Annals of Biomedical Engineering

, Volume 44, Issue 6, pp 1908–1920 | Cite as

Muscle Tissue Engineering Using Gingival Mesenchymal Stem Cells Encapsulated in Alginate Hydrogels Containing Multiple Growth Factors

  • Sahar Ansari
  • Chider Chen
  • Xingtian Xu
  • Nasim Annabi
  • Homayoun H. Zadeh
  • Benjamin M. Wu
  • Ali Khademhosseini
  • Songtao Shi
  • Alireza MoshaveriniaEmail author
Emerging Trends in Biomaterials Research


Repair and regeneration of muscle tissue following traumatic injuries or muscle diseases often presents a challenging clinical situation. If a significant amount of tissue is lost the native regenerative potential of skeletal muscle will not be able to grow to fill the defect site completely. Dental-derived mesenchymal stem cells (MSCs) in combination with appropriate scaffold material, present an advantageous alternative therapeutic option for muscle tissue engineering in comparison to current treatment modalities available. To date, there has been no report on application of gingival mesenchymal stem cells (GMSCs) in three-dimensional scaffolds for muscle tissue engineering. The objectives of the current study were to develop an injectable 3D RGD-coupled alginate scaffold with multiple growth factor delivery capacity for encapsulating GMSCs, and to evaluate the capacity of encapsulated GMSCs to differentiate into myogenic tissue in vitro and in vivo where encapsulated GMSCs were transplanted subcutaneously into immunocompromised mice. The results demonstrate that after 4 weeks of differentiation in vitro, GMSCs as well as the positive control human bone marrow mesenchymal stem cells (hBMMSCs) exhibited muscle cell-like morphology with high levels of mRNA expression for gene markers related to muscle regeneration (MyoD, Myf5, and MyoG) via qPCR measurement. Our quantitative PCR analyzes revealed that the stiffness of the RGD-coupled alginate regulates the myogenic differentiation of encapsulated GMSCs. Histological and immunohistochemical/fluorescence staining for protein markers specific for myogenic tissue confirmed muscle regeneration in subcutaneous transplantation in our in vivo animal model. GMSCs showed significantly greater capacity for myogenic regeneration in comparison to hBMMSCs (p < 0.05). Altogether, our findings confirmed that GMSCs encapsulated in RGD-modified alginate hydrogel with multiple growth factor delivery capacity is a promising candidate for muscle tissue engineering.


Tissue engineering Muscle regeneration Dental mesenchymal stem cells RGD-coupled alginate hydrogel 



This work was supported by grants from the National Institute of Dental, Craniofacial Research (K08DE023825 to A.M. and R01 DE017449 to S.S.). The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.


