The fields of tissue engineering and regenerative medicine have expanded in recent years, with the aim of repairing, regenerating, or replacing damaged tissues and organs.1 Platelet-rich plasma (PRP) is a fraction from the plasma of autologous blood that has an above-average platelet concentration. It has been studied extensively in the fields of plastic surgery and dermatology, with a focus on scar remodeling.2–5 It contains growth factors that stimulate myogenesis and, most importantly, angiogenesis.6,7 It is also known for its cost-efficiency and regenerative capacity and for being an effective wound healing option because of its supraphysiologic concentration of growth factor-rich platelets, which play an important role in the healing process.8,9 Studies have reported novel findings related to this blood element.10
Previous research has favored the use of statins in healing skin lesions. Statins reduce cholesterol, improve endothelial function, increase the stability of atherosclerotic plaques, and inhibit the inflammatory process.11 Studies have demonstrated that statins play a positive immune-modulatory role, improving microvascular function and reducing oxidative stress. However, further clinical studies are needed to evaluate the therapeutic effects of systemic and topical statins in wound healing.12,13 Parson et al14 have demonstrated that rosuvastatin (RSV) therapy positively modifies neurovascular function and basal blood flow to the skin. However, it is unclear whether this effect increases blood flow and decreases neuropathy scores and is the cause of the consequent improvement in the healing process, despite an important reduction in the lipid profile.
This study analyzed the number of collagen fibers in rabbit wounds after PRP treatment, alone or in combination with RSV, to investigate the hypothesis that serial doses of RSV in combination with PRP may improve the amount of newly formed collagen fibers after induced skin injury.
This study was approved by the Ethics Committee on Animal Use of Unoeste, Presidente Prudente, São Paulo, Brazil (protocol no. 3478). Eight adult male New Zealand rabbits (aged 3 ± 1 years) were used in this study. Rabbits were clinically healthy (mean weight, 3.0 ± 1.0 kg) and housed in individual cages with an ambient temperature of 22° ± 2° C and controlled photoperiod (12/12-hour light/dark). The rabbits habituated to their surroundings for 7 days before the study and were kept under standard dietary conditions (Supra Coelho Agro; Alisul Alimentos S.A, Maringá, Paraná, Brazil) with free access to water throughout the experiment.
Rabbits were restrained manually for trichotomy of the right and left dorsal region. The animals were then anesthetized with 2% xylazine hydrochloride and zolazepam hydrochloride at a dose of 15 mg/kg (intramuscular injection).
Investigators collected 8 mL blood from the atrial vein after the anesthetic procedure using a 25-gauge surgical scalp vein needle. Samples were centrifuged for 10 minutes. The plasma fraction and 200 μL red fraction were transferred into a tube for further centrifugation (400g for 10 minutes).
After determining that the platelet concentration was adequate, two tubes were prepared: the first contained 200 μL liquid PRP, 200 μL liquid RSV, and 100 μL 10% calcium gluconate; the second tube contained 400 μL liquid PRP and 100 μL 10% calcium gluconate. The final volume of PRP gel was 0.5 mL in both vials.
Next, researchers prepared a 1.2% RSV solution (Chikkaballapur, Karnataka, India) to incorporate into the PRP gel. The material was subsequently transformed into gel by adding calcium gluconate. This gel was prepared to treat the wounds.
After preparating the materials, the skin of the animals was demarcated with a pen in four locations. Next, 0.1 mL of local anesthetic (2% lidocaine hydrochloride with a vasoconstrictor) was applied to each patch of demarcated skin. An 8-mm punch was used to create surgical wounds at the sites. Fragments were removed with an anatomical tweezers, preserving the muscles.15 The control wound (upper left side; A) was treated with a 0.9% sodium chloride solution. The lesion on the lower left side received the 1.2% RSV gel (C). The upper right-side wound was treated with autologous PRP gel (B), and the lower right wound received both RSV and autologous PRP gels (D). All wounds were dressed with sterile rayon and adhesive dressings.
