Efficacy of Growth Factors Concentration after PRP

Efficacy of growth factors concentration (hGH, IGF-1, FGF-2, PDGF, VEGF) after autologous Platelet-rich plasma injection (PRP) on accelerating healing of proximal hamstring tear for athletes.

Ahmed Gaballah

1- Department of Sports Health Sciences, Damietta University, Damietta, Egypt.

2- Kinesiology and Health Sciences Department, Utah State University, Logan, UT, USA

Abstract

Platelet rich plasma (PRP) become popular biologically method used to accelerate healing in sports medicine and orthopaedic surgery field. PRP is concentrate the human platelets to supra-physiologic levels. It is an autologous producing high level of the platelets concentration centrifuged from the peripheral vein. Then it re-injected under the ultrasound gaudiness during surgery or at a site of injury. METHOD: Seventeen physically active males (age 22.0±0.6) with acute hamstring strain injuries divided to 8 case group and 9 matched controls (age 21.6±2.8) were recruited as research participants. Case group participants were injected with single 3 ml of extracted PRP under ultrasound gaudiness. However, Blood samples were collected by venipuncture at standardized time points: before the injection and 24, 48, 72, and 96 hours after for case group and 4wks. and 8wks for both groups. RESULTS: there was significantly difference between the growth factors results of the case group after 4 weeks compared with the 8 weeks result of the control group. Additionally, the same significant results between the two groups after the 8 weeks. Nevertheless, the physical measurements related with hamstring Strain and Knee flexion range of motion between the two groups were not significant after 4 weeks or 8 weeks. CONCLUTION: a single 3-mL injection of autologous PRP combined with a rehabilitation program was effective in time return to play and reducing the severity of pain after an acute grade 2 hamstring injury. Additionally, increase in circulating concentrations of VEGF, IGF-1, PDGF and FGF-2.

Key Words: Platelet rich plasma (PRP), Human Growth Factors, Hamstring Tear.

1. Introduction:

Skeletal muscle injuries are up to 55% of all sports injuries and causes excessive long term pain and physical disability, Muscles strains and contusions representing more than 90% of all sports related injuries and are the most muscular injuries frequent. [1] [2]  Proximal hamstring tear injuries are common in athletes and frequently result in prolonged rehabilitation, time missed from play, and a significant risk of re-injury. Reports of acute hamstring strains without avulsion in dancers have suggested recovery times for return-to-play ranging from 30 to 76 weeks [3].

Get Help With Your Essay

If you need assistance with writing your essay, our professional essay writing service is here to help!
Find out more about our Essay Writing Service

Platelet rich plasma (PRP) become popular biologically method used to accelerate healing in sports medicine and orthopaedic surgery field. PRP is concentrate the human platelets to supra-physiologic levels. It is an autologous producing high level of the platelets concentration centrifuged from the peripheral vein. Then it re-injected under the ultrasound gaudiness during surgery or at a site of injury [4] [5]. As a result of the lack side-effect and the autologous nature of PRP, it has utilized exponentially over the last few years in sports medicine and orthopaedic. Historically, since the 1950s the platelet-rich plasma (PRP) has been used to dermatological conditions and manage maxillofacial as well [6]. Furthermore, Platelet-derived preparations including PRP were first regulated by WADA under the 2010 Prohibited List because of concerns that the elevated concentrations of growth factors in PRP may confer an unfair advantage to treated athletes. However, WADA lifted the ban on PRP in 2011 in recognition of the lack of evidence to support a systemic performance-enhancing effect and to allow further research in the field [7].

Indeed, the blood contains 6% platelets, 1% white blood cells, and 93% red blood cells.  The PRP technique aims to reverse the concentration of the platelet in lieu of red cells to increase the growth factors that more useful in accelerating the healing. [8] However, Platelet rich plasma (PRP) is a centrifuged blood product that contains a supraphysiologic amount of platelets. Therefore, the preparation process to product concentrative platelet above the baseline values have started with an autologous extraction of patients` blood, then by plasmapheresis centrifuged to obtain a concentrated suspension of platelets. It then separates the solid and liquid components of the anticoagulated blood after a two-stage of centrifugation process [9]. The initial phase separates the plasma and platelets from the erythrocytes and leucocytes. The second stage concentrates the platelets further into platelet-rich and platelet-poor plasma components [10] [11].

