Comparison of the Effects of Platelet-rich Plasma Prepared in Various Forms on the Healing of Dermal Wounds in Rats

Objective. This study was designed to evaluate the effects of 3 different forms of platelet-rich plasma (PRP) on the healing process, wound healing rate, and histopathological changes that occur during recovery of open dermal wounds. Materials and Methods. A 2 cm x 1 cm full-thickness skin defect was made on the backs of 40 Wistar female rats that were divided into 4 groups of 10. In group 1 (control group), the wounds were cleaned with saline; in group 2, the wounds were covered with PRP gel obtained by single centrifugation; in group 3, the wounds were covered with liquid PRP obtained by double centrifugation; and in group 4, PRP gel obtained by double centrifugation with added thrombin was applied on the dermal wounds. All treatments were applied on postoperative days 1, 4, 7, and 10. Results. In all PRP groups, the wound closure was almost complete on day 14 while the wound contraction progressed more slowly in the control group. The mean histopathological scores of epithelialization, inflammation, and fibrosis were significantly better in all PRP groups than the scores in the control group. Conclusion. In conclusion, although all PRP preparations had positive effects on dermal wound healing, double centrifuged PRP topical treatments (with or without thrombin activation) are more effective than single centrifuged PRP, and double centrifugation methods should be preferred for the preparation of PRP.

Platelet-rich plasma (PRP) is defined as a portion of the plasma fraction of autologous blood with a platelet concentration above baseline. A typical blood specimen comprises 93% red blood cells, 6% platelets, and 1% white blood cells. Platelets are small discoid cells with a life span of approximately 7-10 days. Following injuries that cause bleeding, platelets are activated and aggregate together to release granules containing growth factors which stimulate the inflammatory cascade and healing process. Platelets are responsible for hemostasis, construction of new connective tissue, and revascularization. Most of the recent research has been focused on the effects of platelets on the healing process. Over the past few decades it has been demonstrated that platelet activation in the body releases healing proteins called growth factors. There are numerous growth factors with diverse functions, but in general they accelerate wound healing.¹

In 1987, Ferrari et al² first reported the use of autologous PRP in cardiac surgery. More than 20 years ago, PRP was used in the field of dentistry to promote accelerated wound healing in patients with cancer following jaw reconstruction. Physicians have used PRP to aid bone healing after spinal injury and for soft tissue recovery following plastic surgery. It has also been used for the treatment of sports-related injuries. Platelet-rich plasma injections are currently being used in various applications, including orthopedics, burns, osteoarthritis, pilonidal sinus, cardiovascular surgery, cosmetics, facio-maxillary surgery, urology, and experimental studies. As a result, multiple studies are now underway to understand the PRP mechanism of action, refine the treatment, and formally demonstrate efficacy in placebo-controlled trials^.1,3-10

There are many different techniques for the preparation of platelet concentrates which leads to various products classified as pure PRP, leukocyte-rich  and PRP, (L-PRP), pure platelet-rich fibrin (PRF), and leukocyte-rich and platelet-rich fibrin (L-PRF). However, based on the international scientific literature of the experimental and clinical trends, it is difficult to state which products are actually useful. It appears that published experimental results are difficult to classify and interpret, the clinical results are confusing or at least controversial and, finally, the relevance of using these products is debatable.¹¹ In addition to these dilemmas, although there are lots of in vitro studies that have evaluated the content and efficiacy of these different products and many clinical and experimental studies that have investigated the effects of these platelet concentrates, there are very few experimental or clinical studies in literature that compare the in vivo effects of these different products.

The aim of this study, which was designed using an open dermal wound model in rats, was to evaluate the effects of 3 different forms of PRP on the healing process, wound healing rate, and histopathological changes that occur during recovery of the wounds. There is no definitive data in literature about the comparison of these forms of PRPs on open dermal wounds.

Animals. Fifty-eight Wistar female rats, weighing 250g ± 25g, were allowed to adapt to laboratory conditions for 1 week before experimental use. The animals had free access to water and standard laboratory chow. They were kept individually in wire cages at a constant temperature (21°C ± 2°C) under a 12-hour light/dark cycle. Twelve hours before anesthesia, the animals were deprived of food but had free access to water until 2 hours before anesthesia. No enteral or parenteral antibiotics were administered at any time. Rats that died during the experiment were excluded from the study and no new rats were included. The procedures in this experimental study were performed in accordance with the Guide for the Care and Use of Laboratory Animals, and the Animal Ethics Committee of Ankara Education and Research Hospital granted approval for the study.

