Loofah Sponge as an Interface Dressing Material in Negative Pressure Wound Therapy: Results of an In Vivo Study
Since the introduction of negative pressure wound therapy (NPWT), the physiological effects of various interface dressing materials have been studied. The purpose of this experimental study was to compare the use of loofah sponge to standard polyurethane foam or a cotton gauze sponge. Three wounds, each measuring 3 cm x 3 cm, were created by full-thickness skin excision on the dorsal sides of 24 New Zealand adult white rabbits. The rabbits were randomly divided into four groups of six rabbits each. In group 1 (control), conventional saline-moistened gauze dressing was provided and changed at daily intervals. The remaining groups were provided NPWT dressings at -125 mm Hg continuous pressure.
This dressing was changed every 3 days for 9 days; group 2 was provided polyurethane foam, group 3 had conventional saline-soaked antimicrobial gauze, and group 4 had loofah sponge. Wound area measurements and histological findings (inflammation, granulation tissue, neovascularization, and reepithelialization) were analyzed on days 3, 6, and 9. Wound area measurements at these intervals were significantly different between the control group and study groups (P <0.05). Granulation and neovascularization scores were also significantly different between the control and treatment groups at day 3 (P = 0.002). No differences in any of the healing variables studied were observed between the other three dressing materials. According to scanning electron microscopy analysis of the three interface materials, the mean pore size diameter of foam and gauze interface materials was 415.80±217.58 µm and 912.33±116.88 µm, respectively. The pore architecture of foam was much more regular than that of gauze. The average pore size diameter of loofah sponge was 736.83±23.01 µm; pores were hierarchically located — ie, the smaller ones were usually peripheral and larger ones were central. For this study, the central part of loofah sponge was discarded to achieve a more homogenous structure of interface material. Loofah sponge study results were similar to those using gauze or foam, but the purchase price of loofah sponge is lower than that of currently available interface dressings. More experimental, randomized controlled studies are needed to confirm these results.
Potential Conflicts of Interest: This study was supported by a grant from the Scientific Research Projects Unit, Gaziosmanpasa University, Tokat, Turkey. KCI, San Antonio, TX, provided technical and device support.
According to prospective and retrospective clinical and experimental studies,1,2 negative pressure wound therapy (NPWT) has been widely used to facilitate healing of acute and chronic wounds. The method has been shown to provide a moist wound healing environment, increase granulation tissue formation, reduce edema, and stimulate angiogenesis and blood flow to the wound margins.3-8 Torbrand et al’s9 retrospective clinical study attributed these biological effects to the evenly distributed transduction of negative pressure to the wound bed by a vacuum pump.
Pressure transduction is affected by the type and pore size of the interface material and the drainage system.10-12 Since Morykwas and Argenta5 first introduced polyurethane sponge as a wound suction interface, the physiological effects of the interface materials have been investigated in various experimental and clinical studies.5-7,13-15 Polyurethane foam was used widely in the first years; the use of a gauze-based system has been increasing in recent years.16,17 The results of the first use of gauze as a medium was reported in Chariker et al’s case series18 in 1989. Campbell et al19 analyzed gauze-based NPWT on wound healing in a retrospective clinical evaluation including 30 patients, finding the overall rate of wound volume reduction is similar to previously published data from polyurethane foam-based NPWT systems. Malmsjö et al’s in vivo study17 showed that gauze and foam are equally effective at delivering negative pressure and creating mechanical deformation of the wound tissue. In that study, similar wound edge contraction was observed at low (-50 mm Hg) and high (-175 mm Hg) levels of negative pressure. Wilkes et al’s computational study15 showed foam produces greater strain than gauze in the tissue model at -50 and -100 mm Hg.
