Volume 27 Number 2

Key messages

This article discusses the effects of different concentrations of Platelet Rich Plasma on wound healing in terms of wound healing rate and histopathology.

The aim of this study is to answer the question of whether different PRP concentrations are effective in wound healing and which concentration dose is most effective.

The results show that different doses of PRP are effective in wound healing and the most effective dose is the form prepared at a concentration of 1000–1100×10³/ml.

Introduction

The skin is the largest organ of the body, protecting it from the external environment and having immunological and endocrinological activities. The skin plays a role in preventing toxic, thermal and mechanical effects, and maintaining body temperature balance. The skin, which constitutes approximately 16% of the total body weight, has an average weight of 5–6kg. One of the main functions of the skin is to contribute to the body’s defense system with immune system cells located in the dermis.¹ A wound is the disruption of tissue integrity due to trauma, surgery or various diseases. The occurrence of healing depends on the proper extracellular matrix (ECM), angiogenesis and epithelialisation. In the wound healing process, inflammatory cells, fibroblasts, keratinocytes, cytokines and growth factors that facilitate communication between these cells play important roles.²

Many growth factors and cytokines have been reported to have positive effects on wound healing in animal experiments. These substances are expensive, and their pharmacokinetics are not fully known. The goal of all these efforts is to make wound healing faster and more perfect. Rapid and effective wound healing reduces hospital stay, lowers costs and has positive effects on morbidity and mortality.^2-4

Platelets are the main source of the growth factor complex that plays a fundamental role in natural wound healing. Platelets not only form clots to prevent local blood and lymph loss but also contain growth factors and cytokines that initiate wound healing. Growth factors are released by separating platelets through proteins, such as thrombin. A small volume of plasma containing a high concentration of platelets is called platelet-rich plasma (PRP).^5,6 In clinical studies, PRP is prepared by various methods such as double centrifugation, without thrombin activation, and with or without leukocytes. However, the effectiveness of these PRP forms has not been compared.

This study aimed to reveal the effects of PRPs prepared in three different concentrations on the healing process using open wound models created in rats and to evaluate the effects on wound healing speed and histopathological changes during healing. This study, therefore, provides a scientific contribution regarding which PRP concentration can be used more safely and effectively in the clinic.

Materials and methods

This study was conducted at the Experimental Animal Breeding and Experimental Research Center of Ankara Training and Research Hospital, with the approval of the Local Ethics Committee for Animal Experiments of T.C. Ministry of Health Ankara Training and Research Hospital, meeting number 21, and approval number 345. Study protocols were conducted in accordance with the institution’s guidelines or humane care criteria of the National Research Council as outlined in the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication No. 86-23, 1985).

Experimental animals and experimental plan

In this study, 55 female Wistar Albino rats weighing 250±25 grams were used. Fifteen of these animals were used as donors only during the preparation of PRP, and surgical procedures were performed on the other 40 animals. The rats were housed in appropriate cages at 22±2°C, with a 12-hour dark and 12-hour light cycle. The ambient humidity was maintained between 40–50%. The animals’ nutritional needs were regularly met with standard laboratory feed and water. A 5-day quarantine period was applied after the rats were admitted to the laboratory.

General anesthesia was provided to 40 animals in the study group with 50mg/kg ketamine hydrochloride (Ketalar^®, Eczacıbaşı, Turkey) administered intramuscularly (IM). The anesthetised animals were placed in a prone position, and their back hair was shaved carefully without damaging the skin. After cleaning the back area of each rat with povidone-iodine (Betadine®), full-thickness, standardised rectangular wounds of 2x1cm including the skin, subcutaneous tissue, and panniculus carnosus were created using a number 15 scalpel blade guided by a template, and the wounds were covered with gauze moistened with physiological saline. To obtain PRP, 15 donor rats, which were not subjected to any surgical procedure, were sacrificed with high-dose phenobarbital, and their total body blood was collected from the inferior vena cava (Figure 1a).

All surgical procedures were performed by the same surgeon using standard surgical techniques. The animals were placed in appropriate positions on specially designed animal fixation boards for surgical interventions (Figure 1b).

