Biomaterials 35 (2014) 4805e4814

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Ultrashort peptide nanofibrous hydrogels for the acceleration of healing of burn wounds Yihua Loo a, Yong-Chiat Wong b, Elijah Z. Cai c, Chuan-Han Ang c, Ashvin Raju c, Anupama Lakshmanan a, Alvin G. Koh a, Hui J. Zhou d, Thiam-Chye Lim c, Shabbir M. Moochhala b, Charlotte A.E. Hauser a, * a

Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, #04-01, Singapore 138669, Singapore Combat Care and Performance Laboratory, Defence Medical Research Institute, DSO National Laboratories, 27 Medical Drive #12-01, Singapore 117510, Singapore c Department of Surgery (Division of Plastic, Aesthetic and Reconstructive Surgery), Yong Loo Lin School of Medicine, National University of Singapore, NUHS Tower Block, Level 8, 1E Kent Ridge Road, Singapore 119228, Singapore d Biostatistic Unit, Dean’s Office, Level 1, Block MD11, 10 Medical Drive, Singapore 117597, Singapore b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 15 January 2014 Accepted 23 February 2014 Available online 15 March 2014

There is an unmet clinical need for wound dressings to treat partial thickness burns that damage the epidermis and dermis. An ideal dressing needs to prevent infection, maintain skin hydration to facilitate debridement of the necrotic tissue, and provide cues to enhance tissue regeneration. We developed a class of ‘smart’ peptide hydrogels, which fulfill these criteria. Our ultrashort aliphatic peptides have an innate tendency to self-assemble into helical fibers, forming biomimetic hydrogel scaffolds which are non-immunogenic and non-cytotoxic. These nanofibrous hydrogels accelerated wound closure in a rat model for partial thickness burns. Two peptide hydrogel candidates demonstrate earlier onset and completion of autolytic debridement, compared to MepitelÒ, a silicone-coated polyamide net used as standard-of-care. They also promote epithelial and dermal regeneration in the absence of exogenous growth factors, achieving 86.2% and 92.9% wound closure respectively, after 14 days. In comparison, only 62.8% of the burnt area is healed for wounds dressed with MepitelÒ. Since the rate of wound closure is inversely correlated with hypertrophic scar formation and infection risks, our peptide hydrogel technology fills a niche neglected by current treatment options. The regenerative properties can be further enhanced by incorporation of bioactive moieties such as growth factors and cytokines. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: Ultrashort peptide hydrogels Nanofibers Self-assembly Partial thickness burns Wound healing

1. Introduction Burn injuries, particularly partial and full thickness burns, are complex acute wounds which are difficult to assess and manage [1]. With the damage extending well into the dermal tissue, very few viable stem cells remain in the epidermis, resulting in slow regeneration of the burnt tissue by cells migrating from the adjacent healthy tissue. Recovery typically occurs between 2 and 10 weeks with an increased risk of scarring as the healing duration is prolonged [2]. The formation of hypertrophic scars can be inhibited by early wound closure. However, the necrotic eschar tissue impedes healing and often has to be excised. Tissue regeneration is further hampered by compromised blood flow and inflammatory response stimulated by the initial burn injury. While full thickness * Corresponding author. Tel.: þ65 6824 7108; fax: þ65 6478 9080. E-mail address: [email protected] (C.A.E. Hauser). http://dx.doi.org/10.1016/j.biomaterials.2014.02.047 0142-9612/Ó 2014 Elsevier Ltd. All rights reserved.

burns are treated with skin grafts that provide cells and/or biological scaffold for tissue regeneration, there is currently no good dressing or scaffold to treat partial thickness burns [3]. The standard-of-care for burn wounds is the application of a silver-based antibiotic to prevent infection [3], and coverage with dressings that do not traumatize the regenerating epidermis during dressing changes, such as MepitelÒ. Hydrogels and hydrocolloid dressings are of great interest as dressings for burn wounds, as they maintain an ideal hydration environment for wound healing, while preserving the gaseous permeability of traditional gauze dressings [4]. Additional advantages include their ability to absorb wound exudate and provide transparency, which facilitates wound observation [4]. Very often, they can also serve as a matrix for the controlled release of antibiotics and drugs impregnated during manufacturing [4e6]. In this study, we evaluated the efficacy of a unique class of selfassembling peptide hydrogels as primary dressings for partial

