Scaffold derived from GGTA1 and CMAH double knockout pigs elicits only slight inflammation in a gene-edited pig model

Abstract

Acellular extracellular matrix (ECM) contains α-Gal and N-glycolylneuraminic acid (Neu5Gc) antigens that cause clinical side effects. We prepared porcine cornea and small intestinal submucosa ECM (SIS-ECM) from either wild-type (WT) or knockout (KO) pigs, including GGTA1/CMAH double KO (dKO) and CMAH KO (CMAHKO), through gene editing to eliminate α-Gal and/or Neu5Gc antigens. The quality of the SIS-ECM was confirmed by assessing residual DNA, glycosaminoglycan content and histology. The effects of histopathological and IL-6 responses of SIS-ECM from WT, CMAHKO and dKO pigs were compared to those shown by implanting SIS-ECM into the longissimus of WT and dKO pigs. In the histopathological study, WT and dKO SIS-ECM were simultaneously implanted into WT or dKO pigs. Results indicated granulocyte infiltration but no significant difference between the implanted WT and dKO SIS-ECM, however, multinuclear giant cells were significantly more abundant around WT than dKO SIS-ECM if implanted into dKO pigs. In the IL-6 response study, its concentration was significantly higher in WT SIS-ECM than in dKO SIS-ECM; meanwhile, a moderate level of IL-6 was observed in CMAHKO or WT SIS-ECM implanted into dKO or CMAHKO pigs, respectively. Since dKO SIS-ECM elicits only slight inflammation, we conclude that dKO pigs lacking α-Gal and Neu5Gc can serve as biocompatible SIS-ECM donors or as animal models for testing anthropomorphic immune responses to biomedical devices. Statement of SignificanceXenograft‐derived scaffolds, even acellular scaffolds, encounter the challenges of α-Gal and Neu5Gc nonprotein antigens present in the ECM, including the SIS- and corneal-ECM. Gene-edited pigs with depleted α-Gal and nullified Neu5Gc may represent a valuable source for the procurement of tissues for biomaterial manufacturing. This value is indicated by the evidence that the SIS-ECM prepared from these pigs and not wild-type (WT) animals elicited only slight inflammation histopathologically and serologically if implanted into a double gene-knockout (dKO) pig model with α-Gal and Neu5Gc absent. The ECM prepared from dKO pigs provides a further improvement in biocompatibility, and those animals could be used as a testing model and for precisely evaluating Gal and non-Gal antigen immune responses, regardless of biomaterial or biomedical devices.