The animals were monitored postoperatively in standard laboratory conditions under the supervision of a specialist veterinarian, and housed in groups of five in cages. Their feed and water needs were regularly met (Figure 1c).

 

Figure 1. a) Collection of the blood sample from the inferior vena cava; b) preparation of rats before surgery; c)  placement of rats into cages

 

Study groups

Each rat was assigned a numerical number. By drawing lots a total of 40 rats were divided into 4 groups of 10 rats each. Full-thickness wounds of 2x1cm, including the panniculus carnosus, were created in the middle of the back of the rats  that had completed their preoperative preparations. The wounds were covered with gauze moistened with isotonic saline. This procedure was performed uniformly on all rats in the groups (Figure 2).

 

Figure 2. Performing a 2x1cm incision, removal of the panniculus carnosus muscle, created wound, covering the wound with gauze moistened with isotonic saline

 

Group 1 (Control) (n=10): The wound area was cleaned with isotonic saline on days 1, 4, 7, and 10, and then covered with gauze moistened with isotonic saline.

Group 2 (PRP with 200,000–300,000/ml platelet concentration obtained by double centrifugation) (n=10): Blood obtained from donor rats was placed into a tube containing citrate-phosphate-dextrose (CPD) solution and first centrifuged at 1500rpm for 10 minutes. After this centrifugation, a clear yellow plasma was observed at the top, with erythrocytes settled at the bottom, and a thin intermediate layer (buffy coat) formed by platelets and white blood cells. The plasma collected at the top was transferred to another tube and centrifuged again at 3000rpm for 10 minutes. Following this centrifugation, platelet-poor plasma (PPP) was obtained in the upper layer, and PRP was obtained in the lower layer. The obtained PRP, with a concentration of 200,000–300,000/ml, was applied to the wound area of each rat in a volume of 100µL on days 1, 4, 7, and 10. Afterward, gauze moistened with physiological saline was placed over the wound.

Group 3 (Liquid PRP obtained by double centrifugation at a concentration of 600,000–700,000/ml) (n=10): The PRP, prepared in the same manner as Group 2, was applied to the wound area of each rat in a volume of 100µL on days 1, 4, 7, and 10. Afterward, gauze moistened with physiological saline was placed over the wound.

Group 4 (Liquid PRP obtained by double centrifugation at a concentration of 1,000,000–1,100,000/ml) (n=10): The PRP, prepared in the same manner as Group 2, was applied to the wound area of each rat in a volume of 100µL on days 1, 4, 7, and 10. Afterward, gauze moistened with physiological saline was placed over the wound.

On days 1, 4, 7, 10, and 14, the wound areas were photographed, and the remaining wound areas were traced onto acetate paper. These tracings were scanned and analysed using the Image J^® program to measure the wound surface areas on a computer. All rats in the groups were sacrificed on the 14th postoperative day, and samples were taken from the wound areas for histopathological examination.

Preparation of liquid PRP by double centrifugation

The blood required to separate the platelets from the other blood components and form PRP was obtained on days 1, 4, 7, and 10 of the study by collecting the entire body blood from the inferior vena cava of a total of 15 rats, selected as donors, after they were sacrificed with a high dose of phenobarbital.

After 10Ml of blood containing citrate-phosphate-dextrose (CPD) solution was collected from the donor rats into a tube, the sample was centrifuged at 1500rpm for 10 minutes at room temperature (22°C). Following this first centrifugation, a thin intermediate layer (the buffy coat) composed of platelets and white blood cells was observed between the pale yellow plasma that had accumulated at the top and the erythrocytes that had settled at the bottom. The plasma collected at the top was then transferred into a separate tube and centrifuged for another 10 minutes at 3000rpm. As a result of this second centrifugation, platelet-poor plasma (PPP) with a pale yellow color was obtained in the upper layer, while PRP was obtained from the lower layer (Figure 4). The PPP was then separated, and both PPP and PRP were collected into different tubes. A complete blood count was performed on the obtained PRP, and the platelet count was measured as 1434×10³/Ml. The obtained PRP was diluted with PPP and then subjected to another complete blood count. The platelet counts were adjusted to 200–300×10³/Ml, 600–700×10³/Ml, and 1000–1100×10³/Ml; the leukocyte count was set to 0.09–0.11×10³/Ml, and the erythrocyte count was also adjusted to 0.09–0.11×10³/Ml. Finally, the diluted liquid PRP was applied to the wounds of each subject according to group allocation on days 1, 4, 7, and 10 of the study.

