Identification of novel osteogenic compounds that act synergistically with BMP-2

Background and aims: In reconstructive surgery, repair of bone defects is a major reparative problem. Bone morphogenetic proteins (BMPs) have shown significant potential in bone formation, however, there are many inherent drawbacks in the use of BMPs in orthopaedic indications. Successful application of BMP-2 for clinical use may be affected by its bioavailability at the implantation site; therefore, large amounts of BMPs are needed to induce clinically useful bone regeneration. The objective of this research project is to identify compounds which act synergistically with BMP-2 to enhance the expression of putative markers associated with an osteogenic phenotype. The second aim of this thesis is to determine the mechanism of action underlying NMP and BMP-2 synergy. Methods: In total, over 500 test compounds were screened in the primary assay for Alkaline phosphatase (ALP) activity. After the primary screening, the positive findings were validated using qPCR method to measure the ALP and Osterix (Osx) gene-expression. Mineralization assay with Alizarin Red (AR-S) staining was used to assess the expression of final-stage markers of osteogenesis. Finally, the positive findings were confirmed on a protein level with phosphorylated Smad1/2/5 via western blotting. The toxicity of the test compounds was assessed with an MTT assay, which measures cell viability and proliferation. For the mode of action study, a mutation was introduced into BMP-R1A receptor; to create a constitutively active receptor that can signal without requirement for BMP-2-ligand. Results: The data produced in this study presents a novel finding: N, N, dimethylacetamide, which in combination with sub-threshold concentration of BMP-2 enhances putative markers associated with osteogenesis. The ALP activity was induced 220% and Osx gene expression was significantly increased following exposure of N, N dimethyacetamide in C2C12 cells. Additionally, N, N dimethylacetamide improved AR-S staining in MC3T3-E1 cells, indicating that this compound also enhanced the expression of final-stage markers of osteogenesis. The BMP-R1A receptor mutagenesis was the beginning of the mechanism of action studies revealing how NMP exhibits osteogenic properties. The result of this experiment was negative, because the loss-of-function experiment proved to have no effect on osteogenesis in C2C12 cells. Conclusions: The data presented in this study provides strong evidence to demonstrate that N, N, dimethylacetamide has osteogenic properties when combined with BMP-2. Hence, when BMP-2 is present at the site of fracture in low concentrations, no additional exogeneous BMP-treatment is required to accelerate bone growth and patient rehabilitation