  1. 1.
    Abou-Khalil, R., F. Yang, S. Lieu, A. Julien, J. Perry, C. Pereira, F. Relaix, T. Miclau, R. Marcucio, and C. Colnot. Role of muscle stem cells during skeletal regeneration. Stem Cells. 33:1501–1511, 2015.CrossRefPubMedGoogle Scholar
  2. 2.
    Adnot, S., M. Desmier, N. Ferry, J. Hanoune, and T. Sevenet. Forskolin (a powerful inhibitor of human platelet aggregation). Biochem. Pharmacol. 31:4071–4074, 1982.CrossRefPubMedGoogle Scholar
  3. 3.
    Ansari, S., A. Moshaverinia, S. H. Pi, A. Han, A. I. Abdelhamid, and H. H. Zadeh. Functionalization of scaffolds with chimeric anti-BMP-2 monoclonal antibodies for osseous regeneration. Biomaterials. 34:10191–10198, 2013.CrossRefPubMedGoogle Scholar
  4. 4.
    Beier, J. P., F. F. Bitto, C. Lange, D. Klumpp, A. Arkudas, O. Bleiziffer, et al. Myogenic differentiation of mesenchymal stem cells co-cultured with primary myoblasts. Cell. Biol. Int. 35:397–406, 2011.CrossRefPubMedGoogle Scholar
  5. 5.
    Boontheekula, T., H. J. Kongc, and D. J. Mooney. Controlling alginate gel degradation utilizing partial oxidation and bimodal molecular weight distribution. Biomaterials. 26:2455–2465, 2005.CrossRefGoogle Scholar
  6. 6.
    Borselli, C., C. A. Cezar, D. Shvartsman, H. H. Vandenburgh, and D. J. Mooney. The role of multifunctional delivery scaffold in the ability of cultured myoblasts to promote muscle regeneration. Biomaterials. 32:8905–8914, 2011.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Borselli, C., H. Storrie, F. Benesch-Lee, D. Shvartsman, C. Cezar, J. W. Lichtman, H. H. Vandenburgh, and D. J. Mooney. Functional muscle regeneration with combined delivery of angiogenesis and myogenesis factors. Proc. Natl. Acad. Sci. USA 107:3287–3292, 2010.CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Bouhadir, K. H., K. Y. Lee, E. Alsberg, K. L. Damm, K. W. Anderson, and D. J. Mooney. Degradation of partially oxidized alginate and its potential application for tissue engineering. Biotechnol. Prog. 17:945–950, 2001.CrossRefPubMedGoogle Scholar
  9. 9.
    Bristow, M. R., R. Ginsburg, A. Strosberg, W. Montgomery, and W. Minobe. Pharmacology and inotropic potential of for- skolin in the human heart. J. Clin. Invest. 74:212–223, 1984.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Cantu, D. A., P. Hematti, and W. J. Kao. Cell encapsulating biomaterial regulates mesenchymal stromal/stem cell differentiation and macrophage immuno- phenotype. Stem. Cell. Transl. Med. 1:740–749, 2012.CrossRefGoogle Scholar
  11. 11.
    Chen, F. M., J. Zhang, M. Zhang, Y. An, F. Chen, and Z. F. Wu. A review on endogenous regenerative technology in periodontal regenerative medicine. Biomaterials. 31:7892–7927, 2010.CrossRefPubMedGoogle Scholar
  12. 12.
    Dezawa, M., H. Ishikawa, Y. Itokazu, T. Yoshihara, M. Hoshino, S. Takeda, et al. Bone marrow stromal cells generate muscle cells and repair muscle degeneration. Science. 309:314–317, 2005.CrossRefPubMedGoogle Scholar
  13. 13.
    Engler, A. J., S. Sen, H. L. Sweeney, and D. E. Discher. Matrix elasticity directs stem cell lineage specification. Cell. 126:677–689, 2006.CrossRefPubMedGoogle Scholar
  14. 14.
    Evangelista, M. B., S. X. Hsiong, R. Fernandes, P. Sampaio, H. Kong, C. C. Barrias, et al. Upregulation of bone cell differentiation through immobilization within a synthetic extracellular matrix. Biomaterials. 28:3644–3655, 2007.CrossRefPubMedGoogle Scholar
  15. 15.
    Goncalves, M. A., J. Swildens, M. Holkers, A. Narain, G. P. van Nierop, M. J. van de Watering, et al. Genetic complementation of human muscle cells via directed stem cell fusion. Mol. Ther. 16:741–748, 2008.CrossRefPubMedGoogle Scholar
  16. 16.
    Gonzalez, A., E. Aranda, D. Mezzano, and J. Garrido. Effects of diterpene forskolin on the release reaction and protein phosphorylation of human platelets. Cell. Biochem. Funct. 1:179–185, 1983.CrossRefPubMedGoogle Scholar
  17. 17.
    Gronthos, S., M. Mankani, J. Brahim, P. G. Robey, and S. Shi. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc. Natl. Acad. Sci. USA 97:13625–13630, 2000.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Huebsch, N., P. R. Arany, A. S. Mao, D. Shvartsman, O. A. Ali, S. A. Bencherif, J. Rivera-Feliciano, and D. J. Mooney. Harnessing traction-mediated manipulation of the cell/matrix interface to control stem-cell fate. Nat. Mater. 9:518–526, 2010.CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Iwata, T., M. Yamato, Z. Zhang, S. Mukobata, K. Washio, T. Ando, et al. Validation of human periodontal ligament-derived cells as a reliable source for cytotherapeutic use. J. Clin. Periodontol. 37:1088–1099, 2010.CrossRefPubMedGoogle Scholar
