| Objective To prepare and detect selectively decellularised porcine skin (SDPS) which has the characteristics of the lower antigen, continuous epidermis and the dermal matrix without any cellular components, and to verify the biological safety. To explore the clinical efficacy of micro-autografts overlaid with SDPS in the repair of deep burn wounds so as to resolve the problem of the shortage and risk of cadaveric allograft skin. Through Ad-VEGF transfecting UCMSCs in vitro, VEGF-expressing UCMSCs plus SDPS were transplantated to cover relevant full-thickness defect wounds in animal models, to improve wound healing quality, which would lay the experimental foundation for clinical study.Methods 1 Healthy porcine skin was treated with glutaraldehyde, trypsin and detergent to prepare SDPS. Gross observation and histological examination of SDPS were used to explore the optimal concentration of the reagents and action time in different stage. Bacteria, fungi and virus were tested in microbiological examination. Ultimate tensile strength and fracture tensile strain rate were measured by Instron tensile machine. Concentration of glutaraldehyde in SDPS leaching liquor was assayed by HPLC. The effect of SDPS leaching liquor on NCTC clone 929 cell proliferation was determinated by MTT method for evaluating its cytotoxicity according to U.S. Pharmacopeia. The possible irritant reactions of SDPS were observed after intradermal injection of SDPS leaching liquor in rabbits. Seventy-two adult Wistar rats were randomly divided into three groups:SDPS group, fresh porcine skin group and rat allogeneic skin group. One square centimeter of relevant skingraft was subcutaneously implanted into rat back in each group. Gross observation and histological examination were used to evaluate biological biocompability of SDPS on week 1,2,4,8,12 and 16 postoperatively.2 The clinical study protocol was approved by the ethics committee of the first hospital affiliated to general hospital of PLA. Written informed consents were obtained from the patients and/or their family members before surgery. They were 18 men and 13 women, with a mean age of (37.29±11.33) years (range,21 to 60). The total burn area was (65.51±17.43)% TBSA (range,45% to 99%). All patients underwent escharectomy followed by resultant wound being closed with micro-autografts plus SDPS or micro-autografts plus cryopreserved human cadaver skin (CHCS) between 4 and 6 days post burn. The following parameters in all cases were investigated:time of rejection and exfoliation, wound healing rate 3 weeks after surgery, and number of cases with residual wound 3 months after surgery. Hypertrophic scars were graded by Vancouver scar scale (VSS) 3 months after surgery. Wound sample from one case was harvested for pathological examination 12d and 3 months after surgery. Appearance and function in all patients were observed following up 1 to 2 years.3 Adenoviral vector containing hVEGF165 gene was constructed and UCMSCs were passaged and expanded. The infection efficiency of adenovirus vector to UCMSCs was tested by Ad.GFP infection procedure. GFP expression efficiency was observed using the fluorescence microscope and flow cytometry. Ad.VEGF were transfected into the UCMSCs by recombinant adenovirus vector. VEGF expression in UCMSCs were measured by immunohistochemical staining, RT-PCR, and Western blotting, while its secretion levels in culture medium were measured by ELISA. Influence of recombinant adevirous on cell proliferation were evaluated by MTT method.4 Forty-five adult Wistar rats were randomly divided into 3 groups (n=15). Acute full-thickness skin defects were inflicted on back of all the rats as a wound model, followed by covering with equal size SDPS plus micro-autografts, and then injecting underneath with 3 different agents respectively, including VEGF genetically modified UCMSCs (A group), UCMSCs (B group) and PBS (C group). Wound condition was observed at 7d,14d,21d,28d after operation. The rejection and losing time of SDPS on wound were observed and recorded, and wound healing rate was determined at 28d postoperation. Excising transplanted tissue was carried out at 3d, 7d,14d, and 28d postoperation. HE staining of specimen was performed at 7d,14d, 28d postoperation. The expression of VEGF and CD34 at 3d,7d and 14d postoperation were detected by Western blotting and IHC respectively. The microvessel density (MVD) was measured. Statistical