Development of Responsive Chitosan–Genipin Hydrogels for the

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Article Cite This: ACS Appl. Bio Mater. 2019, 2, 2879−2888

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Development of Responsive Chitosan−Genipin Hydrogels for the Treatment of Wounds Abitha M. Heimbuck,†,# Tyler R. Priddy-Arrington,†,# Madison L. Padgett,† Claire B. Llamas,‡ Haley H. Barnett,§ Bruce A. Bunnell,‡ and Mary E. Caldorera-Moore*,† †

Department of Biomedical Engineering, Louisiana Tech University, Ruston, Louisiana 71272, United States Department of Pharmacology, Center for Stem Cell Research and Regenerative Medicine, Tulane University School of Medicine, New Orleans, Louisiana 70118, United States § School of Biological Sciences, Louisiana Tech University, Ruston, Louisiana 71272, United States Downloaded via BUFFALO STATE on July 18, 2019 at 15:15:47 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

‡

S Supporting Information *

ABSTRACT: Chronic wounds are characterized by an increased bacterial presence, alkaline pH, and excessive wound drainage. Hydrogel biomaterials composed of the carbohydrate polymer chitosan are advantageous for wound healing applications because of their innate antimicrobial and hemostatic properties. Here, genipin-cross-linked−chitosan hydrogels were synthesized and characterized, and their in vitro and in vivo performances were evaluated as a viable wound dressing. Characterization studies demonstrate that the developed chitosan−genipin hydrogels were able to neutralize an environmental pH, while averaging ∼230% aqueous solution uptake, demonstrating their use as a perfusive wound dressing. Bacterial activity studies demonstrate the hydrogels’ ability to hinder Escherichia coli growth by ∼70%, while remaining biocompatible in vitro to fibroblast and keratinocyte cells. Furthermore, chitosan−genipin hydrogels promote an enhanced immune response and cellular proliferation in induced pressure wounds in mice. All together, these results reflect the potential of the developed hydrogels to be used as a proactive wound dressing. KEYWORDS: chitosan, genipin, responsive hydrogels, chronic wound healing, fibroblast cytocompatibility and attachment, wound dressing

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INTRODUCTION Nonhealing wounds, also referred to as chronic wounds, affect 6.5 million people in the USA with over $25 billion spent annually in health care costs.1 Chronic wounds, including pressure ulcers and diabetic foot ulcers (DFUs), can lead to decreased function, lower quality of life, less activity for the patient, and eventually amputation or death.2 These wounds compromise the integrity of the epidermis, dermis, underlying adipose tissue, muscle, and bone.3 Chronic wounds are plagued with many disrupting factors that inhibit wound healing including increased bacterial burden, excessive wound drainage, oxidation, elevated pH,4,5 and increased tissue enzyme activity, specifically matrix metalloproteinase (MMP).6 Additionally, bacterial infections within the wound environment can lead to the release of endotoxins resulting in elevated pro-inflammatory cytokines, or exotoxins, which can cause tissue necrosis.7 When left untreated, this may inhibit wound healing processes such as angiogenesis and epithelialization.7 An ideal wound dressing should maintain a local moist environment, stimulate growth factors, absorb wound fluids © 2019 American Chemical Society

and exudates, protect the site from infection, and be biocompatible.2,8 Current treatment options for chronic wounds include moist dressings, bioactive dressings,9,10 scaffolds,11,12 protease modulating ointment,13 and iodine loaded matrices.14 These treatment options tend to have a singular function: reduce bacterial growth, absorb large volumes of wound exudate, or regulate tissue enzyme activity to combat commonly encountered issues. However, chronic wounds often require a combination of different treatment options to provide effective patient care; a need the current treatment methods do not address. Additionally, current treatment methods only treat the symptoms of chronic wounds instead of treating the underlying pathophysiological disturbances that prevent proper wound healing and tissue reorganization. Furthermore, current treatment methods are expensive, painful, and highly time intensive due to replacing the bandages multiple times per day, removing necrotic tissue Received: March 29, 2019 Accepted: June 18, 2019 Published: June 18, 2019 2879

DOI: 10.1021/acsabm.9b00266 ACS Appl. Bio Mater. 2019, 2, 2879−2888

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ACS Applied Bio Materials

hydrogel capable of absorbing large volumes of fluids for chronic wound applications. The effects of the chitosan molecular weight (MW, 50−190 kDa called HC for “high” MW and 15 kDa called LC for “low” MW) and cross-linker percentage on hydrogel properties were investigated to determine the hydrogel’s ability to perform as a wound dressing. Cell viability and attachment studies using adult human foreskin fibroblasts (HFF) and adult immortalized keratinocytes (HaCaTs) were performed to determine the in vitro performance of the developed hydrogel for use as a cell scaffold. The chitosan−genipin hydrogel’s antibacterial capabilities were evaluated by performing colony forming unit (CFU) studies with Escherichia coli (E. coli). Lastly, preliminary in vivo biocompatibility and wound healing efficacy of developed chitosan−genipin hydrogels were investigated using healthy 4-week-old female C57BL/6 wild-type mice with induced stage 1 pressure ulcers.

