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Negative Pressure Wound Therapy-associated Tissue Trauma and Pain: A Controlled In vivo Study Comparing Foam and Gauze Dressing Removal by Immunohistochemistry for Substance P and Calcitonin Gene-related Peptide in the Wound Edge
Abstract
Pain upon negative pressure wound therapy (NPWT) dressing removal has been reported and is believed to be associated with the observation that granulation tissue grows into foam. Wound tissue damage upon removal of the foam may cause the reported pain. Calcitonin gene-related peptide (CGRP) and substance P are neuropeptides that cause inflammation and signal pain and are known to be released when tissue trauma occurs. The aim of this controlled in vivo study was to compare the expression of CGRP and substance P in the wound bed in control wounds and following NPWT and foam or gauze dressing removal. Eight pigs with two wounds each were treated with open-pore structure polyurethane foam or AMD gauze and NPWT of 0 (control) or -80 mm Hg for 72 hours. Following removal of the wound filler, the expression of CGRP and substance P was measured, using arbitrary units, in sections of biopsies from the wound bed using immunofluorescence techniques. Substance P and CGRP were more abundant in the wound edge following the removal of foam than of gauze dressings and least abundant in control wounds. The immunofluorescence staining of the wound edge for CGRP was 52 ± 3 au after the removal of gauze and 97 ± 5 au after the removal of foam (P <0.001). For substance P, the staining was 55 ± 3 au after gauze removal and 95 ± 4 au after foam removal (P <0.001). CGRP and substance P staining was primarily located to nerves and leukocytes. The increase in CGRP and substance P immunofluorescence was especially prominent in the dermis but also was seen in subcutaneous and muscle tissue. Using gauze may be one way of reducing NPWT dressing change-related pain. New wound fillers designed to optimize granulation tissue formation and minimize pain issues presumably will be developed in the near future.
Potential Conflicts of Interest: This study was supported by the Swedish Medical Research Council, Lund University Faculty of Medicine, the Swedish Government Grant for Clinical Research, Lund University Hospital Research Grants, the Swedish Medical Association, the Royal Physiographic Society in Lund, the Ake Wiberg Foundation, the Anders Otto Swärd Foundation/Ulrika Eklund Foundation, the Magn Bergvall Foundation, the Crafoord Foundation, the Anna-Lisa and Sven-Erik Nilsson Foundation, the Jeanssom Foundation, the Swedish Heart-Lung Foundation, Anna and Edvin Berger’s Foundation, the Märta Lundqvist Foundation, Lars Hierta’s Memorial Foundation, and Prospera (Forth Worth, TX).
The most commonly used wound filler materials for negative pressure wound therapy (NPWT) are open-pore polyurethane foam or cotton gauze (eg, Kerlix, AMD gauze, Covidien, Mansfield, MA) dressings.1 The results of in vivo studies have shown that dressing choice can affect the healing process, because both the amount and characteristics of granulation tissue are different when foam or gauze dressings are used.2-4 In a preclinical experimental wound models in dogs and pigs, newly formed granulation tissue was found to be thick but fragile; when gauze was used, the newly formed tissue was thinner and more stable.2,5 Thick granulation tissue is beneficial for rapid wound healing, but results from a clinical study of patients indicate this may lead to problems such as fibrosis, scarring, and contractures as the wound heals.4
Gauze often is used for NPWT because of its moldability and ease of application, advantages described by Jeffery et al6 in a review of treatment modalities for wounds in military personnel caused by landmines and other explosive devices. A retrospective review of 24 pediatric patients7 also suggests that gauze has become especially popular among plastic surgeons for wound bed preparation before grafting in the clinical setting. These observations suggest that gauze may be a good choice when the cosmetic result is of importance or in cases where scar tissue may restrict movement — for example, over joints. On the other hand, as described by Gustafsson et al8 in a overview article describing the surgical technique for NPWT in sternotomy wounds, foam may be more suitable for wounds that benefit from development of thick granulation tissue and where scarring does not pose a problem — for example, in sternotomy wounds. Foam also may be more suitable than gauze in fasciotomy wounds in upper or lower limb compartment syndrome where contraction is beneficial,9 or to provide a bridging technique in patients with acute wounds with large amounts of tissue loss.10,11 Other currently available, but less commonly used, wound fillers for NPWT include the Bio-Dome™ Wound Dressing (ConvaTec, Skillman, NJ).
