Tracheal tissue engineering in rats

Abstract

Tissue-engineered tracheal transplants have been successfully performed clinically. However, before becoming a routine clinical procedure, further preclinical studies are necessary to determine the underlying mechanisms of in situ tissue regeneration. Here we describe a protocol using a tissue engineering strategy and orthotopic transplantation of either natural decellularized donor tracheae or artificial electrospun nanofiber scaffolds into a rat model. The protocol includes details regarding how to assess the scaffolds' biomechanical properties and cell viability before implantation. It is a reliable and reproducible model that can be used to investigate the crucial aspects and pathways of in situ tracheal tissue restoration and regeneration. The model can be established in <6 months, and it may also provide a means to investigate cell-surface interactions, cell differentiation and stem cell fate.

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Figure 1: Scaffolds for tissue-engineered rat trachea.
Figure 2: Biomechanical testing of rat tracheal scaffolds.
Figure 3: Two-photon imaging of live cells and LIVE/DEAD staining on the artificial graft 1 h after implantation.
Figure 4: Implantation of tissue-engineered tracheae in rats.

References

  1. 1

    Grillo, H.C. Tracheal replacement: a critical review. Ann. Thorac. Surg. 73, 1995–2004 (2002).

  2. 2

    Rose, K., Sesterhenn, K. & Wustrow, F. Tracheal allotransplantation in man. Lancet 1, 433 (1979).

  3. 3

    Macchiarini, P. et al. Experimental tracheal and tracheoesophageal allotransplantation. Paris-Sud University Lung Transplantation Group. J. Thorac. Cardiovasc. Surg. 110, 1037–1046 (1995).

  4. 4

    Lenot, B., Macchiarini, P., Dulmet, E., Weiss, M. & Dartevelle, P. Tracheal allograft replacement. An unsuccessful method. Eur. J. Cardiothorac. Surg. 7, 648–652 (1993).

  5. 5

    Levashov, Y. et al. One-stage allotransplantation of thoracic segment of the trachea in a patient with idiopathic fibrosing mediastinitis and marked tracheal stenosis. Eur. J. Cardiothorac. Surg. 7, 383–386 (1993).

  6. 6

    Shaari, C.M., Farber, D., Brandwein, M.S., Gannon, P. & Urken, M.L. Characterizing the antigenic profile of the human trachea: implications for tracheal transplantation. Head Neck 20, 522–527 (1998).

  7. 7

    Kunachak, S., Kulapaditharom, B., Vajaradul, Y. & Rochanawutanon, M. Cryopreserved, irradiated tracheal homograft transplantation for laryngotracheal reconstruction in human beings. Otolaryngol. Head Neck Surg. 122, 911–916 (2000).

  8. 8

    Hisamatsu, C., Maeda, K., Tanaka, H. & Okita, Y. Transplantation of the cryopreserved tracheal allograft in growing rabbits: effect of immunosuppressant. Pediatr. Surg. Int. 22, 881–885 (2006).

  9. 9

    Fonkalsrud, E.W. & Sumida, S. Tracheal replacement with autologous esophagus for tracheal stricture. Arch. Surg. 102, 139–142 (1971).

  10. 10

    Fonkalsrud, E.W., Martelle, R.R. & Maloney, J.V. Surgical treatment of tracheal agenesis. J. Thorac. Cardiovasc. Surg. 45, 520–525 (1963).

  11. 11

    Wurtz, A. et al. Tracheal replacement with aortic allografts. N. Engl. J. Med. 355, 1938–1940 (2006).

  12. 12

    Wurtz, A. et al. Surgical technique and results of tracheal and carinal replacement with aortic allografts for salivary gland-type carcinoma. J. Thorac. Cardiovasc. Surg. 140, 387–393 e2 (2010).

  13. 13

    Ranu, H. & Madden, B.P. Endobronchial stenting in the management of large airway pathology. Postgrad. Med. J. 85, 682–687 (2009).

  14. 14

    Grillo, H.C. The history of tracheal surgery. Chest Surg. Clin. N. Am. 13, 175–189 (2003).

