INTRODUCTION:

Among several therapeutic methods for wound healing, skin graft is one of the most frequently used options nowadays, especially in plastic surgery. Skin graft is a reconstructive surgical technique that transplants healthy skin to cover the defect or wound area to accelerate healing. This technique is still the gold standard for treating burn reconstruction.^1,2 Data from the Healthcare Cost and Utilization Project shows that approximately 160,000 skin graft procedures are performed annually on 1 in 3 patients with burns.³ One hundred ninety-three skin graft procedures were performed in Indonesia on 240 burn patients admitted to the Burn Centre of Cipto Mangunkusumo Hospital from 2013-2015.⁴

Wound healing of skin graft is influenced by the formation and growth of blood vessels into the graft tissue from the base of the recipient site. These new blood vessels will supply the nutrients and oxygen necessary for the survival of the skin graft tissue. This process is also known as angiogenesis.^5-7 Fibroblast activity also plays a vital role in wound healing of skin grafts. Fibroblast proliferation contributes to angiogenesis by secreting proangiogenic factors.⁸ Moreover, fibroblasts will degrade the fibrin clots and replace them with new extracellular matrix components such as collagen.^9-11 Collagen density determines tissue strength to prevent the possibility of wound dehiscence.¹² The extracellular matrix deposition will influence the increased motility of keratinocytes for re-epithelialization.¹³

Lots of factors can impair the wound healing of skin graft, including fluid accumulation between the donor and the recipient site, hematoma, seroma, pus, tissue necrosis, infection, and mechanical displacement preventing the donor and recipient site from attaching.¹⁴Postoperative skin graft treatments are needed to avoid some of these disrupting factors. Topical agents or modern dressings are typically used for postoperative skin graft treatment.¹⁵ These treatments only prevent infection and do not induce the healing process.¹⁶ Current treatments are also limited, have numerous side effects, and are costly.^12,17 Based on these problems, many studies have developed new strategies using natural materials as alternative treatments to improve wound healing of skin graft.^1,14 Natural materials are associated with advantages, such as high therapeutic effects, fewer side effects, and lower costs.¹⁸ The potential therapeutic effects of natural materials are obtained from the content of active ingredients within them, such as polyphenols, flavonoids, alkaloids, triterpenes, and saponins.^19,20 Coffee is one of the natural ingredients that has the potential to be an alternative treatment.

Coffee is a widely consumed plant and has long been used as a traditional medicine to treat various wounds, especially by Indonesians.^21,22 Robusta coffee is one type of widely cultivated coffee in Indonesia.²³ Our previous study showed the promising results of robusta coffee extract in accelerating 2^nd degree burn wound healing by increasing the number of fibroblast cells and collagen density in the proliferation phase.¹² Robusta coffee contains some active ingredients such as CGA, flavonoids, and saponins, which are anti-inflammatory, antioxidant, and antimicrobial.^24-27 These active substances will suppress proinflammatory cytokines and neutralize free radicals, thus accelerating wound healing. The antimicrobial effect will reduce the risk of infection by working to destroy cell membranes and denature microorganism cell proteins.^28,29 Robusta coffee has a higher CGA content than the other types of coffee.^23,30,31As far as we know, no studies that have investigated the effectiveness of robusta coffee extract on the wound healing process of skin grafts. This study is conducted to prove that topical therapy of robusta coffee extract gel can enhance wound healing of skin graft. The authors performed histopathological evaluation to assess angiogenesis, fibroblasts, collagen density, and epithelial thickness. We hypothesize that robusta coffee extract gel can enhance wound healing by improving angiogenesis, fibroblasts, collagen density, and epithelial thickness.

METHODS:

Robusta coffee extraction:

Robusta coffee beans were acquired from Indonesian Coffee and Cocoa Research Institute in Jember. The preparation of coffee extract was carried out by maceration method. 10 kg of coffee beans were crushed and then dried. The dried coffee beans were blended to achieve 1 kg of simplicia powder. 200 g of simplicia powder was extracted using 96% ethanol and then covered with aluminum foil for 18 hours. The maceration results were filtered using a Buchner funnel. Subsequently, the residue was re-extracted once. The extraction results were then combined and concentrated with a rotary evaporator at 30-40°C to obtain a thick extract.

