Wound healing is a complex phenomenon that involves a series of interlocking and relatively independent processes and is most often described as four phases: hemostasis, inflammation, proliferation (including fibroplasia and angiogenesis), and remodeling [1, 2]. Complex wounds such as serious soft tissue defects, tendon- and bone-exposed wounds, and chronic and pressure ulcers associated with systemic diseases often cannot self-heal because of severe tissue damage or wound infection, which require external conditions to assist in healing.

Negative-pressure wound therapy (NPWT), a technology whereby an adjustable sub-atmospheric pressure is applied to a wound via a gauze dressing or open-cell foam under an occlusive drape, has been introduced for the treatment of various types of complex wounds since the 1990s [3, 4]. Based on the excellent clinical effects of NPWT, such as diminishing the frequency of dressing changes and shortening the time of wound care, it has been widely applied in acute and chronic wound healing [5–8]. NPWT could enhance wound healing by creating a moist environment, decreasing tissue edema, removing exudates, stimulating angiogenesis and granulation tissue formation, attenuating the bacterial burden of wound edges, and promoting local blood perfusion [9, 10]. Multiple growth factors including vascular endothelial growth factor (VEGF) [11], basic fibroblast growth factor (bFGF)[12], and angiotensin (Ang) 2 [13] are involved in controlling and regulating the process of angiogenesis. Seswandhana et al [14]. showed that NPWT treatment resulted in the upregulation of VEGF and Ang 2. Another report showed that NPWT resulted in the upregulation of proliferating cell nuclear antigen (PCNA) [15], which is closely related to DNA synthesis and cell proliferation [16].

The therapeutic effect of NPWT varies with different negative pressure values [17, 18]. In a swine model, wounds treated with NPWT at a negative pressure of 125 mmHg displayed an obvious enhancement in granulation tissue formation compared to those treated at 25 or 500 mmHg [19]. Considering the effect of NPWT at various negative pressures, we hypothesize that it might impact the expression of cytokines related to wound healing. In this work, Sprague-Dawley rats were used to construct a skin wound model in vivo. The rats were treated by NPWT at different negative pressure values (75, 125, and 300 mmHg) for various time periods (4, 7, 10, and 14 days), and the wound healing-related indicators were evaluated to explore the therapeutic effects of NPWT at different negative pressures.

Experiment animal model and gross wound measurement. 80 male Sprague-Dawley rats aged 8 weeks (weighing 200–250 g) were purchased from the Laboratory Animal Centre, Huazhong Agriculture University and housed with food and water ad libitum at 22 ± 2°C. The full-thickness skin defect model was constructed as mentioned previously [20]. The hair on the back of the rats were clipped and depilated after they were anesthetized with 1% pentobarbital sodium at 50 mg/kg. After 24 h, the rats were anesthetized with isoflurane inhalation, followed by disinfecting the backs with alcohol patches. Then, a 2 cm × 2 cm full-thickness defect was created on the back. The rats were randomly divided into four groups (n = 20 per group). Rats in the control group (CON) were covered with gauze; those in the experimental groups were treated with an NPWT device containing standard dressing (MKY1485, Maikeyi (Beijing) technology co, ltd, Beijing, China) under negative pressure values of 75 mmHg (-75 mmHg), 125 mmHg (-125 mmHg), and 300 mmHg (-300 mmHg) for 4, 7, 10, and 14 days (the day that surgery was carried out was considered to be the first day). Dressing was changed every two days and gauze was changed every day under isoflurane inhalation. Wound closure was observed from the beginning to the end of the treatment period. At the designated time (on the 4rd, 7rd, 10th, and 14th days after the surgery), anesthesia was administered by intraperitoneal injection of 3% pentobarbital sodium at a dose of 40 mg/kg, and the wound tissue on the back was rapidly collected. If the animal has not yet died and is subjected to excessive anesthesia with pentobarbital sodium (100mg/kg), and the animal shows no breathing or heartbeat after anesthesia, it is determined that the animal is dead. All experimental procedures were approved by the Animal Ethics Committee of the Laboratory Animal Centre, Huazhong Agriculture University, and approval number is HZAURA-2018-009. All experiment were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the State Scientific and Technological Commission of China.

Wound blood perfusion analysis. The PeriCam PSI (Perimed, Beijing, china) apparatus was utilized to measure the wound blood flow after 4, 7, 10, and 14 days of treatment. The distance between the wound and the probe is 15 cm, and the scanning window size is 100 mm× 100 mm. The detection results were recorded in terms of perfusion units (PU).

