Bone Regenerative Engineering Using a Protein Kinase A-Specific Cyclic AMP Analogue Administered for Short Term Okechukwu Clinton Ifegwu 1,2,3 & Guleid Awale 1,2,3,4 & Ho Man Kan 1,3 & Komal Rajpura 1 & Edward O’Neill 1,2,5 & Chia-Ling Kuo 6 & Kevin W.-H. Lo 1,2,4,5,6 Received: 18 July 2017 /Accepted: 31 May 2018 # The Regenerative Engineering Society 2018 Abstract Small molecule-mediated bone regeneration is emerging as a promising strategy for replacing or enhancing the therapeutic protein- based growth factors. However, unknown non-specific toxicity of small molecules on non-target cells or organs due to the long-term exposure has been a concern. We previously demonstrated that the continuous treatment of osteoblast-like MC3T3-E1 cells with small molecule cyclic AMP analogue N 6 -benzoyladenosine-3′ ,5′ -cyclic monophosphate (6-Bnz-cAMP) was capable of inducing in vitro osteogenesis via the protein kinase A (PKA) signaling pathway. In this study, we investigate the effect of short-term 6-Bnz-cAMP treatment, i.e., 1-day treatment, as compared to continuous treatment, on in vitro osteogenesis in osteoprogenitor cells. It is hypothe- sized that the proposed short-term 6-Bnz-cAMP treatment scheme would result in osteogenesis as in the case of continuous 6-Bnz- cAMP treatment. Our results showed that both short-term and continuous 6-Bnz-cAMP treatments elicited osteoblastic differentiation and mineralization of osteoblast-like MC3T3-E1 cells. Short-term treatment using small molecule 6-Bnz-cAMP can serve as a highly promising strategy for bone regeneration while mitigating potential non-specific side effect risks associated with small molecules. Lay Summary The goal of this work is to develop a simple, inexpensive, effective, and safe method to heal bone defect. We would like to treat the bone defects with a small molecule-based therapeutic agent in a short-term treatment so that undesirable side effects from the therapeutics would be significantly minimized. Our work may also result in novel bone graft materials that can potentially become a viable alternative to existing grafts. Keywords Cyclic AMP . Small molecules . Regenerative engineering . Musculoskeletal tissue . Drug discovery . Osteogenesis Introduction With the ever-growing need of better musculoskeletal regen- erative therapies, small molecules are fast emerging as highly promising alternatives for conventional treatment options [1–8]. The magnitude and overwhelming impact of these mus- culoskeletal diseases on the US population and economy can- not be overemphasized. Over the years, grafting techniques have been on the front burner of musculoskeletal disorder treatment options [9–12]. Although these techniques are still very much in use, they are often plagued with donor morbidity and disease transmission, which has aroused genuine concern for patients and clinicians alike [13]. Ideally, regenerating a damaged, diseased, or lost musculoskeletal tissue is consid- ered a better clinical strategy when compared to repairing the Okechukwu Clinton Ifegwu and Guleid Awale contributed equally to the work. * Kevin W.-H. Lo wlo@uchc.edu 1 Institute for Regenerative Engineering, University of Connecticut Health, Farmington, CT 06030, USA 2 Raymond and Beverly Sackler Center for Biomedical, Biological, Physical and Engineering Sciences, University of Connecticut Health, Farmington, CT 06030, USA 3 Department of Orthopaedic Surgery, University of Connecticut Health, Farmington, CT 06030, USA 4 Department of Chemical and Biomolecular Engineering, University of Connecticut, Storrs, CT 06268, USA 5 Department of Medicine, Division of Endocrinology, University of Connecticut Health Center, School of Medicine, Farmington, CT 06030, USA 6 Connecticut Institute for Clinical and Translational Science, University of Connecticut Health Center, Farmington, CT 06030, USA Regenerative Engineering and Translational Medicine https://doi.org/10.1007/s40883-018-0063-1