A biomechanical characterisation of acellular porcine super flexor tendons for use in anterior cruciate ligament replacement: Investigation into the effects of fat reduction and bioburden reduction bioprocesses

Abstract

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.

The effects of irradiation on the biological and biomechanical properties of an acellular porcine superflexor tendon graft for cruciate ligament repair

“…The tendon was cleaned of connective tissue in situ and dissected from the foot. One branch of the isolated pSFT was removed before storage at −80°C on filter article moistened with phosphate buffered saline (PBS; Oxoid).pSFT were decellularized in batches of 12–24 following a previously established method . Briefly, the frozen pSFTs were thawed and then subjected to two freeze thaw cycles (−80°C) in hypotonic buffer (10 m M tris, 2.7 m M EDTA, 10 KIU mL −1 aprotinin [Mayfair House, Leeds, UK], pH 8.0).…”

“…One branch of the isolated pSFT was removed before storage at 2808C on filter article moistened with phosphate buffered saline (PBS; Oxoid).pSFT were decellularized in batches of 12-24 following a previously established method. 18 Briefly, the frozen pSFTs were thawed and then subjected to two freeze thaw cycles (2808C) in hypotonic buffer (10 mM tris, 2.7 mM EDTA, 10 KIU mL 21 aprotinin [Mayfair House, Leeds, UK], pH 8.0). Tendons were then washed in acetone (50 mL) three times for 1 h at 428C with agitation (120 rpm, PSU-10i Orbital shaking platform, Grant Instruments, UK) to remove fat before rehydration in five changes of PBS (50 mL).…”

“…The process uses low concentration sodium dodecyl sulphate (SDS) and proteinase inhibitors and has been successfully applied to cardiovascular tissues, musculoskeletal tissues, and dermis, with evidence of the mechanisms of action and clinical utility of this approach now emerging . The process has been adopted to the development of a decellularized porcine superflexor tendon (pSFT) . The resultant biological scaffold has been shown to possess the mechanical properties necessary for deployment as an “off the shelf” alternative to current methods of ACL reconstruction.…”

“…16,17 The process has been adopted to the development of a decellularized porcine superflexor tendon (pSFT). 18 The resultant biological scaffold has been shown to possess the mechanical properties necessary for deployment as an "off the shelf" alternative to current methods of ACL reconstruction.…”

The effects of irradiation dose and storage time following treatment on the viscoelastic properties of decellularised porcine super flexor tendon

“…Decellularisation was achieved using a 0.1% w/v SDS (sodium dodecyl sulphate) process refined to incorporate bioprocesses including bioburden reduction, fat reduction and terminal chemical sterilisation using 0.1% w/v peracetic acid ( Jones et al, 2016 ), with least disruption to the biomechanical properties ( Herbert et al, 2015 ). Following successful completion of the decellularisation process, tendons were transferred aseptically to foil packaging and stored at −80 °C.…”