Study Groups. Of the total 58 rats, 18 were used as donors for the preparation of PRP. The surgical procedures were performed on the remaining 40 rats. These rats were randomized and divided into 4 groups of 10. All animals were anesthetized with an intramuscular injection of 50 mg/kg ketamine hydrochloride (Ketalar, Parke-Davis, Istanbul) and 5 mg/kg xylazine (Rompun, Bayer, Istanbul). The operation sites were shaved and disinfected with povidone-iodine. A 2 cm × 1 cm rectangle-shaped incision was made on the back of the rats, centered on the midline. Afterwards, a standard full-thickness skin defect, including panniculus carnosus, was created on this site. This study was designed for comparing the effects of different PRP solutions on treatment of acute and open full-thickness dermal wounds. Therefore, the skin defects were not closed primarily by suturization. The treatment protocols of the groups were organized as follows:

Group 1 (control group): The wounds were cleaned with saline on postoperative days 1, 4, 7, and 10 and covered with saline-moistened dressings. 

Group 2 (PRP gel obtained by single centrifugation): The wounds were cleaned with saline on postoperative days 1, 4, 7, and 10, then PRP gel was applied to the wounds and the wounds were covered with saline-moistened dressings.

Group 3 (liquid PRP obtained by double centrifugation): The wounds were cleaned with saline on postoperative days 1, 4, 7, and 10, then liquid PRP was applied to the wounds and the wounds were covered with saline-moistened dressings.

Group 4 (PRP gel obtained by double centrifugation with added thrombin): The wounds were cleaned with saline on postoperative days 1, 4, 7, and 10, then double centrifuged PRP gel with added thrombin was applied to the wounds and the wounds were covered with saline-moistened dressings. 

All surgical procedures were performed by the same surgeon. The wounds were observed for 14 days and no complications developed during this period. The open wounds were drawn on graph acetate paper with a marker pen on days 1, 4, 7, 10, and 14 and were photographed with a digital camera. All rats were euthanized with high-dose ketamine hydrochloride on postoperative day 14. The remaining wound areas were drawn on acetate papers on postoperative days 1, 4, 7, 10, and 14. The drawings were scanned, and the surface areas were measured using ImageJ, an open source image processing program for scientific multidimensional images. In all groups, the rats were euthanized on postoperative day 14, and tissue samples of the wound areas were taken for histopathological examination.

Preparation of platelet-rich plasma. The 18 rats selected as donors for preparation of PRP underwent no surgical procedure. These rats were euthanized by high-dose ketamine hydrochloride injection, and whole body blood was taken from the inferior vena cava. Platelet-rich plasma was obtained for each group using the following techniques.

Platelet-rich plasma gel obtained by single centrifugation. The 10 mL of blood obtained from the donor rats was collected in gel-free biochemistry tubes without any anticoagulant. After centrifugation for 10 min at 2500 rpm, the resulting PRP gel was obtained. The complete blood count results of this PRP were as follows: platelet count was 671 x 10³/mm³, leukocyte count was 0.20 x 10³/mm³, and the number of erythrocytes was 0.22 x 10³/mm³. This PRP gel was applied to the wounds of the rats in group 2 on postoperative days 1, 4, 7, and 10. 

Liquid platelet-rich plasma obtained by double centrifugation. The blood obtained from the donor rats was put into biochemistry tubes containing citrate-phosphate-dextrose solution and centrifuged for 10 min at 1500 rpm. After centrifugation, 3 layers were obtained: the top layer including plasma with a light yellow color; the bottom layer mainly including erythrocytes; and a thin intermediate layer between these layers including platelets and white blood cells (ie, buffy coat). The plasma layer at the top was collected in another centrifugation tube and subjected to a second centrifugation for 10 min at 3000 rpm, after which 2 layers were obtained; the upper layer was platelet-poor plasma (PPP) and the lower was PRP. The PPP and PRP were carefully separated into different tubes. The blood count of PRP showed platelet counts of 1434 x 10³/mm³. Since the ideal platelet count has been defined in previous studies as approximately 2.5 fold of blood platelet count, the PRP in the current experiment was diluted with PPP at a ratio of 2:1 and the complete blood count was repeated. The results of the complete blood count were as follows: platelet count was 588 x 10³/mm³, leukocyte count was 0.10 x 10³/mm³, and the number of erythrocytes was 0.09 x 10³/mm³. This final form of liquid PRP was applied to the wounds of the rats in group 3 on postoperative days 1, 4, 7, and 10. 