Gel formulations:

The gel base consists of 2% carbopol, 15% propylene glycol, 4% triethanolamine (TEA), nipagin, nipasol, and distilled water. The gel base was used as a negative control. Preparation of the gel base started with 2% carbopol and 15% propylene glycol developed with distilled water. Triethanolamine (TEA) 4% was added dropwise to the developed mixture. Nipagin and nipasol were used as preservatives to prevent microbial contamination. 27.6 grams of gel base and 5 grams of dry extract of robusta coffee beans were put into a mortar and then crushed until homogeneous to obtain a concentration of 5% robusta coffee extract in the gel dosage form. A 1% kojic acid gel was used as a positive control to compare the effect of coffee extract. The preparation of 1% kojic acid gel uses the same method as the preparation of robusta coffee extract gel. Once the gel base was formed, 1 gram of kojic acid was combined with 99 grams to obtain a concentration of 1% kojic acid in 100 grams of the gel dosage form.

Animals and skin graft models:

This study was conducted after securing approval from the Health Research Ethics Commission of the Faculty of Dentistry, University of Jember (No. 1657UN25.8/KEPK/DL/2022). We used 30 male Long Evans rats aged 2-3 months (200-250 grams). The rats were randomly divided into three experimental groups: negative control (gel base), positive control (1% kojic acid gel), and 5% robusta coffee extract gel (RCEG). We acclimatized the rats in cages (1 rat per cage) for seven days before the experiment. The cages were maintained at a temperature of around 27°C ± (3°C), and the lighting period was 12 hours light and 12 hours dark. All rats were fed and watered ad libitum.

Our skin graft model was an autologous split-thickness skin graft. After seven days of acclimatization, all rats were anesthetized with ketamine-xylazine (35 mg/kg ketamine and 5 mg/kg xylazine, IP). The dorsal hair of the rats was shaved. The shaved area was disinfected with povidone-iodine and normal saline. Then, a 2 x 2 cm split-thickness skin graft was harvested from the rat dorsal skin using a humby knife. The harvested graft was used to cover the split-thickness wound at the donor collection site. Immediately after surgery, the skin graft area was lubricated with a gel base, 1% kojic acid gel, and 5% RCEG according to each group. The skin graft was covered with transparent film, sterile gauze, and plaster. Topical treatment in each group was carried out every three-four day for 21 days. Furthermore, antibiotics were administered for three days to prevent infection and death of the rats.

Histopathological evaluation

Skin graft tissues were taken by biopsy on days 7, 14, and 21 for histopathological evaluation. Biopsy samples were stored in 10% formalin solution, processed, and embedded in paraffin blocks. Paraffin blocks were cut to 4 µm thickness. Afterward, the prepared slides were stained with hematoxylin-eosin and Masson trichrome. Histopathological evaluation in this study was a quantitative assessment of angiogenesis, fibroblasts, collagen density, and epithelial thickness. We performed the evaluation under a light microscope at 400x magnification connected to an optical camera. Angiogenesis parameters were measured by calculating the total blood vessels found in 6 regions.³² Fibroblasts were measured by counting the number of fibroblast cells in 6 regions.¹² The number of blood vessels and fibroblast cells was counted by manual marker using ImageJ software.³² Collagen density on Masson trichrome stained slides was assessed using ImageJ software. For this purpose, we used four regions for each sample.¹² The blue color in Masson trichrome staining, which indicates collagen fibers, was separated from the background color by a particular thresholding value. Manual thresholding values were adjusted: Hue (135–195), Saturation (25–255), and Brightness (20–255) in ImageJ. The percentage of total collagen was figured as the percentage of gray value.³³ The authors modified the epithelial thickness evaluation method based on the study by Apriasari et al. and Mendonça et al.^34,35 The epithelial area in each of 3 regions was divided into 3 areas and separated by measurement lines. Then, the assessment results on each measurement line were summed up, and the average value was calculated. Evaluation of epithelial thickness was carried out using ImageJ software.³⁴

