The Scaffold Derived from GGTA1 and CMAH Gene Knockout Pigs Generated by Gene Editing Provoked Little Inflammatory Responses in a Humanized Pig Model

Abstract

BackgroundThe porcine small intestinal submucosa ECM (SIS-ECM) has been used as a supportive scaffold for healing in a variety of tissues. However, the outcomes of its application are far from satisfactory. ResultsThe possibility of generating a porcine small intestinal submucosa extracellular matrix (SIS-ECM) that is fully biocompatible was investigated. Samples of SIS-ECM were prepared from either domestic wildtype (WT) or double-gene knockout (dKO) pigs without antigenic responses to α-Gal and Nglycolylneuraminic acid (Neu5Gc). The scaffolds, which were sutured into cuts made in the longissimus muscle of the dKO pigs, were expected to exhibit xeno-reactions through natural antibodies as in a human response. A process was established to manufacture ameliorating acellular porcine SIS-ECM implants with consistent quality characteristics, which were assured by analyzing the level of residual DNA, the glycosaminoglycan content, and histochemical stains. Once implanted, the acellular SIS-ECM from the WT pigs caused a significant increase in serum IL-6 levels in the dKO recipient pigs, indicating a host defense through immune reactions. The levels remained unchanged when preparations from the dKO pigs were used. The pathological score of the multinuclear giant cells of the dKO (WT) group (2.3±0.5) was significantly greater than that of the dKO (dKO) group (0.7±0.3). ConclusionThe IL-6 levels and pathological evidences suggested that dKO pigs without α-Gal or Neu5Gc antigenic causing lower inflammatory response can serve as biocompatible SIS-ECM donors or as animal models for testing anthropomorphic immune responses to biomedical devices. BackgroundThe extracellular matrix (ECM) is a noncellular structure required for the process of forming a new networks and tissues and has now become a successful and widely used medical material in various forms. The porcine small intestinal submucosa ECM (SIS-ECM) has been used as a supportive scaffold for healing in a variety of tissues, including arterial and venous tissues, tendons and wound closures 3 [1-4]. The porcine SIS-ECM consists of collagen, proteoglycan glycosaminoglycan, glycoprotein, and growth factors, and it has been approved as a commercial biomaterial for a variety of clinical applications [5,6]. However, the outcomes of its application are far from satisfactory; prominent noninfectious swelling and severe pain at the site of implantation often accompany its use because of the contamination of porcine DNA [7] and the Gal epitope, which both evoke immune responses [8].In general, matrix decellularization can be achieved by processing efforts; the side effects from using the SIS-ECM in clinical applications is approached based on the consequences of a deleterious innate antigen response.The α-Gal epitope is the chief antigen responsible for hyperacute rejection in xenotransplantation but there is a further non-Gal antigenicity concern. In vertebrates, glycans usually have terminal sialic acids that function as markers in normal cells and are recognized by a variety...