Efficacy of Growth Factors Concentration after PRP

Growth factor

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Abbreviation

Functions

Transforming growth factor

TGF-β

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A mitogen for fibroblasts, smooth muscle cells, osteoblasts Angiogenesis promotion, extracellular matrix production

Platelet-derived growth factor

PDGF

Chemotactic effect on monocytes, neutrophils, fibroblasts, mesenchymal

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stem cells and osteoblasts

A mitogen for fibroblasts and smooth muscle cells Angiogenesis promotion, formation of fibrous tissue, re-epithelialization

Vascular endothelial growth factor

VEGF

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Angiogenesis promotion Chronic wound healing promotion Inhibition of bone formation

Epidermal growth

EGF

factor A mitogen for fibroblasts, endothelial cells, keratinocytes Chronic wound healing promotion

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Insulin-like growth factor

IGF-1

Regulation of bone maintenance Modulator of cell apoptosis Stimulation of bone tissue regeneration

Platet derived endothelial growth factor

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PDEGF

Promotes wound healing by stimulating the proliferation of keratinocyes and dermal fibroblasts

Endothelial Growth Factor

EGF

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Cellular proliferation Differentiation of epithelial cells

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:

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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).

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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.

Table 1. Data Summary for the Growth Factors after PRP Injectiona

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

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