Wound healing is a dynamic and complex process controlled by interacting signals that regulate a myriad of cellular and molecular events. Therefore, no single agent can efficiently mediate all aspects of the wound healing process  . Platelets not only assist in clot formation, but also are a rich source of a host of growth factors and cytokines essential to wound healing  . Platelets activation by proteins as thrombin causes the α-granules to fuse with the platelet cell membrane releasing these growth factors, such as transforming growth factor-b (TGF-b), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), fibroblast growth factor (FGF) and insulin-like growth factor (IGF)  . These growth factors are crucial in attraction of mesenchymal cells into the wound, enhancing their proliferation and promoting extracellular matrix deposition during the healing process  .
When the natural healing process becomes interrupted by either wound factors (i.e. infection) or patient factors (i.e. comorbidities), the standard wound care is not enough to improve the healing outcome and advanced therapeutic modalities are always required, such as negative pressure therapy, hyperbaric oxygen therapy, low level laser therapy, growth factors and platelet-rich plasma (PRP)   . Platelet-rich plasma is a blood derivative that contains a higher concentration of platelets, about 3 to 5 times the normal value  . PRP also contains a variable number of white blood cells and red blood cells according to the preparation method  .
PRP mechanism of action is still questionable; however, it may act by enhancing the natural healing process, as the molecules contained within the PRP preparation act as adjuvant in the inflammatory and proliferative phases  . Although PRP applications are now widely used in many medical fields, including orthopedics, maxillofacial surgery, cardiothoracic surgery, plastic surgery, trauma surgery and wound healing, their efficacy in human subjects is still debated  . We aimed in this study to evaluate the safety and the efficacy of autologous platelet-rich plasma (PRP) as an aid in the healing process of acute wounds; the donor site of the split-thickness skin graft (STSG) was used as a model of acute wounds.
2. Materials and Methods
This prospective study enrolled forty adult patients, of both sexes (28 males and 12 females), aged between 18 - 50 years (mean―28.6 years), who were admitted to the Plastic Surgery Department, Tanta University Hospitals between August 2013 and August 2015. All patients had post-traumatic raw areas and were in need for STSG obtained from the thigh were included in our study. Patients with chronic diseases such as hepatic insufficiency and diabetes, those on steroids or immunosuppressive therapy, or those with blood and collagen diseases as well as smokers were excluded from the study. All materials and procedures were approved by the Ethics Committee of the University. Informed consent was obtained, after detailed description of the procedure, from all patients.
2.1. Preparation of Platelet-Rich Plasma (PRP)
Before the surgical phase, 50 ml of the autologous venous blood was withdrawn from every patient and collected in a sterile tube containing 5 ml of citrate phosphate dextrose (CPD) as anticoagulant. The blood sample was centrifuged at room temperature for 5 min at 2500 r.p.m in the centrifuge machine (eppendorf centrifuge 5804). After the 1st centrifugation, the blood was separated in red blood cells and plasma. The red cells were removed, and the remaining plasma was centrifuged at 3500 r.p.m for 5 min. After the 2nd centrifugation, the centrifuge was separated into platelet-rich plasma (PRP) at the bottom layer; constituting 10% of the withdrawn blood volume and platelet-poor plasma (PPP) at the upper layer. Measurement of the platelet count and the transforming growth factor-B1 concentration using the DRG TGF-B1 ELISA kit in each PRP preparation and the whole blood were done for all patients.
2.2. Surgical Procedure
All surgical procedures were done under general anaesthesia. A STSG was harvested from the thigh using the Humby’s Knife. The donor sites were randomly divided into two equal halfs: the PRP side, which was injected with recently activated PRP by mixing with 2% calcium chloride at a ratio 7:1; and the control side, in which the conventional method of dressing was used. Vaselinized gauze and secondary absorbant layer dressing were used to cover the donor sites in all patients. The time needed from preparation till injection of the activated PRP was recorded for all patients.
2.3. Postoperative Care and Monitoring
All patients were discharged on the 2nd postoperative day and recalled again once weekly for one month and once monthly for 3 months. Clinical monitoring of the donor sites was done every 7 days for 3 weeks, regarding pain perception, epithelialization surface area and possible side effects of the PRP (reaction to PRP-infection-hypertrophic scar-hyperpigmentation). The pain was measured using the visual analogue scale (0 - 10). Histopathological monitoring was done on the 7th postoperative day. Under local anaesthesia (0.5% xylocaine), a 3-mm punch biopsy was taken from the PRP and the control sides then fixed with 10% paraformaldehyde. The paraffin fixed specimens were stained using the H&E stain. Thereafter, examination of the specimen was done regarding keratin formation, epidermal thickening, infiltration of the dermis with inflammatory cells, neovascularization and collagen deposition.
The data collected for statistical analysis were expressed as means and standard error of the means (SEM). Student’s t-test and Chi-square test were used for comparative analysis. Statistical significance was defined as p value of <0.05.
Over a two-year period, forty patients subjected to STSG and PRP application to one half of the donor site. As shown in Table 1, there were significant increase in the mean concentrations of the platelets and TGF-B1 in the PRP compared to the patients’ blood (p < 0.001), with the platelets being about 3.5-fold higher, and TGF-B1 about 2.4-fold higher, than in the serum. The time needed from preparation till injection of the activated PRP ranged from 45 to 82 min (mean―68.3 min).
