International Wound Journal ISSN 1742-4801
ORIGINAL ARTICLE
A comparative study to evaluate the effect of limited access dressing (LAD) on burn wound healing Thittamaranahalli M Honnegowda1 , Pramod Kumar2,* , Echalasara G Padmanabha Udupa3 , Anurag Sharan4 , Rekha Singh5 , Hemanth K Prasad6 & Pragna Rao7 1 Department of Plastic Surgery and Burns, Kasturba Medical College, Manipal, India 2 Department of Plastic Surgery and Burns, Kasturba Medical College, Manipal, India 3 Department of Biochemistry, Kasturba Medical College, Manipal, India 4 Department of Plastic Surgery and Burns, Kasturba Medical College, Manipal, India 5 Department of Pathology, Vydehi Institute of Medical Sciences and Research Center, Bangalore, India 6 Department of Biochemistry, Kasturba Medical College, Manipal, India 7 Department of Biochemistry, Kasturba Medical College, Manipal, India ∗ Present
address: King Abdul Aziz Specialist Hospital, Sakaka, Al-Jouf, Saudi Arabia
Key words Antioxidants (GSH, GPx); Burn wound healing; LAD; MDA; Negative pressure; Oxidative stress Correspondence to Dr P Kumar, MS, MCh, DNB, MBA Consultant Plastic Surgeon King Abdul Aziz Hospital Sakaka Al-Jouf Saudi Arabia E-mail: [email protected]
Honnegowda TM, Kumar P, Padmanabha Udupa EGP, Sharan A, Singh R, Prasad HK, Rao P. A comparative study to evaluate the effect of limited access dressing (LAD) on burn wound healing. Int Wound J 2015; doi: 10.1111/iwj.12384 Abstract Biochemical and histopathological analyses are commonly used objective parameters in research and clinical fields to assess the healing status of burn wounds. In this study, the effect of newer intermittent negative-pressure wound therapy in combination with moist environment [limited access dressing (LAD)] on burn wound healing is studied. Various biochemical parameters like hydroxyproline, hexosamine and total protein, and antioxidants like reduced glutathione (GSH), glutathione peroxidase (GPx) and oxidative biomarker malondialdhyde (MDA) were measured in the granulation tissue. Histopathologically, necrotic tissue, amount of inflammatory infiltrate, angiogenesis and extracellular matrix deposition (ECM) were studied to determine wound healing. A total of 55 patients were divided into two groups as follows: LAD group (n = 28) and conventional dressing group (n = 27). Patients treated with LAD have shown significant increase in the mean levels of (±SD) hydroxyproline (75⋅2 ± 26⋅30 versus 27⋅8 ± 15⋅5; P = 0⋅010), hexosamine (9⋅0 ± 1⋅99 versus 8⋅0 ± 1⋅18; P = 0⋅038), total protein (15⋅6 ± 8⋅23 versus 10⋅26 ± 4⋅94; P = 0⋅003), GSH (7⋅40 ± 1⋅91 versus 5⋅1 ± 1⋅28; P = 0⋅037), GPx (112⋅6 ± 46⋅4 versus 92 ± 32⋅4; P = 0⋅016), and decrease in MDA (6⋅5 ± 2⋅24 versus 1 0⋅6 ± 3⋅8; P = 0⋅002). Histopathologically, between LAD and conventional dressing groups, there was a significant difference after 10 days of treatment (mean±SE) in necrotic tissue of (LAD versus conventional dressing groups = 10 ± 1⋅8 versus 11⋅9 ± 2⋅6; P = 0⋅033), inflammatory cells (8⋅4 ± 1⋅9 versus 13 ± 3⋅46; P = 0⋅021), new blood vessels (12⋅5 ± 2⋅87 versus 9⋅4 ± 1⋅7; P = 0⋅047), ECM deposit (12⋅9 ± 2⋅41 versus 9⋅68 ± 1⋅3; P = 0⋅018) and showed comparatively fewer inflammatory cells, increased and well-organised extracellular matrix deposit, more angiogenesis in LAD group as compared with that in conventional dressing group. To conclude, LAD exerts its beneficial effects on wound healing by reducing oxidative stress, decreasing necrotic tissue and amount of inflammatory infiltrate, and increasing ECM deposition and angiogenesis.
© 2015 Medicalhelplines.com Inc and John Wiley & Sons Ltd doi: 10.1111/iwj.12384
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Effect of LAD on burn wound healing
Introduction
Deep and extensive wounds are difficult to treat, particularly in patients with multiple injuries connected with major burns (1,2). Thermal injury is accompanied by complex events that exert deleterious effects on various organs in different systems distant from the original burn wound. Although the pathophysiological mechanisms of tissue injury remain unclear, there is increasing evidence that oxidative stress has an important role in the development of multiorgan failure after thermal injury (3,4). After thermal trauma, all tissues are subjected to ischaemia, and consequently, especially during burn resuscitation, reperfusion injury occurs (5,6). Recently, several studies (5,7) suggested that oxidative stress initiates an inflammatory cascade that includes acute-phase protein synthesis, upregulation of inflammatory adhesion molecules and pro-inflammatory cytokines, such as interleukin-1 (IL-1) and tumour necrosis factor-α (TNF-α), release in ischaemia reperfusion and burn injury. This activated inflammatory cascade causes local and systemic neutrophil sequestration, which is the source of reactive oxygen species (ROS) and reactive nitrogen species (RNS) (8) and contributes to delay in burn wound healing (9). It has been shown that burn injury, associated with lipid peroxidation, mediated by ROS, is an important cause of oxidative damage to cellular membranes and eventually cell death (10). Increased plasma malondialdehyde (MDA) levels and concentrations of lipid peroxides in the systemic circulation have been described after burn trauma (11–13). Enzymatic antioxidants that include superoxide dismutase (SOD), catalase, and glutathione reductase/glutathione peroxidase (GR/GPx) system offer a defence mechanism against ROS. A non-protein sulfhydryl containing non-protein glutathione (GSH) compound is one of the major constituents of cellular defence mechanisms against oxidative stress. The intracellular GSH/glutathione disulfide (GSSG) ratio is usually kept high through the reduction of GSSG to GSH by GR, enabling GSH to act as an antioxidant (14,15). Tissue ischaemia after burn depletes intracellular antioxidant levels (16), and oxidative stress induces apoptosis in fibroblasts during wound healing (17). Despite recent advances in the management of burn care, thermal trauma is difficult to manage (18). Negative-pressure wound therapy (NPWT) is the use of subatmospheric pressure as non-invasive adjuvant therapy to treat burn wounds. It increases blood supply to the wound; induces the formation of granulation tissue; causes rapid healing by stimulating re-epithelialisation, formation of granulation tissue and proliferation of endothelial, fibroblasts cells, thereby causing angiogenesis and collagen reorganisation; decreases bacterial load; eliminates exudate and slough; reduces oedema and swelling; and maintains an optimal moist environment for good healing (19). The clinical evidence supporting the use of continuous NPWT on burn wounds has been based largely on clinician perception, case series, small cohort studies and weakly powered randomised trials that constitute a substantial number of publications but a overall less amount of evidence. Evidences are lacking on use of intermittent negative-pressure dressing that is 2
Key Messages
• we model two dressing methods which are used for burn wound dressing • we examine the changes in the level of biochemical and histological parameters for wound healing • we examine the effect of intermittent negative pressure in moist environment (LAD) on biochemical and histological parameters • increasing information about this method will increase the quality of burn wound care • increasing information will also improve the outcome
more economical and acceptable clinically (20) than continuous NPWT. This study evaluates the role of intermittent negative pressure using limited access dressing (LAD, cycle of 30-min suction and 3⋅5 hours of rest) on burn wounds by measuring and comparing the biochemical parameters [levels of hydroxyproline, hexosamine, antioxidants (GSH, GPx) and oxidative biomarker (MDA)] and histopathological analysis of amount of necrotic tissue, inflammatory cells, new blood vessels and extracellular matrix deposition (ECM) deposit in the granulation tissue of burn wound patients using LAD with those of conventional group. Materials and methods Ethical approval and informed consent
This prospective randomised study was carried out in the department of plastic surgery, Kasturba Hospital, Manipal. Institutional Ethics committee of Kasturba Medical College and Hospital, Manipal, reviewed and approved the study protocol. Informed consent was obtained from all patients or their next of kin before including them into the study. Clinical trail design
Seventy-five patients of 12–65 (mean age, 38⋅5) years ailing from burn wounds were enrolled in the study. After examining inclusion criteria (10–30% TBSA burn wounds) and exclusion criteria (patients with collagen disorders, diabetes, leprosy, liver cirrhosis and HIV-positive status and pregnant women), 55 patients were randomised of whom 27 and 28 were assigned to the LAD (n = 27) and conventional dressing (n = 28) groups, respectively, by simple randomisation (Figure 1). From the biopsies taken on day 0 from both groups, it was observed that patients of LAD group were treated with LAD with intermittent negative pressure and those of conventional closed dressing group were dressed daily with 5% povidone iodine solution-soaked gauze. In the LAD group, wounds were washed daily using povidone iodine solution. Of 55 patients, 5 participants (two in the LAD group and three in the conventional dressing group) were withdrawn from the study before biopsy was taken. In the 50 patients, biopsies were taken on day 10 and were analysed for biochemical and histopathological parameters under study. © 2015 Medicalhelplines.com Inc and John Wiley & Sons Ltd
