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Figure 1

Figure 1

PRP sample collection and preparation method. (A) blood draw, (B) centrifugation and collection, (C) mechanical activation by passing through a 0.22 µm filter.

Figure 2

Figure 2

A comparison of the cytokine and growth factor concentrations in thrombin or mechanically activated human PRP samples. Concentrations of interleukin (IL)-4, IL-10, IL-13, basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF), and vascular endothelial growth factor (VEGF) were compared in PRP samples activated by thrombin (thrombin) or by applying mechanical force through a 0.22 µm filter (filtered). Data are expressed as the mean ± standard error of the mean (SEM). Two-tailed unpaired t-tests showed no significant difference between the methods. ns = not significant.

Figure 3

Figure 3

A comparison of the cytokine and growth factor concentrations in the mechanically activated human PRP samples freshly collected or stored for nine months at -80°C. Concentrations of (IL)-4, IL-10, IL-13, basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF), vascular endothelial growth factor (VEGF), transforming growth factor beta 1 (TGFb1) and insulin-like growth factor (IGF1) were compared in mechanically activated human PRP samples freshly collected or stored at -80°C for nine months previously. Data are expressed as the mean ± standard error of the mean (SEM). Two-tailed unpaired t-tests showed no significant difference between the methods. ns = not significant.

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Abstract

Platelet-rich plasma (PRP) therapy is employed to treat damaged connective tissues and osteoarthritis. PRP is collected in the presence of an anticoagulant to avoid premature activation. The PRP is then activated by various activation methods that all have regulatory or cost drawbacks. Additionally, activated PRP can only be stored for a limited time. The purpose of this study was to assess the biological stability of a PRP composition obtained from platelets of healthy volunteers using a mechanical activation by passing PRP through a 0.22 µm filter and stored for up to nine months. The PRP fraction was isolated and then activated using either mechanical or thrombin techniques: nine samples were evaluated in each experiment. The concentrations of interleukin (IL)-4, IL-10, IL-13, platelet derived growth factor, transforming growth factor beta 1, insulin-like growth factor 1, and vascular endothelial growth factor were compared in samples that were freshly collected and samples that were previously stored at -80°C for nine months. Protein concentration analysis showed no statistically significant differences in the composition of the PRP when the platelets were activated by thrombin or mechanical activation. There was also no statistically significant difference in the concentration of cytokines and growth factors in the PRP autologous composition after storage for nine months at -80°C. Mechanical activation is an efficient method to activate PRP, and the PRP-derived autologous composition is capable of being stored for up to nine months ithout affecting the concentration of the analyzed proteins.

Introduction

Autologous platelet-rich plasma (PRP) treatment appears to provide a safe method for the treatment of osteoarthritis and other musculoskeletal conditions.1x1Patel, S, Dhillon, MS, Aggarwal, S, Marwaha, N, and Jain, A. Treatment with platelet-rich plasma is more effective than placebo for knee osteoarthritis: a prospective, double-blind, randomized trial. Am J Sports Med. 2013; 41: 356–364

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Inflammatory cytokines, including interleukin (IL)-1, IL-6, IL-8, and tumor necrosis factor-alpha (TNF-α), play a role in the pathogenesis of osteoarthritis by exerting catabolic effects on the surrounding joint tissues, such as cartilage and bone.4x4Fernandes, JC, Martel-Pelletier, J, and Pelletier, JP. The role of cytokines in osteoarthritis pathophysiology. Biorheology. 2002; 39: 237–246

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This catabolic effect can be successfully blocked by platelet-derived inhibitory cytokines, such as IL-4, IL-10, and IL-13.4x4Fernandes, JC, Martel-Pelletier, J, and Pelletier, JP. The role of cytokines in osteoarthritis pathophysiology. Biorheology. 2002; 39: 237–246

PubMed
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It has been reported that PRP is also a source of various bioactive regenerative proteins, including growth factors such as platelet derived growth factor (PDGF), fibroblast growth factor (FGF), and transforming growth factor beta 1 (TGFβ-1).5x5Eppley, BL, Woodell, JE, and Higgins, J. Platelet quantification and growth factor analysis from platelet-rich plasma: implications for wound healing. Plast Reconstr Surg. 2004; 114: 1502–1508

