Effects of autologous platelet-rich plasma in the repair of soft tissue defects of rabbits with free flap

Abstract

Objective: To explore the effects of autologous platelet-rich plasma (PRP) in the repair of soft tissue defects of rabbits with free flap. Methods: Thirty 6-month-old New Zealand white rabbits, male and female unlimited, were used to harvest blood from the heart. PRP was prepared by Aghaloo method, then free flap model with size of 5 cm×3 cm was reproduced on each ear of the rabbit. According to the random number table, one ear of each rabbit was recruited to PRP group, and the other ear was recruited to normal saline group. The base of flap on rabbit ear in PRP group was evenly spread with 1.0 mL autologous PRP, and equivalent volume of normal saline was applied to that in normal saline group. Then, the flap was replanted in situ. On post surgery day (PSD) 2, 3, 5, 7, and 14, 6 rabbits in each group were taken. The survival of flap was observed and recorded. The morphology of the basal tissue of flap was observed by hematoxylin-eosin staining. The expressions of CD31 and α smooth muscle actin (α-SMA) in the basal tissue of flap were detected by immunofluorescence method. Another 6-month-old male New Zealand white rabbit without making flap under the same experimental conditions was used for harvesting whole blood and preparing PRP. Then blood platelet count in whole blood and PRP was determined, and the content of vascular endothelial growth factor (VEGF) and transforming growth factor β (TGF-β) was detected by double-antibody sandwich enzyme-linked immunosorbent assay. Data were processed with analysis of variance of factorial design, paired sample t test, and Bonferroni correction. Results: (1) On PSD 2, the flaps of wounds of rabbits in PRP group were reddish and adhered well to the basal tissue; the flaps of wounds of rabbits in normal saline group were dark red and poorly attached to the basal tissue. On PSD 3, the flaps of wounds of rabbits in PRP group were ruddy and closely adhered to the basal tissue; the flaps of wounds of rabbits in normal saline group were scattered in the plaque-like dark red and generally attached to the base. On PSD 5, the flaps of wounds of rabbits in PRP group were reddish and closely adhered to the basal tissue, and the flaps were alive; while flaps of wounds of rabbits in normal saline group were rosy and closely adhered to the basal tissue. On PSD 7, the surface of flaps of wounds of rabbits in PRP group was covered with a medium amount of rabbit hair. The color of flap was similar to that of the surrounding skin. The flaps of wounds of rabbits in normal saline group were generally attached to the base, and the surface was only covered with a small amount of fluff. On PSD 14, the incisions were healed well in PRP group, while small wounds in normal saline group were not healed. (2) On PSD 2, inflammatory cell infiltration was observed in flaps of wounds of rabbits in both groups. On PSD 3, the flaps of wounds of rabbits in PRP group showed neovascularization, with less interstitial hemorrhage; while there were less neovascularization in the flaps of wounds of rabbits in normal saline group. On PSD 5, a medium number of inflammatory cell infiltration and a small amount of new microvessels were observed in flaps of wounds of rabbits in normal saline group. Many fibroblasts, a small amount of inflammatory cells, and scattered new microvessels were observed in flaps of wounds of rabbits in PRP group. On PSD 7, the number of new microvessels in normal saline group was significantly lower than that in PRP group. On PSD 14, the new microvessels in the flaps of wounds of rabbits in PRP group gradually matured, and a large number of fibroblasts distributed around them. Some of the newly formed microvessels in the flaps of wounds of rabbits in normal saline group were mature, and the healing was slower than that of PRP group. (3) On PSD 2, 3, 5, 7, and 14, the expressions of CD31 and α-SMA in the basal tissue of flaps of wounds of rabbits in PRP group were significantly higher than those in normal saline group (t=10.133, 5.444, 9.450, 6.986, 8.394, 14.896, 10.328, 9.295, 13.902, 10.814, P<0.01). (4) The platelet count in activated PRP of rabbits was (2 863±962)×10(9)/L, which was significantly higher than (393±49)×10(9)/L in whole blood (t=7.690, P<0.05). (5) The content of VEGF and TGF-β in activated PRP of rabbits was (564.3±3.2) and (1 143±251) pg/mL, which was significantly higher than (99.7±0.4) and (274±95) pg/mL in whole blood, respectively (t=287.390, 9.648, P<0.05 or P<0.01). Conclusions: PRP of rabbits contains high concentrations of VEGF and TGF-β. Therefore, PRP can effectively promote microvascular regeneration in free flap tissue and accelerate the survival of free flap.