Effects of a Hydrofiber Dressing on Inflammatory Cells in Rat Partial-Thickness Wounds

Disclosure: This study was financially supported by grants from the Dutch Burns Foundation and ConvaTec, Deeside, United Kingdom. Introduction Wound healing starts with an inflammatory phase mediated mainly by neutrophils and macrophages. While the neutrophils are mainly responsible for host defense and cleansing of the wound, macrophages provide cytokines and growth factors that activate keratinocytes, fibroblasts, and endothelial cells to close the defect.[1] In the past, many approaches and wound dressings have been developed to improve the healing process, especially for deeper wounds, chronic wounds, and burn wounds. A moist environment is initially beneficial for the outgrowth of keratinocytes as was first described by Winter, et al.[2] This was confirmed by many other investigators using various types of dressings capable of creating a moist wound healing environment.[3–5] Aquacel® (ConvaTec, A Bristol-Myers Squibb Co., United Kingdom) is a hydrofiber wound dressing consisting of carboxymethyl cellulose fibers that possess a high fluid absorption capacity.[6] When applied to wounds, each fiber gels as it hydrates with wound exudate, resulting in a moist environment. In addition, the hydrofiber dressing has been shown to absorb and retain microorganisms in vitro.[7] The hydrofiber wound dressing is indicated for management of exuding wounds. These include chronic wounds, such as pressure and leg ulcers, and acute wounds, such as abrasions, lacerations, and incisions. Recently, the hydrofiber dressing was tested on patients with partial-thickness burns after wound debridement. It was observed that the clinical performance of the hydrofiber dressing resembled that of cadaver skin.[8] The hydrofiber dressings that adhered to the wounds were not changed during the entire course of wound healing in the majority of the patients. Only the additional cotton gauzes were replaced to check the adherence of the hydrofiber dressings. During the healing process, the fibers dry out due to evaporation, and the dressing could be easily removed like a dry “crust.” This could be done without disturbance to the new epidermis. In this noncomparative patient study,[8] the effects of the hydrofiber dressing on wound healing were in general good as analyzed using the Vancouver Scar Scale. The aim of the experiments described in the present study was to investigate possible mechanisms by which the hydrofiber dressing causes the observed clinical effects. Partial-thickness wounds were prepared on rats in which the effects of the hydrofiber dressing were compared to the effects of Jelonet™ (paraffin-impregnated gauze, Smith & Nephew, United Kingdom), a dressing that is also used for donor sites.[9] Materials and Methods Wound dressing materials. The hydrofiber dressing consists of 100-percent sodium carboxymethyl cellulose fibers with a low degree of carboxymethylation. The paraffin-impregnated cotton gauze is impregnated with paraffin BP (normal load), a greasy wax. Operation procedure. Male Wistar rats (60 animals) weighing 180 to 200g, 8 to 10 weeks old, were obtained from Harlan CPB (Zeist, The Netherlands). The animals were kept under routine laboratory conditions in accordance with the Dutch animal welfare guidelines. They had free access to food (normal rat chow, Hoppfarm, The Netherlands) and water. Approval of the Vrije Universiteit Medical Centre Animal Welfare Committee was achieved to perform the experiments. Animals were housed separately after operation. Temperature of the room was kept at 22?C with a 12/12 light-dark cycle. The animals were anesthetized using 0.05mL/100g Hypnorm® (fentanylcitrate 0.315mg/mL and fluanison 10mg/mL, Janssen, Beerse, Belgium) and 0.08mL/100g Dormicum® (midazolan 5mg/mL, Genton, Nijmegen, The Netherlands). These agents were injected subcutaneously. Thereafter, the animals were shaved and cleaned with chlorhexidine. A partial-thickness wound of 1x2cm was made on the back using a scalpel blade (No. 10, Swann Morton, Sheffield, United Kingdom). Wounds were covered with the hydrofiber dressing or the paraffin-impregnated gauze, which was fixed using hypoallergenic leukosilk tape (Leukosilk®, BDF Beiersdorf, Hamburg, Germany). After the operation, a single dose of 0.01mL/100g Temgesic® (0.3mg/mL buprenorfine, Shering Plough, Maarsen, The Netherlands) was given intramuscularly to diminish post-operative pain. Sixty rats were included in the experiment and sacrificed at 1, 2, 3, 4, 7, and 10 days after wounding. For each time point, five rats received the hydrofiber dressing and five others received the paraffin-impregnated gauze. Both types of dressings were totally covered with a secondary dressing (cotton gauze). This was further fixated with leukosilk tape, which was placed around the body of the animal to prevent it from removing the dressings. In the patient study,[8] the hydrofiber dressings adhered to the wounds in most cases and were not changed.