Both foam and gauze have been shown to create micro-mechanical deformation of the wound bed, which is believed to stimulate cell proliferation and granulation tissue formation.3 Ingrowth of tissue into the foam during NPWT may cause pain during dressing removal.12 Ingrowth has been examined on a cellular level in histological sections of the wound bed filled with gauze or foam.2 Foam adheres to the wound bed, and it has been shown in porcine wound models that more force is required to remove foam than gauze after NPWT.2,13 Ingrowth into foam may result in pieces of foam becoming embedded in the wound bed; if left there, they may act as foreign bodies that may hinder wound healing. Also, changing foam dressings results in wound bed disruption and mechanical tissue damage when the foam and adhering tissue are torn from the wound bed. Literature reviews12,14 on the subject indicate that pain associated with foam dressing changes is common and requires vigorous treatment, including the systemic administration of morphine or the application of local anesthetics. However, the latter are known to be cytotoxic and may interfere with tissue regeneration.15
Several clinical problems are associated with dressing changes when using foam in NPWT. No controlled study has yet been performed to examine the degree of tissue trauma and pain associated with foam compared to gauze dressing removal during NPWT.
Calcitonin gene-related peptide (CGRP) and substance P are released from sensory nerve endings and inflammatory cells when local tissue trauma and injury occur. Both are known to signal pain; they also initiate a local inflammatory reaction.16 CGRP and substance P are believed to contribute to pain transmission and inflammation mechanisms — for example, those associated with migraine and peripheral pain.16,17
The aim of this controlled, in vivo study was to examine the expression of CGRP and substance P in the wound bed following NPWT and foam or gauze dressing removal. 
Material and Methods
Porcine peripheral wounds were treated with NPWT at -80 mm Hg for 72 hours, and the effects of foam and gauze on the wound bed were compared by examining sections of biopsies from the wound bed after hematoxylin-eosin and immunofluorescence staining for CGRP and substance P.
Animals. Eight healthy domestic pigs of both genders (mean body weight of 70 kg), were fasted overnight and provided free access to water. The experimental protocol for this study was approved by the Ethics Committee for Animal Research, Lund University, Sweden. All animals received humane care in compliance with the European Convention on Animal Care.
Anesthesia and surgical procedure. Premedication was provided using an intramuscular injection of xylazine (Rompun® vet. 20 mg/mL; Bayer AG, Leverkusen, Germany; 2 mg/kg) mixed with ketamine (Ketaminol® vet. 100 mg/mL; Farmaceutici Gellini S.p.A., Aprilia, Italy; 20 mg/kg). Two peripheral veins in the pigs’ ears were cannulated for induction and maintenance of anesthesia and for fluid administration. Anesthesia was maintained with a continuous infusion of ketamine (0.4–0.6 mg/kg/hour). Complete neuromuscular blockade was achieved with a continuous infusion of pancuronium bromide (Pavulon; N.V. Organon, Oss, The Netherlands; 0.3–0.5 mg/kg/hour). To compensate for fluid loss, a continuous infusion of Ringer’s acetate at a rate of 200 mL/kg/hour was provided for the first 24 hours, followed by 110 mL/hour for the remainder of the experiment. The animals received total parenteral nutrition (Kabiven; Fresenius Kabi AB, Uppsala, Sweden). Antibiotics were given once daily as intravenous bolus injections (Streptocillin® vet. 250 mg/mL + 200 mg/mL; Boehringer Ingelheim Vetmedica, Malmö, Sweden; 10 mL).
The animals were orally intubated with cuffed endotracheal tubes. Mechanical ventilation was established with a Siemens-Elema ventilator (Siemens-Elema AB, Solna, Sweden) in the volume-controlled mode (65% nitrous oxide, 35% oxygen). Ventilation settings were identical for all animals (respiratory rate, 15 breaths/minute; minute ventilation, 12 L/minute). A positive end-expiratory pressure of 5 cm water was applied. A Foley catheter was inserted into the urinary bladder through a suprapubic cystostomy. After the experiments were completed, the animals were euthanized with a lethal dose (60 mmol) of intravenous potassium chloride.