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thickness burns. Consisting of three to seven aliphatic amino acids in a characteristic motif, these ultrashort peptides self-assemble into macromolecular nanofibrous networks that resemble extracellular matrix [7]. As such, they can be applied as biomimetic scaffolds to enhance tissue regeneration, providing nanotopographical cues for cell migration and proliferation. The dense nanofiber network may also act as a barrier against infection and abrasion. Due to their amphiphilic nature, these peptide networks can entrap up to 99.9% of water, forming hydrogels when a critical gelation concentration is exceeded. We propose that these hydrogels can maintain tissue hydration and thus facilitate autolytic debridement of necrotic eschar tissue in burn wounds. Two hydrogel candidates were applied to partial thickness burn injuries in a rat burn model. The onset and completion of autolytic debridement, as well as the rate of tissue regeneration were measured to evaluate efficacy in enhancing wound healing, compared to MepitelÒ, a silicone coated polyamide net used as the standard-of-care. We carefully checked for wound closure to evaluate, if this hydrogel technology could be an attractive solution to fulfill unmet clinical needs for better burn wound dressings. The rate of wound closure is an important indicator, since it is inversely correlated with hypertrophic scar formation and the risk of infection. 2. Materials and methods 2.1. Peptide synthesis and stability characterization The ultrashort peptides were synthesized manually, using solid phase peptide chemistry. Briefly, L-lysine residues conjugated to polystyrene beads via a Rinkamide linkage (GL Biochem, Shanghai, China) were swelled in dimethylformamide, de-protected and each succeeding amino acid residue added sequentially. After the terminal amino acid had been added, the N-terminus amine group was acetylated using acetic anhydride (Sigma Aldrich, St. Louis, MO, USA). The peptides were then cleaved using trifluoroacetic acid, precipitated and washed with diethyl ether. After drying under vacuum for 2 h, the crude products were dissolved in dimethylsulfoxide and purified using reverse-phase high-performance liquid chromatography mass spectrometry. The purified peptide solution was subsequently lyophilized. The temperature stability was determined using thermogravimetric analysis to determine the mass change with time. The chemical stability of the peptide following autoclave sterilization (121  C, 15 min) and UV exposure (1 h) was determined using high-performance liquid chromatography mass spectrometry.

bandages were used to secure the dressings. Rats were housed individually and monitored daily for eating and drinking. To evaluate the rate of healing, the wound size, granulation and re-epithelialization were quantitatively assessed using digitalized planimetry every other day, for 14 days. Photographs of the wounds were taken and the images analyzed using the program PictZarÒ Pro to determine the area of re-epithelialization and granulation at different time points. The general linear model for repeated measures was performed using the re-epithelialization and granulation areas (absolute and percentage) at days 10, 12 and 14. This model enabled us to explore the time trend of re-epithelialization and granulation and the effect on this time trend caused by different treatments while adjusting for total wound size. After 7 or 14 days, the animals were euthanized and skin samples were collected from the various burn injuries. Half of each sample was preserved in 10% formalin while the other half was flash frozen in liquid nitrogen. 2.5. Histological examination The formalin-fixed tissue samples were dehydrated in ascending series of alcohol, cleared with xylene and embedded in paraffin wax. Paraffin sections (4 mm thick) were then cut and dried in the oven overnight. The sections were then stained in hematoxylin and eosin and visualized with 3,30 -diaminobenzidine, dehydrated and mounted for viewing under an Olympus BX51 microscope. 2.6. Cytokine multiplex ELISA The frozen skin samples were individually homogenized using sterile metal beads in the Procarta Cell Lysis buffer. The samples were subsequently centrifuged and the supernatant isolated for analysis. The Procarta immunoassay (Affymetrix Inc, CA, USA) was performed according to the manufacturer’s instructions. Briefly, the tissue lysate were incubated with antibody magnetic beads in duplicate. The expression of ten different cytokines and growth factors were assessed, namely, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon gamma (IFNg), interleukin 1-alpha (IL-1a), interleukin 1-beta (IL-1b), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 6 (IL-6), transforming growth factor beta (TGF-b), tumor necrosis factor alpha (TNF-a), and vascular endothelial growth factor A (VEGF-A). After applying the detection antibodies and Streptavidin-conjugated RPhycoerythrin (the detector molecule), the fluorescence intensity were read using a MasterPlex CT v1.0.0.2 (MiraiBio, CA, USA). The concentration (pg/ml) of cytokine was calculated using MasterPlex QT v2.0.0.59 (MiraiBio, CA, USA) using 4-parameter logistics curve fitting against known standards. The cytokine concentration was normalized against the total protein content for a given sample, as measured using the bicinchoninic acid (BCA) assay. One-way analysis of variance (ANOVA) was separately performed for the normalized cytokine expression on days 7 and 14 for each of the cytokines tested. The mean cytokine expressions were compared among three treatment groups to obtain a p-value. Groups for which p < 0.05, namely GMCSF, IL-4, IL-6 and TNF-a, additional post-hoc tests were carried out to highlight statistical differences between any two samples. 2.7. Statistical analysis

2.2. Peptide hydrogel preparation The lyophilized peptide powders were first dissolved in cold milliQ water and mixed by vortexing for 30 s to obtain a homogenous solution, which was subsequently added to phosphate-buffered saline. Gelation was facilitated by sonication for 15 min at room temperature. Hydrogel dressings were prepared in polydimethylsiloxane moulds to obtain 20.0 mm discs of approximately 1.5 mm thick. 2.3. Field-emission scanning microscopy Thin hydrogels samples were snap frozen at 80  C and subsequently lyophilized to remove their water content. The lyophilized samples are mounted onto sample holders using conductive tape, and then sputter-coated with platinum using a JEOL JFC-1600 High Resolution Sputter Coater at 30 mA to improve their conductibility. The coated samples are then examined with a JEOL JSM-7400 fieldemission scanning microscopy system.

