Effects of Negative Pressure Wound Therapy on FGF2 and PDGF Expression in Deep Dermal Burn Wounds: A Comparative Study with Conventional Treatments

Study design

Ethical approval for this study was granted by the Ethical Committee of Universitas Gadjah Mada (approval number: KE/FK/0203/EC/2023). Six male Yorkshire pigs (Sus scrofa domesticus), aged 3 months and weighing approximately 12 kg, were selected as subjects. Pigs that developed illnesses or died during the treatment period were excluded. These animals were fed a standard diet and had access to water ad libitum. Prior to the burn procedure, the pigs were acclimated to their environment for 3 days and fasted overnight.

To make deep dermal wounds on the pigs, we used a custom-made burn device developed in our lab. The device consists of a stainless-steel cylindrical plate with a diameter of 2 cm, connected to an electric heating unit capable of reaching temperatures between 82 °C and 100 °C. The plate temperature was monitored with a thermometer, with the sensor attached directly to the plate.

The pigs were sedated and anesthetized before the burn procedure. Initially, they were given an intramuscular injection of atropine (0.06 mg/kg). After sedation, the hair on the pigs’ bodies was shaved. Anesthesia was then induced with a combination of xylazine (2 mg/kg) and ketamine (20 mg/kg) and maintained with 2% isoflurane administered via a nasal cone. Twelve burn wounds were created on the back of each pig using the heated cylindrical plate applied perpendicularly to the skin with a mechanical force of 1 kgf, while elliptical incisions were used to collect tissue samples from the wounds for biopsy and to facilitate primary closure with suturing. We used similar animal models to those in our previous study [4].

The wounds were then divided into three treatment groups: NaCl, SSD, and NPWT, with each treatment group comprising of four wounds (observed and analyzed on days 3, 7, 14, and 21) for each of the six pig samples, resulting in a total of 72 wounds on six pigs. The NaCl group included wounds treated with 0.9% NaCl solution dressings. The dressings in all groups were changed on days 3, 7, 14, and 21. The SSD group wounds were treated with SSD gauze. The NPWT treatment was regulated to achieve a pressure of –125 mmHg with a vacuum-assisted closure device continuously for 24 hours a day.

Data collection

The burn wounds were evaluated macroscopically on days 3, 7, 14, and 21 as the dressings were changed. Wounds were documented using a Nikon D5300 with an 18-55 mm lens. A ruler was placed on the side of the wounds and included in the pictures taken for scale. With visual estimation, the camera was placed parallel to the wound surface. The pictures were then uploaded and opened with ImageJ (Image J bundled with 64-bit Java 8 for Windows) for size calculations. For every photo, the scale is calibrated based on the ruler at the bottom of the image. On day 14 and day 21, we measured the areas of the wound and applied the numbers to the following formula of wound contraction:

At the same time points, the wounds were biopsied at different sites, followed by primary closure of the wounds. A detailed explanation on the biopsy method was published in our previous study [4]. Tissue samples were also collected from wound margins and examined using the enzyme-linked immunosorbent assay (ELISA) method to determine the concentrations of FGF2 and PDGF. We used an ELISA reagent set from Novus Biologicals, LLC to quantitatively analyze biomolecular samples from wound fluid using a sandwich ELISA format. After preparing all reagents, standards, and samples per manufacturer instructions, and bringing them to room temperature, we began by selecting the necessary number of strips, with unused strips stored at 2–8 °C.

We added 50 μL of standard to the standard wells (no additional antibodies needed) and 40 μL of sample to the sample wells, followed by the specific antibody for each target protein (interleukin-33, matrix metalloproteinase-9, transforming growth factor beta 1 [TGF-β1], keratinocyte growth factor [KGF]). We then added 50 μL of streptavidin-horseradish peroxidase to both sample and standard wells. After a 60-minute incubation at 37 °C, the plate was washed five times, followed by the addition of 50 μL each of substrate solutions A and B. Incubating the plate at 37 °C in the dark for 10 minutes, we then added the stop solution, changing the color from blue to yellow. Optical density was measured at 450 nm within 10 minutes. Protein levels were finally measured in ng/L. The samples were euthanized immediately after the final sampling and clinical observation, which took place on day 21. Subsequently, the research subjects were transferred to the Prof. Soeparwi Veterinary Hospital, Universitas Gadjah Mada, for the euthanasia procedure.