Platelet-rich plasma gel obtained by double centrifugation with added thrombin. The 180 µL of PRP obtained by the same method used in the third group was mixed with 400 µL of thrombin (Human Thrombin Lyophilized, Baxter, New Providence, NJ) activated with 30 µL of calcium chloride. The tube was shaken gently for a short time and a gel form of PRP was obtained. The complete blood count results of this PRP were as follows: platelet count was 525 x 10³/mm³, leukocyte count was 0.20 x 10³/mm³, and the number of erythrocytes was 0.21 x 10³/mm³. This gel was applied to the wounds of the rats in group 4 on postoperative days 1, 4, 7, and 10.

Evaluation of wound contraction rates. The wounds were observed for 14 days. On days 1, 4, 7, 10, and 14, the wound areas were drawn on graph acetate paper with a marker pen and photographed with a digital camera. The drawings were scanned and then surface areas were measured using the image processing program on the computer. 

Histopathological evaluation. The histopathological analyses were performed in the Pathology Department of Aksaray State Hospital, Aksaray, Turkey. At the end of the study, the remaining wound areas were excised en bloc with the scar tissue and normal skin. All specimens were fixed in 10% phosphate-buffered formaldehyde solution for 2 days at room temperature. The specimens were washed in running tap water and dehydrated with graded concentrations of ethanol (50%, 75%, 96%, and 100%, respectively). After dehydration, the specimens were put into xylene to obtain transparency and were then infiltrated with and embedded in paraffin. The embedded tissues were cut into 3-µm thick sections using a microtome (Leica RM 2125 RT, Leica Biosystems, Buffalo Grove, IL) and counter-stained with hematoxylin and eosin (H&E) and Masson’s trichrome. The same pathologist performed histopathological assays in a blind manner with a light microscope (BX51TF System Miscroscope, Olympus Corp, Center Valley, PA) using x4, x10, x20, x40, and x100 magnifications. Epithelialization, inflammation, and formation of granulation tissue were evaluated in H&E-stained sections and fibrosis/degree of healing were evaluated in H&E-stained and Masson’s trichrome-stained sections using a semi-quantitative scoring system. The scoring systems are given in Tables 1, 2, and 3. 

Statistical analysis. Data analysis was performed using the SPSS version 15.0 for Windows (SPSS Inc, Chicago, IL). All variables were normally distributed about the mean, data were presented as mean ± SD, and the differences between the groups were evaluated by one-way analysis of variance or Kruskal-Wallis variance analysis, whichever was appropriate. When the P values from the variance analysis were statistically significant, the Tukey honestly significant difference or Mann-Whitney U multiple comparison test was used to determine which group was different from the others. A value of P < 0.05 was considered to be statistically significant. 

One rat from the control group died during the study. This rat was excluded from the study and no new rat was included. The remaining rats were euthanized on postoperative day 14.

Wound contraction rates. The mean wound areas of the groups on days 1, 4, 7, 10, and 14 are given in Table 4. The wound contraction rates were evaluated in the form of mean and standard deviation. When statistical analyses were performed, no difference was found between the groups on postoperative day 1 (P > 0.05 for all pairwise comparisons). In all PRP groups, the wound closure was almost complete on day 14. In the control group, the wound contraction progressed more slowly. The results of the statistical analysis determined that the wound contraction rates were significantly higher in the PRP groups than the control group both during and at the end of the experiment (P < 0.05 for all PRP groups). These results demonstrated that all PRP forms used in this experimental study had statistically significant positive effects. 

