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

Paola Romina Amable, Rosana Bizon Vieira Carias, Marcus Vinicius Telles Teixeira, Italo da Cruz Pacheco, Ronaldo José Farias Corrêa do Amaral, José Mauro Granjeiro, Radovan Borojevic, Paola Romina Amable, Rosana Bizon Vieira Carias, Marcus Vinicius Telles Teixeira, Italo da Cruz Pacheco, Ronaldo José Farias Corrêa do Amaral, José Mauro Granjeiro, Radovan Borojevic

Abstract

Introduction: Platelet-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.

Methods: PRP 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.

Results: Our 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 × 10(6) to 1.9 × 10(6) 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.

Conclusions: Our 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.

Figures

Figure 1
Figure 1
Platelet yield, recovery and platelet-rich plasma volume after the first blood centrifugation step. (A) Platelet yield, (B) recovery and (C) PRP1= platelet-rich plasma after the first blood centrifugation step. Values expressed as mean ± standard deviation. Different letters indicate statistically significant differences (analysis of variance followed by Tukey post-hoc test, n = 3, α = 0.05). Condition 1: 240 × g, 8 minutes, 16°C; condition 2: 360 × g, 16 minutes, 16°C; condition 3: 300 × g, 5 minutes, 12°C; condition 4: 300 × g, 12 minutes, 5°C; condition 5: 300 × g, 12 minutes, 12°C.
Figure 2
Figure 2
Temperature effect on platelet loss in platelet-poor plasma fraction after the second centrifugation step. First centrifugation was performed at the corresponding temperature: (A) 12°C and (B) 18°C. *Statistically significant differences (analysis of variance followed by Tukey post-hoc test, n = 3, α = 0.05). PPP, platelet-poor plasma.
Figure 3
Figure 3
Individual and daily variation of a platelet-rich plasma preparation at 18°C. (A) PRP2 platelet recovery and (B) PRP2 platelet yield. Samples belong to different donors and the procedure was performed by different researchers on different days. PRP2, platelet-rich plasma volume after the second blood centrifugation step.
Figure 4
Figure 4
Growth factor concentrations secreted after platelet activation. (A) Platelet-derived growth factor (PDGF)-AA, (B) PDGF-AB, (C) PDGF-BB, (D) endothelial growth factor (EGF), (E) transforming growth factor (TGF)-β1, and (F) TGF-β2. *Statistically significant differences from the plasmatic concentration (analysis of variance followed by Dunnett’s multiple comparison test, n = 6, α = 0.05). PPP, platelet-poor plasma; PRP1, platelet-rich plasma after the first blood centrifugation step; PRP2, platelet-rich plasma after the second centrifugation step; PRP2-Ca, calcium-activated PRP2; PRP2-Thr, calcium plus human thrombin-activated PRP2.
Figure 5
Figure 5
Anti-inflammatory cytokine concentration. Concentrations of (A) IL-4, (B) IL-13, (C) IFNα. *Statistically significant differences from the plasmatic concentration (analysis of variance followed by Dunnett’s multiple comparison test, n = 6, α = 0.05). PPP, platelet-poor plasma; PRP1, platelet-rich plasma after the first blood centrifugation step; PRP2, platelet-rich plasma after the second centrifugation step; PRP2-Ca, calcium-activated PRP2; PRP2-Thr, calcium plus human thrombin-activated PRP2.
Figure 6
Figure 6
Proinflammatory cytokine concentration. Concentrations of (A) IL-8, (B) IL-17, and (C) TNFα. *Statistically significant differences from the plasmatic concentration (analysis of variance followed by Dunnett’s multiple comparison test, n = 6, α = 0.05). PPP, platelet-poor plasma; PRP1, platelet-rich plasma after the first blood centrifugation step; PRP2, platelet-rich plasma after the second centrifugation step; PRP2-Ca, calcium-activated PRP2; PRP2-Thr, calcium plus human thrombin-activated PRP2.

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