TY - JOUR
AU - PhD, Robert J Christy,
AB - Abstract While early excision and grafting has revolutionized burn wound care, autologous split-thickness skin grafts are sometimes unavailable. Tissue-engineered skin substitutes have generated great interest but have proven inadequate. Therefore, the development of novel biomaterials to replace/augment skin grafting could improve burn patient outcomes. Herein, we establish the effects of debridement on deep-partial thickness burns and subsequently examine the effects of 3 different hydrogels on healing. Burns were created on the dorsum of pigs and 4 days after, the eschar was either left intact or debrided for treatment with collagen, PEGylated fibrinogen (PEG-fibrin) or PEGylated autologous platelet-free plasma (PEG-PFP) hydrogels. Wounds were photographed, scored, and biopsied for histology on postburn days 7, 10, 14, and 28. Compared with nondebrided wounds, debridement improved wound color and suppleness but accelerated contraction. Debridement also significantly reduced the number of neutrophils in the wound bed at days 10 and 14 postburn. Treatment with any hydrogel transiently mitigated contraction, with the PEG-fibrin group displaying less contraction on day 28. All hydrogels were visible histologically for up to 10 days, with significant cellular and blood vessel infiltration observed in PEG-fibrin hydrogels. Collagen and PEG-fibrin hydrogels reduced neutrophils and macrophages in surrounding granulation tissue on day 7, while PEG-fibrin hydrogels contained less immune cells. These data suggest that a single hydrogel application at the time of debridement has immunomodulatory properties that aid in wound healing. Ultimately, these hydrogels may be combined with other biomaterials, cells, or biologics for replacing/augmenting skin substitutes. Burns are an exceptionally common injury with approximately 11 million people worldwide seeking treatment annually.1 Although superficial burns heal spontaneously within 2 to 3 weeks, deeper second-degree and third-degree wounds require surgical debridement and grafting. Indeed, since this realization by Janzekovic2 in the 1970s, burn wounds have become increasingly survivable and outcomes have improved. However, there are still complications experienced by burn patients due to inadequate healing outcomes, including excessive scarring and associated problems (e.g., pain, reduced range of motion, psychological stress) that lead to impaired quality of life and high medical treatment costs.3 Mechanistically, it has been shown that debridement ameliorates inflammation-mediated wound progression by altering the initial innate immune response to burn injury.4 However, to prevent infection, the open wound that exists after surgical debridement requires coverage with grafts. This is normally performed with autografts that display minimal rejection due to their inherent biocompatibility. Unfortunately, autografts not only create an additional wound, they are not always feasible for patients with large TBSA burns.5 To combat this, tissue-engineered substitutes (ranging from epidermal, dermal, and even complete skin equivalents) have garnered considerable attention.6 Skin equivalents have the potential to aid in graft integration or replace the need for autografting altogether. Despite early successes, skin substitutes are associated with high costs and have proven to be inadequate, in part, due to their immunogenicity6,7 and lack of vascularization.8,9 A promising avenue for improving immune acceptance and vascularization are hydrogel-based therapies. These biomaterials contain 70 to 90% water and provide a moist environment to improve the rate of wound healing.10 Moreover, hydrogels are highly versatile biomaterials that can be engineered to possess tunable mechanical and biochemical properties, incorporate various cell types, and stimulate tissue behaviors ideal for appropriate wound healing.11 Furthermore, natural polymers are an attractive source for generating hydrogels due to their biocompatibility, ability to direct cell behavior, and positive effects on tissue repair. Specifically, collagen and fibrin are 2 naturally occurring biopolymers, which have been examined for their ability to aid cutaneous wound healing.12,13 Collagen type I is the predominant structural protein in human skin and is the major component of several commercially available dressings and skin equivalents (e.g., Integra® and Alloderm®). Fibrin is ubiquitous during the early phases of wound repair, comprising much of the provisional matrix that supports hemostasis, inflammatory cell recruitment, angiogenesis, initiation granulation tissue deposition.4 Indeed, fibrin-based wound therapies, (e.g., fibrin glue) have experienced initial successes in terms of blood clot formation and angiogenesis.14 With the added advantages of being autologous, plasma has also generated interest as a source of fibrinogen for use in potential treatment of burn wounds.15 While the role of endogenous fibrin and collagen on the inflammatory and proliferative phases of wound healing has driven their development, the immunomodulatory properties of their exogenous application have not been fully investigated. To this end, we have employed a porcine, deep partial-thickness burn wound model with sharp debridement to examine how application of fibrin and collagen-based hydrogels alter inflammation and healing. Swine models are regarded as the closest surrogate for human studies, as pig skin closely resembles human skin in both structure (epidermal/dermal thicknesses, hair distribution, and blood vessel patterns) and wound healing (reepithelialization as opposed to contraction).16,17 After first determining the effect of late debridement on the innate immune response to burns, we hypothesized that collagen- and fibrin-based hydrogels would alter the initial inflammatory phase, leading to long-term effects on wound healing. We demonstrate the successful delivery of PEGylated-fibrinogen (PEG-fibrin) hydrogels via a dual-syringe applicator in situ. Furthermore, we show that fibrin-based hydrogels possess immunomodulatory effects that prevent contraction by incorporation within wound tissue. METHODS Animals Female Yorkshire swine (n = 5) (Midwest Research Swine, Gibbon, MN) weighing 43.4 ± 1.7 kg at the time of thermal injury were used in this study. Animals had ad libitum access to food and water and were allowed to acclimate to the facilities for at least 7 days before anesthesia. This protocol was approved by the Institutional Animal Care and Use Committee, U.S. Army Institute of Surgical Research. This study has been conducted in compliance with the Animal Welfare Act, the implementing Animal Welfare Regulations, and the principles of the Guide for the Care and Use of Laboratory Animals. Anesthesia On the day before anesthesia, animals were fasted and a transdermal fentanyl patch (100 µg/hr) was placed on 1 ear of the pig for analgesia. Before anesthesia for the burn and debridement procedures (days 0 and 4, respectively), the pig was premedicated with an intramuscular (IM) injection of glycopyrrolate (0.01 mg/kg, IM) to minimize salivation and bradycardia. Anesthesia was induced with an IM injection of tiletamine-zolazepam (Telazol, 6 mg/kg). Pigs were then intubated with