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 N6-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 hypothesized 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.
Similar content being viewed by others
References
Lo KW-H, Ashe KM, Kan HM, Laurencin CT. The role of small molecules in the musculoskeletal regeneration. Regen Med. 2012;7:1–15.
Lo KW, Jiang T, Gagnon KA, Nelson C, Laurencin CT. Small-molecule based musculoskeletal regenerative engineering. Trends Biotechnol. 2014;32:74–81.
Laurencin CT, Ashe KM, Henry N, Kan HM, Lo KW. Delivery of small molecules for bone regenerative engineering: preclinical studies and potential clinical applications. Drug Discov Today. 2014;19:794–800.
Lo KW, Ulery BD, Deng M, Ashe KM, Laurencin CT. Current Patents on Osteoinductive Molecules for Bone Tissue Engineering. Recent Patents on Biomedical Engineering. 2011;4:153–67.
Lo KW-H, Ulery BD, Ashe KM, Laurencin CT. Studies of bone morphogenetic protein based surgical repair. Adv Drug Deliv Rev. 2012;64:1277–91. https://doi.org/10.1016/j.addr.2012.1003.1014.
Awale G, Wong E, Rajpura K, Lo KW. Engineered bone tissue with naturally-derived small molecules. Curr Pharm Des. 2017;23:3585–94. https://doi.org/10.2174/1381612823666170516145800.
Carbone EJ, Jiang T, Nelson C, Henry N, Lo KW. Small molecule delivery through nanofibrous scaffolds for musculoskeletal regenerative engineering. Nanomedicine. 2014;10:1691–9.
Carbone EJ, Rajpura K, Allen BN, Cheng E, Ulery BD, Lo KW. Osteotropic nanoscale drug delivery systems based on small molecule bone-targeting moieties. Nanomedicine. 2017;13:37–47.
Laurencin C, Khan Y, El-Amin SF. Bone graft substitutes. Expert Rev Med Devices. 2006;3:49–57.
Hartigan BJ, Makowiec RL. Use of bone graft substitutes and bioactive materials in treatment of distal radius fractures. Hand Clin. 2009;23:241–6.
Finkemeier CG. Bone-grafting and bone-graft substitutes. J Bone Joint Surg Am. 2002;84-A:454–64.
De Long WG Jr, Einhorn TA, Koval K, McKee M, Smith W, Sanders R, et al. Bone grafts and bone graft substitutes in orthopaedic trauma surgery. A critical analysis. J Bone Joint Surg Am. 2007;89:649–58.
Rogers GF, Greene AK. Autogenous bone graft: basic science and clinical implications. J Craniofac Surg. 2012;23:323–7.
Laurencin CT, Khan Y. Regenerative engineering. Sci Transl Med. 2012;4:160ed169.
Laurencin CT, Nair LS. Regenerative engineering: approaches to limb regeneration and other grand challenges. Regen Eng Transl Med. 2015;1:1–3.
Laurencin CT, Nair LS. The Quest toward limb regeneration: a regenerative engineering approach. Regen Biomater. 2016;3:123–5.
Cui Q, Dighe AS, Irvine JN Jr. Combined angiogenic and osteogenic factor delivery for bone regenerative engineering. Curr Pharm Des. 2013;19:3374–83.
Segar CE, Ogle ME, Botchwey EA. Regulation of angiogenesis and bone regeneration with natural and synthetic small molecules. Curr Pharm Des. 2013;19:3403–19.
Bessa PC, Casal M, Reis RL. Bone morphogenetic proteins in tissue engineering: the road from laboratory to clinic, part II (BMP delivery). J Tissue Eng Regen Med. 2008;2:81–96.
Bessa PC, Casal M, Reis RL. Bone morphogenetic proteins in tissue engineering: the road from the laboratory to the clinic, part I (basic concepts). J Tissue Eng Regen Med. 2008;2:1–13.
