| Background:Traditional debridement,dressing change,negative pressure suction and other methods have certain therapeutic effects on chronic wounds,but they often fail to achieve satisfactory results for chronic refractory wounds with poor basic conditions and a course of more than 3 months.Plateletrich plasma(PRP)is a platelet concentrate prepared after blood centrifugation.After activation of PRP,it can release a variety of growth factors(such as PDGF,VEGF,EGF,FGF,etc.)to stimulate cell growth and differentiation,angiogenesis,and thus promote wound healing.Chronic refractory wounds need to be treated with PRP many times.However,the preparation of fresh PRP not only increases the pain of patients but also consumes the energy and time of medical personnel.Therefore,to explore the appropriate storage temperature and storage time of PRP without any cryopreservation agent,and to analyze the therapeutic effect of stored PRP on chronic refractory wounds can provide a more convenient application mode for clinical application of PRP.Objective:(1)To prepare effective concentration of PRP,analyze the changes of growth factors in PRP under different storage conditions and the effects of PRP on cell proliferation and migration,so as to explore the most suitable storage conditions of PRP;(2)To analyze the therapeutic effect of PRP on patients with chronic refractory wounds who suffer from ineffective traditional treatment;(3)To compare the therapeutic effects of frozen PRP and fresh PRP on chronic refractory wounds.Methods:(1)Peripheral blood of healthy volunteers was extracted,and PRP was prepared by secondary centrifugation legal system,and platelet enrichment multiple and platelet recovery rate were calculated.(2)The levels of a platelet-derived growth factor(PDGF),vascular endothelial growth factor(VEGF),fibroblast growth factor(FGF)and epidermal growth factor(EGF)in PRP stored at 4 ℃,-20 ℃ and-80 ℃ for 0,7,14 and 28 days were detected by ELISA;(3)The effects of fresh PRP at 0.5%、1%、5% concentration and stored PRP at 4 ℃,-20 ℃ and-80 ℃ on proliferation and migration of human dermal fibroblasts were analyzed by the MTS method.(4)Patients with chronic refractory wounds whose course of the disease is more than 3 months were selected;individualized sequential comprehensive treatment were applied;patients with chronic refractory wounds who suffered from ineffectiveness in treatment were screened out,and further given fresh or storage PRP treatment once a week.(5)After 7,14,21 and 28 days of PRP treatment,the area of chronic refractory wounds,the reduction rate of the wound area,the improvement rate of the wound bed,the time of complete wound healing,VAS pain score and the adverse reactions related to PRP treatment were analyzed.(6)Fresh autologous or allogeneic PRP,storage autologous or allogeneic PRP were used to treat chronic refractory wounds for 28 days,and the reduction rate of wound area and wound bed improvement rate were analyzed and compared.Results:(1)The average platelet enrichment ratio of PRP prepared by secondary centrifugation legal system is 4.85 ± 0.22 times,and the platelet recovery rate is 72.06 ± 10.21%.(2)After 7 days of storage,the growth factors in PRP stored at all temperatures do not decrease significantly.The contents of PDGF and VEGF in PRP stored at 4 ℃ for 14 days are significantly lower than those of fresh PRP(p<0.01),The contents of VEGF in PRP stored at 4°C was significantly lower than that of PRP stored at-20°C and-80°C(P<0.05);The contents of four growth factors in PRP stored at 4 ℃ for 28 days are significantly lower than those of fresh PRP,PRP stored at-20 ℃ and PRP stored at-80 ℃(p < 0.01).Compared with PRP stored at-80 ℃ for 14 days,28 days and-20 ℃,the four growth factors do not decrease significantly(p > 0.05).(3)The proliferative effect of 1% and 5% fresh(0 day)PRP group is significantly higher than that of the 10% FBS group(p < 0.05).The effect of PRP stored at-20 ℃ and-80 ℃ for 7,14 and 28 days on fibroblast proliferation is significantly better than that of PRP stored at 4 ℃ and 10% FBS(p < 0.05),but there is no significant difference between-20 ℃ and-80 ℃ on fibroblast proliferation(p > 0.05).(4)The cell mobility of PRP cells stored at-20 ℃ or-80 ℃ for 14 days and 28 days for 12 hours is significantly higher than that of PRP stored at 4 ℃ and 10% FBS groups(all p < 0.01),but there is no significant difference in cell mobility between PRP stored at-20 ℃ and-80 ℃(p > 0.05).(5)The clinical study includes 67 patients with 74 chronic wounds.After individualized sequential comprehensive treatment,19 patients are screened out and 22 chronic refractory wounds with poor therapeutic effects are treated with PRP,of which 13 wounds are treated with fresh PRP and 9 wounds are treated with-20 ℃ stored PRP.(6)After 28 days of PRP treatment,81.82% of chronic refractory wounds have wound area reduction rate ≥ 50%,and 90.91% of chronic refractory wounds have wound bed improvement rate ≥ 50%;28 days after PRP treatment,the area of chronic refractory wounds is significantly reduced compared with that before treatment(p < 0.01).The VAS score drops from 6.68 ± 1.32 before treatment to 1.86 ± 1.67(p < 0.01).No adverse events related to PRP treatment has occurred during treatment.(7)There is no significant difference in wound reduction rate and wound bed improvement rate between fresh PRP treatment for 28 days and storage PRP treatment for 28 days(p > 0.05).Conclusion:(1)PRP still has biological activity when stored at-20 ℃ and-80 ℃ for 4 weeks,and there is no difference in its effectiveness when stored between-20 ℃ and-80 ℃;(2)PRP has a good therapeutic effect on chronic refractory wounds that are ineffective after individualized sequential comprehensive treatment;(3)Fresh autologous or allogeneic PRP,storage autologous or allogeneic PRP can effectively treat chronic refractory wounds.The effective storage and application of PRP and the application of allogeneic PRP expand the application mode of PRP and provide new references and means for the treatment of refractory wounds. |
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