The cytokine variables were presented as mean  standard deviation as descriptive statistics. One-way analysis of variance (ANOVA) was performed separately to compare mean cytokine values of three different treatment groups for day 7 and day 14. As for re-epithelialization and debridement data, considering the intrasubject correlation over observation time, general linear model for repeated measures was conducted to explore and compare the time trend among three groups. The model results were presented as adjusted means and standard error. P value less than 0.05 was considered statistically significant. SPSS version 19 performed the analysis.

3. Results and discussion 3.1. Ultrashort peptides self-assembly into transparent nanofibrous hydrogels

2.4. Rat model of partial thickness burns As burn infliction techniques are poorly described in rat models [5,6], a procedure to generate consistent partial thickness burns on shaved rat dorsum was evaluated and optimized by varying temperature and contact duration with a heated stainless steel rod. Adult male Sprague Dawley rats (325e350 g) were acclimatised for one week before experiments. The animals were anesthetized with inhalational anesthetic of isoflurane and their dorsal surface was shaved with clippers. The experimental procedures were approved by DSO National Laboratories Institutional Animal Care and Use Committee. Briefly, 10 s exposure to a 1 cm diameter stainless steel rod heated to 100  C in boiling water for 5 min, generated consistent mid partial thickness burns on shaved rat dorsum. Three injuries inflicted on each rat (n ¼ 11) were dressed with ultrashort peptide hydrogels Ac-ILVAGK-NH2 and AcLIVAGK-NH2, and silicone-coated polyamide net MepitelÒ. Polydimethylsiloxane supports were used to maintain the integrity of the soft disc-shaped hydrogels. After positioning the dressings, water-proof TegardermÔ adhesive films, gauze and crepe

The building blocks of the hydrogels are ultrashort amphiphilic peptides with a defined motif of an aliphatic tail of an overall decreasing hydrophobicity towards a hydrophilic polar amino acid head group at the C-terminus, as exemplified by Ac-ILVAGK-NH2. The N-terminus amine is acetylated for facile self-assembly [7]. In comparison to other self-assembling peptides used as biomaterials in regenerative medicine [8,9], these peptides are extremely shorte the smallest members in this class contain only 3 amino acids, while the longest are heptamers. Furthermore, these ultrashort peptides spontaneously self-assemble in aqueous conditions, forming macromolecular scaffolds without the need for gelators, cross-linkers, and mechanical stimulation (such as ultra-

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sonication). During self-assembly, nanofibers are formed via ahelical intermediate structures [7]. As the peptide concentration is increased, the secondary structure of the peptides changes from random coil to a-helical intermediates stacking in anti-parallel fashion to subsequently arrange to b-turn fibrils (Fig. 1a). Further aggregation and condensation of the fibrils into nanofibers (Fig. 1b) gives rise to dense three-dimensional networks (Fig. S1). These fibrillar networks can entrap water up to 99.9% by weight, giving rise to hydrogels with excellent mechanical properties [10]. At low peptide concentration, the resulting hydrogels are colorless and transparent (Fig. 1c). When applied as burn wound dressings, their optical clarity will facilitate the evaluation of the underlying wounds. The peptides are extremely stable and relatively chemically inert, making them ideal for developing products with long shelf life at ambient conditions. To date, we have kept peptide and hydrogel samples for more than three years without compromising their gelation properties or chemical nature. Thermogravimetric studies reveal that the decomposition temperature is approximately 290  C (Fig. S2c). The heat stability enables the use of autoclaving as a facile sterilization technique (Fig. S2d). Thus, when developing them as biomaterials for medical applications, special processing techniques would not be needed to ensure sterility. The mechanical properties of the ultrashort peptide hydrogels are suited to applications such as wound dressings. The rigidity of these hydrogels are two orders of magnitude above that of collagen

