|Year : 2020 | Volume
| Issue : 6 | Page : 294-300
Systemic effects of platelet-rich plasma local injection on serum and urinary anabolic metabolites: A prospective randomized study
Shu-Jui Kuo1, Wen-Yi Chou2, Chieh-Cheng Hsu2, Guo-Ping Chang-Chien3, Su-Fan Lin3, Kai-Kit Siu2, Tsai-Chan Tsai4, Jih-Yang Ko5, Yi Chih Sun4
1 School of Medicine, China Medical University; Department of Orthopedic Surgery, China Medical University Hospital, Taichung, Taiwan
2 Department of Orthopedic Surgery, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan
3 Super Micro Mass Research and Technology Center, Cheng Shiu University, Kaohsiung, Taiwan
4 Center for Shockwave Medicine and Tissue Engineering, Department of Medical Research, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan
5 Department of Orthopedic Surgery; Center for Shockwave Medicine and Tissue Engineering, Department of Medical Research, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung; Department of Orthopedic Surgery, Xiamen Chang Gung Hospital, Fujian, China
|Date of Submission||25-Oct-2020|
|Date of Decision||20-Nov-2020|
|Date of Acceptance||25-Nov-2020|
|Date of Web Publication||26-Dec-2020|
Dr. Jih-Yang Ko
No. 123, Dapi Road, Niaosong District, Kaohsiung City 833
Source of Support: None, Conflict of Interest: None
Platelet-rich plasma (PRP) is widely utilized in the treatment of sports injuries. However, potential systemic effects after localized PRP injection are unclear at present. In this prospective randomized study, 24 Taiwanese male athletes with tendinopathy were randomized into a PRP group (n = 13) or a saline group (n = 11). The concentrations of serum and urine biomarkers were quantified by enzyme-linked immunosorbent assay assessment as well as gas chromatographic and mass spectrometric analysis, respectively. The results showed no significant differences in serum levels of growth hormone, insulin-like growth factor-1, insulin-like growth factor-binding protein 3, vascular endothelial growth factor, platelet-derived growth factor-BB, or serum substance P (SP) between the two groups before intervention, nor at 1, 2, or 7 days after intervention. However, a significant decrease in the serum SP level 1 and 7 days after PRP injection was observed. Regarding urinary concentrations of metabolites of anabolic androgenic steroids (AAS), no between-group differences before intervention, nor at 1, 2, or 7 days after intervention, were observed. Our study failed to observe significant surge of serum anabolic molecules and urinary excretion of anabolic AAS metabolites after PRP injection.
Keywords: Anabolic androgenic steroids, platelet-rich plasma, substance P
|How to cite this article:|
Kuo SJ, Chou WY, Hsu CC, Chang-Chien GP, Lin SF, Siu KK, Tsai TC, Ko JY, Sun YC. Systemic effects of platelet-rich plasma local injection on serum and urinary anabolic metabolites: A prospective randomized study. Chin J Physiol 2020;63:294-300
|How to cite this URL:|
Kuo SJ, Chou WY, Hsu CC, Chang-Chien GP, Lin SF, Siu KK, Tsai TC, Ko JY, Sun YC. Systemic effects of platelet-rich plasma local injection on serum and urinary anabolic metabolites: A prospective randomized study. Chin J Physiol [serial online] 2020 [cited 2021 Jul 28];63:294-300. Available from: https://www.cjphysiology.org/text.asp?2020/63/6/294/304862
| Introduction|| |
Tendinopathy is a significant problem in sport and can interfere with and in some instances, end an athletic career. As a result, the treatment of tendinopathy is of pivotal importance for the health and career of the athletes. Platelet-rich plasma (PRP) is widely-utilized in the treatment of sports injuries, including tendinopathy. The administration of PRP can change the tissue microenvironment by providing a pool of regenerative molecules, thereby enhancing angiogenesis and activating the chemotaxis and proliferation of the regenerative cells. These beneficial anabolic molecules include, but are not limited to, growth hormone (GH), insulin-like growth factor-1 (IGF-1), IGF-BP3, platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), granulocyte-colony stimulating factor, hepatocyte growth factor, and transforming growth factor-beta 1 (TGF-β1). TGF-β1 has been known to exert anabolic effects on cartilage and anti-inflammatory effects on synovial tissues.
