Skip to main content

Bone Regenerative Engineering Using a Protein Kinase A-Specific Cyclic AMP Analogue Administered for Short Term


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.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7


  1. 1.

    Lo KW-H, Ashe KM, Kan HM, Laurencin CT. The role of small molecules in the musculoskeletal regeneration. Regen Med. 2012;7:1–15.

    Article  Google Scholar 

  2. 2.

    Lo KW, Jiang T, Gagnon KA, Nelson C, Laurencin CT. Small-molecule based musculoskeletal regenerative engineering. Trends Biotechnol. 2014;32:74–81.

    CAS  Article  Google Scholar 

  3. 3.

    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.

    CAS  Article  Google Scholar 

  4. 4.

    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.

    CAS  Article  Google Scholar 

  5. 5.

    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.

    CAS  Article  Google Scholar 

  6. 6.

    Awale G, Wong E, Rajpura K, Lo KW. Engineered bone tissue with naturally-derived small molecules. Curr Pharm Des. 2017;23:3585–94.

    CAS  Article  Google Scholar 

  7. 7.

    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.

    CAS  Article  Google Scholar 

  8. 8.

    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.

    CAS  Article  Google Scholar 

  9. 9.

    Laurencin C, Khan Y, El-Amin SF. Bone graft substitutes. Expert Rev Med Devices. 2006;3:49–57.

    CAS  Article  Google Scholar 

  10. 10.

    Hartigan BJ, Makowiec RL. Use of bone graft substitutes and bioactive materials in treatment of distal radius fractures. Hand Clin. 2009;23:241–6.

    Google Scholar 

  11. 11.

    Finkemeier CG. Bone-grafting and bone-graft substitutes. J Bone Joint Surg Am. 2002;84-A:454–64.

    Article  Google Scholar 

  12. 12.

    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.

    Article  Google Scholar 

  13. 13.

    Rogers GF, Greene AK. Autogenous bone graft: basic science and clinical implications. J Craniofac Surg. 2012;23:323–7.

    Article  Google Scholar 

  14. 14.

    Laurencin CT, Khan Y. Regenerative engineering. Sci Transl Med. 2012;4:160ed169.

    Article  Google Scholar 

  15. 15.

    Laurencin CT, Nair LS. Regenerative engineering: approaches to limb regeneration and other grand challenges. Regen Eng Transl Med. 2015;1:1–3.

    Article  Google Scholar 

  16. 16.

    Laurencin CT, Nair LS. The Quest toward limb regeneration: a regenerative engineering approach. Regen Biomater. 2016;3:123–5.

    Article  Google Scholar 

  17. 17.

    Cui Q, Dighe AS, Irvine JN Jr. Combined angiogenic and osteogenic factor delivery for bone regenerative engineering. Curr Pharm Des. 2013;19:3374–83.

    CAS  Article  Google Scholar 

  18. 18.

    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.

    CAS  Article  Google Scholar 

  19. 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.

    CAS  Article  Google Scholar 

  20. 20.

    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.

    CAS  Article  Google Scholar 

  21. 21.

    Jeon OH, Elisseeff J. Orthopedic tissue regeneration: cells, scaffolds, and small molecules. Drug Deliv Transl Res. 2017;6:105–20.

    Article  Google Scholar 

  22. 22.

    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.

    CAS  Article  Google Scholar 

  23. 23.

    Ifegwu OC, Awale G, Rajpura K, Lo KW, Laurencin CT. Harnessing cAMP signaling in musculoskeletal regenerative engineering. Drug Discov Today. 2017;22:1027–44.

    CAS  Article  Google Scholar 

  24. 24.

    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.

    CAS  Article  Google Scholar 

  25. 25.

    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.

    CAS  Article  Google Scholar 

  26. 26.

    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.

    CAS  Article  Google Scholar 

  27. 27.

    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.

    CAS  Article  Google Scholar 

  28. 28.

    Sefcik LS, Petrie Aronin CE, Botchwey EA. Engineering vascularized tissues using natural and synthetic small molecules. Organogenesis. 2008;4:215–27.

    Article  Google Scholar 

  29. 29.

    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.

    CAS  Article  Google Scholar 

  30. 30.

    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.

    CAS  Article  Google Scholar 

  31. 31.

    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.

    CAS  Article  Google Scholar 

  32. 32.

    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.

    CAS  Article  Google Scholar 

  33. 33.

    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.

    Article  Google Scholar 

  34. 34.

    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.

    CAS  Article  Google Scholar 

  35. 35.

    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.

    CAS  Article  Google Scholar 

  36. 36.

    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.

    CAS  Article  Google Scholar 

  37. 37.

    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.

    CAS  Article  Google Scholar 

  38. 38.