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PRP is a heterogeneous term encompassing different preparations of platelets due to the varied production protocols. Although PRP treatment has been proven effective in various medical conditions,1x1Patel, S, Dhillon, MS, Aggarwal, S, Marwaha, N, and Jain, A. Treatment with platelet-rich plasma is more effective than placebo for knee osteoarthritis: a prospective, double-blind, randomized trial. Am J Sports Med. 2013; 41: 356–364

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it remains controversial in the broader medical community due to a lack of standardization and optimization with respect to its preparation methods, detailed cytokine analysis, and treatment protocols. Platelet activation – the release of bioactive molecules secreted by platelet α-granules – can be triggered by the production protocols; therefore, there may be varying concentrations of growth factors and cytokines in the PRP dependent on the production protocol utilized.11x11Anitua, E, Andia, I, Ardanza, B, Nurden, P, and Nurden, AT. Autologous platelets as a source of proteins for healing and tissue regeneration. Thromb Haemost. 2004; 91: 4–15

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,12x12Ehrenfest, D, Rasmusson, L, and Albrektsson, T. Classification of platelet concentrates: from pure platelet-rich plasma (P-PRP) to leucocyte- and platelet-rich fibrin (L-PRF). Trends Biotechnol. 2009; 27: 158–167

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PRP fractions are collected in the presence of an anticoagulant, such as sodium citrate, to avoid premature activation of the platelets13x13Anitua, E, Prado, R, Sánchez, M, and Orive, G. Platelet-rich-plasma: Preparation and formulation. Oper Tech Orthop. 2012; 22: 25–32

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. Platelet activation can then be achieved through several methods, such as addition of calcium chloride (CaCl) or thrombin, freezing and thawing of platelets, and the use of ultrasonic waves11x11Anitua, E, Andia, I, Ardanza, B, Nurden, P, and Nurden, AT. Autologous platelets as a source of proteins for healing and tissue regeneration. Thromb Haemost. 2004; 91: 4–15

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, 12x12Ehrenfest, D, Rasmusson, L, and Albrektsson, T. Classification of platelet concentrates: from pure platelet-rich plasma (P-PRP) to leucocyte- and platelet-rich fibrin (L-PRF). Trends Biotechnol. 2009; 27: 158–167

Abstract | Full Text | Full Text PDF | PubMed | Scopus (1051)
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, 13x13Anitua, E, Prado, R, Sánchez, M, and Orive, G. Platelet-rich-plasma: Preparation and formulation. Oper Tech Orthop. 2012; 22: 25–32

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, 14x14Davey, G and Lüscher, F. Actions of thrombin and other coagulant and proteolytic enzymes on blood platelets. Nature. 1967; 216: 857–858

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, 15x15Cavallo, C, Roffi, A, Grigolo, F, Mariani, E, Pratelli, L, Merli, G et al. Platelet-Rich Plasma: The Choice of Activation Method Affects the Release of Bioactive Molecules. Biomed Res Int. 2016; 2016: 6591717

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; however, each of these activation methods has drawbacks. Thus, the addition of CaCl changes the chemical composition of the final PRP product. Thrombin is a key regulator of robust platelet activation in response to vascular injury: is perhaps the most effective activator of platelets ex vivo14x14Davey, G and Lüscher, F. Actions of thrombin and other coagulant and proteolytic enzymes on blood platelets. Nature. 1967; 216: 857–858

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, however, homologues or xenoproteins of thrombin can lead to an allergic reaction or be a source of pathogens. Additionally, repeated freeze-thaw cycles can lead to the degradation of biologically active proteins, such as PDGF and TGFβ-1.15x15Cavallo, C, Roffi, A, Grigolo, F, Mariani, E, Pratelli, L, Merli, G et al. Platelet-Rich Plasma: The Choice of Activation Method Affects the Release of Bioactive Molecules. Biomed Res Int. 2016; 2016: 6591717

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Finally, there are regulatory issues with the use of ultrasonic waves since this technique does not meet the minimum manipulation regulatory requirements applicable in Canada and the United States of America for PRP treatment approval. Here we compare a mechanical PRP activation method using a small pore cell filter with effective ex vivo platelet activator thrombin14x14Davey, G and Lüscher, F. Actions of thrombin and other coagulant and proteolytic enzymes on blood platelets. Nature. 1967; 216: 857–858

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to create an efficient protocol for activating platelets.