STATISTICAL ANALYSIS:

We used SPSS 25.0 software for statistical analysis. All data in this study were presented as mean ± standard deviation (SD). Data were analyzed using the one-way ANOVA test followed by the Bonferroni post hoc test. The p-value <0.05 indicates that the results are significant. All graphs were created using SPSS 25.0 software.

RESULT:

We performed histopathological evaluation, including angiogenesis, the number of fibroblast cells, percentage of collagen density, and epithelial thickness. The results of histopathological evaluation can be seen in (figure 1).

Figure 1: Histopathological evaluation in each group. * p<0.05, ** p<0.01, *** p<0.001

Angiogenesis was evaluated by assessing the amount of blood vessels. The mean number of blood vessels in the 5% RCEG group was significantly improved than the negative control group on days 7 (10.7±2.58 vs 6.3±2.35, p=0.004), 14 (13.2±2.09 vs 8.7±1.56, p=0.000), and 21 (16.2±1.54 vs 10.5±1.77, p=0.000), but there was no significant difference when compared to the positive control group (p>0.05). (Figure 2) shows the histopathological images of the angiogenesis assessment. Based on (figure 1), the average number of fibroblast cells was significantly higher after being given 5% RCEG compared to the negative control group on days 7 (121.83±16.97 vs 60.28±8.86, p=0.000), 14 (188.6±13.48 vs 95.96±13.83, p=0.000), and 21 (194.33±30.94 vs 116.91±14.56, p=0.000) as well as positive control on days 7 (121.83±16.97 vs 99.48±11.32, p=0.002), 14 (188.6±13.48 vs 142.15±11.6, p=0.000), and 21 (194.33±30.94 vs 151.13±22.73, p=0.001). On hematoxylin-eosin staining, fibroblast cells will appear with a spindle-shaped cell shape, round or oval (figure 2).

Figure 2: Histopathological images of angiogenesis and fibroblast evaluation in each group. Negative control group on day 7 (A), 14 (B), 21 (C), positive control group on day 7 (D), 14 (E), 21 (F), and 5% RCEG on day 7 (G), 14 (H), 21 (I). Black arrows are indicating blood vessels and blue arrows are indicating fibroblast cells. Using Hematoxylin-eosin staining with 400x magnification.

Regarding collagen density, our evaluation showed that the mean percentage (%) of collagen density on days 7, 14, and 21 was significantly improved in the 5% RCEG group compared to the negative control group (57.33±10.89 vs 39.5±10.08, p=0.000; 79.49±11.01 vs 59.43±12.38, p=0.001; 89.01±4.86 vs 80.88±6.34, p=0.003). Furthermore, the percentage of collagen density in the 5% RCEG group also had better results than the positive control group on days 7, 14, and 21 but was significantly different only on day 7 (57.33±10.89 vs 45.35±4.53, p=0.01). A histopathological description of collagen density assessment is shown in (figure 3). According to (figure 1), the group with the highest average epithelial thickness (µm) on days 7, 14, and 21 was the 5% RCEG group (4.81±0.4; 5.69±0.58; 8.56±0.7), followed by the positive control group (4.41±0.62; 5.29±0.64; 7.84±0.57) and the lowest was the negative control group (4.28±0.33; 4.9±0.57; 6.81±0.48). (Figure 4) shows the photomicrograph of epithelial thickness in each experimental group. However, the one-way ANOVA test reported no significant difference in the epithelial thickness evaluation between all experimental groups on day 7(p>0.05). The 5% RCEG group showed a significant difference in epithelial thickness compared to the negative control group on days 14(p=0.02) and 21 (p=0.000). The epithelial thickness in the 5% RCEG group was significantly different from the positive control group only on day 21(p=0.03).