T. M. Honnegowda et al.
Effect of LAD on burn wound healing
Figure 1 Consort flow chart.
Randomisation
On simple randomisation, patients were randomised by generating tables of random numbers through www.random.org. Numbers were assigned to each treatment group and sealed in opaque envelopes containing labelled paper with treatment and patient ID. Chemicals
The following chemicals were purchased from Sigma Aldrich Chemical Company (Bangalore, India): standard glutathione, l-chloro-2,4-dinitrobenzene, nictoinamide adeninedinucleotide phosphate (reduced form), GR (type III, Baker’s yeast), cumene hydrogen peroxide, thiobarbituric acid, trichloroaceticacid, 1,1,3,3-tetramethoxypropane (malonaldehyde-bis-dimethylacetal) and all inorganic salts and organic reagents, graded alcohol, haematoxylin and eosin stain.
to a concentration of 20 mg/ml of dried granulation tissue in the final hydrolysate with distilled water. The hydrolysate was used for the estimation of hydroxyproline and hexosamine levels. Tissue preparation for estimation of lipid peroxidation and antioxidant
Wet weight of granulation tissue samples was noted and they were homogenised using Rotex homogenizer in ice-cold 0⋅2 M phosphate buffer (pH 7⋅4). Homogenates were centrifuged at 15.500 g for 30 minutes in cooling centrifuge, and supernatant was then used to determine total protein, lipid peroxidation, GSH and GPX activities. Protein concentration was determined according to Lowry et al. (21) using purified bovine serum albumin as a standard. Biochemical study parameters
Hydroxyproline Estimation of biochemical parameters
Tissue preparation for estimation of levels of hydroxyproline and hexosamine The granulation tissues were dried at 60∘ C for 24 hours. It was
weighed and kept in glass-stopper test tubes; 6 N HCl was added to each tube so that it contained 40 mg of the dried granulation tissue per millilitre of acid. The tubes were kept on boiling water bath for 24 hours (12 hours each for 2 days) for hydrolysis. The hydrolysate was then cooled, and excess of acid was neutralized by 10 N NaOH using phenolphthalein/methyl red as an indicator. The volume of neutral hydrolysate was diluted © 2015 Medicalhelplines.com Inc and John Wiley & Sons Ltd
The neutralised acid hydrolysate of the dry tissue was used for the determination of hydroxyproline levels. The reaction mixture contains 0⋅05 M copper sulphate, 2⋅5 N sodium hydroxide, 6% hydrogen peroxide, 3 N sulphuric acid, and 5% p-dimethylaminobenzaldehyde using l-hydroxyproline as a standard. The absorbance was measured at 540 nm and expressed in μg/mg as dry tissue weight (22). Hexosamine
The hexosamine content of granulation tissues was estimated as described previously with minor modifications (23). The 3
T. M. Honnegowda et al.
Effect of LAD on burn wound healing
diluted solution was mixed with acetylacetone solution and heated to 96∘ C for 40 minutes. The mixture was cooled, and 95% ethanol was added, followed by the addition of p-dimethyl aminobenzaldehyde solution (Ehrlich’s reagent). The solution was thoroughly mixed and kept at room temperature for 1 hour, and the absorbance was measured at 540 nm. The amount of hexosamine was determined via comparisons with a standard curve and expressed in μg/mg as dry tissue weight. Total protein
Total protein content of the tissue homogenate was estimated using the method of Lowry et al. The absorbance was measured at 540 nm and expressed in mg/g of tissue. Standards were treated similarly using bovine serum albumin (BSA) at concentrations of 0, 20, 40, 60, 80, and 100 μg/ml in 0⋅1 M phosphate buffer at pH 7⋅4 (21). Reduced glutathione
To the assay system, 50-μl tissue supernatant was mixed with 0⋅2 M sodium phosphate buffer of pH 7⋅00 and 10 mM 5, 5′ -dithiobis 2-nitrobenzoic acid (DTNB) solution. The yellow-coloured substance formed as a result of the reaction between GSH and DTNB was measured at 412 nm. The results were expressed as GSH μg/g tissue weight (24). Glutathione peroxidase
GPx activity was determined by using cumene hydrogen peroxide as a substrate. The standard assay mixture contained in 1⋅0 ml final volume: 50 mM/l Tris buffer, pH 7⋅4; 0⋅14 mM/l β nictoinamide adenine dinucleotide phosphate (reduced form, NADPH); 1⋅0 mM/l glutathione; 1⋅0 kU/l GR; 0⋅5 mM/l cumene hydrogen peroxide; and a rate-limiting amount of glutathione peroxidase (tissue homogenate supernatant fraction). For determining the non-enzymatic GPx activity, the value obtained was subtracted from the rate observed in the presence of GPx activity. One unit of GPx activity is expressed as the amount of enzyme required to oxidise 1⋅0 μM/minute of NADPH under the assay conditions (25).
embedded (Leica EG1150 H) in paraffin wax (m.p. 56∘ C). Serial sections of 5-μm thickness were cut using microtome (Leica RM2255) and stained using haematoxylin-eosin. Each section of each wound was examined using light microscopy. The areas of the wounds were evaluated in all sections in eight microscopic fields (100× magnification) by two observers blinded to the experimental protocol. The means ± standard errors were calculated. Wound granulation tissue was graded in a blind manner using a modified 0 to 4 Ehrlich and Hunt numerical scale, modified and internally validated in our laboratory, with 1 representing necrosis; 2, inflammatory cell infiltration (white blood cell and fibroblast count); and 3, ECM deposition. A five-point scale was used to evaluate necrosis and inflammatory cell infiltration (0, no evidence; 1, occasional evidence; 2, light scattering; 3, abundant evidence; and 4, confluent cell) as previously described (27). A four-point scale was also used to evaluate ECM deposition (0, no evidence; 1, little ECM deposition; 2, moderate ECM deposition; and 3, confluent ECM deposition) as previously described (27).
