Preparation of low immunogenic porcine dermal scaffold and evaluation of its cytocompatibility / 中华烧伤杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSIONS</b>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.</p>

Effects of Sophora tonkinensis Gapnep on neurobehavioral function in rats / 中华神经医学杂志

Objective To investigate the effects of Sophora tonkinensis Gapnep on neural behavior and cerebral pathology of rats. Based on the observation and quantitative analysis of neural behavior, a new type of animal models with dystonia is expected to be demonstrated. Methods Twenty-four female SD rats, weighting 250 g, were equally randomized into control and experimental groups. The animals in the experimental group were treated with 1.5 ml Sophora tonkinensis Gapnep once daily for consecutive 10 d. For rats in the control group, the same volume of saline (0.9%) was delivered by the same method. Neurobehavioral changes were observed; and quantitative analysis of neurobehavior and balancing ability was performed by using Noldus and Rota Rod systems. After behavioral experiments, the rats were sacrificed and the brains were got for pathological observations,especially the striatum, midbrain and hippocampus. Results Three to 7 d after the treatment, rats in the experimental group presented different degrees of neurobehavioral changes, mainly manifested as hypokinesia, decreased balanced capacity and postural dystonic abnormalities of the neck and limbs.Behavioral analysis showed that the distance, mean velocity and value of activity index in rats of the experimental group were significantly decreased as compared with those in the control group (P＜0.05).The experimental group showed a more serious dysfunction of the coordinating movement in the tested by rotating rod, a significantly shorter time of staying in the rod as compared with the control group (P＜0.05). Pathological investigation clearly demonstrated that the experimental group had decreased neurons in the corpus striatum and midbrain, especially in the hippocampus, and some neural pathological findings were observed. Conclusion Sophora tonkinensis Gapnep can induce significant changes of neural behavior in rats, which is similar to the symptoms of patients with dystonia caused by Sophora tonkinensis Gapnep poisoning, mainly manifested as hypokinesia, dysfunction of the coordinating movement, indicating that rats in the experimental group maybe a new type of animal model of dystonia, which is important in the study of dystonia.