[Preparation of low immunogenic porcine dermal scaffold and evaluation of its cytocompatibility].

OBJECTIVE: To observe the changes in the structure and cytocompatibility of porcine acellular dermal matrix, which was prepared with dermal reticular layer, treated with matrix metalloproteinase 7 (MMP-7), genipin, and vacuum freeze-drying.

METHODS: Fifty-four pieces of porcine dermal reticular layer, prepared with lateral abdominal skin were obtained from healthy large Yorkshire pig with mechanical method under sanitary condition, each 10.0 mm×5.0 mm in size and 0.5 - 0.6 mm in thickness. They were divided into normal control group (A(1), without treatment, n = 6), decellularization group (B, decellularized, n = 12), decellularization + MMP-7 group (C, treated with MMP-7 after decellularization, n = 12), decellularization + MMP-7 + genipin group (D, treated with MMP-7 and genipin after decellularization, n = 12), and decellularization + MMP-7 + genipin + vacuum freeze-drying group (E, treated with MMP-7, genipin, and vacuum freeze-drying after decellularization, n = 12) according to the random number table. Meanwhile, 6 pieces of human acellular dermal matrix, with the same size and thickness as listed above, were taken as control group (A(2), without treatment) in the cytocompatibility tests. HE staining and scanning electron microscope were used to detect the cell number and the change in tissue structure in dermal scaffold in groups A(1) and B-E. Immunohistochemical staining was used to determine residual vimentin, laminin and collagen IV in groups A(1), B, and C. Cytotoxicity tests were employed to test the cytotoxicity of the leaching solutions of groups B-E. Human fibroblasts were seeded on the surface of dermal scaffold in groups A(2) and B-E. The proliferation of fibroblasts were determined on post culture day (PCD) 3, 7, and 14, and the content of IL-6 and IL-8 in the supernatant were determined on PCD 3 and 7 with enzyme-linked immunosorbent assay. Data were processed with two-way analysis of variance and LSD- t test.

RESULTS: Granular structure with hair follicle in pale yellow color was observed in group A(1). Small amount of hair, epithelial root sheath, nuclei, cell debris-like structure, vimentin, laminin, and collagen IV were observed in group B but not in group C, D, or E, which had been treated with MMP-7. The toughness of dermal scaffold was stronger in groups D, E than in groups B and C as observed in gross condition observation. The collagen fibers of dermal scaffold in groups C-E maintained their structural integrity with similar arrange as that of group A(1). The interspaces among collagen fibers in groups C-E were all increased, while those of groups C and D were similar but larger than that in group B; the interspace in group E was the largest. Groups B-E scored level 0 or 1 in the cytotoxicity test. Fibroblasts could proliferate on the surface of dermal scaffold in groups A(2) and B-E. Furthermore, with the extension of culture time, fibroblasts gradually became to be stratified to form multiple layers, and they proliferated toward the dermis. High density of fibroblasts was observed on the surface in groups D and E and in the deep layer in groups A(2) and C. On PCD 7, the contents of IL-6 [(132 ± 14), (104 ± 9), (122 ± 14), (120 ± 12), (128 ± 17) pg/mL] and IL-8 [(135 ± 18), (102 ± 17), (127 ± 18), (134 ± 23), (141 ± 24) pg/mL] in the supernatant in groups A(2) and B-E were significantly higher than those on PCD 3 [(55 ± 13), (34 ± 8), (48 ± 8), (50 ± 13), (49 ± 12) pg/mL] and [(93 ± 19), (63 ± 11), (82 ± 15), (82 ± 16), (89 ± 16) pg/mL], with F values respectively 98.869, 184.038, 125.531, 93.237, 87.265 and 15.694, 23.451, 22.801, 19.607, 18.808, P values below 0.05. The differences among groups A(2) and B-E in the levels of IL-6 and IL-8 at each time point were statistically significant (with F values respectively 2.809, 3.301 and 3.757, 3.266, P values below 0.05). The differences among groups A(2), C, D, and E in amount of IL-6 and IL-8 at each time point were not statistically significant (with t values respectively 0.058 - 1.905 and 0.034 - 1.295, P values above 0.05), but they were all higher than those in group B (with t values respectively 3.707 - 5.612 and 2.785 - 4.079, P values below 0.05).

CONCLUSIONS: The low immunogenic porcine dermal scaffold treated with MMP-7, genipin, and vacuum freeze-drying after decellularization, has good cytocompatibility. The growth of only a few fibroblasts in the dermal scaffold may be correlated with genipin, which increases tissue toughness.