关键词: 猪真皮, 组织再生支架, 生物材料, 细胞外基质, 脱细胞, 病毒灭活, 伽马辐照, 灭菌

Abstract: BACKGROUND: Process integration of decellularization, antigen removal, viral inactivation and sterilization is essential for producing regenerative biomaterials that meet clinical requirements. 
OBJECTIVE: To systematically compare the effects of two combined processes of decellularization, antigen removal, virus inactivation and sterilization on the physicochemical properties of porcine dermal matrix. 
METHODS: Fresh porcine dermis was obtained and processed by two processes into acellular matrices. (1) Method A: 1% Triton X-100, DNase and RNase, and 1% tributyl phosphate were applied for decellularization, 1% peracetic acid and 25% ethanol for viral inactivation, and gamma-irradiation for terminal sterilization. (2) Method B: Dispase II, 0.5% Triton X-100 and DNase were used for decellularization, α-galactosidase for the antigen removal, 0.1% peracetic acid for viral inactivation and gamma-irradiation terminal sterilization. Porcine dermal matrices prepared by two methods were characterized. 
RESULTS AND CONCLUSION: (1) The collagen content of group A was lower than that of group B. The contents of elastin and sugar of group A were higher than those of group B. The material hardness value of group A was higher than that of group B. The drapability of the matrix material in group A was poor and the flexibility of the matrix material in group B was better. (2) The decellularization of group A did not affect the thermal stability of the matrix material. In group B, decellularization slightly reduced the thermal stability of the matrix material. After gamma ray sterilization, the thermal stability of the matrix material in group A decreased greatly and the thermal stability of the matrix material in group B decreased very little. (3) Compared with group B, the tensile strength and elasticity of the matrix material in group A were smaller. In vitro enzymatic degradation experiments showed that the matrix materials in group A had strong resistance to collagenase degradation, were sensitive to trypsin and were easily degraded; the matrix materials in group B were the opposite. (4) Scanning electron microscope showed that after sterilization by gamma rays, the three-dimensional structure of collagen could not be seen in the matrix material of group A; the collagen fibers were densely arranged, and there was a non-fibrous structural gel-like substance between the collagen fibers. In group B, the structure of collagen fibers in the matrix material was clear, and there was no non-fibrous structural glue-like substance between the collagen fibers. Hematoxylin-eosin staining and trichrome staining showed that after sterilization by gamma rays, a few nuclei were visible in the matrix material of group A; the collagen fibers were closely arranged, and the material was dense. The matrix material in group B had no nucleus and retained a good three-dimensional spatial structure of collagen fibers. (5) The results suggest that different processes have great influences on the physical and chemical properties of derived acellular matrices. Method B is effective in decellularizing and de-antigening and retains intact natural tissue matrix. Method A is more destructive to the material and results in lower collagen content, scratchability, thermal stability and poorer biomechanical properties compared with the Method B. 

Key words: porcine dermis, regenerative tissue scaffold, biomaterial, extracellular matrix, decellularization, viral inactivation, gamma irradiation, sterilization