  15. 15

    Macchiarini, P. et al. Clinical transplantation of a tissue-engineered airway. Lancet 372, 2023–2030 (2008).

  16. 16

    Baiguera, S. et al. Tissue engineered human tracheas for in vivo implantation. Biomaterials 31, 8931–8938 (2010).

  17. 17

    Gonfiotti, A. et al. The first tissue-engineered airway transplantation: 5-year follow-up results. Lancet 383, 238–244 (2013).

  18. 18

    Jungebluth, P. & Macchiarini, P. Airway transplantation. Thorac. Surg. Clin. 24, 97–106 (2014).

  19. 19

    Go, T. et al. Both epithelial cells and mesenchymal stem cell-derived chondrocytes contribute to the survival of tissue-engineered airway transplants in pigs. J. Thorac. Cardiovasc. Surg. 139, 437–443 (2010).

  20. 20

    Haag, J. et al. Biomechanical and angiogenic properties of tissue-engineered rat trachea using genipin cross-linked decellularized tissue. Biomaterials 33, 780–789 (2012).

  21. 21

    Jungebluth, P. et al. The concept of in vivo airway tissue engineering. Biomaterials 33, 4319–4326 (2012).

  22. 22

    Elliott, M.J. et al. Stem-cell-based, tissue engineered tracheal replacement in a child: a 2-year follow-up study. Lancet 380, 994–1000 (2012).

  23. 23

    Berg, M. et al. Replacement of a tracheal stenosis with a tissue-engineered human trachea using autologous stem cells: a case report. Tissue Eng. Part A 20, 389–397 (2013).

  24. 24

    Jungebluth, P. et al. Tracheobronchial transplantation with a stem-cell-seeded bioartificial nanocomposite: a proof-of-concept study. Lancet 378, 1997–2004 (2011).

  25. 25

    Jungebluth, P. et al. Verification of cell viability in bioengineered tissues and organs before clinical transplantation. Biomaterials 34, 4057–4067 (2013).

  26. 26

    Seguin, A. et al. Tracheal regeneration: evidence of bone marrow mesenchymal stem cell involvement. J. Thorac. Cardiovasc. Surg. 145, 1297–1304 (2013).

  27. 27

    Schrepfer, S. et al. Experimental orthotopic tracheal transplantation: the Stanford technique. Microsurgery 27, 187–189 (2007).

  28. 28

    Ajalloueian, F. et al. Biomechanical and biocompatibility characteristics of electrospun polymeric tracheal scaffolds. Biomaterials 35, 5307–5315 (2014).

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Acknowledgements

This work was supported by European Project FP7-NMP-2011-SMALL-5: BIOtrachea, Biomaterials for Tracheal Replacement in Age-related Cancer via a Humanly Engineered Airway (no. 280584); by ALF medicine (Stockholm County Council): Transplantation of bio-engineered trachea in humans (no. LS1101–0042); by Vetenskapsrådet: Klinisk tillämpning av biokonstruerade organ med särskild betoning på trachea (No. K2013-99X-22252-01-5); by Dr. Dorka-Stiftung (Hannover, Germany): Bioengineering of tracheal tissue; and a Mega grant of the Russian Ministry of Education and Science (agreement no. 11.G34.31.0065).

Author information

The concept of tracheal tissue engineering for both biological and artificial scaffolds was developed by P.M. P.J. and S.B. developed the decellularization concept for human trachea. J.C.H. and S.B. developed the decellularization concept for rodent trachea. The animal orthotopic transplantation model was designed by P.J. and conducted by P.J., J.C.H. and S.S. Cell seeding and scaffold evaluation was done by M.L.L., Y.G. and A.B.R. The mathematical model was developed by G.L. The mechanical evaluation setting was developed and conducted by C.D.G. and A.B. I.D. and P.U. were responsible for two-photon image acquisition and processing. The manuscript was approved by all coauthors before submission.

Correspondence to Paolo Macchiarini.

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Jungebluth, P., Haag, J., Sjöqvist, S. et al. Tracheal tissue engineering in rats. Nat Protoc 9, 2164–2179 (2014) doi:10.1038/nprot.2014.149

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