Effects of negative-pressure wound therapy combinedwith microplasma on treating wounds of ulcer and the expression of heat shock protein 90.

The effects of negative pressure wound therapy (NPWT) combined with microplasma on treating wounds of ulcer, and blood perfusion of wound-healing of interface, angiogenesis and the expressions of heat shock protein 90 (HSP90) were explored. We selected continuously 64 patients with wounds of ulcer. The patients were divided into the conventional treatment group (just medical foam dressing and 1% silver sulfadiazine cream for dressing changes) (n=20 cases), the NPWT group (n=22 cases) and the combination group (NPWT combined with microplasma) (n=22 cases), and compared the effects. It was found that in the 7 and 14 day combination group, maturity of granulation tissues and growth degree of epithelium were significantly higher than those in other two groups, and the areas of ulcer reduced significantly, the healing rate increased significantly (P<0.05). In the 7 and 14 day combination group, blood perfusion of wounds and density of new vessels were significantly higher than the other two groups (P<0.05). In the 7 and 14 day combination group, the expression of HSP90 was significantly higher than the other two groups (P<0.05). In conclusion, NPWT combined with microplasma can improve the healing of woulds of ulcers, and it is related to the upregulated expression of HSP90.

RCT Entities:   Population Interventions Outcomes

Introduction

In clinics, the incidence rate of diabetic foot ulcer, deep burn, bedsore, severe varicosity, obliterans of lower extremities, ulcers caused by severe trauma and others, especially chronic refractory skin ulcer has increased significantly which greatly influence the patients survival rate (1). After debridement, medical dressing for periodic dressing change is a common treatment, but the effective rate is only 40–60%, and the infection rate still reaches 10–30% (2). Therefore, actively finding ways to treat ulcer more effectively is a research hotspot. Negative pressure wound therapy (NPWT) is also called vacuum sealing drainage (VAC). It treats wounds by taking advantage of medical foam dressing and semi-transparent biological films to cover the wound area, then connecting wound dressing with negative-pressure device by drainage (3). Microplasma excites nitrogen in the air to microplasma by excited monopolar radiofrequency and forms several micropore on the surface of the skin, which stimulates collagen fiber proliferation and angiogenesis by exfoliation and heat effects. It has marked effects on the treatment of scars (4). This study explores the effects and the possible mechanisms of NPWT combined with microplasma on treating wounds of ulcer.

Patients and methods

Patients

We continuously selected 64 patients with ulcer admitted to Jinan Central Hospital from October 2012 to October 2015, and all of them were cases of first treatment. The excluded patients were those with ulcer canceration, gangrene, serious infections, necrosis that needs amputation, serious complications like pyemia, receiving immunosuppressants, glucocorticoid and chemotherapy, severe hypoproteinemia (<30 g/l), moderate and severe anemia (hemoglobin <90 g/l), uncontrolled blood sugar level (fasting blood-glucose >7.4 mmol/l), poor compliance and incomplete information.

This study was approved by the Ethics Committee of Jinan Central Hospital and written informed consent of the patients or their families was obtained. According to the treatment methods the patients were divide into the conventional treatment group (just medical foam dressing and 1% silver sulfadiazine cream for dressing changes) with 20 cases, the NPWT group with 22 cases and the combination group (NPWT combined with microplasma) with 22 cases. In the conventional treatment group, there were 13 males and 7 females, aged from 19 to 68 years, with a median age of 43.5 years; their disease duration ranged from 1 day to 4 months with the median time of 1.3 months; for ulcer causes, there were 7 cases of trauma, 6 cases of operation, 3 cases of diabetic foot, 3 cases of burn and 1 case of bedsore; there were 2 cases in abdomen, 6 cases in upper limb, 8 cases in lower limb, 4 cases in foot and 2 cases in gluteal region; the maximum diameter was 2.3–11.5 cm with an average of 5.5±1.5; the areas were 5.0–25.0 cm2 with an average of 13.2±3.2; the depth was 1.1–2.8 cm with an average of 2.0±0.5. In comparison to the baseline of the three groups, the differences did not have statistical significance (P>0.05).

Treatment methods

Local bacterial culture was used and the drug sensitivity tests for all wounds of ulcer, and sensitive antibiotics were used according to the results. The wounds were debrided conventionally, necrotic tissues and eschar on the surface of wounds were removed, unobstructed drainage was provided for purulent fluid under the eschar, the important vessels, nerves and tendon were preserved and 3% perhydrol and normal saline was used to wash wounds repeatedly after surgical debridement. In the conventional treatment group, only medical foam dressing (Chinese Shanxi iLSino Medical Instrument Co., Ltd.) and 1% silver sulfadiazine cream was used, and dressing was changed once a day according to the principle that 10 g of dosage is applied to the wounds every 100 cm2 of the wounds.

