Supervisors info:
Νικόλαος Δρακούλης, Αναπληρωτής Καθηγητής Φαρμακολογίας, Τμήμα Φαρμακευτικής, ΕΚΠΑ
Summary:
Wound healing is a complex process that can be divided into 4 continuous and overlapping phases, which are strictly regulated and involve the orchestrated interaction of multiple growth factors, cytokines, chemokines and cell types released at the site of injury.
Optimal healing of a skin wound requires a well-coordinated complex biological and molecular events of cell migration and proliferation, and precise cellular responses. However, this organized healing process is exacerbated by chronic wounds, including those caused by diabetes. The majority of chronic wounds are characterized by a prolonged or excessively inflammatory phase, persistent infections, and an inability of skin or epidermal cells to respond to repulsive stimuli. Managing ulcers is a huge challenge for healthcare due to their poor response to treatment. Diabetic foot is a serious chronic diabetic complication consisting of deep tissue damage associated with neurological disorders and peripheral vascular disease in the lower extremities. The incidence of diabetic foot has increased due to the global prevalence of diabetes and the prolonged life expectancy of diabetic patients. Complications of diabetic foot lead to huge costs for both society and the patients themselves. Treatments for chronic / non-chronic wounds are expensive as they require repeated treatments. Platelet-rich plasma therapy-PRP, for the healing of chronic trauma has been the subject of intense research with promising results.
Autologous PRP is prepared from the patient's blood by the process of centrifugation and platelet activation. When applied to the wound, growth factors and cytokines are introduced directly, thus normalizing the metabolic process, promoting neoangiogenesis, improving cellular metabolism and activating local immunity. This dissertation is an open 12-week study in which we monitored the effect of three PRP applications in patients with chronic and incurable wounds or ulcers. Our ultimate goal is to develop a methodology for developing an autologous PRP preparation for therapeutic purposes. Initially, both the space and the equipment used were sterilized. The materials used (pipettes, eppendorf vials) were wet sterilized and then dry sterilized (Siemens) and finally sterilized in a UV oven. (UV Stratalinker 1800, Stratagene, USA).
The laboratory was also sterilized with a UV lamp (Philips UV254nm). The centrifuge used was the Megafuge 1.0R refrigerated centrifuge. On the first day, all 10 vials of whole blood and sodium citrate of each volunteer / patient were centrifuged for 20 'at 600 rcf at 4 ° C. The supernatant was then carefully collected from each vial with a disposable pasteurized pipette, and transferred to 3 tubes of 15mL [TPPR Centrifuge Tube, Gamma Sterilized-polypropylene], taking care not to disturb the buffer. The underlying red blood cells were discarded. The total amount of plasma per patient was approximately 20 - 25 mL. The plasma vials were then placed at -80°C [SANYO ULTRA-LOW TEMPERATURE FREEZER, MDF-U5186S] and after 2 hours were transferred to + 4°C for 24 hours. On the second day, the plasma falcons were centrifuged for 15 'at 1600g. 2/3 of the supernatant was discarded and the precipitate was collected, homogenizing to the remaining 1/3. 3ml PRP were transferred to the syringe [BD EmeraldTM 5ml STERILE], ready to use.
The syringes were storred in the refrigerator for one day, or at -20°C for a longer period, up to 1 month. For longer, they should be stored at -80°C. Prior to application, it was kept for about 45 minutes in RT.
The sample was small and not large enough to be statistically processed. However, calculating the percentage reduction of the area of the ulcers, it was found in the 4 cases a significant reduction or even complete healing, as well as an improved clinical picture. The 5th case, up to week 12 had not improved, on the contrary we saw a slight increase in its size, most likely due to poor patient compliance. When the work was completed, his follow-up was pending at least until week 16, as well as the follow-up of incidents 6 and 7 that had a baseline of 30/9/20. In conclusion, the first indications were satisfactory and promising, but in the future, this protocol should be applied to a larger sample of patients, so that we can be sure of the beneficial results of PRP.
Keywords:
PRP, platelets, chronic wound, healing, ulcers