Statistical analysis
Statistical analysis for biochemical parameters was performed by Student’s t-test and data were expressed as mean ± standard deviation (SD). For histopathological study, each result between the groups was compared by Mann–Whitney U-test using the SPSS software (15th version package). The data were expressed as mean ± standard error (SE). A P value of < 0⋅05 was considered as significant. When appropriate, statistical uncertainty was expressed by 95% confidence levels.
Results Biochemical parameters
Comparison of the results of hydroxyproline, hexosamine, total protein, GSH, GPx and MDA in granulation tissues of burn wound patients in LAD group with those in conventional dressing group is shown in Table 1.
Lipid peroxidation
MDA, a measure of lipid peroxidation, was measured spectrophotometrically in homogenate, according to the method of Ohkawa et al. (26). MDA levels were determined by adding 1 ml of trichloroacetic acid (TCA, 10%) to 1 ml of thiobarbituric acid (TBA, 0⋅67%) ⋅and then heating the mixture in a boiling water bath at 100∘ C for 30 minutes. It was then cooled under tap water and centrifuged at 12.200 g for 10 minutes. Thiobarbituric acid reactive substances (TBARS) were determined by measuring the absorbance level at 535 nm and were expressed as nM/mg protein (26).
Hydroxyproline
LAD group has significantly high hydroxyproline level (75⋅2 ± 26⋅30 μg/mg dry tissue weight) than the conventional group (27⋅8 ± 15⋅5 μg/mg dry tissue weight) (P = 0⋅010). Hexosamine
LAD group has significantly high hydroxyproline level (9⋅0 ± 1⋅99 μg/mg dry tissue weight) than the conventional group (8⋅0 ± 1⋅18 μg/mg dry tissue weight) (P = 0⋅038).
Tissue preparation for histology
Total protein
Wound biopsies on days 0 and 10 were collected, fixed in 10% buffered formalin, dehydrated through graded alcohol series (50%, 70%, 90% and 100%), cleared in xylene and then
LAD group has high total protein level (15⋅6 ± 8⋅23 mg/g wet tissue weight) than the conventional group (10⋅26 ± 4⋅94 mg/g wet tissue weight) (P = 0⋅003).
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Effect of LAD on burn wound healing
Table 1 Levels of hydroxyproline, total protein, MDA, GSH, GPX in granulation tissue of burn wound in LAD and conventional dressing groups
Parameters
Hydroxyproline (μg/mg of dry weight of tissue) Hexosamine (μg/mg of dry weight of tissue) Total protein (mg/g of wet weight of tissue) GSH (μg/mg protein) GPx ( μM NADPH oxidised/minute/mg protein) MDA (nM/mg protein)
Conventional dressing group (n = 28) (mean ± SD)
LAD group (n = 32) (mean ± SD)
P value
Day 0
Day 10
Days (0–10)
Day 0
Day 10
Days (0–10)
61⋅7 ± 11⋅7
136⋅9 ± 24⋅2
75⋅2 ± 26⋅30
69⋅8 ± 9⋅7
97⋅6 ± 17⋅2
27⋅8 ± 15⋅5
0⋅010
6⋅0 ± 1⋅4
15⋅0 ± 2⋅4
9⋅0 ± 1⋅99
6⋅64 ± 1⋅56
14⋅64 ± 2⋅7
8⋅0 ± 1⋅18
0⋅038
10⋅2 ± 2⋅9
25⋅8 ± 7⋅2
15⋅6 ± 8⋅23
11⋅74 ± 2⋅6
22⋅0 ± 5⋅0
10⋅26 ± 4⋅94
0⋅003
15⋅1 ± 4⋅1 261⋅0 ± 64⋅1
22⋅5 ± 3⋅1 373⋅6 ± 65⋅4
7⋅40 ± 1⋅91 112⋅6 ± 46⋅4
15⋅8 ± 3⋅41 251⋅1 ± 76⋅0
20⋅9 ± 4⋅01 343⋅1 ± 78⋅6
5⋅1 ± 1⋅28 92 ± 32⋅4
0⋅037 0⋅016
19⋅3 ± 6⋅66
12⋅8 ± 6⋅62
6⋅5 ± 2⋅24
17⋅2 ± 5⋅6
6⋅6 ± 3⋅7
10⋅6 ± 3⋅8
0⋅002
GSH, glutathione; GPx, glutathione peroxidase; MDA, malondialdehyde.
Figure 2 Histopathology of 0- and 10-day-old granulation tissue in limited access dressing (LAD) group. A) Pre-LAD (day 0, arrow): numerous neutrophils infiltration (a), minimum number of fibroblasts (b), fewer collagen fibres (c). B) Post-LAD (day 10, arrow): maximum number of fibroblasts (a), fewer inflammatory cells (b), more proliferating blood capillaries (neovascularisation) (c), collagen bundles organised well between the cells (d). (Photographed with Olympus PM20 photomicroscope 20× magnification.)
Reduced glutathione
Histopathological study
LAD group has high GSH level (7⋅40 ± 1⋅91 μg/mg protein) than the conventional group (5⋅1 ± 1⋅28 μg/mg protein) (P = 0⋅037).
Histological scoring of day-0 and day-10 biopsies of granulation tissue was analysed. On day 0 of treatment LAD and conventional groups showed a necrotic tissue with increased cellular infiltration rate (Figures 2A and 3A). On day 10, LAD group (Figure 2B) showed an increase in epithelialisation and ECM deposition; decreased cellular infiltration rate and increased angiogenesis than that of the conventional dressing group were also observed (Figure 3B). The results of histological grading scale on day 0 (before treatment) and day 10 are shown in Table 2. Histological evaluation of the wound region showed that the necrotic tissue score on day 10 was significantly lower in the LAD group (P = 0⋅033). The inflammatory cell infiltration score on day 10 was significantly lower in the LAD group (P = 0⋅021) than in conventional dressing group. The ECM deposits on day 0 were not well defined in both groups, but on day 10 they were significantly higher in the LAD group (P = 0⋅018). The number of blood vessels on day 10 was significantly higher in the LAD group compared with that of conventional dressing group (P = 0⋅047).
Glutathione peroxidase
LAD group exhibit high activity of GPx (112⋅3 ± 46⋅4 μM/ minute/mg protein) than that of conventional group (92 ± 32⋅4 μM/minute/mg protein) (P = 0⋅016). Lipid peroxidation (MDA)
MDA was significantly high in the conventional dressing group (10⋅6 ± 3⋅8 nM/mg protein) compared with that of LAD group (6⋅5 ± 2⋅24 nM/mg protein) (P = 0⋅002). The data in the present study showed that in the LAD group there is significant increase in the levels of hydroxyproline, hexosamine, total protein, reduced GSH and GPx (P < 0⋅05) and decrease in the lipid peroxidation byproduct MDA as compared with those of conventional group (Table 1). © 2015 Medicalhelplines.com Inc and John Wiley & Sons Ltd
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Effect of LAD on burn wound healing
Figure 3 Histopathology of 0- and 10-day-old granulation tissue in the conventional group. A) Preconventional (day 0 arrow): numerous neutrophils infiltration (a), minimum number of fibroblasts (b), poor collagen fibres (c). B) Post-conventional (day 10, arrow): numerous neutrophils infiltration (a), poorly developed matrix, minimum number of fibroblasts (b), poor collagen bundles (c). (Photographed with Olympus PM20 photomicroscope 20× magnification.) Table 2 Histological grading score* Parameters
Necrotic tissue Inflammatory cells New blood vessels ECM deposit
LAD group (n = 25) (mean ± SE)
Conventional dressing group (n = 25) (mean ± SE)
Day 0
Day 10
Day (0–10)
Day 0
Day 10
Day (0–10)
24 ± 3⋅8 18 ± 2⋅2 11 ± 3⋅7 9⋅1 ± 2⋅2
14 ± 4⋅7 9⋅6 ± 3⋅3 16 ± 3⋅2 22 ± 3⋅3
10 ± 1⋅8 8⋅4 ± 1⋅9 12⋅5 ± 2⋅87 12⋅9 ± 2⋅41
25⋅9 ± 2⋅2 26 ± 2⋅7 8 ± 2⋅9 8⋅32 ± 2⋅9
14 ± 1⋅3 13 ± 2⋅9 12 ± 3⋅5 18 ± 4⋅4
11⋅9 ± 2⋅6 13 ± 3⋅46 9⋅4 ± 1⋅7 9⋅68 ± 1⋅3
P value
0⋅033 0⋅021 0⋅047 0⋅018
ECM, extracellular matrix deposition. *Histological scale: 0, no evidence; 1, occasional evidence; 2, light scattering; 3, abundant evidence; and 4, confluent cell or fibres. Each parameter was assessed individually in the eight microscopic fields per slide. The scores of eight microscopic fields were totaled, and the mean ± standard errors were calculated.