In the sporting competitions, the term “doping” is defined as the use of prohibited athletic enhancing agents by the competitive athletes. Blood and urine samples are frequently utilized for the doping tests. Due to concerns surrounding unfair enhancement of athletic performance in competitive sports, the use of PRP among athletes is under dispute. Platelet-derived preparations were included on the 2010 World Anti-Doping Agency prohibited list, but were later removed in 2011 due to a lack of strong evidence regarding performance benefits. In addition to the controversy surrounding the labeling of PRP use as doping, the systemic effect of localized PRP injection is far from clear at present. Despite the fact that there is high-quality study demonstrating that PRP could trigger an increase in circulating growth factors after injection, no prospective randomized study with placebo group has demonstrated differential systemic effects after localized PRP injection compared to the sham injection. There is no evidence to support or refute whether the use of PRP could lead to misinterpretation of blood or urinary doping tests at present.
The purpose of the research was to quantify the impact of local PRP injection on the serum concentrations of anabolic molecules and on the urinary excretion of anabolic androgenic steroids (AASs) metabolites, the frequently measured markers in the urinary doping tests. This prospective randomized study tried to determine PRP injection could exert different effects on the expression of serum concentrations of anabolic molecules and on the urinary excretion of anabolic AAS metabolites than the placebo saline injection, in order to determine whether PRP injection is a concern for the blood and urinary doping tests.
| Materials and Methods|| |
This double-blinded, placebo-controlled trial was performed from January 1, 2017, to December 31, 2017 in accordance with CONSORT guideline. Ethical approval was granted by the Institutional Review Board of the Chang Gung Memorial Hospital (No: 201700133A3). Patients were recruited from a sports medicine clinic held in the Department of Orthopedic Surgery, Kaohsiung Chang Gung Memorial Hospital. All of the authors identify the committee that approved the research and confirm that all research was performed in accordance with relevant guidelines/regulations, and the details of the study were reported referencing upon the Minimum Information for Studies Evaluating Biologics in Orthopaedics guideline proposed by Murray et al. Informed consent was obtained and that the rights of participants were protected for all of the participants.
Recruitment of participants
Male Asian athletes between 18 and 40 years of age with upper or lower limb tendinopathy for at least 3 months were enrolled in the study with the details of tendinopathy summarized in [Table 1]. The diagnosis of tendinopathy was confirmed through the provocation test, ultrasonography, or magnetic resonance imaging by at least two of three orthopedic surgeons (J. Y. K, C. C. H and W. Y. C).
|Table 1: Demographic data of the participants in the platelet-rich plasma group and saline groups|
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The exclusion criteria were as follows: (1) nutritional disorders, (2) hematologic or systemic diseases (e.g., anemia, metabolic disease…etc.), (3) history of hormone therapy, (4) use of anti-inflammatory agents, anti-platelet agents, or traditional Chinese herbs within the past month, (5) surgical history for current injury, and (6) biologic treatment for current injury. Patients were required to be available for all scheduled appointments during the follow-up period.
The minimum sample size required for each group was calculated before the study. The priori power calculation (G*Power 18.104.22.168 software: http://www.gpower.hhu.de/en.html) utilized a 1-tailed Wilcoxon signed-rank test to calculate the sample size of at least 9 for each group (calculated effect size: 1.2; α level: 0.05; power: 80%; allocation ratio: 1).
