Systemic effects after local injection of platelet-rich plasma: a prospective randomized study

Background 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. Design: prospective randomized study Results The results showed no signicant 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 between the two groups at baseline, nor at 1, 2, or 7 days after intervention. However, a signicant decrease in the serum substance P level 1 and 7 days after PRP injection was observed. Regarding urinary concentrations of metabolites of anabolic androgenic steroids (AAS), no between-group differences at baseline, nor at 1, 2, or 7 days after intervention, were observed. Conclusions Our study failed to observe signicant surge of serum anabolic molecules and urinary excretion of anabolic AAS metabolites after PRP injection. 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), DHEA (dehydroepiandrosterone), 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 further derivatized with 50 µL of N-methyl-N-trimethyl-silyltriuoroacetamide (MSTFA) solution for 30 min at 60 °C. Finally, the to gas chromatographic analysis (7890A Network GC system) and mass spectrometric analysis (5975C Network Mass Selective


Background
Tendinopathy is a signi cant problem in sport and can interfere with and, in some instances, end an athletic career (1). 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 (2). 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 (3). These bene cial anabolic molecules include, but are not limited to, growth hormone (GH), insulin-like growth factor-1 (IGF-1), insulin growth factor-BP3 (IGF-BP3), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), basic broblast growth factor (bFGF), granulocyte-colony stimulating factor (G-CSF), hepatocyte growth factor (HGF), and transforming growth factor-beta 1 (TGF-β1) (4). TGF-β1 has been known to exert anabolic effects on cartilage and anti-in ammatory effects on synovial tissues (5).
In the sporting competitions, the term "doping" is de ned 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 (WADA) prohibited list, but were later removed in 2011 due to a lack of strong evidence regarding performance bene ts. 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. No prospective randomized study has demonstrated systemic effects after localized PRP injection, and there is no evidence to support or refute whether the use of PRP could lead to misinterpretation of blood or urinary doping tests.
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 (AAS) 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.

Methods
This double-blinded, placebo-controlled trial was performed from Jan 1st, 2017 to Dec 31st, 2017 in accordance with CONSORT guideline. Ethical approval was granted by the Institutional Review Board of the Chang Gung Memorial Hospital (CGMH-IRB 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 con rm 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 (MIBO) guideline proposed by Murray IR et al (6).
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 con rmed through the provocation test, ultrasonography or MRI by at least two of three orthopedic surgeons (J.Y.K, C.C.H and W.Y.C).

Process of randomization
The unblinded independent research assistant (Y.T.Z) 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 after intervention. After 1 hour 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 (PRP) Autologous platelet-rich plasma (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 revolutions per minute (rpm) for 8 minutes, 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 to 10 times. Finally, we collect 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 minutes 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-in ammatory medications during the rst 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.

Quanti cation of the concentrations of serum biomarkers
A blood sample of approximately 20 mL was taken from the brachial vein of the untreated limb 1 hour 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 hours after eating or training [12,18].

Results
Thirty male Taiwanese athletes ful lling 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-in ammatory 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 nal analysis. The owchart of our study is summarized in FIGURE 1. The gender, age, body mass index, maximal heart rate, training frequency, and chronic diseases were similar in the two groups ( Table 1).
The serum levels of selected biomarkers, including GH, IGF-1, IGFBP-3, VEGF, PDGF-BB, and SP, showed no between-group differences at baseline, nor at 1, 2, or 7 days after intervention (Table 2).  (Table 4). These results suggested that injection of PRP may lead to signi cant changes in the serum level of SP.  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 at baseline, nor at 1, 2, or 7 days after intervention (  (Table 6). These results indicated that PRP injection did not lead to signi cant changes in the urinary excretion of AAS metabolites.  Regarding clinical parameters, no signi cant 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).

Discussion
In this study, no signi cant 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 at baseline, nor at 1, 2, or 7 days after intervention. However, signi cant 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 at baseline, 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 (8 (10). 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 concentration ratio during PRP preparation, the volume of injected PRP, 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, betweengroup differences could not be demonstrated at baseline, 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 (11). 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 (11). 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 labelling local PRP injection as doping.
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 ndings of our study need to be supplemented by the performance data in the following studies.

Conclusions
Our study failed to observe signi cant surge of serum anabolic molecules and urinary excretion of anabolic AAS metabolites after PRP injection. Signi cant decrease in the serum substance P level could The authors declare that they have no competing interests. Funding: CMRPG8B1301-CRRPG8B1303 and CRRPG8F0461 -CRRPG8F0463 from Chang Gung Medical Foundation.
Authors' contributions: SJ and CC wrote the manuscript. WY, KK, and JY recruited the patients. GP, SF, and TC performed the laboratory experiments. Figure 1 The ow diagram of the study. The processed of recruitment, randomization, and intervention of the participants were summarized in this diagram.