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Introduction
Every year, millions of laparotomies are performed worldwide due to trauma, malignancies, acute abdomen syndrome, infections, vascular pathologies, and gynecological and urogenital diseases. Unfortunately, peritoneal adhesions inevitably occur after these operations. Peritoneal adhesions are fibrous tissues formed between the peritoneum and intra-abdominal organs such as the small intestine, colon, and uterus and play an essential role in post-operative morbidity. Adhesions after pelvic surgery are the leading cause of complications such as chronic abdominopelvic discomfort, pain, and infertility. The remarkable increase in morbidity and mortality due to pelvic adhesions has revealed the necessity of developing more safe and effective anti-adhesion auxiliary models1-3. Numerous pharmaceutical studies have recently investigated the trial of drugs with anti-inflammatory effects on various animal models and their usability in clinical settings4-7.
Peritoneal adhesions begin to form on the peritoneal and visceral surfaces concerning post-operative trauma. The vascular permeability of the tissue increases, followed by the exudation of inflammatory cells. The fibrin matrix is gradually organized and replaced by tissue fibroblasts, macrophages, and giant cells. In the next step, fibrin bands are modeled, and connections are formed between the injured tissue and the peritoneum. The fibrinolytic system destroys fibrin bands, and peritoneal healing is achieved. If this system is insufficient, fibrin bands become permanent8. Tissue response may be exaggerated after surgical interventions, and tissue regeneration and remodeling may result in adhesion formation9.
Platelet-rich Plasma (PRP) obtained from the patient’s blood contains growth factors and biomolecules necessary for wound healing. Using autologous PRP eliminates the risks of cross-contamination, disease transmission, or immune reactions. Another significant advantage is that it is simple and fast to prepare, and the preparation cost is low (approximately 30 minutes from blood collection to administration). PRP is prepared from the centrifugation of autologous whole blood and combined with thrombin and calcium chloride to produce a viscous coagulation gel7. PRP contains many growth factors and various proteins that can stimulate the healing process. Therefore, it has widespread clinical use. PRP accelerates neovascularization and increases blood flow and nutrient flow required for cell regeneration in damaged tissues. It also stimulates the proliferation and differentiation of cells involved in the healing process10.
Transverse incision on the uterus is applied in many surgical interventions such as myomectomy and cesarean section in surgical practice. This study was designed to investigate the effect of PRP on the healing process and intra-abdominal adhesion at gynecologic surgery.
Material and methods
Preparation of PRP
Locally applied PRP was used in this study. Three milliliters of blood collected from the rabbits’ ear veins were slowly injected into a tube containing 10:1 sodium citrate. 0.5 ml of blood was taken into a separate tube, and platelet count (489.000/μl) was measured with a complete blood count panel (Mindray BC-6800). The blood was centrifuged at 160 G for 10 min. The blood components were divided into two: the clear supernatant forming PRP and the other part containing erythrocytes and leukocytes. A portion of the clear supernatant is centrifuged again at 300 G for 10 min. With the complete blood count, it was determined that the platelet count reached approximately three times (1463.000/μl). Calcium chloride (10:1) was added for platelet activation before injection7.
Animal experiments
The authors declare that the procedures followed were in accordance with the regulations of the relevant clinical research ethics committee and those of the Code of Ethics of the World Medical Association (Declaration of Helsinki).
In the research within this project’s scope, 16 adults, an average of 20 weeks old, unmated, female, 2500-3000 g white New Zealand rabbits were used as experimental animals. Rabbits were left to acclimate to laboratory conditions for 1 week after purchase. Rabbits are kept as single individuals in a cage. The animals were kept in the laboratory’s standard animal housing conditions with 12 h of light-dark, 21°C temperature, 50-60% humidity, and optionally standard pellet rabbit food and water. All rabbits fasted for 12 h before surgery.