Eventually, 30 male Taiwanese athletes fulfilling the inclusion criteria were evaluated in terms of their eligibility for enrollment in this study, and 6 were excluded due to recent use of anti-inflammatory agents, abnormal biochemical studies, or recent injuries. The remaining 24 participants were randomized into the PRP group (n = 13) or the saline group (n = 11). Two participants in the saline group failed to complete all the surveys, and so the results of the remaining 22 participants were included for final analysis. The flowchart of our study is summarized in [Figure 1]. The gender, age, body mass index, training frequency, and chronic diseases were similar in the two groups [Table 1].
|Figure 1: The flow diagram of the study. The processes of recruitment, randomization, and intervention of the participants were summarized in this diagram.|
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Process of randomization
One independent research assistant randomized the eligible participants into the PRP group or the saline group using suitable computer software. The participants randomized into the PRP group were treated with 4 mL PRP, while the participants in the saline group received a saline injection of equal volume. On the day of intervention, each subject donated a 20 mL blood sample and a 50 mL urine sample before intervention. After 1 h of preparation, the research assistant selected the correct syringe and blinded the content with the use of a covering sheath to surround the syringe and hub of the needle. To ensure concealment of the subject's group allocation, data on allocation were stored in a secret location. The content of the injection was blinded for the orthopedic physicians, researchers, and patients. Treatment-related complications and assessment of pain intensity using the Numeric Rating Scale (NRS) were recorded by the independent research assistant.
Preparation of platelet-rich plasma
Autologous PRP was prepared using the RegenKit THT system (RegenLab SA, Le Mont-sur-Lausanne, Switzerland) following the manufacturer's instructions. Medical technicians, who had been well-trained by the manufacturer, were responsible for the process of PRP preparation. For each patient, 8–10 mL of venous blood was drawn and collected to the commercial RegnLab THT tube, which contained 1 mL sodium citrate. After single centrifugation at 3400 rpm for 8 min, 4–5 mL of PRP was yielded with leukocytes maintained at physiological levels and red blood cells depleted. Then, the blood components were separated, with the platelet pellet resting on the separating gel. PRP for later application was obtained by re-suspending the platelet pellet in the plasma supernatant by gently inverting the unopened RegenKit THT tube 5–10 times. Finally, we collected the supernatant fraction using the syringe equipped with a 5 mL Luer Lok syringe without any activating agent. All the steps were performed in room air and were completed within 60 min before the injection. The whole blood characters were counted in Kaohsiung Chang Gung Memorial Hospital.
The three orthopedic surgeons (J. Y. K, W. Y. C and C. C. H) delivered 4 mL PRP or saline to the respective intratendinous lesions. Patients were advised not to take anti-inflammatory medications during the first 7 days after the injection. Physical therapy or heavy training was forbidden for 7 days after the intervention, and the daily activities were allowed. Individualized rehabilitation was instructed and supervised by a physical therapist 7 days after the intervention.
Quantification of the concentrations of serum biomarkers
A blood sample of approximately 20 mL was taken from the brachial vein of the untreated limb at 1 h before and at 1, 2, and 7 days after PRP injection. To mitigate the confounding effects of diurnal variation and the metabolic effects of diet and acute bouts of exercise, blood was drawn at precisely the same time each morning between 8 and 10 am and at least 6 h after eating or training.
The blood specimen was centrifuged at 3000 × 3 for 10 min and then stored at −80°C until enzyme-linked immunosorbent assay (ELISA) assessment. The concentrations of target proteins were assessed using a Quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA) following the manufacturer's instructions. Results were calculated by interpolation from a standard curve established from graded concentrations of GH (DGH00, R&D Systems), IGF-1 (DG100, R&D Systems), IGFBP-3 (DGB300, R&D Systems), PDGF-BB (DBB00, R&D Systems), VEGF (DVE00, R&D Systems), and substance P(SP) (KGE007, R&D Systems).