  20. 20.
    Koning, M., M. C. Harmsen, M. J. van Luyn, and P. M. Werker. Current opportunities and challenges in skeletal muscle tissue engineering. J. Tissue Eng. Regen. Med. 3:407–415, 2009.CrossRefPubMedGoogle Scholar
  21. 21.
    Krauss, R. S., F. Cole, U. Gaio, G. Takaesu, W. Zhang, and J. S. Kang. Close encounters: regulation of vertebrate skeletal myogenesis by cell-cell contact. J. Cell Sci. 118:2355–2362, 2005.CrossRefPubMedGoogle Scholar
  22. 22.
    Kuang, S., M. A. Gillespie, and M. A. Rudnicki. Niche regulation of muscle satellite cell self-renewal and differentiation. Cell Stem Cell. 2:22–31, 2008.CrossRefPubMedGoogle Scholar
  23. 23.
    Litosch, I., T. H. Hudson, I. Mills, S. Y. Li, and J. N. Fain. Forskolin as an activator of cyclic AMP accumulation and lipolysis in rat adipocytes. Mol. Pharmacol. 22:109–115, 1982.PubMedGoogle Scholar
  24. 24.
    Lu, H. H., J. M. Vo, H. S. Chin, J. Lin, M. Cozin, R. Tsay, et al. Controlled delivery of platelet-rich plasma growth factors for bone formation. J. Biomed. Mater. Res. A. 86:1128–1136, 2008.CrossRefPubMedGoogle Scholar
  25. 25.
    Lubeck, D. P. The costs of musculoskeletal disease: health needs assessment and health economics. Best. Pract. Res. Clin. Rheumatol. 17:529–539, 2003.CrossRefPubMedGoogle Scholar
  26. 26.
    Markusen, J. F., C. Mason, D. A. Hull, M. A. Town, A. B. Tabor, M. Clements, C. H. Boshoff, and P. Dunnill. Behavior of adult human mesenchymal stem cells entrapped in alginate-GRGDY beads. Tissue Eng. Part A. 12:821–830, 2006.CrossRefGoogle Scholar
  27. 27.
    Mikos, A. G., S. W. Herring, P. Ochareon, J. Elisseeff, H. H. Lu, R. Kandel, et al. Engineering complex tissues. Tissue Eng. 12:3307–3339, 2006.CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Miura, M., S. Gronthos, M. Zhao, B. Lu, L. W. Fisher, P. G. Robey, et al. SHED: stem cells from human exfoliated deciduous teeth. Proc. Natl. Acad. Sci. USA 100:5807–5812, 2003.CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Morosetti, R., M. Mirabella, C. Gliubizzi, A. Broccolini, L. De Angelis, E. Tagliafico, et al. MyoD expression restores defective myogenic differentiation of human mesoangioblasts from inclusion-body myositis muscle. Proc. Natl. Acad. Sci. USA 103:16995–17000, 2006.CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Moshaverinia, A., S. Ansari, C. Chen, X. Xu, K. Akiyama, M. L. Snead, et al. Co-encapsulation of anti-BMP2 monoclonal antibody and mesenchymal stem cells in alginate microspheres for bone tissue engineering. Biomaterials. 34:6572–6579, 2013.CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Moshaverinia, A., C. Chen, K. Akiyama, S. Ansari, X. Xu, W. W. Chee, et al. Alginate hydrogel as a promising scaffold for dental-derived stem cells: an in vitro study. J. Mater. Sci: Mater. Med. 23:3041–3051, 2012.Google Scholar
  32. 32.
    Moshaverinia, A., C. Chen, K. Akiyama, X. Xu, W. W. Chee, S. R. Schricker, and S. Shi. Encapsulated dental-derived stem cells in an injectable and biodegradable scaffold for applications in bone tissue engineering. J. Biomed. Mater. Res. Part. A. 101:3285–3294, 2013.Google Scholar
  33. 33.
    Moshaverinia, A., C. Chen, X. Xu, K. Akiyama, S. Ansari, H. H. Zadeh, and S. Shi. Bone regeneration potential of stem cells derived from periodontal ligament or gingival tissue sources encapsulated in RGD-modified alginate scaffold. Tissue Eng. Part A. 20:611–621, 2013.PubMedPubMedCentralGoogle Scholar
  34. 34.
    Moshaverinia, A., C. Chen, X. Xu, S. Ansari, H. H. Zadeh, S. R. Schricker, et al. Regulation of the stem cell-host immune system interplay using hydrogel coencapsulation system with an anti-inflammatory drug. Adv Funct Mater. 15:2296–2307, 2015.Google Scholar
  35. 35.
    Moshaverinia, A., X. Xu, C. Chen, S. Ansari, H. H. Zadeh, M. L. Snead, et al. Application of stem cells derived from the periodontal ligament or gingival tissue sources for tendon tissue regeneration. Biomaterials. 35:2642–2650, 2014.CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Murphy, W. L., T. C. McDevitt, and A. J. Engler. Materials as stem cell regulators. Nat. Mater. 13:547–557, 2014.CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Pirskanen, A., J. C. Kiefer, and S. D. Hauschka. IGFs, insulin, Shh, bFGF, and TGF-beta1 interact synergistically to pro- mote somite myogenesis in vitro. Dev. Biol. 224:189–203, 2000.CrossRefPubMedGoogle Scholar
  38. 38.