Experimental studies using different biomaterials suggest foam porosity is a critical parameter that can affect cellular activities, morphology, and depth of ingrowth into biomaterials.20,21 Devices that induce microdeformation on wound beds may directly stimulate dermal cell proliferation, promoting granulation tissue formation. An in vivo study22 using porous degradable collagen-glycosaminoglycan materials demonstrated pore size is a critical parameter for inducing wound healing and skin regeneration. In vitro and in vivo studies have shown that mechanical forces are able to stimulate cell proliferation and differentiation.23 In an in vivo study, Pietramaggiori et al24 found application of tension increases vascular area and epidermal cell proliferation, both critical for enhanced repair of tissue defects. Mechanical forces stimulate cell proliferation and vascular remodeling in living skin.25
Loofah sponge is a hydrophilic material with a highly porous structure of interconnecting pores, a structural and physical appearance closely resembling gauze. This natural material, a member of the Cucurbitaceae family, consists of cellulose and lignin.26 The struts of this natural sponge are characterized by microcellular architecture with continuous hollow microchannels that form vascular bundles and yield a multimodal hierarchical pore system.27 Loofah is produced abundantly in many developing countries within the tropical and subtropical zones and primarily used for bathing and washing.26 Recently, loofah sponges also have been applied as cell carriers in bioreactors,28 for scaffolds in tissue engineering,29 and for the development of biofiber-reinforced composites.30,31
Owing to loofah’s fibrous vascular network that can mimic polyurethane foam and cotton gauze sponge, it was hypothesized that the complex canal system within loofah could create a porous environment for cellular integration in wound healing and effectively deliver negative pressure to the wound bed. The purpose of this experimental study was to compare vascularization, reepithelialization, and collagen deposition rates between loofah sponge and interface materials already in use as interface dressings for NPWT.
Materials and Methods
This study was approved by the ethical committee on animal experiments in Gaziosmanpasa University, Tokat, Turkey. A total of 24 New Zealand adult white rabbits weighing an average of 3.323±0.54 (range 2.4–4) kg were used. Anesthesia was induced with intramuscular injection of 20 mg/kg ketamine hydrochloride and 2 mg/kg xylasine hydrochloride. In all rabbits, three wounds were created by performing a 3 cm x 3 cm full-thickness skin excision that included the panniculus carnosus on their dorsal sides. Continuous sutures were applied to the wound edges to prevent wound contraction. The rabbits then were randomly divided into four groups of six rabbits each. Group 1 (control) wounds were dressed with conventional saline-moistened gauze, covered with elastic bandage, and changed at daily intervals. In the remaining three groups, NPWT was provided by a standard vacuum pump (V.A.C., Kinetic Concepts, Inc, San Antonio, TX) at a continuous negative pressure of 125 mm Hg using three different interface materials for a total of 9 days and assessed at three-day intervals (see Figure 1). For group 2, the interface material was hydrophobic polyurethane foam with a pore size 400–600 µm (V.A.C. Kinetic Concepts, Inc, San Antonio, TX). For group 3, saline-moistened antimicrobial gauze (Kerlix-AMD; Tyco, Gosport, UK) was applied and covered by a semi-occlusive polyurethane adhesive drape (OpSite Flexigridt, Smith & Nephew, UK). A wound drain (V.A.C. T.R.A.C. Pad, Kinetic Concepts Inc) was connected to the negative pressure device. In group 4, loofah sponge was used as the interface material. Loofah sponge was purchased from a local specialty shop in Turkey and prepared for use by removing the skin of the fruit and immersing in 2% NaOH solution for approximately 60 minutes. The loofah fibers then were washed with distilled water until a neutral pH was reached and dried at 60˚ C for 24 hours.32 The inner part of the loofah sponge, which contains larger pores, was removed, and the remaining outer shell containing more homogenous smaller pores was cut open on one longitudinal side and converted from a cylindrical to a rectangular shape (see Figure 2). Finally, the sponges were sterilized in an autoclave at 120˚ C for 1 hour. Before application, the sponges were moistened with saline (see Figure 3). A drainage tube was placed over the sponge and connected to the negative pressure pump (see Figure 4). Sleeves and swivels were used to protect the tubing and avoid twisting. On day 9, all rabbits were sacrificed.
The surface area of the wounds was measured at 3-day intervals using the wound tracing method. A transparency was placed directly over the wound, and wound margins were traced with a pen. The surface area for the tracing was determined by counting the squares manually on graph paper.
Scanning electron microscopy (SEM) was performed to analyze the pore size of loofah sponge, foam, and gauze (see Figure 5), and the pore size measurements of interface materials were made on the SEM photographs by using TPSDIG 2.00 software (FJ Rohlf, tpsDig, Digitize Landmarks and Outlines, Version 2.0, State University of New York-Stoney Brook, Stoney Brook, NY). One of each three wounds with its surrounding skin and underlying tissue was excised en bloc at days 3, 6, and 9 for histological evaluation. The tissue samples, including wound ulcer border and center, were fixed in 10% formaldehyde solution and processed for paraffin embedding; 5-µm thick sections were obtained and stained with hematoxylin and eosin (H&E) for light microscopic analysis. Inflammation, granulation tissue, neovascularization, and reepithelialization were evaluated and semiquantitatively scored according to the system suggested by Abramov et al.33 Each parameter was scored independently, and a grade between 0 and 3 was given (see Table 1).