Quantification of doping substances in urine
Doping substances in urine, mainly metabolites of AAS, were quantified in the laboratory of the Super-Micro Mass Research and Technology Center, Cheng Shiu University, Taiwan. The metabolites of AAS included testosterone (17β-hydroxyandrost-4-en-3-one), epitestosterone (17α-hydroxy-4-androsten-3-one), androsterone (4-androsten-3,17-dione), etiocholanolone (3α-hydroxy-5β-androstan-17-one), dehydroepiandrosterone (DHEA), dihydroandrosterone (5α-androstane-3α,17β-diol), and etiocholane-3α,17β-diol (5β-androstane-3α,17β-diol). Each urine sample (6 mL) was mixed with 50 μL standard solution (5000 ng/mL methyltestosterone and androsterone-D4 6000 ng/mL) and 1 mL of phosphate buffer, and the mixture was heated for 60 min at 50°C. After cooling at room temperature, liquid–liquid extraction was performed, and phase separation was achieved. The organic extract was evaporated to dryness, and the dried residue was further derivatized with 50 μL of N-methyl-N-trimethyl-silyltrifluoroacetamide solution for 30 min at 60°C. Finally, the sample was subjected to gas chromatographic analysis (7890A Network GC system) and mass spectrometric analysis (5975C Network Mass Selective Detector) for quantification of doping substances of interest.
All data were expressed as the median (lower quartile and upper quartile). Categorical variables were compared using the Chi-square test. Due to the small sample size and the assumption of normality is not met even under normal probability plot and data transformation, the non-parametric tests were employed for our study. The Mann–Whitney U-test was utilized to compare inter-group differences. The Friedman test was utilized for repeated-measures analysis of repeated within-group comparisons for continuous variables, and the Wilcoxon signed-rank test was used for post hoc analysis. The corrections for multi-comparisons were performed for all pertinent tests. Statistical analysis was performed using PASW Statistics Statistical software (version 10.0; SPSS, Chicago, IL, USA). A result was considered statistically significant at P < 0.05.
| Results|| |
The serum levels of selected biomarkers, including GH, IGF-1, IGFBP-3, VEGF, PDGF-BB, and SP, showed no between-group differences before intervention, nor at 1, 2, or 7 days after intervention [Table 2].
|Table 2: Comparison of serum biomarkers between the platelet-rich plasma and the saline groups at all time points|
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The P values of the Friedman test for GH, IGF-1, IGFBP-3, VEGF, PDGF-BB, and SP in the PRP group were 0.819, 0.764, 0.403, 0.059, 0.231, and <0.001, respectively [Table 3]. The Wilcoxon signed-rank test was used for post hoc analysis of the serum level of SP in the PRP group, and the P values for baseline–Day 1, baseline–Day 2, baseline–Day 7, Day 1–Day 2, Day 1–Day 7, and Day 2–Day 7 were 0.016, 0.423, <0.001, 0.027, 0.034, and <0.001, respectively [Table 4]. These results suggested that injection of PRP may lead to significant changes in the serum level of SP.
|Table 3: Comparison of serum biomarkers with baseline values after platelet-rich plasma injection|
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|Table 4: The P value for the post hoc analysis of serum level of substance P for the platelet-rich plasma group by Wilcoxon signed-rank test|
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With regards to the urine concentrations of selected AAS metabolites, including testosterone, epitestosterone, androsterone, etiocholanolone, DHEA, dihydroandrosterone, and etiocholane-3α,17β-diol, no between-group differences were observed before intervention, nor at 1, 2, or 7 days after intervention [Table 5]. The P values of the Friedman test for testosterone, epitestosterone, androsterone, etiocholanolone, DHEA, dihydroandrosterone, and etiocholane-3α,17β-diol were 0.873, 0.742, 0.123, 0.270, 0.819, 0.753, and 0.896, respectively [Table 6]. These results indicated that PRP injection did not lead to significant changes in the urinary excretion of AAS metabolites.
|Table 5: Comparison of urine anabolic androgenic steroid metabolites between platelet.rich plasma and saline group at each time point|
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|Table 6: Comparison of urine anabolic androgenic steroid metabolite with baseline values after platelet-rich plasma injection|
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Regarding clinical parameters, no significant between-group difference in NRS was observed between the PRP and saline group. There were no adverse responses associated with the injection in the two groups [Table 7].