Statistical analysis

Levels of FGF2 and PDGF along with the area of wound contraction at all time points were tested for normality using the Kolmogorov Smirnov test and further analyzed using repeated analysis of variance. All data were presented as mean±standard deviation. Post-hoc analyses were performed to determine the significance of differences between treatment groups. A P-value of <0.05 was considered statistically significant. IBM SPSS Statistics version 26 was used for all statistical analyses.

Macroscopic evaluation of the wounds

Direct observation of the wounds (Fig. 1) revealed that on day 3, the wounds in the NPWT group had eschars with more moisture than the other treatment groups. Since the first day, the NPWT group had experienced continuous exfoliation, followed by the NaCl and SSD groups which started exfoliating on day 7. On day 14, eschar removal was performed on each treatment group. The NaCl group exhibited wound contraction characterized by uneven granulation tissue growth, resulting in a burn wound surface that was not level with the surrounding tissue. The group treated with SSD also exhibited wound contraction comparable to that observed in the NaCl dressing group, but granulation tissue was not level with the surrounding tissue. Additionally, there was more crusting in the SSD group than in the other treatment groups. The NPWT group demonstrated superior granulation, characterized by an even wound surface that was level with the surrounding tissue. On day 21, epithelialization in the NaCl treatment group had nearly completely covered the burn area, with prominent wound contractions also forming. In the SSD group, epithelialization nearly fully covered the wound, showing hyper-granulation where the granulation tissue rose above the surrounding skin. By the end of the observation period, the NPWT group had not achieved complete coverage with epithelialization and granulation; however, the granulation tissue in this group was more evenly distributed, despite minimal wound contraction. In contrast, the NaCl group showed incomplete epithelialization by day 21, with prominent areas of granulation protruding from the wound bed. Although the wounds in both the NPWT and NaCl groups remained partially uncovered, the more uniform granulation in NPWT suggested improved wound bed quality. Further analysis showed a significant difference in average wound contraction between groups, with a P-value of 0.001 on day 14 and <0.001 on day 21 (Table 1).

Fig. 1.

Macroscopic photographs of burn wounds in three treatment groups. NaCl, 0.9% sodium chloride; SSD, silver sulfadiazine.; NPWT, negative-pressure wound therapy.

Table 1.

Wound contraction percentage of each group as analyzed using ANOVA

Fibroblast growth factor-2

The results of FGF2 levels in deep dermal burn wounds are described in Table 1. The FGF2 levels in the NPWT group were the highest on both day 7 (780.62±353.88 ng/mL) and day 21 (504.31±254.25 ng/mL). The data in Table 2 shows that the concentration of FGF2 in the NaCl group significantly decreased from day 3 to day 21 (P=0.028). Similarly, a significant decrease was observed in the SSD group (P<0.001). The NPWT group, while showing a decrease, did not exhibit significant differences (P=0.051). This demonstrated that NPWT potentially maintained more stable FGF2 than the other therapies (Fig. 2). Error bars of the analysis are shown in Fig. 3.

Table 2.

Mean FGF2 concentration values between groups as analyzed using ANOVA

Fig. 2.

FGF2 levels at all time points. The NPWT group maintains higher FGF2 levels compared to the others, particularly noticeable on days 7 and 21. FGF2, fibroblast growth factor-2; NaCl, 0.9% sodium chloride; NPWT, negative pressure wound therapy; SSD, silver sulfadiazine.

Fig. 3.

Error bars of FGF2 levels at all time points. FGF2, fibroblast growth factor-2; NaCl into 0.9% sodium chloride; NPWT, negative- pressure wound therapy; SSD, silver sulfadiazine; SD, standard deviation.

In terms of delta (Δ) values between time points (Table 3), the NPWT group exhibited significant changes in FGF2 levels over time (P=0.042), with an increase from day 14 to day 21 (+160.2 ng/mL). This suggests a potential benefit of NPWT in enhancing FGF2 levels during the later stages of wound healing.

Table 3.