from the wound site, and other required maintenance actions. Altogether, high cost of care, low patient compliance, and high wound maintenance time have amplified the need for a low cost, low maintenance, multifunctional treatment option. A range of biopolymer hydrogels have been explored for wound healing applications due to their innate properties and biocompatibility.15−19 Hydrogels are 3D cross-linked polymeric matrices capable of exchanging large volumes of fluid. Over the past three decades, a variety of hydrogel-based biomaterials have been developed to be environmentally responsive to stimuli such as biomolecules, pH, temperature, or a combination of these factors.20 Of these biopolymers, chitosan is an attractive option for chronic wound healing due to its antimicrobial, mucoadhesive, and hemostatic properties.21 In addition, chitosan is pH-responsive, due to its cationic nature, and can indirectly regulate tissue enzymatic activity, which is elevated22 at the alkaline pH of chronic wounds.5 Furthermore, researchers have demonstrated chitosan scaffolds were found to accelerate the closure of pressure ulcer wounds in old mice.6 Covalent cross-linking of chitosan yields a mechanically robust, uniform hydrogel network capable of absorbing large amounts of aqueous solution without dissolving.23 Covalently cross-linked chitosan hydrogels allow free water diffusion, and the permanent nature of the cross-linked hydrogels is conducive for their use as a scaffold for tissue engineering.24 Glutaraldehyde-cross-linked collagen− chitosan hydrogels have been explored for wound healing11 and adipose tissue engineering applications.25 However, glutaraldehyde’s toxicity and severe skin irritancy properties make its use problematic in medical applications.26 Alternatively, genipin, a biologically derived substance, has a low in vivo toxicity with a median lethal dose (LD50) of 382 mg/kg in mice models, which is 1000 times less toxic than glutaraldehyde, making genipin a more ideal cross-linker.27 Several studies have explored the various medical applications of chitosan−genipin biomaterials.21,28−30 In these studies, chitosan and genipin were mixed at room temperature or at 37 °C to understand the mechanism of cross-linking,30−32 and to determine the gelation point.33 Additional studies have developed solution cast chitosan− genipin stents at 37 °C that were mechanically superior than standard metallic stents.34 In these studies, dried chitosan films were either soaked in genipin solution,30,34 genipin was added to the chitosan solution then dried,21,31−33 or chitosan and genipin were mixed with other polymers then solution cast, electrospun into fibers, or directly lyophilized.28,29 These studies lacked parameter controls, such as regulating reaction temperature, polymer MW, and length of drying/reaction time; thus, they are limited in their ability to be reproduced or scaled into implementation. Conversely, this work systematically explores the optimal methods and components for synthesis of chitosan−genipin hydrogels in a controlled, reproducible, and scalable fashion via thermal cross-linking to reduce the variability between batches. Understanding the roles chitosan molecular weight and cross-linking density have on the resulting hydrogel’s properties would improve the tunability of chitosan−genipin hydrogels as these effects have yet to be characterized. Here we report on the development of responsive hydrogels that integrate the beneficial properties of chitosan with the absorbent, biomimetic nature of a hydrogel to develop a multifunctional wound dressing platform. Chitosan was crosslinked using genipin for the development of a pH-responsive

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EXPERIMENTAL SECTION

Materials. Hydrogel Synthesis and Characterization. Chitosan of 15 kDa MW (termed LC) and 85% degree of deacetylation (DD) was obtained from Polysciences, USA. Chitosan of MW 50−190 kDa (termed HC) and DD of 75−85%, genipin, glucosamine hydrochloride, and ninhydrin reagent were obtained from Sigma-Aldrich, USA. Phosphate buffered saline (PBS, 1× tablets that contain 0.01 M phosphate buffer, 0.0027 M potassium chloride, and 0.137 M sodium chloride), sodium hydroxide, and glacial acetic acid were obtained from Fisher Scientific, USA. Unless otherwise stated, all materials were analytical grade and used as received. In Vitro Performance Studies. Trypsin-ethylenediaminetetraacetic acid (trypsin-EDTA), Dulbecco’s modified Eagle medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640, sterile phosphate buffered saline solution (PBS at 1× concentration), fetal bovine serum (FBS), and L-glutamine were obtained from Fisher Scientific, USA. MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) cell proliferation assay, actin green 488 ready probes reagent, and Hoechst trichloride trihydrate solution were obtained from Thermo Fisher Scientific, USA. Luria− Bertani agar was obtained from Sigma-Aldrich, USA. Sylgard 184 silicone elastomer kits were obtained from Ellsworth Adhesives, USA. Human foreskin fibroblast (HFF) cells were provided by Dr. Bodily’s laboratory at Louisiana State University Health, Shreveport. Immortalized human keratinocytes (HaCaTs) were generously provided by Dr. Sapp’s laboratory at Louisiana State University Health, Shreveport. Human monocytes (THP-1) were provided by Dr. Jain’s laboratory at Louisiana State University Health, Shreveport. E. coli was provided by Dr. Giorno-McConnell’s lab at Louisiana Tech University. Unless otherwise stated, all materials were analytical grade and used as received. In Vivo Performance Studies. Female C57BL/6 wild-type mice (4 weeks old) were purchased from Charles River Laboratories (Wilmington, MA). The mice were housed in a pathogen-free environment, monitored regularly for maladies, and provided with food and water as needed by the vivarium staff at Tulane University. All animal studies were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committees (IACUC) at Tulane University. Mice were acclimated for 1 week prior to pressure ulcer induction using magnets.35 Isoflurane, formalin, tissue path IV tissue cassettes, 3 M vetbond tissue adhesive, 3 M Tegaderm transparent tissue dressing, and hematoxylin and eosin stains were obtained from Thermo Fisher Scientific, USA, and used according to the manufacturer’s protocol. Methods. Hydrogel Synthesis. The hydrogel synthesis process was performed using two different MWs of chitosan denoted by low molecular weight chitosan (LC), 15 kDa, and high molecular weight chitosan (HC), 50−190 kDa. Hydrogels were cross-linked with genipin at different concentrations: 1%, 2%, or 3% weight (wt), with respect to chitosan, to characterize the most efficient cross-linker 2880

