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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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


  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


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Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Shulamit Levenberg or Robert Langer.

Supplementary information

  1. Supplementary Fig. 1

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

  2. 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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