Platelet-Rich Plasma (PRP) is a low-cost procedure to deliver high concentrations of autologous growth factors (GFs). Platelet activation is a crucial step that might influence the availability of bioactive molecules and therefore tissue healing. Activation of PRP from ten voluntary healthy males was performed by adding 10% of CaCl2, 10% of autologous thrombin, 10% of a mixture of CaCl2 + thrombin, and 10% of collagen type I. Blood derivatives were incubated for 15 and 30 minutes and 1, 2, and 24 hours and samples were evaluated for the release of VEGF, TGF-
Tissue repair in musculoskeletal injuries is often a slow and sometimes incomplete process, with patient suffering pain and limited function, and therefore it is accompanied by high costs to society, in terms of both money spent on healthcare and also loss of work. Thus, many efforts have been made in order to investigate new approaches to increase the regenerative potential and favour tissue healing. Since several studies have underlined the role of growth factors (GFs) in the regulation of normal tissue structure and the reaction to tissue damage, their use is thought to be useful in clinical practice to promote rapid healing with high quality tissue and allow an early and safe return to unrestricted activity [
Platelets constitute a reservoir of critical GFs and cytokines which may govern and regulate the tissue healing process. The bioactive molecules secreted by platelet
However, despite the numerous benefits ascribed to PRP and the promising results reported for its therapeutic potential, the clinical outcomes are heterogeneous and sometimes contradictory. These controversial findings are due to both the different clinical protocols applied, making it difficult to compare results and draw conclusions about its real efficacy, and even more so the lack of standardization in PRP preparation procedures. This has led to the availability of a huge number of products differing in terms of cell types and quantity and therefore GF and cytokine content and release times. Among the several variables affecting PRP releasate, platelet activation is a crucial step that might influence the availability of bioactive molecules and therefore tissue healing [
The term “activation” refers to 2 key processes that are initiated during PRP preparation: (1) degranulation of platelets to release GFs from
The aim of the present study is therefore to compare different strategies to activate PRP, by evaluating the content of both GFs and cytokines, as well as their release kinetics.
This study was approved by the local Ethics Committee and the Institutional Review Board, and each donor signed an informed written consent. PRP, Platelet-Poor Plasma (PPP), and autologous thrombin were obtained from ten voluntary healthy men (mean age ± SD:
PRP, PPP, and autologous thrombin were prepared by a whole blood separator (Angel, Cytomedix Inc., Gaithersburg, MD). For PRP preparation, 150 mL of venous blood was drawn from each donor and transferred into an Angel centrifuge chamber and centrifuged for 25 min at two different speeds: at 3500 rpm for the first 3 minutes and at 3000 rpm for the remaining time. Then, PRP was extracted from the buffy-coat into an empty sterile syringe. PPP was collected from another bag and transferred into a new syringe. Autologous thrombin was prepared starting from PPP according to the manufacturer’s instructions.
The platelet concentration and the white blood cell (WBC) number of PRP, PPP, and peripheral blood (PB) were analysed with an automated blood cell counter (COULTER LH 750 Haematology Analyzer Beckman Coulter SRL, Milan, Italy). Linearity was 5–1000 × 103/
Activation of PRP was performed by adding 10% of CaCl2 (final concentration 22.8 mM), 10% of autologous thrombin, 10% of a mixture of CaCl2 + thrombin, and 10% of collagen type I (final concentration 4
PRP and PPP were evaluated for the release of VEGF, TGF-
All continuous normally distributed data were expressed in terms of the mean and the standard deviation of the mean; the median was used for not normally distributed ones. The Kolmogorov Smirnov test was performed to test normality of continuous variables. The area under the curve of release at every time of measurement was calculated for each activation method to quantify the amount and kinetics of the released molecules. The Repeated Measures General Linear Model (GLM) with Sidak test for multiple comparisons was performed to assess the differences at different follow-up times in each activation method. The Repeated Measures GLM was also used to assess the influence of the different activation methods.
For all tests,
All statistical analyses were performed using SPSS v. 19.0 (IBM Corp., Armonk, NY, USA).
The median number of platelets per cubic millimeter was
CaCl2, thrombin, CaCL2/thrombin, and collagen type I induced a different platelet aggregation. In particular, PRP activated with CaCl2, thrombin, and CaCL2/thrombin formed clots detected in the 15-minute evaluation and persisting up to 24 hours (thrombin and CaCL2/thrombin already macroscopically stable at 15 minutes, CaCl2 starting at 15 and visually stabilized at 30 minutes), whereas in collagen-type-I-activated samples no clot formation was noticed for any of the time points evaluated (Figure
Clot formation at 24 h in the different activation groups.
No detectable levels of IL-1
Significantly lower amounts of GFs were detected in nonactivated PRP and PPP compared to the differently activated PRP (
The effects of the different activation methods on PRP GF release are shown in Figure
TGF, PDGF, and VEGF release kinetics according to the activation method.