General evaluation wound contraction measurements In all groups, the progression of wound healing and the extent of wound contraction in the rats during the postoperative period were documented on days 1, 4, 7, 10, and 14 by drawing on acetate paper with a permanent marker and capturing the images with a digital camera. The acetate papers were then scanned, and the wound surface areas were measured in square millimeters (mm²) using the Image J^® program on a computer.

 

Figure 3. a) First centrifugation; b)second centrifugation; c) the form of PRP ready for application after dilution with PPP; d) the method of applying the liquid PRP

 

Histopathological evaluation Tissue samples measuring 2×1cm, obtained from the skin of rats, were fixed in a 10% formaldehyde solution for 2 days. Following fixation, the tissues were dehydrated in ethanol (in sequential concentrations of 50%, 75%, 96%, and 100%) and cleared with xylene, after which they were embedded in paraffin. From the paraffin-embedded tissues, 3µm sections were obtained using a Leica RM 2125 RT microtome. The tissue sections were then stained with Hematoxylin & Eosin (H&E) and Mason’s Trichrome for examination. In the H&E-stained sections, epithelialisation and inflammation/inflammatory granulation tissue were evaluated, while in both H&E and Trichrome-stained sections, the presence of fibrosis/healing was assessed using a semi-quantitative scoring system. The histopathological examination was performed using an OLYMPUS BX51TF microscope equipped with x4, x10, x20, x40, and x100 objective leffnses.

Statistical analysis

For the statistical analysis, the SPSS 15.0 for Windows program was used. Numerical values are expressed as n (number of rats), mean ± standard deviation, mean, and percentage (%). To evaluate the differences between groups, either one-way ANOVA or the Kruskal-Wallis statistical analysis was used, whichever was appropriate. When the p-values obtained from the variance analyses were found to be statistically significant, Tukey or Mann-Whitney U tests were used for pairwise comparisons between groups. In the analysis results, p-values less than 0.05 were considered statistically significant.

Results

Wound contraction area measurements

Throughout the experimental period, the subjects were examined macroscopically on a daily basis. No antibiotics were administered to the rats during the experiment. After full-thickness wounds measuring 2×1cm—including the mid-dorsal region and involving the panniculus carnosus—were created, the wound areas were recorded in all groups on days 1, 4, 7, 10, and 14 using a digital camera (Figure 4). In addition, the wound outlines were drawn onto acetate paper with a permanent marker on those same days. The acetate papers were then scanned, and the wound surface areas were calculated in square millimeters (mm²) using the Image J® program on a computer. The mean wound areas and standard deviations for each group on days 1, 4, 7, 10, and 14 were determined. For example, in group 1 (control) the mean wound area was 205.5mm² on day 1 and 58.6mm² on day 14; in group 2, the mean wound area was 212.4mm² on day 1 and 47.1mm² on day 14; in group 3, the mean wound area was 186.6mm² on day 1 and 47.9mm² on day 14; and in group 4 the mean wound area was 186.6mm² on day 1 and 35.2mm² on day 14. All the values are presented in Table 1.

 

Figure 4. Macroscopic images of the wound areas on days 1, 4, 7, 10 and 14

 

Table 1. Wound contraction areas (With the help of Image J ® program, the wound contraction areas of the subjects were calculated in square millimeters (mm²) and the averages and standard deviations were tabulated.)

 

Statistical analysis revealed that on day 14 the wound areas in Groups 2, 3, and 4 were significantly smaller compared to group 1 (control), indicating that the wounds in the PRP-treated groups healed faster (p<0.05). Furthermore, when comparing Groups 2 and 3, the wound area in Group 3 was significantly smaller and exhibited a more rapid healing process than group 2 (p<0.05). Likewise, the analysis between Groups 2 and 4 demonstrated that the wound area in Group 4 was significantly smaller and healed faster than in Group 2 (p<0.05). Additionally, a comparison between Groups 3 and 4 showed that wounds in Group 4 were significantly smaller and healed faster than those in Group 3 (p<0.05).