Discussion
Thermal burn initiates systemic inflammatory reactions producing burn toxins, oxygen radicals and finally leads to peroxidation. The relationship between the amount of oxidative metabolism products and the natural scavengers of free radicals determines the outcome of tissue damage (28). This can be determined by assessing the levels of enzymatic antioxidants namely SOD, catalase and GPx/GST. The study on lipid peroxidation product and the MDA gives us the extent of free radical damage in the cells (29). The formation of well-vascularised granulation tissue in the wound bed is a prerequisite for burn wound healing (30). Granulation tissue is composed of fibroblasts, collagen, edema and new small blood vessels. Extracellular matrix of granulation tissue contains mostly collagen which assists in wound healing and plays an important role in homeostasis (31,32). Collagen not only confers strength and integrity to the tissue matrix, but also plays an important role in homeostasis and epithelialisation during wound healing. Collagen is composed of hydroxyproline, an amino acid that has been used as a biochemical marker for tissue collagen (32). Various studies on human wound models have shown that dressing techniques such as moist wound dressing (33) and 6
continuous NPWT (34) increased the level of hydroxyproline in wound granulation tissue. In this study, the mean (±SD) hydroxyproline level in the LAD group was significantly higher (77⋅32 ± 30⋅19 μg/mg of dry weight of tissue) than that of conventional dressing group (32⋅33 ± 16⋅18) (P = 0⋅026). Hexosamine is an important part of the extracellular matrix and one of the main glycosaminoglycans secreted during tissue repair. Increased hexosamine content reflects the stabilisation of collagen molecules by enhancing electrostatic and ionic interactions with it, which in turn reflects remodelling of the new extracellular matrix produced (35). Hence, enhanced synthesis of hydroxyproline and hexosamine provides strength to repaired tissue and stimulates healing. In this study, the mean hexosamine (±SD) in LAD versus conventional dressing groups was 9⋅0 ± 1⋅99 versus 8⋅0 ± 1⋅18; P = 0⋅038. Protein is another important constituent of extracellular matrix. High protein content confirms positive effects towards cellular growth and proliferation, granulation tissue formation and epithelisation (36). In this study, in the LAD group, mean (±SD), total protein was 15⋅6 ± 8⋅23 which was significantly higher than that of the conventional dressing group (10⋅26 ± 4⋅94 mg/g wet tissue weight) (P = 0⋅003). © 2015 Medicalhelplines.com Inc and John Wiley & Sons Ltd
T. M. Honnegowda et al.
GSH (tripeptide) and GPx, which are normally present at high concentrations intracellular, constitute the major reducing capacity of the cytoplasm and protect the cellular system against the toxic effects of lipid peroxidation (37). In this study, mean (±SD) GSH level (7⋅40 ± 1⋅91 μg/mg protein) in the LAD group was higher than that of the conventional dressing group (5⋅1 ± 1⋅28 μg/mg protein; P = 0⋅037). In the LAD group, mean GPx (±SD) level was significantly higher (112⋅3 ± 46⋅4 μM/minutes/mg protein) than that of the conventional dressing group (92 ± 32⋅4 μM/minutes/mg protein) (P = 0⋅016). MDA is produced during the attack by free radicals to membrane lipoproteins and polyunsaturated fatty acids (38). In this study, MDA was significantly high in the conventional dressing group (10⋅6 ± 3⋅8 nM/mg protein) compared with that in LAD group (6⋅5 ± 2⋅24 nM/mg protein) (P = 0⋅002). Results of histopathological analysis after 10 days of treatment showing mean ± SE of necrotic tissue (LAD versus conventional dressing group = 10 ± 1⋅8 versus 11⋅9 ± 2⋅6; P = 0⋅033), inflammatory cells (8⋅4 ± 1⋅9 versus 13 ± 3⋅46; P = 0⋅021), new blood vessels (12⋅5 ± 2⋅87 versus 9⋅4 ± 1⋅7; P = 0⋅047), ECM deposit (12⋅9 ± 2⋅41 versus 9⋅68 ± 1⋅3; P = 0⋅018) (Table 2) indicated that there was increase in epithelialisation, ECM deposition and angiogenesis and decrease in cellular infiltration in the LAD group than in the conventional dressing group. Conclusion
LAD exerts its beneficial effects on burn wound healing by significantly reducing oxidative stress, decreasing necrotic tissue score and amount of inflammatory infiltrate, and increasing ECM deposition and angiogenesis than the conventional dressings.
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© 2015 Medicalhelplines.com Inc and John Wiley & Sons Ltd
Effect of LAD on burn wound healing
10. Cetinkale O, Senel O, Bulan R. The effect of antioxidant therapy on cell-mediated immunity following burn injury in an animal model. Burns 1999;25:113–8. 11. Daryani R, LaLonde C, Zhu D, Weidner M, Knox J, Demling RH. Effect of endotoxin and a burn injury on lung and liver lipid peroxidation and catalase activity. J Trauma 1990;30:1330–4. 12. Demling RH, Lalonde C. Systemic lipid peroxidation and inflammation induced by thermal injury persists into the post-resuscitation period. J Trauma 1990;30:69–74. 13. Demling R, Ikegami K, Lalonde C. Increased lipid peroxidation and decreased antioxidant activity correspond with death after smoke exposure in the rat. J Burn Care Rehabil 1995;16:104–10. 14. Schafer FQ, Buettner GR. Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple. Free Radic Biol Med 2001;30:1191–212. 15. Jacob C, Giles GI, Giles NM, Sies H. Sulfur and selenium: the role of oxidation state in protein structure and function. Angew Chem Int Ed Engl 2003;42:4742–58. 16. Sener G, Sehirli AO, Satiro˘glu H, Keyer-Uysal M, Ye˘gen BC. Melatonin improves oxidative organ damage in a rat model of thermal injury. Burns 2002;28:419–25. 17. Takahashi A, Aoshiba K, Nagai A. Apoptosis of wound fibroblasts induced by oxidative stress. Exp Lung Res 2002;28:275–84. 18. Church D, Elsayed S, Reid O, Winston B, Lindsay R. Burn wound infections. Clin Microbiol Rev 2006;19:403–34. 19. González Alaña I, Torrero López JV, Martín Playá P, Gabilondo Zubizarreta FJ. Combined use of negative pressure wound therapy and Integra® to treat complex defects in lower extremities after burns. Ann Burns Fire Disasters 2013;26:90–3. 