The Effect Of Crosslinking Agent Genipin On The Decellularized Porcine Liver Tissue Immunogenicity

Objective: The purpose of this study was to produce immunogen-reduced and biocompatible matrices from porcine liver and to investigate the crosslinking agent genipin on the immunogenicity of decellularized porcine liver tissue and the host immune rejection. Methods: Whole porcine livers were perfusion-decellularized by 1% SDS +1%Triton X-100 and cross-linked with 0.625% glutaraldehyde and 0.625% genipin. For evaluation the structural integrity and cell removal, fractions of the native liver and decellularized livers were fixed in 4% paraformaldehyde and embedded in paraffin. Sections were stained with hematoxylin and eosin(H&E), Masson’s trichrome staining kits and immunohistochemistry, SEM examinations, DNA agarose gel electrophoresis and PCR examinations, additionally. Proteins were extracted and the migratory response of human mononuclears toward protein extracts was examined using an in vitro migration chamber. In addition, biopsy specimens of decellularized scaffolds were implanted subcutaneous into rodents for 4 weeks to investigate scaffold immunogenicity, scaffolds were explanted after 3, 7, 14 and 28 days and fixed for histological analysis. Results: Staining confirmed cellular clearance from pig livers, with removal of nuclei and cytoskeletalcomponents and widespread preservation of structural extracellular molecules. Scanning electron microscopy confirmed preservation of an intact liver capsule and a porous acellular lattice structure. There was no evidence of porcine DNA remnants within the decellularized matrices by agarose gel electrophoresis. The PCR analysis showed that galactose-α-1,3-galactose-β-1,4-N-acetylgluc- osamine(1,3 gal), swine leukocyte antigen(SLA), and porcine endogenous retrovirus(PERV) were completely removed in the matrices. After crosslinking, the GP-treated one was dark blue, the GA-treated material turned to be brownish close to native liver. Materials became slightly smaller and stiffer after crosslinking. Histological analysis indicated that three-dimensional microarchitectures were not changed after crosslinking. As shown in SEM micrographs, more interconnected porous structures in decellularized liver materials compared to the native one and the porosities both in GA- and GP-treated liver materials were no different from the uncrosslinked decellularized one. Migration of human monocytes in vitro showed that migration number of human monocytes in native, decellularized, GA-and GP-treated tissues were(108.8±15.33) ×103,(64.33±6.936)×103,(31.67±2.906)×103 and(34.67±4.33) ×103 cells, respectively. Decellularization significantly reduced the migration of monocytes compared to native porcine tissue. Although the proportion of transmigrating lymphocytes was much lower, cross-linked again reducedthe migratory response. In the native and decellularized groups, acute rejection was occurred after implanted 3 days, and obvious attenuation and lessening were seen at 14 days. After implanted 28 days, the decellularized and native samples were degraded, and the glutaraldehyde-treated group occurred severe inflammatory reaction, however, minimal inflammatory cells infiltration was seen in the genipin-treated group during the investigation periods. Conclusions: 1. 1%SDS+1%Triton X-100 perfusion method can effectively remove the cells and antigen in liver tissues of integrity; 2. GP and GA did not change the porous material and the three-dimensional structure of decellularized liver tissue; 3. Crosslinking agent genipin can effectively reduce the decellularized liver scaffold biomaterials immunogenicity.