The Use And The Probable Mechanism Of Autologous Platelet Gel To Treat Refractory Diabetic Dermal Ulcer

Part one:The use of autologous platelet gel to treat refractory diabetic dermal ulcer—A preliminary studyObjective: To evaluate the feasibility, efficacy and security of autologous platelet gel (APG) in the treatment of refractory diabetic dermal ulcer.Methods: APG was produced by platelet-rich plasma (PRP) with thrombin and calcium gluconate. 13 cases with refractory diabetic dermal ulcers were enrolled in treatment with APG from July 2005 to April 2007. APG treatment consisted of wound dressing with APG, followed by topical washing and cleaning. The APG was then covered with Vaseline gauze and left for 72 hours, after which the ulcers were treated by standard care. The standard care consisted of daily topical washing, cleaning, debridement and dressing changing of the wounds. Patients underwent antibiotic therapy were required. The clinical endpoints of the study were the healing rate, and/or the time required to bring about adequate tissue regeneration in order to undergo reconstructive plastic surgery.Results: 69.2% ulcers were cured, 15.4% of patients were no response; the ulcers area were reduced significantly in the first 3 weeks. No adverse reactions were observed.Conclusion: Topical therapy with APG may be considered as an effective adjuvant method in treating refractory diabetic dermal ulcer.Part two:The preparation of autologous platelet gel and the platelet quantification in PRP and the growth factors analysis from APGObjective:①To compare the platelet enrich ratio in the PRP prepared by the different centrifgual methods, search for the best effective centrifgual method.②To analyse the growth factors released from APG.Methods:①Preparation of PRP: The patient's venous blood samples were drawn into steriled centrifuge tube. The superstratum plasma was sucted after the first centrifugalization, and the superstratum plasma was centfifugated again, the plasma of underlayer was defined of PRP. 3 centrifgual methods were selected: A: 2600rpm for 4 minutes and 3800rpm for 6 minutes. B: 2000rpm for 4minutes and 4000rpm for 6 minutes. C: 1500rpm for 5 minutes and 4000rpm for 5 minutes, counting the blood cell before and after centrifugalization.②Preparation of APG: The PRP mixed with Thrombin-calcium for APG.③To measure the PDGF-BB, VEGF, IGF-1, EGF and TGF-β1 in the APG and the whole blood using the Enzyme-linked immunoadsordent assay method.Results:①The B method was the best centfifgual method, the average platelet concentration was 1363.80×10~9/L and the platelet recovery rate was 75.2%.②The average platelet concentration in PRP was 957.63×10~9/L, an average 4.35-fold increase in platelet concentration was found in the PRP compared with that of whole blood.③The concentration of growth factors all increased with increasing platelet number. However, growth factor concentration varied from patient to patient. On average for the whole blood as compared with PRP, the IGF—1 concentration increased from 14.54±35.34 ng/ml to 110.56±84.36 ng/ml, the EGF concentration increased from 160.73±71.10pg/ml to 265.95±138.43pg/ml, the PDGF-BB concentration increased from 145.94±133.24pg/ml to 503.81±197.86pg/ml, the TGF—β1 concentration increased from 3.31±2.27ng/ml to 5.67±4.80 ng/ml. No increase was found for VEGF.④There were positive correlation between the platelet concentration and PDGF-BB, TGF—β1.Conclusion: The B method is the best centrifgual method. A variety of therapeutic growth factors were detected and released from the platelets in significant levels in APG. There were positive correlation between the platelet concentration and PDGF-BB, TGF—β1.Part three:The probable mechanism of autologous platelet gel to treat the refractory diabetic dermal ulcerObjective: By measuring the growth factors concentration in the granulation tissue pre-treatment and post-treatment of APG, to investigate the probable mechanism of APG to treat refractory diabetic dermal ulcer.Methods: The PDGF-BB, VEGF, IGF-1, EGF and TGF-β1 in the granulation tissues were detected by ELISA of pre-treatment and 3, 6, 9, 15 days after APG treatment.Results: The concentration of 5 growth factors all began to increase in the granulation tissues after 3 days treatment, on average for pre-treatment as compared with post-treatment, the VEGF concentration increased 2.1-fold, the IGF-1 concentration increased 1.95-fold, the EGF concentration increased 1.75-fold, the PDGF-BB concentration increased 1.89-fold and the TGF-β1 concentration increased 1.67-fold. The ulcers areas were reduced obviously in 3 days and in 9-15 days after APG treatment.Conclusion: The one of the main mechanism of APG to treat the refractory diabetic dermal ulcer may be that the growth factors of granulation tissue increase after APG treatment.