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[10] and they are also considerably stiffer than other short peptide hydrogels [11e15] despite their shorter length. Nonetheless, being a soft biomaterial, these hydrogels are easily and painlessly cleaned away by gentle swabbing during the frequent dressing changes necessitated by the severity of the burn injury [16]. This is advantageous as dressings like gauze often entangle with the regenerating skin tissue, causing discomfort and further injury during removal. The nanofibrous architecture of the ultrashort peptide hydrogels resemble the extracellular matrix (ECM) [17], which predispose them as scaffolds for skin regeneration [18e20]. In particular, they can potentially serve as a synthetic skin substitute for deep, partial and full thickness burns. Since the peptides are chemically synthesized, they are chemically well-defined and will not cause immunogenic problems associated with donor and cadaveric skin grafts [1]. They are also a good alternative to collagen I matrices. Collagen I is a major component of the ECM, which facilitates wound healing [21]. However, as collagen I is typically purified from animal sources, concerns regarding its potential immunogenicity have limited its clinical applications [22]. While other nanofibrous materials such as silk [23] and electrospun polymers [24] have been used as wound dressings, they do not offer the advantages of hydrogels, which provide a moisture-rich environment for autolytic debridement and cell proliferation. On the other hand, hydrogels prepared from natural (such as chitosan, hyaluronic acid and alginate) and synthetic (such as polyethylene

Fig. 1. Ultrashort peptides self-assemble into macromolecular nanofibrous hydrogels. (a) Ultrashort peptides consisting of 3e7 natural aliphatic amino acids have a characteristic defining motif of an aliphatic tail of decreasing hydrophobicity capped by a hydrophilic polar amino acid, as exemplified by Ac-LIVAGK-NH2. Self-assembly in aqueous conditions occurs when the amino acids pair and subsequently stack into a-helical fibrils. As the peptide concentration increases, conformational changes from random coil (black dotted line) to a-helical intermediates (red dashed line) to b-fibrils (blue solid line) are observed. (b) The fibrils further aggregate to form nanofibers that readily condense into sheets, as shown in this field emission scanning micrograph of lyophilized Ac-ILVAGK-NH2 (2 mg/mL). The nanofibers and sheet-like structures resembles extracellular matrix. (c) Due to their amphiphilic nature, the peptide network entraps a significant amount of water, forming clear and colorless hydrogels exemplified by 5 mg/mL Ac-ILVAGK-NH2 and 7.5 mg/mL AcLIVAGK-NH2. The optical clarity is demonstrated by a 1.5 mm thick 5 mg/mL Ac-ILVAGK-NH2 in the polydimethylsiloxane mould used in this experiment. This gel (stained with methylene blue for visibility and balanced on a metal spatula) is sufficiently rigid for easy handling. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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glycol) polymers [22] typically do not form nanofibers during selfassembly. Our ultrashort peptide hydrogels thus combine the advantages of nanofibrous scaffolds and polymer hydrogels. We can further engineer the peptide hydrogel microenvironment through intelligent materials design to stimulate tissue regeneration [22]. Cell-adhesion motifs such as RGD can be conjugated to stimulate keratinocyte migration, proliferation and differentiation [25]. The encapsulation of small molecule drugs [4], anti-microbials, analgesics, nucleic acids, cytokines [26], growth factors [24,27] and autologous cells can potentially reduce the inflammatory response, induce cell migration into the injury site and stimulate tissue regeneration. Thus, in addition to its ECM-like topography and high water content, we can incorporate a combination of bioactive moieties to enhance wound healing. 3.2. Peptide candidate selection and formulation design Two ultrashort peptide candidates Ac-ILVAGK-NH2 and AcLIVAGK-NH2 were formulated into hydrogel patches of 25.0 mm diameter and 1.5 mm thick. The peptide candidates chosen for this study contained lysine as a polar head group, which could potentially facilitate hemostasis of open wounds. Analogs and derivatives of lysine, such as ε-aminocaproic acid and tranexamic acid, are antifibrinolytics that prevent excessive blood loss by competitively inhibiting proteolytic enzymatic activity [28] to prevent the breakdown of blood clots. Furthermore, as both peptides bind DNA, the hydrogel can act as a sponge to scavenge proinflammatory extracellular oligonucleotides and attenuate their immune stimulatory effects [29]. Immunostimulatory nucleic acids such as RNA and unmethylated DNA released by damaged host cells and invading pathogens are recognized by toll-like receptors which in turn activates the innate immune system. In sequestering these

oligonucleotides, we can reduce the risk of developing autoimmune diseases [30] and partially modulate the inflammatory response elicited by the burn injury. Hexameric peptides were chosen for this study in lieu of shorter candidates as they form hydrogels at substantially lower concentrations. Ac-ILVAGK-NH2 and Ac-LIVAGK-NH2 forms hydrogels in phosphate-buffered saline (PBS) at 3 and 7.5 mg/mL respectively (Fig. 1c), while trimer Ac-IVK-NH2 and tetramer Ac-IVAK-NH2 have minimum gelation concentrations exceeding 20 mg/mL. This translates to significant cost savings. Another advantage to choosing the subclass of peptides containing lysine is that they demonstrate salt-enhanced gelation and greater rigidity in the presence of physiological buffers such as normal saline and PBS. In other words, the minimum gelation concentration is reduced in the presence of salts and more rigid hydrogels are obtained with increasing salt concentration. Taking this into consideration, PBS was used to prepare the hydrogel dressings so as maintain the physiological pH and increase the capacity of the dressing to absorb wound exudates. In a clinical setting, saline (0.9% sodium chloride) is used to irrigate wounds as it does not irritate the wound when applied. In contrast, water is a hypotonic solution which can cause cellular edema and pain when used to flush raw wounds. For this study we selected peptide concentrations which form hydrogels sufficiently rigid for easy handling e 5 mg/mL AcILVAGK-NH2 and 7.5 mg/mL Ac-LIVAGK-NH2. Peptides dissolved in sterile water were mixed with 10 times concentrated PBS, and subsequently aliquoted into polydimethylsiloxane (PDMS) moulds to obtain circular patches of 25.0 mm diameter and 1.5 mm thick (Fig. 1c). The hydrogels were then incubated at 37  C, wherein gelation occurred within minutes, producing stable and uniformlysized hydrogels amendable to handling.