The effects of different forms of PRP on wound healing were also compared with each other. The results of statistical analyses of the wound areas of groups 2, 3, and 4 on postoperative day 14 were as follows: the wound areas of the rats in groups 3 and 4 were significantly smaller than the wound areas of the rats in group 2 (P = 0.023 for both comparisons). These results showed that although the PRP used in group 2 (PRP gel obtained by single centrifugation) accelerated wound healing when compared with the control group, the PRP used in group 3 (liquid PRP obtained by double centrifugation) and group 4 (PRP gel obtained by double centrifugation with added thrombin) were more effective than the PRP used in group 2. When the wound areas of the rats in groups 3 and 4 were compared, no statistically significant difference was found between these groups (P > 0.05). However, the mean wound area value of group 4 was smaller than the mean wound area of group 3. The graph of alteration in wound areas of the groups during the experimental process is shown in Figure 1. 

Histopathological results. The mean scores of epithelialization, inflammation, and fibrosis are given in Table 5. There was a statiscally significant difference between the mean epithelialization, inflammation, and fibrosis scores of the control and all PRP groups (P < 0.05 for all comparisons). No statistically significant difference was found between the PRP used in groups 2, 3, and 4 (P > 0.05 for all histopathological score comparisons). 

The samples taken from various tissue sections showing the histopathological scoring system of epithelialization, inflammation, and fibrosis are given in Figures 2 and 3. 

Blood is an important source of essential therapeutic products that comprise both cellular and protein products and that cannot be obtained from other sources. The bioactive molecules, which are involved in the wound healing process, are contained in the α-granules of circulating platelets. Platelets are anucleated, discoid-shaped blood cells that accomplish a variety of critical functions. Although the most widely known function of platelets is coagulation, it was soon discovered that platelets contain more than 1,100 proteins including growth factors, enzymes, enzyme inhibitors, immune system messengers, and different bioactive compounds involved in various aspects of tissue repair.^12,13 Activated platelets release numerous growth factors and cytokines that play various roles supporting cell growth and tissue repair.^14-16 Platelets contain high quantities of key growth factors, such as transforming growth factor (TGF)-β, platelet-derived growth factors (PDGF-AA, PDGF-AB, and PDGF-BB), insulin-like growth factor (IGF), vascular endothelial growth factors (VEGFs), epidermal growth factor (EGF), and fibroblast growth factor (FGF)-2, which are able to stimulate cell proliferation, matrix remodeling, and angiogenesis.^1,17,18  

Platelet-rich plasma has been described as “a volume of autologous plasma that has a platelet concentration above baseline.”¹⁸ The aim of PRP treatment is to utilize a patient’s own platelets and growth factors to improve healing at the site of injury. The concept of being able to concentrate autologous platelets into a volume of plasma to deliver increased levels of bioactive factors is not new, and autologous PRP has been used extensively in dentistry, orthopedics, cardiovascular surgery, ophthalmology, neurosurgery, urology, maxillofacial surgery, and cosmetic surgery for more than 30 years.^14-16,19 

In an experimental rat model, Li et al²⁰ evaluated whether PRP could improve skin flap survival; they found that PRP significantly improved flap survival rates when compared with the PPP treatment and nontreatment groups.²⁰ 

Setta et al²¹ compared the efficiency of PRP and PPP on the healing of 24 patients with chronic diabetic ulcers. The results of this study showed the healing of chronic diabetic foot ulcers by PRP was significantly faster than by PPP. 

Nakamura et al²² investigated the effects of PRP on resorption and adipocyte survival in autologous fat grafts of rats prepared with isogenous PRP and found that normal adipocytes were obviously decreased in the control group from 20 days, while the PRP group demonstrated increased granulation tissue and capillary formation and good maintenance of normal adipocytes for at least 120 days.