20. Kumar P. Exploiting potency of negative pressure in wound dressing using limited access dressing and suction-assisted dressing. Indian J Plast Surg 2012;45:302–15. 21. Lowry OH, Rosebrough MH, Farr L, Randell RJ. Protein measurement with the folin-phenol reagent. J Biol Chem 1951;93:265–75. 22. Neuman RE, Logan MA. The determination of collagen and elastin in the tissues. J Biol Chem 1950;186:549–56. 23. Johansen PG, Marshall RD, Neuberger A. Carbohydrates in protein. 2. The hexose, hexosamine, acetyl and amide-nitrogen content of hen’s-egg albumin. Biochem J 1960;77:239–247. 24. Beutler E, Duron O, Kefly BM. Improved method for the determination of blood glutathione. J Lab Clin Med 1963;61:882–8. 25. Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterisation of erythrocyte glutathione peroxidase. J Lab Clin Med 1967;70:158–169. 26. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979;95: 351–58. 27. Turan M, Sarayd´yn SU, Bulut HE, Elagöz S, Cetinkaya O, Karadayi K, Canbay E, Sen M. Do vascular endothelial growth factor and basic fibroblast growth factor promote phenytoin’s wound healing effect in rat? An immuno-histochemical and histopathologic study. Dermatol Surg 2004;30:1303–9. 28. Cetinkale O, Bele A, Konukoglu D, Senyuva C, Gumustas MK, Tas T. Evaluation of lipid peroxidation and total antioxidant status in plasma of rats following thermal injury. Burns 1997;23:114–6. 29. Latha B, Babu M. The involvement of free radicals in burn injury: a review. Burns 2001;27:309–17. 30. Edraki M, Akbarzadeh A, Hosseinzadeh M, Tanideh N, Salehi A, Koohi-Hosseinabadi O. Healing effect of sea buckthorn, olive oil, and their mixture on full-thickness burn wounds. Adv Skin Wound Care 2014;27:317–23. 31. Li J, Chen J, Kirsner R. Pathophysiology of acute wound healing. Clin Dermatol 2007;25:9–18. 32. Broughton G II, Janis JE, Attinger CE. Wound healing: an overview. Plast Reconstr Surg 2006;117:1e-S–32e-S. 33. Balakrishnan B, Mohanty M, Umashankar PR, Jayakrishnan A. Evaluation of an in situ forming hydrogel wound dressing based on oxidized alginate and gelatin. Biomaterials 2005;26:6335–42. 7
Effect of LAD on burn wound healing
34. Molnar JA. Applications of negative pressure wound therapy to thermal injury. Ostomy Wound Manage 2004;50:17–9. 35. Ricard Blum S, Ruggiero F. The collagen super family: from the extracellular matrix to the cell membrane. Pathol Biol 2005;53:430–442. 36. James O, Victoria IA. Excision and incision wound healing potential of Saba florida (Benth) leaf extract in Rattus novergicus. Int J Pharm Biomed Res 2010;1:101–7.
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© 2015 Medicalhelplines.com Inc and John Wiley & Sons Ltd
ORIGINAL ARTICLE
A comparative study to evaluate the effect of limited access dressing (LAD) on burn wound healing Thittamaranahalli M Honnegowda1 , Pramod Kumar2,* , Echalasara G Padmanabha Udupa3 , Anurag Sharan4 , Rekha Singh5 , Hemanth K Prasad6 & Pragna Rao7 1 Department of Plastic Surgery and Burns, Kasturba Medical College, Manipal, India 2 Department of Plastic Surgery and Burns, Kasturba Medical College, Manipal, India 3 Department of Biochemistry, Kasturba Medical College, Manipal, India 4 Department of Plastic Surgery and Burns, Kasturba Medical College, Manipal, India 5 Department of Pathology, Vydehi Institute of Medical Sciences and Research Center, Bangalore, India 6 Department of Biochemistry, Kasturba Medical College, Manipal, India 7 Department of Biochemistry, Kasturba Medical College, Manipal, India ∗ Present
address: King Abdul Aziz Specialist Hospital, Sakaka, Al-Jouf, Saudi Arabia
Key words Antioxidants (GSH, GPx); Burn wound healing; LAD; MDA; Negative pressure; Oxidative stress Correspondence to Dr P Kumar, MS, MCh, DNB, MBA Consultant Plastic Surgeon King Abdul Aziz Hospital Sakaka Al-Jouf Saudi Arabia E-mail: [email protected]
Honnegowda TM, Kumar P, Padmanabha Udupa EGP, Sharan A, Singh R, Prasad HK, Rao P. A comparative study to evaluate the effect of limited access dressing (LAD) on burn wound healing. Int Wound J 2015; doi: 10.1111/iwj.12384 Abstract Biochemical and histopathological analyses are commonly used objective parameters in research and clinical fields to assess the healing status of burn wounds. In this study, the effect of newer intermittent negative-pressure wound therapy in combination with moist environment [limited access dressing (LAD)] on burn wound healing is studied. Various biochemical parameters like hydroxyproline, hexosamine and total protein, and antioxidants like reduced glutathione (GSH), glutathione peroxidase (GPx) and oxidative biomarker malondialdhyde (MDA) were measured in the granulation tissue. Histopathologically, necrotic tissue, amount of inflammatory infiltrate, angiogenesis and extracellular matrix deposition (ECM) were studied to determine wound healing. A total of 55 patients were divided into two groups as follows: LAD group (n = 28) and conventional dressing group (n = 27). Patients treated with LAD have shown significant increase in the mean levels of (±SD) hydroxyproline (75⋅2 ± 26⋅30 versus 27⋅8 ± 15⋅5; P = 0⋅010), hexosamine (9⋅0 ± 1⋅99 versus 8⋅0 ± 1⋅18; P = 0⋅038), total protein (15⋅6 ± 8⋅23 versus 10⋅26 ± 4⋅94; P = 0⋅003), GSH (7⋅40 ± 1⋅91 versus 5⋅1 ± 1⋅28; P = 0⋅037), GPx (112⋅6 ± 46⋅4 versus 92 ± 32⋅4; P = 0⋅016), and decrease in MDA (6⋅5 ± 2⋅24 versus 1 0⋅6 ± 3⋅8; P = 0⋅002). Histopathologically, between LAD and conventional dressing groups, there was a significant difference after 10 days of treatment (mean±SE) in necrotic tissue of (LAD versus conventional dressing groups = 10 ± 1⋅8 versus 11⋅9 ± 2⋅6; P = 0⋅033), inflammatory cells (8⋅4 ± 1⋅9 versus 13 ± 3⋅46; P = 0⋅021), new blood vessels (12⋅5 ± 2⋅87 versus 9⋅4 ± 1⋅7; P = 0⋅047), ECM deposit (12⋅9 ± 2⋅41 versus 9⋅68 ± 1⋅3; P = 0⋅018) and showed comparatively fewer inflammatory cells, increased and well-organised extracellular matrix deposit, more angiogenesis in LAD group as compared with that in conventional dressing group. To conclude, LAD exerts its beneficial effects on wound healing by reducing oxidative stress, decreasing necrotic tissue and amount of inflammatory infiltrate, and increasing ECM deposition and angiogenesis.