In the NPWT group, the medical foam dressing was used to cover and semi-transparent films used to close the wounds. The drainage was used to connect foam of the wounds with RNPT-1 negative-pressure device (provided by Chinese Shanxi iLSino Medical Instrument Co., Ltd.). The wounds were treated with continuous negative pressure at 120 mmHg for 4 h, for 7 days continuously. In the combination group, the microplasma (provided by Alma Lasers, Ltd. Buffalo Grove, IL, USA) was adopted at the same time, and there were two kinds of treatment methods. One was roller-type (perimeter was 25 mm, ring-width was 10 mm, the row number of image bundles was 6 and output power was 70–90 W). The other method was a point-type (diameter was 12 mm, and the distance between points of image bundles was 1 mm). Before irradiation, 5% compound xylocaine cream (provided by Beijing Tsinghua Unisplendour Pharmaceutical Factory, Beijing, China) was used to apply on the treatment region evenly, covering with preservative film to have superficial anesthesia for ~40 min. The lateral, vertical and oblique scanning was performed 3 times to make plasma action uniformly. After treatment, the compound polymyxin B ointment (provided by Beijing Tsinghua Unisplendour Pharmaceutical Factory) was applied to the surface to prevent postoperative infection, and ice bags were used as cold compress for 15 min to prevent blisters. The surgical areas were recommended to be kept away from contact with water for 1 week post-surgery until the crust came off naturally, and attention was paid to sunscreen. The second treatment was carried out with an interval of two weeks.

Observational index

The maturity of granulation tissues, growth degree of epithelium, ulcer areas and the healing rate after treating was compared at 7 and 14 days. The growth conditions of granulation tissues were obtained with digital camera (Canon, Inc., Tokyo, Japan) and were calculated by applying Image-Pro Plus v6.0 image analysis software (Microsoft Corporation, Redmond, WA, USA) by calculating (original wound area - the wound area with no granulation covering)/original wound area × 100%.

The evaluation method for the growth of epithelial tissues was the use of transparent tracing paper to trace the original wound area of ulcer before treatment, after treatment the wound was traced with the same method, and scanned into the computer by calculating (original wound area - no wound area with no epithelization)/original wound area × 100%.

The blood perfusion of wounds and density of new vessels were compared after treating for 7 and 14 days. The blood perfusion of wounds was measured by PeriScan PIM 3 Laser Doppler Perfusion imager (produced by Perimed AB, Stockholm, Sweden), during the detection, distance from the sensing probe to the wound was set as 14 cm, the scan window size was 100×100 mm, and the test results were indicated as perfusion units (PU). The detection method of density of new vessels was to cut the wound, wounded tissues of the healing tissue interface with diameter ~0.3 cm, was fixed in 4% paraformaldehyde solution, the section was embedded with conventional paraffin, then immunohistochemical staining with CD34 (CD34 antibody was offered by the R&D Systems, Minneapolis, MN, USA), diaminobenzidine (DAB) color kit was provided by the Beijing Zhongshan Biotechnology Co., Ltd., Beijing, China). PBS was used to replace primary antibody as the negative control, observing by light microscopy, the claybank cytoplasm was positive. Based on the expression of CD34, three sections were selected in each group, to observe and count the vessel numbers by using FSX100 biological image navigator (Olympus Corporation, Tokyo, Japan) at ×100 magnification. The images were taken from five directions for each section, microvessel density was calculated by adopting the Pareek method. The results were expressed as one/horizon.

The expression levels of heat shock protein 90 (HSP90) were compared at 7 and 14 days of treatment. For western blotting, the wound was extracted with conventional method - total protein in wounded tissues of healing tissue interface. SDS-PAGE electrophoresis was performed and proteins were transferred to the membrane. The membrane was blocked with 50 g/l skim milk for 1 h, the primary antibodies were polyclonal rabbit anti-human HSP90 antibody (dilution, 1:250; cat. no. BA1638) and rabbit polyclonal β-actin antibody (dilution, 1:400; cat. no. BA2305), and the secondary antibodies were goat anti-rabbit antibody (dilution, 1:5,000; cat. no. BA1039) (all from Wuhan Boster Biological Technology, Ltd., Wuhan, China) labeled by horseradish peroxidase. After staining with DAB (Beijing Zhongshan Biotechnology Co., Ltd.), the grayscale intensity of bands were measured by using Bandscan analysis software for semi-quantitative comparative analysis. The grayscale values were used for correction, and the band grayscale of target gene and β-actin grayscale rate was used for expression of proteins.