[8] Therefore, the authors also did not change them on the rats. The choice of the leukosilk tape was dictated by observations made previously; it was the only material with which the rats were unable to interfere. The animals were able to remove other types of cover dressings and cause disturbance to the wounds. Animals were sacrificed using a mixture of O2/CO2 followed by CO2 inhalation. The leukosilk tape was removed carefully. After macroscopical observation, the wounds were excised and cut into three pieces. Thereafter, the tissue was frozen into liquid nitrogen in preparation for microscopical analyses. Microscopical analysis. Cryosections of 7mm thickness were prepared from each of the three tissue pieces of all rats and stained with the following procedures to study the wound healing process. Hydrofiber dressing adhesion to the wound was studied using phosphotungstic acid haematoxylin (PTAH) staining for fibrin. To investigate the involvement of fibronectin in the adhesion process, a rabbit anti-human antibody (Dako, Glostrub, Denmark) against fibronectin cross-reacting with rat fibronectin was used. Haematoxylin-eosin (H&E) staining was used to study the morphology of the wound and parameters, such as reepithelization and granulation tissue formation. The H&E sections were also used to assess whether each wound had the same depth. The antibody ED-2 was used to recognize mature macrophages (10) and HIS-48 (Serotec, Oxford, United Kingdom) to identify rat neutrophils. For each staining, five representative cryosections from the three pieces of tissue of each wound were evaluated. Quantitative analysis. A grid in the ocular of the microscope was used to quantify the numbers of HIS-48 positive (HIS-48+) cells per mm2 wound bed. The numbers of cells in five representative sections per rat were quantified. For each time point, the results were expressed as the mean number of cells of the five rats. Reepithelization and the amount of granulation tissue that had been formed were quantified on the H&E stained sections. The percentage of the wound bed that was covered with keratinocytes and the percentage of an area of 1mm2 underneath the new epidermis occupied by granulation tissue were determined using the ocular grid. Statistical differences between the hydrofiber dressing treated and the paraffin-impregnated gauze treated rats were determined using the student t-test, with pHistological staining procedures. For the PTAH staining, the sections were fixed in Bouin solution. After washing in tap water, the sections were incubated in five-percent Fe(NH4)2SO4 (Merck, Darmstadt, Germany) for 30 minutes. This was followed by a 10-minute incubation in five-percent oxalic acid solution (Merck) and further stained in haematoxylin with tungstophosphoric acid and KMnO4 (all from Merck). This staining procedure results in purple colored connective tissue, blue nuclei, and dark-blue fibrin. Haematoxylin and eosin were obtained from Gurr (BDH Ltd. Poole, United Kingdom). Sections were fixated in Baker’s formol. Immunocytochemistry. After 10-minute fixation in acetone, the slides were washed with phosphate-buffered saline and incubated for 45 minutes with the primary antibodies (ED-2, HIS-48, antifibronectin). After washing, this was followed by rabbit antimouse F(Ab)2 coupled with biotin (Dakopatts, Glostrup, Denmark) or swine anti-rabbit biotin for 45 minutes. Normal rat serum was added at a final dilution of 1:50 in order to block nonspecific binding. Thereafter, the slides were incubated for 30 minutes with streptavidin coupled to alkaline phosphatase (Vector, Burlingame, California). Labelled cells were stained with alkaline phosphatase substrate containing Napthol AS-BI phosphate (Sigma Chemicals, St Louis, Missouri) and New Fuchsine (Gurr). One millimeter of levamisole (Sigma) was added to block endogenous alkaline phosphatase activity. Labelled cells stain red in this procedure. Sections were counterstained using haematoxylin. Control slides, in which the primary antibody was omitted or replaced by an irrelevant antibody of the same isotype, did not show any reactivity. Results Macroscopic results. At the first time points, the gelled fibers were adherent on the wound whereas the paraffin-impregnated gauze was initially not adherent. The hydrofiber dressing was still adherent to the wounds up to Day 4. Thereafter, on Days 7 and 10, there was some variation in between the rats; the hydrofiber dressing dried out and detached on one out of five rats but was still