After the surgical procedure, the animals received tramadol hydrochloride (IM; 0.5 mg/kg) twice daily for 3 consecutive days to minimize initial discomfort. The first dressing was changed 3 days after wound induction, and the second dressing remained for an additional 4 days. As described by Vendramin et al,16 treatments were carried out every 4 days for 16 days. The lesions were biopsied at days 0, 7, 14, and 17 for histologic analysis. On days 7 and 14, fragments were harvested using a 4-mm punch, covering an integral part and the healing part. On days 0 and 17, the 8-mm punch was used, and the entire wound length was harvested. The skin samples were fixed in 10% buffered formalin solution for 24 hours. After fixation, tissue was sectioned in paraffin blocks to obtain four 4-μm histologic sections for each animal. Sections were stained with hematoxylin-eosin solution and picrosirius to measure the number of collagen fibers.
The picrosirius red F3BA polarization technique was used to quantify and evaluate the number of collagen fibers. This technique allows for the determination of mature and immature collagen. In the RGB (red, green, blue) system, the thicker, strongly birefringent type I mature collagen fibers appear in yellow, orange, and red, whereas the finer, dispersed, weakly birefringent type III immature collagen fibers appear in green.17
Microscopy and Imaging
Images were obtained using an optical microscope (Leica DMLB, Buffalo Grove, Illinois), coupled with a camera (Leica DFC300 FX), and installed with the Leica Qwin software plus Leica Qwin Colour (RGB image). Three fields were chosen randomly, and images were captured at 400×. Collagen fiber quantification was obtained using the same software. This generated a frequency histogram of red, green, and blue color intensity and recorded only regions that were stained vibrant red. Three fields per wound were evaluated, captured just below the epidermis. The results were expressed as percentage of total collagen fibers (mature and immature).
The epidermis was evaluated for degeneration, necrosis, and regeneration. The dermis was evaluated for edema, hemorrhage, degree of neovascularization, fibrosis, and type of inflammatory infiltrate. The following scoring system was applied for all parameters: (0) absence, (1) mild alteration, (2) moderate alteration, and (3) severe alteration. All samples were evaluated by a single observer blinded to the experimental conditions.18
The wound area was measured using a digital caliper (DC-60 Western, São Paulo, Brazil) at days 0, 7, 14, and 17, and the percentage of contraction (Pc) of each lesion was calculated using a mathematical model proposed by Oliveira et al.19 In this model,
where Fa is the final area, and Ia is the initial area.
All variables were assessed for normality using the Shapiro-Wilk test before any further analyses were conducted, which revealed a nonparametric distribution. Researchers compared differences between groups using the Kruskal-Wallis test. Within-group differences were compared using the Student-Newman-Keuls test. Results were considered statistically significant if P < .05.
At day 17, the percentage of wound contraction in RSV and RSV and PRP wounds was 85.4% and 87.4%, respectively (Figure 1). Wounds treated with PRP alone showed a contraction of 90.9%, which was similar to the control wounds (93.3%; P > .05).
There were no macroscopic differences in wound closure. The wounds remained pink throughout the experiment, with no macroscopic characteristics of contamination, excess granulation, pain, or presence of exudate. None of the wounds closed completely, but PRP treatment resulted in a more homogenous closure of wound edges, as illustrated in Figure 2.
The histopathologic evaluation after 17 days of treatment revealed partial reepithelialization in 100%, 50%, and 75% of the wounds treated with PRP, RSV, and PRP and RSV, respectively (P > .05; Figure 3).
Edema was noted in 50% of the experimentally treated wounds, compared with 12.5% of the control wounds. The PRP and RSV treatment inhibited blood loss, as demonstrated by the lack of hemorrhagic characteristics in this group. The use of PRP alone resulted in a 62.5% reduction in blood loss, compared with 50% loss in wounds treated with PRP and RSV.
At day 17, all control wounds exhibited mild to severe polymorphonuclear cell infiltration, which is a marker of inflammatory infiltration. Wounds treated with PRP alone or in combination with RSV were predominantly composed of mononuclear cells.
All experimentally treated wounds had a higher number of collagen fibers than the control wounds (78.27% ± 4.69%). The wounds treated with RSV alone had the lowest number of collagen fibers (85.98% ± 3.51%), although this finding was not statistically significant. The mean number of collagen fibers in wounds treated with PRP alone and PRP and RSV was 90.07% ± 6.20% and 90.76% ± 3.51%, respectively (Figure 4).
Mansoub et al20 studied wound healing in diabetic rats and reported that wound contraction started earlier in groups treated with PRP or keratinocytes than in the control group. However, when applied in combination, wound healing was delayed. In this study, the level of wound contraction in rabbits was higher in the control group than in the treatment groups.