an endotracheal tube and placed on an automatic ventilator adjusted to maintain an end tidal PCO2 of 40 ± 5 mm Hg. Anesthesia was maintained with 1 to 3% isofluorane in 100% oxygen. On postburn days 7, 10, 14, and 28, each pig was briefly anesthetized in order to obtain punch biopsies and photographs of wound beds (see below). For these timepoints, animals were sedated with ketamine (10–20 mg/kg, IM) and maintained under mask anesthesia (3–5% isofluorane). Thermal Injury Before burning, hair was removed from the dorsum of the swine with clippers, and the skin was sterilized with chlorhexidine. A room-temperature 3-cm brass cylinder template was used to mark burn placement, which was 1.5 cm from the spine, with 2.5 cm between adjacent burns. The perimeter of each wound area was tattooed with an electric tattoo machine (Spaulding and Rogers, Voorhesville, NY) before thermal injury. Deep partial-thickness burn wounds were produced with a device described previously.18 Briefly, 3-cm brass probes with stainless steel posts (to allow for handling) were heated in a warm bath incubator to 100°C (Fig. 1A). These probes lock into a spring-loaded Delrin®-insulated device (Fig. 1B) that ensures a consistent pressure when probes are applied to the animal (Fig. 1C). In this study, the heated brass probes were applied to the dorsum of the animal within the tattoo markings for ~25 seconds (Fig. 1D), a time previously shown to create deep partial-thickness burns.19 Five animals were used to create a total of 56 wounds. Wounds were then covered with a nonadherent gauze (Telfa, Tyco Healthcare, Mansfield, MA) secured in place with Elastikon surgical tape (Johnson and Johnson, New Brunswick, NJ) and finally covered with antibiotic Ioban™ (3M, St. Paul, MN). Figure 1. View largeDownload slide Burn instrumentation and design. (A) Three centimeter brass probes were heated in a dry bath incubator custom-fitted with hard anodized aluminum blocks. (B) The custom-made device used to retrieve the brass probes allows for a constant pressure to be applied, independent of user-applied force. (C) An example of application of a heated probe to the dorsum of pigs. (D) The dorsum of a pig showing the burn wounds within the tattooed area. Perforated line in C and D indicate location of the spine. Figure 1. View largeDownload slide Burn instrumentation and design. (A) Three centimeter brass probes were heated in a dry bath incubator custom-fitted with hard anodized aluminum blocks. (B) The custom-made device used to retrieve the brass probes allows for a constant pressure to be applied, independent of user-applied force. (C) An example of application of a heated probe to the dorsum of pigs. (D) The dorsum of a pig showing the burn wounds within the tattooed area. Perforated line in C and D indicate location of the spine. Hydrogel Preparation Collagen Hydrogel. Three milliliters of collagen hydrogels were prepared the day before treatment using previously published protocols.20 Briefly, Dulbecco’s phosphate buffered saline (Sigma, St. Louis, MO) and 1N NaOH were added to type 1 collagen (5 mg/mL; Trevigen, Gaithersburg, MD) to achieve a final collagen concentration of 4.5 mg/mL at a pH of 6.8 to 7.0. Collagen gels were allowed to polymerize for 30 to 40 minutes at 37°C, 5% CO2. PEG-Fibrin Hydrogel. PEG-fibrin hydrogels were prepared as previously described, with modifications for in situ delivery.21 Succinimidyl glutarate–modified polyethylene glycol (NOF America Corporation, White Plains, NY) was dissolved with tris-buffered saline (pH, 7.8; Sigma-Aldrich) to a concentration of 8 mg/mL and subsequently sterilized by passing through a 0.22-µm filter. The PEG stock solution was added to 40 mg/mL fibrinogen (Sigma-Aldrich) in a 1:10 molar concentration ratio and incubated for 20 minutes at 37°C. Fibrinogen-PEG solution mixture was drawn into a 10-mL syringe of a Fibrijet dual syringe applicator (Nordson Micromedics Inc., St. Paul, MN). For gelation, a stock solution of 1000 U/mL thrombin (Sigma-Aldrich) in deionized water was diluted with 40 mM of calcium chloride to a final working thrombin concentration of 12.5 U/mL, which was drawn into the separate 1-mL syringe of the dual syringe applicator. PEGylated Platelet-Free Plasma Hydrogel. On the day of thermal injury (i.e., 4 days before treatment), ~14 mL of blood was collected into vacutainer tubes containing sodium citrate (BD Biosciences, Franklin Lakes, NJ) via a percutaneous stick of the vena cava. The blood was centrifuged at 4300 ×g for 10 minutes at room temperature (21–23°C) with no brake. After centrifugation, the supernatant (platelet-free plasma [PFP]) layer was carefully collected without touching the buffy coat interface and subsequently stored at −20°C until the day of treatment. To prepare PEG-PFP hydrogels, 1.375 mL of the 8 mg/mL PEG stock solution described above was added to 8.625 mL of PFP and incubated for 20 minutes at 37°C. The plasma-PEG solution was drawn into a 10-mL syringe of a Fibrijet dual syringe applicator similar to the PEG-fibrin. The thrombin activator stock solution (1 KU/mL in deionized water) was diluted with 40-mM calcium chloride to a final working concentration of 12.5 U/mL and was drawn into a 1-mL syringe of the dual syringe applicator. Surgical Debridement, Treatment, and Biopsies On day 4 postinjury, dressings were removed to expose the wounds. In the first set of experiments, half of the wounds on each pig underwent surgical debridement. Prominent zones of hyperemia served as the initial point of excision (Fig. 2A). Surgical excision was performed tangentially with a scalpel until viable tissue was reached, as indicated by bleeding. Wounds were then individually covered as described above with the exception of using a hydrocolloid dressing (Duoderm, ConvaTec, Inc., Skillman, NJ) in place of Telfa to prevent desiccation of the wound bed. In the second set of experiments, all wounds were debrided on day 4 postburn as described above. Wounds were randomized to receive no hydrogel treatment (debrided), collagen hydrogel (approximately 3 mL), PEG-fibrin, or PEG-PFP. Randomization was performed to ensure each treatment group was represented equally along the craniocaudal axis of the animal. Collagen gels were added with a sterile spatula, while the PEG-fibrin and PEG-PFP hydrogels were applied by filling the wound using the dual syringe applicator, resulting in a total volume of approximately 2.2 mL per wound. Following treatment application, wounds were individually covered with Duoderm, as described above. Figure 2. View largeDownload slide Gross morphology of burn wounds over time. (A) Photo documentation of representative debrided and nondebrided wounds immediately postburn (day 0), immediately postdebridement (day 4), and on postburn days 7, 10, 14, and 28. Asterisks indicate biopsy sites that were taken at the previous timepoint. (B) Wound scoring on day 28 revealed a significantly lower scores after debridement (red bars) compared with nondebrided wounds (black bars). (C) Tattoo markings were traced at each timepoint (dashed lines on days 0, 7, and 28 in [A]) and normalized to original day 4 area. Quantification of wound contraction reveals decreased wound area in debrided wounds (red lines) compared with nondebrided wounds (black lines).