Negative Pressure Wound Therapy Using a New Pressure Monitoring Device for Wound Treatment: Results of an Animal Model

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NPWT is a widely used and efficacious modality for wound management. In 1993, Fleischmann et al¹ published their innovative amalgamation of traditional negative pressure drainage with contemporary closed dressing, ushering in a novel drainage technology that was initially used to treat infected wounds with remarkable success. Subsequently, in 1997, Argenta and Morykwas² introduced a porous polyurethane sponge as a negative pressure sealing material, enhancing drainage efficacy and garnering US Food and Drug Administration approval, leading to widespread adoption in North America and Europe. This technology has been widely applied in treating infected wounds, burn wounds, flap transplantation, and postoperative wounds.^3-6 NPWT demonstrates multifaceted benefits in wound care, protecting wounds and expediting healing processes by diminishing wound secretions and fostering a moist environment. It alleviates edema, enhances local blood flow, stimulates vascularization and granulation formation, accelerates epithelial cell growth, and facilitates wound epithelialization.^7,8 Additionally, some researchers have shown that NPWT serves to prevent microbial invasion and infection from the external environment.⁹ In the context of flap transplantation, NPWT plays a pivotal role in promoting vascularization of the root and securing the skin.^10,11 Furthermore, it enhances patient comfort by reducing the frequency of dressing changes, mitigating pain associated with dressing changes, and managing wound exudation and odor.¹² Importantly, NPWT contributes to reducing the workload for nursing staff, shortening hospital stays, and mitigating complications.¹³ 

Materials used for wound coverage in NPWT generally fall into 2 categories: PVA and polyurethane. PVA materials exhibit dense pores ranging in size from 100 µm to 300 µm.¹⁴ Some researchers augment polyurethane materials with additional components, such as silver ions or silicone, creating polyurethane foam materials with silver ion or silicone content.^15,16 The efficacy of NPWT is closely tied to the applied negative pressure, necessitating precise calibration based on patient- and wound-specific conditions. Argenta and Morykwas² conducted a study examining the effect of negative pressure ranging from −400 mm Hg to 0 mm Hg in NPWT. Their observations indicated that a negative pressure of −125 mm Hg maximally increased local blood supply. It is recommended that NPWT be used within a therapeutic range of −40 mm Hg to −150 mm Hg.¹⁷ Excessive negative pressure poses risks of tissue ischemia, particularly in patients with peripheral vascular disease or diabetic foot conditions, and during the early stages of burns or trauma. Caution is warranted to avoid bleeding in patients with suspected coagulopathy. Conversely, high negative pressure may be used for the initial management of heavily contaminated and edematous wounds, and adjustments in negative pressure should be made for large application areas or inadequately sealed wounds.

NPWT uses negative pressure machines or hospital central vacuum systems (the latter are not used in North America, Europe, or Oceania) as its pressure source. However, it is crucial to note that the “negative pressure value” displayed on the gauge does not accurately reflect the actual pressure experienced by the wound surface. Throughout the course of NPWT, the pressure on the wound surface dynamically changes. 

The present study endeavors to elucidate the real-time fluctuations in actual negative pressure values on the wound surface. To achieve this, a pressure monitoring dressing equipped with a novel, self-developed pressure sensor chip at a preset pressure value of −125 mm Hg or −75 mm Hg was used.

Development of the WPMD

In this study, the negative pressure sensor chip (BY-002) was integrated with a polyvinyl glycol foam dressing, resulting in the development of a WPMD (Chinese patent No. ZL202223032996.3). This innovative dressing is equipped with the capability to display wound pressure values and wirelessly transmit data. The chip’s primary components include a pressure sensor, a pressure calibration display showing values expressed in millimeters of mercury, an independent power supply, wireless transmission equipment, and a polyvinyl chloride sensor probe with an outer diameter of 5 mm and an inner diameter of 4 mm. The sensor probe was precisely positioned at the mid-level of the PVA foam dressing, maintaining a distance of 2 mm from the wound surface and 0.5 cm to 1.0 cm from the negative pressure drainage tube. This strategic arrangement ensures optimal monitoring of real-time pressure changes in the wound dressing during treatment. Subsequently, the obtained pressure data are transmitted to a computer recording terminal using Bluetooth technology (Bluetooth SIG, Inc), as depicted in Figure 1A. This process facilitates a comprehensive analysis of dynamic pressure changes throughout the treatment duration.