Figure 3. Photomicrograph of the percentage of density collagen in each group. Negative control group on day 7 (A), 14 (B), 21 (C), positive control group on day 7 (D), 14 (E), 21 (F), and 5% RCEG on day 7 (G), 14 (H), 21 (I). Using Mason trichrome staining with 400x magnification.

Figure 4. Epithelial thickness of each group. Negative control group on day 7 (A), 14 (B), 21 (C), positive control group on day 7 (D), 14 (E), 21 (F), and 5% RCEG on day 7 (G), 14 (H), 21 (I). Using Hematoxylin-eosin staining with 400x magnification.

DISCUSSION:

Postoperative skin graft treatment is crucial to reducing the risk of skin graft failure. Natural ingredients such as plants are essential alternative sources with therapeutic potential and have long been used to help treat numerous diseases, including treating wounds.^36,37 Robusta coffee might enhance wound healing with active ingredients within, such as CGA, flavonoids, and saponins that are anti-inflammatory, antioxidant, and antimicrobial.^22,24-26 Based on our histopathological evaluation, topical application of 5% RCEG can enhance wound healing of skin graft by increasing angiogenesis, fibroblasts, collagen density, and epithelial thickness. Kenisa et al. have investigated the effect of robusta coffee extract on full-thickness wound healing. According to their findings, robusta coffee extract can increase the amount of capillary blood vessels and fibroblast cells, resulting in faster full-thickness wound healing compared to the control group.²⁴ Two other studies strengthened these results.^38,39 Hurmayanto and Rahman found that 15% robusta coffee extract can improve wound healing by increasing the number of fibroblast cells in the proliferation phase.³⁸ Meanwhile, the study by Sulistiawati et al. reported that 40% robusta coffee extract gel significantly increased the number of fibroblast cells in gingival wound healing compared to the control group.³⁹

Angiogenesis is generated from a combination of a hypoxic environment, growth factors, endothelial cells, pericytes, smooth muscle cells, and extracellular matrix.^40-42 The content of CGA, flavonoids, and saponins in robusta coffee has angiogenic effects. They can increase the expression of the proangiogenic factor such as VEGF.^43-45 When VEGF binds to its receptor, a signal will be sent to the nucleus of endothelial cells. The signal will generate new molecules by endothelial cell organelles, namely protease enzymes (MMP), to destroy the extracellular matrix and release perithelial cells so that endothelial cells can migrate and proliferate as new blood vessel branching points.^40,46 According to Wuttimongkolchai et al., CGA has been shown to increase Ang-2 expression from endothelial cells to promote angiogenesis.⁴⁷ Ang-2 is involved in angiogenesis by redeploying pericytes and smooth muscle cells after being released by MMPs to stabilize new blood vessels.^40,48 Otherwise, CGA is a potent antioxidant. Two studies by Bagdas et al. showed that CGA works as a free radical scavenger, as proved by decreased levels of NO and MDA and increased levels of SOD and GSH in skin flap tissue. Free radicals are associated with ischemic damage. The formation of ROS is one of the mechanisms that cause necrosis in tissues, which will undoubtedly inhibit wound healing. GSH and SOD are antioxidant defense systems as protective mechanisms against free radicals.^49,50 Antioxidants also have an effect in reducing inflammatory cytokines, thereby accelerating the end of the inflammatory phase to the following process, namely the proliferation phase.⁵¹

