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The porcine superflexor tendon (SFT) was identified as having appropriate structure and properties for development of a decellularized device for use in anterior cruciate ligament reconstruction. SFTs were decellularized using a combination of freeze–thaw and washes in hypotonic buffer and 0.1% (w/v) sodium dodecyl sulfate in hypotonic buffer plus proteinase inhibitors, followed by nuclease treatment and sterilization using peracetic acid. The decellularized biological scaffold was devoid of cells and cell remnants and contained only 13 ng/mg (dry weight) residual total DNA. Immunohistochemistry showed retention of collagen type I and III and tenascin-C. Quantitative analysis of sulfated sugar and hydroxyproline content revealed a loss of glycosaminoglycans compared with native tissue, but no loss of collagen. The decellularized SFT was biocompatible in vitro and in vivo following implantation in a mouse subcutaneous model for 12 weeks. Uniaxial tensile testing to failure indicated that the gross material properties of decellularized SFTs were not significantly different to native tissue. Decellularized SFTs had an ultimate tensile strength of 61.8 ± 10.3 MPa (±95% confidence limits), a failure strain of 0.29 ± 0.04, and a Young's modulus of the collagen phase of 294.1 ± 61.9 MPa. Analysis of the presence of the α-Gal (galactose-α-1,3-galactose) epitope by immunohistochemistry, lectin binding, and antibody absorption assay indicated that the epitope was reduced, but still present post decellularization. This is discussed in light of the potential role of noncellular α-Gal in the acceleration of wound healing and tissue regeneration in the presence of antibodies to α-Gal.

The decellularisation of xenogenic and allogeneic biological grafts offers a promising solution to replacement of the anterior cruciate ligament (ACL). The purpose of this investigation was to determine the biomechanical effects of additional fat reduction and bioburden reduction steps in the decellularisation of porcine super flexor tendon (pSFT). Study 1 investigated the use of acetone or chloroform–methanol as a fat reduction agent. The most effective of these was then carried forward into Study 2, which investigated the use of antibiotics or peracetic acid (PAA) as a bioburden reduction agent. Stress relaxation data was analysed using a Maxwell–Wiechert viscoelastic model and, in addition to classical material properties, the tangent modulus of the toe-region was determined from strength testing data. In both studies, the majority of decellularised groups demonstrated no statistical differences for material properties such as tensile strength and Young’s modulus compared to native controls. Different trends were observed for many of the viscoelastic parameters, but also for the tangent modulus in the toe-region indicating a change in performance at low strains. The most severe deviations from the profile of the native tangent modulus were found to occur in Study 2 when PAA was used for bioburden reduction. Classic material properties (E, UTS etc.) are often used to compare the characteristics of native and decellularised tissues, however they may not highlight changes occurring in the tissues at low strains. In this study, this represented the physiological strains encountered by substitute acellular ACL grafts. Acetone was chosen as the fat reduction step whereas, antibiotics was preferable over PAA as a bioburden reduction step.

Acellular xenogeneic tissues have the potential to provide ‘off‐the‐shelf’ grafts for anterior cruciate ligament (ACL) repair. To ensure that such grafts are sterile following packaging, it is desirable to use terminal sterilization methods. Here, the effects of gamma and electron beam irradiation on the biological and biomechanical properties of a previously developed acellular porcine superflexor tendon (pSFT) were investigated. Irradiation following treatment with peracetic acid was compared to peracetic acid treatment alone and the stability of grafts following long‐term storage assessed. Irradiation did not affect total collagen content or biocompatibility (determined using a contact cytotoxicity assay) of the grafts, but slightly increased the amount of denatured collagen in and decreased the thermal denaturation temperature of the tissue in a dose dependant fashion. Biomechanical properties of the grafts were altered by irradiation (reduced ultimate tensile strength and Young's modulus, increased failure strain), but remained superior to reported properties of the native human ACL. Long term storage at 4°C had no negative effects on the grafts. Of all the conditions tested, a dose of minimum 25 kGy of gamma irradiation had least effect on the grafts, suggesting that this dose produces a biocompatible pSFT graft with adequate mechanical properties for ACL repair. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2477–2486, 2017.