Activated PRP can typically only be stored for about six hours at room temperature before losing biological activity18x18Ho, CH and Chan, IH. The influence of time of storage, temperature of storage, platelet number in platelet-rich plasma, packed cell, mean platelet volume, hemoglobin concentration, age, and sex on platelet aggregation test. Ann Hematol. 1995; 71: 129–133

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; However, some PRP therapy methods require multiple blood draws (at each treatment visit), which is invasive, time-consuming, and costly. To mitigate these concerns, mainly to enhance treatment safety by significantly reducing the risk of insertion-site infection and potential contamination of collected blood, we propose a new PRP preparation method allowing storage (up to nine months) of mechanically activated PRP at -80°C for future use. Previous research has compared growth factor and cytokine concentrations in fresh and frozen PRP stored for an extended period of time and reported a significant decrease in insulin-like growth factor 1 (IGF1), PDGF and TGFβ-1 concentrations in frozen storage conditions.19x19McClain, AK and McCarrel, TM. The effect of four different freezing conditions and time in frozen storage on the concentration of commonly measured growth factors and enzymes in equine platelet-rich plasma over six months. BMC Vet Res. 2019; 15: 292

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,20x20Roffi, A, Filardo, G, Assirelli, E, Cavallo, C, Cenacchi, A, Facchini, A et al. Does Platelet-Rich Plasma Freeze-Thawing Influence Growth Factor Release and Their Effects on Chondrocytes and Synoviocytes?. Biomed Res Int. 2014; 2014: 692913

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The aim of this study is to evaluate the effect of prolonged storage at -80°C of mechanically activated PRP protein concentrations compared to baseline concentrations.

Material and Methods

Ethics Statement

this study was approved by the Chesapeake International Review Board (CR00082114). The study population included healthy males or females between the ages of 21 and 60 with a body weight of at least 110 pounds. 12 patients were recruited voluntarily, and informed consents were signed.

PRP sample collection and preparation

Venous blood samples (9.5cc) were collected in polystyrene tubes (B.D.) containing 0.5cc 4% sodium citrate (Baxter, JB7747) (Figure 1A) and centrifuged at 7500 rpm for 30 S (Figure 1B). A standard PRP activation method includes the use of a single dose of thrombin or CaCl, stimulating a rapid release of cytokines and growth factors. Normal platelets are 1.5–3 µm in diameter; therefore, applying mechanical force by passing the platelet concentrate through a 0.22 µm pore size polyethersulfone membrane will physically damage the platelet membranes initiating the release of the granule content. Harvested PRP was passed through a 0.22 µm filter (Millipore, SLGP033RS) (Figure 1C). Bovine thrombin (Cedarline, 528-50-EA) was added as a control to inactivated samples: 100 units per 1cc PRP. Samples were aliquoted as 2.5 mL portions under sterile, laminar flow conditions and stored at -80°C for nine months. Nine samples were used for each assay. All samples were analyzed in triplicate. The effect of storing the mechanically activated PRP for nine months at -80°C on the concentrations of IL-4, IL-10, IL-13, VEGF, PDGF, bFGF, TGFβ-1 and IGF1 levels was analyzed by comparing frozen samples to fresh mechanically activated PRP.

Figure 1 Opens large image

Figure 1

PRP sample collection and preparation method. (A) blood draw, (B) centrifugation and collection, (C) mechanical activation by passing through a 0.22 µm filter.

Cytokines quantification

Concentrations of IL-4, IL-10, IL-13, vascular endothelial growth factor (VEGF), PDGF, IGF1, bFGF, and TGFβ-1 were quantified in experimental and control samples using an optimized multiplex test (BIO-PLEX Assay, Bio-Rad) in a Multiplex reader (Bio-Plex MAGPIX, Bio-Rad) according to the supplier's protocol. The results were analyzed using the xPONENTTM software. The absolute concentrations of the samples were determined by the construction of a standard curve for each analyte.