© 2015 Medicalhelplines.com Inc and John Wiley & Sons Ltd doi: 10.1111/iwj.12384
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Introduction
Deep and extensive wounds are difficult to treat, particularly in patients with multiple injuries connected with major burns (1,2). Thermal injury is accompanied by complex events that exert deleterious effects on various organs in different systems distant from the original burn wound. Although the pathophysiological mechanisms of tissue injury remain unclear, there is increasing evidence that oxidative stress has an important role in the development of multiorgan failure after thermal injury (3,4). After thermal trauma, all tissues are subjected to ischaemia, and consequently, especially during burn resuscitation, reperfusion injury occurs (5,6). Recently, several studies (5,7) suggested that oxidative stress initiates an inflammatory cascade that includes acute-phase protein synthesis, upregulation of inflammatory adhesion molecules and pro-inflammatory cytokines, such as interleukin-1 (IL-1) and tumour necrosis factor-α (TNF-α), release in ischaemia reperfusion and burn injury. This activated inflammatory cascade causes local and systemic neutrophil sequestration, which is the source of reactive oxygen species (ROS) and reactive nitrogen species (RNS) (8) and contributes to delay in burn wound healing (9). It has been shown that burn injury, associated with lipid peroxidation, mediated by ROS, is an important cause of oxidative damage to cellular membranes and eventually cell death (10). Increased plasma malondialdehyde (MDA) levels and concentrations of lipid peroxides in the systemic circulation have been described after burn trauma (11–13). Enzymatic antioxidants that include superoxide dismutase (SOD), catalase, and glutathione reductase/glutathione peroxidase (GR/GPx) system offer a defence mechanism against ROS. A non-protein sulfhydryl containing non-protein glutathione (GSH) compound is one of the major constituents of cellular defence mechanisms against oxidative stress. The intracellular GSH/glutathione disulfide (GSSG) ratio is usually kept high through the reduction of GSSG to GSH by GR, enabling GSH to act as an antioxidant (14,15). Tissue ischaemia after burn depletes intracellular antioxidant levels (16), and oxidative stress induces apoptosis in fibroblasts during wound healing (17). Despite recent advances in the management of burn care, thermal trauma is difficult to manage (18). Negative-pressure wound therapy (NPWT) is the use of subatmospheric pressure as non-invasive adjuvant therapy to treat burn wounds. It increases blood supply to the wound; induces the formation of granulation tissue; causes rapid healing by stimulating re-epithelialisation, formation of granulation tissue and proliferation of endothelial, fibroblasts cells, thereby causing angiogenesis and collagen reorganisation; decreases bacterial load; eliminates exudate and slough; reduces oedema and swelling; and maintains an optimal moist environment for good healing (19). The clinical evidence supporting the use of continuous NPWT on burn wounds has been based largely on clinician perception, case series, small cohort studies and weakly powered randomised trials that constitute a substantial number of publications but a overall less amount of evidence. Evidences are lacking on use of intermittent negative-pressure dressing that is 2
Key Messages
• we model two dressing methods which are used for burn wound dressing • we examine the changes in the level of biochemical and histological parameters for wound healing • we examine the effect of intermittent negative pressure in moist environment (LAD) on biochemical and histological parameters • increasing information about this method will increase the quality of burn wound care • increasing information will also improve the outcome
more economical and acceptable clinically (20) than continuous NPWT. This study evaluates the role of intermittent negative pressure using limited access dressing (LAD, cycle of 30-min suction and 3⋅5 hours of rest) on burn wounds by measuring and comparing the biochemical parameters [levels of hydroxyproline, hexosamine, antioxidants (GSH, GPx) and oxidative biomarker (MDA)] and histopathological analysis of amount of necrotic tissue, inflammatory cells, new blood vessels and extracellular matrix deposition (ECM) deposit in the granulation tissue of burn wound patients using LAD with those of conventional group. Materials and methods Ethical approval and informed consent
This prospective randomised study was carried out in the department of plastic surgery, Kasturba Hospital, Manipal. Institutional Ethics committee of Kasturba Medical College and Hospital, Manipal, reviewed and approved the study protocol. Informed consent was obtained from all patients or their next of kin before including them into the study. Clinical trail design
Seventy-five patients of 12–65 (mean age, 38⋅5) years ailing from burn wounds were enrolled in the study. After examining inclusion criteria (10–30% TBSA burn wounds) and exclusion criteria (patients with collagen disorders, diabetes, leprosy, liver cirrhosis and HIV-positive status and pregnant women), 55 patients were randomised of whom 27 and 28 were assigned to the LAD (n = 27) and conventional dressing (n = 28) groups, respectively, by simple randomisation (Figure 1). From the biopsies taken on day 0 from both groups, it was observed that patients of LAD group were treated with LAD with intermittent negative pressure and those of conventional closed dressing group were dressed daily with 5% povidone iodine solution-soaked gauze. In the LAD group, wounds were washed daily using povidone iodine solution. Of 55 patients, 5 participants (two in the LAD group and three in the conventional dressing group) were withdrawn from the study before biopsy was taken. In the 50 patients, biopsies were taken on day 10 and were analysed for biochemical and histopathological parameters under study. © 2015 Medicalhelplines.com Inc and John Wiley & Sons Ltd
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Effect of LAD on burn wound healing
Figure 1 Consort flow chart.
Randomisation
On simple randomisation, patients were randomised by generating tables of random numbers through www.random.org. Numbers were assigned to each treatment group and sealed in opaque envelopes containing labelled paper with treatment and patient ID. Chemicals
The following chemicals were purchased from Sigma Aldrich Chemical Company (Bangalore, India): standard glutathione, l-chloro-2,4-dinitrobenzene, nictoinamide adeninedinucleotide phosphate (reduced form), GR (type III, Baker’s yeast), cumene hydrogen peroxide, thiobarbituric acid, trichloroaceticacid, 1,1,3,3-tetramethoxypropane (malonaldehyde-bis-dimethylacetal) and all inorganic salts and organic reagents, graded alcohol, haematoxylin and eosin stain.
to a concentration of 20 mg/ml of dried granulation tissue in the final hydrolysate with distilled water. The hydrolysate was used for the estimation of hydroxyproline and hexosamine levels. Tissue preparation for estimation of lipid peroxidation and antioxidant
Wet weight of granulation tissue samples was noted and they were homogenised using Rotex homogenizer in ice-cold 0⋅2 M phosphate buffer (pH 7⋅4). Homogenates were centrifuged at 15.500 g for 30 minutes in cooling centrifuge, and supernatant was then used to determine total protein, lipid peroxidation, GSH and GPX activities. Protein concentration was determined according to Lowry et al. (21) using purified bovine serum albumin as a standard. Biochemical study parameters
Hydroxyproline Estimation of biochemical parameters
Tissue preparation for estimation of levels of hydroxyproline and hexosamine The granulation tissues were dried at 60∘ C for 24 hours. It was
weighed and kept in glass-stopper test tubes; 6 N HCl was added to each tube so that it contained 40 mg of the dried granulation tissue per millilitre of acid. The tubes were kept on boiling water bath for 24 hours (12 hours each for 2 days) for hydrolysis. The hydrolysate was then cooled, and excess of acid was neutralized by 10 N NaOH using phenolphthalein/methyl red as an indicator. The volume of neutral hydrolysate was diluted © 2015 Medicalhelplines.com Inc and John Wiley & Sons Ltd
The neutralised acid hydrolysate of the dry tissue was used for the determination of hydroxyproline levels. The reaction mixture contains 0⋅05 M copper sulphate, 2⋅5 N sodium hydroxide, 6% hydrogen peroxide, 3 N sulphuric acid, and 5% p-dimethylaminobenzaldehyde using l-hydroxyproline as a standard. The absorbance was measured at 540 nm and expressed in μg/mg as dry tissue weight (22). Hexosamine
The hexosamine content of granulation tissues was estimated as described previously with minor modifications (23). The 3
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Effect of LAD on burn wound healing
diluted solution was mixed with acetylacetone solution and heated to 96∘ C for 40 minutes. The mixture was cooled, and 95% ethanol was added, followed by the addition of p-dimethyl aminobenzaldehyde solution (Ehrlich’s reagent). The solution was thoroughly mixed and kept at room temperature for 1 hour, and the absorbance was measured at 540 nm. The amount of hexosamine was determined via comparisons with a standard curve and expressed in μg/mg as dry tissue weight. Total protein
Total protein content of the tissue homogenate was estimated using the method of Lowry et al. The absorbance was measured at 540 nm and expressed in mg/g of tissue. Standards were treated similarly using bovine serum albumin (BSA) at concentrations of 0, 20, 40, 60, 80, and 100 μg/ml in 0⋅1 M phosphate buffer at pH 7⋅4 (21). Reduced glutathione
To the assay system, 50-μl tissue supernatant was mixed with 0⋅2 M sodium phosphate buffer of pH 7⋅00 and 10 mM 5, 5′ -dithiobis 2-nitrobenzoic acid (DTNB) solution. The yellow-coloured substance formed as a result of the reaction between GSH and DTNB was measured at 412 nm. The results were expressed as GSH μg/g tissue weight (24). Glutathione peroxidase
GPx activity was determined by using cumene hydrogen peroxide as a substrate. The standard assay mixture contained in 1⋅0 ml final volume: 50 mM/l Tris buffer, pH 7⋅4; 0⋅14 mM/l β nictoinamide adenine dinucleotide phosphate (reduced form, NADPH); 1⋅0 mM/l glutathione; 1⋅0 kU/l GR; 0⋅5 mM/l cumene hydrogen peroxide; and a rate-limiting amount of glutathione peroxidase (tissue homogenate supernatant fraction). For determining the non-enzymatic GPx activity, the value obtained was subtracted from the rate observed in the presence of GPx activity. One unit of GPx activity is expressed as the amount of enzyme required to oxidise 1⋅0 μM/minute of NADPH under the assay conditions (25).