Fig. 2. Self-assembled ultrashort peptide hydrogels maintain tissue hydration and facilitate autolytic debridement of necrotic eschar tissue in burn wounds. (a) Ac-ILVAGK-NH2 and Ac-LIVAGK-NH2 hydrogel candidates demonstrated earlier onset and completion of autolytic debridement, compared to Mepitel, as observed during gross examination. (b) Gross histological analysis of skin sections taken at day 7 confirmed the completion of autolytic debridement for wounds dressed with peptide hydrogels. The loss of the necrotic epidermal layer in peptide hydrogel-treated burns, is contrasted with remnants of epidermal tissue adhered to the dermis (marked by the black arrow) for the Mepitel-dressed wounds.

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The peptides’ propensity for self-assembly and stimuliresponsive nature facilitate the design of different formulations e from membranes that can be rehydrated, to topical gels, and to spray formulations (Fig. S3a). By carefully drying the hydrogel, we obtained membranes which were subsequently rehydrated by adding a fixed volume of clean water (Fig. S3b). This re-hydration process can be accelerated by warming and sonication. Viscous fluidic gels were prepared using peptide concentrations just below

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the minimum gelation concentration. We also developed a spray formulation using a dual-compartment dispersal unit. The peptide solution is initially separated from the gelation-trigger agent, a high concentration salt solution. Upon dispersal, the solutions mix and gelation occurs. We are currently in the process of developing justadd-water formulations with long shelf-life at room temperature. This formulation design will greatly reduce transportation costs, thereby facilitating adoption as part of basic first aid kits

Fig. 3. Ultrashort peptide hydrogels enhance wound contracture. (a) Both Ac-ILVAGK-NH2 and Ac-LIVAGK-NH2 hydrogels accelerated the regeneration of new epidermal tissue, as denoted by the area of re-epithelialization, E. The granulation, G, also decreased over time. All the images in this Figure show the progression of wound healing for the same animal (H24). The burn injury treated with Ac-LIVAGK-NH2 hydrogel completely regenerated its epidermis by day 14. (b) Quantitative evaluation of wound healing using digital planimetry revealed that the peptide hydrogels stimulated re-epithelialization and reduced granulation, compared to Mepitel. The error bars denote the standard error of the mean (n ¼ 6).

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particularly in war zones and third world countries. By exploiting the stimuli-responsive self-assembling properties, we can tailor the formulation design to meet the needs of different markets. 3.3. Autolytic debridement of eschar tissue The efficacy of ultrashort peptide hydrogels as wound dressings was evaluated in a rat partial thickness burn model. Consistent partial thickness burns were inflicted by bringing a hot rod in contact with the shaved dorsal skin of anesthetized animals. To minimize the number of animals and to allow us to compare the efficacy of the different dressings in the same animal, three burns were made per animal to evaluate the rate of wound healing (Fig. S4). A standard-of-care dressing, MepitelÒ, was used as a control. This dressing consists of a flexible polyamide net coated with silicone to discourage adhesion of the regenerating tissue and prevent abrasion of the recovering tissue.

The key advantage of using hydrogels as burn wound dressings is their propensity to induce autolytic debridement of the necrotic eschar tissue. Autolytic debridement is preferred over physical methods to remove the necrotic eschar, which could potentially abrade the recovering tissue [16]. Both peptide hydrogels stimulated autolytic debridement. The onset of debridement of hydrogel-treated wounds was observed by day 8 for all the animals, compared to day 10 for Mepitel-treated wounds (Fig. 2a). Complete debridement of the eschar occurred over the course of one to four days, completing by day 12 for both hydrogel-treated groups and by day 14 for the Mepitel-treated. For all the six animals evaluated, the peptide hydrogel-treated burn injuries demonstrated earlier onset and completion of debridement. Histological analysis of skin samples taken at day 7 corroborated the clinical observation of complete debridement for wounds dressed with peptide hydrogels. The loss of the necrotic epidermal layer for burnt areas treated with peptide hydrogels

