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Exercise and the platelet activator calcium chloride affect the growth factor content of platelet-rich plasma (PRP): overlooked biochemical factors that may affect PRP therapy.

There is strong evidence that exercise affects platelet hemostasis factors, but this potential effect on growth factor concentrations in platelet-rich plasma (PRP) has never been studied. In addition, there are few studies focusing on the effects of activating agents used with PRP. The first aim of this study was to examine the effect of exercise on the concentrations of platelet and platelet-derived growth factors (PDGF)-AB, hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), and vascular endothelial growth factor and PRP (VEGF). is to evaluate. It was also to examine the effect of the activating agent calcium chloride (CaCl2) on growth factor concentration in relation to different exercise states. Methods Controlled laboratory study. Ten healthy participants performed a maximum of six 1-hour exercises in which blood was withdrawn immediately before, after, and 18 hours later. PRP was prepared in both active CaCl2 and inactive form under all conditions. PDGF AB, HGF, IGF-1 and VEGF concentrations were evaluated using standard ELISA systems.
Exercise had no significant effect on platelet concentration, but significantly suppressed both VEGF and PDGF-AB concentrations. Exercise status had no significant effect on IGF-1 or HGF concentration. Activation by CaCl2 resulted in a significant increase in PDGF-AB and IGF-1 concentrations, unchanged VEGF and significantly reduced HGF concentrations. Results Exercise significantly affects PDGFs in PRP with significantly reduced concentrations of VEGF and PDFG-AB. Moreover, activation of PRP with CaCl2 results in a differentiated GF release from platelets. These relevant factors could potentially affect the outcome in daily clinical practice and are recommended to be taken into account in future study design.

A recent meta-analysis of the efficacy of autologous platelet-rich plasma (PRP), widely used for 14 musculoskeletal indications, showed conflicting evidence regarding its use, mainly due to deficiencies in standardization of study protocols and confounding factors affecting platelet administration. The authors strongly recommend basic science studies focusing on optimal preparation, dosage, effects of activating agents, and timing of injecting autologous blood products. Despite its apparent clinical popularity and an increasing number of clinical trials, there is still only limited understanding of the role of cellular and extracellular elements, optimal platelet concentrations, leukocytes, and released growth factor (GF) dose, timing, and activation. Platelet-derived GF (PDGF) is stored in α-granules within platelets and is selectively released upon activation. Platelet activation is dependent on specific platelet membrane glycoproteins that bind to ligands, kinase activation, and cytoplasmic calcium influx from both the dense tubular system and the extracellular environment, and can be initiated in vivo by a number of factors such as thrombin, calcium, collagen and shear stress. Each platelet contains approximately 80 α-granules containing, in addition to GF, adhesive proteins, chemokines, fibrinolytic proteins and anticoagulant molecules. In vitro, calcium and thrombin are routinely used to induce GF release from PRP; In clinical practice, pre-activation of PRP is widely used. However, there is no evidence and consensus on the therapeutic necessity for pre-injection activation. GF released from the α-granules of platelets is hypothesized to provide the regenerative benefits of PRP. Hematological studies have shown that exercise can affect platelet function by increasing the release of platelet-derived procoagulant microparticles following exercise. In conclusion, it is possible that exercise may also affect the release of GF from platelets, thereby affecting the clinical efficacy of PRP. The first aim of this study was to evaluate the effect of exercise on platelet and PDGFs PDGF-AB hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF-1) and vascular endothelial growth factor (VEGF) concentrations in PRP. . The second aim was to examine the effect of the activating agent calcium chloride (CaCl2) on GF concentration in relation to different exercise states.

Eleven white male patients were recruited after a thorough explanation of all the risks and benefits of participation in the study. Patients were then excluded from the analysis if they suffered from any type of injury or were taking any medication that could affect platelet function. The study protocol was approved by our Institutional Medical Ethics Committee. All patients gave written informed consent.

Experimental overview
Each participant was asked to report not exercising for 36 hours and fasting for 12 hours (excluding water) to the exercise laboratory. On arrival at the controlled environment laboratory (~21°C, 40–60% RH, 760–770 mm Hg), body mass and participation in sports were recorded. Resting blood pressure was measured and venous blood (10 ml) was drawn from an antecubital vein. Participants then performed a modified26 six-maximum bike test on an electronically braked bicycle ergometer (Lode Excalibur Sport, Groningen, The Netherlands) to estimate maximum aerobic capacity and peak power output (PPO). A single researcher oversaw all cycling tests. After 7 days, the first laboratory procedure was repeated with the same fasting and exercise avoidance protocol. Before the exercise, 54 ml of blood was drawn from the participants and this blood was prepared just as described below. Participants were then asked to exercise for 1 hour at 50% of their estimated PPO (based on previous testing). An additional 54 ml of blood was sampled immediately after cessation of exercise. Participants were advised to avoid exercise; 18 hours after the exercise bout, another 54 ml of blood was drawn from each subject for analysis.