Platelet rich plasma (PRP) contains some biologic factors which have been enhanced the proliferation and collagen secretion of tenocytes. These factors including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF2), and transforming growth factor β (TGF β) [12] [13]. There is an increasing the stimulus response of PDGF and TGF-β in the early stages of tendon and muscles healing after PRP injection resulting in new vessel formation and collagen synthesis. [14]. In addition to decrease oxidative stress that could lead to cell apoptosis, PRP has been promoted tendon and muscles cell growth [15]. This is evidenced and reinforced by release of inflammatory meiators such as COX-1 and2, PGE-2 [16] [17].

Recently, there are various approaches reported the benefits of treating the soft tissue injuries such as muscles tears and tendinosis by injecting platelet rich plasma (PRP). Despite this popularization and increasing use in soft tissue injuries, its efficacy still not clear and remains controversial. It has been previously established that platelets provide regenerative potential by the process of chemo-taxis [18] [19] [20]. The use of PRP in order to accelerate recovery time after muscle injury has become a relatively common practice in sports medicine. Several studies represent that PRP can improve skeletal muscle healing after acute injury. In particular, local PRP which increased expression of several myogenic factors at mRNA level acting on modlating the inflammatory response and myogenesis in the early stages after acute injury [21] [22]. Rossi L, et al. reported the effects of an autologous PRP injections on time to return to play in randomized controlled study conducted on 75 patients. The study represented time to return to play for recreational and competitive athletes and recurrence rate after acute muscle injuries as well. The main result in the study that PRP injection significant reduction of re-injury rates at 2 years. Additionally, it was decreased the pain severity score and significantly decreased the time of return to sports as well [23]. One more study reported that 14 professional athletes were treated with ultrasound-guidance injections of PRP after acute muscle injuries. The athletes showed a quick return to activity and improved healing in muscle tears [8]. Similar results have represented in Sanchez et al study, which conducted on 20 athletes. These results supported the benefits of PRP and its role in muscle healing. The patients recovered in half of the expected time [24].

Figure 1. Process of platelet activation (PDGF, platelet derived growth factor). Source [25]

Platelet Activity in muscles:

(Figure 1,) represents the released serotonin contributes to vasoconstriction. The conversion of ATP into ADP releases the energy necessary to establish and maintain the aggregation. The release of the calcium ions inside the platelet makes the myofibril within it contract, thus allowing the aggregation and release of the content of the granules. This is serum calcium, which is necessary for the formation of the fibrin network [26]. The presence of the Ca2+ ions in the plasma makes the coagulation factors activate and group, forming the fibrin network, which is stabilized by factor XIII and transformed in a stable clot. The calcium ions also inhibit the anticoagulant activity of heparin, preserving the clot [27].

The PRP and the growth factors:

The functions of these growth factors are presented in Table 1. It should be noted that the mechanism of action of platelet-rich plasma does not differ from the physiological healing process, but allows for obtaining higher concentrations of growth factors. As a result, the process of tissue regeneration is accelerated [28] [29] [30]. Platelet α-granules are comprised of haemostatic factors, regulators inflammation, and wound healing. Substances stored in dense granules are thrombocyte-activating factors. Platelets also contain lysosomal granules, which secrete acid hydrolases [31] [32]. Platelet activation results in growth factor release. Platelet growth factors include platelet-derived growth factor (PDGF), transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and insulin-like growth factor 1 (IGF-1) [33] [8].

Table 1. Growth Factors function

Figure 2. Platelet degranulation and action of the released cytokines in the process of formation of new bone and muscle tissue (VEGF, vascular endothelial growth factor; PDGF, platelet derived growth factor; BMP, bone morphogenetic protein; TGF-b, transforming growth factor).