Table 2 shows that, there was no significant difference between the PRP side and the control side in the pain scores at day 0 and day 21 postoperative, but the PRP side had significantly lower pain scores at days 7 and 14 postoperative (p = 0.002 and p = 0.004 respectively). Furthermore, there was improvement in the wound healing in the PRP side as evident by the significant increase in the epithelialization surface area in the PRP side at days 7 and 14 postoperative (p < 0.001 and p = 0.039 respectively), while at day 21 postoperative there was no significant difference between both sides. One patient had infection and another developed hypertrophic scar at both sides, while two patients had hyperpigmentation and hypertrophic scar at the PRP side only. There was no significant difference between both sides regarding the incidence of complications.
As regards histopathological monitoring, the PRP side (Figure 1) showed differentiation of the epidermal keratinocytes with intact epithelium, differentiation of the cells in stratum spongiosum and stratum granulosum, neovascularization and beginning of collagen deposition, while the control side (Figure 2) showed minimal epithelial covering with extensive areas of ulceration, edema below the epidermis with extensive perivascular inflammatory infiltrate with acute inflammatory cells mainly neutrophils, undifferentiated keratinocytes in stratum spongiosum and stratum granulosum and minimal collagen deposition.
Table 1. Laboratory data (mean ± SEM).
Table 2. Outcome clinical data.
a,bMean ± SEM.
Figure 1. Light micrograph of PRP side at day 7 using H&E stain. (a) Regenerated intact epithelium with differentiated epidermal keratinocyes (×100); (b) Differentiated polyhydral cells in stratum spongiosum and cells in stratum granulosum (×400); (c) Collagen deposition with minimal infiltration of inflammatory cells (×200).
Figure 2. Light micrograph of control side at day 7 using H&E stain. (a) Minimal and partial epithelial covering with extensive areas of ulceration (×100); (b) Thin epidermal covering in high power field (×400); (c) Extensive perivascular inflammatory infiltrate and minimal collagen deposition (×200).
Platelet-rich plasma (PRP) has been largely used in several clinical settings. Moreover, it is considered to promote tissue regeneration due to presence of growth factors and essential cytokines. Nevertheless, many studies fail to deliver conclusive evidence concerning the benefits of PRP in the field of wound healing. This study was undertaken to evaluate the safety and the efficacy of autologus platelet-rich plasma (PRP) in acute wound healing.
In our study, we chose the donor sites of STSGs as model of acute wounds. Similarly, Danielsen et al.  tested the effect of PRP on the epithelialization of the donor sites of STSGs. In other studies, Hom et al.  and Kazakos et al.  evaluated the effect of PRP on full thickness skin punch wounds and acute traumatic wounds as open fracture tibia respectively.
Sommeling et al.  in their systematic review of 15 randomised controlled trials and 25 case control studies found that there is no standard technique of PRP preparation. In this study, we adapted the double spin technique and observed that the platelet count was increased about 3.5 folds and TGF-B1 was increased 2.4 folds. In a similar study, Marukawa et al.  used the double spin technique for PRP preparation and noticed that the platelet count was increased about 3 times and the platelet released growth factors increased about 2 - 3 times. Conversely, Pietrzak et al.  suggested that a four to five fold increase in the baseline of platelet count is needed but they show no clear evidence that lower or higher concentration may decrease or increase the positive effect of PRP.
We found that the mean time needed from preparation till injection of the activated PRP was 68.3 min. Unlike us, Kazakos et al.  reported a relatively shorter time (mean―52 min), which could be attributed to the single centrifugation protocol adapted by them. This series witnessed improvement in the wound healing in the PRP side as evident by the significant increase in the epithelialization surface area in the PRP side at days 7 and 14 postoperative (p < 0.001 and p = 0.039 respectively). Similar to our study and findings, Kakudo et al.  studied the effect of PRP on the donor site of STSG and noticed macroscopic epithelialization on the 5th day of PRP application. In another study, Spyridakis et al.  noted that complete wound closure was statistically faster in the PRP treated wounds. Contrary to our results, Danielsen et al.  observed no significant difference in the macroscopic epithelialization between PRP and control groups in their study.
In our series, the pain scores, while not differing at day 0 and 21 postoperative, significantly decreased by day 7 and 14 postoperative in the PRP side compared to the control side (p = 0.002 and p = 0.004 respectively). In similar studies, Englert et al.  found that postoperative pain was significantly reduced for the PRP treated wounds and Khalifi et al.  observed that intravenous narcotic use was statistically lower in the PRP treated subjects. In another study, Kazakos et al.  noticed that there was no pain difference between both groups at the end of the 1st week, while there was lower pain scores in the PRP treated group at the end of the 2nd week.
This study demonstrated that the PRP side exhibits intact epithelium with thicker epidermis, differentiated epidermal keratinocytes, neovascularization and beginning of collagen deposition. These data are in accordance with that of Hom et al.  , who reported thicker epidermis at the 7th day of PRP application to acute wounds and Marx et al.  , who noticed thicker epithelium and newly formed blood vessels in the PRP side. In experimental study, Carter et al.  tested the effect of PRP gel on equine wounds and observed that PRP gel induced accelerated epithelial differentiation and early collagen deposition.
We observed that there was no significant difference between both sides regarding the incidence of complications which is consistent with Wang-Saegusa et al.  who studied the effect of PRP injection into the knee joint of over 800 patients and noticed no adverse effects, and is also consistent with Mazzucco et al.  who used PRP gel to treat heal wounds and reported no serious adverse events. Moreover, Powell et al.  demonstrated that wound treatment with PRP gel can reduce the incidence of ecchymosis and the formation of edema. In our point of view, the small sample size and the single injection time are the main limitations of our study.
We can conclude that the platelet-rich plasma is safe and effective adjuvant in the management of acute wounds. However, we recommend for larger clinical trials for standardized method for PRP preparation and better understanding of the efficacy and the safety of this blood product.
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