embedded (Leica EG1150 H) in paraffin wax (m.p. 56∘ C). Serial sections of 5-μm thickness were cut using microtome (Leica RM2255) and stained using haematoxylin-eosin. Each section of each wound was examined using light microscopy. The areas of the wounds were evaluated in all sections in eight microscopic fields (100× magnification) by two observers blinded to the experimental protocol. The means ± standard errors were calculated. Wound granulation tissue was graded in a blind manner using a modified 0 to 4 Ehrlich and Hunt numerical scale, modified and internally validated in our laboratory, with 1 representing necrosis; 2, inflammatory cell infiltration (white blood cell and fibroblast count); and 3, ECM deposition. A five-point scale was used to evaluate necrosis and inflammatory cell infiltration (0, no evidence; 1, occasional evidence; 2, light scattering; 3, abundant evidence; and 4, confluent cell) as previously described (27). A four-point scale was also used to evaluate ECM deposition (0, no evidence; 1, little ECM deposition; 2, moderate ECM deposition; and 3, confluent ECM deposition) as previously described (27).
Statistical analysis
Statistical analysis for biochemical parameters was performed by Student’s t-test and data were expressed as mean ± standard deviation (SD). For histopathological study, each result between the groups was compared by Mann–Whitney U-test using the SPSS software (15th version package). The data were expressed as mean ± standard error (SE). A P value of < 0⋅05 was considered as significant. When appropriate, statistical uncertainty was expressed by 95% confidence levels.
Results Biochemical parameters
Comparison of the results of hydroxyproline, hexosamine, total protein, GSH, GPx and MDA in granulation tissues of burn wound patients in LAD group with those in conventional dressing group is shown in Table 1.
Lipid peroxidation
MDA, a measure of lipid peroxidation, was measured spectrophotometrically in homogenate, according to the method of Ohkawa et al. (26). MDA levels were determined by adding 1 ml of trichloroacetic acid (TCA, 10%) to 1 ml of thiobarbituric acid (TBA, 0⋅67%) ⋅and then heating the mixture in a boiling water bath at 100∘ C for 30 minutes. It was then cooled under tap water and centrifuged at 12.200 g for 10 minutes. Thiobarbituric acid reactive substances (TBARS) were determined by measuring the absorbance level at 535 nm and were expressed as nM/mg protein (26).
Hydroxyproline
LAD group has significantly high hydroxyproline level (75⋅2 ± 26⋅30 μg/mg dry tissue weight) than the conventional group (27⋅8 ± 15⋅5 μg/mg dry tissue weight) (P = 0⋅010). Hexosamine
LAD group has significantly high hydroxyproline level (9⋅0 ± 1⋅99 μg/mg dry tissue weight) than the conventional group (8⋅0 ± 1⋅18 μg/mg dry tissue weight) (P = 0⋅038).
Tissue preparation for histology
Total protein
Wound biopsies on days 0 and 10 were collected, fixed in 10% buffered formalin, dehydrated through graded alcohol series (50%, 70%, 90% and 100%), cleared in xylene and then
LAD group has high total protein level (15⋅6 ± 8⋅23 mg/g wet tissue weight) than the conventional group (10⋅26 ± 4⋅94 mg/g wet tissue weight) (P = 0⋅003).
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Effect of LAD on burn wound healing
Table 1 Levels of hydroxyproline, total protein, MDA, GSH, GPX in granulation tissue of burn wound in LAD and conventional dressing groups
Parameters
Hydroxyproline (μg/mg of dry weight of tissue) Hexosamine (μg/mg of dry weight of tissue) Total protein (mg/g of wet weight of tissue) GSH (μg/mg protein) GPx ( μM NADPH oxidised/minute/mg protein) MDA (nM/mg protein)
Conventional dressing group (n = 28) (mean ± SD)
LAD group (n = 32) (mean ± SD)
P value
Day 0
Day 10
Days (0–10)
Day 0
Day 10
Days (0–10)
61⋅7 ± 11⋅7
136⋅9 ± 24⋅2
75⋅2 ± 26⋅30
69⋅8 ± 9⋅7
97⋅6 ± 17⋅2
27⋅8 ± 15⋅5
0⋅010
6⋅0 ± 1⋅4
15⋅0 ± 2⋅4
9⋅0 ± 1⋅99
6⋅64 ± 1⋅56
14⋅64 ± 2⋅7
8⋅0 ± 1⋅18
0⋅038
10⋅2 ± 2⋅9
25⋅8 ± 7⋅2
15⋅6 ± 8⋅23
11⋅74 ± 2⋅6
22⋅0 ± 5⋅0
10⋅26 ± 4⋅94
0⋅003
15⋅1 ± 4⋅1 261⋅0 ± 64⋅1
22⋅5 ± 3⋅1 373⋅6 ± 65⋅4
7⋅40 ± 1⋅91 112⋅6 ± 46⋅4
15⋅8 ± 3⋅41 251⋅1 ± 76⋅0
20⋅9 ± 4⋅01 343⋅1 ± 78⋅6
5⋅1 ± 1⋅28 92 ± 32⋅4
0⋅037 0⋅016
19⋅3 ± 6⋅66
12⋅8 ± 6⋅62
6⋅5 ± 2⋅24
17⋅2 ± 5⋅6
6⋅6 ± 3⋅7
10⋅6 ± 3⋅8
0⋅002
GSH, glutathione; GPx, glutathione peroxidase; MDA, malondialdehyde.
Figure 2 Histopathology of 0- and 10-day-old granulation tissue in limited access dressing (LAD) group. A) Pre-LAD (day 0, arrow): numerous neutrophils infiltration (a), minimum number of fibroblasts (b), fewer collagen fibres (c). B) Post-LAD (day 10, arrow): maximum number of fibroblasts (a), fewer inflammatory cells (b), more proliferating blood capillaries (neovascularisation) (c), collagen bundles organised well between the cells (d). (Photographed with Olympus PM20 photomicroscope 20× magnification.)
Reduced glutathione
Histopathological study
LAD group has high GSH level (7⋅40 ± 1⋅91 μg/mg protein) than the conventional group (5⋅1 ± 1⋅28 μg/mg protein) (P = 0⋅037).