Robusta coffee is also known to have antimicrobial activity. The presence of microorganisms can lead to infection, thus inhibiting wound healing. Bacteria can release various endotoxins, reducing the proliferation capacity of fibroblasts and epithelial cells. Some studies mention that flavonoids and saponins are reported to have antimicrobial properties.^22,52 A study showed that robusta coffee powder has potent inhibition against methicillin-resistant Staphylococcus aureus bacteria, helping to accelerate the shortening of the inflammatory phase of the wound healing process.²⁸ Moreover, antimicrobial properties are also associated with keratinocyte proliferation and fibroblast migration to enhance collagen synthesis.⁵³ Flavonoids in robusta coffee have anti-inflammatory effects that play a role in wound healing by inducing TGF-β.⁵⁴ In the wound healing process, TGF-β contributes to the formation of extracellular matrix by regulating fibroblast proliferation, migration, and collagen synthesis, which further affects keratinocyte activity in the epithelialization process.^55-57Collagen synthesis is also known to be regulated by CGA. The study by Chen et al. found that CGA improved the healing process of excisional wounds by increasing collagen synthesis through the upregulation of TNF-α.⁵⁸ This reason may explain why our study's fibroblast cell count, collagen density percentage, and epithelial thickness were better than the other groups.

This study assessed the wound on days 7, 14, and 21 because the proliferative phase of wound healing occurs during these periods. In the proliferation phase, there is an increase in angiogenesis activity, fibroblast migration and proliferation, collagen and extracellular matrix synthesis, keratinocyte production, and re-epithelialization.¹⁷ Angiogenesis involves the proliferation, migration, and branching of endothelial cells to form new blood vessels with the help of angiogenetic factors such as VEGF, PDGF, FGF, and Ang.^59,60 Along with endothelial cell proliferation, pericytes cells are activated. Pericytes will surround the endothelial cells in blood vessels and are responsible for destabilizing and stabilizing the microvasculature, regulating blood flow, and forming a vascular barrier for bacteria.⁶⁰ While new blood vessels are formed, fibroblasts proliferate in response to growth factors such as PDGF, TGF-β, and FGF secreted by platelets and macrophages.^61,62 Fibroblasts will then secrete proteinases, such as MMPs, to degrade the surrounding matrix and replace it with collagen as the new extracellular matrix.⁶² Once the matrix is formed, keratinocytes will migrate and begin proliferation to cover the wound layer by layer. In epithelialization, keratinocytes are essential to restoring the epidermis after injury.⁶³

In this study, we used a gel dosage form. The gel can provide a cooling sensation and reduce pain. Acute pain after injury can cause hemodynamic changes and feelings of anxiety in experimental animals, which further inhibit healing. Pain can also inhibit macrophage activity, reducing TGF-β secretion to regulate fibroblast proliferation.¹² In addition, the gel can also facilitate the application of drugs on the wound and moisturize the wound to accelerate healing.⁶⁴ Our study only investigated the effect of robusta coffee extract gel with a single concentration. Many previous studies on wound healing have investigated topical agents in various concentrations. We suggest further preclinical studies on wound healing of skin grafts using various concentrations to determine the concentration that produces the best results. In addition, further studies using immunohistochemistry are needed for a more detailed evaluation.

CONCLUSION:

The results of this study confirm that topical application of 5% robusta coffee extract gel can enhance wound healing of skin graft by increasing angiogenesis, fibroblasts, collagen density, and epithelial thickness. These data show promising results and may open new avenues to consider coffee extract as a topical therapy for the healing process of skin graft. Further research with various concentrations of extract and immunohistochemistry is needed.

CONFLICT OF INTEREST:

The authors declared no potential conflicts of interest associated with this study.

ACKNOWLEDGMENTS:

The authors would like to acknowledge those who contributed to the completion of this study.

ABBREVIATIONS:

RCEG, robusta coffee extract gel; CGA, chlorogenic acid; MMP, matrix metalloproteinase; VEGF, vascular endothelial growth factor; Ang, angiopoietin; Ang-2, angiopoietin-2; NO, nitric oxide; MDA, malondialdehyde; SOD, superoxide dismutase; GSH, glutathione; ROS, reactive oxygen species; TGF-β, Transforming growth factor-β; TNF-α, tumor necrosis factor-α; PDGF, platelet-derived growth factor; FGF, fibroblast growth factor

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Received on 30.07.2023 Modified on 26.10.2023

Research J. Pharm. and Tech 2024; 17(6):2788-2794.

DOI: 10.52711/0974-360X.2024.00438