Immunological Risk Assessment of Xenogeneic Dural Patch by Comparing with Raw Material via GTKO Mice

Abstract

Objective. In this study, α-Gal epitope-deficient (GGTA1 knockout (GTKO)) mice were used to assess the immunological risks of xenogeneic dural patch by comparing with raw material. Methods. The xenogeneic dural patch (T2) was prepared from bovine pericardium (T1, raw material) through decellularization and carboxymethyl chitosan (CMCS) coating. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were used to characterize the collagen fibers and surface microstructural changes in the T1 and T2 samples. The remnant α-Gal epitopes and DNA of implants were detected by standardized method. T1 and T2 were implanted subcutaneously into GTKO mice for 4 and 12 weeks, respectively, and the negative control group (Con) was only performed sham operation. The total serum antibody, anti-Gal antibody, and splenic lymphocyte subtypes were analyzed by ELISA or flow cytometry, and histological analysis of implant-tissue was performed by H&E and Masson stain. Results. TEM and Sirius red staining showed that the collagen fibers in the dural patch were closely arranged, and SEM showed that a loose three-dimensional structure was successfully constructed on the surface of the dural patch after CMCS coating. The remnant DNA in T2 was 24.64 ± 8.73 ng / mg (dry weight), and clearance of α-Gal epitope was up to 99.83% compared to T1. The significant increases in serum total IgM, anti-Gal IgG, and anti-Gal IgM at 4 weeks and the significant changes in anti-Gal IgG and spleen lymphocyte at 12 weeks were observed in the T1 group, but no significant change was observed in the T2 group, compared to the control group. Histological semiquantitative analysis showed severe cell and tissue responses at 4 weeks and a moderate response at 12 weeks in the T1 group, while a moderate response at 4 weeks and a slight response at 12 weeks in the T2 group. Conclusions. The results demonstrated that the xenogeneic dural patch has a lower and acceptable immunological risk compared to the raw material and control, respectively. On the other hand, it was suggested that GTKO mice are useful experimental model for immunological risk assessment of animal tissue-derived biomaterials.

Fish scale derived hydroxyapatite incorporated 3D printed PLA scaffold for bone tissue engineering

“…Semi-quantitative histological evaluation in experimental animals for local tissue responses, in comparison with that of reference material, is the gold standard for assessing the potential safety of any biomaterial. 47 Pure 3D-printed PLA scaffolds serve as the reference material for the evaluation. 34 The semi-quantitative parameters evaluated were summarized in Table 2.…”

GTKO rabbit: A novel animal model for preclinical assessment of decellularized xenogeneic grafts via in situ implantation

“…Owing to the development of gene-editing technology, GTKO mice have been established and used to study the specific mechanism of xenograft rejection [ 11 , 12 ]. We have successfully established a GTKO mouse model using gene targeting techniques (homologous arm recombination) [ 10 , [12] , [13] , [14] ] and revealed that anti-Gal antibody levels increased significantly after implantation of xenogeneic bone matrix or xenogeneic decellularized cornea in GTKO mice. However, due to the small size of GTKO mice, it is difficult to assess the biocompatibility and effectiveness of xenografts via in situ implantation.…”