DOI: 10.1021/acsabm.9b00266 ACS Appl. Bio Mater. 2019, 2, 2879−2888

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Figure 1. Hydrogel synthesis process. Schematic illustrating the chitosan−genipin hydrogel synthesis process and the different hydrogel groups synthesized by varying chitosan molecular weights and genipin weight percentages and investigated. percent as summarized in Figure 1. First, a 2% (w/v) chitosan solution was prepared by dissolving chitosan powder into 0.075 M glacial acetic acid aqueous solution by mixing for 8−12 h. A stock solution of genipin was then prepared by dissolving genipin powder into 100% ethanol at a ratio of 5 mg/mL. The desired final volume of genipin was then added to the chitosan solution and mixed for 30 min to form the hydrogel precursor solution. Next, the precursor solution was sonicated for 30 min in the Branson 2800 Ultrasonic Bath to thoroughly mix the solution, while also removing air bubbles. After sonication, the precursor solution was incubated for 24 h at 50 °C in an oven for uniform heating. Absorbance spectral scans in the visible range (300−800 nm) were used to monitor the rate of chitosan− genipin cross-linking throughout the reaction by monitoring the intensity of the 610 nm peak. Sample absorbance spectra between 300−800 nm were measured at the start of the cross-linking reaction (0 h) and during the reaction at 50 °C at different time intervals (3, 6, 12, and 24 h) using the Cytation 5 Biotek Microplate reader (see Supporting Information). After cross-linking, hydrogels were placed in a chemical fume hood, uncovered, and allowed to air dry for 5 days. Dried samples were rinsed in deionized (DI) water for 24 h prior to use. Un-cross-linked control chitosan films (termed LC and HC) were synthesized identical to the cross-linked chitosan−genipin hydrogels but without genipin. Hydrogel Characterization. The resulting chitosan−genipin hydrogels’ chemical composition was evaluated using solid-state nuclear magnetic resonance spectroscopy (NMR) and the ninhydrin assay to determine the optimal chitosan−genipin ratio and chitosan MW. 13C NMR spectra of all genipin-cross-linked−chitosan hydrogel formulations and the raw materials were collected using a Bruker AV console and a Bruker triple resonance 4 mm MAS probe at a resonance frequency of 100.6 MHz (see Supporting Information). A ninhydrin assay was used to quantify the concentration of unreacted amines in the post-cross-linked chitosan hydrogels as a function of genipin. The morphology of each hydrogel was characterized by environmental scanning electron microscopy (ESEM). The synthesized hydrogels’ swelling and pH profiles were investigated from short- and long-term swelling studies in PBS (see Supporting Information) and in cell-conditioned media. Ninhydrin Assay. A ninhydrin assay was used to measure the concentration of unreacted amines on the chitosan in the post genipin

cross-linked chitosan hydrogels as a function of genipin concentration introduced to the precursor solution. A D-glucosamine stock solution of 50 μM in 0.05% v/v glacial acetic acid was used to prepare the standard curve. Two mg of air-dried hydrogel samples from each group (Figure 1) were swollen overnight in 0.5 mL of DI water. Ninhydrin reagent (0.5 mL) was then added to the hydrogel samples and heated to 100 °C in a water bath for 10 min. The solution was then cooled to room temperature, centrifuged at 16 000g for 5 min, and diluted 10 times using 95% ethanol. The absorbance at 570 nm, proportional to the amount of free amine groups present in the sample, was recorded using the Biotek Cytation 5 Microplate reader. The concentration of free amines in each group was calculated using the D-glucosamine standard curve. For each chitosan molecular weight, the moles of free amines in the control chitosan film were assumed to be the total moles of amine groups available for genipin cross-linking. The cross-linking degree (CD) was calculated using eq 1: ij C − C t yz zz × 100 cross‐linking degree (CD) = jjj c j Cc zz k {