2. Methods

2.1. Participants:

This study was approved by Damietta University, Egypt, Alexandria University, Egypt and Utah state University, UT, USA. Twenty-three physically active males with acute grade 2 hamstring tear were voluntarily recruited for data collection (age 21.8 ± 2.64y, mass 71.52±2.74 Kg, height 175.4±2.32). All patients receiving local ultrasound-guided intratendinous PRP injection at our institution between September 2014 and December 2016 were screened for eligibility to participate in the study, and 17 patients were ultimately enrolled. Exclusion criteria included five participants with previous injury or diagnoses in hamstring. 8 physically active males (age 22.0±0.6) with acute hamstring strain injuries and 9 matched controls (age 21.6±2.8) were recruited as research participants. The case and control groups were performed rehabilitation program included aquatic exercise for 8 weeks. The history of pain data and the daily hours of using the smartphones were collected by survey. Furthermore, the procedures were explained to the subjects and their written signatures were obtained on the informed consent.

2.2. Platelet rich plasma preparation and injection:

In accordance of GPSTM III Systems instruction the blood collected for PRP was prepared by (Biomet Biologics, Inc., Warsaw, Ind) and standard 60 ml GPSTM III kit. Approximately of 7 ml of PRP was prepared in 30 minutes. Furthermore, single 3 ml of extracted PRP were injected under ultrasound gaudiness after adding 8.4% sodium bicarbonate buffered PRP for increasing the pH to normal physiological levels. The sodium was added in a ratio 0.05 ml to 1 ml of PRP. All the participants blood samples were stored in -25° Celsius and were analyzed to determine the concentration of the growth factors.

The PRP injection of the current study were injected directly into the injured area under aseptic technique. The case group participants only received the single autologous PRP combined with the rehabilitation program. The participants were kept under observation for 96 hours and were performed the rehabilitation program after 5-7 days of PRP injection. Blood samples were collected by venipuncture at standardized time points: before (baseline) and 24, 48, 72, and 96 hours to 4wks. and 8wks. after administration of PRP. blood was drawn at precisely the same time each morning and at least 3 hours after eating and exercising per WADA standards

Figure 3. PRP set up.

2.3. Growth Factor Quantification:

Six growth factors and related molecules that are concentrated in PRP preparations were quantified in PRP and blood by direct immunoassay using the Quantikine enzymelinked immunosorbent assay (ELISA kit), as outlined  the Growth factors studied were: human growth hormone (hGH), insulin-like growth factor-1 (IGF-1), insulin-like growth factor binding protein-3 (IGFBP-3), basic fibro blast growth factor (bFGF or FGF-2), vascular endothelial growth factor (VEGF), and platelet-derived growth factor- BB (PDGF-BB). Because bFGF is present in the blood only at very low concentrations, a high-sensitivity ELISA kit was used to ensure accurate detection [34] [35] [5].

Figure 4. PRP injection under ultrasound guidance.

2.4. Rehabilitation program:

A Physical rehabilitation program was performed for six weeks and designed for lower limb. In particular, hamstring muscles. both groups participants (8 case and 9 control) performed the exercises protocol once a day for 55 minutes for each training session and 5 times a week (275 M. / week). The exercise protocol was consisted of aquatic exercise and strength exercises and was divided to tree stages, the first stage was focused on the flexibility and isotonic strength exercise with 5 sets and 12-15 1RM intensity. While, the second stage was designed for strength exercises with 3 sets and 8-10 1RM intensity. While the third stage for endurance and exercise related of activity performance. Aquatic pool, Machine weights and The Thera-Band resistance bands exercises were used during the 6 weeks especially the colors (red, blue, black, silver). The red and blue bands were used in the first stage and the black and silver used in second stage. Furthermore, all the exercises were performed by stretching the band between 75 – 100 %. knowing that, the weight of stretching in Thera-Band between 75-100% is red 3.3-3.9kg, blue 5.9-7.1kg, black 8.1-9.7, and silver 11.1-13.2kg.

