Small Molecule-Mediated Enhanced Osteogenesis of Human Mesenchymal Stem Cells: a Probable Alternate for BMP-2

Purpose The use of bone morphogenic protein-2 (BMP-2) to accelerate the healing of bone defects has resulted in side effects such as ectopic bone formation, soft tissue swelling, and cyst-like bone void formation. An equipotential and a cost-effective replacement for BMP-2 is therefore required. In this study, different combinations of small molecules like FK506 (F), an immunosuppressant; purmorphamine (P), a hedgehog agonist; and Ku-0063794 (K), an mTOR inhibitor, were evaluated on human mesenchymal stem cells (hMSCs) to assess the osteoinduction behavior. Methods Dose optimization and various combinations of small molecules were evaluated using MTT and ALP assay. The gene and protein expression of osteogenic markers RunX2, Osteopontin (OPN), and collagen type 1 on treatment with small molecules was evaluated and compared with BMP-2 expression. The confirmation of the osteogenic potential of small molecules was calcium deposits formed in hMSCs after staining with Alizarin Red S. Results Optimum concentration of individual small molecule (F 5 nM, K 10 nM, and P 2 mu M) was determined based on the MTT and ALP activity. Small molecules (F + K + P) with osteogenic media (OS) showed 1.5-fold higher ALP activity with respect to OS. The synergistic effect of F + K + P and BMP-2/OS demonstrated an upregulation of RUNX2, OPN, and collagen 1 proteins and genes. Small molecules alone (F- 5 nM, K- 10 nM, and P- 2 mu M) expressed the formation of calcium ions stained by Alizarin Red S which confirmed their osteoinductive effect. Conclusion Thus, small molecules may have the potential to substitute or enable the use of reduced dose of BMP-2 for enhanced osteogenesis of progenitor cells. Lay Summary The use of Bone Morphogenic Protein-2 (BMP-2) to accelerate the healing of bone defects has resulted in side effects such as ectopic bone formation, soft tissue swelling, and cyst-like bone void formation. We show that cost-effective small molecules, in combination with osteogenic differentiation cocktail, have the potential to substitute BMP-2 for enhanced osteogenesis of progenitor cells.