Jeon OH, Elisseeff J. Orthopedic tissue regeneration: cells, scaffolds, and small molecules. Drug Deliv Transl Res. 2017;6:105–20.
Park KW, Waki H, Kim WK, Davies BS, Young SG, Parhami F, et al. The small molecule phenamil induces osteoblast differentiation and mineralization. Mol Cell Biol. 2009;29:3905–14.
Ifegwu OC, Awale G, Rajpura K, Lo KW, Laurencin CT. Harnessing cAMP signaling in musculoskeletal regenerative engineering. Drug Discov Today. 2017;22:1027–44.
Alves H, Dechering K, Van Blitterswijk C, De Boer J. High-throughput assay for the identification of compounds regulating osteogenic differentiation of human mesenchymal stromal cells. PLoS One. 2011;6:e26678.
Brey DM, Motlekar NA, Diamond SL, Mauck RL, Garino JP, Burdick JA. High-throughput screening of a small molecule library for promoters and inhibitors of mesenchymal stem cell osteogenic differentiation. Biotechnol Bioeng. 2011;108:163–74.
Han CY, Wang Y, Yu L, Powers D, Xiong X, Yu V, et al. Small molecules with potent osteogenic-inducing activity in osteoblast cells. Bioorg Med Chem Lett. 2009;19:1442–5.
Doorn J, Leusink M, Groen N, van de Peppel J, van Leeuwen JP, van Blitterswijk CA, et al. Diverse effects of cyclic AMP variants on osteogenic and adipogenic differentiation of human mesenchymal stromal cells. Tissue Eng A. 2012;18:1431–42.
Sefcik LS, Petrie Aronin CE, Botchwey EA. Engineering vascularized tissues using natural and synthetic small molecules. Organogenesis. 2008;4:215–27.
Siddappa R, Martens A, Doorn J, Leusink A, Olivo C, Licht R, et al. cAMP/PKA pathway activation in human mesenchymal stem cells in vitro results in robust bone formation in vivo. Proc Natl Acad Sci U S A. 2008;105:7281–6.
Nohria A, Prsic A, Liu PY, Okamoto R, Creager MA, Selwyn A, et al. Statins inhibit Rho kinase activity in patients with atherosclerosis. Atherosclerosis. 2009;205:517–21.
Woo SM, Lim HS, Jeong KY, Kim SM, Kim WJ, Jung JY. Vitamin D promotes odontogenic differentiation of human dental pulp cells via ERK activation. Mol Cells. 2015;38:604–9.
Tintut Y, Parhami F, Bostrom K, Jackson SM, Demer LL. cAMP stimulates osteoblast-like differentiation of calcifying vascular cells. Potential signaling pathway for vascular calcification. J Biol Chem. 1998;273:7547–53.
Wong E, Sangadala S, Boden SD, Yoshioka K, Hutton WC, Oliver C, et al. A novel low-molecular-weight compound enhances ectopic bone formation and fracture repair. J Bone Joint Surg Am. 2011;95:454–61.
Fan J, Guo M, Im CS, Pi-Anfruns J, Cui ZK, Kim S, et al. Enhanced mandibular bone repair by combined treatment of bone morphogenetic protein 2 and small-molecule phenamil. Tissue Eng A. 2017;23:195–207.
Fan J, Im CS, Cui ZK, Guo M, Bezouglaia O, Fartash A, et al. Delivery of phenamil enhances BMP-2-induced osteogenic differentiation of adipose-derived stem cells and bone formation in calvarial defects. Tissue Eng A. 2015;21:2053–65.
Lo KW, Ulery BD, Kan HM, Ashe KM, Laurencin CT. Evaluating the feasibility of utilizing the small molecule phenamil as a novel biofactor for bone regenerative engineering. J Tissue Eng Regen Med. 2014;8:728–36.
Lo KW, Kan HM, Gagnon KA, Laurencin CT. One-day treatment of small molecule 8-bromo-cyclic AMP analogue induces cell-based VEGF production for in vitro angiogenesis and osteoblastic differentiation. J Tissue Eng Regen Med. 2016;10:867–75.