    Beavo JA, Brunton LL. Cyclic nucleotide research—still expanding after half a century. Nat Rev Mol Cell Biol. 2002;3:710–8.

    CAS  Article  Google Scholar 

  39. 39.

    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.

    CAS  Article  Google Scholar 

  40. 40.

    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.

    CAS  Article  Google Scholar 

  41. 41.

    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.

    CAS  Article  Google Scholar 

  42. 42.

    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.

    CAS  Article  Google Scholar 

  43. 43.

    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.

    CAS  Article  Google Scholar 

  44. 44.

    Baek D, Villen J, Shin C, Camargo FD, Gygi SP, Bartel DP. The impact of microRNAs on protein output. Nature. 2008;455:64–71.

    CAS  Article  Google Scholar 

  45. 45.

    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.

    CAS  Article  Google Scholar 

  46. 46.

    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.

    CAS  Article  Google Scholar 

  47. 47.

    Landis WJ. Mineral characterization in calcifying tissues: atomic, molecular and macromolecular perspectives. Connect Tissue Res. 1996;34:239–46.

    CAS  Article  Google Scholar 

  48. 48.

    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.

    CAS  Article  Google Scholar 

  49. 49.

    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.

    CAS  Article  Google Scholar 

  50. 50.

    Aubin JE. Bone stem cells. J Cell Biochem Suppl. 1998;30-31:73–82.

    CAS  Article  Google Scholar 

  51. 51.

    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.

    CAS  Article  Google Scholar 

  52. 52.

    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.

    Article  Google Scholar 

  53. 53.

    Lee DJ, Tseng HC, Wong SW, Wang Z, Deng M, Ko CC. Dopaminergic effects on in vitro osteogenesis. Bone Res. 2015;3:15020.

    Article  Google Scholar 

  54. 54.

    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.

    CAS  Article  Google Scholar 

  55. 55.

    Standal T, Borset M, Sundan A. Role of osteopontin in adhesion, migration, cell survival and bone remodeling. Exp Oncol. 2004;26:179–84.

    CAS  Google Scholar 

  56. 56.

    Seibel MJ. Biochemical markers of bone turnover: part I: biochemistry and variability. Clin Biochem Rev. 2005;26:97–122.

    Google Scholar 

  57. 57.

    Delmas PD, Malaval L, Arlot ME, Meunier PJ. Serum bone Gla-protein compared to bone histomorphometry in endocrine diseases. Bone. 1985;6:339–41.

    CAS  Article  Google Scholar 

  58. 58.

    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.

    Google Scholar 

  59. 59.

    Wang YX, Yan SX. Biomedical imaging in the safety evaluation of new drugs. Lab Anim. 2008;42:433–41.

    CAS  Article  Google Scholar 

  60. 60.

    Guengerich FP. Mechanisms of drug toxicity and relevance to pharmaceutical development. Drug Metab Pharmacokinet. 2011;26:3–14.

    CAS  Article  Google Scholar 

  61. 61.

    Marquis ME, Lord E, Bergeron E, Drevelle O, Park H, Cabana F, et al. Bone cells-biomaterials interactions. Front Biosci. 2009;14:1023–67.

    CAS  Article  Google Scholar 

  62. 62.

    Gronowicz G, McCarthy MB. Response of human osteoblasts to implant materials: integrin-mediated adhesion. J Orthop Res. 1996;14:878–87.

    CAS  Article  Google Scholar 

  63. 63.

    Garcia AJ. Get a grip: integrins in cell-biomaterial interactions. Biomaterials. 2005;26:7525–9.

    CAS  Article  Google Scholar 

  64. 64.

    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.

    CAS  Article  Google Scholar 

  65. 65.

    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.

    Article  Google Scholar 

  66. 66.

    Rosales C, O’Brien V, Kornberg L, Juliano R. Signal transduction by cell adhesion receptors. Biochim Biophys Acta. 1995;1242:77–98.

    Google Scholar 

  67. 67.

    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.

    CAS  Article  Google Scholar 

  68. 68.

    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.

    CAS  Article  Google Scholar 

  69. 69.

    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.

    CAS  Article  Google Scholar 

  70. 70.

    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.

    CAS  Article  Google Scholar 

Download references


We wish to thank Dr. Cato T. Laurencin, the Director of the Institute for Regenerative Engineering (IRE), for his leadership.


This work was supported by the Project Fund from the Connecticut Institute for Clinical and Translational Science (CICATS) to Dr. Kevin Lo.

Author information



Corresponding author

Correspondence to Kevin W.-H. Lo.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

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).

Download citation


  • Cyclic AMP
  • Small molecules
  • Regenerative engineering
  • Musculoskeletal tissue
  • Drug discovery
  • Osteogenesis