Statistical analysis

SPSS 19.0 statistical software (IBM, Armonk, NY, USA) was used to analyze data, mean ± standard deviation was used to indicate quantitative data, and one-way ANOVA for the comparisons among groups. The case number or percentage was used to indicate qualitative data, and χ2 test was used for the comparisons among groups. The difference among values with P<0.05 was considered to indicate a statistically significant difference.

Results

Comparisons of the healing states of wounds

In the 7 and 14 day combination group, maturity of granulation tissues and growth degree of epithelium were significantly higher than in the other two groups, the area of the ulcer was reduced and the healing rate increased significantly (P<0.05) (Table I).

Table I.

The comparisons of the healing states of wounds.

	After treatment for 7 days				After treatment for 14 days
Groups	Maturity of granulation tissues (%)	Growth degree of epithelium (%)	Areas of ulcer (cm2)	Healing rate, cases (%)	Maturity of granulation tissues	Growth degree of epithelium	Areas of ulcer	Healing rate
Conventional treatment (n=20)	27.4±5.3	1.5±0.3	7.2±2.4	3 (15.0)	43.8±7.2	5.4±0.9	5.3±1.4	9 (45.0)
NPWT (n=22)	33.9±6.6	3.3±0.8	6.6±1.3	6 (27.3)	61.6±7.4	9.6±1.3	3.7±0.7	15 (68.2)
Combination (n=22)	46.5±7.2	7.6±1.1	4.3±1.2	11 (50.0)	72.7±8.3	23.3±4.2	0.8±0.2	19 (86.4)
F-value (χ2)	5.624	5.768	5.832	6.220	5.937	6.302	6.425	8.145
P-value	0.026	0.025	0.024	0.045	0.024	0.017	0.015	0.017

NPWT, negative pressure wound therapy.

Comparisons of blood perfusion of wounds and density of new vessels

Before treatment, comparison of blood perfusion in wounds had no statistical significance (P>0.05). In the 7 and 14 day combination group, blood perfusion and density of new vessels were significantly higher than other in the other two groups (P<0.05). After treating for 14 days, vessels of wounds in the combination group and the NPWT group were arranged well and uterine cavities were large, while vessels in the conventional treatment group were arranged irregularly and the uterine cavities were tortuous (Table II and Figs. 1 and 2).

Table II.

Comparisons of blood perfusion of wounds and density of new vessels.

	Blood perfusion (PU)		Density of vessels (one/horizon)
Groups	Day 0	Day 7	Day 14	Day 7	Day 14
Conventional treatment	146.8±32.3	223.6±42.2	253.4±54.7	12.3±3.4	26.4±5.6
NPWT group	143.5±36.5	254.8±46.7	288.6±56.5	15.6±3.8	38.7±5.7
Combination group	142.7±37.4	297.3±52.3	343.5±63.2	19.2±4.4	52.3±5.9
F-value	0.634	5.327	5.956	5.768	6.421
P-value	0.428	0.035	0.024	0.025	0.015

NPWT, negative pressure wound therapy; PU, perfusion units.

Figure 1.

Blood perfusion of the wounds in each of the three groups.

Figure 2.

CD34 immunohistochemical staining of the wound in each of the three groups (x200).

Comparison of the expression of HSP90 in wound tissues

In the 7 and 14 day combination group, the expression of HSP90 was significantly higher than in the other two groups (P<0.05) (Fig. 3).

Figure 3.

HSP90 protein expression of the wound tissues in each of the three groups was detected by western blotting. HSP90, heat shock protein 90.

Discussion

The wound was prepared by removing necrotic tissue, controlling wound inflammation effect and infection, reducing wound exudation and stimulating the granulation tissues of the wound. Moreover, the microenvironment of wound was improved for increasing treatment efficiency for wound and promoting wound healing (5). The wound healing progresses in stages, the inflammatory reaction phase, proliferating phase of cells and tissues, remodeling phase of tissue structure and function (6). The wound forming abundant new vessels and with fine functional status was the foundation of survival in free flap grafting and skin flap transplantation operations (7). Wet dressing is the most common handling method after clinical debridement, frequent wound dressing may stimulate the wound, consuming a long time with poor effect, and increase the suffering of the patients. For some complex wounds, such as tendon or bone exposed wounds, wet dressing often could not foster ideal granulation tissue and results in delayed healing and increased infection probability (8).