Inflammation was defined as acute (including neutrophils) or chronic (including lymphocytes and plasma cells) and scored as 0 = no inflammatory cells, 1 = scant, 2 = moderate, and 3 = severe inflammation.
The amount of granulation tissue was scored as 0 = none, 1 = scant, 2 = moderate, and 3 = marked. This tissue also was noted as to whether it was smooth or coarse.
Neovascularization was evaluated by counting the vessels in four adjacent areas at high power (x400), and the average vessel count was determined for each tissue. Neovascularization was scored as 0 = no neovascularization, 1 = less than five vessels, 2 = six to 10 vessels, and 3 = more than 10 vessels.
Reepithelialization was scored as score 0 = no reepithelialization; 1 = partial reepithelialization of the wound surface, initiation of squamous reepithelialization with creating epithelial buds from the wound border; 2 = complete but immature reepithelialization of the wound surface and thin, irregular, full reconstruction of epithelium with inadequate quality and organization; and 3 = complete and mature reepithelialization of the wound surface and regular, complete reconstruction of epithelium with adequate quality and organization like normal squamous epithelium.
To detect fibrosis, specimens were stained with Masson’s trichrome (MTC). Fibrosis was scored as 0 = no fibrosis; 1 = minimal, loose fibrous tissue; 2 = moderate; and 3 = severe and dense fibrosis.
Statistical collection and analysis. Wound areas were presented as mean ± standard deviation. Pearson’s chi-square test was used to compare the categorical data among groups. Kruskal-Wallis analysis of variance was used to compare the wound areas among groups. For post-hoc comparisons between the pair-wise groups, the Bonferroni-adjusted Mann-Whitney U test was used. The Friedman test was used to compare the four periods for each groups. For post-hoc comparisons between the pairs of follow-up periods, the Bonferroni-adjusted, Wilcoxon rank sum test was used. Cochran’s Q test was used to compare the three control periods for each group. For post-hoc comparisons between the pairs of follow-up periods, the McNemar Test was used. Categorical variables were presented as count and percentages. P <0.05 was considered significant. Analyses were performed using commercial software (IBM SPSS Statistics 19, SPSS Inc, an IBM Company, Somers, NY).
The application of NPWT was well-tolerated by the rabbits, and no complications with NPWT or wound fillers were encountered during the study.
Surface area. Wound surface area was significantly reduced in all groups when compared with the initial wound (9 cm2). Wound area differences were statistically different at each time point for the control versus each of the other three groups (P = 0.002, P = 0.001, and P = 0.002 for days 3, 6, and 9, respectively), but no statistical significance was noted between the three study groups. At day 3, average surface area was 8.35±0.49 cm2 in the control group, 5.02±0.36 cm2 in group 2 (polyurethane foam), 5.17±0.71 cm2 in group 3 (antibacterial gauze), and 4.57±0.62 cm2 in group 4 (loofah sponge). At day 6, average surface area of the wounds was 7.14±1.01 cm2 in group 1, 3.29±0.57 cm2 in group 2, 4.07±0.88 cm2 in group 3, and 3.04±0.65 cm2 in group 4. At day 9, the average surface area of the wounds was 5.79±1.15 cm2 in the control group, 0.70±0.27 cm2 for group 2, 0.87±0.24 cm2 for group 3, and 0.75±0.28 cm2 for group 4 (see Table 2, Figures 6, 7).
Granulation. The amount of granulation tissue increased significantly in all study groups at day 3 compared to the control group (P = 0.002). After day 3, the amount of granulation tissue was similar, with no significant difference among the groups. Reepithelialization increased after wounding in all groups and was complete by day 9 (see Figure 8). In the control group, reepithelialization was immature and incomplete; in the study groups, it was mature and complete.
Neovascularization. Neovascularization scores increased after wounding in all study groups (see Figure 9). The number of blood vessels in the control group was <3; in the study groups, it was 6 to 10. The difference between the control and study groups was statistically significant at day 3 (P = 0.002). No significant difference among the groups was found at days 6 and 9.
Collagen. Collagen deposition increased after wounding in all groups at days 6 and 9, but no significant difference was noted among the groups (see Figure 10). However, the collagen bundles were dense and oriented in the study groups and loose and sparse in the control group.
SEM. SEM observations confirmed similarities and differences among the interface materials. According to SEM analysis, the mean pore size diameter of foam and gauze interface materials was 415.80±217.58 µm and 912.33±116.88 µm, respectively. However, the pore architecture of foam was much more regular than that of gauze. Loofah sponges had medium-sized pores (736.83±23.01 µm) among the interface materials used in the study.
