[Simplified preparation and relative evaluation of decellularized porcine aortic scaffold]

Abstract

Objective: To investigate the feasibility of applying enzymatic method to prepare decellularized porcine aorta and to evaluate its biomechanical properties, immunogenicity and cell compatibility.

Methods: 0.1% trypsin-0.01% EDTA was applied to extract cells from porcine aorta under 37 degrees C continuously vibrating condition and its histology and microstructure were observed. The thickness, stress-strain curve, ultimate tension stress (UTS) and strain of failure (SOF) were compared before and after decellularization for 48, 96 and 120 hours under uniaxial tensile tests, respectively. The histological change was observed at 1, 3 and 6 weeks after the decellularized tissue was implanted subcutaneously in 3 dogs. According to the HE stains and a semi-quantitative Wakitani grading method, gross changes, category and amounts of infiltrated cells and neo-capillaries were compared between pre- and post-decellularization of porcine aortae. Endothelial cells from canine external jugular vein were seeded onto the decellularized patches to observe the cell compatibility.

Results: Microscopy and electron microscopies examination identified that cell components was completely removed from the fresh porcine aorta and Masson's trichrome showed that the structure of matrix (fibrins) was maintained intact at 96 hours using the decellularization method. There were no significant differences in the thickness, UTS and SOF between before and after decellularization (P > 0.05). However, The UTS values showed a decrease tendency and SOF showed an increase tendency. The stress-strain curve also verified a decrease tendency in mechanical intensity and an increase one in ductility after decellularization. After implanting the acellularized matrix subcutaneously in canine, moderately lymphocyte infiltration was seen at the 1st week and the infiltration was replaced by fibroblasts accompanied by neocapillary formation at the 6th week. A semi-quantity histological evaluation showed that there were differences in gross observation, category and the numbers of the infiltrated cells between decellularized and non-decellularized tissues (P < 0.05). A cell monolayer was identified by HE staining and scanning electron microscopy when the endothelial cells were seeded onto the inner luminal surface of the scaffold, aligned at the same direction on the whole.

Conclusion: The decellularized porcine aortic scaffold, prepared by trypsin-EDTA extraction under continuously vibrating condition, could meet the requirements of tissue-engineering graft in biomechanical properties, immunogenicity and cell compatibility.