Abstract: Background and Objective: Platelet-rich plasma is the portion of the blood plasma which contains platelet concentration beyond usual levels. It is a new method to stimulate tissue regeneration response as it contains a wide range of growth factors that enhance wound healing and tissue repairing mechanism. This study was aimed to investigate the impact of concurrent application of topical and subcutaneous (S/C) infiltration of PRP at the excised wound boundaries upon epidermal surgical lesions. Materials and Methods: A clean epidermal incision of 5 mm in diameter was made in the back region of each mouse (n = 20). Animals were divided in 2 groups, group 1 (n = 10) were left untreated (control group), while group 2 received bovine fetal. Wounds were topically and subcutaneously infiltrated with PRP for 21 days. Wound healing measurement, percentage of wound contraction and histopathological investigation of the wounds from skin biopsy were evaluated, both in treated and un-treated mice at 3rd day, 7th day, 14th day and 21st day post wounding. Results: There was an obvious progress in wound healing response at 14th day of PRP treatment and complete healing after 21 days of PRP treatment, comparing to the control group. Also, the percentage of epithelization and wound contraction were significantly increased in PRP-treated mice at any period of time, comparing to control group. Histopathological results showed higher granulation tissue formation, new blood vessel formation and collagen synthesis. Conclusion: The PRP could be useful to enhance wound repair and reduces scar tissue formation in surgically-induced excised skin wounds.
INTRODUCTION
Wound healing is a physiological process that empowers the injured tissue to renovate its integrity and replace the lost tissues1. Wound is caused by a disruption in the cellular continuity of dermal tissue by chemical, physical, thermal, microbial, or immunological injury to the tissue, which leads to distraction of the structure and function of underlying normal tissue2. There are a wide range of blood cells that play an essential role in the process of wound healing including cytokines and growth factors that play vital role in the renewal of normal structure and function of the injured tissue3. There are 3 major stages that involve in the process of wound healing which include inflammation, new tissue formation (granulation and angiogenesis) and tissue restoration4. In addition, these 3 stages comprise of disciplined collaborations between numerous cell types such as leukocytes, fibroblasts and keratinocytes and are controlled by several factors, including cytokines, chemokines, growth factors and enzymes. After the early hemostasis, inflammation plays a vital role in the normal process of wound healing. However, it has been shown that sustained inflammation prevents entering into the proliferative phase and delays wound healing process4,5.
Furthermore, it has been shown that various factors play an essential role in delaying the process of wound healing through influencing of broken tissue, such as repeated injury, infection, oxygenation and free radical creation. The process of wound healing could be delayed and negatively affected by the presence of free radicals, which can destroy wound neighboring cells, or by infectious microorganisms6. Recently, in vivo studies have shown that Platelet-rich plasma (PRP) can significantly suppress the expression of pro-inflammatory genes, suggesting that PRP would potentially impede the inflammation7. It has also been shown that PRP plays a key role in therapeutic application in tissue regeneration and engineering as it contains high amount of growth factors with potential mitogenic and anti-inflammatory activity8,9. In addition, PRP is a congregation of platelets (3-5 fold the plasma baseline level), which contains many growth factors including transforming growth factor β1 (TGF-β1) and TGF-β2, platelet derived growth factors (PDGFAA, PDGF-BB, PDGF-AB), insulin-like growth factor 1 (IGF-I), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and hepatocyte growth factor (HGF) that are very important for regeneration processes. Moreover, these growth factors synergistically increase the infiltration of neutrophils and macrophages to enhance angiogenesis, fibroplasia, matrix deposition and eventually re-epithelialization, enhancing the subsequent tissue regeneration8,9. Likewise, the presence of anti-inflammatory substances, including HGF, reinforces the capability of PRP to prevent inflammatory process10.