At 15 and 30 minutes, thrombin and CaCl2/thrombin produced a significantly higher amount of PDGF with respect to that of CaCl2 (
At 15 minutes, thrombin and CaCl2/thrombin showed a greater amount of TGF-
At 15 and 30 minutes, thrombin and CaCl2/thrombin produced a significantly higher amount of VEGF with respect to that of CaCl2 and collagen type I (
The release pattern of PRP activated with CaCl2 was similar for all the GFs evaluated, with a significant and progressive release of GFs starting from 15 minutes and increasing up to 24 hours (
The main finding of our study is that the activation modality influences PRP clot formation, leading to differences in terms of both amount and release kinetics of platelet-derived GFs.
The most commonly used activation methods in the current clinical practice [
Collagen is a weak platelet activator, which results in a lower amount of GFs released with respect to the other activation methods. This is a key aspect to bear in mind, since GFs are potent molecules and even small variations might affect the results in the tissue healing process [
Besides the overall higher amount of released GFs with the other activation strategies used in the clinical practice, the comparison of the amount of molecules detected at each time point underlined another key factor related to PRP activation: the different release kinetics. This is of major importance and may also affect the treatment outcome. In fact, a rapid activation has been associated with a decrease in the total amount of GFs available at the tissue site over time [
The study results highlighted that thrombin alone and in combination with CaCl2 and collagen type I (even if at lower level in this case) presented similar kinetics, stimulating a rapid release of GFs that remains stable up to 24 h. Similarly, comparing the PRP releasate induced by thrombin or collagen type I, Fufa et al. [
Conversely, CaCl2 showed a gradual release over time, with a lower initial level followed by a progressively increasing amount of GFs released, reaching similar or even higher levels at the 24-hour evaluation.
Another important aspect for the clinical application of blood derivatives is their physical form, which may range from liquid to solid gel allowing both surgical augmentations as well as minimally invasive injective PRP delivery. Concerning this, the study underlined how different activators influence platelet aggregation. In particular, the use of CaCl2 induced clot formation within 30 minutes of its addition, whereas thrombin and CaCl2/thrombin caused a more rapid clot formation, which was already detectable at the 15-minute evaluation. Interestingly, collagen-type-I-activated platelet concentrates exhibited far less aggregation, with no visible clots up to 24 hours. This result is partially in contrast to that obtained by Fufa et al. [
Finally, besides the differences in clot formation and GFs amount and release kinetics, another interesting finding that emerged from the study analysis regards the lack of influence of the activation methods on the inflammatory molecules in the releasate. The PRP used in this experimental setting is a leukocyte-rich PRP, which is currently being debated for the potentially deleterious effects of proteases and reactive oxygen species released by the white blood cell component. In fact, whereas some authors consider leukocytes to be a beneficial source of cytokines and enzymes that may be important for the prevention of infection, others attribute better results to formulations with leukocyte depletion [
The study results showed that, even in this leukocyte-rich PRP, none of the selected activators was able to induce an inflammatory releasate, as demonstrated by the lack of IL-1
This study has some limitations that need to be discussed. In fact, today little is known about the concentration of calcium, thrombin, or collagen needed to trigger the optimal release of GFs, and different concentrations may lead to different results. For example, it has been reported that high concentrations of calcium and thrombin trigger an immediate and significant increase in TGF-
The results of this study confirm the importance of the method chosen to activate PRP, by determining both its physical form and the amount and release kinetics of GFs. It is not only the presence of GFs that dictates the level of healing response, but also the ability of targeting the treatment area, thus modulating cells with an appropriate dosage and in a timely manner [
Giuseppe Filardo is consultant and receives institutional support from Finceramica Faenza SpA (Italy), Fidia Farmaceutici SpA (Italy), and CartiHeal (2009) Ltd. (Israel). He is a consultant for EON Medica SRL (Italy). He receives institutional support from IGEA Clinical Biophysics (Italy), BIOMET (USA), and Kensey Nash (USA). Elizaveta Kon is a consultant for CartiHeal (2009) Ltd. (Israel) and has stocks of CartiHeal (2009) Ltd. (Israel). She is a consultant and receives institutional support from Finceramica Faenza SpA (Italy). She receives institutional support from Fidia Farmaceutici SpA (Italy), IGEA Clinical Biophysics (Italy), BIOMET (USA), and Kensey Nash (USA). Maurilio Marcacci receives royalties and research institutional support from Fin-Ceramica Faenza SpA (Italy). He receives institutional support from Fidia Farmaceutici SpA (Italy), CartiHeal (2009) Ltd. (Israel), IGEA Clinical Biophysics (Italy), BIOMET (USA), and Kensey Nash (USA). All the other authors declare that there are no competing interests regarding the publication of this paper.
The authors thank Dr. Elettra Pignotti for statistical assistance and Mr. Keith Smith for English editing, Rizzoli Orthopaedic Institute, Bologna, Italy. This work was supported by grants from “