(1)

where Cc is the moles of free amines in the control chitosan film and Ct is the amine group concentration in the cross-linked hydrogels. Hydrogel Morphology. Environmental scanning electron microscopy (ESEM) was used to evaluate the chitosan−genipin hydrogel innate morphology in comparison to control chitosan films. A FEI Quanta 3D FEG FIB/SEM system in ESEM mode at 100 Pa and 20 kV was used to image the samples in the relaxed state. Samples were mounted to the sample stub using double-sided carbon tape, and images were obtained quickly to prevent sample shrinkage from drying. Swelling and pH Profiles in Cell-Media. Hydrogel swelling and pH response in cell-conditioned media (CM), harvested from individual cell cultures of HFF, HaCaT, and THP-1 cells 2 days post 100% confluence, were evaluated to determine the synthesized chitosan− genipin hydrogel swelling profile and ability to neutralize pH in a physiologically relevant environment. The CM was centrifuged at 1500 rpm for 5 min to remove dead cell debris and stored at 4 °C prior to use. A mixture of 1:1:1 of HFF CM, HaCaT CM, and THP-1 CM was used to determine the hydrogel swelling behavior in cell2881

DOI: 10.1021/acsabm.9b00266 ACS Appl. Bio Mater. 2019, 2, 2879−2888

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and tissue culture plates as controls at a density of 5.2 × 104 cells/cm2 in triplicates. Wells with and without hydrogels were treated identically. To allow the cells to adhere, the wells were incubated for 48 h after cell seeding. The cells were washed twice with sterile PBS (1×) and fixed using 50 μL of 10% formalin for 10 min. Formalin was removed from the wells and rinsed twice with PBS (1×). The cells were permeabilized using 200 μL per well of 0.2% Triton X-100 in PBS for 10 min. The cells were rinsed twice with PBS (1×) and stained using 200 μL of a 2 drop per mL stock solution of Actin Green Ready probes for 30 min. Plates were wrapped in aluminum foil for the duration of the staining process. Each well was then stained with 200 μL of Hoechst dye, diluted to 8.10 μM, 20 min after the start of Actin Green staining. The stains were removed, and the wells were rinsed twice with PBS (1×). The hydrogels were then moved to new wells and covered with fresh PBS for imaging. Plates were covered in aluminum foil and stored at 4 °C until imaging. Samples were imaged using either the EVOS microscope system or the Biotek Cytation 5 plate reader in fluorescent imaging mode. Antibacterial Activity. Escherichia coli (E. coli) was used to study the antibacterial activity of the hydrogels against Gram (−) organisms. Air-dried, cross-linked chitosan−genipin hydrogels (HC2% and LC2%) and control chitosan films (HC and LC) were punched out at a diameter of 5.9 mm and UV sterilized for 15 min prior to equilibration in sterile PBS for 3 h under sterile conditions. For each trial, a streak plate of glycerol stock was prepared to isolate a single colony. The single E. coli colony was added to 5 mL of Luria− Bertani (LB) broth and incubated overnight at 37 °C. The culture was diluted to obtain the optical density (OD) of a 0.5 McFarland standard (0.08−0.1) using sterile PBS (1×), approximately equal to 107 − 108 CFUs/mL. A 200 μL aliquot of a diluted bacterial culture was then added directly to chitosan samples and incubated for 4 h at 37 °C. The bacterial culture was diluted 10−4, 10−5, 10−6, and 10−7 times using PBS (1×), and 100 μL of each dilution was plated in triplicate onto LB-agar plates. The plates were incubated at 37 °C overnight to determine the colony forming units from each treatment; then, the colony forming units were counted. The bactericidal activity of the hydrogel films was calculated as a ratio of the CFUs from hydrogel-treated cultures to untreated, control cultures. The experiment was performed in triplicate, with n = 3 plates for each dilution and each dilution repeated 3 times for a total of 9 culture plates for each hydrogel group. In Vivo Performance Studies. In vivo biocompatibility and wound healing efficacy of developed chitosan−genipin hydrogels were investigated using healthy 4-week-old female C57BL/6 wild-type mice with induced stage 1 pressure ulcers. All animal studies were conducted in accordance with protocols approved by the IACUC at Tulane University. Pressure ulcers were induced as previously described.39 After a one-week acclimation period, two magnets, 12 mm in diameter, were placed within the thoracic area between the neck and the humerus, post hair removal, while under anesthesia. The magnets were applied for two on−off 12 h cycles. The left side was marked as the control and treated with gauze, while the right wound was treated with a sterilized chitosan−genipin hydrogel (LC2%). Prior to scaffold application, the wounds were photographed, and the length and width of the wounds were measured using digital Vernier calipers. Hydrated hydrogel or gauze was applied to each wound and sealed tight using a Tegaderm adhesive dressing. Four groups, 3, 7, 14, and 21 days, with five mice per group were studied. At each time point, the mice were sacrificed via 2 min exposure to carbon dioxide (CO2) followed by cervical dislocation. The wound was digitally imaged, and wound length and width were measured. Wound size was calculated based on the area of an ellipse from the length and width measurements. Wounds were harvested with a 5 mm rim of unwounded skin tissue, fixed in 10% neutral buffer formalin for at least 48 h before embedding in paraffin and processed on the Lecia TP1020 in 12 steps. The processed tissue was placed vertically in a mold with melted paraffin and allowed to fully solidify. The tissue blocks were then sectioned into 5 μm slices, rehydrated, and stained with hematoxylin and eosin (H&E) using an automated slide stainer (Varistain Gemini ES). Coverslips were mounted on stained tissue