Furthermore, platelets are non-nucleated cell elements in the blood that results from megakaryocytes fractionation in the bone marrow11. Intracellularly, there are a wide range of reservoir organelles in platelets, including lysosomes, alpha granules and dense granule. The alpha granules, which are found in copious supply, contain various proteins that play a vital role in stimulating platelet adhesion and aggregation, antimicrobial activity and tissue healing12 whilst the dense granules are considered to play a crucial role in platelet function. In addition, the alpha granules of platelets play a vital role in the activation and secretion of important biomolecules in the clot, including growth factors, coagulation factors, platelet-specific proteins, adhesion particles, cytokines, angiogenic factors, proteoglycans and cytokines/chemokines13. Recent study has shown that secretion of cytokines, chemokines and growth factors from platelets persuade proliferation and activation of cells that are essential for wound healing, such as fibroblasts, neutrophils, monocytes, smooth muscle cells and mesenchymal stem cells (MSC)14. Approximately 14 million surgical operations are performed in the field of obstetric, musculoskeletal, urological and gastrointestinal tract each year; thus wound healing will remains to be most crucial both for the surgeon and patient12,14. Most recent study has shown that platelet-rich plasma, or platelet concentrate, is a potential adjuvant therapy to enhance the process of wound healing15. Research on wound healing mediators is one of the developing areas in modern biomedical sciences and many studies have been conducted using different wound healing models7. Thus, the specific aim of this study was to investigate the healing activity of PRP on the induced excised wound in animal model.
MATERIALS AND METHODS
This study was conducted from January to July, 2019 in College of Veterinary Medicine, University of Sulaimani, Kurdistan region, Iraq.
Animals: Twenty adult healthy mice (about 3 months old, male, body weight approximately 30 g) were used in this study for wound healing model experiments16. The animals were obtained from animal house laboratory, College of Veterinary Medicine, University of Sulaimani, Kurdistan region, Iraq and housed, in standard cages and hygienically maintained in veterinary teaching hospital. All animals had free access to standard chow and drinking water and were maintained on a 12 h light/dark cycle with adequate ventilation. This study was carried out in strict accordance with the recommendations approved by the committee on the ethics of animal, University of Sulaimani, College of Veterinary Medicine. All surgeries were performed under ketamine (30 mg kg–1)/xylazine (20 mg kg–1) anesthesia, which was administered as a mixture at a dose of 0.01 mL g–1 body weight via intraperitoneal injection and all efforts were made to minimize pain. An excision wound, scarred areas 5 mm in diameter, by a longitudinal incision of the skin on back of each mouse was created. The animals will randomly allocate into four groups of 5 animals/each group.
Preparation of platelets rich plasma (PRP): In this study, cow’s fetal umbilical cords (CUC) were collected from slaughtered cows in Sulaimani slaughterhouse on March-Jun, 2019 in Sulaimani city-Kurdistan region, Iraq. Then the umbilical cord blood was taken directly from cow umbilical cord artery, using sterile syringes and needle and collected into a sterile bag containing an anticoagulant to prevent clotting.
The platelet-rich plasma (PRP) was synthesized from the collected umbilical cord blood and concentrated using a series of centrifugations in the research center of college of Veterinary Medicine-University of Sulaimani. The cord blood was collected in acid-citrate dextrose+adenine (AcD-A) coated collection tubes and initially centrifuged at 2000 RPM for 5 min to separate the red blood cell (RBC) portion from the platelet-rich plasma. The upper layer of the RBC portion, a PRP layer (buffy coat), was comprised as the platelets containing the largest amount of growth factors. Then, the plasma-enriched platelets were pelleted by a hard centrifugation of buffy coat plasma at 3875 RPM for 5 min. The prepared PRP was carefully collected and aliquoted, then stored at -80°C for future use15-17.
Administration of PRP, in treated animals with excised wound, was performed by application of 0.25 mL of PRP from the aliquot onto the wounds, both by directly applying topically onto the wounds (first half of 0.25 mL) and via subcutaneous injection (second half of 0.25 mL) in to the margin of the wounds (Fig. 1).