Animals were randomly divided into two groups, 8 for each, as the control group and the PRP application group. Animals were anesthetized by intramuscular injection of 10 mg/kg xylazine (Rompun; Bayer, Turkey) and 30-35 mg/kg ketamine (Ketalar; Parke Davis, Turkey, Istanbul) and allowed to breathe spontaneously. A rectal tube was inserted into the animals and placed on a heating pad to maintain their body temperature at 37°C. All surgical procedures were performed by the same surgeon. Rabbits were fixed in the supine position. The surgery was limited to 10 min per rabbit to control the effect of tissue drying in room air, with care being taken to avoid large amounts of bleeding from the tissues. The midline lower abdomen of each rabbit was shaved and disinfected with an iodine solution. The abdomen was entered with an approximately 5 cm long midline vertical incision. In each group, a transverse incision was made on the uterus for wound healing and adhesion evaluation. The abdominal wall was opened, and a typical lesion was created by making a 10 mm long transverse incision on the antimesenteric face of one of the uterine horns with a scalpel. The uterus was duly closed with 4-0 vicryl. After the bleeding control was achieved with cautery, the PRP prepared on the wound lips was applied equally to the PRP group with an insulin syringe. Since we injected PRP into the sutured uterine incision, local injection with saline was applied as a placebo fluid for the control group, considering the damage that the injection needle would cause to the uterine tissue. Then, the abdominal wall was closed with two layers of 4/0 vicryl. Antibiotic prophylaxis was administered during and after the surgery. Relaparotomy was performed in both groups 21 days after the first operation. The uterine incision site was excised and preserved for histopathological examination.
Macroscopic adhesion scoring
Adhesions formed after relaparotomy on the 21st day were evaluated macroscopically by two independent researchers unaware of the applications. The mean score of both researchers was used for that rabbit. Grading of adhesions was evaluated in terms of adhesion prevalence and severity, with scores from 0 to 4. The total adhesion score (0-8) was found for each rabbit by summing the adhesion prevalence and severity scores11 (Table 1).
Table 1 Macroscopic Adhesion Scoring11
| Score | Definition |
|---|---|
| Adhesion prevalence (0-4) | |
| 0 | 0% |
| 1 | < 25% |
| 2 | 25-50% |
| 3 | 50-75% |
| 4 | > 75% |
| Adhesion severity (0-4) | |
| 0 | Absent |
| 1 | Self detached |
| 2 | Separated by withdrawal |
| 3 | Separated by blunt dissection |
| 4 | Separated by sharp dissection |
| Total adhesion score (range 0–8) | Total adhesion prevalence and severity score |
Histopathological evaluation
The uterine incision site was excised 21 days after the first operation. These tissues were fixed in 10 % neutral buffered formaldehyde solution at room temperature for 24 h. In the 15-h follow-up process with the tissue tracking device (Sacura), dehydration, transparency, and tissue hardening processes were completed by passing through alcohol, xylene, and paraffin stages. Then, the tissues were made into blocks by embedding paraffin in a tissue embedding device (Thermo Shandon). After the blocks were cooled in the refrigerator, 3-micron-thick sections were taken on the microtome device (Leica). Some of these sections were taken from standard slides for hematoxylin-eosin (HE) and Mason’s Trichrome (MT) staining, and some of them were taken on a positively charged slide for anti-vascular endothelial growth factor-1 (VEGF-1) immunohistochemistry staining. Sectioned preparations for HE (Facepath) staining were stained in an automatic staining-off device (Sacura) and closed. MT staining (Facepath) was applied to the slides manually with the kit. VEGF-1 antibody (Abcam, Clone: Y103, 1/100 dilution, 32 min incubation time) immunohistochemical staining was performed on the preparations taken on positively loaded slides in an automatic Ventana Benchmark XT device. Then, the slides were closed by dripping entellan. After staining procedures, the samples were assessed in a blinded manner by two pathologists to avoid bias with a light microscope (Leica DM 750). Inflammation and fibrosis were evaluated in HE-stained preparations9,12,13 (Table 2).