The primary role of the interface material in wound healing is to provide optimal wound healing conditions. In general practice, polyurethane foam or moistened gauze is used as wound filler material.34-38 Many experimental and clinical studies have sought to determine which interface material is more effective in difficult-to-heal wounds and whether interface materials affect changes in wound characteristics such as depth, size, and exudate.16,17,20,21,38 Different indications provide some guidance on using negative pressure with foam or gauze and depend on types of wounds, patients, and anatomical location.38 Malmsjö et al’s experimental studies16,17 showed that gauze and foam are equally effective at delivering negative pressure and creating mechanical deformation of the wound. According to a large review of the literature,16-20 no differences in the degree of blood flow or wound contraction in small wounds was observed with either foam or gauze, although polyurethane foam was found to result in more contraction than gauze in large wounds. In the current study, wound diameter was 9 cm2. Thus, wounds were small and created on rabbits, where it is known wounds heal faster than in larger animals or humans.
Current study results regarding the control, gauze, and foam correlate with the literature on wound contraction, granulation, and neovascularization. The use of loofah sponge, a natural product similar in form to foam or gauze, was found to have the same results. It has been shown in a literature review39 and an in vivo study40 that the open-pore wound filler plays a crucial role in suction device-induced wound healing. Foam porosity is a critical parameter that can affect cellular activities such as binding, migration, proliferation, morphology, and ingrowth into biomaterials.20 O’Brien et al21 suggested pore sizes between 20 and 120 µm were optimal and that pores should be large enough to allow cell migration but small enough to allow cell adhesion. Heit et al’s41 similar experimental study utilized polyurethane foam in large (300 µm), medium (130 µm), and small (70 µm) pore size diameters to treat full-thickness wounds in diabetic mice. In that study, the thickest granulation tissue was obtained after large pore size treatment compared with medium and small pore size treatments. Angiogenesis was induced only in small and medium pore size groups. Small and medium pore size-treated wounds showed a greater number of proliferating cells compared with large pore size-treated wounds. In addition, tissue ingrowth into small pore size polyurethane foam was much lower than that of medium and large pore size. According to Klinge et al’s in vivo study,42 the pore size of an interface material appears to be an important parameter in tissue integration. In that study, the authors concluded that small diameters theoretically will lead to increased flow resistance and to impaired passive exchange of the soluble components, particularly if the surface tension of the tissue liquids is considered. Thus, it can be speculated that tiny pores may markedly inhibit fluid transport through the interface material. Per experimental studies,43-45 cellular ingrowth within a sponge depends on the porosity and the presence of fibrous structure. In an experimental study,46 larger pores were shown to increase wound surface strain, tissue ingrowth, and transformation of contractile cells. The medium and small pore size can be superior to large pore size to enable the reorganization and clustering of the cells in a granulating wound. In the current study, loofah sponge (736 µm) stimulated granulation tissue formation and neovascularization by days 6 and 9 as well as gauze (912 µm) and foam (415 µm), with no difference among study groups.
Granulation tissue formation was observed to be more coarse in the control and smooth in the study groups. Wound biopsies showed similar surface undulations and small tissue blebs, which is the result of pulling effect of NPWT into the pores of the wound filler in all study groups. These mechanical effects (microdeformations) are thought to result in shearing forces of the wound dressing materials with NPWT, which affects the cytoskeleton and stimulation of angiogenesis and leads to promotion of granulation tissue formation and accelerated wound healing.47 Thus, loofah sponge exacted the same end result as available wound fillers on the market today.
In humans, NPWT helps decrease wound volume through active contraction and generation of granulation tissue; in rabbits, where the skin is loose, the primary mode for wound closure is contraction and epithelialization, even though some granulation tissue formation occurs, usually after about 4 to 6 days.48 In animal studies with NPWT, the time to complete closure is between 8 to 12 days depending on the size of the wound.49 In the present study, the optimum follow-up period was 9 days. At that time, the surface area of the wounds in the study groups was significantly smaller than that of control group, and no differences between the study groups were seen.