Measuring the percentage of wound contraction: The measurement of wound contraction was performed, using imageJ software, on the ruler-calibrated images16,17 (Fig. 2).
| Fig. 1: | Application of PRP |
| Fig. 2: | Steps required for measurement the size or area of the wound in imageJ. After setting the scale, the freehand selection tool was selected to outline the wound area (circle). By selecting the measure tool from the analyze tool bar the outlined area of each wound was determined |
| Table 1: | Histological grading criteria for wound healing according to Shafer criteria17 |
The image was opened or dragged into the ImageJ software and then the straight-line selection tool was used to drag a line across the calibrated ruler on the image in order to specify a distance (e.g., 1000 μm). Then, setting the scale by clicking the Analyze tool, the set scale window was opened that show the length of the dragged line (e.g., 1000 μm) in pixels, which could then be converted to a specified micrometers (μm),
The size of the wounds (areas) was measured using the freehand selection tool and outlining the wounds (Fig. 2). By selecting the measuring tool the outlined area of each wound was determined. The wounds were measured from the 1st-21st day in both control and treated groups. Then the percentage of wound contraction were taken from the 3rd day by subtracting the measured size of the wounds from the 3rd, 7th, 14th and 21st from the size of the wounds from the 1st day of the operation and the area of each lesion was calculated. Percent contraction of area was calculated, using the following equation18,19:
Histopathological examination: Histopathological analysis are performed within tissue specimens and skin samples were collected from treated and control groups on day 3, 7, 14 and 21. The samples were fixed in 10% PBS buffered formalin solution more than 7 days for histopathology study. Six-micron-thick sections of the surrounding skin were stained with H and E for light microscope. Histological state of the healing wound was analyzed by assessing a wide range of wound healing parameters of skin cells, including collagen content, vascularity or number of capillaries, granulation tissue, abscess formation, necrotic epithelium. The total healing score for wound healing in each case was calculated by adding the scores of individual criteria according to Shafer et al.18 and Aragon-Sanchez et al.19. Accordingly, the grading system was ranged from grade 1 (very light healing), grade 2 (moderate healing), grade 3 (advanced healing) and 4 grade (well-organized) (Table 1).
Statistical analysis: All results are presented as Mean±SD where n is the number of experiments. For data presentation and statistical analysis, ImajeJ and GraphPad Prism 4 software (GraphPad Software, USA) were used for statistical analysis and either a Student’s t-test (unpaired), one-way ANOVA (with Tukey’s multiple comparison post-test) used where applicable. The p<0.05 were considered statistically significant.
RESULTS
PRP enhances the skin wound closure: The physical condition of the animals were good in both control and treatment groups. The physical activities of the animals, including feeding, drinking, defecation, urination, alert and brightness after creation of the wounds were approximately normal and similar both in control and treated groups.
Digital photographs of wounds were taken at days 0, 3, 7, 10, 14, 21 and 28. Time to wound closure was defined as the time at which the wound bed was completely re-epithelialized and filled with new tissue. Moreover, postoperative complications such as inflammation and abscess formation were not observed in all control and treated mice. The induced skin wounds appeared dry, crusted, erythematous without exudate and regressed slowly until loss of surface layer and epithelialization. Healing of the wounds was passed through the principles steps of wound healing, where, the size of the wound started to decrease from the 3rd day after the operation in PRP treated mice comparing to control group. However, from the 3rd-7th day post treatment with PRP, there was no obvious difference in wound healing size between the treated and control groups.
However, there was remarkable improvement in skin wound healing after 14th days of treatment in PRP treated mice the comparing to the control group. Interestingly, at the end of experiments on the 21st day of treatment, there was complete closure of the skin wound in PRP treated mice comparing to control mice, where a small amount of scar tissue was left at the site of the wound (Fig. 3), the visual scar area was carefully circled on the images of wound. This implies that PRP play an important role in the healing process of induced excised wound in mice after 21st days of treatment with PRP.
PRP augmented the percentage of skin wound healing: In this study, the percentage of wound contraction was assessed during the different periods of wound treatment with PRP. According to ImageJ criteria on the basis of measuring the size of the wounds from 1st-21st day of study, it was found that the size of the wound were decreased progressively in each group from the 3rd-21st day.
