In Situ "Humanization" of Porcine Bioprostheses: Demonstration of Tendon Bioprostheses Conversion into Human ACL and Possible Implications for Heart Valve Bioprostheses

Abstract

This review describes the first studies on successful conversion of porcine soft-tissue bioprostheses into viable permanently functional tissue in humans. This process includes gradual degradation of the porcine tissue, with concomitant neo-vascularization and reconstruction of the implanted bioprosthesis with human cells and extracellular matrix. Such a reconstruction process is referred to in this review as "humanization". Humanization was achieved with porcine bone-patellar-tendon-bone (BTB), replacing torn anterior-cruciate-ligament (ACL) in patients. In addition to its possible use in orthopedic surgery, it is suggested that this humanization method should be studied as a possible mechanism for converting implanted porcine bioprosthetic heart-valves (BHV) into viable tissue valves in young patients. Presently, these patients are only implanted with mechanical heart-valves, which require constant anticoagulation therapy. The processing of porcine bioprostheses, which enables humanization, includes elimination of α-gal epitopes and partial (incomplete) crosslinking with glutaraldehyde. Studies on implantation of porcine BTB bioprostheses indicated that enzymatic elimination of α-gal epitopes prevents subsequent accelerated destruction of implanted tissues by the natural anti-Gal antibody, whereas the partial crosslinking by glutaraldehyde molecules results in their function as "speed bumps" that slow the infiltration of macrophages. Anti-non gal antibodies produced against porcine antigens in implanted bioprostheses recruit macrophages, which infiltrate at a pace that enables slow degradation of the porcine tissue, neo-vascularization, and infiltration of fibroblasts. These fibroblasts align with the porcine collagen-fibers scaffold, secrete their collagen-fibers and other extracellular-matrix (ECM) components, and gradually replace porcine tissues degraded by macrophages with autologous functional viable tissue. Porcine BTB implanted in patients completes humanization into autologous ACL within ~2 years. The similarities in cells and ECM comprising heart-valves and tendons, raises the possibility that porcine BHV undergoing a similar processing, may also undergo humanization, resulting in formation of an autologous, viable, permanently functional, non-calcifying heart-valves.

Keywords: anterior cruciate ligament reconstruction; anti-Gal antibody; anti-non gal antibody; bioprosthesis humanization; heart valve bioprosthesis; porcine tendon bioprosthesis; α-gal epitope; α-galactosidase.

Figure 1

Anti-Gal and anti–non-Gal IgG titers at various time points post implantation of porcine patellar-tendon in rhesus monkeys (n = 13). Anti-Gal titers were measured by ELISA with synthetic α-gal epitopes linked to bovine serum albumin (BSA) as solid-phase antigen. Anti-non gal antibodies measured by ELISA with homogenate of unprocessed porcine patellar-tendon as solid-phase antigen and with sera that were depleted of anti-Gal. The implanted tendon was unprocessed or was a bone-patellar-tendon-bone (BTB) bioprosthesis processed by treatment with recombinant α-galactosidase and partial crosslinking with glutaraldehyde. Error bars represent standard deviation of titers (modified from [53]).

Figure 2

Anti-non gal antibody analysis by Western blots with porcine patellar-tendon and kidney proteins, or human patellar-tendon proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). (A) Pre-implantation serum of patient P-10. (B) Serum of patient P-10, six months post-implantation of porcine BTB. (C) Serum of patient P-03, six months post-implantation of porcine BTB. In this analysis, the sera were diluted 1:10, and depleted of anti-Gal by adsorption on glutaraldehyde fixed rabbit red blood cells (RBC) (modified from [71]).

Figure 3

Hypothesis on humanization of porcine BTB or BHV bioprostheses implanted into patients with torn ACL or with impaired heart valve, respectively: Stage 1. Elimination of α-gal epitopes from the bioprosthesis by incubation with recombinant α-galactosidase prevents accelerated destruction by anti-Gal and by the macrophages it recruits. Stage 2. Partial crosslinking with glutaraldehyde creates “speed bumps” that slow macrophage infiltration following recruitment of the macrophages into the bioprosthesis by anti-non-gal antibodies binding to many porcine protein antigens. Colors of the antibody molecules vary because of different specificities of anti-non-gal antibodies. The macrophages bind via Fc-receptors to the Fc “tail” of anti-non-gal IgG molecules that interact with the porcine antigens of the ECM, and of non-viable cells in the implanted bioprosthesis. Stage 3. The porcine tissue is gradually degraded by the infiltrating macrophages. Fibroblasts that follow the macrophages align with the porcine collagen fibers scaffold and secrete their collagen and other ECM components. This concomitant destruction and reconstruction (remodulation) results in humanization of the bioprosthesis by gradual replacement of the porcine tissue with autologous permanently functional ACL or heart valve.

Figure 4

Bioprosthesis prepared from porcine bone-patellar-tendon-bone (length of ~10 cm and width of ~1 cm) for reconstructing torn ACL in humans. Note the two bone-plugs of the patella and tibia bones.

Figure 5

Histopathology demonstrating humanization stages in patients with implanted porcine BTB bioprostheses. Black arrows: blood vessels, white arrows: macrophages. (A) Pre-implantation porcine BTB bioprosthesis. (B) Infiltration of macrophages into the implanted bioprosthesis by extravasation. Elongated cells are endothelial cells of a blood vessel. (C) Vascularization of the implanted BTB in a region near macrophage infiltrates. (D) Repopulation of a section of the bioprosthesis by the recipient’s fibroblasts that aligned with the porcine collagen fiber scaffold (above the dashed line). Porcine collagen fibers and no cells, seen under the dashed line. (E) An advanced stage of humanization, with repopulating fibroblasts secreting their own ECM. (F) De novo produced collagen fibers, stained blue in Mason-trichrome staining. H&E, (×200) (modified from [71]).

Figure 6

Anti-non gal IgG antibody response in three of the patients implanted with processed porcine BTB bioprosthesis for the reconstruction of torn ACL. Anti-non gal antibody activity at various time-points post-implantation was determined with anti-Gal depleted sera, by ELISA. Homogenate of fragmented porcine tendon was used as solid-phase antigen. The figure describes antibody binding at serum dilution of 1:640 (based on data from [71]).