Mean delta values of FGF2 concentrations in treatment groups as analyzed using ANOVA

Platelet-derived growth factor

Higher PDGF concentration levels were observed in the early phase of wound healing (days 3 and 7) (Table 4). However, none of the treatment groups differed significantly from the other groups over time. As for the delta values in Table 5, there were significant changes in PDGF levels in the SSD group (P=0.018) with a significant increase from day 14 to day 21 (late stages of wound healing), suggesting that SSD may negatively impact burn wound healing during the later stages compared to NPWT and NaCl treatments (Fig. 4). Error bars of the analysis are shown in Fig. 5.

Table 4.

Mean PDGF concentration values between groups as analyzed using ANOVA

Table 5.

Mean delta values of PDGF concentrations in treatment groups as analyzed using ANOVA

Fig. 4.

PDGF levels at all time points. PDGF levels fluctuate over time within each treatment group (NaCl, NPWT, SSD), but the changes are not significant. PDGF, platelet-derived growth factor; NaCl, 0.9% sodium chloride; NPWT, negative pressure wound therapy; SSD, silver sulfadiazine.

Fig. 5.

Error bars of PDGF levels at all time points. PDGF, platelet-derived growth factor; NaCl, 0.9% sodium chloride; NPWT, negative pressure wound therapy; SSD, silver sulfadiazine.

Fibroblast growth factor-2

The FGF family consists of 22 subtypes in humans and mice, each contributing to various biological functions. Research by Komi-Kuramochi et al. [7] observed that FGF-7, known as KGF, and FGF10 are elevated during the healing process and stimulate keratinocyte proliferation in both normal and wounded skin. FGF2, also known as basic FGF, activates fibroblasts, endothelial cells, osteoblasts, and chondrocytes, thereby accelerating wound closure [8].

In our study, the concentration of FGF2 in the NaCl group and the SSD group significantly decreased from day 3 to day 21. Meanwhile, in the NPWT group, the FGF2 concentration did decrease, but not to a significant degree. Although the results were not statistically significant, the NPWT group maintained the highest FGF2 concentration among all groups on days 7 and 21. This trend persisted despite a slight decrease in FGF2 levels observed across all groups on day 21. Such results can be aligned with the proposed theory that NPWT provides higher levels of growth factors, such as FGF2, VEGF, and PDGF, in wound healing processes compared to other treatments [8]. This finding indicates that while levels of FGF2 in burn wounds decrease as the healing progresses, NPWT might help in maintaining FGF2 levels.

The vacuum system of NPWT is believed to stimulate cell function, protein synthesis, and gene expression, leading to the production of matrix molecules that increase the proliferation of endothelial cells in newly formed granulation tissue. Literature suggests that FGF2 is a key factor for fibroblast formation in wound healing. However, in this study, the FGF2 concentration did not show a significant difference in the NPWT group, unlike the findings of Kopp et al. [9], who observed a significant increase in growth factor concentrations in wound exudate samples from neuropathic diabetic foot ulcer patients treated with NPWT.

Our results, which lacked statistical significance, may differ from the findings of Seswandhana [5], who reported significantly elevated levels of KGF (FGF7) in the wound fluid of dermal burns treated with NPWT. These findings suggest that NPWT may primarily influence the wound surface environment, as evidenced by the higher concentration of KGF (FGF7) reported in previous studies. However, its direct impact on the wound site might be limited, as indicated by the non-significant changes in FGF2 levels observed in our study. We assume that this is correlated to the mechanisms of action of NPWT on wound healing, which include macrodeformation, microdeformation, fluid removal and alteration of the wound environment, all mainly located on the wound surface. Macrodeformation is shrinkage of the wound when suction is applied to the foam, causing pore collapse and pulling wound margins closer to the center. Microdeformation is a mechanism by which NPWT applies suction to create mechanical forces at a microscopic scale, activating cascades that stimulate granulation tissue formation. NPWT also provides exudate control by removing excess fluid through the lymphatic system at the wound edges. The foam and drape used in NPWT also stabilize osmotic and oncotic gradients at the wound surface, making a sterile and moist environment surrounding the wound [10,11].