| Fig. 3(a-b): | Experimental wound on the back of the mouse in (a) Control and (b) Treatment groups at different stages of healing from the 1st-21st day |
There is a noticeable progress in wound healing from the 14th day and complete healing on 21st day in the treatment group when compared to the control group, n = 10 for each group |
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When these measurements in both groups compared, there was a significant difference in the percentage of wound contraction in the treated group (n = 3, p<0.05) comparing to the control group (n = 3). As shown in Fig. 4, results showed that the average percentage of wound contraction with their standard deviation was significantly increased in the treated group on the 3rd day (23±2.10%), 7th day (54.13±11.23%), 14th day (80.72± 5.60) and 21st day (100%), respectively, using Mann Whitney unpaired T-test (p<0.05), comparing to the percentage of wound contraction in control group on the 3rd day (11.86±0.86%), 7th day (22.83±4.14%), 14th day (45.77±7.44%) and the 21st day(87.02±6.08%), respectively. In order to further emphasize the statistically significant differences (p<0.05) in the percentage of wound contraction, in different days, between PRP treated and untreated mice, histological examination and scoring of the wound were under taken.
Microscopic observation of wound healing from histological sections: The histological analysis for wound healing after 3rd day of PRP treatment in mice with excised skin wound showed almost no healing with the same histological grade comparing to positive control group. Histological results showed that there is no collagen content, no capillaries, absence of granulation tissue, severe abscess formation, severe necrotic epithelium, both in the PRP treated and untreated mice. In addition, histological observation, both in the PRP treated and un treated mice, at 7th days post wounding showed grade one of wound healing (very light healing) with low collagen content, scarce vascularity or low number of capillaries, absence of granulation tissue, abscess formation and necrotic epithelium (Fig. 5).
| Fig. 4: | Percentage of wound contraction between the control (n = 3) and the treatment group (n = 3) at 3rd, 7th, 14th and the 21st days |
There were a significant difference in the percentage of wound contraction in treatment group when compared to the control group using Mann-Whitney unpaired t-test (p<0.05) (Error bars = Standard deviation), n = 10 for each group |
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| Fig. 5(a-d): | Histopathological changes of healed skin wounds at various period of post wounding in PRP treated and un-treated mice. (a) Control group (3rd day post wounding), (b) Treated group (3rd day post wounding) (c) Control group (7th day post wounding) and (d) Treated group (7th day post wounding) |
White arrow (a-b): Ulcer site with necrotic epithelium covered by fibrinopurulent material, no collagen content, no capillaries, absence of granulation tissue, White arrow (c-d): New capillary formation, low collagen content, low number of capillaries, abscess formation |
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| Fig. 6(a-d): | Histopathological changes of healed skin wounds in PRP treated and un-treated mice. (a) Control group (after 14th day) shows low collagen content, low number of capillary, (b) Treated group (after 14th day) shows moderate collagen content moderate collagen content, moderate number of capillaries, onset of granulation tissue (white arrow), (c) Control group (after 21st day) shows moderate collagen content, moderate number of capillaries and onset of granulation tissue formation (white arrow) and (d) Treated group (after 21st day) show abundant collagen fibers, abundance of capillaries, well organized granulation tissue (white arrow) |
However, histological sections from the 14th days of wounds healing in PRP treated group showed grade 2 of wound healing (moderate healing) with moderate collagen content, moderate number of capillaries, onset of granulation tissue formation, no abscess formation and no necrotic epithelium. In contrast, the untreated mice showed grade one (very light healing) of wound healing after 14th days of operation with low collagen content, scarce vascularity or low number of capillaries, absence of granulation tissue, no abscess formation, no necrotic epithelium (Fig. 6). In addition, histopathological examinations from the day 21st of wound healing have showed grade 3 and 4 of wound healing in PRP treated mice with abundant collagen fibers, capillaries, well organized granulation tissue, comparing to control mice where they showed grade 2 of wound healing with moderate content of collagen and capillaries, onset of granulation tissue formation (Fig. 6).
DISCUSSION
The results of this current study have shown that the skin wound healing, by application of bovine fetal PRP, was enhanced following 14 days of treatment with PRP comparing to un-treated mice. Likewise, bovine fetal PRP produced a potent wound healing effects at the end of experiments on the 21st day of treatment as PRP application caused a complete closure of the excised skin wound in treated mice, comparing to un-treated mice. This effect could potentially be through the influence of alpha granules in platelets that can secret a wide range of growth factors which play an important role in the process of soft tissue wound healing20,21. It is also consistent with previous results which suggested that PRP has been used in combination with stem cell therapy to augment the tissue healing response18. Results of this study also supported by previous works that showed human PRP plays an important role in promoting of wound healing responses, potentially through its fast and effective influence on fibroblast proliferation, which are responsible for producing most of the extracellular matrix tissue to enhance tissue repair and wound healing22,23.
