pp 1-11 | Cite as

Decellularization of Large Tendon Specimens: Combination of Manually Performed Freeze-Thaw Cycles and Detergent Treatment

Protocol
Part of the Methods in Molecular Biology book series

Abstract

Reliable decellularization techniques applicable to tendon tissue play a critical role in the field of current tissue engineering. Particularly, an application as three-dimensional culture model for in vitro research and translational approaches to establish graft-based tendon repair as a routine clinical tool represent two main application fields for decellularized tendon scaffolds. Considering methodological issues of tendon decellularization, one of the major challenges lies in the preservation of the tendon-specific extracellular matrix (ECM) architecture to reflect natural tissue characteristic as best as possible. Concurrently, further requirements for high-quality decellularized biological tendon scaffolds include not only the reduction of resident cells, but also an ensured cytocompatibility.

To date, a large number and a wide variety of decellularization protocols for natural tendon tissue have already been investigated and usually, physical as well as chemical and/or enzyme-based treatments are used for the purpose of decellularization. However, to the best of our knowledge, there is a lack of evidence-based protocols for the processing of full-thickness large tendon samples, such as the equine flexor tendons.

Therefore, the here presented protocol describes a reliable procedure to decellularize equine superficial digital flexor tendons by using a combined treatment of physical decellularization in the form of repetitive freeze-thaw cycles, and of chemical decellularization with the non-ionic detergent Triton X-100. The decellularization effectiveness evaluated by reduction of cell and DNA content, the influence of decellularization on the morphology of the tendon extracellular matrix (ECM) as well as the cytocompatibility of the decellularized tendon scaffolds obtained have been investigated previously. Based on this previous study, the here present protocol is an effective procedure, particularly applicable for large tendon specimens.

Keywords:

Regenerative medicine Tissue engineering Decellularization Scaffold Freeze-thaw cycles Triton X-100 Detergent treatment Tendon Horse 

References

  1. 1.
    Cheng CW, Solorio LD, Alsberg E (2014) Decellularized tissue and cell-derived extracellular matrices as scaffolds for orthopaedic tissue engineering. Biotechnol Adv 32(2):462–484CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Youngstrom DW, Barrett JG (2016) Engineering tendon: scaffolds, bioreactors, and models of regeneration. Stem Cells Int 2016:3919030CrossRefPubMedGoogle Scholar
  3. 3.
    Lovati AB, Bottagisio M, Moretti M (2016) Decellularized and engineered tendons as biological substitutes: a critical review. Stem Cells Int 2016:7276150CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Schulze-Tanzil G, Al-Sadi O, Ertel W, Lohan A (2012) Decellularized tendon extracellular matrix—a valuable approach for tendon reconstruction? Cell 1(4):1010–1028CrossRefGoogle Scholar
  5. 5.
    Burk J, Erbe I, Berner D, Kacza J, Kasper C, Pfeiffer B et al (2014) Freeze-thaw cycles enhance decellularization of large tendons. Tissue Eng Part C Methods 20(4):276–284CrossRefPubMedGoogle Scholar
  6. 6.
    Youngstrom DW, Barrett JG, Jose RR, Kaplan DL (2013) Functional characterization of detergent-decellularized equine tendon extracellular matrix for tissue engineering applications. PLoS One 8(5):e64151ADSCrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Bottagisio M, Pellegata AF, Boschetti F, Ferroni M, Moretti M, Lovati AB (2016) A new strategy for the decellularisation of large equine tendons as biocompatible tendon substitutes. Eur Cell Mater 32:58–73CrossRefPubMedGoogle Scholar
  8. 8.
    Dowling BA, Dart AJ (2005) Mechanical and functional properties of the equine superficial digital flexor tendon. Vet J 170(2):184–192CrossRefPubMedGoogle Scholar
  9. 9.
    Thorpe CT, Clegg PD, Birch HL (2010) A review of tendon injury: why is the equine superficial digital flexor tendon most at risk? Equine Vet J 42(2):174–180CrossRefPubMedGoogle Scholar
  10. 10.
    Patterson-Kane JC, Rich T (2014) Achilles tendon injuries in elite athletes: lessons in pathophysiology from their equine counterparts. ILAR J 55(1):86–99CrossRefPubMedGoogle Scholar
  11. 11.
    Roth SP, Glauche SM, Plenge A, Erbe I, Heller S, Burk J (2017) Automated freeze-thaw cycles for decellularization of tendon tissue—a pilot study. BMC Biotechnol 17(1):13CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Gilbert TW, Sellaro TL, Badylak SF (2006) Decellularization of tissues and organs. Biomaterials 27(19):3675–3683PubMedGoogle Scholar
  13. 13.
    Azuma C, Tohyama H, Nakamura H, Kanaya F, Yasuda K (2007) Antibody neutralization of TGF-β enhances the deterioration of collagen fascicles in a tissue-cultured tendon matrix with ex vivo fibroblast infiltration. J Biomech 40(10):2184–2190CrossRefPubMedGoogle Scholar
  14. 14.
    Omae H, Zhao C, Sun YL, An K-N, Amadio PC (2009) Multilayer tendon slices seeded with bone marrow stromal cells: a novel composite for tendon engineering. J Orthop Res 27(7):937–942CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Stewart AA, Barrett JG, Byron CR, Yates AC, Durgam SS, Evans RB et al (2009) Comparison of equine tendon-, muscle-, and bone marrow-derived cells cultured on tendon matrix. Am J Vet Res 70(6):750–757CrossRefPubMedGoogle Scholar
  16. 16.
    Gage AA, Baust J (1998) Mechanisms of tissue injury in cryosurgery. Cryobiology 37(3):171–186CrossRefPubMedGoogle Scholar
  17. 17.
    Crapo PM, Gilbert TW, Badylak SF (2011) An overview of tissue and whole organ decellularization processes. Biomaterials 32(12):3233–3243CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Gilbert TW (2012) Strategies for tissue and organ decellularization. J Cell Biochem 113(7):2217–2222CrossRefPubMedGoogle Scholar
  19. 19.
    Smeak DD, Olmstead ML (1984) Infections in clean wounds: the roles of the surgeon, environment, and host. The compendium on continuing education for the practicing veterinarian. Comp Cont Educ 6:626Google Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Susanne Pauline Roth
    • 1
    • 2
  • Ina Erbe
    • 1
  • Janina Burk
    • 1
    • 3
  1. 1.Faculty of Veterinary Medicine, Equine Clinic and Hospital LeipzigUniversität LeipzigLeipzigGermany
  2. 2.Saxonian Incubator for Clinical TranslationUniversität LeipzigLeipzigGermany
  3. 3.Faculty of Veterinary Medicine, Institute of Veterinary PhysiologyUniversität LeipzigLeipzigGermany

Personalised recommendations