Skip to main content
SpringerLink
Log in

A Comparative Study of the Effects of Different Decellularization Methods and Genipin-Cross-Linking on the Properties of Tracheal Matrices

  • Original Article
  • Published:
Tissue Engineering and Regenerative Medicine Aims and scope

Abstract

Background:

Different decellularization methods can affect the integrity and the biomechanical and biocompatible properties of the tracheal matrix. Natural cross-linking with genipin can be applied to improve those properties. The goals of this study were to evaluate the effects of different decellularization methods on the properties of genipin-cross-linked decellularized tracheal matrices in rabbits.

Methods:

The tracheas of New Zealand rabbits were decellularized by the Triton-X 100-processed method (TPM) and the detergent-enzymatic method (DEM) and were then cross-linked with genipin. Mechanical tests, haematoxylin–eosin staining, Masson trichrome staining, Safranin O staining, DAPI staining, scanning electronic microscopy (SEM), and biocompatibility tests were used to evaluate the treatment. The bioengineered trachea and control trachea were then implanted into allogeneic rabbits for 30 days. The structural and functional analyses were performed after transplantation.

Results:

The biomechanical tests demonstrated that the biomechanical properties of the decellularized tracheas decreased and that genipin improved them (p < 0.05). The histological staining results revealed that most of the mucosal epithelial cells were removed and that the decellularized trachea had lower immunogenicity than the control group. The analysis of SEM revealed that the decellularized trachea retained the micro- and ultra-structural architectures of the trachea and that the matrices cross-linked with genipin were denser. The biocompatibility evaluation and in vivo implantation experiments showed that the decellularized trachea treated with the DEM had better biocompatibility than that treated with the TPM and that immunogenicity in the cross-linked tissues was lower than that in the uncross-linked tissues (p < 0.05).

Conclusions:

Compared with the trachea treated with the TPM, the rabbit trachea processed by the DEM had better biocompatibility and lower immunogenicity, and its structural and mechanical characteristics were effectively improved after the genipin treatment, which is suitable for engineering replacement tracheal tissue.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

Similar content being viewed by others

References

  1. Sukumaran R, Nair RA, Jacob PM, Koshy SM, Mathew AP. Extramedullary plasmacytoma of the trachea. Head Neck Pathol. 2014;8:220–4.

    Article  PubMed  Google Scholar 

  2. Kojima K, Vacanti CA. Tissue engineering in the trachea. Anat Rec (Hoboken). 2014;297:44–50.

    Article  CAS  Google Scholar 

  3. Luo X, Liu Y, Zhang Z, Tao R, Liu Y, He A, et al. Long-term functional reconstruction of segmental tracheal defect by pedicled tissue-engineered trachea in rabbits. Biomaterials. 2013;34:3336–44.

    Article  CAS  PubMed  Google Scholar 

  4. Elliott MJ, De Coppi P, Speggiorin S, Roebuck D, Butler CR, Samuel E, et al. Stem-cell-based, tissue engineered tracheal replacement in a child: a 2-year follow-up study. Lancet. 2012;380:994–1000.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Ott LM, Weatherly RA, Detamore MS. Overview of tracheal tissue engineering: clinical need drives the laboratory approach. Ann Biomed Eng. 2011;39:2091–113.

    Article  PubMed  Google Scholar 

  6. Panseri S, Russo A, Cunha C, Bondi A, Di Martino A, Patella S, et al. Osteochondral tissue engineering approaches for articular cartilage and subchondral bone regeneration. Knee Surg Sports Traumatol Arthrosc. 2012;20:1182–91.

    Article  PubMed  Google Scholar 

  7. Rowland CR, Lennon DP, Caplan AI, Guilak F. The effects of crosslinking of scaffolds engineered from cartilage ECM on the chondrogenic differentiation of MSCs. Biomaterials. 2013;34:5802–12.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Cortijo J, Dixon JS, Foster RW, Small RC. Influence of some variables in the Triton X-100 method of skinning the plasmalemmal membrane from guinea pig trachealis muscle. J Pharmacol Methods. 1987;18:253–66.

    Article  CAS  PubMed  Google Scholar 

  9. Kutten JC, McGovern D, Hobson CM, Luffy SA, Nieponice A, Tobita K, et al. Decellularized tracheal extracellular matrix supports epithelial migration, differentiation, and function. Tissue Eng Part A. 2015;21:75–84.

    Article  CAS  PubMed  Google Scholar 

  10. Wallis JM, Borg ZD, Daly AB, Deng B, Ballif BA, Allen GB, et al. Comparative assessment of detergent-based protocols for mouse lung de-cellularization and re-cellularization. Tissue Eng Part C Methods. 2012;18:420–32.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Evaristo TC, CruzAlves FC, Moroz A, Mion W, Acorci-Valério MJ, Felisbino SL, et al. Light-emitting diode effects on combined decellularization of tracheae. A novel approach to obtain biological scaffolds. Acta Cir Bras. 2014;29:485–92.