Furthermore, results of this study provided further insight into the role of bovine PRP in promoting wound healing process, because there was an obvious increase in the percentage of wound closure following bovine PRP treatment at 7th, 14th and 21st day post wounding in mice, comparing to control mice without treatment. One potential explanation for this apparent result could be that PRP can secrete platelet-derived growth factor (PDGF), which stimulates the production of fibronectin, a cell adhesion molecule, used in cellular proliferation and migration during healing response that accelerate soft-tissue healing through enhancement of wound contraction and restoration. Another possible explanation could be that PDGF derived from platelets play an essential role in angiogenesis and neo-vascularization via stimulating increased levels of vascular endothelial growth factor at the site of wound, this leads to increase in wound contraction and closure21. In addition, results of this study were consistent with most recent works by Farghali et al.24 and Pastar et al.25, who showed that direct application of PPR on to the excised wound in dogs, for 3 sequential weeks, caused obvious enhancement of re-epithelization and wound contraction throughout the study period26.
Histopathological investigations of the healed wounds in PRP treated mice showed obvious improvement in wound healing especially after 14th and 21st day of skin wounding, comparing to control mice without treatment. There was higher collagen fibers, large numbers of capillaries (angiogenesis), well organized granulation tissue formation in PRP treated wound in time dependent manner comparing to control group. Angiogenesis play a vital role to supply a sufficient amount of oxygen and nutrients to the newly formed granulating tissue in the wound. As soon as the granulation tissue cot is arranged, the process of re-epithelialization can take place23,26. The most likely explanation would be that PRP contains copious amount of growth factors such as transforming growth factor (TGF)-β, which decreases the proliferation of basal keratinocyte whilst persuades the differentiation of supra-basal cell, this leads to stimulation of the epidermal regeneration associated with skin wound healing27,28. In addition, increase in granulation tissue formation and re-epithelization could be attributed to significant increase in the activity and expression of matrix metalloproteinase (MMP)-9 in PRP treated wound22 as MMP plays an essential role in cell migration and re-epithelization during skin wound healing24.
CONCLUSION
From the current data, it could be concluded that application of bovine fetal PRP using topical and subcutaneous infiltration route, simultaneously, at the wound margins showed significant improvement in wound re-epithelization and healing with reduced scar formation. This route of PRP administration re-enforced the novel use of the bovine fetal PRP as a biological wound healing enhancer. Future experiments are needed to assess the effect o f PRP on the skin tensile strength and elasticity after wounding, through measuring the levels of collagen within the wound via real-time PCR (qPCR) and immunohistochemistry evaluation of COLIA2 gene expression in skin biopsies of PRP treated and untreated wound in mice.
SIGNIFICANCE STATEMENT
This study discovered the potential effect of the fetal plasma, which can be beneficial for the dermal wound healing because of its positive influence on various phases of the healing process. Recently, it has been documented that the umbilical fetal plasma is effective in view of its content with different types of growth factors that may help the tissue to regenerate itself. Although, the mechanism of wound healing has been delineated but its pathophysiological process is still unclear and different materials were used in order to enhance the process of wound healing. This study will help the researcher to uncover the critical area of wound healing therapy by using fetal plasma to improve the process of wound healing and resolved impaired or non-healed wounds, that many researchers were not able to explore using a recent new therapeutic method to facilitate the process of wound healing. Thus, a new theory on using fetal plasma is to design an effective wound healing therapy protocol, which may be arrived at if it supported by continuing with enormous importance of researches in the future.
ACKNOWLEDGMENT
We would like to express the profound sense of gratitude to all the doctors and technicians in the Histopathology Department of Shorsh Hospital for their cooperation and assistance, for their helpful assistance and generous support during this research study. This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.