Several techniques have been developed to process synthetic and natural scaffold materials into porous structures.50 These conventional fabrication techniques are defined herein as processes that create scaffolds having a continuous, uninterrupted pore structure that lacks any long-range channeling microarchitecture. The fibrous network of loofah sponge is mainly comprised of cellulose (60%), hemicelluloses (30%), and lignin (10%).26,27 The tensile strength of the fibers is due to cellulose and its compression strength to lignin.51 The netting-like, fibrovascular loofah sponge has approximately 800 µm macropores, created by rough and indented fibers with continuous hollow microchannels.52 Cellulose, as a bioaffinity carrier, exhibits good chemical stability, recoverability, reproducibility, and mechanical strength.53 In plant cell walls, lignins are closely related with hydrophilic polysaccharides; they are amorphous, hydrophobic heteropolymers. Because of their hydrophobic character, lignins make plant cells impermeable to water.53 The presence of lignin in the cell walls or between the fibers is known to hinder the chemical reactions of cellulose and hemicelluloses as it prevents the permeation of water across the cell walls. Thus, loofah sponge may be a suitable biostructure for cell immobilization and bioprocess activities. The sponge structure follows the “tensegrity” principle — ie, an architectonical system in which structures stabilize themselves owing to equilibrium between opposite forces of traction and compression.54 The surface of the fibers is rough, due to the presence of small longitudinal and transverse stripes. In longitudinal section, the sponge appears like a network of fibers of different diameters, more or less close together.55
The fibrous and porous network of loofah sponge is similar to gauze and foam. It is similar to gauze in hydrophilic character, and its absorption capacity has been found to be 13.6 g/g.56
NPWT has been used for many difficult-to-heal wounds during the last decade; however, it is still an expensive treatment modality. Wounds present a substantial cost to patients as well as the healthcare system. The most important determinant of cost appears to be wound complications that require hospitalization or delay hospital discharge. Reducing costs requires a systematic focus on effective and timely diagnosis, on planning an appropriate wound treatment, and on taking measures to prevent complications and wound-related hospitalization.57 According to Albert et al’s prospective comparison,58 gauze- and foam-based NPWT systems both can provide comparable wound healing; nurse perceptions of ease of dressing changes when working with these devices and direct costs associated with the use of the devices (dressings and chargeable equipment) did not significantly differ. The total cost of interface materials in the present study was $1,580 for polyurethane foam, $1,000 for cotton gauze, and $20 for loofah sponge. Because it was the same for all three interface materials, the cost of the canister (collection unit) was not taken into consideration. The price advantage of using loofah is derived from its more basic route of supply — ie, a local store. Manufactured products cost more, and loofah sponge can be prepared locally for use as an interface material. It should be noted that raw material costs of gauze or foam dressings are probably relatively a small fraction of their commercial price after manufacturing. Even though the business costs account for a significant portion of the final price, it can be concluded that loofah sponge can be an inexpensive interface material option as compared to foam and gauze. In addition, because it dissolves easily, it has organic (“green”) implications.
An acute wound model was used in this study; it does not resemble a chronic, exudating wound with bacterial contamination, which could be the case in a clinical setting. Also, performing daily dressing changes with moist gauze is known to delay wound healing in rabbits, which also was observed in this study. A group where no wound dressing is used or a moisture retentive dressing could have been added to yield more clinically relevant information. In this study, a continuous 125 mm Hg negative pressure was used in all experimental groups, a pressure proposed by the negative pressure device company. Additional studies with application of intermittent pressure at different values may be useful, as would a comparison between NPWT and more modern (moisture retentive) dressings.
Loofah sponge was observed to have characteristics similar to current NPWT interface materials when used in rabbits. Because of its widespread cultivation around the world and low supply price when compared with currently marketed interface materials, loofah sponge appears to provide an accessible, less expensive alternative as an NPWT interface dressing. Before conducting clinical studies, more experimental research is needed on larger animal models with different negative pressure values in order to provide more evidence that loofah sponge can replace the conventional interface materials.
The authors thank the KCI for technical and device support.
Drs. Tuncel, Turan, and Markoc are Assistant Professors, Department of Plastic Reconstructive and Aesthetic Surgery, Gaziosmanpasa University, Faculty of Medicine, Tokat, Turkey. Dr. Erkormaz is an Assistant Professor, Sakarya University, Faculty of Medicine, Department of Biostatistics, Sakarya, Turkey. Dr. Elmas is an Associate Professor, Gazi Medical School, Department of Histology and Embryology, Ankara, Turkey. Dr. Kostakoglu is a Professor and Head, Department of Plastic Reconstructive and Aesthetic Surgery, Gaziosmanpasa University. Please address correspondence to: Dr. Umut Tuncel, Faculty of Medicine, Plastic Reconstructive and Aesthetic Surgery, Gaziosmanpasa University, 60100, Tokat, Turkey; email: email@example.com.
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