Hematological analysis
For immediate analysis of the whole blood platelet count, a blood sample was taken from the antecubital vein with 10 ml drawn into an EDTA-coated tube. All platelet analyzes were completed using the CELL DYN 3700 SL analyzer (Coulter Count; Abbot Diagnostics, Chicago, USA). PRP was prepared from the GPS III centrifugal separation system (Biomet Recover, GPS III Platelet Separation System) using the manufacturer's instructions. In a 60 ml syringe, 54 ml of whole blood was added to 6 ml of ACD-A anticoagulant and immediately centrifuged at 3200 rpm for 15 minutes. Extraction of PRP and platelet-poor plasma (PPP) was completed according to the method specified in the commercially available separation system. Volumes of PRP and PPP were recorded and each sample was aliquoted into 500 µl with an aliquot used for platelet concentration (PC) analysis as shown above. The remaining samples were either stored immediately at -80°C or activated with CaCl 2 (25 mM) for 1 hour at 37°C. After activation, the samples were centrifuged at 4000 rpm at 4°C for 10 minutes and the fibrin clot was separated from the supernatant. The supernatant was aliquoted and frozen at -80°C until analysis. Following both inactive and in vitro activation of PDGF-AB, HGF, IGF-1 and VEGF in PRP, GF levels were assessed by commercially available ELISA (R&D systems, Oxon, UK). All inter- and intra-assay coefficients of variation were <10%.

Statistical analysis
All statistical analyzes were performed using SPSS (V.19.0). The data were scanned for outliers and deviation from normality and therefore no transformation was applied. In addition, using a linear mixed model with fixed effect terms for different GFs (HGF, IGF, PDGF-AB and VEGF), activation state (active or inactive) and time group (0 hour) in PPP and PRP, respectively. analyzed. (rest), immediately after exercise and 18 hours after exercise) and random effect terms for participants and residuals. The effect size (ES) was calculated as partial η2. ES of 0.01, 0.06, and 0.14 indicate small, medium, and large association, respectively. Where a significant effect was found, post hoc pairwise comparisons were made. Statistical significance was accepted as p<0.05.

One participant was excluded from the study due to concomitant use of a platelet-suppressing drug (clopidogrel), and then data from 10 participants (mean age 34.3 ± 3.2 years; mean body mass index 26.1±4.28 kg/m2) were analysed. All participants were physically fit and regularly engaged in moderate to intense physical exercise. Mean PC in whole blood increased significantly in expected ranges (271.9 ±54.0×103 ml), PRP (1065.3 ±4144.8×103 ml) and PPP (43.2 ±16.3 ml) ×103 ml) was significantly reduced. Mean white blood cell concentration (WBC) increased significantly from baseline whole blood levels (7.34 ±1.72×103 ml) in PRP (34.06±11.06 × 103 ml) and increased in PPP (0, 04±0.04 × 103 ml) decreased significantly. In PRP, it was not associated with concentrations of PC, HGF, VEGF, or IGF-1. PC was positively associated with PDGF-AB (p<0.001); Each unit increase in PC is associated with an increase of 25.2 units in PDGF.
Effect of exercise status on PRP
There was no significant change from rest to post-exercise conditions in PRP PC (Fig. 1). Analysis, exercise status PDGF-AB (F (2.41.9)=4.0; p=0.025; ES=0.311) and VEGF (F (2.42.3)=3.7; p=0.034; ES) =0.456), with significant suppression in both VEGF (rest vs 1 hp=0.024; and vs 18 hp=0.001) and PDGF-AB (rest vs 1 hp=0.021; and vs 18 hp=0.003) concentration (fig. 2).

PRP Kalsiyum aktivatör, CGF Sistemi, CGF Nedir, PRP Nedir, Platelet activation with calcium chloride, Kalsiyum Klorür aktivasyonu PRP
PRP, CGF, Trombosit aktivasyonu

Activation status on PDGF-AB (F (1.41.5)=63.6; p<0.001; ES=0.312) and VEGF (F (2.42.1)=4.1; p=0.023; ES) There was a significant interaction effect between and duration. =0.398).

