Effect of human cancellous bone graft soluble molecules onto differentiated human adipose derived mesenchymal stem cells towards osteogenic lineages

The involvement of cellular and molecular mechanisms of bone tissue sometimes fail during bone repair, resulting in either delayed-union or non-union. Numerous signaling molecules near the fracture site have a major role in mesenchymal stem cell adhesion, proliferation and differentiation towards osteogenic lineages. The aim of this study was to determine potential effects of human cancellous bone chip secretion (BCPs) onto differentiated human adipose-derived mesenchymal stem cells (hAD-MSCs) into osteogenic lineage. The cells within the bone chips secrete proteins and soluble molecules that inhibit the process of osteogenesis. In the previous studies, the effect of the BCPs on hAD-MSCs in bone repair has not been discussed before. In this study, hAD-MSCs were cultured in either basal medium (BM) or osteogenic medium (OM) in the presence of the BCPs. Results from this study demonstrated that proliferation and viabilities of hAD-MSCs were decreased by the BCPs. Furthermore, BCPs distinctly led to a reduction of alkaline phosphatase activity in comparison with the control cells. In the same way, BCPs caused an inhibition of mineralized bone nodules and a reduction in calcium concentration of extracellular matrix mineralization during bone formation. Besides, a significant decrease in the level of bone-related genes, including osteocalcin (OCN), collagen type one (Col1A1), osteonectin (ON), and osteopontin (OPN) were observed in hAD-MSCs with BCPs. Expression of osteoprotegerin (OPG) was also significantly decreased during osteoblasts maturation. Nevertheless, expression of Bone Morphogenetic Protein-2 (BMP-2) was constantly induced by BCPs during bone regeneration. Moreover, a significant increase in the level of sclerostin expression was also observed in hAD-MSCs cultured in OM at the fracture site under the influence of BCPs. Nevertheless, BCPs did not have any significant effect on the expression of Dkk-1. Our results have demonstrated that production of pro-inflammatory cytokines such as TNF-α, IL-6 and IL-1β was stimulated by BCPs. Altogether, our findings demonstrated that the secretions of bone chips inhibit bone formation process during fracture healing.