moist and adherent on the other four rats. In some cases, the paraffin-impregnated gauze became incorporated into the wound. This was the case on 2 out of 5 rats at Day 4 and on 1 out of 5 rats at Day 7 after wounding. At Day 10, the paraffin-impregnated gauze was not adherent to any of the rats, and the wounds showed a moist appearance. The authors did not observe any signs of wound infection in the rats. Microscopic results. The wounds were excised with the hydrofiber dressing (in case it was still adherent) or paraffin-impregnated gauze (in case it was incorporated) still attached to the wound and then frozen into liquid nitrogen. By using cryosections, these attached dressings were not lost during the process of cutting and staining. Therefore, the authors were able to study the adherence of the hydrofiber dressing to the wound bed. Adherence was both by fibrin (Figure 1A) and fibronectin (Figure 1B). There was no dead space between the hydrofiber dressing and the wound bed. A crust with both fibrin and fibronectin was present on the wounds covered with paraffin-impregnated gauze. All the hydrofiber dressing fibers were fully gelled and the fibers did not dissolve, as can be observed in Figure 2. Effects on inflammatory cells. As the preparation method for the histological samples did not disturb the adhesion of the hydrofiber dressing to the wound bed, the authors were able to observe an interesting phenomenon. It appeared that there were many cells in between the fibers (Figure 2). These cells were mainly neutrophils as identified by morphology on H&E stainings and by positivity for the HIS-48 antibody. Some keratinocytes and ED2+ macrophages were also observed. The numbers of cells in between the fibers increased during the first three days after wounding. Thereafter, the wounds were almost completely reepithelized, concomitant with no further increase of cell numbers into the hydrofiber dressing. In contrast to the hydrofiber dressing on the wound, the wound bed itself contained relatively low numbers of neutrophils throughout the experimental period. On wounds covered with the paraffin-impregnated gauze, HIS-48+ neutrophils were present in the crust and in the wound bed. During the first days after wounding, the dermis contained large numbers of HIS-48+ cells. More cells were present in these wounds when compared to the dermis of wounds covered with the hydrofiber dressing (Figures 3A and B). When the authors quantified the numbers of HIS-48+ cells in the dermis, it appeared that at Days 1 and 2 after wounding, the numbers were significantly higher in the the paraffin-impregnated gauze-covered wounds (Figure 4) in comparison to the hydrofiber dressing covered wounds. At Days 3 and 4, the numbers of neutrophils in the dermis had decreased, both in the hydrofiber dressing and in the paraffin-impregnated gauze-covered wounds. At Day 7, a slight increase of neutrophils was observed in both groups. At all time points studied, few ED-2 macrophages were observed between the hydrofiber dressing fibers. There were more macrophages present in the crust on the wounds covered with the paraffin-impregnated gauze. There were no striking differences between the hydrofiber dressing and the paraffin-impregnated gauze in the numbers of ED-2 positive macrophages present in the dermal parts of the wounds. Therefore, the authors did not quantify the numbers of cells present. Both in the hydrofiber dressing and the paraffin-impregnated gauze-covered wounds, the number of macrophages peaked at Day 4 after wounding. Effects on the wound healing process. Epithelization. The wounds were prepared using a scalpel blade, removing the epidermis and only part of the dermis. Hair follicles are not completely removed in this way, therefore reepithelization takes place from the follicles and from the borders of the wound. In the H&E-stained sections, the authors first assessed the depth of the wounds. Although there were small differences within the wounds, all wounds were of partial thickness. Thereafter, the percentage of the wound bed that was covered with keratinocytes was determined using the ocular grid. Significant differences in wound depth were not observed in any of the wounds. Reepithelization of the wounds was quantified using the grid in the ocular of the microscope. There was no clear migration or proliferation of the keratinocytes at Day 1, only activation as the nuclei of the cells were euchromatic. Therefore, this time point was not quantified. At Day 2, reepithelization was significantly (pGranulation tissue. The H&E stained sections were used to examine the formation of granulation tissue. This started at Day 3 post-wounding, both in wounds covered with the hydrofiber dressing and the paraffin-impregnated