Statistical analysis

12 samples were analyzed for each experiment. 3 separate measurements were conducted for 3 sets of samples (triplicates). A power analysis was performed using GPower 3.0.software. The observed power results variated from 0.68 to 0.99 for statistically significant effects (p<0.05) and from 0.08 to 0.7 for p>0.05 results. Two-tailed unpaired t-tests were performed. A p-value less than 0.05 indicated significance. All statistical tests were performed using GraphPad PrismTM version 5.01. Data are expressed as the mean ± standard error of the mean (SEM).

Results

Comparative analysis of cytokine and growth factor concentrations in PRP fractions obtained by human thrombin or mechanical activation

Previously published data has shown that the release of cytokines and growth factors from platelets could also be achieved by disruption of the platelet cell membrane.16x16Wu, Y, Kanna, MS, Liu, C, Zhou, Y, and Chan, CK. Generation of Autologous Platelet-Rich Plasma by the Ultrasonic Standing Waves. IEEE Trans Biomed Eng. 2016; 63: 1642–1652

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To test this hypothesis, we compared the concentrations of IL-4, IL-10, IL-13, VEGF, PDGF, and bFGF in PRP samples activated by the two different methods: thrombin activation or by applying mechanical force via a 0.22 µm filter. As shown in Figure 2 no statistical differences were observed in any of the cytokine or growth factor concentrations between the samples; confirming the efficacy of using mechanical activation as part of the final PRP preparation.

Figure 2 Opens large image

Figure 2

A comparison of the cytokine and growth factor concentrations in thrombin or mechanically activated human PRP samples. Concentrations of interleukin (IL)-4, IL-10, IL-13, basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF), and vascular endothelial growth factor (VEGF) were compared in PRP samples activated by thrombin (thrombin) or by applying mechanical force through a 0.22 µm filter (filtered). Data are expressed as the mean ± standard error of the mean (SEM). Two-tailed unpaired t-tests showed no significant difference between the methods. ns = not significant.

The effect of prolonged storage of mechanically activated PRP without preservation agents on the concentration of anti-inflammatory cytokines and growth factors

Previous studies have pointed out differences in the protein profiles of fresh or frozen stored PRP19x19McClain, AK and McCarrel, TM. The effect of four different freezing conditions and time in frozen storage on the concentration of commonly measured growth factors and enzymes in equine platelet-rich plasma over six months. BMC Vet Res. 2019; 15: 292

Crossref | PubMed | Scopus (15)
| Google ScholarSee all References
,20x20Roffi, A, Filardo, G, Assirelli, E, Cavallo, C, Cenacchi, A, Facchini, A et al. Does Platelet-Rich Plasma Freeze-Thawing Influence Growth Factor Release and Their Effects on Chondrocytes and Synoviocytes?. Biomed Res Int. 2014; 2014: 692913

Crossref | PubMed | Scopus (64)
| Google ScholarSee all References
. Figure 3 shows that there was no change in any of the cytokine and growth factor concentrations between the fresh or previously frozen samples of mechanically activated PRP. TGFβ-1 and IGF1 are two potentially strong therapeutic agents due to their ability to suppress a chronic inflammation pathway and promote collagen synthesis.21x21Ge, R, Mo, L, Wu, R, Liu, J, Zhang, H, Liu, Z et al. Insulin-like growth factor-1 endues monocytes with immune suppressive ability to inhibit inflammation in the intestine. Sci Rep. 2015; 5: 7735

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| Google ScholarSee all References
,22x22Roberts, AB, McCune, BK, and Sporn, MB. TGF-Β1-β: regulation of extracellular matrix. Kidney Int. 1992; 41: 557–559

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. Unfortunately, previously described freeze-thawing PRP protocols were found to lead to a significant decrease in TGFβ-1 and IGF1 protein levels. Intriguingly, as shown in Figure 3 no significant differences were observed, suggesting that mechanical activation of PRP samples can provide a novel method for allowing longer term storage of PRP.