Fig. 4. Histological evaluation of burn wound healing at days 7 and 14. (a) The tissue damage penetrated the deep dermis tissue as examined on day 7. Necrotic epidermal tissue was observed for the Mepitel-dressed samples, while complete debridement of the eschar epidermal tissues was observed for peptide hydrogel-dressed injuries. At the boundary of the injured and healthy tissue (denoted by the black triangles), infiltration of the healthy basal cells from the epidermisedermis junction and the adjacent undamaged hair shafts, into the damaged tissue was observed for the peptide hydrogel-dressed injuries. At this interface, the basal cells were rapidly proliferating and were packed into more layers compared to healthy epidermis. (b) Recovery of the damaged tissue at day 14, as compared to day 7. The peptide hydrogel-treated wounds almost completely regenerated their epidermis. At the boundary of the injury site, significantly higher extent of cell replication has occurred amongst basal cells located in the hair follicles, for wounds dressed with peptide hydrogels.

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(Fig. 2b and S5), is contrasted with remnants of epidermal tissue adhered to the dermis for the wounds dressed with Mepitel. By accelerating autolytic removal of the necrotic tissue, the ultrashort peptide hydrogels create room for cells to infiltrate the damage region and commence tissue regeneration. 3.4. Wound closure Following the onset of autolytic debridement, the reepithelialization and granulation of each wound can be quantified using digital planimetry (Fig. 3a). Re-epithelialization is important as the skin plays a major barrier function in protecting the host against pathogens. When the skin barrier is compromised, the immune system reacts by producing cytokines to repel invading pathogens. The overproduction of cytokines (a cytokine storm) in severe burn injuries causes excessive inflammation, which can trigger organ failure and cause patient mortality. By accelerating reepithelialization to close the wound and restore the skin barrier, the risk of prolonged inflammation is dampened. Wound closure is in part facilitated by autolytic debridement. As such, it was not surprising that wound contracture was significantly faster for injuries treated with peptide hydrogels as compared to those treated with Mepitel (Fig. 3b). By day 10, 58.6% and 55.0% of the injured area had re-epithelialized for Ac-ILVAGK-NH2 and AcLIVAGK-NH2-treated wounds respectively. In contrast, only 47.3% of the wound area had healed for Mepitel-dressed wounds. The difference between three groups did not reach statistical significance at this time (p ¼ 0.342). By day 14, 86.2% and 92.9% reepithelialization was achieved for the hydrogels, which was dramatically higher than the 62.8% for Mepitel dressings (p ¼ 0.001). Adjusting for total wound size, the rate of reepithelialization was significantly greater for hydrogel-treated burn injuries (p ¼ 0.026), as confirmed by general linear model for repeated measures fitting the consecutive observational data (Table S1). Digital planimetry also enabled us to measure the area of granulation e the formation of new connective tissue and extracellular matrix. During granulation, vascularization of the regenerating tissue occurs, giving rise to tiny blood vessels on the surface of the wound. This manifests as characteristic light red shading beginning from the edges of the wound (Fig. 3a and S6). A greater (but not statistically significant) extent of granulation

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was initially observed at day 10 for both peptide hydrogels, which eventually declined as the wound re-epithelialized (Fig. 3b). When the wound was completely re-epithelialised, as exemplified by the Ac-LIVAGK-NH2 treated burn injury in Fig. 3a at day 14, the granulation score was zero. For Mepitel-treated burn injuries, the granulation initially increased from 7.3% to 13.8% and then dropped to 7.7%, which was higher than the averaged values obtained for the hydrogel-treated wounds (p ¼ 0.352). Histological analysis of the injury suggests that the technique used consistently generated deep partial thickness burns (Fig. 4). The structural integrity of the tissue was significantly altered, giving rise to more disorganized extracellular matrix, loss of hair-shaft follicles in the dermis, and detachment of the epidermis in most specimens. There was also extensive lymphocyte infiltration at the interface of injured and healthy tissue (Fig. 4a). The damage extended more than 50% through the dermis even after 7 days. As illustrated in Fig. 4a, necrotic epidermal tissue was observed for the Mepitel-dressed samples, while complete debridement of the eschar epidermal tissues was observed for peptide hydrogeldressed injuries. While extracellular matrix re-modeling in the dermis is incomplete, there are some signs of recovery, as demonstrated by the proliferation of hair shaft follicles in the surrounding healthy tissue. The infiltration of healthy basal cells from the epidermisedermis junction and the adjacent undamaged hair shafts, into the damaged tissue was observed for the peptide hydrogel-dressed injuries, under higher magnification. These stem cells are responsible for skin epithelial and hair follicle regeneration following injury. By day 14, the regeneration of the epidermis was almost complete for peptide hydrogel treated wounds (Fig. 4b). The injured tissue was predominantly (>90%) covered by healthy epithelial tissue and the new epidermis was almost as thick as that of healthy skin. Basal cells at the epidermisedermis interface and stem cells from the adjacent intact hair follicles have also begun to penetrate the injured dermis to form precursors of new hair shaft follicles [31] (Fig. 4b). In comparison, the epidermal tissue in Mepitel-dressed wounds covered a smaller area, was thinner and more fragile. Hydrogel-treated wounds also demonstrated a greater degree of matrix remodeling in the dermis, as well as significantly higher proliferation of basal cells in the hair follicles. In contrast, hair shaft follicle regeneration in

