Drug Delivery and Translational Research

, Volume 6, Issue 2, pp 121–131 | Cite as

In vivo comparison of biomineralized scaffold-directed osteogenic differentiation of human embryonic and mesenchymal stem cells

  • Cai Wen
  • Heemin Kang
  • Yu-Ru V. Shih
  • YongSung Hwang
  • Shyni Varghese
Research Article


Human pluripotent stem cells such as embryonic stem cells (hESCs) and multipotent stem cells like mesenchymal stem cells (hMSCs) hold great promise as potential cell sources for bone tissue regeneration. Comparing the in vivo osteogenesis of hESCs and hMSCs by biomaterial-based cues provides insight into the differentiation kinetics of these cells as well as their potential to contribute to bone tissue repair in vivo. Here, we compared in vivo osteogenic differentiation of hESCs and hMSCs within osteoinductive calcium phosphate (CaP)-bearing biomineralized scaffolds that recapitulate a bone-specific mineral microenvironment. Both hESCs and hMSCs underwent osteogenic differentiation responding to the biomaterial-based instructive cues. Furthermore, hMSCs underwent earlier in vivo osteogenesis compared to hESCs, but both stem cell types acquired a similar osteogenic maturation by 8 weeks of implantation.


Embryonic stem cells Mesenchymal stem cells In vivo osteogenic differentiation Biomineralized scaffold Bone tissue engineering Calcium phosphate 



This work was supported by National Institutes of Health (NIH; Grant 1 R01 AR063184-01A1). The hMSCs used in this study were provided by Texas A&M University from the NIH (Grant P40RR017447).

Conflict of interest

Authors declare no conflict of interest.