In vivo full-thickness wound model

To assess the efficacy of the WPMD in wound healing, Sprague-Dawley male rats aged 9 weeks to 11 weeks were procured from Si Pei Fu Biotechnology Co, Ltd. All procedures were conducted at the Kang Mei Hua Da in adherence with its Animal Research Committee (IACUC-20200604-06). A total of 36 male rats were randomly divided into 3 groups (12 rats per group). Prior to surgery, all animals were anesthetized using isoflurane administered through a gas anesthesia machine (R640; RWD Life Science Co, Ltd). The dorsal skin of each rat was depilated and sanitized with 75% ethanol. Full-thickness round wounds measuring 2.0 cm in diameter were surgically induced. Subsequently, the WPMD apparatus was applied over the wounds and covered with a Tegaderm membrane (3M). To prevent the rats from removing their dressings, the authors of the present study designed a special sleeve that was placed over the torso of each rat, making sure the dressing, arm, leg, head, and tail were outside the sleeve. Each rat was placed in its own cage and had no problem moving, eating, drinking, or defecating. 

Continuous negative pressure suction (VSD-M-B; Wuhan VSD Medical Technology) was maintained at a level of −75 mm Hg or −125 mm Hg. In the control group, wounds were covered with medical PVA foam dressing and the biological membrane, with dressing changes every 48 hours. The WPMD dressings were changed dependent on the wound drainage (approximately 4–5 days after wounding), and no re-wounding was observed during removal. Wound closure progression was documented through photographic records on days 0, 3, 5, and 7 (4 rats in each group). The photographer was unaware of the healing treatments. The wound closure dynamics were quantified and analyzed using ImageJ software. The wound closure rate (percentage) was calculated as (initial wound area − indicated wound area) / initial wound area × 100.

Histological analysis and immunostaining

Cutaneous wound repair areas were dissected on day 5 and day 7 after wounding, 4 rats from each group were killed at each time point, and the wound bed with adjacent healthy skin tissue was collected for histological analysis. The harvested skin tissues were fixed using 4% paraformaldehyde, followed by embedding in paraffin and subsequent perpendicular sectioning into 5-µm–thick longitudinal sections. H&E staining was used for histological analysis of wound repair. Immunohistochemical staining of the specimens was conducted using anti-VEGF-R2 antibodies (ab9698S; Abcam Limited), anti-Ki67 antibodies (ab16667; Abcam Limited), anti-CD31 antibodies (ab182981; Abcam Limited), and anti-TGF-β1 antibodies (ab215715; Abcam Limited). The histological sections were reviewed by an observer trained in histopathology and masked to all treatments received. For immunohistochemical staining slides, the study authors defined the brown cells as positive cells and quantified the number of positive cells to evaluate the histological findings.

Statistical analysis

Statistical analysis was performed using Prism version 8.0.2 (GraphPad). Statistical significance between the 2 groups was calculated using a 2-tailed t test. Multiple comparisons were assessed using 1-way analysis of variance with Tukey post-test. Four independent experiments were carried out, and data were expressed as mean (standard deviation). 

Dynamics of negative pressure tracked by the WPMD throughout wound healing

To assess the efficacy of the WPMD in tracking changes in negative pressure values, the authors of the present study used Sprague-Dawley rats as the experimental wound model and measured negative pressure values at various time intervals following application of the WPMD. Initially, the stability of the WPMD was tested by continuously monitoring negative pressure values every 10 minutes over the course of 1 hour. Figure 1B illustrates the consistent and unvarying nature of negative pressure values across different preset levels during this monitoring period. Subsequently, the investigators conducted an extensive data collection spanning 7 consecutive days during rat wound healing, maintaining preset negative pressure values at −75 mm Hg in 1 group of rats and −125 mm Hg in another group. Monitoring data revealed pressure variations during wound healing ranging from −128 mm Hg to −63 mm Hg in the −125 mm Hg preset group, and from −72 mm Hg to −37 mm Hg in the −75 mm Hg preset group. The results revealed a gradual decline in negative pressure values over the duration of NPWT, as depicted in Figure 1C.

Macroscopic observation of skin wound and calculation of wound closure 

Wound closure data were also collected to further assess the efficacy of the intervention. Images of wounds subjected to varying preset negative pressure values at different time intervals are depicted in Figure 2A. Notably, no signs of apparent infection were observed on the wound surfaces throughout the postoperative stages, and a consistent reduction in wound area was noted over time. The progression of wound closure in each group is illustrated in Figure 2B. Observationally, both the −125 mm Hg and −75 mm Hg groups exhibited significantly higher rates of wound closure compared with the control group. Furthermore, at day 5 and day 7, the closure rate of the −75 mm Hg group surpassed that of the −125 mm Hg group (Figure 2C).