This research shows that exercise can affect the concentration of GF in PRP and that activation of PRP by CaCl2 results in a differentiated GF release. These are important observations because of the assumption that GF in PRP provides regenerative stimulation to tissues. However, we still have limited understanding of the multitude of factors that may influence GF concentrations and subsequent outcomes of PRP treatment. The platelet counts, WBC, and platelet increase factor observed in this study are comparable to previous reports using the same separation system.28 29 600 000– 1,000 000 platelet/ml levels are considered appropriate for PRP, but there is little clinical evidence of which level is optimal. . Contrary to previous studies, our finding that platelet count in PRP was not associated with three out of four GF concentrations casts doubt on the clinical significance of absolute platelet count. Remarkably, increased WBC was observed in this PRP preparation. The benefits of high or low WBC in PRP remain clinically unclear and may ultimately depend on clinical indication. While previous authors reported a 25% increase in whole blood PC immediately following strenuous exercise, no significant changes were found in either whole blood or PRP PC following our exercise protocol. However, we observed that exercise had a significant effect on GF concentrations in PRP. It is well known that platelet a-granules are heterogeneous in their content and that different stimuli may cause different granule and factor release. Furthermore, it has been previously shown that there will be an increase in platelet-derived factors involved in hemostasis in response to exercise-induced shear stress. Our finding that exercise significantly reduces circulating PDGF, VEGF, and PDFG-AB concentrations, but has no effect on HGF concentrations, supports this differential content release from α-granules. IGF-1, which is not the primary component of platelet α-granules, was also unaffected by strenuous exercise. Based on this finding, it appears that exercise can confuse α-granule release and exercise affinity should be considered when preparing PRP and reporting clinical results. Whether a change in GF concentration of the magnitude shown would be clinically significant remains to be determined. Although quality evidence for the clinical use of PRP for chronic degenerative tendon indications is lacking, the potency of GFs in acute muscle injuries has not yet been studied and remains unknown. Factors such as timing, dose, and relative rates of GF may be relevant; Anything that changes the GF content of PRP can have a confusing effect on the results. Both the necessity and rationale for preinfiltration activation of PRP in the clinical setting are controversial. One school of thought suggests that activation prior to administration produces optimal levels of GF release,37 while the opposite argument suggests that platelets will respond to the in vivo environment and release appropriate GF. While each of these arguments has a theoretical basis, there is no clinical evidence to describe an answer. Our data imply that activation of PRP by CaCl2 resulted in a specific cellular response with increased PDGF-AB and IGF-1 plasma concentrations but paradoxically decreased HGF concentrations and had no effect on VEGF concentrations. The reason for the paradoxical decrease in HGF concentration is unknown. Although it cannot be excluded that the release of HGF-containing α-granules is inhibited by CaCl2, this seems highly unlikely. We cannot rule out a technical error and further research is required to test the reproducibility of the data presented. Similarly, IGF-1 is not typically considered a component of α-granules. The mechanism by which activation with CaCl2 increases IGF-1 concentration has not yet been determined, but it can be considered as a non-platelet effect. From a clinical point of view, differential release of GF has no relation to the clinical setting in which PRP will subsequently leak. Further work is required to delineate the consequences of prefilter activation relative to the in vivo demands of the tissue. An obvious limitation of this study is that only four GFs were evaluated in 10 male participants. Another confounding factor in this study may be that some GFs remain in the activated clot and are therefore not evaluated in the supernatant, thus an underestimation of actual GF release. Different concentrations of GF and a wide range of concentrations in response to both exercise and activation by CaCl2 suggest that further research is needed in this area. Its validity in the female population is unknown, as platelet function and activity may be affected by hormonal factors. We recommend that exercise status be clearly defined in any future study design.

This study demonstrates that exercise can affect GF concentrations in PRP with significantly reduced VEGF and PDFG-AB concentrations. Furthermore, activation of PRP with CaCl2 (Calcium Chloride) resulted in a differentiated GF release from platelets. These potentially confounding factors may affect clinical outcomes and should be considered both in future study design and when evaluating clinical efficacy.

New Findings from this Study;

▸ This study demonstrates that exercise can suppress both vascular endothelial growth factor and platelet-derived growth factor-AB concentrations in PRP, thus demonstrating the importance of knowing the exercise status of subjects and patients when observing and reporting clinical outcomes.
▸ Furthermore, activation by CaCl2 resulted in a distinct, inhomogeneous change in GF concentration. This latest finding is an important consideration for researchers trying to establish the mechanisms behind any PRP activity and highlights the complexity of this biological tool.
▸ This new study adds important information for clinicians and researchers using PRP and highlights the need for more detailed in vivo and in vitro research in a wider population.

In summary;

In the future, administration of GF in the form of autologous blood products should target specific GFs at precise times during the healing process. This study highlights that exercise may affect GF concentration and thus the efficacy of PRP, and this may need to be considered in the future.

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