[Preparation of platelet-rich plasma gel and its effect on skin flap survival of rat].

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 x g centrifuge for 15 minutes, and 500 x g centrifuge for 10 minutes; in group B, 312 x g centrifuge for 10 minutes, and 1 248 x g centrifuge for 10 minutes; and in group C, 200 x g centrifuge for 15 minutes, and 200 x 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 beta1 (TGF-beta1) 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 x 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-beta1 and PDGF-BB were significantly higher in PRP than in PPP and serum (P < 0.05). The flap survival rate was 61.2% +/- 9.1% in PRP group, showing significant differences (P < 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 < 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 < 0.05).

CONCLUSION

200 x g centrifuge for 15 minutes and 500 x 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.