Wound preparation. Two circular wounds, extending 1.5 cm below the wound surface into the subcutaneous tissue, were created on each pig’s back. Kerlix, AMD gauze (Covidien, Mansfield, MA) or open-pore structure (400–600 micronpore size) polyurethane foam (V.A.C. Therapy® black GranuFoam®, KCI, San Antonio, TX) was used as wound filler. The gauze was soaked with saline. The gauze used is impregnated with polyhexamethylene biguanide (PHMB) to provide antimicrobial control. Wound filler, estimated at surgery, was approximately one-and-a-half times wound volume to allow volume reduction during negative pressure application. This process was the same for both the gauze and foam dressing used; the two different wound fillers were compared in each pig.
A drainage tube was inserted into the wound filler and connected to a vacuum source (Prospera PRO-III®, Prospera Technologies LLC, Fort Worth, TX). The wound then was sealed with a transparent adhesive drape, which overlapped the wound margins by 10 cm. The wounds were sealed for NPWT and either left at atmospheric pressure (0 mm Hg) or treated with NPWT at -80 mm Hg. The level of negative pressure was continuous — ie, left at a constant pressure for the entire duration of the therapy. After 72 hours, the adhesive drape covering the wound was cut, using a scalpel, along the borderline between the tissue and the wound filler. The cut was superficial enough to only slice the thin drape and not affect the underlying tissue structures or dressings, which was confirmed by inspection of the wound and the wound filler. After the drain was removed, the wound filler was attached to a force measurement device and a motor-driven traction device set to withdraw the wound filler at a constant speed of 4 mm/second.
Histology. For the control group, histology samples were taken from fresh tissue. For the treatment group, a strip of the wound filling material (1 cm x 1 cm x 2 cm) was sewn onto the bottom of each wound. After NPWT was completed and the wound filler removed, the strip and the underlying wound bed tissue were excised with a scalpel. Both control and treatment group tissue then was treated in 4% paraformaldehyde, dehydrated, embedded in paraffin, and left overnight. Sectioning was performed with a rotary microtome HM 355 (ThermoFisher Scientific, MA).
Hematoxylin-eosin staining was performed for morphological analysis. Slides with 4 µm-thick tissue sections were deparaffinized for 2 x 4 minutes in xylene, 2 x 3 minutes in 99.5% ethanol, and 3 minutes in tap water. The slides were stained for 12 minutes in Mayer’s hematoxylin (Histolab AB, Rockville, MD) and then treated/rinsed with tap water for 8 minutes, erythrosin (1.5 g in 500 mL water) for 6 minutes, tap water for 3 minutes, 99.5% ethanol for 2 x 3 minutes, and xylene for 4 minutes.
For immunostaining, the sections were blocked in incubation buffer (5% goat serum in PBS with 1% bovine serum albumin and 0.25% Triton X100) for 1 hour at room temperature and incubated overnight at +4˚ C with 1:400 rabbit anti-CGRP (B47-1, Santa Cruz Biotechnology Inc, CA) or 1:400 rabbit antisubstance P (B45-1, Santa Cruz Biotechnology Inc.) diluted in incubation buffer. Slides were washed three times for 10 minutes in PBS with 0.25% Triton X100, followed by incubation for 2 hours at room temperature with 1:400 goat anti-rabbit IgG FITC (Cayman Chemical, MI) diluted in PBS with 0.25% Triton X100. The slides were washed three times for 10 minutes in PBS with 0.25% Triton X100 and mounted with Vectashield mounting medium (Vector Laboratories Inc., CA). Microscopy was performed using a Zeiss Axiophoto microscope equipped with an AxioCam camera (Carl Zeiss, Oberkochen, Germany). The morphology of the biopsy sections was examined, and the intensity of CGRP and substance P staining was quantified and expressed as arbitrary units (au).
