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Engineering vascularized skeletal muscle tissue

Nature Biotechnology volume 23, pages 879–884 (2005)Cite this article

Abstract

One of the major obstacles in engineering thick, complex tissues such as muscle is the need to vascularize the tissue in vitro. Vascularization in vitro could maintain cell viability during tissue growth, induce structural organization and promote vascularization upon implantation. Here we describe the induction of endothelial vessel networks in engineered skeletal muscle tissue constructs using a three-dimensional multiculture system consisting of myoblasts, embryonic fibroblasts and endothelial cells coseeded on highly porous, biodegradable polymer scaffolds. Analysis of the conditions for induction and stabilization of the vessels in vitro showed that addition of embryonic fibroblasts increased the levels of vascular endothelial growth factor expression in the construct and promoted formation and stabilization of the endothelial vessels. We studied the survival and vascularization of the engineered muscle implants in vivo in three different models. Prevascularization improved the vascularization, blood perfusion and survival of the muscle tissue constructs after transplantation.

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Figure 1: In vitro vascularization of engineered skeletal muscle tissue
Figure 2: Quantitative analysis of endothelial vessels in muscle constructs, in vitro.
Figure 3: In vivo analysis of engineered muscle constructs (a) Differentiation of engineered muscle in vivo.

References

  1. Saxena, A.K., Marler, J., Benvenuto, M., Willital, G.H. & Vacanti, J.P. Skeletal muscle tissue engineering using isolated myoblasts on synthetic biodegradable polymers: preliminary studies. Tissue Eng. 5, 525–532 (1999).

    Article  CAS  Google Scholar 

  2. Neumann, T., Nicholson, B.S. & Sanders, J.E. Tissue engineering of perfused microvessels. Microvasc. Res. 66, 59–67 (2003).

    Article  CAS  Google Scholar 

  3. Bach, A.D., Stem-Straeter, J., Beier, J.P., Bannasch, H. & Stark, G.B. Engineering of muscle tissue. Clin. Plast. Surg. 30, 589–599 (2003).

    Article  CAS  Google Scholar 

  4. Wigmore, P.M. & Evans, D.J. Molecular and cellular mechanisms involved in the generation of fiber diversity during myogenesis. Int. Rev. Cytol. 216, 175–232 (2002).

    Article  CAS  Google Scholar 

  5. Buckingham, M. Skeletal muscle formation in vertebrates. Curr. Opin. Genet. Dev. 11, 440–448 (2001).

    Article  CAS  Google Scholar 

  6. Zisch, A.H., Lutolf, M.P. & Hubbell, J.A. Biopolymeric delivery matrices for angiogenic growth factors. Cardiovasc. Pathol. 12, 295–310 (2003).

    Article  CAS  Google Scholar 

  7. von Degenfeld, G., Banfi, A., Springer, M.L. & Blau, H.M. Myoblast-mediated gene transfer for therapeutic angiogenesis and arteriogenesis. Br. J. Pharmacol. 140, 620–626 (2003).

    Article  CAS  Google Scholar 

  8. Lu, Y. et al. Recombinant vascular endothelial growth factor secreted from tissue-engineered bioartificial muscles promotes localized angiogenesis. Circulation 104, 594–599 (2001).

    Article  CAS  Google Scholar 

  9. Levenberg, S., Golub, J.S., Amit, M., Itskovitz-Eldor, J. & Langer, R. Endothelial cells derived from human embryonic stem cells. Proc. Natl. Acad. Sci. USA 99, 4391–4396 (2002).

    Article  CAS  Google Scholar 

  10. Levenberg, S. et al. Differentiation of human embryonic stem cells on three-dimensional polymer scaffolds. Proc. Natl. Acad. Sci. USA 100, 12741–12746 (2003).

    Article  CAS  Google Scholar 

  11. Darland, D.C. & D'Amore, P.A. TGF beta is required for the formation of capillary-like structures in three-dimensional cocultures of 10T1/2 and endothelial cells. Angiogenesis 4, 11–20 (2001).

    Article  CAS  Google Scholar 

  12. Carmeliet, P. Angiogenesis in health and disease. Nat. Med. 9, 653–660 (2003).

    Article  CAS  Google Scholar 

  13. Jain, R.K. Molecular regulation of vessel maturation. Nat. Med. 9, 685–693 (2003).

    Article  CAS  Google Scholar 

  14. Sieminski, A.L., Padera, R.F., Blunk, T. & Gooch, K.J. Systemic Delivery of Human Growth Hormone Using Genetically Modified Tissue-Engineered Microvascular Networks: Prolonged Delivery and Endothelial Survival with Inclusion of Nonendothelial Cells. Tissue Eng. 8, 1057–1069 (2002).

    Article  CAS  Google Scholar 

  15. Koike, N. et al. Tissue engineering: creation of long-lasting blood vessels. Nature 428, 138–139 (2004).

    Article  CAS  Google Scholar 

  16. Black, A.F., Berthod, F., L'Heureux, N., Germain, L. & Auger, F.A. In vitro reconstruction of a human capillary-like network in a tissue-engineered skin equivalent. FASEB J. 12, 1331–1340 (1998).