Histological scoring of day-0 and day-10 biopsies of granulation tissue was analysed. On day 0 of treatment LAD and conventional groups showed a necrotic tissue with increased cellular infiltration rate (Figures 2A and 3A). On day 10, LAD group (Figure 2B) showed an increase in epithelialisation and ECM deposition; decreased cellular infiltration rate and increased angiogenesis than that of the conventional dressing group were also observed (Figure 3B). The results of histological grading scale on day 0 (before treatment) and day 10 are shown in Table 2. Histological evaluation of the wound region showed that the necrotic tissue score on day 10 was significantly lower in the LAD group (P = 0⋅033). The inflammatory cell infiltration score on day 10 was significantly lower in the LAD group (P = 0⋅021) than in conventional dressing group. The ECM deposits on day 0 were not well defined in both groups, but on day 10 they were significantly higher in the LAD group (P = 0⋅018). The number of blood vessels on day 10 was significantly higher in the LAD group compared with that of conventional dressing group (P = 0⋅047).
Glutathione peroxidase
LAD group exhibit high activity of GPx (112⋅3 ± 46⋅4 μM/ minute/mg protein) than that of conventional group (92 ± 32⋅4 μM/minute/mg protein) (P = 0⋅016). Lipid peroxidation (MDA)
MDA was significantly high in the conventional dressing group (10⋅6 ± 3⋅8 nM/mg protein) compared with that of LAD group (6⋅5 ± 2⋅24 nM/mg protein) (P = 0⋅002). The data in the present study showed that in the LAD group there is significant increase in the levels of hydroxyproline, hexosamine, total protein, reduced GSH and GPx (P < 0⋅05) and decrease in the lipid peroxidation byproduct MDA as compared with those of conventional group (Table 1). © 2015 Medicalhelplines.com Inc and John Wiley & Sons Ltd
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Figure 3 Histopathology of 0- and 10-day-old granulation tissue in the conventional group. A) Preconventional (day 0 arrow): numerous neutrophils infiltration (a), minimum number of fibroblasts (b), poor collagen fibres (c). B) Post-conventional (day 10, arrow): numerous neutrophils infiltration (a), poorly developed matrix, minimum number of fibroblasts (b), poor collagen bundles (c). (Photographed with Olympus PM20 photomicroscope 20× magnification.) Table 2 Histological grading score* Parameters
Necrotic tissue Inflammatory cells New blood vessels ECM deposit
LAD group (n = 25) (mean ± SE)
Conventional dressing group (n = 25) (mean ± SE)
Day 0
Day 10
Day (0–10)
Day 0
Day 10
Day (0–10)
24 ± 3⋅8 18 ± 2⋅2 11 ± 3⋅7 9⋅1 ± 2⋅2
14 ± 4⋅7 9⋅6 ± 3⋅3 16 ± 3⋅2 22 ± 3⋅3
10 ± 1⋅8 8⋅4 ± 1⋅9 12⋅5 ± 2⋅87 12⋅9 ± 2⋅41
25⋅9 ± 2⋅2 26 ± 2⋅7 8 ± 2⋅9 8⋅32 ± 2⋅9
14 ± 1⋅3 13 ± 2⋅9 12 ± 3⋅5 18 ± 4⋅4
11⋅9 ± 2⋅6 13 ± 3⋅46 9⋅4 ± 1⋅7 9⋅68 ± 1⋅3
P value
0⋅033 0⋅021 0⋅047 0⋅018
ECM, extracellular matrix deposition. *Histological scale: 0, no evidence; 1, occasional evidence; 2, light scattering; 3, abundant evidence; and 4, confluent cell or fibres. Each parameter was assessed individually in the eight microscopic fields per slide. The scores of eight microscopic fields were totaled, and the mean ± standard errors were calculated.
Discussion
Thermal burn initiates systemic inflammatory reactions producing burn toxins, oxygen radicals and finally leads to peroxidation. The relationship between the amount of oxidative metabolism products and the natural scavengers of free radicals determines the outcome of tissue damage (28). This can be determined by assessing the levels of enzymatic antioxidants namely SOD, catalase and GPx/GST. The study on lipid peroxidation product and the MDA gives us the extent of free radical damage in the cells (29). The formation of well-vascularised granulation tissue in the wound bed is a prerequisite for burn wound healing (30). Granulation tissue is composed of fibroblasts, collagen, edema and new small blood vessels. Extracellular matrix of granulation tissue contains mostly collagen which assists in wound healing and plays an important role in homeostasis (31,32). Collagen not only confers strength and integrity to the tissue matrix, but also plays an important role in homeostasis and epithelialisation during wound healing. Collagen is composed of hydroxyproline, an amino acid that has been used as a biochemical marker for tissue collagen (32). Various studies on human wound models have shown that dressing techniques such as moist wound dressing (33) and 6
continuous NPWT (34) increased the level of hydroxyproline in wound granulation tissue. In this study, the mean (±SD) hydroxyproline level in the LAD group was significantly higher (77⋅32 ± 30⋅19 μg/mg of dry weight of tissue) than that of conventional dressing group (32⋅33 ± 16⋅18) (P = 0⋅026). Hexosamine is an important part of the extracellular matrix and one of the main glycosaminoglycans secreted during tissue repair. Increased hexosamine content reflects the stabilisation of collagen molecules by enhancing electrostatic and ionic interactions with it, which in turn reflects remodelling of the new extracellular matrix produced (35). Hence, enhanced synthesis of hydroxyproline and hexosamine provides strength to repaired tissue and stimulates healing. In this study, the mean hexosamine (±SD) in LAD versus conventional dressing groups was 9⋅0 ± 1⋅99 versus 8⋅0 ± 1⋅18; P = 0⋅038. Protein is another important constituent of extracellular matrix. High protein content confirms positive effects towards cellular growth and proliferation, granulation tissue formation and epithelisation (36). In this study, in the LAD group, mean (±SD), total protein was 15⋅6 ± 8⋅23 which was significantly higher than that of the conventional dressing group (10⋅26 ± 4⋅94 mg/g wet tissue weight) (P = 0⋅003). © 2015 Medicalhelplines.com Inc and John Wiley & Sons Ltd
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GSH (tripeptide) and GPx, which are normally present at high concentrations intracellular, constitute the major reducing capacity of the cytoplasm and protect the cellular system against the toxic effects of lipid peroxidation (37). In this study, mean (±SD) GSH level (7⋅40 ± 1⋅91 μg/mg protein) in the LAD group was higher than that of the conventional dressing group (5⋅1 ± 1⋅28 μg/mg protein; P = 0⋅037). In the LAD group, mean GPx (±SD) level was significantly higher (112⋅3 ± 46⋅4 μM/minutes/mg protein) than that of the conventional dressing group (92 ± 32⋅4 μM/minutes/mg protein) (P = 0⋅016). MDA is produced during the attack by free radicals to membrane lipoproteins and polyunsaturated fatty acids (38). In this study, MDA was significantly high in the conventional dressing group (10⋅6 ± 3⋅8 nM/mg protein) compared with that in LAD group (6⋅5 ± 2⋅24 nM/mg protein) (P = 0⋅002). Results of histopathological analysis after 10 days of treatment showing mean ± SE of necrotic tissue (LAD versus conventional dressing group = 10 ± 1⋅8 versus 11⋅9 ± 2⋅6; P = 0⋅033), inflammatory cells (8⋅4 ± 1⋅9 versus 13 ± 3⋅46; P = 0⋅021), new blood vessels (12⋅5 ± 2⋅87 versus 9⋅4 ± 1⋅7; P = 0⋅047), ECM deposit (12⋅9 ± 2⋅41 versus 9⋅68 ± 1⋅3; P = 0⋅018) (Table 2) indicated that there was increase in epithelialisation, ECM deposition and angiogenesis and decrease in cellular infiltration in the LAD group than in the conventional dressing group. Conclusion
LAD exerts its beneficial effects on burn wound healing by significantly reducing oxidative stress, decreasing necrotic tissue score and amount of inflammatory infiltrate, and increasing ECM deposition and angiogenesis than the conventional dressings.
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