Xenogeneic bone matrix immune risk assessment using GGTA1 knockout mice

Abstract

Homeotransplantation of bones for replacement therapy have been demonstrated reliably in clinical data. However, human donor bones applicable for homeotransplantation are in short supply, which facilitates the search for suitable alternatives, such as xenografts grafts. The α-Gal antigen-related immune risk of xenografts directly affects the safety and effectiveness of the biomaterials and limits their applications in the clinic. The immune risk can be prevented by depletion or breaking anti-Gal antibody prior to transplant. Therefore, how to assess the immune risk of the bone substitutes and select the reliable animal research model become extremely important. In this study, we prepared lyophilized bone substitutes (T1) and Guanghao Biotech bone substitutes (T2, animal-derived biomaterials with α-Gal antigen decreased), aimed to assess the immune risk of xenografts bone substitutes on GGTA1 knockout mice. The α-Gal antigen contents of T1 and T2 were firstly detected by ELISA method in vitro. The bone substitutes were then implanted subcutaneously into GGTA1 knockout mice for 2, 4 and 12 weeks, respectively. The total serum antibody levels, anti-α-Gal antibody levels, inflammatory cytokine and splenic lymphocyte surface molecules were detected and histology analysis of skin and thymus were performed to systematically evaluate the immune response caused by the T1 and T2 bone substitutes in mice. In vitro results showed that the amount of α-Gal epitopes in T1 bone substitutes was significantly higher than T2 bone substitutes, and the clearance rate of α-Gal antigen in T2 bone substitutes achieved about 55.6%. Results of antibody level in vivo showed that the T1 bone substitutes group possessed significantly higher total IgG, IgM, IgA and anti-α-Gal IgG levels than T2 and control group, while T2 group showed no significant changes of these indexes compared with control. In terms of inflammatory cytokines, T1 bone substitutes showed evidently higher levels of IL-4, IL-12P70 and IL-10 than T2 and control, while T2 group was comparable to control. No changes in the levels of splenic lymphocyte surface molecules were found in the three groups (T1, T2 and control group) during the experimental periods. The pathological results demonstrated that the inflammatory response in T2 group was lighter than the T1 group, which was in accordance with the inflammatory cytokines levels. The above results indicated that the process of antigen removal effectively reduced the α-Gal antigens content in T2 bone substitutes, which caused little immune response in vivo and could be used as bone healing materials. This study also demonstrated that GGTA1 knockout mice can be used as a routine tool to assess the immune risk of animal-derived biomaterials.

GGTA1/iGb3S Double Knockout Mice: Immunological Properties and Immunogenicity Response to Xenogeneic Bone Matrix

“…Our results demonstrated that T1 stimulate a higher antiα-Gal antibody expression level in G/i DKO mice compared with the T2 group, which was similar to our previous findings using GGTA1 KO mice [17]. However, T1 implantation did not cause a significant increase in anti-Gal IgM and IgA levels in GGTA1 KO mice [17].…”

“…Sun et al assessed the immunotoxicity of xenogeneic bone by embedded materials into the intermuscular space of Balb/c mice was insufficiently scientific [29]. Third, to further verify the applicability of G/i DKO mice as a model for the assessment of immune responses of xenoimplants, G/i DKO mice were implanted with raw lyophilized bone substitutes (T1, containing high Gal antigens, ð8:14 ± 3:17Þ × 10 12 /mg) and Guanhao Biotech bone substitutes (T2,~50% decreased α-Gal antigen relative to T1 [17]).…”

“…The highest levels of IgM and IgG antibodies were detected in fresh porcine posterior corneal lamellar grafts in GGTA1 KO mice while reduced IgG deposition was observed in fresh porcine posterior corneal lamellar grafts in WT mice and decellularized porcine posterior corneal lamellar grafts in GGTA1 KO mice [16]. Our previous study showed that implantation of raw lyophilized bone substitutes into GGTA1 KO mice induced a significant increase in anti-α-Gal antibody and Th2 immune reaction-related inflammatory factors while α-Gal antigendecreased bone substitutes (Guanhao Biotech bone substitutes) displayed slight changes relative to the control [17]. These findings support the suitability of GGTA1 KO mice as a model for the analysis of anti-Gal antibody-mediated immunogenicity.…”

Immune risk assessment of residual αGal in xenogeneic decellularized cornea using GTKO mice

“…The reported immune effects induced by xenogeneic material implantation in GTKO mice also included the elevation of cytokine levels. Xenogeneic bone tissue had induced more than 10 times increase in anti-Gal IgG level of GTKO mice compared with the blank control, accompanied by a significant increase in serum IL-12p70 and IL-4 content [ 20 ]. However, at 4 weeks after implantation of the DCM, there were no significant differences in serum IFN-γ, IL-4 and IL-12p70 levels between the GTKO mice and the blank control group, although the elevation of the anti-Gal IgG antibody stimulated by residual αGal antigen contained in the DCM occurred.…”

“…The αGal contents of fresh corneal matrix and DCM were quantitatively detected by an inhibitory enzyme-linked immunosorbent assay (ELISA) [ 20 ]. Firstly, the lysates of corneal matrix were prepared by homogenized in lysis buffer containing 1% protease inhibitor PMSF using a homogenizer (Benchmark D1000-E, USA), incubating at room temperature for 1–3 h, and making sure that there were no obvious solid matters and the α-Gal antigen was completely exposed.…”