In another study, it was shown that wounds treated with PRP gel exhibited more rapid epithelial differentiation and enhanced organization of dermal collagen compared to controls in an equine model.²³ 

The positive effects of PRP on intestinal and colonic anastomotic healing have also been demonstrated in experimental studies.^5,6

Apart from these studies on the evaluation of the efficacy of PRP on wound healing and skin flap survival, it has been widely used for different indications such as chronic tendinopathy, rotator cuff repair, Achilles tendon repair, anterior cruciate ligament repair, muscle injuries, acute sports injuries, joint repair, and cartilage repair.²⁴ 

Although the definition of PRP suggests a pure mixture of plasma and platelets, the generic term “platelet-rich plasma” has recently expanded to include a variety of final products that can vary markedly, not only in the final concentration of platelets they produce, but also in the amount of red blood cells and/or white blood cells included in the final preparation. All PRP preparation techniques are not the same and the platelet concentration of a given PRP preparation can vary greatly among patients. In other words, PRP preparations vary depending on characteristics such as the number of platelets, the speed and duration of centrifugation, the presence or absence of white blood cells, and the use of activators such as exogenous thrombin and calcium chloride. In addition, the natural variations in platelet concentration among individuals as well as the daily variation in platelet parameters observed within individuals can further affect the consistency and efficacy of the final product.^15,16,19 

The broad variability in PRP-preparation equipment and techniques used in different studies may affect clinical outcomes, making interpretation of the results challenging. The variability in the preparation methods and the composition of PRPs poses a dilemma in making true comparisons between different studies and their relative effectiveness. Even documenting the final platelet count in the plasma fraction may still make it impossible to ensure what precisely is being administered in a given volume of PRP because studies have shown a poor correlation between platelet concentration and growth factor concentration in some PRP preparations.^1,15 

Following the debates about the contents and the role of the various components of these preparations, a simple classification system that separated the products according to the cell content and the fibrin architecture was proposed in 2009.¹⁷ This separation allowed for the definition of 4 main groups: 1) pure PRP, also known as leukocyte-poor PRP), 2) leukocyte-rich PRP and PRP, 3) pure platelet-rich fibrin (P-PRF, also known as leukocyte-poor PRF), and 4) leukocyte-rich fibrin and platelet-rich fibrin (L-PRF).¹¹ 

In addition to their extensive, successful use for various clinical application, several studies have investigated the mechanism of action, clinical benefits, and ingredients (eg, growth factors) of these different forms of PRP.^2,4-9,12,13 However, there are not enough in vivo clinical or experimental studies that have investigated which preparations are more efficient than the others. No definitive data is found in literature about the comparison of different forms of PRPs on dermal wounds. In light of all these features, the aim of this study was to compare the effects of 3 different forms of PRP on the recovery of open dermal wounds in rats. 

The PRP forms used in this study were prepared with different techniques: PRP gel obtained by single centrifugation, liquid PRP obtained by double centrifugation, and PRP gel obtained by double centrifugation with added thrombin. When globally determined, it was clear that all PRP preparations had significant positive effects on wound healing when compared with the control group. In all PRP groups, the wound closure was almost complete on day 14 while wound contraction progressed more slowly in the control group. Statistical analysis determined the wound contraction rates were significantly higher in the PRP groups than the control group both during and at the end of the experiment (P < 0.05 for all PRP groups). The mean histopathological scores of epithelialization, inflammation, and fibrosis were significantly better in all PRP groups than the scores in the control group (P < 0.05 for all comparisons).

The effects of different forms of PRP on wound healing were also compared against each other. Although no significant difference was found between the histopathological scores of the 3 PRP groups, there was a statistically significant difference between the wound contraction rates of these groups. The wound areas of the rats in groups 3 and 4 were significantly smaller than the wound areas of the rats in group 2.

All available PRP techniques have some common points. Blood is collected with anticoagulant and immediately subjected to centrifugation steps depending on the desired character of the final product. The initial centrifugation (ie, the “soft” spin) separates the plasma and platelets from the red and white cells. After centrifugation, 3 layers appear: the top layer including plasma with a light yellow color, the bottom layer mainly comprised of erythrocytes, and a thin intermediate layer between these layers of platelets and white blood cells (ie, the buffy coat). The resulting plasma supernatant, which contains the suspended platelets and may contain a portion of the white cell buffy coat, should be subjected to a second, longer centrifugation step (ie, the “hard” spin), further concentrating the platelets. After the second centrifugation, 2 layers form: the upper layer is PPP and the lower is PRP. Platelet-poor plasma and PRP should be separated carefully and collected in different tubes.^15,17,25 The liquid PRP used in group 3 of the current study was obtained by this technique and applied to the wounds of the rats on postoperative days 1, 4, 7, and 10.