analysis was performed finallyResults 1 The SDPS, produced by the glutaraldehyde-trypsin-detergent method, had well-conserved continuous epidermis and dermal matrix without any cellular components, skin appendage and blood vessels, and collagen fibers arranged regularly. Pathogenic microorganism was undetectable. Ultimate tensile strength was (8.80±0.03) Mpa, Fracture tensile strain rate (77.60±1.60)%. No residual glutaraldehyde was detected in SDPS leaching liquor. The cytotoxicity was proved to be first grade by biocompatibility test, according with national standards of biological evaluation of medical devices. The primary irritation was mild 24h and 48h after intradermal injection of SDPS leaching liquor, and very slight 96h after injection. The experimental results of subdermal implantation demonstrated that inflammatory reaction appeared in all 3 groups. In SDPS group and allogeneic skin group, inflammatory reactions were moderate and alleviated in a time-dependent manner, fresh vessels and fibroblasts appearing in dermal matrix, the implanted skin grafts being degraded and absorbed gradually. However, in fresh porcine skin group, severe inflammatory reaction occurred and lasted longer than that in the other two groups, the implanted skin grafts becoming stiff and hard to be degraded.2 Clinical data showed that SDPS and allogeneic skin survived on wound at 10d postoperation in 31 cases, with epidermis basically existing, base turning red, without obvious exudates. With the growth and flakiness of autologous skin particles, both covering materials gradually losed. The rejection and exfoliation time of the SDPS was (17.5±3.3) days and that of the CHCS was (14.2±2.5) days (P<0.05). The wound healing rate were (70.16±23.15)% and (75.45±22.38)% at 3 weeks after operation (P>0.05) respectively. Postoperation 3 months, cases having residual wound were 4 and 5 respectively (P>0.05), the mean score of hypertrophic scar being (7.8±3.6) and (8.1±3.3) respectively (P>0.05). Inflammatory reaction appeared in both skin substitutes 12d postoperation. Histopathological observation showed regenerative epidermis, and fibroblasts, small blood vessels and collagen in dermis 3 month postoperation. There was no significant difference in appearance and function between the two groups following up 1 to 2 years.3 UCMSCs could be effectively infected with adenovirus containing GFP gene in vitro. The transfection efficiency has dose-effect relationship with MOI. When MOI was 100, the infection efficiency was over 95%. In UCMSCs'lysate, The expression of VEGF was traced at 2d after infection. In culture medium, the expression of VEGF reached peak on 7d, and could be observed even on 13d. Recombinant adevirus transfection had no effect on the growth and proliferation of UCMSCs.4 The exclusion and losing time of SDPS on wound was longer in A group than that in B group and C group (P<0.01). Wound healing rate was higher in A group than that in B group and C group at 28d postoperation (P<0.01). But there were no differences in both indexes between B group and C group (P>0.05). Postoperation 7d, wound bed under SDPS was observed. Angiogenesis in A group was more clearly than that in B group and C group. Postoperation 3d,7d and 14d, compared with the other two groups, the expression of VEGF and CD34 were increased in A group. Postoperation 7d and 14d, MVD in A group was higher than that in the other two groups (P<0.01). But there was no statistical difference between B and C group in expression of VEGF and CD34, and MVD (P>0.05).Conclusion SDPS, produced by the glutaraldehyde-trypsin-detergent method, has morphological advantages of possessing well-conserved continuous epidermis and dermal matrix without any cellular components. Its biological safety was verified by microbiology, physical and chemical properties and biological testing, meeting national standards of biological evaluation of medical devices. The clinical effect of microskin autografts overlaid with SDPS in the repair of deep burn wounds is similar to that of microskin autograft overlaid with CHCS. So it could be selected as a new skin substitute for allo-skin to cover extensive deep burn wounds. Further, combined transplantation of SDPS plus VEGF-expressing UCMSCs augments early angiogenesis of wound, reduces wound healing time, and improves the wound healing quality. |
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