Table 2 Histopathological evaluation9,12,13
| The degree of inflammation | |
|---|---|
| 0 | |
| 1 | No inflammation |
| 2 | Rarely presence of giant cells, lymphocytes, and plasma cells |
| 3 | Presence of giant cells, plasma cells, eosinophils, and neutrophils Presence of many inflammatory cells and microabscesses |
| The degree of fibrosis | |
| 0 | No fibrosis |
| 1 | Mild |
| 2 | Moderate |
| 3 | Severe |
| Percentages of VEGF-1 immunoreactive cells | |
| 0 | Negative for staining |
| 1 | 33% positive staining |
| 2 | 33-66% positive staining |
| 3 | 66% recorded as positive staining |
VEGF: vascular endothelial growth factor.
Results
A veterinarian checked the rabbits daily, and no problems were encountered throughout the study. The total adhesion score of the control group was 38, and the total adhesion score of the PRP group was 32 (Table 3). A statistically significant relationship was found between the two groups regarding total adhesion score (p = 0.005) (Table 4). The histopathological findings of both groups were compared, and the results were scored (Table 5).
Table 3 Macroscopic adhesion scoring in groups
| Macroscopic adhesion | Control group | PRP group |
|---|---|---|
| Adhesion prevalence | 3, 3, 2, 3, 3, 2, 3, 2 | 2, 2, 2, 2, 2, 2, 1, 1 |
| Adhesion severity | 3, 3, 3, 3, 2, 2, 3, 3 | 2, 3, 2, 2, 3, 2, 2, 2 |
| Total adhesion score | 6, 6, 5, 6, 5, 4, 6, 5 | 4, 5, 4, 4, 5, 4, 3, 3 |
PRP: platelet-rich plasma.
Table 4 Statistical comparison of macroscopic adhesion scoring
| Macroscopic adhesion | Control group | PRP group | p-value |
|---|---|---|---|
| Adhesion prevalence | 2.62 ± 0.51 (min-2, max-3) | 1.75 ± 0.46 (min-1, max-2) | 0.009 |
| Adhesion severity | 2.75 ± 0.46 (min-2, max-3) | 2.25 ± 0.46 (min-2, max-3) | 0.051 |
| Total adhesion score | 5.37 ± 0.74 (min-4, max-6) | 4.00 ± 0.75 (min-3, max-5) | 0.005 |
Results were given as mean ± standard deviation (n = 8 for each group). PRP: platelet-rich plasma.
Table 5 Scoring of histopathological findings
| Histopathological findings | Control group | PRP group |
|---|---|---|
| Inflammation | 2, 1, 1, 2, 1, 1, 2, 1 | 1, 1, 1, 1, 1, 0, 1, 0 |
| Fibrosis | 1, 2, 1, 1, 2, 1, 1, 2 | 1, 1, 1, 1, 1, 0, 1, 0 |
| MT | 1, 2, 1, 1, 2, 1, 1, 2 | 1, 1, 1, 1, 1, 0, 1, 0 |
| VEGF-1 | 3, 2, 3, 3, 3, 3, 3, 3 | 3, 2, 2, 2, 3, 2, 2, 2 |
PRP: platelet-rich plasma; MT: Mason’s Trichrome staining; VEGF: vascular endothelial growth factor.
When the control group and PRP group were evaluated in terms of inflammation (Fig. 1), there was a significant difference between the two groups (p = 0.041). When the control and PRP groups were evaluated in terms of MT staining and fibrosis, There was a significant difference between groups (p = 0.041) (Figs. 2 and 3). When the control and PRP groups were evaluated in terms of VE GF-1, there was also a statistically significant difference between the groups (p = 0.020) (Table 6) (Figs. 4 and 5).
Figure 1 Control group. A: moderate fibrosis, inflammation with giant cells and signs of increased vascularization (hematoxylin-eosin [HE] × 200). B: mild fibrosis (HE × 200).
Figure 2 Platelet-Rich Plasma group. A: no fibrosis (hematoxylin-eosin [HE] × 200). B: mild fibrosis, mild signs of inflammation (HE × 100).