    Article  CAS  Google Scholar 

  17. Shinoka, T. Tissue engineered heart valves: autologous cell seeding on biodegradable polymer scaffold. Artif. Organs 26, 402–406 (2002).

    Article  Google Scholar 

  18. Flamme, I., Frolich, T. & Risau, W. Molecular mechanisms of vasculogenesis and embryonic angiogenesis. J. Cell. Physiol. 173, 206–210 (1997).

    Article  CAS  Google Scholar 

  19. Rossant, J. & Hirashima, M. Vascular development and patterning: making the right choices. Curr. Opin. Genet. Dev. 13, 408–412 (2003).

    Article  CAS  Google Scholar 

  20. Hirschi, K.K., Rohovsky, S.A. & D'Amore, P.A. PDGF, TGF-beta, and heterotypic cell-cell interactions mediate endothelial cell-induced recruitment of 10T1/2 cells and their differentiation to a smooth muscle fate. J. Cell Biol. 141, 805–814 (1998).

    Article  CAS  Google Scholar 

  21. Darland, D.C. et al. Pericyte production of cell-associated VEGF is differentiation-dependent and is associated with endothelial survival. Dev. Biol. 264, 275–288 (2003).

    Article  CAS  Google Scholar 

  22. Lammert, E., Cleaver, O. & Melton, D. Induction of pancreatic differentiation by signals from blood vessels. Science 294, 564–567 (2001).

    Article  CAS  Google Scholar 

  23. Matsumoto, K., Yoshitomi, H., Rossant, J. & Zaret, K.S. Liver organogenesis promoted by endothelial cells prior to vascular function. Science 294, 559–563 (2001).

    Article  CAS  Google Scholar 

  24. Yaffe, D. & Saxel, O. Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle. Nature 270, 725–727 (1977).

    Article  CAS  Google Scholar 

  25. Blau, H.M. et al. Plasticity of the differentiated state. Science 230, 758–766 (1985).

    Article  CAS  Google Scholar 

  26. Holder, W.D. Jr. et al. Cellular ingrowth and thickness changes in poly-L-lactide and polyglycolide matrices implanted subcutaneously in the rat. J. Biomed. Mater. Res. 41, 412–421 (1998).

    Article  CAS  Google Scholar 

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Acknowledgements

The authors thank the MIT division of comparative medicine for excellent assistance in tissue embedding and processing, Adam Kapur for help with data analysis, and Justin S. Golub for help with RT-PCR assays. We would like to thank Joseph Itskovitz-Eldor for assistance and cooperation in conducting this research. This work was supported by National Institutes of Health grants HL60435 (R.L. and S.L.) and EY05318 (P.A.D. and D.C.D.).

Author information

Authors and Affiliations

  1. Department of Biomedical Engineering, Technion, Haifa, 32000, Israel

    Shulamit Levenberg

  2. Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, 02139, Massachusetts, USA

    Shulamit Levenberg, Mara Macdonald & Robert Langer

  3. Institute for Biomedical Technology, Twente University, Prof. Bronkhorstlaan 10-D, Bilthoven, 3723MB, The Netherlands

    Jeroen Rouwkema & Clemens A van Blitterswijk

  4. Department of Surgery, Brigham and Women's Hospital, 75 Francis St., Boston, 02115, Massachusetts, USA

    Evan S Garfein

  5. Department of Pediatrics, Massachusetts General Hospital, 55 Fruit St., Boston, 02114, Massachusetts, USA

    Daniel S Kohane

  6. The Schepens Eye Research Institute and Department of Ophthalmology, 20 Staniford St., Boston, 02114, Massachusetts, USA

    Diane C Darland & Patricia A D'Amore

  7. Division of Comparative Medicine, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, 02139, Massachusetts, USA

    Robert Marini

  8. Department of Molecular Medicine, Children's Hospital 300 Longwood Avenue, 02115, Harvard Medical School, Boston, Massachusetts, USA

    Richard C Mulligan

Authors
  1. Shulamit Levenberg
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  2. Jeroen Rouwkema
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  3. Mara Macdonald
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  4. Evan S Garfein
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  5. Daniel S Kohane
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  6. Diane C Darland
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  7. Robert Marini
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  8. Clemens A van Blitterswijk
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  9. Richard C Mulligan
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  10. Patricia A D'Amore
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  11. Robert Langer
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Corresponding authors

Correspondence to Shulamit Levenberg or Robert Langer.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

VEGF and PDGF-B expression in 3D constructs. (PDF 873 kb)

Supplementary Fig. 2

Quantitative analysis of number of endothelial vessels in muscle implants seeded with HUVEC or hESC-derived endothelial cells. (PDF 481 kb)

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Levenberg, S., Rouwkema, J., Macdonald, M. et al. Engineering vascularized skeletal muscle tissue. Nat Biotechnol 23, 879–884 (2005). https://doi.org/10.1038/nbt1109

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