Histological and immunohistochemical evaluation

Histological examination was used to further evaluate wound healing. Tissues obtained from the control, −75 mm Hg, and −125 mm Hg groups on day 5 and day 7 were subjected to H&E staining (Figure 3). As shown in Figure 3, on day 5 and day 7, the −75 mm Hg and −125 mm Hg groups displayed new blood vessels with a substantial number of red blood cells and regenerated dermal tissue. In contrast, the control group manifested thick epidermis, dead cell debris, and a high concentration of white blood cells.

Additionally, the expression of key tissue repair markers was assessed through immunohistochemistry. Figure 4 illustrates immunohistochemically stained images and statistical analyses of Ki67, VEGF-R2, CD31, and TGF-β1 expression. Notably, at day 5 and day 7, the NPWT treatment groups exhibited more positive cells for all these markers compared with the control group (Figure 4).

The correlation between negative pressure on the wound surface during NPWT and therapeutic outcomes, as well as complications, is pivotal. Some researchers have shown that variable pressure NPWT was more effective in terms of therapeutic effects than continuous negative pressure.^18,19 The presently endorsed negative pressure values are established through expert consensus and guidelines.²⁰ Previous in vitro simulations have reported on pressure distribution on both foam dressings and wounds.^21,22 Cozza et al²³ used probes to assess pressure at varying distances (0 cm, 1.5 cm, and 3 cm) between the surface of foam dressings and the negative pressure drainage tube in treated patients. In the present study, the developed WPMD integrates real-time wound pressure display and wireless pressure data transmission functionalities (Figure 1A). Over the initial 7 days, the authors of the present study observed a gradual reduction in dressing pressure, reaching less than 50% of the initial value by day 7 (Figure 1C). It has been reported that negative pressure at 1.5 cm and 3 cm from the suction cup significantly decreased in pressure tests on foam dressings in patients who received NPWT.⁴ Owing to the physical deformation of traditional dressings, solidification of wound exudate, and corresponding chemical reactions, the authors of the present study observed shrinkage and drying of the foam by day 7 compared with its initial state. Consequently, the negative pressure treatment device did not exert therapeutic effects. These results may elucidate the temporal changes in internal pressure in the negative pressure wound treatment system dressing.

In gross observations of skin wounds, the WPMD exhibited accelerated wound healing compared with the control group (Figure 2A). Additionally, wound healing was superior under a preset pressure value of −75 mm Hg compared with −125 mm Hg (Figure 2C). H&E staining was performed to assess the healing pathology of wounds (Figure 3). Reepithelialization and granulation tissue formation, identified as typical indicators of the wound healing process, were evident in the NPWT treatment groups, presenting almost complete coverage of the epithelium and an epidermal thickness closely resembling healthy skin at day 7 (Figure 3). On immunohistochemical analysis, CD31, a specific vessel marker, showed increased expression, indicating enhanced neovascularization and beneficial effects on wound healing. TGF-β1, which promotes granulation tissue proliferation and neovascularization, and VEGF-R2, which is crucial in angiogenesis, exhibited higher expression levels in WPMD-treated skin wounds (Figure 4). Increased Ki67 immunostaining suggested highly proliferative epidermis of ulcer margins.²⁴

Wound pressure variation is a dynamic process that is influenced by factors such as pipeline length, quality of semipermeable membrane, and dressing material. Traditional NPWT devices do not accurately represent actual wound pressure during application. The WPMD device used in the present study objectively and sensitively monitors wound pressure changes, preventing deviations from preset negative pressure values. Results indicate that a −75 mm Hg pressure setting is preferable to −125 mm Hg. In this study, an acute wound animal model was used to monitor negative pressure changes under 2 presets. Future in vivo investigations and clinical studies are needed to precisely adjust negative pressure according to different wounds. 

There are 4 major limitations in this study that could be addressed in future research. First, the control group should have received a placebo NPWT device that was inactivated but dressed with the same dressing. Second, the study only focused on traditional PVA dressing on an acute wound model, so the findings cannot be generalized. Third, only 2 preset negative pressure values were investigated, and they may not obtain the best value of preset negative pressure suitable for wound healing. Last, only 1 commercial vacuum sealing drainage device was used to provide NPWT, and the results may be not suitable for other devices.

According to the WPMD findings, both high and low preset negative pressure values were effective for acute wound treatment; however, the low preset negative pressure value provided better tissue repair and wound shrinkage results. The WPMD evaluated in the present study has the potential in clinical application for adjusting the preset negative pressure value according to real-time display data in various wounds for better wound repair. The authors of the present study recommend further assessment studies with more types of wounds that monitor negative pressure value until the wound has completely healed, and the comparison of different preset negative pressure values.