Wound contraction, closure, and the amount of collagen are considered to be related measures. More collagen leads to greater wound contraction; however, other factors, such as collagen quality, can affect this. Wound healing treatments may produce better-quality collagen fibers, which leads to a more homogeneous scar. Another hypothesis is associated with the orientation of the collagen fibers; previous studies have shown that fibers that present in a longitudinal orientation are favorable for the healing process.16
Statins are well-established hypolipidemic drugs; however, they have also been investigated in experimental studies demonstrating pleiotropic effects unrelated to lipid metabolism,21 such as anti-inflammatory,22 antioxidant,23 immunomodulating,24 proliferative,25 antithrombotic, and endothelial protective activities.26
Isoprenoids are intermediate products of the mevalonate pathway, which is inhibited by statins, and show anti-inflammatory activity. Therefore, when isoprenoids are inhibited, there is a reduction in the activity of guanosine triphosphate enzyme (GTPase) proteins. These GTPase proteins are cellular signaling proteins that affect cell proliferation and migration, controlling cellular oxidation-reduction and apoptosis. When statins inhibit the mevalonate cascade, they reduce the action of GTPases by enabling the activity of endothelial nitric oxide synthase (eNOS), suppressing local oxidative stress, decreasing nuclear factor κ-light-chain enhancer of activated B cells activity, and reducing the release of inflammatory cytokines,27 which explains the prevalent mononuclear cell type in lesions receiving RSV in the current study.
Polymorphonuclear cells were more common in the early phases of the repair process. In the intermediate phase, study authors observed a decrease in these cells and a predominance of mononuclear cells. At the end, rare leukocytes were predominant. This distribution implies a slower repair process because of associated inflammation. Santos et al28 have evaluated the healing of standardized wounds in rabbits treated serially with autologous PRP gel and observed more mononuclear cells in wounds treated with PRP. In addition, Barrionuevo et al15 have compared different sources of PRP and verified that autologous or homologous PRPs produce a homogeneous scar with fewer polymorphonuclear cells.
Similar results were obtained by Pradeep et al29 in a controlled and standardized clinical trial. They treated 65 patients with chronic periodontitis who underwent subgingival treatment with 1.2% RSV gel. Clinical and radiologic evaluation were undertaken and compared with the placebo-controlled group for 6 months. This study demonstrated that RSV treatment stimulates the genetic expression of morphogenetic proteins in osteoblasts, thereby reducing local bone reabsorption and improving periodontal inflammatory conditions.
Bao et al30 found that RSV decreases inflammation via a reduction in inflammatory cytokine expression, such as interleukin 6 and tumor necrosis factor α. Further, Al-Kuraishy et al31 demonstrated that RSV, alone or in combination with the antibiotic cefixime, has an antimicrobial effect against Gram-positive and negative bacteria by decreasing the minimum inhibitory concentration in insolation cultures.
In a randomized study using statins, Lin et al25 evaluated the presence of peripheral epithelial progenitor cells and vascular endothelial growth factor (VEGF) in patients at high risk of cardiac problems, diabetes, and hypercholesterolemia for 12 weeks. They quantified systemic biomarkers in vivo, such as total nitric oxide, interleukin 10, tumor necrosis factor α, apolipoprotein A1, and asymmetric dimethylarginine (ADMA), a natural eNOS inhibitor. There was a significant increase in VEGF and a corresponding reduction in ADMA following the use of pitavastatin.
An in vitro angiogenesis study also demonstrated the proliferation of epithelial progenitor cells following treatment with statins, which resulted in a higher rate of proliferation and increased eNOS expression. This study proposed a direct effect of pitavastatin and atorvastatin on epithelial progenitor cell proliferation and eNOS activation, which was associated with an increase and decrease in VEGF and ADMA, respectively. The current study revealed no increase in angiogenesis and reepithelialization; however, statins have been shown to reduce bleeding, as reported by Teshima et al.24
This study had several limitations. Researchers used a small number of animals, which limits the statistical strength of the analysis. In addition, the histopathology analysis was performed by a single observer, which may have affected the results obtained.
Study authors conclude that PRP improves the overall wound healing process in the first 7 days of lesion occurrence. In addition, hypolipidemic treatment alone or in combination with PRP strengthens the other cicatrization phases. It may be applicable, in combination with other agents, as a novel therapeutic agent for treatment of wounds. Further studies on the verification of the molecular elements involved in the healing process when using RSV should be conducted to better elucidate its performance in the tissue healing process.
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