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[Effect of acellular process on small intestinal submucosa cell residue and growth factor content]
1Division of Stem Cell and Tissue Engineering, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu Sichuan, 610041, PR China.
Objective:
To investigate the effect of machine-enzyme digestion method on the residual quantity of small intestinal submucosa (SIS) cell and the content of growth factors.
Methods:
Fresh jejunum of pig within 4 hours after harvesting was prepared into SIS after machine digestion (removing placenta percreta, mucosa, and muscular layer), decrease, trypsinization, abstergent processing, and freeze drying. Samples were kept after every preparation step serving as groups A, B, C, D, and E, respectively (n=4 per group). And the fresh jejunum served as control group (group F, n=4). The histological alteration in each preparation process was reviewed with HE staining and scanning electron microscope (SEM). Nest-polymerase chain reaction (PCR) was used to determine the content of death associated protein 12 (DAP12), and enzyme-linked immunosorbent assay (ELISA) was applied to detect the content of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), transforming growth factor beta (TGF-beta), tumor necrosis factor alpha (TNF-alpha).
Results:
HE staining and SEM observation showed that there were residual cells in groups A and B, and there were no residual cells in groups C, D, and E. Nest-PCR test revealed the occurrence of DAP12 in each group. The contents of DAP12 in groups A, B, C, D, E, and F were (18.01 +/- 9.53), (11.87 +/- 2.35), (0.59 +/- 0.27), (0.29 +/- 0.05), (0.19 +/- 0.04), and (183.50 +/- 120.13) copy x 10(6)/cm2. The content of DAP12 in group F was significant higher than that of other groups (P < 0.05), groups A and B was higher than groups C, D, and E (P < 0.05), there were significant differences among groups C, D, and E (P < 0.05), and there was no significant difference between groups A and B (P > 0.05). The ELISA test showed the content of VEGF, bFGF, TGF-beta, and TNF-alpha in group A was significantly higher than that of groups B, C, D, and E (P < 0.05), and there was no significant difference among groups B, C, D, and E (P > 0.05).
Conclusion:
SIS prepared by simple mechanical method has more residual cells, while the machine-enzyme digestion method can effectively remove the cells and significantly reduce the DAP12 content. This approach can not obviously reduce the growth factor content in SIS.
