[Experimental study on the effect of three-dimensional porous structures on the vascularization rate of artificial dermis].

Abstract

Objective: To explore the effects of orienting three-dimensional porous network (type A) and honeycomb briquette-shaped vertically penetrating three-dimensional porous network (type B) on the vascularization rate of artificial dermis. Methods: The experimental research method was used. The artificial dermis was composed of a double layer of silicone layer and scaffold layer. Based on the difference of scaffold layer, they were divided into type A and type B artificial dermis (type A dermis and type B dermis, for short) containing type A and type B structure, respectively. The type A and type B structures were prepared by gradient freeze-drying technique and physical pore-making technique, respectively. The micro-morphology of two kinds of dermis scaffold was observed by scanning electron microscopy. The porosity of two kinds of dermis scaffold was measured by the Pyrex method. According to the method of national medical industry standard, the hydroxyproline content in degradation liquids and their residues in two kind of dermis were determined after degradation at 4, 8, 13, and 24 h, reflecting the degradation rates of two kinds of dermis. According to the random number table, L929 cells were divided into type A dermis group, type B dermis group, negative control group, and positive control group. The positive control group was added with minimum essential medium (MEM) containing 5% dimethyl sulfoxide, The negative control group was added with high-density polyethylene extract, and the other two groups were added with the corresponding extract. At 24 hours after culture, the growth rate of L929 cells was detected by methyl thiazolyl tetrazolium, and the cytotoxicity was graded. L929 cells and human umbilical vein endothelial cells (HUVECs) were inoculated into pore plates with two kinds of dermis preinstalled. On 1, 4, 7, and 14 d after inoculating, the adhesion and growth of L929 cells on the surfaces of the two kinds of scaffolds were detected by immunofluorescence method. On 7 d after inoculating, the migration of the above two kinds of cells into the two kinds of dermal scaffolds was detected by immunofluorescence and hematoxylin-eosin (HE) staining. Three full-thickness skin defect wounds of 5.0 cm×5.0 cm were created on both sides of the back of three 6-month-old healthy male Ba-Ma mini pigs. According to the random number table, six columns of wounds were divided into type A dermis two-step method group, type B dermis two-step method group, and type B dermis one-step method group. The wounds in type A dermis two-step method group and type B dermis two-step method group were transplanted with type A or type B dermis respectively before, and with autologous split-thickness skin grafting later. The wounds in type B dermis one-step method group were transplanted in a synchronous procedure including type B dermis (without silicone layer) and autologous skin grafting simultaneously. The bleeding, exudation, and infection of the wounds on the back in type A dermis two-step method group and type B dermis two-step method group on the 7th day after the second transplantation and in type B dermis one-step method group on the 14th day after the first transplantation were generally observed. The area of autologous skin graft was measured by the transparent film grid method, and the survival rate of autologous skin was calculated. On 4, 7, and 14 d after the first transplantation, the inflammatory cells, fibroblasts (Fbs), and capillary infiltration into the scaffolds of the three groups were detected by HE staining. On 7, 14 d after the first transplantation, the vascularization of the scaffolds was further observed by immunohistochemistry. On 28, 90 d after the first operation, the degradation of the scaffolds of type A dermis and type B dermis was observed by HE staining. Data were statistically analyzed with one-way analysis of variance, independent sample t test, and Bonferroni correction. Results: A large number of round and oval micropores were evenly distributed on the surface of type A scaffold, and the cylindrical hole walls could be observed arranging in a parallel direction in the longitudinal section. The honeycomb briquette-shaped penetrating macropores on the surface of type B scaffold were arranged in an orderly matrix. The pore walls of the honeycomb briquette-shaped penetrating macropores were connected by micropores to form a network structure. The porosity of type A dermis was (93.21±0.72)%, which was similar to (95.88±1.00)% of type B dermis (t=4.653, P>0.05). The degradation rates of type A dermis at 4, 8, 13, and 24 h were similar to those of type B dermis at the corresponding time point (t=0.232, 0.856, 0.258, 7.716, P>0.05). At 24 h after culture, the proliferation rates of L929 cells in the type A dermis group, type B dermis group, and negative control group were significantly higher than those of the positive control group (t=2 393.46, 2 538.27, 1 077.77, P<0.01). The cytotoxicity rating of cells in positive control group was grade 4, while that of the other three groups was grade zero. On 1, 4, 7, and 14 d after inoculation, both L929 cells and HUVECs proliferated in a time-dependent manner in two kinds of dermal scaffolds. The adhesion growth and proliferation rate of the two kinds of cells on the surface of type B dermis was higher than that of type A dermis. On 7 d after inoculation, both L929 cells and HUVECs covered the surface of type B dermis and migrated into one side of the silicone layer. However, the above two kinds of cells migrated slowly into type A dermis, and only a few cells were found on one side of the silicone layer. There was no bleeding, exudation, or infection in the wounds repaired by type A and type B dermis. The survival rate of autologous skin grafting of 6 wounds in each group was 100%. On 4, 7, and 14 d after the first operation, inflammatory cells, Fbs, and capillaries gradually infiltrated into the scaffold layer, and the cell infiltration rate from high to low was type B dermis one-step method group, type B dermis two-step method group, and type A dermis two-step method group. The scaffold in wound in the type B dermis one-step method group gradually collapsed on 28 d after the first operation, and completely degraded in 3 months after the first operation. The scaffold degradation rate of type A dermis two-step method group was similar to that mentioned above. Conclusions: The honeycomb briquette-shaped vertically penetrating three-dimensional porous network structure of type B scaffold can accelerate its vascularization process, which is beneficial to autogenous split-thickness skin in one-step procedure to repair full-thickness skin defects wound in Ba-Ma mini pigs. Compared with the two-step method of staged transplantation of type A scaffold and autologous split-thickness skin, and one-step transplantation has equal efficacy and can provide a better choice for wound treatment.