|Table 7: Comparison of numerical rating scale between platelet-rich plasma and saline groups|
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| Discussion|| |
In this study, no significant differences in the serum levels of GH, IGF-1, IGFBP-3, VEGF, PDGF-BB, or SP were observed between the PRP group and saline group before intervention, nor at 1, 2, or 7 days after intervention. However, significant decreases in the serum SP level were observed 1 and 7 days after PRP injection. Regarding urinary AAS metabolites, including testosterone, epitestosterone, androsterone, etiocholanolone, DHEA, dihydroandrosterone, and etiocholane-3α,17β-diol, no between-group differences before intervention, nor at 1, 2, or 7 days after intervention, were observed. These results have not been reported before and merit note.
PRP has been utilized in the treatment of musculoskeletal diseases, such as osteoarthritis, with favorable functional outcomes of the injected site, despite the fact that the pertinent studies were not free from bias. However, the systemic effects after PRP injection are inconclusive at present. Banfi et al. observed significant modifications of serum VEGF, EGF, and CCL2 levels 30 min after PRP injection, which returned to baseline within 24 h. Wasterlain et al. centrifuged 30–60 mL whole blood to yield 3–6 mL PRP. Serum levels increased significantly for IGF-1 at 24 and 48 h, bFGF at 72 and 96 h, and VEGF at 3, 24, 48, 72, and 96 h after PRP injection. In addition, VEGF was increased in all 25 patients after PRP treatment. These studies were all longitudinal observational studies without control groups. In our study, we observed no between-group differences in the serum levels of all selected biomarkers nor in the urinary excretion of AAS metabolites. We did not observe increases in the serum levels of IGF-1, bFGF, or VEGF after PRP injection. The discrepancies between the results of our study and previous studies may be due to differences in the brand-specific characteristics, including range of concentration ratio and the volume of injected PRP, as well as in the subject-specific characteristics, including the vascularity of the injection sites and the differences in the demographic compositions.
We did observe a substantial decrease in the serum SP level after local PRP injection. However, between-group differences could not be demonstrated before intervention, nor at 1, 2, or 7 days after intervention. A decrease in the serum level of SP has not been reported previously. Previous study has suggested a positive correlation between the systemic SP level and pain nociception. Lisowska et al. found that among patients with rheumatoid arthritis, the extent of pain after total knee arthroplasty was positively correlated with the serum level of SP. The decrease in the serum SP level after PRP injection could be partially explained by decreased nociception due to the healing process after PRP injection. Reviewing the literature, there is no evidence of a correlation between the serum SP level and athletic performance. As a result, the decreased serum SP level after PRP injection does not constitute evidence in support of labeling local PRP injection as doping.
The AAS are the chemical, synthetic derivatives of testosterone with enhanced anabolic potentials. The anabolic effects of AAS involve muscles, bones, heart, and kidneys. These organs possess little 5α-reductase activity and thus AAS enhance protein synthesis, muscle fiber formation, erythropoiesis, and bone growth. AAS could also antagonize glucocorticoid receptors and inhibit muscle protein catabolism, leading to the muscle building effect. However, AAS could exert side effects on cardiovascular, hepatic, endocrine/reproductive, and psychological aspects, and is on the International Olympic Committee's list of banned substances. Urine sample collection was a routine part of the doping control procedure for AAS overuse since 1970s, and any therapeutics for the athletes should not interfere with the interpretations of urinary doping tests. The current study failed to demonstrate significant changes in the urinary excretion of AAS metabolites after PRP injection. Our results should offer peace of mind for the athletes requiring PRP treatment in terms of urinary doping tests.