Figure 3 Opens large image

Figure 3

A comparison of the cytokine and growth factor concentrations in the mechanically activated human PRP samples freshly collected or stored for nine months at -80°C. Concentrations of (IL)-4, IL-10, IL-13, basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF), vascular endothelial growth factor (VEGF), transforming growth factor beta 1 (TGFb1) and insulin-like growth factor (IGF1) were compared in mechanically activated human PRP samples freshly collected or stored at -80°C for nine months previously. Data are expressed as the mean ± standard error of the mean (SEM). Two-tailed unpaired t-tests showed no significant difference between the methods. ns = not significant.

Discussion

The first goal of this study was to investigate a new PRP activation protocol utilizing mechanical activation. We approached this using a 0.22 µm pore filter to activate PRP since applying a mechanical force physically damages the platelet membranes initiating the release of the granule content. Previously, researchers have successfully used freeze-thaw cycles as an activation method for PRP by rupturing the platelet membrane.18x18Ho, CH and Chan, IH. The influence of time of storage, temperature of storage, platelet number in platelet-rich plasma, packed cell, mean platelet volume, hemoglobin concentration, age, and sex on platelet aggregation test. Ann Hematol. 1995; 71: 129–133

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However, the application of this approach in clinical medicine can cause certain complications due to the presence of membrane fragments in the final product that can stimulate the inflammatory process. Here we show data confirming that filtered PRP comprises of the same active protein concentrations as PRP activated by thrombin, presenting the advantage of being potentially clinically safer due to its acellular nature. Platelet activation is a step that might affect the PRP biological activity in healing processes; however, there are inconsistent reports in the literature describing the therapeutic potential of PRP endogenous (surrounded tissue) versus exogenous (thrombin, CaCl, etc.) activation23x23Zhu, Y, Yuan, M, Meng, Y, Wang, A, Guo, Q et al. Basic science and clinical application of platelet-rich plasma for cartilage defects and osteoarthritis: a review. Osteoarthritis and Cartilage. 2013; : 1627–1637

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,24x24Foster, E, Puskas, B, Mandelbaum, M, Gerhardt, M, and Rodeo, S. Platelet-rich plasma: from basic science to clinical applications. The American Journal of Sports Medicine. 2009; 37: 2259–2272

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. Although some published studies suggest that PRP does not need to be activated prior to the treatment25x25Smith, OJ, Talaat, S, Tomouk, T, Jell, G, and Mosahebi, A. An Evaluation of the Effect of Activation Methods on the Release of Growth Factors from Platelet-Rich Plasma. Plast Reconstr Surg. 2022; 149: 404–411

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,26x26Gentile, P, Cole, JP, Cole, MA, Garcovich, S, Bielli, A, Scioli, MG et al. Evaluation of Not-Activated and Activated PRP in Hair Loss Treatment: Role of Growth Factor and Cytokine Concentrations Obtained by Different Collection Systems. Int J Mol Sci. 2017; 18: 408

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, define concurrent data highlight positive effect of activation protocol employment24x24Foster, E, Puskas, B, Mandelbaum, M, Gerhardt, M, and Rodeo, S. Platelet-rich plasma: from basic science to clinical applications. The American Journal of Sports Medicine. 2009; 37: 2259–2272

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. Future in vivo studies should aim at further investigating the effect of the mechanical activation strategy to analyze the clinical outcomes.

The effect of PRP storage conditions, for example the optimal duration and temperature of freezing, on the PRP protein concentrations compared to fresh PRP has been previously investigated.19x19McClain, AK and McCarrel, TM. The effect of four different freezing conditions and time in frozen storage on the concentration of commonly measured growth factors and enzymes in equine platelet-rich plasma over six months. BMC Vet Res. 2019; 15: 292

Crossref | PubMed | Scopus (15)
| Google ScholarSee all References
,22x22Roberts, AB, McCune, BK, and Sporn, MB. TGF-Β1-β: regulation of extracellular matrix. Kidney Int. 1992; 41: 557–559