  1. 1.
    Gamie Z, MacFarlane RJ, Tomkinson A, Moniakis A, Tran GT, Gamie Y, et al. Skeletal tissue engineering using mesenchymal or embryonic stem cells: clinical and experimental data. Expert Opin Biol Ther. 2014;14(11):1611–39.CrossRefPubMedGoogle Scholar
  2. 2.
    Marolt D, Knezevic M, Novakovic GV. Bone tissue engineering with human stem cells. Stem Cell Res Ther. 2010;1(10):1–10.Google Scholar
  3. 3.
    Seong JM, Kim B-C, Park J-H, Kwon IK, Mantalaris A, Hwang Y-S. Stem cells in bone tissue engineering. Biomed Mater. 2010;5(6):062001.CrossRefPubMedGoogle Scholar
  4. 4.
    Varghese S, Hwang NS, Ferran A, Hillel A, Theprungsirikul P, Canver AC, et al. Engineering musculoskeletal tissues with human embryonic germ cell derivatives. Stem Cells. 2010;28(4):765–74.CrossRefPubMedGoogle Scholar
  5. 5.
    Hwang NS, Varghese S, Lee HJ, Zhang Z, Elisseeff J. Biomaterials directed in vivo osteogenic differentiation of mesenchymal cells derived from human embryonic stem cells. Tissue Eng A. 2013;19(15-16):1723–32.CrossRefGoogle Scholar
  6. 6.
    de Peppo GM, Marcos-Campos I, Kahler DJ, Alsalman D, Shang L, Vunjak-Novakovic G, et al. Engineering bone tissue substitutes from human induced pluripotent stem cells. Proc Natl Acad Sci U S A. 2013;110(21):8680–5.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Karp JM, Ferreira LS, Khademhosseini A, Kwon AH, Yeh J, Langer RS. Cultivation of human embryonic stem cells without the embryoid body step enhances osteogenesis in vitro. Stem Cells. 2006;24(4):835–43.CrossRefPubMedGoogle Scholar
  8. 8.
    Ahn SE, Kim S, Park KH, Moon SH, Lee HJ, Kim GJ, et al. Primary bone-derived cells induce osteogenic differentiation without exogenous factors in human embryonic stem cells. Biochem Biophys Res Commun. 2006;340(2):403–8.CrossRefPubMedGoogle Scholar
  9. 9.
    Hwang NS, Zhang C, Hwang YS, Varghese S. Mesenchymal stem cell differentiation and roles in regenerative medicine. Wiley Interdiscip Rev Syst Biol Med. 2009;1(1):97–106.CrossRefPubMedGoogle Scholar
  10. 10.
    Shimko DA, Burks CA, Dee KC, Nauman EA. Comparison of in vitro mineralization by murine embryonic and adult stem cells cultured in an osteogenic medium. Tissue Eng. 2004;10(9-10):1386–98.CrossRefPubMedGoogle Scholar
  11. 11.
    Marolt D, Campos IM, Bhumiratana S, Koren A, Petridis P, Zhang G, et al. Engineering bone tissue from human embryonic stem cells. Proc Natl Acad Sci. 2012;109(22):8705–9.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Both SK, van Apeldoorn AA, Jukes JM, Englund MC, Hyllner J, van Blitterswijk CA, et al. Differential bone-forming capacity of osteogenic cells from either embryonic stem cells or bone marrow-derived mesenchymal stem cells. J Tissue Eng Regen Med. 2011;5(3):180–90.CrossRefPubMedGoogle Scholar
  13. 13.
    Bilousova G, Jun DH, King KB, De Langhe S, Chick WS, Torchia EC, et al. Osteoblasts derived from induced pluripotent stem cells form calcified structures in scaffolds both in vitro and in vivo. Stem Cells. 2011;29(2):206–16.CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Levi B, Hyun JS, Montoro DT, Lo DD, Chan CK, Hu S, et al. In vivo directed differentiation of pluripotent stem cells for skeletal regeneration. Proc Natl Acad Sci U S A. 2012;109(50):20379–84.CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Peng Y, Kang Q, Luo Q, Jiang W, Si W, Liu BA, et al. Inhibitor of DNA binding/differentiation helix-loop-helix proteins mediate bone morphogenetic protein-induced osteoblast differentiation of mesenchymal stem cells. J Biol Chem. 2004;279(31):32941–9.CrossRefPubMedGoogle Scholar
  16. 16.
    Friedman MS, Long MW, Hankenson KD. Osteogenic differentiation of human mesenchymal stem cells is regulated by bone morphogenetic protein-6. J Cell Biochem. 2006;98(3):538–54.CrossRefPubMedGoogle Scholar
  17. 17.
    Kawaguchi J, Mee PJ, Smith AG. Osteogenic and chondrogenic differentiation of embryonic stem cells in response to specific growth factors. Bone. 2005;36(5):758–69.CrossRefPubMedGoogle Scholar
  18. 18.
    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(1):163–74.CrossRefPubMedGoogle Scholar
  19. 19.