*P < 0.05, **P < 0.01. N = 10/group. Figure 2. View largeDownload slide Gross morphology of burn wounds over time. (A) Photo documentation of representative debrided and nondebrided wounds immediately postburn (day 0), immediately postdebridement (day 4), and on postburn days 7, 10, 14, and 28. Asterisks indicate biopsy sites that were taken at the previous timepoint. (B) Wound scoring on day 28 revealed a significantly lower scores after debridement (red bars) compared with nondebrided wounds (black bars). (C) Tattoo markings were traced at each timepoint (dashed lines on days 0, 7, and 28 in [A]) and normalized to original day 4 area. Quantification of wound contraction reveals decreased wound area in debrided wounds (red lines) compared with nondebrided wounds (black lines).*P < 0.05, **P < 0.01. N = 10/group. Photodocumentation of Wound Healing On postburn days 7, 10, 14, 21, and 28, dressings were removed and digital pictures of each wound (Nikon D3000, Nikon Inc. Melville, NY) were taken to assess contraction. Each image was acquired with a 4-sided ruler for calibration during analysis. Image analysis was performed with ImagePro software version 6.2 (Media Cybernetics, Bethesda, MD) by spatial calibration and use of the polygon measurement tool. The tattoo area on each follow-up day was normalized to the tattoo area of the same wound on day 4 (i.e., day of debridement) to determine percent wound contraction for that particular wound. Also, on day 28, each wound was assessed by 2 independent, blinded scorers for pigmentation, pliability, and thickness on a scale from 1 (normal skin) to 10 (worst scar imaginable). Histological Analysis and Immunohistochemistry On postburn days 7, 10, 14, and 28, 8-mm diameter punch biopsies (minimum n = 5 samples/group/timepoint) were taken and fixed in 10% buffered formalin for 48 hours. Each wound was biopsied 2 times at different locations—once on the lateral aspect and once on the medial aspect of the wound. Importantly, each treatment group underwent the same biopsy schedule wherein the same number of biopsies (and the same location within the wound) was taken in each treatment group. Biopsies were then processed, embedded in paraffin, and cut into 6-μm thick cross-sectional slices. Slides were deparaffinized in xylene, rehydrated to water, and stained with Masson’s trichrome (Masson Kit, Sigma-Aldrich). Immunohistochemistry was performed as follows: heat-mediated antigen retrieval with 0.01 M citrate buffer at 95 to 98°C for 15 minutes was used for all antibodies, except for the MAC387 antibody recognizing Calprotectin, which underwent enzymatic antigen retrieval with 0.6 U/mL Proteinase K (SigmaAldrich) for 20 minutes at 37°C. Endogenous peroxidase activity was blocked with 0.3% H2O2 for 20 minutes at room temperature. Nonspecific ImmunoglobulinG was blocked with 10% horse serum in Hanks’ balanced salt solution for 30 minutes at room temperature. Then tissue sections were incubated with the following primary antibodies diluted in 3% horse serum: α-smooth muscle actin (α-SMA; Abcam, Cambridge, MA; ab7817, mouse monoclonal, 1:50 dilution), myeloperoxidase (MPO) (Hycult, Plymouth Meeting, PA; HP9048, rabbit polyclonal, 1:50 dilution), MAC387 (Abcam, ab22506, mouse monoclonal, 1:100 dilution), for 60 minutes at room temperature or overnight at 4°C. Following primary antibody incubation, slides were washed with Hanks’ balanced salt solution and treated with either biotinylated anti-mouse (Vector Labs, Burlingame, CA) or biotinylated anti-rabbit (Vector Labs) secondary antibodies for 60 minutes. Finally, immunostaining was completed with 30-minute incubation with Vectastain-RTU Kit solution (Vector labs, PK-7100) followed by 5 to 10–minute incubation with ImmPACT DAB (Vector Labs) at room temperature. Slides were then counterstained with hematoxylin and dehydrated for cover slipping. Images of entire wound biopsies stained for MPO and MAC387 were obtained using an AxioScan Z1 slide scanner (Carl Zeiss, Inc., Thornwood, NY) at 10× magnification. After image export, the entire biopsy was used for quantification of MPO and MAC387 expression, which was performed with ImagePro software version 6.2 (Media Cybernetics, Rockville, MD). Two regions of interest were traced in each histological section: one of the forming granulation tissue (excluding the hypodermal layer and surrounding normal skin) and one of any visible hydrogel (apparent on days 7 and 10). Isolation of the blue color channel was performed to account for the darkest staining areas. The color counting tool was utilized to set a histogram range from 0 to 48, and limit the size of objects limited to be between 10 and 250 pixels. A count was performed and the total number of positively stained pixels in the region of interest was counted and subsequently normalized to the total number of pixels in the wound bed. Statistical Analysis Statistical evaluations were performed using GraphPad Prism software (GraphPad Software Inc., San Diego, CA). For all analyses, either a 1 or 2-way analysis of variance (ANOVA) with t tests or Bonferonni post hoc testing being used only if an effect of treatment was found. For the analysis of immune cells within hydrogels, only the treatment groups were included due to the lack of hydrogel in no debridement and no-treatment groups. P values less that 0.05 were considered significant in all cases. Unless otherwise stated, all results are expressed as the arithmetic mean ± SEM. RESULTS The Effect of Debridement on Wound Healing Representative pictures from nondebrided and debrided wounds are shown in Figure 2A. For the first 4 days postburn, wounds appeared pale with an increasingly prominent zone of hyperemia, characteristic of deep burns (Fig. 2A). By day 7, noticeable granulation tissue had formed after debridement, while the eschar in nondebrided wounds was necrotic, with a yellowish-brown appearance. By day 10, this difference was even more pronounced, with a substantial acceleration of granulation tissue deposition in the debrided wounds. By day 14, nondebrided wounds still showed areas of necrotic tissue; however, red granulation tissue was also present. Additionally, debridement improved overall quality (1 = normal skin, 10 = worst scar imaginable; Fig. 2B), as scores for pigmentation, pliability, and thickness were significantly higher for nondebrided wounds (P < 0.05). Histological analysis of reepithelialization on biopsies that spanned the entire wound bed (day 28) showed that nondebrided wounds were 76.54 ± 5.87% reepithelialized, compared with 94.37 ± 3.54% reepithelialized after debridement (data not shown). Debridement also produced a slight, but significant increase in surface area at day 7 (Fig. 2C) owing to the eschar removal, but subsequently contracted considerably more than nondebrided wounds at the 14- and 21-day timepoints. On day 28 postburn, however, no difference was seen as nondebrided and debrided wounds were contracted to 77.30 ± 2.99% and 76.65 ± 1.84% of the day 4 burn area, respectively. The Effect of Debridement on Immune Cell Infiltration To characterize the changes in neutrophil and macrophage infiltration due to debridement, tissue sections were immunostained using antibodies against MPO and MAC387, respectively. On day 7 postburn, there was no