Histopathological evaluation findings

Tissue samples (measuring 2×1cm) obtained from the skin of the rats were fixed in a 10% formaldehyde solution for two days. Following fixation, the tissues underwent ethanol dehydration in increasing concentrations (50%, 75%, 96%, and 100%) and were cleared with xylene. The cleared tissues were then embedded in paraffin, and 3µm sections were obtained using a Leica RM 2125 RT microtome. These sections were stained with Hematoxylin & Eosin (H&E) and Mason’s Trichrome. In the H&E-stained sections, epithelialisation and the formation of inflammatory granulation tissue were evaluated, whereas both the H&E and Trichrome-stained sections were assessed for the presence of fibrosis/healing using a semi-quantitative scoring system. The histopathological examination was performed with an OLYMPUS BX51TF microscope using objective lenses at magnifications of x4, x10, x20, x40 and x100.

Epithelialisation scoring system

Score 0: No epithelial formation on the wound surface.

Score 1: Incomplete re-epithelialisation with focal epidermal hyperplasia.

Score 2: Complete re-epithelialisation.

Inflammation scoring system

Score 0: Absent inflammation.

Score 1: Only a few inflammatory cells (lymphocytes, plasma cells, and giant cells) present in the dermis.

Score 2: Noticeable vascular proliferation in the dermis and presence of plasma cells, eosinophils, neutrophils and giant cells.

Score 3: A marked inflammatory response with numerous inflammatory cells, extensive vascular proliferation and microabscess formation.

Fibrosis scoring system

Score 0: No fibrosis.

Score 1: Mild fibrosis, characterised by sparse collagen deposition.

Score 2: Moderate fibrosis.

Score 3: Severe or intense fibrosis.

When comparing all groups, the average scores for fibrosis, epithelialisation, and inflammation were determined as follows:

Group 1: Fibrosis=0.88; Epithelialisation=0.33; Inflammation=2.55

Group 2: Fibrosis=1.6; Epithelialisation=1.1; Inflammation=1.6

Group 3: Fibrosis=2.10; Epithelialisation=1.20; Inflammation=1.4

Group 4: Fibrosis=2.3; Epithelialisation=1.4; Inflammation=1.3

The pathological score averages and standard deviations for the groups are summarised in Table 2.

 

Table 2. Pathological score means and standard deviations of the groups

 

Statistical analysis of the epithelialisation, inflammation, and fibrosis scores showed that significant differences existed between Group 1 and Groups 2, 3, and 4 (p<0.05). However, no statistically significant differences were observed between Groups 2 and 3, between Groups 2 and 4, or between Groups 3 and 4 (p>0.05). Histopathological images demonstrating the features of epithelialisation and inflammation are presented in Figure 5, while images illustrating fibrosis are displayed in Figure 6.

 

Figure 5. The appearance of the epidermis at different stages: a) In the ulcerated view, no epithelialisation is observed in the epidermis (Epithelialisation score 0, Hematoxylin & Eosin [H&E] staining). A large number of inflammatory cells and microabscess formations are present (Inflammation score 3). b) In a tissue exhibiting fibrosis, inflammatory granulation tissue composed of a few lymphocytes and plasma cells is seen alongside foreign body-type giant multinucleated cells (Inflammation score 2). Furthermore, histiocytic giant multinucleated cells that have phagocytosed foreign bodies are indicated (arrow). c) Complete re-epithelialisation is observed (Epithelialisation score 2, H&E). The section shows epidermal hyperplasia with a few inflammatory cells and prominent fibrosis/healing features (Inflammation score 1).

