancies (V. N. 3+. ) and nitrogen N-N bonds (N. 4+. ). 3. These surface charges promoted binding of proteins onto the negatively charged Si. 3. N. 4. surface. A dipole-like-charge of V. N. 3+. /N. 4+. and SiO. −. defective sites is proposed as a mechanism to explain the attraction between proteins and the COO. −. and NH. 2+. terminus, respectively. This is analogous to the mechanism occurring in hydroxyapatite where protein groups are displaced by positively charged calcium loci (Ca. +. ) and off-stoichiometry phosphorus sites (PO. 4. 2−. ). Conclusion. Osteoblast proliferation and apatite-growth are important properties in regenerative bone therapies. In general, these properties were pronounced on all of the Si. 3. N. 4. substrates; but achieved maximum values on the N. 2. -annealed Si. 3. N. 4.

Construction of a functional skeleton is accomplished through co-ordination of the developmental processes of chondrogenesis, osteogenesis, and synovial joint formation. Infants whose movement in utero is reduced or restricted and who subsequently suffer from joint dysplasia (including joint contractures) and thin hypo-mineralised bones, demonstrate that embryonic movement is crucial for appropriate skeletogenesis. This has been confirmed in mouse, chick, and zebrafish animal models, where reduced or eliminated movement consistently yields similar malformations and which provide the possibility of experimentation to uncover the precise disturbances and the mechanisms by which movement impacts molecular regulation. Molecular genetic studies have shown the important roles played by cell communication signalling pathways, namely Wnt, Hedgehog, and transforming growth factor-beta/bone morphogenetic protein. These pathways regulate cell behaviours such as proliferation and differentiation to control maturation of the skeletal elements, and are affected when movement is altered. Cell contacts to the extra-cellular matrix as well as the cytoskeleton offer a means of mechanotransduction which could integrate mechanical cues with genetic regulation. Indeed, expression of cytoskeletal genes has been shown to be affected by immobilisation. In addition to furthering our understanding of a fundamental aspect of cell control and differentiation during development, research in this area is applicable to the engineering of stable skeletal tissues from stem cells, which relies on an understanding of developmental mechanisms including genetic and physical criteria. A deeper understanding of how movement affects skeletogenesis therefore has broader implications for regenerative therapeutics for injury or disease, as well as for optimisation of physical therapy regimes for individuals affected by skeletal abnormalities.

Cite this article: Bone Joint Res 2015;4:105–116