Table 1 Normalized average cytokine expression at days 7 and 14. The p-values are obtained following ANOVA analysis of the average cytokine expression for the different treatment groups. The statistically significant (p < 0.05) variations are in bold, and subsequently subjected to post-hoc comparisons. Cytokine [pg/mg] Day 7 GM-CSF IFN-g IL-1a IL-1b IL-4 IL-6 TGF-b TNF-a VEGF-a Day 14 GM-CSF IFN-g IL-1a IL-1b IL-4 IL-6 TGF-b TNF-a VEGF-a

Mepitel (5.61  0.92) (1.16  0.14) 2.90  0.34 2.64  0.97 (5.16  0.33) (6.22  0.58) (6.68  0.95) (2.60  0.30) 0.42  0.14

Ac-ILVAGK-NH2  103  103

   

104 102 103 102

(3.80 ± 0.14)  10L3 (1.70  0.32)  103 3.90  0.34 1.48  0.66 (6.91 ± 0.76)  104 (7.01 ± 0.63)  102 (6.63  1.37)  103 (2.54 ± 0.20)  102 (5.16  0.31)  102

(4.66  1.09) (1.14  0.15) 1.99  0.57 2.77  1.76 (6.09  0.34) (6.60  0.95) (5.29  1.13) (2.32  0.41) 0.15  0.06

Ac-LIVAGK-NH2

 103  103

   

104 102 103 102

(5.42 ± 0.48)  10L3 (1.93  0.22)  103 4.82  0.67 0.86  0.20 (9.42 ± 0.94)  104 (9.97 ± 0.98)  102 (6.99  1.29)  103 (3.45 ± 0.29)  102 (6.07  0.70)  102

(6.62  1.14) (1.59  0.31) 1.78  0.37 4.81  0.93 (6.38  1.87) (7.54  1.10) (7.49  1.56) (2.37  0.43) 0.11  0.02

p-value

 103  103

   

104 102 103 102

(3.93 ± 0.31)  10L3 (1.28  0.14)  103 3.30  0.41 0.72  0.31 (6.14 ± 0.57)  104 (6.79 ± 0.62)  102 4.93  0.91)  103 (2.41 ± 0.19)  102 (5.55  0.92)  102

0.451 0.305 0.216 0.437 0.728 0.587 0.475 0.863 0.067 0.006 0.176 0.116 0.441 0.02 0.015 0.452 0.009 0.654

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Mepitel-dressed wounds was minimal. In conclusion, histological analysis corroborated observations gleaned from gross examinations and digital planimetry. The peptide hydrogel wound dressing promotes reepithelialization, in comparison with the Mepitel control. This translates to faster wound closure, which in turn reduces risk of infection and implicates earlier tissue regeneration. Our results also suggest that the slight amino acid sequence difference between the two ultrashort peptides can potentially influence the rate at which wound healing occurs. In this study, Ac-LIVAGK-NH2 prompted the wounded skin to re-epithelialize at a faster rate, resulting in almost complete epithelial regeneration by day 14. 3.5. Cytokine expression During wound healing, the complex interplay of cytokines and growth factors orchestrate the migration and proliferation of different cells to mediate the overlapping processes of inflammation, granulation, re-epithelialization, matrix formation and remodeling [32,33]. To glean an insight into the cytokines that influence hydrogel-enhanced burn wound healing, multiplex enzyme-linked immunosorbent assay (multiplex ELISA) was carried out on a panel of ten cytokines and growth factors for homogenized skin samples extracted at days 7 and 14 (Table 1).

In view of the small sample volume, the bead-based multiplex assay was chosen for its sensitivity and to minimize the assay volume. The expression levels were mostly detectable, though generally low after normalizing against the total sample protein content. As such, we did not further assay for chemokine expression. At both the day 7 and day 14 time points, the expression of pro-inflammatory, anti-healing interleukin-2 (IL-2) was below the detection limit for all the samples tested, including healthy skin. The low expression of pro-inflammatory cytokines was partially attributed to the declining granulation and increasing reepithelialization (particularly for wounds treated with AcLIVAGK-NH2 hydrogels). Another plausible explanation was the biocompatibility of all the dressings evaluated. The clinicallyapproved, silicone-based Mepitel is chemically inert. The in vivo biocompatibility of our ultrashort peptides have been well-studied in various animal models. These peptides do not elicit cellular and humoral immune responses; the cellular response to implanted hydrogels was minimal to mild and no antibodies were detected even when the peptides were co-administered with an adjuvant. Various assays have also been completed to demonstrate that they are non-mutagenic, non-immunogenic and non-allergic. Thus, the dressings are not expected to exacerbate the inflammatory response and differences in the cytokine profile can be associated with the healing process.