    Puri, P. L., S. Iezzi, P. Stiegler, T. T. Chen, R. L. Schiltz, G. E. Muscat, A. Giordano, L. Kedes, J. Y. Wang, and V. Sartorelli. Class I histone deacetylases sequentially interact with MyoD and pRb during skeletal myogenesis. Mol. Cell. 8:885–897, 2001.CrossRefPubMedGoogle Scholar
  39. 39.
    Re’em, T., O. Tsur-Gang, and S. Cohen. The effect of immobilized RGD peptide in macroporous alginate scaffolds on TGFb1-induced chondrogenesis of human mesenchymal stem cells. Biomaterials. 31:6746–6755, 2010.CrossRefPubMedGoogle Scholar
  40. 40.
    Rowley, J. A., G. Madlambayan, and D. J. Mooney. Alginate hydrogels as synthetic extracellular matrix materials. Biomaterials. 20:45–51, 1999.CrossRefPubMedGoogle Scholar
  41. 41.
    Sachlos, E., and J. T. Czernuszka. Making tissue engineering scaffolds work. Review on the application of solid freeform fabrication technology to the production of tissue engineering scaffold. Euro. Cell. Mater. 5:29–40, 2003.Google Scholar
  42. 42.
    Salani, S., C. Donadoni, F. Rizzo, N. Bresolin, G. P. Comi, and S. Corti. Generation of skeletal muscle cells from embryonic and induced pluripotent stem cells as an in vitro model and for therapy of muscular dystrophies. J. Cell Mol. Med. 16:1353–1364, 2012.CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Seamon, K. B., W. Padgett, and J. W. Daly. Forskolin unique diterpene activator of adenylate cyclase in membranes and in intact cells. Proc. Natl. Acad. Sci. USA 78:3363–3367, 1981.CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Seed, J., and S. D. Hauschka. Clonal analysis of vertebrate myogenesis. VIII. Fibroblasts growth factor (FGF)-dependent and FGF-independent muscle colony types during chick wing development. Dev. Biol. 128:40–49, 1988.CrossRefPubMedGoogle Scholar
  45. 45.
    Seo, B. M., M. Miura, S. Gronthos, P. M. Bartold, S. Batouli, J. Brahim, et al. Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet. 364:149–155, 2004.CrossRefPubMedGoogle Scholar
  46. 46.
    Siebens, H. C. Musculoskeletal problems as comorbidities. Am. J. Phys. Med. Rehabil. 86:69–78, 2007.CrossRefGoogle Scholar
  47. 47.
    Silva, A. K., M. Juenet, A. Meddahi-Pellé, and D. Letourneur. Polysaccharide-based strategies for heart tissue engineering. Carbohydr. Polym. 116:267–277, 2015.CrossRefPubMedGoogle Scholar
  48. 48.
    Tseng, A. S., F. B. Engel, and M. T. Keating. The GSK 3 inhibitor BIO promotes proliferation in mammalian cardiomyocytes. Chem Biol. 13:957–963, 2006.CrossRefPubMedGoogle Scholar
  49. 49.
    Tseng, B. S., P. Zhao, J. S. Pattison, S. E. Gordon, J. A. Granchelli, R. W. Madsen, et al. Regenerated mdx mouse skeletal muscle shows differential mRNA expression. J. Appl. Physiol. 93:537–545, 2002.CrossRefPubMedGoogle Scholar
  50. 50.
    Wang, W., N. Ma, K. Kratz, X. Xu, Z. Li, T. Roch, et al. The influence of polymer scaffolds on cellular behaviour of bone marrow derived human mesenchymal stem cells. Clin. Hemorheol. Microcirc. 52:357–373, 2012.PubMedGoogle Scholar
  51. 51.
    Weintraub, H. The MyoD family and myogenesis: redundancy, networks, and thresholds. Cell. 75:1241–1244, 1993.CrossRefPubMedGoogle Scholar
  52. 52.
    Wright, W. E., D. A. Sassoon, and V. K. Lin. Myogenin, a factor regulating myogenesis, has a domain homologous to MyoD. Cell. 56:607–617, 1989.CrossRefPubMedGoogle Scholar
  53. 53.
    Xu, X., C. Chen, K. Akiyama, Y. Chai, A. D. Le, Z. Wang, and S. Shi. Gingivae contain neural-crest and mesoderm-derived mesenchymal stem cells. J. Dent. Res. 92:825–832, 2013.CrossRefPubMedPubMedCentralGoogle Scholar
  54. 54.
    Zhang, Q., S. Shi, Y. Liu, J. Uyanne, Y. Shi, S. Shi, et al. mesenchymal stem cells derived from human gingiva are capable of immunomodulatory functions and ameliorate inflammation-related tissue destruction in experimental colitis. J. Immunol. 183:7787–7798, 2009.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Biomedical Engineering Society 2016

Authors and Affiliations

  1. 1.Division of Growth and Development, School of DentistryUniversity of CaliforniaLos AngelesUSA
  2. 2.School of Dental MedicineUniversity of PennsylvaniaPhiladelphiaUSA
  3. 3.Ostrow School of DentistryUniversity of Southern CaliforniaLos AngelesUSA
  4. 4.Department of Chemical EngineeringNortheastern UniversityBostonUSA
  5. 5.Weintraub Center for Reconstructive Biotechnology, Division of Advanced Prothodontics, School of DentistryUniversity of CaliforniaLos AngelesUSA
  6. 6.Biomaterials Innovation Research Center, Department of Medicine, Brigham and Women’s HospitalHarvard Medical SchoolCambridgeUSA

Personalised recommendations