Bacterial content analysis. After 4, 7, 10, and 14 days of treatment, the exudates on the surface of the wounds were cleaned using sterile saline solution. Biopsies were harvested under aseptic conditions and the granulation tissues in the center of the wounds were obtained. The extracted tissues were weighed, followed by homogenization and dilution (1:10⁸). The diluted solution was quantitatively injected into a petri dish. After incubating at 37 ^oC with 5% CO₂ for 24 h, the colony forming unit (CFU) was calculated. The bacterial content was calculated as follows: Bacterial content (cfu/g) = CFU × [volume of diluent (ml) + tissue weight] / tissue weight × dilution ratio [21].

Histology and immunohistochemical analysis. 2–3 mm of the tissue from the wound rim was harvested after 7 days of treatment and fixed in 10% formalin. The fixed tissues were rinsed, dehydrated, embedded in paraffin, sliced into 4-µm sections, and placed onto microscope slides. After dewaxing and hydration, the tissues were placed into citrate buffer for 15 min for antigen retrieval, then into 3% H₂O₂ for 10 min to eliminate endogenous peroxidase activity. The tissues were incubated for 1 h with primary antibodies against VEGF (Bioswamp, Myhalic Biotechnology Co., Ltd., Wuhan, China); bFGF (Bioswamp); Ang 1/2 (Bioswamp); α-smooth muscle actin (α-SMA, Bioswamp); CD34 (Bioswamp); B-cell lymphoma 2 (Bcl-2, Abcam, Cambridge, UK); Bcl-2-associated x (Bax, Abcam); caspase 3 (Bioswamp); and PCNA (Bioswamp), then with secondary antibody (MaxVision TM HRP-Polymer anti-Mouse/Rabbit IHC Kit, Bioswamp) for 30 min. The tissues were stained with diaminobenzidine (Bioswamp) until the color changed, followed by counterstaining with hematoxylin for 3 min. After dehydration and mounting, the tissues were photographed using a microscope (Leica, Germany)

Statistical analysis. All data are presented as the mean ± standard deviation, Differences between more than two groups were analyzed using one-way analysis of variance followed by a least significant difference. P < 0.05 was considered to be statistically significant.

NPWT improved wound healing. The wound areas treated with or without NPWT were observed at day 0, 4, 7, 10, and 14 after therapy initiation (Fig. 1). As shown in the results, NPWT obviously promoted wound healing compared to CON group. Meanwhile, there were no apparent inflammation and induration in wounds treated with NPWT. The wounds receiving NPWT became more hyperemic with new granulation tissue, while the wounds in the CON group were covered with some necrotic tissue. The wound healing speed was associated with the negative pressure. NPWT under a pressure of -125 mmHg exhibited the best therapeutic effect in our experiment, the wound treated with which was almost healed after 14 days of therapy.

NPWT accelerated wound blood perfusion. The wound blood perfusion was assessed using the PIM3 type laser Doppler imager and the captured images are shown in Fig. 2A. Area 1 is the wound (red circle), and the values of blood perfusion across the wound are shown in Fig. 2B. The wound blood perfusion in all groups were almost the same on the 4th day of treatment. However, NPWT at the three tested pressures resulted in relatively enhanced wound blood perfusion at the later stages from day 7 to 14 of wound healing compared to that in the CON group. Furthermore, -125 mmHg had a larger effect on wound blood perfusion than that of -75 and − 300 mmHg.

NPWT reduced bacterial content in the wounds. The bacteria content in the wounds was evaluated at day 4, 7, 10, and 14 after NPWT. As shown in Fig. 3, the bacterial content in the wound treated with NPWT was significantly reduced compared to that of the CON group, and NPWT at -125 mmHg showed the most prominent effect. On day 14 after therapy, the bacterial content of wound in the CON group was 463 × 10⁸ cfu/g and was reduced to 400 × 10⁸, 98 × 10⁸, and 258 × 10⁸ cfu/g with NPWT at -75, -125, and − 300 mmHg, respectively. These results indicate that NPWT decreased the bacterial content of the wound, the extent of which was influenced by the value of negative pressure.

NPWT accelerated angiogenesis of the wound. Immunohistochemistry was performed to qualitatively evaluate the protein expression of VEGF, bFGF, Ang2, α-SMA, and CD34 in wound treated with or without NPWT at day 7 after therapy. As shown in Fig. 4, the levels of the above-mentioned proteins were evidently increased by NPWT treatment at -125 and − 300 mmHg compared with those observed in the CON and − 75 mmHg group, with-125 mmHg inducing the highest expression levels. The results collectively illustrated that NPWT accelerated angiogenesis of the wound, the extent of which was influenced by the value of negative pressure.

