Single-Dose Induction of Osteogenic Differentiation of Mesenchymal Stem Cells Using a Cyclic AMP Activator, Forskolin

Small molecule-based bone regenerative engineering has been proposed as a promising approach in repairing bone tissue and circumventing the issues associated with protein-based growth factor treatment. However, off-target effects of small molecules due to their short half-life and non-specificity hinder their wide-spread usage. Previously, we have investigated the capability of various cyclic adenosine monophosphate (cAMP) analogues to induce osteogenic differentiation in vitro using osteoprogenitor MC3T3-E1 cells. A promising strategy to mitigate the side-effect risk of small molecule treatments is to reduce their frequency of administration. However, the capability of these cAMP small molecules to induce osteogenesis in primary cells such as mesenchymal stem cells using the shorter-term or single-dose treatment approach has yet to be fully evaluated. In this study, we performed a phenotypic mini-screen of several cAMP analogues and activating small molecules to compare their osteoinductive effects on rabbit mesenchymal stem cells (MSCs). Our results demonstrated that the treatment of the small molecule forskolin (100 µM) for 24 h significantly increased the osteogenic differentiation and mineralization of rabbit adipose-derived stem cells (rADSCs) and rabbit bone marrow-derived stem cells (rBMSCs). In addition, we compared the effects of single-dose and repeat-dose forskolin treatment towards inducing osteogenic differentiation of rBMSCs. Overall, by inducing the osteogenic differentiation of mesenchymal stem cells with a single-dose of forskolin and without requiring repeated forskolin administration shows great potential for bone regenerative engineering applications. Lay Summary Bone grafting procedures have become increasingly common in the United States, with approximately 500,000 cases occurring each year at a societal cost exceeding $2.5 billion. Recombinant human bone morphogenetic proteins (rhBMPs) are therapeutic agents that have been widely used by orthopaedic surgeons to stimulate bone tissue formation when paired with biomaterials. However, significant limitations such as instability, low solubility, immunogenicity, high production cost, and the potential risk of disease transmission of these therapies remain. Therefore, efforts have been made to discover and repurpose small molecule therapeutics to promote bone regeneration. This study describes the evaluation of cAMP analogues and activating small molecules that can be used as alternative treatment options to rhBMPs in bone repair.