Platelet-rich plasma preparation for regenerative medicine: optimization and quantification of cytokines and growth factors

Abstract

IntroductionPlatelet-rich plasma (PRP) is nowadays widely applied in different clinical scenarios, such as orthopedics, ophthalmology and healing therapies, as a growth factor pool for improving tissue regeneration. Studies into its clinical efficiency are not conclusive and one of the main reasons for this is that different PRP preparations are used, eliciting different responses that cannot be compared. Platelet quantification and the growth factor content definition must be defined in order to understand molecular mechanisms behind PRP regenerative strength. Standardization of PRP preparations is thus urgently needed.MethodsPRP was prepared by centrifugation varying the relative centrifugal force, temperature, and time. Having quantified platelet recovery and yield, the two-step procedure that rendered the highest output was chosen and further analyzed. Cytokine content was determined in different fractions obtained throughout the whole centrifugation procedure.ResultsOur method showed reproducibility when applied to different blood donors. We recovered 46.9 to 69.5% of total initial platelets and the procedure resulted in a 5.4-fold to 7.3-fold increase in platelet concentration (1.4 × 106 to 1.9 × 106 platelets/μl). Platelets were highly purified, because only <0.3% from the initial red blood cells and leukocytes was present in the final PRP preparation. We also quantified growth factors, cytokines and chemokines secreted by the concentrated platelets after activation with calcium and calcium/thrombin. High concentrations of platelet-derived growth factor, endothelial growth factor and transforming growth factor (TGF) were secreted, together with the anti-inflammatory and proinflammatory cytokines interleukin (IL)-4, IL-8, IL-13, IL-17, tumor necrosis factor (TNF)-α and interferon (IFN)-α. No cytokines were secreted before platelet activation. TGF-β3 and IFNγ were not detected in any studied fraction. Clots obtained after platelet coagulation retained a high concentration of several growth factors, including platelet-derived growth factor and TGF.ConclusionsOur study resulted in a consistent PRP preparation method that yielded a cytokine and growth factor pool from different donors with high reproducibility. These findings support the use of PRP in therapies aiming for tissue regeneration, and its content characterization will allow us to understand and improve the clinical outcomes.

Interaction of Components in 3D PRF Matrix and Neuronal Cells In Vitro

“…PDGF (platelet-derived growth factor), TGFβ (transforming growth factor beta), IGF (insulin-like growth factor), VEFG (vascular endothelium growth factor) and EGF (epidermal growth factor) are the most important of them. 4,5 Angiogenesis, cell proliferation, differentiation and formation of extracellular matrix are stimulated by growth factors. An important component of platelet α-granules is also prothrombin that plays an important role in the platelet aggregation.…”

Autogenic platelet-rich plasma: Types, activation methods, and applications

“…Jednym z ich zadań jest udział w pierwotnej i wtórnej hemostazie, zapobiegając wynaczynieniu krwi w czasie urazu tkanek z uszkodzeniem ciągłości naczyń krwionośnych. Aktywacja płytek krwi i uruchomienie kaskady krzepnięcia prowadzą do uformowania w miejscu urazu skrzepu, w którym oprócz trombocytów znajdują się erytrocyty oraz białe krwinki (2,4,24).…”

“…Pobudzają również odbudowę naczyń krwionośnych, co w połączeniu z innymi właści-wościami umożliwia procesy naprawy i regeneracji tkanek organizmu. Działanie to odbywa się na drodze autokrynnej, parakrynnej oraz endokrynnej, a magazynem dla wspomnianych czynników są: cytoplazma trombocytów, ziarnistości alfa, ziarnistości gęste i lizosomy (2,4,5,15,28).…”

“…czynnikom wzrostu, zmagazynowanym głównie w ziarnistościach alfa trombocytów (2,4,28). Czynniki wzrostu są niejednorodną grupą białek wydzielanych poza trombocytami również przez inne tkanki organizmu (tj.…”

Extracellular Vesicles from Platelet-Rich Plasma as Conveyors of Regeneration Potential in Orthopedics

“…For example, the study of Nugraha et al (2013) suggested that the first step of centrifugation should be made by applying the centrifuge acceleration 1300.g (g=9.81 m/s 2 is the earth's gravitational acceleration) for 5 minutes and the second step by applying the centrifuge acceleration 2300.g for 7 minutes to obtain the highest increase of platelet count in the plasma [3]. Both steps are usually performed in refrigerated centrifuge [4]. Platelets can then be activated in shear stress (by augmentation of the surface of contact with the moving PRP) [5] and injected to the site of the lesion.…”