【Abstract】 Objective To find out the best method to prepare platelet-rich plasma (PRP) and to evaluate the effect of PRP gel on skin flap survival and its mechanism. Methods Totally, 72 Wistar rats (aged 12 weeks, weighing 250-300 g) were used for the experiment. The arterial blood (8-10 mL) were collected from the hearts of 24 rats to prepare PRP with three kinds of centrifuge methods: in group A, 200 × g centrifuge for 15 minutes, and 500 × g centrifuge for 10 minutes；in group B, 312 × g centrifuge for 10 minutes, and 1 248 × g centrifuge for 10 minutes；and in group C, 200 × g centrifuge for 15 minutes, and 200 × g centrifuge for 10 minutes. The platelet was counted in the whole blood, PRP, and platelet-poor plasma (PPP) to determine an ideal centrifuge. PRP, PPP, and the serum after first centrifuge were collected. The concentrations of platelet-derived growth factor BB (PDGF-BB) and transforming growth factor β1 (TGF-β1) were measured in the PRP, PPP, and serum using the enzyme-linked immunosorbent assay method, and PRP and PPP gels were prepared. The flaps of 11 cm × 3 cm in size were elevated on the back of 48 rats, which were divided into 3 groups: PRP gel (PRP group, n=16) and PPP gel (PPP group, n=16) were injected, no treatment was given in the control group (n=16). The flap survival rate was measured at 7 days. Histological and real-time PCR were used to count the inflammatory cells and blood vessel density, and to detect the expressions of vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), PDGF-AA, and PDGF-BB mRNA at 8 hours, 24 hours, 3 days, and 7 days. Results Platelet counting showed platelet in group A was the highest. ELISA evaluation showed that the concentrations of TGF-β1 and PDGF-BB were significantly higher in PRP than in PPP and serum (P lt; 0.05). The flap survival rate was 61.2% ± 9.1% in PRP group, showing significant differences (P lt; 0.05) when compared with that in PPP group (35.8% ± 11.3%) and control group (28.0% ± 5.4%). The inflammatory cells were significantly lower and the blood vessel density was significantly higher in PRP group than in PPP group and control group (P lt; 0.05). When compared with PPP group and control group, the expressions of VEGF and PDGF-BB increased at all time after operation in PRP group; the expression of EGF increased within 24 hours; and the expression of PDGF-AA increased after 3 days. There were significant differences in PDGF-AA mRNA at 3 days and 7 days, PDGF-BB mRNA at 8 hours, VEGF mRNA at 24 hours and 3 days, and EGF mRNA at 24 hours between PRP group and PPP and control groups (P lt; 0.05). Conclusion 200 × g centrifuge for 15 minutes and 500 × g centrifuge for 10 minutes is the best PRP preparation method. PRP can improve the skin flap survival by regulating the genes involved in angiogenesis.
ObjectiveTo investigate the effect of natural hirudin combined with hyperbaric oxygen therapy on the survival of transplanted random-pattern skin flap in rats.MethodsA random-pattern skin flap in size of 10.0 cm×2.5 cm was elevated on the dorsum of 72 Sprague Dawley rats. Then the 72 rats were randomly divided into 4 groups (n=18) according to the therapy method. At immediate and within 4 days after operation, the rats were treated with normal saline injection in control group, normal saline injection combined with hyperbaric oxygen treatment in hyperbaric oxygen group, the natural hirudin injection in natural hirudin group, and the natural hirudin injection combined with hyperbaric oxygen treatment in combined group. The flap survival was observed after operation, and survival rate was evaluated at 6 days after operation. The skin samples were collected for histological analysis, microvessel density (MVD) measurement, and evaluation of tumor necrosis factor α (TNF-α) expression level by the immunohistochemical staining at 2 and 4 days after operation.ResultsPartial necrosis occurred in each group after operation, and the flap in combined group had the best survival. The survival rate of flap was significantly higher in hyperbaric oxygen group, natural hirudin group, and combined group than that in control group, and in combined group than in hyperbaric oxygen group and natural hirudin group (P<0.05). There was no significant difference between hyperbaric oxygen group and natural hirudin group (P>0.05). At 2 days, more microvascular structure was observed in hyperbaric oxygen group, natural hirudin group, and combined group in comparison with control group; while plenty of inflammatory cells infiltration in all groups. At 4 days, the hyperbaric oxygen group, natural hirudin group, and the combined group still showed more angiogenesis. Meanwhile, there was still infiltration of inflammatory cells in control group, inflammatory cells in the other groups were significantly reduced when compared with at 2 days. At 2 days, the MVD was significantly higher in hyperbaric oxygen group, natural hirudin group, and combined group than that in control group (P<0.05); the expression of TNF-α was significantly lower in hyperbaric oxygen group, natural hirudin group, and combined group than that in control group (P<0.05). There was no significant difference in above indexes between hyperbaric oxygen group, natural hirudin group, and combined group (P>0.05). At 4 days, the MVD was significantly higher in hyperbaric oxygen group, natural hirudin group, and combined group than that in control group, in natural hirudin group and combined group than in hyperbaric oxygen group (P<0.05). The expression of TNF-α was significantly lower in hyperbaric oxygen group, natural hirudin group, and combined group than that in control group, in combined group than in natural hirudin group and hyperbaric oxygen group (P<0.05).ConclusionHyperbaric oxygen and natural hirudin therapy after random-pattern skin flap transplantation can improve the survival of flaps. Moreover, combined therapy is seen to exhibit significant synergistic effect. This effect maybe related to promotion of angiogenesis and the reduction of inflammation response.