    Phadke A, Shih YRV, Varghese S. Mineralized synthetic matrices as an instructive microenvironment for osteogenic differentiation of human mesenchymal stem cells. Macromol Biosci. 2012;12(8):1022–32.CrossRefPubMedGoogle Scholar
  20. 20.
    Madl CM, Mehta M, Duda GN, Heilshorn SC, Mooney DJ. Presentation of BMP-2 mimicking peptides in 3D hydrogels directs cell fate commitment in osteoblasts and mesenchymal stem cells. Biomacromolecules. 2014;15(2):445–55.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Petrie TA, Raynor JE, Dumbauld DW, Lee TT, Jagtap S, Templeman KL, et al. Multivalent integrin-specific ligands enhance tissue healing and biomaterial integration. Sci Transl Med. 2010;2(45):45ra60.CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Yang C, Tibbitt MW, Basta L, Anseth KS. Mechanical memory and dosing influence stem cell fate. Nat Mater. 2014;13(6):645–52.CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Cameron K, Travers P, Chander C, Buckland T, Campion C, Noble B. Directed osteogenic differentiation of human mesenchymal stem/precursor cells on silicate substituted calcium phosphate. J Biomed Mater Res A. 2013;101(1):13–22.CrossRefPubMedGoogle Scholar
  24. 24.
    Müller P, Bulnheim U, Diener A, Lüthen F, Teller M, Klinkenberg ED, et al. Calcium phosphate surfaces promote osteogenic differentiation of mesenchymal stem cells. J Cell Mol Med. 2008;12(1):281–91.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Kang H, Wen C, Hwang Y, Shih Y-RV, Kar M, Seo SW, et al. Biomineralized matrix-assisted osteogenic differentiation of human embryonic stem cells. J Mater Chem B. 2014;2(34):5676–88.CrossRefGoogle Scholar
  26. 26.
    Kang H, Shih Y-RV, Hwang Y, Wen C, Rao V, Seo T, et al. Mineralized gelatin methacrylate-based matrices induce osteogenic differentiation of human induced pluripotent stem cells. Acta Biomater. 2014;10(12):4961–70.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    LeGeros RZ. Calcium phosphate-based osteoinductive materials. Chem Rev. 2008;108(11):4742–53.CrossRefPubMedGoogle Scholar
  28. 28.
    Chai YC, Roberts SJ, Desmet E, Kerckhofs G, van Gastel N, Geris L, et al. Mechanisms of ectopic bone formation by human osteoprogenitor cells on CaP biomaterial carriers. Biomaterials. 2012;33(11):3127–42.CrossRefPubMedGoogle Scholar
  29. 29.
    Ayala R, Zhang C, Yang D, Hwang Y, Aung A, Shroff SS, et al. Engineering the cell–material interface for controlling stem cell adhesion, migration, and differentiation. Biomaterials. 2011;32(15):3700–11.CrossRefPubMedGoogle Scholar
  30. 30.
    Lin S, Sangaj N, Razafiarison T, Zhang C, Varghese S. Influence of physical properties of biomaterials on cellular behavior. Pharm Res. 2011;28(6):1422–30.CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Zhang C, Aung A, Liao L, Varghese S. A novel single precursor-based biodegradable hydrogel with enhanced mechanical properties. Soft Matter. 2009;5(20):3831–4.CrossRefGoogle Scholar
  32. 32.
    Phadke A, Zhang C, Hwang Y, Vecchio K, Varghese S. Templated mineralization of synthetic hydrogels for bone-like composite materials: Role of matrix hydrophobicity. Biomacromolecules. 2010;11(8):2060–8.CrossRefPubMedGoogle Scholar
  33. 33.
    Oyane A, Kim HM, Furuya T, Kokubo T, Miyazaki T, Nakamura T. Preparation and assessment of revised simulated body fluids. J Biomed Mater Res A. 2003;65(2):188–95.CrossRefPubMedGoogle Scholar
  34. 34.
    Chang C-W, Hwang Y, Brafman D, Hagan T, Phung C, Varghese S. Engineering cell–material interfaces for long-term expansion of human pluripotent stem cells. Biomaterials. 2013;34(4):912–21.CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Deans RJ, Moseley AB. Mesenchymal stem cells: biology and potential clinical uses. Exp Hematol. 2000;28(8):875–84.CrossRefPubMedGoogle Scholar
  36. 36.
    Phadke A, Hwang Y, Hee Kim S, Hyun Kim S, Yamaguchi T, Masuda K, et al. Effect of scaffold microarchitecture on osteogenic differentiation of human mesenchymal stem cells. Eur Cell Mater. 2013;25:114–29.PubMedPubMedCentralGoogle Scholar
  37. 37.