difference in the amount of neutrophils due to debridement as shown in Figure 3. However, the neutrophil population subsided in debrided wounds by day 10 (0.30 ± 0.06% of pixels) when compared with nondebrided wounds (0.93 ± 0.39% of pixels; P < 0.05). While this difference was maintained on day 14 (0.19 ± 0.08% of pixels in debrided wounds vs 1.13 ± 0.54% of pixels for nondebrided wounds, P < 0.05), the amounts were similar by day 28. This same pattern of immune cell staining was seen with macrophages when immunohistochemistry to MAC387 was performed (Fig. 4). While similar levels of macrophages in both groups were apparent at day 7, they decreased more quickly and levels tended to be lower at days 10 and 14 after debridement. Interestingly, macrophages identified at day 28 tended to be present below the neo-epidermis in both groups, as well as in the epidermis adjacent to inflamed tissue, consistent with other reports of chronic skin inflammation.22 Figure 3. View largeDownload slide Quantification of immunohistochemistry to MPO. Representative MPO immunostaining of whole biopsies (center) throughout the course of the experiment, with the associated high magnification images (sides) outlined. Two-way ANOVA revealed an overall effect of debridement (P = 0.0278), with post hoc testing showing lower MPO expression in debrided wounds (red bars) compared with nondebrided wounds (black bars) (*P < 0.05). N = minimum of 5 biopsies/group/timepoint. MPO, myeloperoxidase; ANOVA, analysis of variance. Figure 3. View largeDownload slide Quantification of immunohistochemistry to MPO. Representative MPO immunostaining of whole biopsies (center) throughout the course of the experiment, with the associated high magnification images (sides) outlined. Two-way ANOVA revealed an overall effect of debridement (P = 0.0278), with post hoc testing showing lower MPO expression in debrided wounds (red bars) compared with nondebrided wounds (black bars) (*P < 0.05). N = minimum of 5 biopsies/group/timepoint. MPO, myeloperoxidase; ANOVA, analysis of variance. Figure 4. View largeDownload slide Quantification of immunohistochemistry to MAC387. Representative MAC immunostaining of whole biopsies (center) throughout the course of the experiment, with the associated high magnification images (sides) outlined. Two-way ANOVA revealed an overall effect of debridement (P = 0.0322), with post hoc testing showing lower MAC387 expression in debrided wounds (red bars) compared with nondebrided wounds (black bars) (*P < 0.05). N = minimum of 5 biopsies/group/timepoint. Figure 4. View largeDownload slide Quantification of immunohistochemistry to MAC387. Representative MAC immunostaining of whole biopsies (center) throughout the course of the experiment, with the associated high magnification images (sides) outlined. Two-way ANOVA revealed an overall effect of debridement (P = 0.0322), with post hoc testing showing lower MAC387 expression in debrided wounds (red bars) compared with nondebrided wounds (black bars) (*P < 0.05). N = minimum of 5 biopsies/group/timepoint. Hydrogel Application After determining the effect of debridement on wound healing and the immune response in this model, we then applied hydrogels directly to debrided burns (Fig. 5). The opaque collagen hydrogel is shown placed within the debrided wound bed (Fig. 5A). The PEG-fibrin and PEG-PFP hydrogels were applied and polymerized in situ using a dual-syringe applicator (Fig. 5B, see video, Supplemental Digital Content, http://links.lww.com/BCR/A98), with the final appearance of the PEG-PFP hydrogel shown in Figure 5C. A supplementary movie shows application of the PEG-fibrin hydrogel, which polymerized in roughly 10 to 20 seconds. Two-way ANOVA revealed a significant effect of hydrogels on contraction (Fig. 5D). Post-hoc testing showed that all hydrogels transiently increased wound area early after debridement, but only PEG-fibrin hydrogels mitigated contraction of the wound for the duration of the study. At 28 days, wound areas were 81.19 ± 2.21%, 85.45 ± 2.58%, and 78.08 ± 1.79% of the day 4 burn area for the collagen, PEG-fibrin, and PEG-PFP groups, respectively. In addition, there was a tendency for PEG-fibrin treatment to lower thickness and pliability scores (Fig. 5E); however, this difference did not reach significance. Figure 5. View largeDownload slide The effects of hydrogel application on wound contraction. (A) Collagen hydrogels applied to the wound after debridement on postburn day 4. PEG-fibrin hydrogels (B) and PEG-PFP hydrogels (C) are applied in situ with a dual-syringe applicator and conform to the edges of the wound bed. (D) Quantification of wound contraction using tattoo markings reveals an overall effect of hydrogel application (P < 0.0001) as determined by two-way ANOVA, with post hoc testing showing less contraction in PEG-fibrin (*) and collagen (@) and PEG-PFP (^) hydrogel groups (P < 0.05, P < 0.01, P < 0.001) compared with debrided but untreated wounds. (E) A trend for hydrogel treatment to improve wound scores did not reach statistical significance. N = minimum 9 wounds/group. Figure 5. View largeDownload slide The effects of hydrogel application on wound contraction. (A) Collagen hydrogels applied to the wound after debridement on postburn day 4. PEG-fibrin hydrogels (B) and PEG-PFP hydrogels (C) are applied in situ with a dual-syringe applicator and conform to the edges of the wound bed. (D) Quantification of wound contraction using tattoo markings reveals an overall effect of hydrogel application (P < 0.0001) as determined by two-way ANOVA, with post hoc testing showing less contraction in PEG-fibrin (*) and collagen (@) and PEG-PFP (^) hydrogel groups (P < 0.05, P < 0.01, P < 0.001) compared with debrided but untreated wounds. (E) A trend for hydrogel treatment to improve wound scores did not reach statistical significance. N = minimum 9 wounds/group. Sections from biopsies of hydrogel-treated burns were stained with Masson’s trichrome (Fig. 6). Debrided wounds without hydrogel treatment appeared with a thick fibrinaceous crust on both days 7 (Fig. 6A) and 10 (Fig. 6E). On day 7, all hydrogels were apparent within the wound bed (Fig. 6D); however, PEG-fibrin hydrogels appeared deeper within the wound bed. By day 10, collagen hydrogels (Fig. 6F) appeared superficial to the tissue and were not well integrated within the wound bed, whereas PEG-fibrin hydrogels (Fig. 6G) integrated very well, with 2 distinct zones: a superficial, less-cellularized layer infiltrated with blood vessels, and a deeper cell-rich layer. Histological analysis of wounds treated with PEG-PFP showed less remaining hydrogel than collagen and PEG-fibrin on day 10 (Fig. 6H). However, some PEG-PFP–treated wound showed evidence of reepithelialization at day 10, which was not apparent in other groups. Figure 6. View largeDownload slide Histological appearance of hydrogels in the wound bed. Untreated wounds exhibited a fibrinaceous eschar on days 7 and 10. Collagen hydrogels were apparent on day 7 but did not appear well adhered to the tissue on day 10. In contrast, PEG-fibrin gels showed deeper integration on day 7, and by day 10, the gel was fully incorporated, with 2 distinct layers: 1) a deeper, more cellular layer and 2) a superficial, less cellular layer. Blood vessels (arrows) formed between the 2 layers. While the PEG-PFP group appeared similar to PEG-fibrin on day 7, the gel is mostly absent by day 10, although a leading edge of reepithelialization is present (asterisk). Scale bars for the entire biopsy are 1 mm, smaller inset for day 10 images is 0.5 mm, and higher magnification images are 50 µm. Figure 6. View largeDownload slide Histological appearance of hydrogels in the wound bed. Untreated wounds exhibited a fibrinaceous eschar on days 7 and 10. Collagen hydrogels were apparent on day 7 but did not appear well adhered to the tissue on day 10. In contrast, PEG-fibrin gels showed deeper integration on day 7, and by day 10, the gel was fully incorporated, with 2 distinct layers: 1) a deeper, more cellular layer and 2) a superficial, less cellular layer. Blood vessels (arrows) formed between the 2 layers. While the PEG-PFP group appeared similar to PEG-fibrin on day 7, the gel is mostly absent by day 10, although a leading edge of reepithelialization is present (asterisk). Scale bars for the entire biopsy are 1 mm, smaller inset for day 10 images is 0.5 mm, and higher magnification images are 50 µm. Observed differences in wound contraction were further explored with immunostaining for α-SMA expression on days 10 and 14 (Fig. 7). In debrided wounds, earlier and more diffuse SMA staining was seen, particularly on the edges of the wound bed. This trend was similar with more ubiquitous staining on day 14. On both days 10 and 14, PEG-fibrin–treated wounds displayed reduced expression of SMA. Treatment with all hydrogels seemed to increase the amount of granulation tissue on day 10 compared with debridement alone. There was also a trend for PEG-PFP–treated wounds to display increased reepithelialization on day 14 (data not shown); however, this was not quite significant (P = 0.058). Figure 7. View largeDownload slide α-Smooth muscle actin (SMA) staining on days 10 and 14 from wound biopsies. More widespread SMA expression is seen after debridement, with increased granulation tissue deposition after hydrogel treatment starting on day 10. The amount of SMA expression in deeper areas of granulation tissue is less in the hydrogel-treated groups compared with untreated wounds. Scale bars for the entire biopsy are 1 mm and for higher magnification images are 50 µm. Figure 7. View largeDownload slide α-Smooth muscle actin (SMA) staining on days 10 and 14 from wound biopsies. More widespread SMA expression is seen after debridement, with increased granulation tissue deposition after hydrogel treatment starting on day 10. The amount of SMA expression in deeper areas of granulation tissue is less in the hydrogel-treated groups compared with untreated wounds. Scale bars for the entire biopsy are 1 mm and for higher magnification images are 50 µm. The Effect of Hydrogels on Immune Cell Infiltration To address the immunomodulatory effects of these hydrogels, we performed quantification of MPO and MAC 387 expression in hydrogel-treated wounds (Fig. 8). Two-way ANOVAs revealed a significant effect of the type of hydrogel and time for both MPO (Fig. 8A) and MAC387 (Fig. 8B) expression within newly formed granulation tissue. Post hoc testing, however, only revealed a significant difference of the collagen and PEG-fibrin hydrogels for mitigating infiltration of neutrophils and macrophages on day 7. The expression of MPO and MAC within the hydrogels (combination of days 7 and 10) were also examined (Fig. 8C). There was significantly less infiltration of MPO- and MAC-positive immune cells within the PEG-fibrin hydrogels, as compared with collagen and PEG-PFP hydrogels. Figure 8. View largeDownload slide The effect of hydrogel application on immune cells. Quantification of the amount of positive myeloperoxidase (A) and MAC387 (B) pixels in granulation tissue. Two-way ANOVA revealed that treatment with collagen and PEG-fibrin hydrogels reduced these cells at day 7 compared with untreated debrided wounds (*P < 0.05). N = minimum of 4 biopsies/group/timepoint. (C) Analysis of immune cells within the hydrogels from early timepoints (7 and 10 days) shows a significant decrease in the PEG-F hydrogels. (*P < 0.05). N = 9/group/stain. Figure 8. View largeDownload slide The effect of hydrogel application on immune cells. Quantification of the amount of positive myeloperoxidase (A) and MAC387 (B) pixels in granulation tissue. Two-way ANOVA revealed that treatment with collagen and PEG-fibrin hydrogels reduced these cells at day 7 compared with untreated debrided wounds (*P < 0.05). N = minimum of 4 biopsies/group/timepoint. (C) Analysis of immune cells within the hydrogels from early timepoints (7 and 10 days) shows a significant decrease in the PEG-F hydrogels. (*P < 0.05). N = 9/group/stain. DISCUSSION The current clinical standard of care treatment for severe burns is skin autografting, which is not always feasible and is associated with donor-site morbidity. To overcome these problems, several temporary skin substitutes have been engineered and are commercially available; however, none have proven to give satisfactory cosmetic results in patients with severe burns.6,23 A major consideration of these tissue-engineered products is complications due to the immune response, which has led to the use of acellular biomaterials. As the normal process of wound healing involves a tightly regulated immune response, the immunomodulatory nature of different treatments and biomaterials must be taken into account.4 In the current study, we established the effect of burn eschar debridement on alterations in the cellular populations of the innate immune system and subsequently identified how treating those debrided wounds with 3 different hydrogels affect the immune response and wound healing. While several methods of burn eschar debridement (e.g., enzymatic) have been studied,24,25 none have replaced sharp tangential surgical excision.26 The timing of deep burn excision can be variable, as it is often difficult to distinguish between wound beds that will heal in 2 to 3 weeks from those that will not heal. For these studies, we chose to employ surgical debridement on day 4 postburn to allow for the inflammatory myeloid response to persist during burn progression. Despite this relatively late surgical debridement, we found that debrided wounds reepithelialized more than nondebrided wounds at day 28, which is in agreement with previous studies.27 While faster wound closure is desired for optimizing patient outcomes, increased contraction causes unwanted scar formation and may result in the eventual loss of the patients’ range of motion. We observed accelerated contraction after debridement, which was associated with a more widespread distribution of SMA. However, this difference disappeared by the end of the experiment. An earlier report by Wang et al27 indicated that while the type of dressing used affected contraction, debridement per se did not. This highlights the importance of considering the time-dependent nature of contraction, as their study examined contraction at 6 weeks postburn, while ours ended at 4 weeks. This timeframe does not allow for the assessment of scar formation but rather only wound healing. Additionally, this points out a limitation of these studies in that the wounds are not critically