The second centrifugation step can also include the addition of calcium chloride, which replaces the calcium bound by the anticoagulant and permits the conversion of prothrombin to thrombin, or exogenous thrombin, both of which initiate the clotting cascade and the precipitation of a fibrin scaffold.^15,17 The PRP  gel obtained by this technique was used in group 4 of the current study and was applied to the wounds of the rats on postoperative days 1, 4, 7, and 10.

The need for platelet activation with exogenous thrombin before injection is not clear. Thrombin is a serine protease derived from prothrombin which catalyzes the conversion of fibrinogen to fibrin in addition to activating platelets and consequently causing them to release the contents of their granules. Bovine thrombin is used in some PRP preparation techniques, usually in combination with calcium chloride, to catalyze the conversion of fibrinogen to fibrin. Upon activation, degranulated platelets release growth factors immediately.¹⁹ It has been reported that platelets secreted 70% of their stored growth factors within 10 minutes of activation and close to 100% of release occurs within the first hour.¹⁸ Thus, the PRP forms prepared with the addition of thrombin and calcium chloride must be administered to the wounded tissue immediately after activation, as performed in group 4 of the current study. 

In contrast, Mann²⁶ has declared that when PRP was injected into connective tissues, it came into contact with tissue thromboplastin, which could activate platelets and initiate the formation of a fibrin scaffold. Han et al²⁷ concluded that activation with thrombin was unnecessary, and may even be detrimental to the biological activity of the growth factors. It is also known that since the dose response curves of growth factors were not linear they might be inhibitory at higher concentrations.¹⁵ 

In the current study, no statistically significant difference was found between the wound healing effects of thrombin-activated (group 4) and nonactivated (group 3) groups. When the wound areas of the rats in groups 3 and 4 were compared, no statistically significant difference was found between these groups, although the mean wound area value of group 4 was smaller than the mean wound area of group 3. However, the eventual biological effects of exogenous thrombin activation of PRP on wound healing have yet to be clearly identified.

Choukroun’s²⁸ PRF is the latest development of the PRP-preparation protocols. In this technique, blood is collected in gel-free biochemistry tubes without any anticoagulant and immediately centrifuged. A natural coagulation process occurs and allows for the easy collection of a PRP gel without the need for any biochemical modification of the blood. This means that no anticoagulants, thrombin, or calcium chloride are required. This open-access technique is the most simple and also the least expensive protocol developed thus far.^17,28 The gel form of PRP was used in group 2 of the current study and applied to the wounds of rats on postoperative days 1, 4, 7, and 10.

The results of the current study showed that although the PRP used in group 2 (PRP gel obtained by single centrifugation) accelerated wound healing when compared with the control group, the PRPs used in group 3 (liquid PRP obtained by double centrifugation) and group 4 (PRP-gel obtained by double centrifugation with added thrombin) were more effective than the PRP used in group 2. 

Since this study was designed only to compare the effects of 3 different forms of PRPs on the healing process, there was no evaluation of the mechanism of action, growth factor levels, or other possible influential factors on the wound healing effects of these PRP preparations. 

In conclusion, it can be said that although all PRP preparations had positive effects on dermal wound healing, double-centrifuged (with or without thrombin activation) PRPs are more effective than single-centrifuged PRPs and double-centrifugation methods should be preferred for the preparation of PRP. The present study was conducted to investigate the effects of different PRP forms on acute wounds. Further studies investigating the effects of PRP solutions on chronic wounds should also be designed. However, in addition to more experimental studies, prospective and randomized clinical studies are needed to investigate the role of the various PRP preparations for improving wound healing.

From the Ankara Education and Research Hospital, Department of General Surgery, Ankara, Turkey; Aksaray State Hospital, Department of Pathology, Aksaray, Turkey; and Ankara Education and Research Hospital, Department of Biochemistry, Ankara, Turkey

Address correspondence to:
Kemal Kismet, MD
S.B. Ankara Egitim Ve Arastirma Hastanesi Genel Cerrahi Klinigi
Ulucanlar, Ankara, Turkey
kemalkismet@yahoo.com 

Disclosure: The authors disclose no financial or other conflicts of interest.