    Article  PubMed  Google Scholar 

  12. Peloso A, Ferrario J, Maiga B, Benzoni I, Bianco C, Citro A, et al. Creation and implantation of acellular rat renal ECM-based scaffolds. Organogenesis. 2015;11:58–74.

    Article  PubMed  PubMed Central  Google Scholar 

  13. Den Hondt M, Vanaudenaerde BM, Maughan EF, Butler CR, Crowley C, Verbeken EK, et al. An optimized non-destructive protocol for testing mechanical properties in decellularized rabbit trachea. Acta Biomater. 2017;60:291–301.

    Article  CAS  Google Scholar 

  14. Jungebluth P, Haag JC, Sjöqvist S, Gustafsson Y, Beltrán Rodríguez A, Del Gaudio C, et al. Tracheal tissue engineering in rats. Nat Protoc. 2014;9:2164–79.

    Article  CAS  PubMed  Google Scholar 

  15. Wang Z, Wang Z, Yu Q, Xi H, Weng J, Du X, et al. Comparative study of two perfusion routes with different flow in decellularization to harvest an optimal pulmonary scaffold for recellularization. J Biomed Mater Res A. 2016;104:2567–75.

    Article  CAS  PubMed  Google Scholar 

  16. O’Connor Mooney R, Davis NF, Hoey D, Hogan L, McGloughlin TM, Walsh MT. On the automatic decellularisation of porcine aortae: a repeatability study using a non-enzymatic approach. Cells Tissues Organs. 2016;201:299–318.

    Article  CAS  PubMed  Google Scholar 

  17. Sun F, Pan S, Shi HC, Zhang FB, Zhang WD, Ye G, et al. Structural integrity, immunogenicity and biomechanical evaluation of rabbit decelluarized tracheal matrix. J Biomed Mater Res A. 2015;103:1509–19.

    Article  CAS  PubMed  Google Scholar 

  18. Shan Y, Wang Y, Li J, Shi H, Fan Y, Yang J, et al. Biomechanical properties and cellular biocompatibility of 3D printed tracheal graft. Bioprocess Biosyst Eng. 2017;40:1813–23.

    Article  CAS  PubMed  Google Scholar 

  19. Yang H, Tan Q, Zhao H. Progress in various crosslinking modification for acellular matrix. Chin Med J (Engl). 2014;127:3156–64.

    PubMed  Google Scholar 

  20. Tomizawa Y. Late spontaneous nonanastomotic transgraft hemorrhage from biological material-impregnated fabric vascular graft may be due to autologous tissue detachment: a clinical hypothesis. Artif Organs. 2014;38:1058–60.

    Article  PubMed  Google Scholar 

  21. Pimenta FR, Ferreira MD, Gir E, Hayashida M, Canini SR. Care and specialized clinical follow-up of nursing professionals who have been victims of accidents with biological material. Rev Esc Enferm USP. 2013;47:198–204.

    Article  PubMed  Google Scholar 

  22. Crapo PM, Gilbert TW, Badylak SF. An overview of tissue and whole organ decellularization processes. Biomaterials. 2011;32:3233–43.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Cheng NC, Estes BT, Young TH, Guilak F. Genipin-crosslinked cartilage-derived matrix as a scaffold for human adipose-derived stem cell chondrogenesis. Tissue Eng Part A. 2013;19:484–96.

    Article  CAS  PubMed  Google Scholar 

  24. Tchoukalova YD, Hintze JM, Hayden RE, Lott DG. Tracheal decellularization using a combination of chemical, physical and bioreactor methods. Int J Artif Organs. 2017. https://doi.org/10.5301/ijao.5000648.

    Article  PubMed  Google Scholar 

  25. Seddon AM, Curnow P, Booth PJ. Membrane proteins, lipids and detergents: not just a soap opera. Biochim Biophys Acta. 2004;1666:105–17.

    Article  CAS  PubMed  Google Scholar 

  26. Dong SW, Ying DJ, Duan XJ, Xie Z, Yu ZJ, Zhu CH, et al. Bone regeneration using an acellular extracellular matrix and bone marrow mesenchymal stem cells expressing Cbfa1. Biosci Biotechnol Biochem. 2009;73:2226–33.

    Article  CAS  PubMed  Google Scholar 

  27. Dong X, Wei X, Yi W, Gu C, Kang X, Liu Y, et al. RGD-modified acellular bovine pericardium as a bioprosthetic scaffold for tissue engineering. J Mater Sci Mater Med. 2009;20:2327–36.

    Article  CAS  PubMed  Google Scholar 

  28. Uriel S, Labay E, Francis-Sedlak M, Moya ML, Weichselbaum RR, Ervin N, et al. Extraction and assembly of tissue-derived gels for cell culture and tissue engineering. Tissue Eng Part C Methods. 2009;15:309–21.