conditioned media. The dry mass of the hydrogels was obtained and used in computing the percent swelling as a function of time at 37 °C. The air-dried hydrogels were placed in CM, and the corresponding weight and pH were recorded at various time points over 21 days (0.5 , 1, 2, 4, 6, 12, 24 h, daily for 7 days, and every 2 days after until day 21 of exposure). Swelling behavior of the hydrogels as a function of time was calculated as a percentage using eq 2:

swelling (%) =

WF − Wi × 100 Wi

(2)

where WF is the weight of the hydrogel at each time point and Wi is the initial dry weight of the hydrogel. Un-cross-linked chitosan films (HC and LC) were used as controls for both swelling and pH profiles. Additionally, control samples of cell media solution not exposed to hydrogels were also measured over time to track the pH drift of the media solution. In Vitro Performance Studies. Cell Culture. DMEM complete media was prepared using DMEM (4.5 g/L glucose) supplemented with 10% FBS, 2 mM L-glutamine, and 1% penicillin−streptomycin solution. HFFs and HaCaTs were cultured in DMEM complete medium and plated into 75 cm2 cell culture flasks. THP-1 cells were cultured in RPMI 1640 supplemented with 10% FBS and 1% penicillin−streptomycin in a 25 cm2 cell culture flask. Cells were maintained in a humidified incubator with 5% CO2 at 37 °C. Culture medium was refreshed every 2−3 days, and cells were passaged when they reached ∼80% confluency. Biocompatibility. Re-epithelialization is crucial in defining the healing success of a wound.36 Keratinocytes are responsible for maintaining the epidermal barrier and for epidermal repair postinjury via cues from neutrophils and macrophages recruited to the site of injury.37 Keratinocytes migrate and proliferate to close the wound gap; this process is accelerated by growth factors such as FGF−238 produced by fibroblasts. Owing to the importance of these two cell types in wound remodeling, the biocompatibility of chitosan−genipin hydrogels and control films was tested on adult human foreskin fibroblasts (HFF) and adult human immortalized keratinocytes (HaCaTs) using an MTS cell viability assay with 48 h exposure of cells to hydrogels. Cells were plated on a 48-well plate in triplicate at a density of 5.2 × 104 cells/cm2 and incubated at 37 °C for 24 h to reach confluency. The cells were gently rinsed twice with sterile PBS (1×) to remove cell debris and unattached cells. Air-dried hydrogels and control films were punched to 12.3 mm in diameter and 3 mm in height for use. The hydrogels/control films were UV sterilized for 15 min and then equilibrated in sterile PBS for 3 h under sterile conditions. The hydrogels were then incubated in DMEM complete media overnight at 37 °C to imbue the hydrogels with cell culture media. The following day, sterilized hydrogels were anchored down directly in contact with the adhered cell monolayer using polydimethylsiloxane (PDMS) rings, 12.5 mm outer diameter and 10 mm inner diameter, in the 48-well plate and 400 μL of DMEM complete media was added and incubated at 37 °C and 5% CO2 for 24 and 48 h. At each time point, PDMS rings and hydrogels were removed and cells were gently washed twice using sterile PBS (1×) before 40 μL of MTS reagent was added and incubated for 4 h. The absorbance at 490 nm was collected using the Biotek Cytation 5 microplate reader to determine the metabolic activity of cells in each well. For each cell type and each time point, control wells with cells only were used for reference. The experiment was conducted in triplicate for statistical relevance. The percentage cell viability was calculated as a ratio of average absorbance of cells exposed to hydrogels against the average absorbance of control cells. Fluorescence Imaging of Cells on Hydrogels. Fluorescent imaging of HFF and HaCaTs on sterilized hydrogels was performed to assess cell attachment to the hydrogels to show potential for use as a platform for cells to attach and grow at the wound site. Sterilized 2% cross-linked hydrogels (HC2% and LC2%) and un-cross-linked chitosan films (HC and LC) were anchored to the bottom of each well using PDMS rings and equilibrated in DMEM complete media for 24 h. Cells were seeded directly onto hydrogels in a 48-well plate 2882

DOI: 10.1021/acsabm.9b00266 ACS Appl. Bio Mater. 2019, 2, 2879−2888

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Figure 2. Cross-linking density of chitosan−genipin hydrogels. (a) Reactive amine concentration quantified using the ninhydrin reaction and (b) cross-linking degree for different hydrogel formulations; p < 0.05; n = 9.