Beavo JA, Brunton LL. Cyclic nucleotide research—still expanding after half a century. Nat Rev Mol Cell Biol. 2002;3:710–8.
Ho WC, Greene RM, Shanfeld J, Davidovitch Z. Cyclic nucleotides during chondrogenesis: concentration and distribution in vivo and in vitro. J Exp Zool. 1982;224:321–30.
Lo KW-H, Kan HM, Ashe KM, Laurencin CT. The small molecule PKA-selective cyclic AMP analogue as an inducer of osteoblast-like cells differentiation and mineralization. J Tissue Eng Regen Med. 2012;6:40–8.
Lo KW-H, Ulery BD, Ashe KM, Kan HM, Laurencin CT. Evaluating the feasibility of utilizing small molecule phenamil as a novel biofactor factor for bone regenerative engineering. J Tissue Eng Regen Med. 2014;8:728–36.
Lo KW, Kan HM, Laurencin CT. Short-term administration of small molecule phenamil induced a protracted osteogenic effect on osteoblast-like MC3T3-E1 cells. J Tissue Eng Regen Med. 2016;10:518–26.
Lo KWH, Ashe KM, Kan HM, Lee DA, Laurencin CT. Activation of cyclic AMP/protein kinase A signaling pathway enhances osteoblast cell adhesion on biomaterials for regenerative engineering. J Orthop Res. 2011;29:602–8.
Baek D, Villen J, Shin C, Camargo FD, Gygi SP, Bartel DP. The impact of microRNAs on protein output. Nature. 2008;455:64–71.
Quarles LD, Yohay DA, Lever LW, Caton R, Wenstrup RJ. Distinct proliferative and differentiated stages of murine MC3T3-E1 cells in culture: an in vitro model of osteoblast development. J Bone Miner Res. 1992;7:683–92.
Hoemann CD, El-Gabalawy H, McKee MD. In vitro osteogenesis assays: influence of the primary cell source on alkaline phosphatase activity and mineralization. Pathol Biol (Paris). 2009;57:318–23.
Landis WJ. Mineral characterization in calcifying tissues: atomic, molecular and macromolecular perspectives. Connect Tissue Res. 1996;34:239–46.
Dean DD, Schwartz Z, Bonewald L, Muniz OE, Morales S, Gomez R, et al. Matrix vesicles produced by osteoblast-like cells in culture become significantly enriched in proteoglycan-degrading metalloproteinases after addition of beta-glycerophosphate and ascorbic acid. Calcif Tissue Int. 1994;54:399–408.
Jikko A, Harris SE, Chen D, Mendrick DL, Damsky CH. Collagen integrin receptors regulate early osteoblast differentiation induced by BMP-2. J Bone Miner Res. 1999;14:1075–83.
Aubin JE. Bone stem cells. J Cell Biochem Suppl. 1998;30-31:73–82.
Cheng SL, Shao JS, Charlton-Kachigian N, Loewy AP, Towler DA. MSX2 promotes osteogenesis and suppresses adipogenic differentiation of multipotent mesenchymal progenitors. J Biol Chem. 2003;278:45969–77.
Kulterer B, Friedl G, Jandrositz A, Sanchez-Cabo F, Prokesch A, Paar C, et al. Gene expression profiling of human mesenchymal stem cells derived from bone marrow during expansion and osteoblast differentiation. BMC Genomics. 2007;8:70.
Lee DJ, Tseng HC, Wong SW, Wang Z, Deng M, Ko CC. Dopaminergic effects on in vitro osteogenesis. Bone Res. 2015;3:15020.
Widaa A, Claro T, Foster TJ, O’Brien FJ, Kerrigan SW. Staphylococcus aureus protein A plays a critical role in mediating bone destruction and bone loss in osteomyelitis. PLoS One. 2012;7:e40586.