    Article  CAS  PubMed  Google Scholar 

  29. Baiguera S, Gonfiotti A, Jaus M, Comin CE, Paglierani M, Del Gaudio C, et al. Development of bioengineered human larynx. Biomaterials. 2011;32:4433–42.

    Article  CAS  PubMed  Google Scholar 

  30. Grandi C, Baiguera S, Martorina F, Lora S, Amistà P, Dalzoppo D, et al. Decellularized bovine reinforced vessels for small-diameter tissue-engineered vascular grafts. Int J Mol Med. 2011;28:315–25.

    CAS  PubMed  Google Scholar 

  31. Zang M, Zhang Q, Chang EI, Mathur AB, Yu P. Decellularized tracheal matrix scaffold for tissue engineering. Plast Reconstr Surg. 2012;130:532–40.

    Article  CAS  PubMed  Google Scholar 

  32. Jungebluth P, Bader A, Baiguera S, Möller S, Jaus M, Lim ML, et al. The concept of in vivo airway tissue engineering. Biomaterials. 2012;33:4319–26.

    Article  CAS  PubMed  Google Scholar 

  33. Langmaier F, Mládek M, Mokrejš P. Hydrogels of collagen hydrolysate cross-linked with dialdehyde starch. J Therm Anal Calorim. 2009;98:807.

    Article  CAS  Google Scholar 

  34. Wang GF, Wu SY, Rao JJ, Lü L, Xu W, Pang JX, et al. Genipin inhibits endothelial exocytosis via nitric oxide in cultured human umbilical vein endothelial cells. Acta Pharmacol Sin. 2009;30:589–96.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Yamazaki M, Chiba K. Genipin exhibits neurotrophic effects through a common signaling pathway in nitric oxide synthase-expressing cells. Eur J Pharmacol. 2008;581:255–61.

    Article  CAS  PubMed  Google Scholar 

  36. Baiguera S, Del Gaudio C, Kuevda E, Gonfiotti A, Bianco A, Macchiarini P. Dynamic decellularization and cross-linking of rat tracheal matrix. Biomaterials. 2014;35:6344–50.

    Article  CAS  PubMed  Google Scholar 

  37. Sung HW, Huang RN, Huang LL, Tsai CC, Chiu CT. Feasibility study of a natural crosslinking reagent for biological tissue ixation. J Biomed Mater Res. 1998;42:560–7.

    Article  CAS  PubMed  Google Scholar 

  38. Wang M, Da L, Xie Y, Xie H. Application of genipin for modification of natural biomaterials as a crosslinking agent. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2013;27:580–5.

    CAS  PubMed  Google Scholar 

  39. Haugh MG, Murphy CM, McKiernan RC, Altenbuchner C, O’Brien FJ. Crosslinking and mechanical properties significantly influence cell attachment, proliferation, and migration within collagen glycosaminoglycan scaffolds. Tissue Eng Part A. 2011;17:1201–8.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This study was supported by the National Natural Science Foundation of China (No. 81770018), the Foundation of Taizhou People’s Hospital (No. ZL201830) and the Jiangsu Students’ Innovation Training Program (No. 201811117035Z).

Author information

Authors and Affiliations

  1. Medical College of Yangzhou University, 11 Huaihai Road, Yangzhou, 225001, Jiangsu Province, China

    Yi Zhong, Ai Jiang, Yuanfan Xiao, Ying Gu, Lei Wu, Yujie Zhang & Hongcan Shi

  2. Center of Translational Medicine, Yangzhou University, 11 Huaihai Road, Yangzhou, 225001, Jiangsu Province, China

    Yi Zhong, Ai Jiang, Yuanfan Xiao, Ying Gu, Lei Wu, Yujie Zhang & Hongcan Shi

  3. Peking University Third Hospital, 49 Huayuan Road, Haidian District, Beijing, 100191, China

    Ai Jiang

  4. Taizhou People’s Hospital, 366 Taihu Road, Taizhou, 225300, Jiangsu Province, China

    Fei Sun

Authors
  1. Yi Zhong
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ai Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Fei Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yuanfan Xiao
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ying Gu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Lei Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yujie Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Hongcan Shi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hongcan Shi.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical statement

All animal experiments were approved in compliance with the rules and regulations of Laboratory Animals formulated by the National Ministry of Science (IACUC no. SYXK2016-0041).

Additional information

Yi Zhong and Ai Jiang contributed equally to this work.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhong, Y., Jiang, A., Sun, F. et al. A Comparative Study of the Effects of Different Decellularization Methods and Genipin-Cross-Linking on the Properties of Tracheal Matrices. Tissue Eng Regen Med 16, 39–50 (2019). https://doi.org/10.1007/s13770-018-0170-6

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13770-018-0170-6

Keywords

Search

Navigation