Table 6 Statistical comparison of histopathological scoring
| Histopathological findings | Control group | PRP group | p-value |
|---|---|---|---|
| Inflammation | 1.38 ± 0.51 (min-1, max-2) | 0.75 ± 0.46 (min-0, max-1) | 0.041 |
| Fibrosis | 1.38 ± 0.51 (min-1, max-2) | 0.75 ± 0.46 (min-0, max-1) | 0.041 |
| MT | 1.38 ± 0.51 (min-1, max-2) | 0.75 ± 0.46 (min-0, max-1) | 0.041 |
| VEGF-1 | 2.88 ± 0.35 (min-2, max-3) | 2.25 ± 0.46 (min-2, max-3) | 0.020 |
Results were given as mean ± standard deviation (n = 8 for each group). PRP: platelet-rich plasma; MT: Masson’s trichrome staining; VEGF: vascular endothelial growth factor.
Discussion
Adhesion development after abdominopelvic surgery is a common complication (60-93%). This situation forms the basis of many problems, such as intestinal obstruction, chronic pain, and infertility14.
First of all, in this study, when PRP produced from the rabbits own blood was applied to the uterine incision, it was observed that adhesion was reduced in all the examined parameters, both macroscopically and histopathologically.
PRP is 3-5 times more concentrated than the amount of autologous rabbit platelets in whole blood. It contains growth factors and biomolecules that accelerate wound healing15-19. Using autologous PRP eliminates the risks of cross-contamination, disease transmission, or immune reactions. By accelerating neovascularization, PRP helps the proliferation and differentiation of cells involved in the healing process10.
Many studies have been conducted on using PRP as a gel on the wound to accelerate wound healing. The common conclusion in these studies is that PRP contributes to wound healing20-22. After the acute inflammatory phase, wound healing occurs with increased fibroblastic activity. It continues for several weeks to months to restore tissue strength and maturation. During this continuity, the healing response may occur more than necessary. These results in delayed adhesion formation are related to the time of surgery because post-operative adhesions in the peritoneum most commonly become permanent within the 1st week and after the 2nd week23. Unlike previous studies, in which the degree of adhesion was evaluated at day 7, 10, or 14 postoperatively12,24,25, fibroblastic activity and degree of fibrosis were evaluated at day 21 in this study. When the adhesion scoring was performed macroscopically, it was determined that the extent of adhesion and the total adhesion score were significantly lower in the PRP group, and the adhesion severity was close to the significance level.
The first step in peritoneal adhesion formation is inflammation26. In our study, as expected, inflammation was detected significantly less in the PRP group compared to the control group.
Fibrosis score is another important parameter in the evaluation of peritoneal adhesions, and it is frequently used to evaluate the effectiveness of anti-adhesion studies27. A low fibrosis score is indicative of weak adhesions. Fibroblasts and many growth factors released from them increase during the healing process and contribute to wound healing. It has also been reported that PRP application increases fibroblast migration and proliferation28,29. This study detected a significantly reduced fibrosis score histopathologically in the PRP group compared to the control group.
Another parameter examined immunohistochemically is VEGF-1. High expression levels of VEGF-1 are thought to stimulate angiogenesis and fibrosis. In addition, tissue expression of VEGF-1 will result in adhesion formation secondary to tissue damage. VEGF-1 is a major factor in wound healing and is responsible for adhesion formation30,31. In this study, as in all other parameters, tissue expression of VEGF-1 decreased significantly with PRP application. The strength of this study is the autologous preparation and use of PRP.
Our study is the first to reveal the effect of autologous PRP on uterine wounds. In experimental rat models, the blood volume is too low to produce autologous PRP, so allogeneic preparations are preferred9. We chose rabbits in the study to use autologous PRP and test its effectiveness. Thus, risks such as cross-contamination, disease transmission, or immune reactions are eliminated. In the literature, there is no other intra-abdominal adhesion study using autologous PRP.