sized and will heal spontaneously. Despite this, we were able to identify differences in immune cell populations during the myeloid response (i.e., neutrophils and macrophages) as a function of debridement and treatment. Debridement reduced both neutrophils and macrophages in the wound bed for the first 2 weeks postburn. By 28 days, both cell populations were infrequently identified. Both cell types are integral in the first stage of inflammation, and the neutrophils that are normally cleared within 14 days likely persisted longer due to the burn eschar. The eschar represents a formidable source of debris that must be removed, and these data underscore just one of the reasons why complete removal of necrotic tissue is important, although not always realized clinically.28 Cleaner excisional wounds in swine have been used to evaluate various biomaterials for their healing benefits.29–31 We chose to utilize debrided burn wounds because they likely have a significantly different/amplified inflammatory profile than excisional wounds. There is limited and conflicting information in animal models on the effect of burn wound debridement on inflammation. A recent study in rats concluded that immediate postburn debridement restored neutrophil delivery to infected skin, but debridement 1 week postburn did not lead to any significant effects on neutrophils.32 However, many studies on inflammation have been performed in rodents and the correlation to human inflammation and wound healing remains controversial.33 It is widely accepted that pigs are the closest surrogate to human skin for studying cutaneous healing for a variety of reasons.16,17 However, there have been limited studies in pigs connecting debridement and inflammation. One study showed that several methods of debridement 2 days postburn reduced the amount of infiltrating white blood cells for up to 3 weeks.34 Another report indicated that debridement led to a significant decrease in inflammatory foci seen histologically 6 weeks postburn.35 While these studies did not aim to identify specific phenotypes of these leukocytes, they do emphasize the importance of long-term inflammation in the wound healing process. Indeed, examining the adaptive immune response (i.e., lymphocyte/T-cell subtypes) in the current model warrants further investigation. Hydrogels have been used to coat implants for reducing inflammatory infiltrate, as well as in combination with other immunomodulatory agents.36 Advancements in hydrogels for regenerative medicine purposes have generated both degradable and nondegradable material, as well as synthetic polymer-based biomaterials10; however, we wanted to use naturally occurring polymers. As the major structural protein found in cutaneous tissue, collagen is a logical natural fiber to study for wound healing, and indeed is the main component of many different skin substitutes. Moreover, collagen hydrogels have been shown to possess immunomodulatory properties in vitro.37 Fibrin and fibrinogen-based hydrogels have also been investigated for use in skin substitutes38 and represent another natural polymer source that plays an important role in wound healing. Unfortunately, there are several limitations to using fibrin alone in preparing hydrogels, such as stiffness, shrinkage, and degradation rates.39,40 One strategy to overcome these limitations is to copolymerize fibrin with polyethylene glycol,21,41 which also allows for stem cell encapsulation. For example, it has been shown that these composite gels can induce release of angiogenic growth factors from adipose-derived stem cells in vitro.42 It has also been shown that both fibrin and collagen hydrogels can be combined with adipose-derived stem cells to control their phenotype and differentiation.43 However, no information exists on the immunomodulatory properties of these hydrogels themselves on burn wounds in vivo. Collagen, PEG-fibrin, and PEG-PFP hydrogels all prevented contraction to differing degrees when compared with untreated debrided burns (Fig. 5). Moreover, wounds treated with PEG-fibrin displayed less contraction than untreated controls even out to 28 days postburn. It is unclear from this study whether wounds treated with PEG-fibrin would maintain less contraction over time or whether wounds would eventually become similarly sized. As mentioned earlier, these wounds are not critically sized, and the total amount of contraction (~80%) is also low, which is consistent with an earlier report examining wound healing in swine.44 We did see evidence that PEG-PFP hydrogels assisted with reepithelialization but did not have biopsies at the timepoints to support this statistically. Histological results showed that hydrogels within the wound bed on days 7 and 10, but by day 14, no collagen or fibrin from the hydrogels could be seen (Fig. 6). Blood vessel infiltration seen into the PEG-fibrin hydrogels may prolonged the presence of hydrogels within the wound, possibly increasing the mechanical forces that ultimately mitigate contraction. Moreover, SMA staining also appears to be lower in PEG-fibrin–treated wounds compared with other groups (Fig. 7); however, exact quantification is confounded by expression of SMA in blood vessels. Also evident via histology is that the addition of PEG-fibrin hydrogels accelerated granulation tissue deposition on days 10 and 14 postburn. The addition of collagen or PEG-fibrin hydrogels decreased the amount of both neutrophils and macrophages within granulation tissue 7 days postburn; however, their presence at day 10 postburn did not reduce the amount of inflammatory cells. PEG-PFP hydrogels did not have this effect and instead showed a response similar to untreated debrided burn wounds. Taken together, these data reveal a beneficial role of PEG-fibrin incorporation to burn wounds. Despite the presence of growth factors and other plasma proteins, PEG-PFP hydrogels did not have the same effects on inflammation and contraction. This is potentially due to the overall concentration of fibrinogen within the gel, which is limited to circulating fibrinogen levels. Similarly, perhaps the lack of integration of the collagen hydrogels is due to the concentrations used in this study, as more concentrated gels have been previously shown to limit contraction in vitro.45 Alternatively, the in situ nature of the PEG-fibrin delivery may allow for better conformity to the wound bed than the preformed collagen hydrogel, which must be done due to the use of the alkaline solution needed to repolymerize collagen solutions. This brings to light a couple of potential clinical uses of PEG-fibrin. The ability to form the PEG-fibrin gels at the time of application does allow for tuning of gelation time, raising the possibility of their use instead of, or as adjuncts to acellular skin substitutes. In this regard, PEG-fibrin may be applied to enable primary healing after debridement, whether or not temporary wound coverages are used. These hydrogels can be combined with stem cells, growth factors, or other biologics to maximize their anti-inflammatory and pro-regenerative effects. Alternatively, in a scenario where split-thickness skin grafts are meshed to increase coverage, PEG-fibrin could be added in the interstices to mitigate the contraction that accompanies meshing