2.5. Statistical Analysis

The paired t-test was used to compare the collected data before performing the exercise protocol (Pre-test) and those which were obtained after the 6 and 8-weeks training period (Post-test). The differences between the samples were significant at the t = 1.740 p < 0.05 level. All the analyses were performed by using SPSS 21 software for Windows 7 (SPSS Inc. Chicago, IL, USA). Additionally, all values within the text and table are observed as standard deviation and mean (mean ± SD).

3. Results:

Table 2, 3 illustrate the large variations in growth factor concentrations between participants in the two groups before and after PRP injection. Regarding growth factor (GF) trajectories for the case group participants are shown in Figure 3, and data are summarized in Table 2, the human growth hormone increased dramatically within the first 24 hours after PRP injection while these results were not significant after the 4 weeks and 8 weeks.  Moreover, IGF-1 increased relative to baseline within 24 hours after PRP and remained elevated at all-time points thereafter, and the change was statistically at 24 until 96 hours as well after 4 weeks and 8 weeks. Likewise, VEGF and PDGF were significantly elevated at 24 hours and at all-time points thereafter and were significant after 4 and 8 weeks.  Furthermore, FGF-2 rose at the point between 24 to 96 hours after PRP injection but not significantly, while it was elevated significantly after 4 week and 8 weeks.

It obviously represented in table 3, the spectacular significantly difference between the growth factors results of the case group after 4 weeks compared with the 8 weeks result of the control group. Additionally, Figure 6, reveals the same significant results between the two groups after the 8 weeks. Nevertheless, the physical measurements related with hamstring Strain and Knee flexion range of motion between the two groups were not significant after 4 weeks or 8 weeks.

aPRP, platelet-rich plasma; hGH, human growth hormone; IGF-1, insulin-like growth factor-1; FGF-2, basic fibroblast growth factor; VEGF, vascular endothelial growth factor; PDGF, platelet-derived growth factor.

Table 2. Difference of the Growth Factors concentration between the Case and Control group

PRP, platelet-rich plasma; hGH, human growth hormone; IGF-1, insulin-like growth factor-1; FGF-2, basic fibroblast growth factor; VEGF, vascular endothelial growth factor; PDGF, platelet-derived growth factor; N, Number; * Significant difference P ≤ 0.05, t =1.740 (N= 17).

Table 3. Difference of Hamstring Force and Knee Flexion (ROM) between the Case and Control group

aPRP, platelet-rich plasma; HF, Hamstring Force; KF(ROM), Knee Flexion range of motion. N, Number. P ≤ 0.05, t =1.740 (N= 17).

4. Discussion:

There is little published evidence to support whether a statistically significant increase in growth factors with performance-enhancing potential, including IGF-1, hGH VEGF, PDGF and FGF-2, necessarily leads to clinically relevant ergogenic effects. This is further complicated by evidence from some animal studies that local IGF-1 overexpression enhances local muscle mass and strength without systemic increases in IGF-1. The current study aims to determine the effect of PRP in accelerate the healing of hamstring strain. Moreover, to identify potential molecular markers that could be used to distinguish athletes who have been treated with local PRP injections from those who have not.

Figure 5, The concentration of the growth factors after the PRP injection. PRP, platelet-rich plasma; hGH, human growth hormone; IGF-1, insulin-like growth factor-1; FGF-2, basic fibroblast growth factor; VEGF, vascular endothelial growth factor; PDGF, platelet-derived growth factor.

The performance of the growth factors after a single PRP injection was enhanced and increased significantly from 24 until 96 hours.  Indeed, hGH was peaked within the 24-hour window, although the results were not significant after 4 weeks or 8 weeks. Similarly, IGF-1 is significantly increased by 24 until 96 hours after PRP, while its’ activation was decreased after 4 weeks and 8 weeks but with significantly difference compared with the pretest and the control group 8 weeks’ test. Furthermore, IGF-1 is generated in the liver in response to hGH, is the primary downstream mediator of hGH, and is the most specific marker of supraphysiological hGH exposure [36] [37].

