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Flexor Tendon Tissue Engineering: Acellularization of Human Flexor Tendons Reduces Immunogenicity in-vivo
Shyam S. Raghavan; Colin Woon; Armin Kraus; Kai Megerle; Matthew Choi; Brian Pridgen; Hung Pham; James Chang, MD
Plastic & Reconstructive Surgery, Stanford University, Stanford, CA

Background: In mutilating hand injuries, tissue engineered tendon grafts may provide a reconstructive solution.  We have previously described a method to decellularize cadaveric human flexor tendons while preserving mechanical properties and biocompatibility.  The purpose of this study is to evaluate the immunogenicity and strength of these grafts when implanted into an immunocompetent rat model. 

Methods: Human flexor tendons were decellularized with sodium dodecyl sulfate (SDS), ethylenediaminetetracetic  acid (EDTA), and peractetic acid (PAA) as previously described.  Both untreated and treated tendons were then analyzed for the presence of major-histocompatability complexs (MHC-1) via immunohistochemistry (IHC). Fresh frozen and acellular tendons (pair-matched) were then placed into the dorsal subcutaneous tissue and anchored to the spinal ligaments of Wistar rats for 2 or 4 weeks, and then harvested.  The infiltration of B-cells and macrophages was determined using IHC.  The explants where then subjected to mechanical testing to determine the ultimate tensile strength (UTS) and elastic modulus (EM).  Statistical analysis was performed using a paired student’s t-test. 

Results: The decellularization protocol successfully removed cells and MHC-1 complexes from fresh human cadaveric flexor tendon.  At two weeks after implantation, there was increased infiltration of B-cells in the fresh frozen group compared to the acellular group, both in the capsule and deep into the tendon.  There was no appreciable difference in macrophage infiltration.  There was improved UTS and EM in the acellular group (42.7±8.3 vs. 22.8±7.8 MPa and 830.2±206.7 vs. 421.2±171.3 MPa, respectively; p<0.05).  At four weeks, there was continued B-cell infiltration in the fresh frozen group compared to the acellular group, and no appreciable difference in macrophage infiltration was seen.  The acellular group continued to have improved UTS and EM at 4 weeks (40.5±9.1 vs. 14.6±4.2 MPa, and 454.05±101.5 vs. 204.6±91.3 MPa, respectively; p<0.05).

Conclusions: Human flexor tendons that were decellularized with SDS, EDTA, and PAA resulted in removal of cellular antigens and resulted in a decreased immune response when placed into Wistar rats.  These grafts showed improved mechanical properties at 2 and 4 weeks.  Decellularization is an important step towards the use of tissue engineered flexor tendons in upper extremity reconstruction.

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