Fig. 5. The differential expression of pro-inflammatory cytokines by burn wound injuries dressed with Mepitel and peptide hydrogels after 14 days. The cytokine concentration was normalized against the total protein content. In general, the expression levels are low. The cytokine expression levels of Mepitel and Ac-LIVAGK-NH2 are comparable to that of healthy skin. Several pro-inflammatory cytokines are up-regulated for wounds treated with Ac-ILVAGK-NH2. All these cytokines (IL-1a, IL-6, TNF-a and GM-CSF) are associated with re-epithelialization. The groups which are statistically significant (p < 0.05) are marked with the symbol (*). The line indicates the mean value while the error bars refer to standard error.

Y. Loo et al. / Biomaterials 35 (2014) 4805e4814

One-way ANOVA was conducted comparing the normalized mean of different cytokines for the three treatment groups. There were no statistically significant differences in normalized cytokine expression levels on day 7 (Table 1). By day 14, the cytokine expression of wounds treated with Ac-LIVAGK-NH2 and Mepitel were comparable to that of healthy skin. Ac-ILVAGK-NH2 elicited significantly higher expression of interleukin-4 (IL-4) (p ¼ 0.02), interleukin-6 (IL-6) (p ¼ 0.015), tumor necrosis factor-alpha (TNFa) (p ¼ 0.009) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (p ¼ 0.006) (Fig. 5). The latter three are proinflammatory cytokines. Interleukin-1a (IL-1a) was also slightly elevated. IL-1a is constitutively produced by epidermal keratinocytes to maintain the barrier function. When the skin is damaged, IL-1a is secreted to stimulate the production of collagen precursors and cellular proliferation [32]. IL-1a also acts synergistically with TNF-a to induce inflammation and facilitate reepithelialization [32]. IL-6 is another integral cytokine which indirectly induces leukocyte infiltration, matrix re-modeling, angiogenesis and epithelialization [33]. Likewise, GM-CSF promotes re-epithelialization directly by increasing keratinocyte proliferation and indirectly by up-regulating IL-6 [32]. Since wounds treated with Ac-LIVAGK-NH2 have almost completely reepithelialized, the expression of these pro-inflammatory cytokines have been down-regulated and have comparable expression levels to healthy skin (Fig. 5); whereas for Ac-ILVAGK-NH2, the higher levels of IL-1a, IL-6 and TNF-a mediate the ongoing reepithelialization. Thus, the differing immune responses between the two ultrashort peptides can be attributed to differences in rate of re-epithelialization. 4. Conclusion The application of ultrashort peptide hydrogels expedites the recovery of partial thickness burn wounds by accelerating autolytic debridement, stimulating epithelial cell proliferation and promoting wound closure. They combine the advantages of commercial hydrogel dressings with those of nanofibrous scaffolds in providing topographical cues for tissue regeneration. Their optical transparency facilitates wound observation and their material properties enable painless wound cleaning during the frequent dressing changes necessitated by the severity of injury. Having demonstrated the biocompatibility and efficacy, future prospects include the encapsulation of therapeutics such as antibiotics and anti-inflammatories to forestall infection and accelerate the tissue regeneration. The peptides’ propensity for self-assembly also lends itself amendable to the design of different formulations e from membranes that can be rehydrated, to topical gels, and to spray formulations. The versatility in formulation will facilitate commercialization for different needs. Considering the long shelf-life stability of sealed peptides stored at room temperature, we are particularly keen on developing “just-add-water” formulations. The hydrogels can be reconstituted by adding a fixed volume of clean water to lyophilized peptide powder at the point of application. This formulation design will greatly reduce transportation costs and potentially revolutionize emergency medicine, providing a convenient, easyto-use first-line treatment for partial thickness burn injuries. In doing so, our ultrashort peptide hydrogels will fill a niche that is sorely neglected by commercial skin substitutes and topical hydrogel dressings currently available in the market. Acknowledgments This work is supported by the Institute of Bioengineering and Nanotechnology (Biomedical Research Council, Agency for Science,

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Technology and Research, Singapore) and the Defense Science Organisation. The authors would like to thank Dr Kiat Hwa Chan for his help with FESEM, Ms Nina Ma for her help with thermogravimetric studies, Dr Michael Reithofer for his help with mass spectroscopy analysis, Ms Eileen Hing for her help with coordinating the animal study, Dr Wu Jian for his help with animal handling and Ms Mui Hong Tan for her help with histology.

Appendix A. Supplementary data Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j.biomaterials.2014.02.047.

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