Stone et al. studied the possible elimination of α-Gal epitopes from xenograft cartilage by the use of recombinant α-galactosidase [3] (Fig. 9.2). This enzyme cleaves terminal galactosyl unit from the α-Gal epitope (Gal α1-3Galβ1-4GlcNAc-R) into the carbohydrate structure Galβ1-4GlcNAc-R which is also present on human cells and cannot bind the anti-Gal antibody. Porcine meniscus and articular cartilage specimens treated for 12 h with recombinant α-galactosidase were confirmed to completely lack this epitope. This was demonstrated by the inability of a monoclonal anti-Gal antibody to bind to these treated specimens in comparison to the extensive binding of the antibody to untreated specimens. The α-galactosidase treated cartilage specimens were implanted into the suprapatellar pouch of cynomolgus monkeys, and the immune response to cartilage was monitored by serum evaluation and histology 2 months post-implantation.

The results of this study demonstrated no significant increase in anti-Gal activity after enzymatic elimination of α-Gal epitopes from the treated grafts. The inflammatory response within the α-galactosidase treated xenografts was lower by ~95% than that in untreated cartilage, and the proportion of T lymphocytes within the cellular infiltrates was greatly reduced [3]. However, the removal of α-Gal epitopes did not eliminate the immune response to non-Gal antigens. Most monkeys produced anti-non-Gal antibodies to non-Gal antigens in porcine cartilage. The reason for this anti-non-Gal antibody response is that most porcine proteins differ from homologous proteins in monkeys (as well as in humans); therefore, they elicit antibody response against differences. The production of anti-non-Gal antibodies resulted in induction of a reduced inflammatory response consisting primarily of macrophages infiltrating into the cartilage. These macrophages bind via their Fc receptors to anti-non-Gal antibodies immunocomplexed with the immunogenic porcine cartilage proteins and are likely to cause destruction of the xenograft, albeit at a pace that is much slower than the destruction in the presence of α-Gal epitopes. The anti-non-Gal-mediated destruction of orthopedic xenografts led to developing methods to attenuate this immune response, increasing the clinical utility for using xenograft devices.

The anterior cruciate ligament (ACL) is the key stabilizer of the knee joint and is frequently injured in athletic activities. Over 350,000 patients with damaged ACLs undergo in-patient or out-patient surgical intervention in the US each year (38). Current surgical techniques consist of either the use of the patient’s own tissue to reconstruct the ACL (autologous harvest procedures) or, less frequently, cadaveric tissue grafts (allografts). Grafts used to reconstruct the ACL include constructs of bone-tendon-bone, bone-tendon, and soft tissue tendon. All grafting techniques have disadvantages and risks. Reconstruction utilizing an autologous harvest procedure involves two surgical sites, the primary operative site and the additional harvest site. The harvest procedure for reconstruction often results in larger or additional incisions, increased pain, longer recovery periods, and increased morbidity (5, 16, 17). Adverse effects from the harvest procedure may include patellar fracture, patellar tendon rupture with scar formation, and muscle weakness (2, 22, 35). Cadaveric tissue allografts offer a limited source of ACL replacement tissue due to the scarcity of available tissue from young healthy donors. Variability in tissue quality and performance is also an issue between donors. The risk of transmission of adventitious disease has been another obstacle to the acceptance of cadaveric tissue (25).

Clinical ACL reconstruction (ACLR) with synthetic and nonhuman tissue-based devices has led to failure due to a range of factors including material property mismatch, fatigue, abrasion, particulate shedding, poor fixation, anatomical placement, and immunologic rejection (6, 10, 19, 29, 36.) The cause of immunologic rejection when transplanting animal tissues into humans was identified in multiple studies as a reaction to the carbohydrate antigen called “α-Gal epitope” present in high concentration on animal tissues but completely absent in humans (1, 13, 32, 37). In contrast, humans, apes, and Old World monkeys (monkeys of Asia and Africa) produce large amounts of a natural antibody called “anti-Gal” that binds effectively to α-Gal epitopes (14). Stone and co-workers demonstrated the utility of treating porcine tissues with the glycosidase enzyme, α-galactosidase, effectively attenuating host to graft immune recognition by α-Gal epitope cleavage (3, 13, 24, 31). The objective of these investigations was to develop an immunocompatible, dynamic bio-implant xenograft for ACL reconstruction with characteristics matching homologous human tissue.

A focused effort was undertaken to develop a xenograft ligament device from a section of the porcine patellar tendon with bone blocks and based on the immunogenicity mechanisms learned from the previous series of primate studies. Grafts were sourced from a porcine stifle and processed into a bone-tendon-bone configuration (BTB). The graft was exposed to a series of chemical treatments. Intially, a decellularization treatment to remove intact porcine cells and cellular components was applied, followed by exposure to recombinant α-galactosidase enzyme solution to cleave α-Gal epitopes from the graft. Removal of these α-Gal epitopes was confirmed by ELISA testing with a monoclonal anti-Gal antibody that demonstrated that essentially 100% of these epitopes were removed from the tendon portion of the construct. Following enzyme treatment, low-level glutaraldehyde cross-linking treatment was employed, with the intention of attenuating the anti-non-Gal mediated destruction by the host immune system. We determined empirically incubating tendons in low-level glutaraldehyde followed by glycine quench of aldehyde yields optimal conditions for macrophage infiltration allowing for the gradual remodeling of implants.

The final stage of treatment included packaging and exposure of the hydrated graft to 17.8 kGy of e-Beam irradiation, a low level of irradiation intended to provide graft sterility while minimizing the degradative effects of radiation.

In order to mechanically characterize treated and sterilized porcine grafts, clinically relevant controls for comparative biomechanical evaluation using standardized static testing methods. The two test groups included treated porcine device (pPT) and human bone-patellar tendon-bone allograft (hPT) cut to 9-mm width. Ten grafts were used in each assessment group. All testing used fresh-frozen grafts stored frozen and thawed just before testing. Porcine ligament graft specimens were immunochemically processed, while human patellar tendon grafts were sourced from accredited tissue banks as human use graded specimens.

Structural properties were determined from load displacement curves: ultimate load, yield load, ultimate displacement, yield displacement, and axial stiffness. Axial stiffness was calculated from linear slope. Conversion of these tensile properties was accomplished by normalization of stress vs. strain plots and specimen cross-sectional area. Structural and material properties were derived for all specimens. A retrospective comparison of our porcine and human patellar tendon results to Noyes 1976 study evaluating young and old human anterior cruciate ligaments that is shown for physical, structural, and material properties [4]. The biomechanical characterization of the specimens compared processed xenograft with human allograft. Human ACL construct groups are presented from Noyes 1976 study. Cross-sectional area and bone-to-bone length of the tested graft groups closely approximates the ACL. The structural properties of ultimate load, yield load, ultimate displacement, yield displacement, and stiffness in ten grafts are presented in Table 9.1.