Figure 3. Environmental SEM images of synthesized chitosan−genipin hydrogels showing network porosity. section slides using permount mounting media and dried for 24 h. The slides were viewed and imaged at 4× and 10× magnification using a Leitz Laborlux X microscope fitted with a CCD camera for histological analysis. Statistical Analysis. Results are presented as a mean ± standard deviation. Two-way ANOVA followed by Tukey’s multiple comparison tests were performed using GraphPad Prism version 6.00 for Windows, GraphPad Software, La Jolla California USA. Effects were considered significant if the p value was less than 0.05. Multifactor interaction and ANOVA were determined using R-project. Data was collected in triplicate unless otherwise stated.

tion) and a ninhydrin assay. Resulting hydrogel morphology of each hydrogel was characterized by ESEM. The synthesized hydrogels’ swelling and pH profiles were investigated from short- and long-term swelling studies in PBS (see Supporting Information) and in cell-conditioned media. Ninhydrin Assay. A ninhydrin assay was used to quantify the concentration of unreacted amines present on the chitosan in the genipin-cross-linked−chitosan hydrogels with respect to control chitosan films (HC and LC). The results (Figure 2) validated that increasing the genipin weight percent increased the amount of amine consumption during the cross-linking. The concentration of free amines in the cross-linked hydrogels compared to the control chitosan films (HC and LC) showed that fewer reactive amine groups are available in the crosslinked hydrogel (Figure 2A). These results indicate that some

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RESULTS AND DISCUSSION Hydrogel Characterization. The chemical composition of the synthesized chitosan genipin hydrogels was determined using solid-state 13C NMR spectra (see Supporting Informa2883

DOI: 10.1021/acsabm.9b00266 ACS Appl. Bio Mater. 2019, 2, 2879−2888

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Figure 4. In vitro characterization of chitosan−genipin hydrogels. (A) Swelling (left) and pH profile (right) of the chitosan−genipin hydrogel (LC 2% and HC 2%) and control film (LC and HC) in cell-conditioned media (CM) at 37 °C for a period of 21 days. (B) Cell viability (normalized with respect to control wells) of HFF and K post 24 h hydrogel exposure. (C) Antibacterial activity against Escherichia coli (E. coli) when exposed to the hydrogels. (D, E) Fluorescent microscopy of (D) human adult foreskin fibroblast (HFF) and (E) adult immortalized keratinocytes (HaCaTs) cells with nuclear DAPI (blue) stain and GFP (green) actin stain grown on HC2% hydrogels.

pH when applied as a wound dressing. Initial PBS studies demonstrated that neither chitosan MW, genipin weight percent, nor environmental pH had a significant effect on hydrogel swelling percent. The chitosan−genipin hydrogels averaged 230% fluid uptake with a pH drop of 0.80 ± 0.24 pH units in pH 8.2 PBS and a drop of 0.30 ± 0.11 pH units in pH 7.4 PBS (see Supporting Information). To determine the swelling profile and pH effect in a more physiologically relevant environment, cell media at pH ∼ 7.6 containing metabolites, growth factors and other cell waste was employed to evaluate the synthesized hydrogels’ fluid uptake and pH neutralization capabilities. The fluid uptake of the chitosan− genipin hydrogels achieved ∼150% after 1 h of swelling and maintained this swelling percent over the 21 days studied (Figure 4A). Cross-linking does significantly increase the swelling percent for HC and LC at all time points. However, chitosan MW does not significantly affect swelling of crosslinked hydrogels. No significant change in solution pH with respect to the pH of the control solution was observed. This implies that, at a neutral pH (7.6), the hydrogels do not drastically affect the pH of the cell media in a way that would be harmful to cell growth. In Vitro Performance Studies. Biocompatibility. Hydrogel biocompatibility was determined through an MTS cell viability assay over 48 h of exposure. Neither cross-linking percentage nor molecular weight significantly affected HaCaT or HFF cell viability (Figure 4B). For both cell types utilized, no statistically significant difference was seen between control wells and wells exposed to hydrogels. These results mirror and support the results from Norowski et al., who reported that the cytocompatibility of dermal fibroblasts seeded on electrospun chitosan−genipin cross-linked nanofibrous mats was equivalent to control cell groups.29 These results demonstrate that

of the amine groups are consumed in the genipin−chitosan cross-linking reaction. Results also showed that increasing the genipin weight percent introduced into the precursor hydrogel solution led to a decrease in free amine concentration. The difference in free amines in the 1, 2, and 3% genipin hydrogels (for both the high and low MW chitosan films) indicate an increase in total cross-linking. Additionally, from the ninhydrin assay result, it appears a cross-linking plateau was reached at a genipin percentage of 2%; above which increasing the amount of genipin introduced into the precursor solution had no significant effect on the hydrogel synthesis. The maximum cross-linking degree achieved was 47% and 52% for HC crosslinked with 2% wt genipin and LC cross-linked with 3% wt genipin, respectively (Figure 2B). Increasing the cross-linker amount above 2% wt was not cost-effective, as no significant change in amine utilization was observed. Considering these results, in vitro and in vivo characterization experiments were performed using only hydrogels with 2% wt genipin crosslinker to minimize cost and toxicity effects while maximizing the cross-linking, and thus physical integrity, of the chitosan− genipin hydrogels. Hydrogel Morphology. Environmental scanning electron microscopy (ESEM) was used to obtain images of the hydrogels in the relaxed state (Figure 3). From these images it can be observed that both the control films (HC & LC) exhibit a flat, rigid pattern with no visible pores. The rigid pattern could be a result of water evaporation on the hydrogel surface. Conversely, both cross-linked hydrogel groups clearly exhibit a highly porous network, with no apparent qualitative differences between the different molecular weight samples. Swelling and pH Profiles in Cell Media. Swelling studies were performed to estimate the fluid uptake and holding capacity of the hydrogels and their ability to neutralize a basic 2884