 

Figure 6. Collagen-type fibrosis development with histochemical trichrome staining: (a) Mild fibrosis, b) Moderate fibrosis, c) Severe fibrosis)

 

Discussion

Wound healing is a well-organised reparative process that occurs following surgical procedures or trauma. Many studies have been conducted to determine the most effective, cost-efficient, and physiologically appropriate approaches for wound healing a process in which multiple factors are involved.^7,8 With advancements in technology and the increasing body of information on wound healing, research activities have accelerated. Whereas in past decades most wounds healed in a complicated manner, complications related to wound healing have decreased nowadays. Nonetheless, problems in wound healing and the resulting economic and psychological burdens still remain significant challenges for clinicians.⁹

Despite numerous scientific investigations and advances in technology and medicine, it is still not possible to claim that definitive knowledge about wound healing exists. However, the knowledge base in this field is expanding every day.^5,10 The wound healing process is governed by a complex signaling network comprising various growth factors, cytokines and chemokines. Successful wound healing depends on the integration and transformation of signals released by bioactive molecules—which also play roles in normal tissue repair.^11,12 The absence or deficiency of one or more of these bioactive molecules can result in incomplete and uncoordinated healing.^13,14 Platelets, for example, contain over 1100 proteins including growth factors, immune system mediators, enzymes, enzyme inhibitors and many other bioactive compounds, and they are involved in various phases of tissue repair.¹⁵

The concept of obtaining concentrated autologous platelets from a given volume of plasma to provide high levels of these bioactive molecules is not new. PRP has been used for more than thirty years in dentistry, orthopedics, ophthalmology, neurosurgery, maxillofacial surgery and for cosmetic purposes.¹⁶ It was first employed in 1987 by Ferrari and colleagues following open-heart surgeries to reduce the need for transfusions of homologous blood products.¹⁷ More recently, PRP has been applied in various sports injuries—including chronic tendinopathies, rotator cuff repairs, Achilles tendon repairs, anterior cruciate ligament reconstructions, muscle injuries, and the repair of acute joint and fracture injuries.^18,19 Takikawa et al²⁰ applied PRP complexed with fragmin/protamine microparticles to planned skin flaps on the dorsum of rats. They reported that fragmin binds heparin-linked growth factors and enhances their effect, leading to increased vascularisation, fibrous tissue formation and callus growth. Li et al²¹ investigated the effect of subcutaneous injections of PPP (platelet-poor plasma) versus PRP on the viability of dorsal skin flaps in rats.They found that flaps in the PRP group exhibited significantly lower necrosis rates compared to those in the PPP and control groups, and also reported increased vascular density and elevated growth factor levels. In a similar study, Fındıkçioğlu et al²² performed preoperative subcutaneous applications of PRP and fibrin adhesive on abdominal skin flaps in rats and compared the outcomes with a fibrin adhesive group and a control group, reporting that PRP improved flap viability. In a study on forty rabbits in which they created Achilles tendon transections on the right hind legs, Geng et al²³ observed that although all tendons healed, the PRP-treated group showed significantly greater fibrous tissue formation at the anastomosis site. Histological examination revealed more prominent collagen fibrils in the repair area in the experimental group, ultimately suggesting that PRP accelerates healing and improves the quality of tendon repair in rabbits. Saad et al²⁴ investigated the influence of PRP on tissue healing in 24 patients with diabetic foot ulcers by dividing them into two groups—one treated with PRP and the other with PPP—and found that PRP significantly accelerated the healing process. Marx et al²⁵ compared the use of bone grafting alone with that of combined bone grafting and PRP in treating mandibular defects following tumor surgery, finding that the application of PRP increased both the quantity and speed of bone fill. Na et al²⁶ applied fractional carbon dioxide laser skin resurfacing in 25 patients, comparing the effects of PRP with those of physiological saline; they reported that in the PRP group the healing time was significantly shorter, and that there were notably fewer issues with skin fluid loss, erythema, and inflammation-related hyperpigmentation.