Calculations and statistics. The differences in staining intensity were calculated and analyzed using GraphPad 5.0 software (San Diego, CA). Statistical analysis was performed using the Mann-Whitney test when comparing two groups, and the Kruskal-Wallis test with Dunn’s post-test for multiple comparisons when comparing three groups or more. Results are presented as means ± the standard error of the mean (SEM) (n = 8). 
Results
CGRP and substance P staining. After NPWT, the intensity of staining for substance P and CGRP was higher in the wound edge in foam-dressed compared to gauze-dressed wounds (see Figures 1 and 2). The immunofluorescence staining of the wound edge for CGRP was 52 ± 3 au after the removal of gauze and 97 ± 5 au after the removal of foam (P <0.001). For substance P, the staining was 55 ± 3 au after gauze removal and 95 ± 4 au after foam removal (P <0.001). The least intense staining was observed in both the foam and gauze control wounds that were sealed as for NPWT but not subjected to negative pressure (see Figures 1 and 2).
Tissue morphology. Both CGRP and substance P staining was observed in the wound edge; the staining was most intense in the superficial tissue (75 ± 5 au for CGRP and 78 ± 6 au for substance P) that had been exposed to the forces of the negative pressure and in direct contact with the wound filler and less intense in deeper tissue (23 ± 2 au for CGRP and 31 ± 3 au for substance P). CGRP and substance P staining was especially intense in the dermis, but was also present in the subcutaneous and muscle tissue. Slides stained for CGRP and substance P were compared to adjacent slides stained with hematoxylin-eosin. Staining for both CGRP and substance P were primarily observed located to nerves and leukocytes (see Figures 3 and 4). 
Discussion
The results of the present study show that substance P and CGRP are more abundant in the wound edge of porcine wounds after the removal of foam than the removal of gauze after NPWT. This is in line with clinical observations that dressing changes are more painful when NPWT has been carried out using foam than when using gauze.
A literature review by Krasner et al12 addressed the application and removal of foam as a source of procedural pain. It was suggested that patients who experienced pain during dressing changes be given pain relief 30 to 60 minutes before the procedure, following the World Health Organization’s three-step analgesic ladder, proceeding from nonopioids to opioids. In many cases, it was necessary to administer opioids systemically before changing dressings to ensure an adequate and acceptable (to the patient) level of pain relief. Attempts also have been made to reduce pain by instilling a 1% lidocaine solution through the drain to reach the surface of the wound; the effects were evaluated in a clinical, double blind, prospective, randomized study.14 However, we believe the administration of local anesthetics is undesirable because they may be cytotoxic, as shown in in vitro studies.15
Previously, foam was used as a wound filler in almost all patients treated with NPWT. Today, NPWT that utilizes gauze-type dressings are widely available.
The effects of gauze and foam on the wound bed have been shown to differ substantially in porcine wound models.2,3 The granulation tissue may adhere to the foam or actually grow into the pores of the foam, but in vivo studies of porcine wounds treated with NPWT show no ingrowth into gauze.2,13 On a cellular level, ingrowth has been examined in histological sections of the wound bed treated with overlying gauze or foam wound filler.2 Preclinical in vivo studies2 show that more force is needed to remove foam than to remove gauze after NPWT.
The pain during NPWT dressing changes may be due to the disruption of the granulation tissue in the wound bed resulting from foam removal.2,13 Per clinician observation, it has been suggested that dressing changes when using gauze as the wound filler result in less pain than when using foam. Another strategy may be to use foam of a smaller pore size, such as the polyvinyl alcohol (PVA) white foam dressing, but data examining tissue ingrowth for this type of foam have not been published. It is difficult to conduct comparative studies in the clinical situation because the pain assessment tools (eg, visual analog scales or the Faces scale) are subjective measures and are affected by the individual patient’s pain threshold. Furthermore, each wound is different, and this will affect the level of pain experienced — eg, changing the dressing on a diabetic foot ulcer may be relatively painless as a result of neuropathy as quantified using the above mentioned pain assessment tools.
