Xenotransplantation of pig chondrocytes: therapeutic potential and barriers for cartilage repair.

Transplantation may be the best option for the repair of many cartilage lesions including early osteoarthritis. Currently, autologous and allogeneic chondrocytes are grafted into cartilage defects to treat selected patients with moderate clinical success. However, their limited use justifies exploring novel therapies for cartilage repair. Xenotransplantation could become a solution by offering high cell availability, quality and genetic engineering capabilities. The rejection process of xenogeneic cartilage is thus being elucidated in order to develop counteractive strategies. Initial studies determined that pig cartilage xenografts are rejected by a slow process comprising humoral and cellular responses in which the galactose α1,3-galactose antigen participates. Since then, our group has identified key mechanisms of the human response to pig chondrocytes (PCs). In particular, human antibody and complement contribute to PC rejection by inducing a pro-inflammatory milieu. Furthermore, PCs express and up-regulate molecules which are functionally relevant for a variety of cellular immune responses (SLA-I, the potent co-stimulatory molecule CD86, and adhesion molecules VCAM-1 and ICAM-1). These participate by triggering a T cell response, as well as supporting a prominent role of the innate immune responses led by natural killer (NK) cells and monocytes/macrophages. Human NK cells lyse PCs by using selected NK activating receptors, whereas human monocytes are activated by PCs to secrete cytokines and chemokines. All this knowledge sets the bases for the development of genetic engineering approaches designed to avert rejection of xenogeneic chondrocytes and leads the way to developing new clinical applications for cartilage repair.

Genetic engineering strategies to prevent the effects of antibody and complement on xenogeneic chondrocytes.

Advances in animal transgenesis may allow using xenogeneic chondrocytes in tissue-engineering applications for clinical cartilage repair. Porcine cartilage is rejected by humoral and cellular mechanisms that could be overcome by identifying key molecules triggering rejection and developing effective genetic-engineering strategies. Accordingly, high expression of α1,2-fucosyltransferase (HT) in xenogeneic cartilage protects from galactose α1,3-galactose (Gal)-mediated antibody responses. Now, we studied whether expression of a complement inhibitor provides further protection. First, porcine articular chondrocytes (PAC) were isolated from non-transgenic, single and double transgenic pigs expressing HT and moderate levels of human CD59 (hCD59) and their response to human serum was assessed. High recombinant expression of human complement regulatory molecules hCD59 and hDAF was also attained by retroviral transduction of PAC for further analyses. Complement activation on PAC after exposure to 20 % human serum for 24 hours mainly triggered the release of pro-inflammatory cytokines IL-6 and IL-8. Transgenic expression of HT and hCD59 did not suffice to fully counteract this effect. Nevertheless, the combination of blocking anti-Gal antibodies (or C5a) and high hCD59 levels conferred very high protection. On the contrary, high hDAF expression attained the most dramatic reduction in IL-6/IL-8 secretion by a single strategy, but the additional inhibition of anti-Gal antibodies or C5a did not provide further improvement. Notably, we demonstrate that both hCD59 and hDAF inhibit anaphylatoxin release in this setting. In conclusion, our study identifies genetic-engineering approaches to prevent humoral rejection of xenogeneic chondrocytes for use in cartilage repair.

Inhibition of complement component C5 protects porcine chondrocytes from xenogeneic rejection.

OBJECTIVE: Tissue-based xenografts such as cartilage are rejected within weeks by humoral and cellular mechanisms that preclude its clinical application in regenerative medicine. The problem could be overcome by identifying key molecules triggering rejection and the development of genetic-engineering strategies to counteract them. Accordingly, high expression of α1,2-fucosyltransferase (HT) in xenogeneic cartilage reduces the galactose α1,3-galactose (Gal) antigen and delays rejection. Yet, the role of complement activation in this setting is unknown. DESIGN: To determine its contribution, we assessed the effect of inhibiting C5 complement component in α1,3-galactosyltransferase-knockout (Gal KO) mice transplanted with porcine cartilage and studied the effect of human complement on porcine articular chondrocytes (PAC). RESULTS: Treatment with an anti-mouse C5 blocking antibody for 5 weeks enhanced graft survival by reducing cellular rejection. Moreover, PAC were highly resistant to complement-mediated lysis and primarily responded to human complement by releasing IL-6 and IL-8. This occurred even in the absence of anti-Gal antibody and was mediated by both C5a and C5b-9. Indeed, C5a directly triggered IL-6 and IL-8 secretion and up-regulated expression of swine leukocyte antigen I (SLA-I) and adhesion molecules on chondrocytes, all processes that enhance cellular rejection. Finally, the use of anti-human C5/C5a antibodies and/or recombinant expression of human complement regulatory molecule CD59 (hCD59) conferred protection in correspondence with their specific functions. CONCLUSIONS: Our study demonstrates that complement activation contributes to rejection of xenogeneic cartilage and provides valuable information for selecting approaches for complement inhibition.