After an injury, the first phase of wound healing is inflammation, which is characterized by reactions mediated by cytokines, chemokines, and growth factors. Activation of these mediators induces cell proliferation, migration, and differentiation [12], leading to infiltration of neutrophils, granulocytes, and monocytes, which in turn transform into macrophages [13].

While all groups demonstrated a decrease in FGF2 from day 7 to day 14, only the NPWT group showed a significant difference. This time point may represent a transition from the proliferation stage to the early remodeling stage, during which fibroblasts and deposited proteins contribute to the formation of scar tissue. This indicates the anti-fibrotic function of FGF2 in early remodeling phase, where it prevents myofibroblasts from inducing excessive scarring [12].

In this study, the NPWT group also showed a significant increase in FGF2 from day 14 to 21, indicating a potential benefit of NPWT in increasing FGF2 levels during the later stages of healing. NPWT increases FGF2 during the proliferation phase, facilitating fibroblast migration to wound edges and production of proteoglycan, glycosaminoglycan, and collagen. Growth factors are essential during the late inflammatory phase, extending up to day 21, which aligns with the findings of this study [13].

Platelet-derived growth factor

PDGF is produced by various cell types, including endothelial cells, fibroblasts, smooth muscle cells of blood vessels, osteoblasts, glia, and neurons. PDGF acts as a chemoattractant for early inflammatory cells (neutrophils and macrophages) as well as fibroblasts and smooth muscle cells. Furthermore, PDGF triggers the release of TGF-β from macrophages and increases VEGF secretion. It also independently contributes to angiogenesis, epithelialization, and extracellular matrix synthesis [14].

The levels of PDGF in all treatment groups seemed to be higher on early time points (day 3 and day 7). This shows that PDGF is released in the early phase of wound healing. In the inflammation phase, several cytokines are released, recruiting neutrophils and other leukocytes to the site of injury. T-cells infiltrate the wound and recruit macrophages, which secrete PDGF and TGF-β to stimulate fibroblasts and myofibroblasts, initiating the proliferation phase [15].

Although the difference in PDGF levels across the groups was insignificant, there was a significant increase in PDGF in the SSD group from day 14 to 21, suggesting that SSD might have a negative effect on late-stage wound healing compared to NPWT and NaCl treatments. While PDGF plays a crucial role in the early stages of the wound healing process, the presence of high PDGF concentrations in the late stages might not be beneficial and could potentially indicate or cause complications, such as fibrosis, chronic wound, and impaired remodeling [16,17]. Though PDGF is vital for initiating and progressing the early stages of wound healing, its levels should ideally decrease or be suppressed by treatment as the wound transitions into later stages to ensure proper healing and prevent complications, as shown from the NPWT group.

This finding may be explained by a previous study conducted by Burd et al., who performed a series of in-vitro and in-vivo experiments to assess the impact of five commercially available silver-based dressings on wound healing. Their in-vitro results demonstrated that silver significantly delayed re-epithelialization in an epidermal cell proliferation model. Meanwhile, the use of negative pressure wound therapy has been shown to enhance the expression of PDGF and TGF-β1 in human fibroblast in-vitro models [18]. There is limited data on the correlation between NPWT and PDGF, making it challenging to compare the findings of this study with existing literature.

Conclusion

NPWT has demonstrated promising efficacy in accelerating burn wound healing by creating a sterile, moist environment that supports granulation tissue formation and re-epithelialization while enhancing blood flow and nutrient delivery. This study observed consistent granulation and epithelialization in NPWT-treated wounds with minimal contraction, suggesting effective wound healing progression. Furthermore, analysis of wound-healing biomarkers, including FGF2 and PDGF, highlighted NPWT’s ability to maintain growth factor levels essential for cellular proliferation and tissue formation. Although FGF2 levels did not show significant changes in NPWT-treated wounds, their maintenance at higher levels aligns with the treatment’s role in optimizing the wound environment. PDGF, primarily influential in the early inflammatory phase, showed increased concentrations in the SSD group in later stages, suggesting potential negative implications for fibrosis and chronic wound formation. NPWT’s role in sustaining optimal growth factor levels and controlling wound environment supports its potential as a beneficial therapy in managing complex burn wounds, providing an alternative to conventional treatments, which can sometimes result in excessive scarring.