    Shih Y-R, Phadke A, Yamaguchi T, Kang H, Inoue N, Masuda K, et al. Synthetic bone mimetic matrix-mediated in situ bone tissue formation through host cell recruitment. Acta Biomater. 2015;19:1–9.CrossRefPubMedGoogle Scholar
  38. 38.
    Kang H, Shih Y-RV, Varghese S. Biomineralized matrices dominate soluble cues to direct osteogenic differentiation of human mesenchymal stem cells through adenosine signaling. Biomacromolecules. 2015;16(3):1050–61.CrossRefPubMedGoogle Scholar
  39. 39.
    Barradas A, Yuan H, Blitterswijk CA, Habibovic P. Osteoinductive biomaterials: current knowledge of properties, experimental models and biological mechanisms. Eur Cell Mater. 2011;21:407–29.PubMedGoogle Scholar
  40. 40.
    Yuan H, Fernandes H, Habibovic P, de Boer J, Barradas AM, de Ruiter A, et al. Osteoinductive ceramics as a synthetic alternative to autologous bone grafting. Proc Natl Acad Sci. 2010;107(31):13614–9.CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Yang H, Zeng H, Hao L, Zhao N, Du C, Liao H, et al. Effects of hydroxyapatite microparticles morphology on bone mesenchymal stem cell behavior. J Mater Chem B. 2014;2:4703–10.CrossRefGoogle Scholar
  42. 42.
    Choi S, Murphy WL. A screening approach reveals the influence of mineral coating morphology on human mesenchymal stem cell differentiation. Biotechnol J. 2013;8:496–501.CrossRefPubMedGoogle Scholar
  43. 43.
    Wen L, Wang Y, Wang H, Kong L, Zhang L, Chen X, et al. L-type calcium channels play a crucial role in the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells. Biochem Biophys Res Commun. 2012;424:439–45.CrossRefPubMedGoogle Scholar
  44. 44.
    Barradas A, Fernandes HA, Groen N, Chai YC, Schrooten J, van de Peppel J, et al. A calcium-induced signaling cascade leading to osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells. Biomaterials. 2012;33(11):3205–15.CrossRefPubMedGoogle Scholar
  45. 45.
    Shih Y-RV, Hwang Y, Phadke A, Kang H, Hwang NS, Caro EJ, et al. Calcium phosphate-bearing matrices induce osteogenic differentiation of stem cells through adenosine signaling. Proc Natl Acad Sci. 2014;111(3):990–5.CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Suarez-Gonzalez D, Barnhart K, Migneco F, Flanagan C, Hollister SJ, Murphy WL. Controllable mineral coatings on PCL scaffolds as carriers for growth factor release. Biomaterials. 2012;33(2):713–21.CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Suárez-González D, Lee JS, Lan Levengood SK, Vanderby Jr R, Murphy WL. Mineral coatings modulate β-TCP stability and enable growth factor binding and release. Acta Biomater. 2012;8(3):1117–24.CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Liu Y, Hunziker EB, Layrolle P, De Bruijn JD, De Groot K. Bone morphogenetic protein 2 incorporated into biomimetic coatings retains its biological activity. Tissue Eng. 2004;10(1-2):101–8.CrossRefPubMedGoogle Scholar
  49. 49.
    Yuan H, Zou P, Yang Z, Zhang X, De Bruijn J, De Groot K. Bone morphogenetic protein and ceramic-induced osteogenesis. J Mater Sci Mater Med. 1998;9(12):717–21.CrossRefPubMedGoogle Scholar
  50. 50.
    Murphy W, Simmons C, Kaigler D, Mooney D. Bone regeneration via a mineral substrate and induced angiogenesis. J Dent Res. 2004;83(3):204–10.CrossRefPubMedGoogle Scholar
  51. 51.
    Kaigler D, Wang Z, Horger K, Mooney DJ, Krebsbach PH. VEGF scaffolds enhance angiogenesis and bone regeneration in irradiated osseous defects. J Bone Miner Res : Off J Am Soc Bone Miner Res. 2006;21(5):735–44.CrossRefGoogle Scholar
  52. 52.
    Xiao X, Wang W, Liu D, Zhang H, Gao P, Geng L, et al. The promotion of angiogenesis induced by three-dimensional porous beta-tricalcium phosphate scaffold with different interconnection sizes via activation of PI3K/Akt pathways. Sci Rep. 2015;5:9409.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Controlled Release Society 2015

Authors and Affiliations

  • Cai Wen
    • 1
  • Heemin Kang
    • 2
  • Yu-Ru V. Shih
    • 2
  • YongSung Hwang
    • 2
  • Shyni Varghese
    • 2
  1. 1.School of Chemistry and Chemical EngineeringSoutheast UniversityNanjingPeople’s Republic of China
  2. 2.Department of BioengineeringUniversity of California— San DiegoLa JollaUSA

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