NPWT accelerated cell proliferation. Furthermore, the proliferation-related protein PCNA and apoptosis-related proteins Bax, Bcl-2, and caspase 3 were examined using immunohistochemistry (Fig. 5). We observed that the expression levels of PCNA in the NPWT-treated tissues were remarkably higher than those without NPWT treatment. The expression of Bcl-2 was elevated by NPWT at -300 mmHg and further increased at -125 mmHg compared to that in the CON and − 75 mmHg groups. By contrast, the expression of Bax and caspase 3 in each group showed the opposite trend as that of Bcl-2.

NPWT has exhibited efficacy in accelerating wound healing and has been widely used in clinical settings [22, 23]. It has been reported that the therapeutic effect of NPWT is closely related to the negative pressure value at which it is applied. Previous experimental and clinical research has demonstrated that NPWT showed better therapeutic effect at -125 mmHg compared to other pressures [24, 25]. In this work, we chose three negative pressure values (-75, -125, and − 300 mmHg) to treat wounds in rats. The results showed that application of NPWT at -125 mmHg contributed substantially to wound closure compared to other pressures, especially wounds treated only with gauze. Meanwhile, the wound blood flow induced by -125 mmHg was obviously increased compared to that in the CON and other NPWT-treated groups at the later stages (7–14 days) of wound healing, which was consistent with previous study [26].

Bacterial infection impedes wound healing. Whether NPWT can effectively reduce bacterial content in infected wounds is controversial. Clinical and experimental studies have revealed that NPWT reduced bacterial content in infected wounds and promoted wound healing [27, 28]. However, other clinical investigations have shown that NPWT did not reduce bacterial content in the wound at all [29]. This work showed that NPWT at -75, -125, and − 300 mmHg promoted bacterial content reduction, with significant differences among the different negative pressures.

VEGF and bFGF are involved in the process of angiogenesis. The expression of VEGF and bFGF in cells was increased in response to biaxial cyclic stretching and mechanical stretching, followed by the induction of angiogenesis [30, 31]. Previous studies have demonstrated that NPWT resulted in an upregulation of the expression of VEGF [32] and bFGF [33]. This work showed that the expression VEGF and bFGF was clearly increased in wounds treated with NPWT at -125 mmHg. Meanwhile, Ang 2, which is involved in regulating angiogenesis and vessel maturation [34], was highly expressed after the application of NPWT at -125 and − 300 mmHg compared to that in the other groups. It has been reported that high expression of Ang 2 and VEGF promoted neovascular formation [35], CD34 can serve as marker of capillaries [36, 37]. This work showed that NPWT upregulated CD34 in wounds, with relative higher expression at -125 and − 300 mmHg. α-SMA has been reported to act as a general marker of pericyte-like perivascular mural cells (pericytes) [26], which are a vital factor of mature blood vessels [38, 39]. According to a previous study, upregulation of α-SMA expression increased blood flow perfusion during the process of wound healing [26]. This work showed that wounds treated with NPWT exhibited higher expression of α-SMA compared to non-treated those, and the level of α-SMA was the highest at -125 mmHg. Collectively, these results implicated that NPWT promoted angiogenesis and induced the recruitment of pericytes, resulting in the gradual maturation of vessels and thus enhancing blood flow perfusion and wound healing. The therapeutic effect of NPWT is related to the negative pressure value, which resulted in the regulation of angiogenesis and associated cytokines to various extents.

PCNA plays an important regulatory role in DNA replication and cell proliferation and acts as a marker of cell proliferation [40]. This work demonstrated that the wound treated with NPWT resulted in higher expression of PCNA compared to the wound treated with only gauze. The expression levels at different negative pressures were similar, which is in agreement with a previous report [41]. However, the expression of apoptosis-related factors was different in different groups. Bcl-2 is involved in inhibiting apoptosis, while Bax and caspase 3 are associated with promoting apoptosis [42, 43]. This work showed that NPWT at -300 mmHg increased the expression of Bcl-2 compared to that in the CON and − 75 mmHg groups, and it was further improved at -125 mmHg. The expression of Bax and caspase 3 in each group showed the opposite tendency compared to that of Bcl-2. These results revealed that NPWT at appropriate pressures can accelerated wound healing by modulating apoptosis-relative proteins.

In conclusion, we demonstrated that at appropriate pressures, NPWT improved angiogenesis and tissue proliferation, thereby promoting wound healing. The therapeutic effects of NPWT at different negative pressures were different, which might be associated with the situation that different negative pressures contribute to the regulation of angiogenesis and apoptosis-related cytokines to varying extents.