Silver nanoparticle entrapped soft GelMA gels as prospective scaffolds

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Silver nanoparticle entrapped soft GelMA gels as prospective scaffolds for wound healing Iffat Jahan, Edna George, Neha saxena, and Shamik Sen ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00663 • Publication Date (Web): 15 Apr 2019 Downloaded from http://pubs.acs.org on April 15, 2019

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Silver nanoparticle entrapped soft GelMA gels as prospective scaffolds for wound healing Iffat Jahan1, Edna George1, Neha Saxena2, Shamik Sen1* 1

Department of Biosciences & Bioengineering, IIT Bombay

2

Department of Chemical Engineering, IIT Bombay

*: Correspondence: [email protected]

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Abstract: Gelatin-based hydrogels have received particular attention for tissue engineering applications given their biocompatibility, ease of tuning their physical properties through chemical modifications, and incorporation of antibacterial activity. While several studies have focused on detailed quantification of biomechanical properties of these gels, considerably lesser attention has been paid to understanding how adhesivity of these gels impacts single as well as collective cell migration which directly determine the efficacy of wound healing. In this study, we address this question by quantifying fibroblast motility and antibacterial activity of silver nanoparticle (AgNP) entrapped methacrylated gelatin (GelMA) hydrogels. Using 5 and 15% GelMA gels crosslinked with 1 min UV exposure, we first show that cells spread more and migrate faster on 15% GelMA gels. Next, we show that ~10 nm sized AgNPs entrapped in 15% GelMA gels get released over a time-scale greater than 72 hours and exhibit antibacterial activity against both gram-positive and gram-negative bacteria at concentrations non-toxic to cells. Finally, using a polydimethylsiloxane (PDMS) device for simulating wound healing, we show that closure of ~800 m gaps on GelMA gels is significantly faster compared to other conditions. Together, our findings illustrate the potential of AgNP entrapped soft GelMA gels to be employed as scaffolds for achieving accelerated wound healing of deep dermal wounds by enabling fast fibroblast migration and minimization of microbial infections.

Keywords: methacrylated gelatin (GelMA); silver nanoparticles (AgNPs); antibacterial activity; fibroblasts; migration; wound healing.

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Introduction To minimize and/or eliminate the high chances of infection associated with suturing, tissue adhesives have emerged as an attractive alternate for sealing and healing wounds 1. Tissue adhesives are biomaterials which offer several advantages including non-invasiveness, patient friendliness, minimal risk of infection, accelerated wound repair processes and do not require removal2. Tissue adhesives have been widely used to restore and enhance natural wound healing process, as wound closure devices, homeostatic organs and tissue sealants to control blood loss3. While cyanoacrylate-based synthetic tissue adhesives are widely being used, owing to their cytotoxic effects2, researchers have been exploring biological tissue adhesives as suitable alternatives. Biological tissue adhesives are made up of several natural components including coagulating factors such as fibrinogen and thrombin4, polysaccharides such as chitosan5, protein such as collagen6, gelatin and/or their combination7,8. These sealants are biodegradable, biocompatible, contain bioactive motif in their chemical structure, and do not cause any adverse reactions when they come in contact with body fluids. Despite numerous favourable features, these adhesives have low bonding strength with tissues and may cause blood borne transmission diseases9.

Gelatin, a derivate of collagen- a major component of the extracellular matrix (ECM)-contains arginine-glycine-aspartic acid (RGD)10 and matrix metalloproteinase (MMP) motifs11 which are essential for cell attachment and remodeling12. To improve the mechanical strength of gelatin, several different crosslinking strategies have been explored13–16. Of these, methacrylated gelatin (GelMA) gels have been shown to exhibit efficient gelation kinetics17 in the presence of photointiators18, and provide better mechanical strength. Though several studies have demonstrated the utility of GelMA gels in mediating wound healing19,20, most of these studies have used stiff ~10-100 kPa gels18,21,22. Since porosity of gels scales inversely with gel stiffness for most biopolymer networks23,24 stiff gels with reduced pore sizes may delay infiltration of cells and/or restrict cell spreading and motility. We posit that using softer GelMA gels may be more effective in mediating wound healing. Since stiffness of GelMA gels is dictated not only by GelMA concentration, but also by duration of UV exposure, GelMA gels fabricated by limiting UV exposure may be used not only as adhesive patches but also for in situ gelling applications. However, whether or not these GelMA gels can still support cell adhesion and migration has not been addressed.

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Though wound healing is a well-organized and a complex process, during injury and pathological conditions, it gets affected. During injury, possibility of microbial contamination at the wound site increases and formation of chronic wound occurs. Moreover, implants used in surgery and biomaterials used in biomedical applications may also increase risk of microbial infection because of moist and nutrient rich environments25. Hence, to reduce microbial effects on wound, antibacterial agents such as antiseptics, antibiotics, herbal therapeutics, enzymes and silver have widely been used26. Amongst these, silver has gained prominence in wound dressing/wound healing applications due to its broad spectrum antibacterial activity and to fulfill the demand of drug resistance27. For example, silver sulfadiazine has been extensively used as a burn ointment due to its own antibacterial activity28. Although, silver (Ag+) exhibits potent antibacterial activity against the methicillin resistant Staphylococcus aureus (MRSA), its use is limited due to high cytotoxic effects on mammals29. To overcome these limitations, in recent years, nano-silver has been introduced in the biomedical field. These nanoparticles are less toxic than Ag+30, possess enhanced antibacterial activity31, show anti-inflammatory effects and promote wound healing phenomenon32.