There are limitations to our study. The heterogeneity of the disease composition might jeopardize the comparability of the two groups. The small volume of yielded PRP by the RegenKit THT system did not allow for the component characterization for every participant. The relatively small sample size might make the study underpowered. The findings of the nonsignificant differences of outcome variables between the PRP and saline group may be due to the small sample size. The findings of our study need to be supplemented by the performance data in the following studies.
| Conclusions|| |
Our study failed to observe significant surge of serum anabolic molecules and urinary excretion of anabolic AAS metabolites after PRP injection. Significant decrease in the serum SP level could be observed 1 and 7 days after PRP injection.
We are thankful for the help by the Super Micro Mass Research and Technology Center, Cheng Shiu University for the quantification of doping substances in the urine. We are also thankful for the help by the Center for Shockwave Medicine and Tissue Engineering, Department of Medical Research, Kaohsiung Chang Gung Memorial Hospital, Taiwan as well as the Core Laboratory for Phenomics and Diagnostics, Kaohsiung Chang Gung Medical Foundation, Taiwan.
Financial support and sponsorship
The study was supported by grants CMRPG8B1301-CRRPG8B1303 and CRRPG8F0461-CRRPG8F0463 from Chang Gung Medical Foundation.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Reinking M. Tendinopathy in athletes. Phys Ther Sport 2012;13:3-10.
Fice MP, Miller JC, Christian R, Hannon CP, Smyth N, Murawski CD, et al
. The role of platelet-rich plasma in cartilage pathology: An updated systematic review of the basic science evidence. Arthroscopy 2019;35:961-76.
Andia I, Sanchez M, Maffulli N. Tendon healing and platelet-rich plasma therapies. Expert Opin Biol Ther 2010;10:1415-26.
Foster TE, Puskas BL, Mandelbaum BR, Gerhardt MB, Rodeo SA. Platelet-rich plasma: From basic science to clinical applications. Am J Sports Med 2009;37:2259-72.
Kuo SJ, Liu SC, Huang YL, Tsai CH, Fong YC, Hsu HC, et al
. TGF-β1 enhances FOXO3 expression in human synovial fibroblasts by inhibiting miR-92a through AMPK and p38 pathways. Aging (Albany NY) 2019;11:4075-89.
Wasterlain AS, Braun HJ, Harris AH, Kim HJ, Dragoo JL. The systemic effects of platelet-rich plasma injection. Am J Sports Med 2013;41:186-93.
Murray IR, Geeslin AG, Goudie EB, Petrigliano FA, LaPrade RF. Minimum Information for Studies Evaluating Biologics in Orthopaedics (MIBO): Platelet-rich plasma and mesenchymal stem cells. J Bone Joint Surg Am 2017;99:809-19.
Kuo SJ, Hsu HC, Wang CJ, Siu KK, Hsu YH, Ko JY, et al
. Effects of computer-assisted navigation versus conventional total knee arthroplasty on the levels of inflammation markers: A prospective study. PLoS One 2018;13:e0197097.
Morgan CJ. Use of proper statistical techniques for research studies with small samples. Am J Physiol Lung Cell Mol Physiol 2017;313:L873-7.
Dai WL, Zhou AG, Zhang H, Zhang J. Efficacy of platelet-rich plasma in the treatment of knee osteoarthritis: A meta-analysis of randomized controlled trials. Arthroscopy 2017;33:659-70.
Banfi G, Corsi MM, Volpi P. Could platelet rich plasma have effects on systemic circulating growth factors and cytokine release in orthopaedic applications? Br J Sports Med 2006;40:816.
Lisowska B, Siewruk K, Lisowski A. Substance P
and acute pain in patients undergoing orthopedic surgery. PLoS One 2016;11:e0146400.
Saudan C, Baume N, Robinson N, Avois L, Mangin P, Saugy M. Testosterone and doping control. Br J Sports Med 2006;40 Suppl 1:i21-4.
Mottram DR, George AJ. Anabolic steroids. Baillieres Best Pract Res Clin Endocrinol Metab 2000;14:55-69.
Kuhn CM. Anabolic steroids. Recent Prog Horm Res 2002;57:411-34.
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]