Standal T, Borset M, Sundan A. Role of osteopontin in adhesion, migration, cell survival and bone remodeling. Exp Oncol. 2004;26:179–84.
Seibel MJ. Biochemical markers of bone turnover: part I: biochemistry and variability. Clin Biochem Rev. 2005;26:97–122.
Delmas PD, Malaval L, Arlot ME, Meunier PJ. Serum bone Gla-protein compared to bone histomorphometry in endocrine diseases. Bone. 1985;6:339–41.
Liu SH, Yang RS, Al-Shaikh R, Lane JM. Collagen in tendon, ligament, and bone healing. A current review. Clin Orthop Relat Res. 1995;318:265–78.
Wang YX, Yan SX. Biomedical imaging in the safety evaluation of new drugs. Lab Anim. 2008;42:433–41.
Guengerich FP. Mechanisms of drug toxicity and relevance to pharmaceutical development. Drug Metab Pharmacokinet. 2011;26:3–14.
Marquis ME, Lord E, Bergeron E, Drevelle O, Park H, Cabana F, et al. Bone cells-biomaterials interactions. Front Biosci. 2009;14:1023–67.
Gronowicz G, McCarthy MB. Response of human osteoblasts to implant materials: integrin-mediated adhesion. J Orthop Res. 1996;14:878–87.
Garcia AJ. Get a grip: integrins in cell-biomaterial interactions. Biomaterials. 2005;26:7525–9.
Enserink JM, Price LS, Methi T, Mahic M, Sonnenberg A, Bos JL, et al. The cAMP-Epac-Rap1 pathway regulates cell spreading and cell adhesion to laminin-5 through the alpha3beta1 integrin but not the alpha6beta4 integrin. J Biol Chem. 2004;279:44889–96.
Ribeiro VP, Almeida LR, Martins AR, Pashkuleva I, Marques AP, Ribeiro AS, et al. Modulating cell adhesion to polybutylene succinate biotextile constructs for tissue engineering applications. J Tissue Eng Regen Med. 2016;11:2853–63.
Rosales C, O’Brien V, Kornberg L, Juliano R. Signal transduction by cell adhesion receptors. Biochim Biophys Acta. 1995;1242:77–98.
Poppe H, Rybalkin SD, Rehmann H, Hinds TR, Tang XB, Christensen AE, et al. Cyclic nucleotide analogs as probes of signaling pathways. Nat Methods. 2008;5:277–8.
Carbone EJ, Rajpura K, Jiang T, Kan HM, Yu X, Lo KW-H. Osteotropic nanoscale drug delivery system via a single aspartic acid as the bone-targeting moiety. J Nanosci Nanotechnol. 2017;17:1747–52.
Rooney GE, Knight AM, Madigan NN, Gross L, Chen B, Giraldo CV, et al. Sustained delivery of dibutyryl cyclic adenosine monophosphate to the transected spinal cord via oligo [(polyethylene glycol) fumarate] hydrogels. Tissue Eng A. 2011;17:1287–302.
Siddappa R, Doorn J, Liu J, Langerwerf E, Arends R, van Blitterswijk C, et al. Timing, rather than the concentration of cyclic AMP, correlates to osteogenic differentiation of human mesenchymal stem cells. J Tissue Eng Regen Med. 2010;4:356–65.
Acknowledgements
We wish to thank Dr. Cato T. Laurencin, the Director of the Institute for Regenerative Engineering (IRE), for his leadership.
Funding
This work was supported by the Project Fund from the Connecticut Institute for Clinical and Translational Science (CICATS) to Dr. Kevin Lo.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Ifegwu, O.C., Awale, G., Kan, H.M. et al. Bone Regenerative Engineering Using a Protein Kinase A-Specific Cyclic AMP Analogue Administered for Short Term. Regen. Eng. Transl. Med. 4, 206–215 (2018). https://doi.org/10.1007/s40883-018-0063-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s40883-018-0063-1