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.

Efficient decellularization of equine tendon with preserved biomechanical properties and cytocompatibility for human tendon surgery indications

“…The use of NaCl has the advantage of easy leeching from tissues during the washing steps with less residual product remaining in the matrix that could be a cause for cellular toxicity, as potentiated with the use of strong detergents. The ability to automate F/T decellularization has recently been documented, 29 which, combined with the use of biocompatible GMP-grade human cellular therapies presented here, further supports the development of decellularized SDFT as a future clinical solution for human tendon surgery indications.…”

“…The addition of hFPTs for tendon recellularization would be a potential way to improve tendon matrices for grafting, as hFTPs would be progressively replaced by host tissue after surgical placement, leading to an improved integration and better healing response . To date, studies of reseeding SDFT have used equine adipose‐derived mesenchymal stromal cells, which are a promising cellular therapy for veterinary medicine, but would be not suitable for human cellular therapy. Human fetal cells such as hFTPs are not only able to stimulate healing, but they are also highly proliferative and can be used to create cell banks for a consistent, safe clinical therapy .…”

“…The second decellularization protocol consisted of combined F/T with neutral solutions of hypertonic NaCl 1M (Sigma‐Aldrich), hypotonic ddH2O, or Triton 1%. A modified version of F/T decellularization described by Roth et al was used, where samples were submitted to five cycles of 2‐minute freezing in liquid nitrogen and 10 minutes at 37°C in 1X phosphate‐buffered saline (PBS; Interlaken, Switzerland). Samples were then incubated 48 hours at RT in ddH 2 O followed by 48 hours of incubation in NaCl 1M or ddH2O.…”

Trends in Decellularized Extracellular Matrix Bioinks for 3D Printing: Inspiring a new Kind of Medicine

“…Triton-X-100 is known to damage the ultrastructure of the ECM in addition to removal of some glycosaminoglycans (GAGs) [62,66]. Triton X-100 was shown to leave large quantities of cellular material when used for decellularizing the tendon [65,67,68]. Ammonium hydroxide is one common method used for cellular removal, though it is known to induce some damage to ECM collagens [69][70][71][72].…”

Tenogenic Properties of Mesenchymal Progenitor Cells Are Compromised in an Inflammatory Environment

“…Equine distal limbs were collected at an abattoir, and healthy superficial digital flexor tendons were excised from the metacarpal regions aseptically. Tendons were then subjected to decellularization by combining freeze-thaw cycles and treatment with Triton X-100 (Carl Roth GmbH & Co. KG, Karlsruhe, Germany) as described and evaluated previously [ 49 , 50 ]. 300 µm-thick tendon extracellular matrix scaffolds were then prepared by sectioning the tendons longitudinally using a cryostat (CM 3050 S, Leica Biosystems, Wetzlar, Germany).…”