Decellularization of Large Tendon Specimens: Combination of Manually Performed Freeze-Thaw Cycles and Detergent Treatment.
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.
Publication Date: 2017-07-14 PubMed ID: 28702884DOI: 10.1007/7651_2017_49Google Scholar: Lookup
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- Journal Article
Summary
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This study presents an efficient method for the decellularization of large tendon samples, using a combination of freeze-thaw cycles and treatment with a non-ionic detergent. The technique aims to maintain as much of the original extracellular matrix architecture of the tendon as possible, which is crucial for tissue engineering and graft-based tendon repair.
Introduction to Tendon Decellularization
- Decellularization techniques are important in tissue engineering, especially in the development of tendon scaffolds. These scaffolds can be used in in vitro research and to create grafts for tendon repair operations.
- One of the challenges in tendon decellularization is preserving the extracellular matrix (ECM) architecture. This provides the natural characteristics of the tendon tissue, which is vital for effective grafting and for in vitro studies of cell behavior.
- In addition to ensuring the preservation of ECM, decellularization techniques aim to reduce the presence of resident cells while also ensuring that the scaffold is compatible with other cells, known as cytocompatibility.
Methodology of the Study
- Various protocols exist for tendon decellularization, mainly involving physical treatment or chemical/enzyme-based treatment. However, there are few evidence-based protocols dealing with full-thickness large tendon samples, such as equine flexor tendons.
- This study presents a method that combines physical and chemical decellularization to treat equine superficial digital flexor tendons. The physical treatment involves repeated freeze-thaw cycles, while the chemical treatment makes use of the non-ionic detergent Triton X-100.
Evaluation of Decellularization Effectiveness
- The effectiveness of this decellularization procedure was assessed by measuring the reduction in cell and DNA content. The extent to which the ECM morphology was maintained and the cytocompatibility of the resulting tendon scaffolds were also investigated.
- Based on a previous study, which formed the basis of this protocol, the authors claim this method to be particularly effective for the decellularization of large tendon samples.
Cite This Article
APA
Roth SP, Erbe I, Burk J.
(2017).
Decellularization of Large Tendon Specimens: Combination of Manually Performed Freeze-Thaw Cycles and Detergent Treatment.
Methods Mol Biol, 1577, 227-237.
https://doi.org/10.1007/7651_2017_49 Publication
Researcher Affiliations
- Faculty of Veterinary Medicine, Equine Clinic and Hospital Leipzig, Universität Leipzig, An den Tierkliniken 21, Leipzig, 04103, Germany. susanne.roth@uni-leipzig.de.
- Saxonian Incubator for Clinical Translation, Universität Leipzig, Philipp Rosenthal Straße 55, Leipzig, 04103, Germany. susanne.roth@uni-leipzig.de.
- Faculty of Veterinary Medicine, Equine Clinic and Hospital Leipzig, Universität Leipzig, An den Tierkliniken 21, Leipzig, 04103, Germany.
- Faculty of Veterinary Medicine, Equine Clinic and Hospital Leipzig, Universität Leipzig, An den Tierkliniken 21, Leipzig, 04103, Germany.
- Faculty of Veterinary Medicine, Institute of Veterinary Physiology, Universität Leipzig, An den Tierkliniken 7, Leipzig, 04103, Germany.
MeSH Terms
- Animals
- Buffers
- Detergents / chemistry
- Extracellular Matrix / chemistry
- Freezing
- Horses
- Hypotonic Solutions / chemistry
- Octoxynol / chemistry
- Tendons / chemistry
- Tendons / cytology
- Tissue Engineering / methods
- Tissue Scaffolds / chemistry
Citations
This article has been cited 12 times.- Jin Y, Sun Q, Ma R, Li R, Qiao R, Li J, Wang L, Hu Y. The trend of allogeneic tendon decellularization: literature review. Cell Tissue Bank 2023 Jun 24;.
- Khorsandi L, Orazizadeh M, Bijan Nejad D, Heidari Moghadam A, Nejaddehbashi F, Asadi Fard Y. Spleen extracellular matrix provides a supportive microenvironment for β-cell function. Iran J Basic Med Sci 2022 Sep;25(9):1159-1165.
- Moffat D, Ye K, Jin S. Decellularization for the retention of tissue niches. J Tissue Eng 2022 Jan-Dec;13:20417314221101151.
- Schubert S, Brandt L, Burk J. A 3D Dynamic In Vitro Model of Inflammatory Tendon Disease. Methods Mol Biol 2021;2269:167-174.
- Roth SP, Brehm W, Groß C, Scheibe P, Schubert S, Burk J. Transforming Growth Factor Beta 3-Loaded Decellularized Equine Tendon Matrix for Orthopedic Tissue Engineering. Int J Mol Sci 2019 Nov 3;20(21).
- Aeberhard PA, Grognuz A, Peneveyre C, McCallin S, Hirt-Burri N, Antons J, Pioletti D, Raffoul W, Applegate LA. Efficient decellularization of equine tendon with preserved biomechanical properties and cytocompatibility for human tendon surgery indications. Artif Organs 2020 Apr;44(4):E161-E171.
- Dzobo K, Motaung KSCM, Adesida A. Recent Trends in Decellularized Extracellular Matrix Bioinks for 3D Printing: An Updated Review. Int J Mol Sci 2019 Sep 18;20(18).
- Brandt L, Schubert S, Scheibe P, Brehm W, Franzen J, Gross C, Burk J. Tenogenic Properties of Mesenchymal Progenitor Cells Are Compromised in an Inflammatory Environment. Int J Mol Sci 2018 Aug 28;19(9).
- Li J, Wen M, Zhang S, Du L, Fan X, Liang H, Wang H, Sun J, Ding Y, Ge L, Ma J, Zhang J. Research Progress on the Preparation and Application of Decellularized Tendons. Curr Issues Mol Biol 2025 Apr 6;47(4).
- Nelson AL, O'Hara KM, Nolte PC, Fukase N, Murata Y, Nolte AK, Huard J, Bernholt DL, Millett PJ, Bahney CS. Engineered Decellularized Tendon Matrix Putty Preserves Native Tendon Bioactivity to Promote Cell Proliferation and Enthesis Repair. J Tissue Eng Regen Med 2023;2023:4665795.
- Zhong S, Lan Y, Liu J, Seng Tam M, Hou Z, Zheng Q, Fu S, Bao D. Advances focusing on the application of decellularization methods in tendon-bone healing. J Adv Res 2025 Jan;67:361-372.
- Data K, Kulus M, Ziemak H, Chwarzyński M, Piotrowska-Kempisty H, Bukowska D, Antosik P, Mozdziak P, Kempisty B. Decellularization of Dense Regular Connective Tissue-Cellular and Molecular Modification with Applications in Regenerative Medicine. Cells 2023 Sep 16;12(18).
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