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Figure 5. Chitosan−genipin hydrogel wound healing efficacy. (A) Induction of pressure wounds process and application of hydrogel and gauze treatments. (B) Change in wound size for control and hydrogel (LC2%) treated wounds. (C) Hematoxylin and eosin stained images of control and hydrogel treated wounds over time.

antibacterial activity.13,14 However, no statistical difference was observed between any of the treated groups, thus neither chitosan molecular weight nor cross-linking percent causes a significant change in antibacterial activity of chitosan. All treatment groups (HC, LC, HC2%, and LC2%) were significantly different from the control group that was not exposed to any samples, showing that these chitosan films and genipin-cross-linked−chitosan hydrogels do significantly hinder bacterial growth. These results suggest that chitosan− genipin hydrogels are capable of preventing and reducing bacterial growth of Gram-negative species, such as E. coli, in chronic wound sites. Furthermore, the ability of the chitosan− genipin hydrogels to limit bacterial growth without the use of antibiotics is very promising due to the rising concerns of antibiotic resistance.40,41 In Vivo Performance Studies. The objective of the murine wound model studies (Figure 5A) was to determine the chitosan−genipin hydrogel in vivo biocompatibility and functional efficiency as a wound treatment platform. Measured wound sizes of chitosan−genipin hydrogel treated wounds and control (gauze only) wounds at day 3, 7, 14, and 21 postwounding showed both groups had similar healing rates. Wound size analysis showed a linear progression of wound closure until day 14 for both the control and hydrogel treatment groups (Figure 5B). ANOVA and Tukey’s pairwise comparison showed that time was a significant factor in healing, but no statistically significant difference was observed between the hydrogel treated and control wound diameter during closure.

chitosan−genipin hydrogels do not adversely affect cell health. Furthermore, this indicates that chitosan−genipin hydrogels have the potential to function as chronic wound dressings without risk of inhibiting cell viability. Fluorescent Imaging of Cells on Hydrogels. No qualitative differences in cell attachment were observed between hydrogel groups with different molecular weights or percent crosslinking, and all exhibited excellent cellular adhesion as shown on the HC2% hydrogel (Figure 4C,D). The 2D fluorescent images obtained from this procedure confirm the ability for cellular adhesion to occur on the hydrogels. The promotion of cellular adhesion of HFF and HaCaTs on the chitosan− genipin hydrogel surface establishes the hydrogel matrix to be a proficient cell scaffold. Characterization and in vitro studies showed no statistical difference between hydrogel MW; therefore, to save on cost and time, only one hydrogel type was used in this study. LC2% was chosen due to lower variability in molecular weight and displaying a more porous morphology than HC2% gels in the ESEM images. Antibacterial Activity. The antibacterial activity of the chitosan−genipin hydrogels was tested by counting the colony forming units of bacterial culture exposed to hydrogels. Control films, HC and LC, were observed to reduce CFU growth of E. coli by ∼90% and cross-linked hydrogels cut growth by ∼70% (Figure 4E). The innate antibacterial ability of chitosan is due to the charged amine (−NH2) groups on the chitosan;13,14 however, during the cross-linking process of the hydrogel synthesis, many of these amine groups are consumed, which caused the cross-linked gels to exhibit a slightly lower 2885

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that is more likely to heal.5 In addition, these hydrogels retained their swelling capabilities in cell-conditioned media for 21 days, and they did not cause a significant change in pH of neutral cell-conditioned media. This indicates that their effect on pH is negligible at neutral conditions, meaning they would not cause adverse effects on cell growth. Biocompatibility was confirmed through in vitro and in vivo characterization. An MTS cell-viability assay determined that human fibroblasts and keratinocytes remained viable when exposed to the chitosan−genipin hydrogels, for 48 h, thus indicating biocompatibility. As demonstrated by fluorescence imaging, this platform could also support the attachment of fibroblasts and keratinocytes, two common epithelial cell types. Antimicrobial studies indicated that cross-linked hydrogels were able to reduce E. coli growth compared to control LB agar plates that were not exposed to chitosan hydrogels. Finally, H&E images taken from 4-week-old female mice with induced stage 1 pressure ulcers showed an increase in granulation, indicating a stronger immune response and more cell growth, in wounds treated with chitosan−genipin hydrogels compared to wounds treated by a gauze bandage. Through in vitro and in vivo characterization, we demonstrate the synthesis of biocompatible chitosan−genipin hydrogels that are an attractive option for low cost, low maintenance, and multifunctional wound dressings. Future work will focus on utilizing chitosan−genipin hydrogels in full thickness wound models to better evaluate their potential use in chronic wound healing applications.