Despite the promising potential benefits of growth factors in enhancing tissue repair, the clinical advantages of using PRP to improve functional outcomes have not yet been conclusively demonstrated.²⁷ One reason for the discrepancy between preclinical studies and clinical applications is the significant variability in the PRP obtained.²⁸ A universal classification system for PRP has not yet been established. However, in recent years, a classification system called PAW (platelets, activation, white blood cells) has been proposed. This system takes into account factors such as platelet concentration, the requirement for activation with thrombin, and the inclusion or exclusion of white blood cells. Although the PAW classification helps to highlight important differences among PRP preparation techniques, a complete consensus on the optimal preparation of PRP has not been reached.²⁹

In our study, we prepared PRP using a laboratory centrifuge. We applied PRP at different concentrations to three distinct groups with wound areas created on the dorsum of the rats at various time points. Within the PAW classification framework, we evaluated which of the liquid PRP preparations—obtained by double centrifugation at different concentrations—was most effective in promoting wound healing. To eliminate the potential effects of white blood cells on PRP, we maintained the white blood cell counts in our PRP preparations at similar levels. The optimal platelet concentration necessary for effective wound healing has not been exactly determined. In vitro studies have shown that dose–response curves for growth factors are not linear with increasing platelet concentrations.³⁰ Wound healing appears to be more effective within platelet concentrations ranging from 200–300×10³/mL to 1000–1100×10³/mL. Higher platelet concentrations are not recommended because of concerns regarding biological compatibility.^31,32 The potential effects of higher platelet concentrations depend on the initial whole blood volume, the preparation technique used, and the plasma volume in the final platelet solution.²⁸ Additionally, studies have reported that natural variations in platelet concentration occur depending on the donor’s physiological condition during blood collection; however, the underlying causes of this variability are not fully understood.^30,32 The differing outcomes observed in various studies using PRP might be due to these physiological variations during platelet concentration preparation. In our study, we maintained the platelet counts in our PRP preparations within an optimal and very similar range: 200–300×10³/mL in Group 2, 600–700×10³/mL in Group 3, and 1000–1100×10³/mL in Group 4. This approach allowed us to assess the effect of differences in platelet concentration on the efficacy of PRP. To evaluate the effectiveness of PRP on wound healing, we examined the wound areas of PRP-treated rats macroscopically over a 14-day period and on the final day also performed histopathological sampling of the wounds.

Previous studies employing PRP have consistently shown that PRP significantly reduces wound healing time compared to control groups. Groups treated with high–platelet–concentration PRP exhibited better wound contraction rates than those treated with lower–platelet–concentration PRP—in other words, wounds in the high–platelet groups closed to a greater extent and more rapidly. The similar histopathological scores observed across groups indicate that the wound healing process was still ongoing in the examined samples.

Conclusions

In conclusion, this study demonstrated that the PRP forms prepared by double centrifugation can be safely used for wound healing. In particular, it was observed that the high-concentration liquid PRP form (1000–1100×10³/ml) obtained through double centrifugation was the most effective in enhancing wound healing, as evidenced by the wound area measurements. In order to more clearly assess the efficacy of PRP on wound healing, it is necessary to first conduct clinical, prospective randomised studies with a focus on dose dependency. We believe that as the number of studies demonstrating the positive effects of PRP on wound healing increases in the future, its clinical application for wound healing will become even more effective.

Implications for clinical practice

Different doses of PRP can be used safely in wound healing. Higher doses of PRP are more effective in wound healing.

Authors' contributions

Abdullah Bulgurcu, Veysel G Soylu and Alper Doğu: Writing, review and editing, performing animal testing processes.  Pınar Celebli: Conceptualisation, methodology. Ertuğrul Ertaş:  Review & editing (support)

All authors reviewed the results and approved the final version of the manuscript.

Conflict of interest

The authors declare no conflicts of interest.

Funding

The authors received no funding for this study.

Author(s)

Abdullah Bulgurcu*¹ MD, Veysel G Soylu² Associate Professor, Alper Doğu³ MD, Pınar Celepli² Associate Professor,
Ertuğrul Ertaş⁵, Professor Doctor
¹Department of General Surgery, Polatlı State Hospital, Ankara, Turkey
²Intensive Care Department, Kastamonu University Faculty of Medicine, Kastamonu, Turkey
³Department of General Surgery, Ankara Training and Research Hospital, Ankara, Turkey
⁴Department of Pathology, Ankara Gülhane Health Application and Research Center, Ankara, Turkey
⁵Department of General Surgery, Duzce University Faculty of Medicine, Düzce, Turkey

*Corresponding author email drabulgurcu@hotmail.com

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