The present study aimed to quantify the tissue trauma and pain resulting from the removal of foam and gauze wound fillers. As described in previous literature reviews,16 CGRP and substance P are released from sensory nerve endings and inflammatory cells upon local tissue trauma and injury. These neuropeptides are known to signal pain and also to initiate a local inflammatory reaction.16 The results of the present study show that the use of foam in NPWT leads to greater expression of substance P and CGRP in the wound edge than when using gauze. This is in line with clinical observations that NPWT dressing removal causes pain and that patients treated with NPWT using foam experience more pain during dressing changes than patients treated with NPWT using gauze.17
Multiple strategies often are required to manage pain adequately in patients treated with NPWT. The obvious method is to provide the patient with adequate medication to alleviate pain. However, it is also important to consider the cause of the pain and to try and avoid or minimize it. This may reduce the need for strong medication. Other strategies also should be considered. When the clinician anticipates complication, a wound contact layer can be placed under the wound filler as described in previous clinical case series.18,19 A recent preclinical in vivo study in pigs20 showed that the use of a nonadherent wound contact layer prevents the growth of tissue into foam, thus facilitating dressing changes, although this may slightly reduce the formation of granulation tissue. The degree of wound bed adhesion may be reduced by increasing the frequency of dressing changes, thus reducing the pain during dressing changes. Another frequently employed strategy is to instill saline into the wound, through the wound filler (eg, via the drain) 15 to 30 minutes before gently removing the dressing.
NPWT using foam is known to create thick granulation tissue in the wound bed; gauze creates a thinner but stable granulation tissue in pig wounds.2,5 These differences are believed to be the result of differences in the mechanical shear forces caused by the foam or gauze on the wound bed. The results from the present study show a greater release of CGRP and substance P after treatment of foam than gauze. This may reflect the presence of a greater mechanical forces causing deformation of the wound bed under foam than under gauze.
Study Limitations
One limitation of the present study is that it is was performed in pigs. It would be of clinical relevance to perform a similar study in humans. One option would be to collect biopsies from wounds treated with NPWT using foam or gauze and examine these using immunohistochemistry for CGRP and substance P. Another limitation of the present study is that it was only performed with one type of gauze (ie, the clinically most commonly used Kerlix AMD gauze). It is important to note that nearly all gauze used in NPWT has been a particular type of cotton gauze (Kerlix AMD) to facilitate impregnation with polyhexamethylene biguanide (PHMB) to provide antimicrobial control. The observations from this study may not apply to all types of gauze.
Conclusion
CGRP and substance P are neuropeptides released as a result of tissue trauma and are known to signal pain and initiate a local inflammatory response. In the present study, it was shown that the expression of CGRP and substance P in the wound edge was more pronounced in wounds treated with NPWT than control wounds and more pronounced after removing foam compared to gauze dressings when used in NPWT. This may confirm previous reports that granulation tissue grows into foam but not into gauze and the clinical observation that dressing removal after NPWT using foam is more painful than dressing removal after NPWT using gauze. The use of gauze may be one way of reducing the problem of pain during dressing changes in NPWT.
Dr. Malmsjö is a consultant, Department of Ophthalmology, Lund University and Skåne University Hospital, Lund, Sweden. Dr. Gustafsson is a consultant, Department of Ophthalmology, Lund University. Dr. Lindstet is a consultant, Department of Cardiothoracic Surgery, Lund University and Skåne University Hospital. Dr. Ingemansson is a consultant, Department of Cardiothoracic Surgery, Lund University and Skåne University Hospital. Please address correspondence to: Malin Malmsjö, MD, PhD, BMC A13, SE-221 84 Lund, Sweden; email: malin.malmsjo@med.lu.se.
1. Campbell PE, Smith GS, Smith JM. Retrospective clinical evaluation of gauze-based negative pressure wound therapy. Int Wound J. 2008;5(2):280–286.
2. Borgquist O, Gustafsson L, Ingemansson R, Malmsjö M. Tissue ingrowth into foam but not into gauze during negative pressure wound therapy. WOUNDS. 2009;21(11):302–309.