Abstract | Full Text PDF | PubMed | Scopus (450)
| Google ScholarSee all References
,27x27Shiga, Y, Kubota, G, Orita, S, Inage, K, Kamoda, H, Yamashita, M et al. Freeze-dried human platelet-rich plasma retains activation and growth factor expression after an eight-week preservation period. Asian Spine J. 2017; 11: 329–336

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,28x28Hosnuter, M, Aslan, C, Isik, D, Caliskan, G, Arslan, B, and Durgun, M. Functional assessment of autologous platelet-rich plasma (PRP) after long-term storage at −20°C without any preservation agent. J Plast Surg Hand Surg. 2017; 51: 235–239

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The authors reported that PDGF levels were no different in activated human PRP after four weeks of storage in -80°C but that its concentration decreased after that; additionally, the concentration of TGFβ-1 decreased by eight weeks.21x21Ge, R, Mo, L, Wu, R, Liu, J, Zhang, H, Liu, Z et al. Insulin-like growth factor-1 endues monocytes with immune suppressive ability to inhibit inflammation in the intestine. Sci Rep. 2015; 5: 7735

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Another study analyzed the concentrations of IGF1, PDGF, and TGFβ-1 in human CaCl activated PRP stored for up to 14 days at -20°C28x28Hosnuter, M, Aslan, C, Isik, D, Caliskan, G, Arslan, B, and Durgun, M. Functional assessment of autologous platelet-rich plasma (PRP) after long-term storage at −20°C without any preservation agent. J Plast Surg Hand Surg. 2017; 51: 235–239

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; data showed a significant decrease in all growth factors under these storage conditions.22x22Roberts, AB, McCune, BK, and Sporn, MB. TGF-Β1-β: regulation of extracellular matrix. Kidney Int. 1992; 41: 557–559

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It has previously been shown that the clot formation that accompanies thrombin and CaCl PRP activation might retain substantial concentrations of growth factors,29x29Textor, JA and Tablin, F. Activation of equine platelet-rich plasma: comparison of methods and characterization of equine autologous thrombin. Vet Surg. 2012; 41: 784–794

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which are lost upon freezing. This present study demonstrated, for the first time, that mechanically activated human PRP could be stored at -80°C for up to nine months without diminishing the PRP-derived protein concentrations.

Study limitation: The present study reports the result of sample analysis from 12 subjects. The eligibility criteria included healthy subjects and were age-restricted, which may limit generalizability: there are high variations in serum cytokine levels among individuals depending on age, sex and health status30x30Esposito, K, Nappo, F, Marfella, R, Giugliano, G, Giugliano, F, Ciotola, M et al. Inflammatory cytokine concentrations are acutely increased by hyperglycemia in humans: role of oxidative stress. Circulation. 2002; 106: 2067–2072

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,31x31Bai, MY, Chuang, MH, Lin, MF, Tang, SL, Wong, CC, and Chah, WP. Relationships of Age and Sex with Cytokine Content and Distribution in Human. Platelet Fibrin Gels. Sci Rep. 2018; 8: 10642

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. Other limitations may include the variations in commercially available PRP preparation methods and the storage time. Further studies to evaluate PRP cytokine concentration according to various commercially available methods of preparation in the heterogeneous subject populations are required for the proper PRP analysis. Changes to the protein conformation, such as the partial unfolding of protein molecules during freeze-thawing, which might affect the therapeutic benefit of frozen PRP have been previously described32x32Kueltzo, LA, Wang, W, Randolph, TW, and Carpenter, JF. Effects of solution conditions, processing parameters, and container materials on aggregation of a monoclonal antibody during freeze-thawing. J Pharm Sci. 2008; 97: 1801–1812

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. Therefore, further work will be needed to assess whether this issue affects mechanically activated PRP stored at -80°C. Future clinical studies are required to explore the clinical benefits of the presented PRP activation and storage method.

Uncited References

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Declaration of interests

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

This research was funded by the Antnor LTD Company. Antnor LTD holds a product patent and Anthony Michael Galea is a stockholder.

Appendix. Supplementary materials

References

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