Figure 6. Difference between the case and control group in the concentration of the Growth Factors after 8 weeks. hGH, human growth hormone; IGF-1, insulin-like growth factor-1; FGF-2, basic fibroblast growth factor; VEGF, vascular endothelial growth factor; PDGF, platelet-derived growth factor.

Despite that both groups performed the same rehabilitation program, our study reported a significant increase in the growth factors for the control group after 4 and 8 weeks (Table 3, figure 6). However, the values of the case group after 4 weeks for the case group were more advanced than the 8 weeks’ values of the controlled (Figure 7,8). Therefore, the PRP injection enhanced the concentration of the growth. It is notably that the physical measurements of hamstring force and knee flexion range of motion were not significant at either 4 or 8 weeks. Wallace et al demonstrated that an acute bout of exercise increases total circulating IGF-1 by only about 20% [38] [9].

Figure 7, Difference between the case and control group in (NFROM) Knee flexion range of motion after 8 weeks.

Figure 8, Difference between the case and control group in (HF) Hamstring Force after 8 weeks.      

By comparison, participants in case group who treated with PRP and exercise program. Our study demonstrated a 38% increase in IGF-1 in case group and 9.5% in control group. Relative to baseline, suggesting that PRP treatment activates the hGH-IGF-1 pathway but that a single PRP injection is important to combined with the exercise to maximally stimulate. [39] [9] (Table 3) (Figure 6). We also observed FGF-2 and VEGF also peaked after treatment with PRP.  Fibroblast growth factor contributes to angiogenesis by stimulating the proliferation of endothelial cells to enhance the proliferation of satellite cells, which are the stem cells of mature muscle [40]. Basic fibroblast growth factor may enhance athletic performance by inducing muscle hyper- trophy and increasing oxygen transport. Vascular endothelial growth factor is a powerful stimulator of angiogenesis and could have noteworthy performance-enhancing effects if it entered systemic circulation and exerted its effects in tissues other than the site of injury [41].

The potential effects of autologous biological substances to hasten muscle healing were reported in several case reports [34] [42] [43]. Borrione et al [34] noted that athletes with grade 3 muscle strains treated with PRP showed earlier functional improvement and more complete recovery than those treated nonoperatively. Hamid et al  [44] demonstrated that a single PRP injection was effective in accelerating recovery for grade 2. However, the PRP Group achieved full recovery significantly earlier than controls and returned to play after 27 days while control group returned after 43 days. Another approach successfully treated an athlete with a grade 2 semimembranosus muscle injury with a single 3-mL infiltration of platelet-enriched plasma under ultrasound guidance. The athlete was pain free and allowed to train at the preinjury intensity 21 days after treatment [45]. The effect of a preparation rich in growth factors (PRGF) to hasten muscle recovery was reported in a 35-year-old professional bodybuilder diagnosed with a right adductor longus rupture. The athlete successfully returned to competitive training within 1 week after the third PRGF injection [43]. The effect of PRP in accelerated and associated a hamstring injury was also observed in the current study. The PRP preparation contained a high concentration of several growth factors including TGF-b, FGF-2, and insulin-like growth factor-1, but the amount of platelets and WBCs present was not stated. Additionally, the actual effect of PRP on soft tissue healing is not fully understood,22 our findings supported the possible role of higher growth factors (concentration level) in hastening recovery as postulated by previous researchers [46] [47] [42]. Sanchez et al reported full functional recovery of hamstring and adductor muscle injuries 2 times faster in 20 professional athletes treated with a PRGF [24].

Similar designed study by Rettig et al was investigated the effects of an autologous PRP injection and was retrospective case-control study conducted to determine the effect of the PRP on return time to play after acute hamstring injuries. The study included 10 professional National Football League (NFL) players with acute hamstring injury. The participants were divided equally into PRP and Control groups. Under ultrasound guidance the PRP group patients were injected once with 6 mL of PRP. Both groups were performed the same rehabilitation program. Several differences were identified between the study by Rettig et al and the current study. For instance, the