3. Borgquist O, Gustafsson L, Ingemansson R, Malmsjö M. Micro- and macromechanical effects on the wound bed by negative pressure wound therapy using gauze and foam. Ann Plast Surg. 2010;64(6):789–793.
4. Fraccalvieri M. Negative pressure wound therapy using the gauze and the foam: immunohistological and ultrasonography morphological analysis of the granulation tissue and the scar tissue. Preliminary report of a clinical study. 3rd Congress CO.R.T.E 2010; 4-6 of March (Personal Communication).
5. Demaria MG, Stanley BJ, Hauptman JG, et al. Comparison of Foam and Gauze Based Negative Pressure Wound Therapy on the Healing of Open Wounds in Dogs. Poster presentation at Clinical Symposium on Advances in Skin & Wound Care, San Antonio, Texas. 2009.
6. Jeffery SL. Advanced wound therapies in the management of severe military lower limb trauma: a new perspective. Eplasty. 2009;9:e28.
7. Chariker ME, Gerstle TL, Morrison CS. An algorithmic approach to the use of gauze-based negative-pressure wound therapy as a bridge to closure in pediatric extremity trauma. Plast Reconstr Surg. 2009;123(5):1510–1520.
8. Gustafsson RI, Sjogren J, Ingemansson R. Deep sternal wound infection: a sternal-sparing technique with vacuum-assisted closure therapy. Ann Thorac Surg. 2003;76(6):2048–2053; discussion 2053.
9. Zannis J, Angobaldo J, Marks M, et al. Comparison of fasciotomy wound closures using traditional dressing changes and the vacuum-assisted closure device. Ann Plast Surg. 2009;62(4):407–409.
10. Bollero D, Carnino R, Risso D, et al. Acute complex traumas of the lower limbs: a modern reconstructive approach with negative pressure therapy. Wound Repair Regen. 2007;15(4):589–594.
11. Stannard JP, Robinson JT, Anderson ER, et al. Negative pressure wound therapy to treat hematomas and surgical incisions following high-energy trauma. J Trauma. 2006;60(6):1301–1306.
12. Krasner DL. Managing wound pain in patients with vacuum-assisted closure devices. Ostomy Wound Manage. 2002;48(5):38–43.
13. Morykwas M. Sub-Atmospheric Pressure Therapy: Research Evidence. 1st International Topical Negative Pressure Therapy ETRS Focus Group Meeting. London,UK: ETRS;2003:39–44.
14. Franczyk M, Lohman RF, Agarwal JP, et al. The impact of topical lidocaine on pain level assessment during and after vacuum-assisted closure dressing changes: a double-blind, prospective, randomized study. Plast Reconstr Surg. 2009;124(3):854–861.
15. Boselli E, Duflo F, Debon R, et al. The induction of apoptosis by local anesthetics: a comparison between lidocaine and ropivacaine. Anesth Analg. 2003;96(3):755–756.
16. Fernandes ES, Schmidhuber SM, Brain SD. Sensory nerve-derived neuropeptides: possible therapeutic targets. Handb Exp Pharmacol. 2009;194:393–416.
17. Benemei S, Nicoletti P, Capone JG, et al. CGRP receptors in the control of pain and inflammation. Curr Opin Pharmacol 2009; 9(1):9-14.
18. Hurd T, Chadwick P, Cote J, et al. Impact of gauze-based NPWT on the patient and nursing experience in the treatment of challenging wounds. Int Wound J. 2010;7(6):448–455.
19. Blakely M, Weir D. The innovative use of Safetac soft silicone in conjunction with negative pressure wound therapy: three case studies. Poster presentation at the Symposium on Advanced Wound Care. Tampa, FL. April 28–May 1, 2007.
20. Dunbar A, Bowers DM, Holderness H Jr. Silicone net dressing as an adjunct with negative pressure wound therapy. Ostomy Wound Manage. 2005;51(11 suppl A):21–22.
21. Malmsjo M, Ingemansson R. Similar biological effects of green and black polyurethane foam in negative pressure wound therapy: green foam facilitates monitoring of wound status, bleeding and exudate. 20th Conference of the European Wound Management Association. Geneva, Switzerland. May 26–28, 2010.