Tissue sections were taken at each time point from both hydrogel and control (gauze) wounds for H&E staining to visualize the cellular response within the tissue throughout the wound healing process. Complete re-epithelialization of wounds was observed in H&E stained images of tissue sections for both wounds (Figure 5C). However, samples from the hydrogel treated group exhibited a thicker stratum spinosum, correlated to keratinocyte migration, and increased granulation tissue, which corresponds to cellular infiltration, as early as day 3 postwounding. Similar granulation tissue was not observed in the control group until day 7. This indicates that migration and proliferation of keratinocytes and fibroblasts were increased by the hydrogel alone, and this implies that the hydrogel treatment elicited an overall increased immune response at the wound site. These results show the potential of chitosan−genipin hydrogels to incite a stronger healing response at the tissue level than current gauze-treatment methods and support the use of chitosan−genipin hydrogels as a chronic wound dressing. This preliminary in vivo experiment reports the first results of an in vivo model for a wound dressing of solely chitosan− genipin hydrogels for pressure wound treatment. While the Wagner grade 1 ulcer used in this preliminary experiment did not represent a complex, profusive, chronic wound environment, for which the chitosan−genipin hydrogels were designed, and therefore did not test the hydrogel’s ability to absorb wound exudate, prevent bacterial infection, or regulate the pH environment of the wound, this experiment is still novel as the first reported in vivo model for a chitosan−genipin hydrogel chronic wound dressing. The chitosan−genipin hydrogels were designed to be most effective at treating high level, open chronic wounds with effusive drainage, a high pH environment, and necrotic tissue. Therefore, this preliminary study is a simplistic representation of the potential effectiveness of chitosan−genipin hydrogels for chronic wound healing; however, this study is still an essential first step. Future in-depth studies with full thickness wound models are needed to accurately assess the wound healing properties of the chitosan−genipin hydrogels.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsabm.9b00266. Methods and results of chitosan−genipin hydrogel formation absorbance, solid-state 13C nuclear magnetic resonance spectroscopy of chitosan−genipin hydrogels, and chitosan−genipin hydrogel swelling and pH profile in phosphate buffered saline (PDF)

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CONCLUSIONS The objective of this work was to develop a single chitosan− genipin biomaterial platform with multiple benefits to chronic wound healing such as fluid absorption, pH regulation, and antibacterial activity at the wound site. The effects of chitosan molecular weight and genipin weight percent on hydrogel synthesis and physical properties were evaluated to optimize the formulation. Due to chitosan’s cationic nature and innate antimicrobial, mucoadhesive, and hemostatic properties, it was expected that these hydrogels would be biocompatible and an optimal low cost, multifunctional option for wound healing applications. The synthesized chitosan−genipin hydrogels demonstrated fluid uptake of ∼230% in PBS (Supporting Information) and ∼150% in cell-conditioning media demonstrating their potential use as an absorbent dressing in perfusive chronic wounds. Characterization studies also demonstrated that the chitosan−genipin hydrogels could neutralize an environment with pH 8.2 within 24 h (Supporting Information). This is a beneficial property in wound healing therapies as chronic wounds typically demonstrate an alkaline pH. By neutralizing the pH at the wound site, the oxygen supply is increased and protease activity is decreased, resulting in a healthier wound

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +1(318)257-2207. Fax: +1(318)257-4000. ORCID

Mary E. Caldorera-Moore: 0000-0002-1675-9084 Author Contributions #

A.M.H. and T.R.P.-A. contributed equally. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding

This work was supported by the Louisiana Board Regents Research Competitiveness program [LEQSF (2015-18)-RD-A17], the Louisiana Biomedical Research Network (LBRN), with funds from the National Institute of General Medical Sciences (NIGMS) Grant (8P20GM103424). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank undergraduate students Julie Gaudin and Kelly Kneale for assisting with data collection, 2886

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Anusha Elumalai for microbiology training, Dr. Jonathan Alexander at the Louisiana Health Science Center, Shreveport, for donation of the cells used in the study, and Dr. Rebecca Giorno for the use of lab space to conduct the bacterial studies. We would also like to thank the Louisiana State University NMR facility and Dr. Thomas Weldeghiorghis in Baton Rouge for assisting with NMR data collection and Dr. Dongmei Cai and Dr. Clayton Loehn at the Shared Institute at LSU Institute for Advanced Materials Shared Instrument Facility for help with Environmental SEM of hydrogel samples.

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ABBREVIATIONS MMP, matrix metalloproteinase; E. coli, Escherichia coli; DFU, diabetic foot ulcer; LD50, median lethal dose; MW, molecular weight; kDa, kilodaltons; HC, high MW chitosan films; LC, low MW chitosan films; HFF, human foreskin fibroblasts; HaCaTs, adult immortalized keratinocytes; CFU, colony forming units; DD, degree of deacetylation; PBS, phosphate buffered saline; trypsin-EDTA, trypsin-ethylenediaminetetraacetic acid; DMEM, Dulbecco’s modified Eagle medium; RPMI, Roswell Park Memorial Institute; FBS, fetal bovine serum; THP-1, human monocytes; IACUC, Institutional Animal Care and Use Committees; wt, weight; (w/v), weight-to-volume; DI, deionized; NMR, nuclear magnetic resonance; ESEM, environmental scanning electron microscope; CD, cross-linking degree; CM, cell media; FGF-2, fibroblast growth factor 2; PDMS, polydimethylsiloxane; HC2%, high MW chitosan cross-linked with 2% genipin hydrogels; LC2%, low MW chitosan cross-linked with 2% genipin hydrogels; LB, Luria−Bertani; OD, optical density; H&E, hematoxylin and eosin

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