Tissue-engineered intrasynovial tendons: Optimization of acellularization and seeding

Abstract

The purpose of this research was to develop a tissueengineered intrasynovial flexor tendon construct with the use of an acellularized flexor tendon scaffold repopulated with intrasynovial tendon cells. New Zealand white rabbit intrasynovial flexor tendons were acellularized by the following methods: high concentration NaCl + SDS, Trypsin/EDTA, Trypsin/EDTA + Triton X-100, Triton X-100, Triton X-100 + SDS, and freezing at-70 °C followed by Trypsin/EDTA + Triton X-100. Epitenon and endotenon cells were also isolated from rabbit intrasynovial tendons and expanded in culture. Acellularized tendon scaffolds were then reseeded with these cells. A subset of epitenon and endotenon cells was labeled with green and red fluorescent markers, respectively, to further characterize the preferred location of their attachment. Optimal acellularization was achieved by freezing at-70 °C followed by Trypsin/EDTA + Triton X-100. After reseeding, light microscopy of tendon constructs showed attachment of both epitenon and endotenon to the tendon scaffolds, with endotenon cells more likely to be found in the core of the scaffold. An intrasynovial tendon construct was developed with the use of acellularized intrasynovial tendons repopulated with intrasynovial tenocytes. These constructs grossly resemble normal intrasynovial tendons, and cells were found both on the surface and the core of the construct histologically. This new construct represents an important first step in developing a viable tissue-engineered flexor tendon.

Human Flexor Tendon Tissue Engineering: Decellularization of Human Flexor Tendons Reduces Immunogenicity In Vivo

“…We have subjected constructs to mechanical stimulation using a bioreactor [6][7][8][9] to improve strength and stiffness, and reseeded them with fibroblasts, tenocytes, and adipoderived stem cells. 1,[10][11][12] Although we have shown decellularization with hematoxylin and eosin (H&E) staining of treated tendon, we have yet to prove that this process eliminates major histocompatibility complexes and is nonimmunogenic in vivo. The purpose of this study is to verify that these scaffolds are nonimmunogenic and are biocompatible when implanted into an immunocompetent rat model.…”

“…Reconstruction of minor flexor tendon defects is usually treated with autografts. 1,2 However, in mutilating injuries that lead to significant damage, this approach may not be suitable as these autografts are in limited supply. Our research group has studied tissue engineering of flexor tendons to create a possible solution to this problem.…”

Advances in injured tendon engineering with emphasis on the role of collagen implants

“…However, homogenous cell infiltration by mononuclear, elongated, fibroblast-like host cells was observed at the periphery as well as within the inner matrix of the implanted scaffolds at all time points 43 . Zhang et al 44 tried different methods of acellularization to produce tissueengineered intra-synovial flexor tendon construct with the use of an acellularized flexor tendon scaffold re-populated with intra-synovial tendon cells. The intra-synovial flexor tendons of rabbits were acellularized by high concentration NaCl+SDS, Trypsin/EDTA, Trypsin/ EDTA+Triton X-100, Triton X-100, Triton X-100+SDS, and freezing at −70 °C followed by application of Trypsin/ EDTA+Triton X-100.…”

“…The acellularized tendon scaffolds were then reseeded with these cells. An optimal acellularization was achieved by freezing at −70 °C followed by trypsin/EDTA+Triton X-100 44 …”

Studies in Flexor Tendon Reconstruction: Biomolecular Modulation of Tendon Repair and Tissue Engineering

“…53 This is a clinically useful scaffold because large quantities of human cadaver flexor tendon can be harvested, processed, and stored. In addition, it is hypothesized that tenocytes will more readily repopulate, and mesenchymal stem cells will more readily differentiate, onto acellularized tendon because this scaffold retains the normal collagen architecture of tendon.…”