Mechanisms of NPWT includes the following steps. First, cover the wound surface keeping the wound wet. NPWT utilizes semi-permeable membrane to cover the wound, for prevention of any infection. The function of combining semipermeable membrane and polymer foam dressings was similar in the function of skin dressing which can keep the wound wet, to keep local environment close to physiological environment. It is conducive to wound re-epithelialization, maintain sustain growth factor activity of the wound, stimulate potency of wound healing and create a favorable condition for wound healing (9). The second step is to mitigate wound edema. An observational study on NPWT was applied for the treatment of hands in burn patients (n=7) in comparison with traditional dressing, NPWT largely reduced wound exudate and shorten the required time of tissue edema regression (10). It was found that in the study of defect wound of the pigs back as the model, in comparison to the control group, NPWT can significantly reduce the edema of peripheral part of the wound, narrow the gap between skin-grafting of the wound and the base of the wound for earlier recovery (11). The third step, increasing blood flow volume of the wounds. It has been shown in wound model of the pigs back, with Doppler technology to determine the influence of −25 to −400 mmHg negative pressure to blood flow volume of wound (12). The result was in a certain pressure range, blood perfusion volume of the wound was positively associated with pressure (12). The changes in blood flow volume of the wound of the pigs back were assessed using different detection methods (heat diffusion method, by Piduopule testing and invasive Doppler detection) showing that negative pressure can increase the blood flow volume at a margin of wound for 2.5 cm, and the blood flow volume would increase gradually with the increasing of negative pressure and reach a maximum during −80 to −120 mmHg, but the blood flow volume away from the wound margin of 0.5 cm gradually decreased with increasing pressure (13). The fourth step is cleaning the wound and reducing bacterial colonization of the wound. Taking the pigs back wound as the model, bacterial enumeration in wounds of NPWT treatment group was significantly reduced in the 4th and 5th day, while bacterial colonization volume of the wound in control group was significantly increased at the same time-point (12). A prospective study of thoracic surgery showed that NPWT after thoracotomy in the wound treatment of patients can significantly reduce the occurrence of infection rate of deep tissues (14). The fifth step is the effect of mechanical stress promoting the proliferation of wound cells. It the in vitro experiments, NPWT through foam dressing can apply positive pressure to the wound, and the size of pressure was positively correlated with negative pressure (15). The view that in promoting wound healing mechanism, NPWT may have mechanical stress effect has been gradually recognized. Through the interaction between extracellular matrix and cytoskeleton, mechanical stress can transfer the outer stress of cells into the cells through cytomembrane and cytoskeleton, then motivate intracellular signaling pathways, promote cytokine expression, and ultimately lead to cell proliferation and matrix synthesis (16). Taking the full-thickness dermal wounds of diabetic mice as a model (17), Ki67 was used to detect proliferation activity of cells in the wound tissue, it was been found that Ki67 expression volume of NPWT group was significantly higher than the control group. In addition, another study established VAC for the treatment of computer digital model of wound to discuss the treatment mechanism of VSD. It was considered that negative pressure may intermittently influence the cytoskeleton, activate the intracellular signaling pathways, prompt the release of second messenger substances in cells and induce repair of the cell proliferation, thereby promoting wound healing. The sixth step is the influence on tissue factors of the wound (18). During the healing process, the expression regulation of tissue factors is an important factor. A study has suggested that NPWT can significantly increase the expression of HSP90, while HSP90 is the important regulatory factor in inducing angiogenesis (19).

The core technology of microplasma is the unique dual-stage thermal effects by microplasma peeling off the surface-abnormal skin. It activates the wound repair mechanisms of skin by proliferation, migration and differentiation of epidermis cell of surrounding normal skin, epidermis would be repaired so as to help the lesion site damage close to the normal skin. The treatment was taken by stimulating the neogenesis of collagen and tissue remodeling. Heat effect generated by unipolar ions can not only stimulate fibroblast cells to create new collagen fibers and matrix, but also make disorganized collagen fibers in the tissue rearranged to achieve the effect of tissue remodeling (20).

In conclusion, we found that NPWT and microplasma can significantly improve insertion status of chronic ulcer wounds, and this is related to increased expression of HSP90.