autografts. Regardless of the application, the end goal is an improved functional and cosmetic outcome. We attempted to use autologous plasma as an inexpensive source of fibrinogen for generating hydrogels, which could be done at the bedside. The use of plasma for fabricating hydrogels to treat burn wounds is of recent interest15,46 and has been shown to stimulate wound healing, resulting in increased granulation tissue and collagen deposition.47 It is unclear why PEG-PFP in the current study did not have this same effect; however, it is of note that plasma previously used included platelets, whereas this study utilized PFP. Also possible is that opposite forces are in play, wherein any immunogenic reaction of the plasma was counteracted by the effect of the hydrogel itself. Indeed, the amount of infiltrating cells within the hydrogel itself was highest in the PEG-PFP hydrogels and lowest in the PEG-fibrin (Fig. 8C). The study previously described by Henderson et al47 expected an effect on reepithelialization but did not find one. We did see a trend toward accelerated epithelialization in the PEG-PFP group on day 14, which was not quite significant. We also noticed less of the PEG-PFP hydrogel remained, which may have potentiated the epithelium to recover the wound. All treated wounds were completely reepithelialized by day 28 postburn, and an intermediate timepoint (e.g., 21 days) may have been revealing for different rates of reepithelialization. There are certain limitations to the current study. During the acquisition of photographs, we noticed areas of red to pink mottled appearance (Fig. 1D). This suggests a certain degree of heterogeneity in our burn wounds that has been documented by others previously.48 Additionally, and consistent with previous studies,44 there was some anatomical variability on the cranio-caudal axis in terms of wound healing, which was addressed by randomizing each treatment group down the spine (i.e., anatomical normalization). Additionally, this study does not examine any differences in the adaptive immune system (i.e., lymphocytes). Pan macrophage markers are also not ideal, as they do not allow for the differentiation between M1 and M2 macrophage phenotypes. Specifically, M2 macrophages are more involved with regeneration and remodeling, which would be more indicative that the wound was in the proliferative stage of healing. Unfortunately, the lack of pig-specific antibodies precludes the ability to characterize the phenotype of macrophage populations in detail. The current study uses a burn model with sharp debridement to test the beneficial effects of 3 different hydrogels. We demonstrate that a dual syringe applicator can be used for the in situ delivery of fibrinogen-based hydrogels, including one that uses the patient’s own plasma. The use of autologous plasma remains an attractive option, and more work should be done to elucidate the properties that induce immunogenicity vs immunosuppression. Furthermore, we show that hydrogel application can prevent contraction and accelerate granulation tissue deposition in debrided burn wounds without impeding reepithelialization. The effects of these hydrogels are, in part, driven by immunomodulatory properties by the hydrogels themselves. Specifically, PEG-fibrin hydrogels warrant further investigation on the mechanisms of immunomodulation. Ultimately, these gels can be combined with stem cell therapies or other biologics for the purpose of enhancing coverage of burn wounds with skin substitutes. The opinions or assertions contained herein are the private views of the author and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense. The authors declare no personal or financial conflicts of interest. Medical Research and Materiel Command provided funding for this project. Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Web site. ACKNOWLEDGMENTS We thank Dr. Rodney Chan for input and the US Army Institute of Surgical Research Veterinary Staff for their technical assistance with surgeries REFERENCES 1. Peck MD . Epidemiology of burns throughout the world. Part I: distribution and risk factors . Burns 2011 ; 37 : 1087 – 100 . Google Scholar CrossRef Search ADS PubMed 2. Janzekovic Z . A new concept in the early excision and immediate grafting of burns . J Trauma 1970 ; 10 : 1103 – 8 . Google Scholar CrossRef Search ADS PubMed 3. Hop MJ , Polinder S , van der Vlies CH , Middelkoop E , van Baar ME . Costs of burn care: a systematic review . Wound Repair Regen 2014 ; 22 : 436 – 50 . Google Scholar CrossRef Search ADS PubMed 4. Singer AJ , Clark RA . Cutaneous wound healing . N Engl J Med 1999 ; 341 : 738 – 46 . Google Scholar CrossRef Search ADS PubMed 5. Gibran NS , Wiechman S , Meyer W , et al. . American Burn Association consensus statements . J Burn Care Res 2013 ; 34 : 361 . Google Scholar CrossRef Search ADS PubMed 6. Shahrokhi S , Arno A , Jeschke MG . The use of dermal substitutes in burn surgery: acute phase . Wound Repair Regen 2014 ; 22 : 14 – 22 . Google Scholar CrossRef Search ADS PubMed 7. Benichou G , Yamada Y , Yun SH , Lin C , Fray M , Tocco G . Immune recognition and rejection of allogeneic skin grafts . Immunotherapy 2011 ; 3 : 757 – 70 . Google Scholar CrossRef Search ADS PubMed 8. Cuttle L , Kempf M , Phillips GE , et al. . A porcine deep dermal partial thickness burn model with hypertrophic scarring . Burns 2006 ; 32 : 806 – 20 . Google Scholar CrossRef Search ADS PubMed 9. Kant CD , Akiyama Y , Tanaka K , et al. . Primary vascularization of allografts governs their immunogenicity and susceptibility to tolerogenesis . J Immunol 2013 ; 191 : 1948 – 56 . Google Scholar CrossRef Search ADS PubMed 10. Toh WS , Loh XJ . Advances in hydrogel delivery systems for tissue regeneration . Mater Sci Eng C Mater Biol Appl 2014 ; 45 : 690 – 7 . Google Scholar CrossRef Search ADS PubMed 11. Boateng JS , Matthews KH , Stevens HN , Eccleston GM . Wound healing dressings and drug delivery systems: a review . J Pharm Sci 2008 ; 97 : 2892 – 923 . Google Scholar CrossRef Search ADS PubMed 12. Cui F , Li G , Huang J , et al. . Development of chitosan-collagen hydrogel incorporated with lysostaphin (CCHL) burn dressing with anti-methicillin-resistant Staphylococcus aureus and promotion wound healing properties . Drug Deliv 2011 ; 18 : 173 – 80 . Google Scholar CrossRef Search ADS PubMed 13. Zamora DO , Natesan S , Becerra S , et al. . Enhanced wound vascularization using a dsASCs seeded FPEG scaffold . Angiogenesis 2013 ; 16 : 745 – 57 . Google Scholar CrossRef Search ADS PubMed 14. Ceccarelli J , Putnam AJ . Sculpting the blank slate: how fibrin’s support of vascularization can inspire biomaterial design . Acta Biomater 2014 ; 10 : 1515 – 23 . Google Scholar CrossRef Search ADS PubMed 15. Marck RE , Middelkoop E , Breederveld RS . Considerations on the use of platelet-rich plasma, specifically for burn treatment . J Burn Care Res 2014 ; 35 : 219 – 27 . Google Scholar CrossRef Search ADS PubMed 16. Middelkoop E , van den Bogaerdt AJ , Lamme EN , Hoekstra MJ , Brandsma K , Ulrich MM . Porcine wound models for skin substitution and burn treatment . Biomaterials 2004 ; 25 : 1559 – 67 . Google Scholar CrossRef Search ADS PubMed 17. Sullivan TP , Eaglstein WH , Davis SC , Mertz P . The pig as a model for human wound healing . Wound Repair Regen 2001 ; 9 : 66 – 76 . Google Scholar CrossRef Search ADS PubMed 18. Gaines C , Poranki D , Du W , Clark RA , Van Dyke M . Development of a porcine deep partial thickness burn model . Burns 2013 ; 39 : 311 – 9 . Google Scholar CrossRef Search ADS PubMed 19. Burmeister DM , Ponticorvo A , Yang B , et al. . Utility of spatial frequency domain imaging (SFDI) and laser speckle imaging (LSI) to non-invasively diagnose burn depth in a porcine model . Burns 2015 ; 41 : 1242 – 52 . Google Scholar CrossRef Search ADS PubMed 20. Helary C , Zarka M , Giraud-Guille MM . Fibroblasts within concentrated collagen hydrogels favour chronic skin wound healing . J Tissue Eng Regen Med 2012 ; 6 : 225 – 37 . Google Scholar CrossRef Search ADS PubMed 21. Zhang G , Wang X , Wang Z , Zhang J , Suggs L . A PEGylated fibrin patch for mesenchymal stem cell delivery . Tissue Eng 2006 ; 12 : 9 – 19 . Google Scholar CrossRef Search ADS PubMed 22. Paquet P , Jennes S , Rousseau AF , Libon F , Delvenne P , Piérard GE . Effect of N-acetylcysteine combined with infliximab on toxic epidermal necrolysis. A proof-of-concept study . Burns 2014 ; 40 : 1707 – 12 . Google Scholar CrossRef Search ADS PubMed 23. Shakespeare PG . The role of skin substitutes in the treatment of burn injuries . Clin Dermatol 2005 ; 23 : 413 – 8 . Google Scholar CrossRef Search ADS PubMed 24. Rosenberg L , Krieger Y , Silberstein E , et al. . Selectivity of a bromelain based enzymatic debridement agent: a porcine study . Burns 2012 ; 38 : 1035 – 40 . Google Scholar CrossRef Search ADS PubMed 25. Singer AJ , Taira BR , Anderson R , McClain SA , Rosenberg L . The effects of rapid enzymatic debridement of deep partial-thickness burns with Debrase on wound reepithelialization in swine . J Burn Care Res 2010 ; 31 : 795 – 802 . Google Scholar CrossRef Search ADS PubMed 26. Kirshen C , Woo K , Ayello EA , Sibbald RG . Debridement: a vital component of wound bed preparation . Adv Skin Wound Care 2006 ; 19 : 506 – 17; quiz 517–9 . Google Scholar CrossRef Search ADS PubMed 27. Wang XQ , Kempf M , Liu PY , et al. . Conservative surgical debridement as a burn treatment: supporting evidence from a porcine burn model . Wound Repair Regen 2008 ; 16 : 774 – 83 . Google Scholar CrossRef Search ADS PubMed 28. Gurfinkel R , Rosenberg L , Cohen S , et al. . Histological assessment of tangentially excised burn eschars . Can J Plast Surg 2010 ; 18 : e33 – 6 . Google Scholar CrossRef Search ADS PubMed 29. Ghosh K , Ren XD , Shu XZ , Prestwich GD , Clark RA . Fibronectin functional domains coupled to hyaluronan stimulate adult human dermal fibroblast responses critical for wound healing . Tissue Eng 2006 ; 12 : 601 – 13 . Google Scholar CrossRef Search ADS PubMed 30. Gomer RH , Pilling D , Kauvar LM , et al. . A serum amyloid P-binding hydrogel speeds healing of partial thickness wounds in pigs . Wound Repair Regen 2009 ; 17 : 397 – 404 . Google Scholar CrossRef Search ADS PubMed 31. Kim H , Son D , Choi TH , et al. . Evaluation of an amniotic membrane-collagen dermal substitute in the management of full-thickness skin defects in a pig . Arch Plast Surg 2013 ; 40 : 11 – 8 . Google Scholar CrossRef Search ADS PubMed 32. Tchervenkov JI , Epstein MD , Silberstein EB , Alexander JW . Early burn wound excision and skin grafting postburn trauma restores in vivo neutrophil delivery to inflammatory lesions . Arch Surg 1988 ; 123 : 1477 – 81 . Google Scholar CrossRef Search ADS PubMed 33. Seok J , Warren HS , Cuenca AG , et al. . Genomic responses in mouse models poorly mimic human inflammatory diseases . Proc Natl Acad Sci U S A 2013 ; 110 : 3507 . Google Scholar CrossRef Search ADS PubMed 34. Nusbaum AG , Gil J , Rippy MK , et al. . Effective method to remove wound bacteria: comparison of various debridement modalities in an in vivo porcine model . J Surg Res 2012 ; 176 : 701 – 7 . Google Scholar CrossRef Search ADS PubMed 35. Wang XQ , Phillips GE , Wilkie I , Greer R , Kimble RM . Microscopic inflammatory foci in burn scars: data from a porcine burn model . J Cutan Pathol 2010 ; 37 : 530 – 4 . Google Scholar CrossRef Search ADS PubMed 36. Vishwakarma A , Bhise NS , Evangelista MB , et al. . Engineering immunomodulatory biomaterials to tune the inflammatory response . Trends Biotechnol 2016 ; 34 : 470 – 82 . Google Scholar CrossRef Search ADS PubMed 37. Yuan T , Zhang L , Li K , et al. . Collagen hydrogel as an immunomodulatory scaffold in cartilage tissue engineering . J Biomed Mater Res B Appl Biomater 2014 ; 102 : 337 – 44 . Google Scholar CrossRef Search ADS PubMed 38. Hojo M , Inokuchi S , Kidokoro M , et al. . Induction of vascular endothelial growth factor by fibrin as a dermal substrate for cultured skin substitute . Plast Reconstr Surg 2003 ; 111 : 1638 – 45 . Google Scholar CrossRef Search ADS PubMed 39. Jockenhoevel S , Zund G , Hoerstrup SP , et al. . Fibrin gel—advantages of a new scaffold in cardiovascular tissue engineering . Eur J Cardiothorac Surg 2001 ; 19 : 424 – 30 . Google Scholar CrossRef Search ADS PubMed 40. Mosesson MW . Fibrinogen and fibrin structure and functions . J Thromb Haemost 2005 ; 3 : 1894 – 904 . Google Scholar CrossRef Search ADS PubMed 41. Liu SQ , Ee PL , Ke CY , Hedrick JL , Yang YY . Biodegradable poly(ethylene glycol)-peptide hydrogels with well-defined structure and properties for cell delivery . Biomaterials 2009 ; 30 : 1453 – 61 . Google Scholar CrossRef Search ADS PubMed 42. Chung E , Rytlewski JA , Merchant AG , Dhada KS , Lewis EW , Suggs LJ . Fibrin-based 3D matrices induce angiogenic behavior of adipose-derived stem cells . Acta Biomater 2015 ; 17 : 78 – 88 . Google Scholar CrossRef Search ADS PubMed 43. Natesan S , Zamora DO , Wrice NL , Baer DG , Christy RJ . Bilayer hydrogel with autologous stem cells derived from debrided human burn skin for improved skin regeneration . J Burn Care Res 2013 ; 34 : 18 – 30 . Google Scholar CrossRef Search ADS PubMed 44. Wang XQ , Liu PY , Kempf M , et al. . Burn healing is dependent on burn site: a quantitative analysis from a porcine burn model . Burns 2009 ; 35 : 264 – 9 . Google Scholar CrossRef Search ADS PubMed 45. Helary C , Abed A , Mosser G , Louedec L , Meddahi-Pellé A , Giraud-Guille MM . Synthesis and in vivo integration of improved concentrated collagen hydrogels . J Tissue Eng Regen Med 2011 ; 5 : 248 – 52 . Google Scholar CrossRef Search ADS PubMed 46. Pallua N , Wolter T , Markowicz M . Platelet-rich plasma in burns . Burns 2010 ; 36 : 4 – 8 . Google Scholar CrossRef Search ADS PubMed 47. Henderson JL , Cupp CL , Ross EV , et al. . The effects of autologous platelet gel on wound healing . Ear Nose Throat J 2003 ; 82 : 598 . Google Scholar PubMed 48. Kempf M , Cuttle L , Liu PY , Wang XQ , Kimble RM . Important improvements to porcine skin burn models, in search of the perfect burn . Burns 2009 ; 35 : 454 – 5 . Google Scholar CrossRef Search ADS PubMed Copyright © 2017 by the American Burn Association
TI - In Situ Delivery of Fibrin-Based Hydrogels Prevents Contraction and Reduces Inflammation
JF - Journal of Burn Care & Research
DO - 10.1097/BCR.0000000000000576
DA - 2017-12-27
UR - https://www.deepdyve.com/lp/oxford-university-press/in-situ-delivery-of-fibrin-based-hydrogels-prevents-contraction-and-wdAOnhZ0BT
SP - 1
EP - 53
VL - Advance Article
IS - 1
DP - DeepDyve
ER -