gauze. Using the ocular grid, the percentage of an area of 1mm2 underneath the new epidermis, which was covered with granulation tissue, was quantified. The average areas of granulation tissue were not statistically different, however, as shown in Figure 6. In some cases, a few hydrofiber dressing fibers were incorporated in the granulation tissue at 7 and 10 days after wounding. Some macrophages were present near these fibers. On Day 10, the number of incorporated fibers was lower, indicating that the fibers did not induce further inflammation. The authors did not observe giant cell formation. Discussion When used for chronic wounds, the hydrofiber dressings are in most cases changed at regular intervals, dependent on the wound situation. In the treatment of partial-thickness burns, however, the hydrofiber dressing showed a good adherence to the wound and was left on the wounds until it came off spontaneously.8 At that time, the fibers had dried out completely and could be easily removed from the reepithelized wounds as a dry “crust.” In the present study, using an acute, partial-thickness wound rat model, histological stainings show that the hydrofiber dressing adhered to the wound by fibrin and fibronectin. The hydrofiber dressing adhered to the wounds without dead space. Furthermore, the hydrofiber dressing absorbed neutrophils into its hydrated fiber structure. The presence of these cells in the hydrofiber dressing could be the result of active migration of cells or passive movement. Due to the uptake, the number of neutrophils in the dermal part of the wound is significantly lower when compared to wounds covered with the paraffin-impregnated gauze. There were no differences in macrophage numbers. The uptake of neutrophils by the hydrofiber dressing fibers reduces the number of these cells in the wound bed itself and may, therefore, contribute to improved healing. Although neutrophils are important in the control of infection by microorganisms, their enzymes can also harm the surrounding tissue.[11,12] By trapping a significant number of these neutrophils in the dressing fibers, the presence of tissue-damaging enzymes in the wound tissue may be reduced. Later on, the new epithelium itself will protect the underlying tissue. The fibers gel after contact with wound exudate and seal the wound, keeping it moist. This moist environment is initially beneficial for the outgrowth of the keratinocytes.[2–5] Reepithelization was significantly faster at Day 2 after wounding when compared to wounds dressed with the paraffin-impregnated gauze. Due to the occlusive nature of the leukosilk tape, wounds covered with the paraffin-impregnated gauze were also moist. This suggests that the beneficial effect of the hydrofiber dressing on the outgrowth of the epithelium is not only caused by the moist environment but also by its uptake of neutrophils. In contrast, the paraffin-impregnated gauze is sometimes incorporated into the wound bed, resulting in loss of dermal tissue. At Day 4, wounds were almost completely re-epithelized in both groups, but the rate of epithelization was reduced again at Day 7. The wounds remained moist and showed some maceration, especially the hydrofiber dressing-covered wounds. The hydrofiber dressing did not become dry and nonadherent as was observed in the patient study.[8] Apparently, the exudate present in the fibers could not evaporate properly. When the new epidermis is closed, the keratinocytes can only differentiate normally if the wound is allowed to dry gradually. Presumably, the leukosilk tape (which the authors used for fixation and which is the only material rats are not able to interfere with) seems to be too occlusive to achieve this purpose. Due to this unexpected technical difficulty, it was not possible to assess the influence of both wound dressings on the final outcome of the healing process in this experiment. These results, however, emphasize the importance of the covering materials used to keep the dressings on the wound, especially when using the hydrofiber dressing, indicating that these should not be too occlusive. Outgrowing epithelium needs a moist environment followed by a more dry environment for proper differentiation and keratinization.[13] In conclusion, the hydrofiber dressing adheres to the wound by fibrin and fibronectin. The fibers gel after contact with wound exudate. The uptake of neutrophils by the fibers reduces the numbers of these cells in the wound bed. Most probably, this creates more optimal circumstances for the outgrowth of the keratinocytes. When additional dressings, such as cotton gauzes, that allow fibers to dry and detach after the new epidermis is closed are used the hydrofiber dressing can give good results as shown for partial-thickness burns.[8]