Histopathological study of chronic wounds modulated by intermittent negative pressure therapy under limited access dressing

Introduction

Negative pressure wound therapy (NPWT) is a bio-stimulating treatment for chronic wounds. In recent years, it has become one of the most frequently adopted treatments for complex wounds worldwide. While it is generally believed that NPWT improves wound healing and is cost-effective, there is a persistent demand for scientific evidence.

Several recent studies support the use of NPWT to enhance wound healing by removing excess extracellular fluid and decreasing tissue edema, which leads to increased blood flow^[1] and stabilization of the wound environment. A reduction in systemic (e.g. interleukins, monocytes) and local mediators of inflammation has been demonstrated in experimental models,^[2] while decreased matrix metalloproteinase activity^[3] and bacterial burden have been documented clinically.^[4]In vivo, NPWT has been shown to increase fibroblast proliferation and migration, collagen organization, and to increase the expression of vascular endothelial growth factor and fibroblast growth factor-2, thereby enhancing wound healing.^[5]

Limited access dressings (LADs) have been shown to be more effective in the treatment of chronic wounds.^[6] The present study evaluates the role of intermittent negative pressure in a moist environment using LAD (cycle of 30-min suction and 3.5-h rest) on chronic wounds by quantitative and comparative analysis of histopathological parameters including the amount of necrotic tissue, the number of inflammatory cells, extent of angiogenesis, and the level of extracellular matrix (ECM) deposit.

Methods

Clinical study design

Two hundred and fifteen patients with chronic wounds of more than 4 weeks duration were enrolled and assessed for eligibility. After examination of inclusion criteria (nonhealing chronic wounds, 12-65 years of age) and exclusion criteria (collagen disorders, diabetes, leprosy, liver cirrhosis, HIV positive status, and pregnant women), 140 patients were randomized into the LAD group (n = 64) or conventional dressing group (n = 76) [Figure1]. In the LAD group, the mean age was 38.3 ± 10.56 years(range: 12-60 years), with a mean wound size at the time of admission of 19 cm² (range: 28-40 cm²). In the conventional dressing group, the mean age was 35.3 ± 14.0 years (range: 17-65 years) with a mean wound size at the time of admission of 18 cm² (range: 26-39 cm²) [Table1].

Figure 1. Consort flow chart

Table 1

Patient demographics and baseline wound characterization

Details	LAD group	Conventional group
Number of patients	42	42
Age, years (mean ± SD, range)	(38.3 ± 10.56, 12-60)	(35.3 ± 14.0, 17-65)
Mean wound size (cm2)	28 (range: 19-40)	26 (range: 18-39)
Female (%)	52	42.8
Male (%)	48	57.1

LAD: Limited access dressing, SD: Standard deviation

The LAD group was treated with intermittent negative pressure in a moist environment. The conventional closed dressing group was treated with daily dressing changes using a squeezed 5% povidone-iodine gauze which is a routine protocol in our burn unit.^[7]

Wounds were washed daily both the LAD group and the conventional group prior to dressing with 5% povidone-iodine solution. Of these 140 patients, 56 participants (22 in the LAD group and 34 in the conventional dressing group) were lost to follow-up. Of 42 patients in the LAD group, 22 (52%) were women, and 20 (48%) were men. In the conventional dressing group, 18 (42.8%) were women, and 24 (57.1%) were men. Biopsies were taken on days 0 and 10 and were analyzed for the histopathologic parameters under study.

Results

On day 0, both the LAD and conventional groups showed necrotic tissue with increased inflammatory infiltrates [Figure 2A and Figure 3A]. On day 10, the LAD group [Figure 2B] showed an increase in ECM deposition and angiogenesis with a decrease in inflammatory infiltrate when compared to the conventional group [Figure 3B]. The results of the histopathologic scoring are shown in Table 2. Histopathology revealed that in LAD group after 10 days of treatment, the scores of necrotic tissue (P = 0.007) and inflammatory cell infiltrate (P = 0.018) were significantly lower than those of the conventional treatment group. The score of ECM deposits and number of blood vessels on day 0 were not well-defined in either group, but on day 10 ECM deposits (P= 0.001) and number of blood vessel (P = 0.005) were significantly higher in the LAD group than in the conventional group [Table 2, Figure 4].

Figure 2. Histopathology of granulation tissue on days 0 and 10 in the limited access dressing (LAD) group. A: (a) LAD group - day 0 - (arrow) abundant inflammatory cells, (b) poorly developed matrix, (c) minimal blood capillaries. (photographed with an Olympus PM20 photomicroscope ×20); B: (a) LAD group - day 10 - (arrow) increased number of fibroblast cells, (b) fewer inflammatory cells, (c) increased proliferating blood capillaries, (d) collagen bundles organized well between the cells (photographed with an Olympus PM20 photomicroscope ×20)

Figure 3. Histopathology of granulation tissue on days 0 and 10 in the conventional group. A: (a) Conventional group - day 0 - (arrow) abundant inflammatory cells, (b) minimal blood capillaries, (c) poor matrix (photographed with an Olympus PM20 photomicroscope ×20); B: (a) Conventional group - day 10 - (arrow) More number of fibroblast cells, (b) poorly developed matrix with a minimal number of fibroblasts, (c) minimal blood capillaries. (photographed with an Olympus PM20 photomicroscope ×20)

Figure 4. Histopathologic scores of necrotic tissue, inflammatory cells, new blood vessels, and extracellular matrix deposit in limited access dressing versus conventional groups (mean ± SE, P < 0.05)

Discussion

Wound healing is a complex and dynamic process which involves cell-cell interactions and cell-matrix interaction. The proliferative phase of wound healing is marked by angiogenesis, collagen deposition, granulation tissue formation, epithelialization, and wound contraction resulting in less scar tissue.^[10]

A study by Nain et al.^[11] showed a decrease in the amount of necrotic tissue in chronic wounds treated with NPWT. Histopathological analysis showed significantly less necrotic tissue in the LAD group compared to a conventional dressing group after 10 days of treatment (mean ± SE, 11.5 ± 0.48 vs. 10.1 ± 0.30, P = 0.007). The ECM is made up of collagen fibers and glycosaminoglycons, which form the principal component of connective tissue and play a central role in wound healing by providing a structural framework for regenerating tissue.^[12]

Borgquist et al.^[13] showed that chronic wounds treated with NPWT promote the formation of granulation tissue with an increase in ECM. After 10 days of treatment, there was a significant increase in the ECM deposit of a LAD group compared to a conventional group (mean ± SE, 13.3 ± 0.50 vs. 9.6 ± 0.24, P = 0.001).

Angiogenesis improves circulation to the wound bed by providing oxygen and essential nutrients for the healing process.^[14] Xia et al.^[15] conducted a study on chronic wounds treated with NPWT, and showed that NPWT-treated wounds had increased angiogenesis and blood flow, with the number of new blood vessels significantly higher in a LAD group when compared to a conventional dressing group (mean ± SE, 12.8 ± 0.58 vs. 9.3 ± 0.29, P = 0.005).

Huang et al.^[16] showed that NPWT promotes healing by modulating inflammation and cell migration. There were significantly fewer inflammatory cells in a LAD group as compared to a conventional dressing group (mean ± SE, 12.6 ± 0.60 vs. 8.63 ± 0.35; P = 0.018).

In conclusion, the present study demonstrates that intermittent NPWT in a moist environment with a LAD to chronic wounds accelerates the rate of wound healing by significantly increasing ECM deposits and the formation of new blood vessels while decreasing the number of inflammatory cells and the amount of necrotic tissue.