Since effective wound healing should not only hasten healing but also protect against external infections, combining the advantages of GelMA and silver nanoparticles (AgNPs) is likely to enhance wound healing keeping infection at bay. Indeed, this approach of incorporating AgNPs in wound dressing applications has been adopted in several studies using various different systems including collagen33, gelatin nanofibers34, collagen/chitosan scaffolds35, alginate hydrogels36, and chemically modified GelMA hydrogels37,38. While soft GelMA gels are expected to provide an advantage vis-à-vis cell infiltration, the extent of loading of AgNPs within these soft gels, correlation of their release kinetics with antibacterial activity and cell toxicity, and the impact of these AgNPs on cell motility if any, remains unclear. We hypothesize that AgNP-entrapped soft GelMA gels polymerized in situ by UV exposure may serve as prospective tissue adhesives by protecting against bacterial infections, stimulating fibroblast migration into the gels, promoting fibroblast proliferation, and subsequent healing through remodeling of the gels (Fig. 1). We first show that soft ~150 Pa GelMA gels indeed support attachment, spreading, proliferation and motility of NIH 3T3 fibroblasts. We then show that ~10 nm AgNPs entrapped within GelMA gels at high enough concentrations exhibit robust antibacterial activity but are non-toxic to cells.

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Finally, we show that the GelMA gels mediate fast wound healing independent of AgNP concentration. Collectively, our results illustrate the potential of AgNP entrapped soft GelMA gels to be employed as scaffolds for wound healing. Extension of these studies in animal models is further required to fully establish the wound healing capability of these gels.

Materials and Methods 1. Fabrication and characterization of methacrylated gelatin and collagen hydrogels: For synthesis of methacrylated gelatin (GelMA), type B gelatin (Nitta Gelatin India, Ltd) was dissolved in 50 mL of phosphate buffered saline (PBS, 1x, Himedia) at 40 °C. Next, 10 ml of methacrylic anhydride was added in gelatin-PBS solution and the reaction was continued for 2 hours at 50 °C to allow for formation of GelMA. The solution was then dialyzed (membrane cut off size ~ 12kDa) at 40 °C for at least 4 days to remove unreacted methacrylic anhydride. Finally, the dialyzed solution was lyophilized and stored at -20 °C for further analysis10,18. Modification of gelatin was characterized using 1H-NMR spectroscopy to determine the extent of methacrylation. For 1H-NMR, 10 mg lyophilized GelMA was dissolved in 1 mL deuterium oxide (D2O) at 37 °C and spectra was recorded at a frequency of 300 MHz using VARIAN, Mercury Plus NMR spectrometer with 5 nm auto-switchable probe.

For preparing GelMA hydrogels, 5 and 15% (w/v) of lyophilized GelMA was dissolved in distilled water at 37 °C and then 1.5% (w/v) of photo initiator (Irgacure 2959, Ciba Chemicals) was added as a crosslinking agent. Crosslinking reaction was carried out with exposure of ultra violet (UV) light (365nm) for 1 min. During the crosslinking UV lamp (6W) was placed at ~2 cm distance from the gel solution. Pore size of GelMA gels was determined using Field Emission Gun Scanning Electron Microscopy (Cryo-FEG-SEM) microscopy. Hydrogel was placed on a copper grid, coated with conductive material graphite, and then air dried at room temperature. Upon drying, the specimen was frozen at (-200 to -192) oC with liquid nitrogen and fractured. After fracturing, specimen was sublimated at -85 oC and coated with platinum for 60 second at 10 mA to make it conductive. All the above sample preparation steps were performed on a Cryo Preparation System (PP3000T). For imaging, 5 kV voltage was applied and images were acquired in SEI mode at 20,000 to 40,000X magnification by using FEG-SEM (JSM-7600F).

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Collagen hydrogels (rat tail collagen type I, Corning) were prepared by mixing 3D collagen solution with ice cold 10× PBS and DMEM followed by incubation at 37 ºC for 1 hour to achieve a final concentration of 1.5 mg/ml39. The prepared gels were used for both 2D motility and wound healing studies.

Rheology of GelMA gels: Rheological measurement of GelMA gels was carried out using a parallel plate (25 mm diameter) rheometer (Physica MCR 301, Anton Paar). GelMA gels (25 mm diameter and 1.8 mm height) were placed between two parallel plates and temperature was maintained at 25 οC throughout the experiment. Frequency sweep (0.01- 10 Hz) and strain sweep (0.1-200%) was carried out at a constant strain of 0.05% and constant frequency 1 Hz respectively. 2. Cell Culture and Reagents: For experiments, NIH-3T3 cells (purchased from NCCS, Pune) were cultured in DMEM (Dulbecco’s Modified Eagle’s Medium, from Gibco), 10% FBS (Fetal bovine serum- US origin, Himedia), and 1% antibiotic (Penicillin-Streptomycin, Himedia) and kept at 37 ºC with 5% CO2. When cells were ~80% confluent, cells were trypsinized and processed for various experiments. 3. Cell spreading, proliferation and 2D motility of fibroblasts on GelMA gels: For experiments, 3T3 fibroblasts were cultured on GelMA hydrogels (seeding density ̴ 0.5×104 in 24 well plates) and incubated for 24 hours at 37 ºC in 10% FBS. After 24 hours, images were captured using a phase contrast microscope (Olympus IX 71) at 20x magnification for measuring the spreading area of cells. For quantifying cell proliferation, cells were cultured on 5 and 15% GelMA gels for 24 hours and 48 hours in media containing either 2% or 10% FBS. At these time-points, cells were fixed and stained with Hoechst 33342 (Thermo) and calcein AM (Life Scientific), and fluorescence images acquired using an inverted microscope (Olympus IX 71) at 10x magnification. Cell proliferation was quantified by counting the average number of viable cells per frame, with cells positive for both Hoechst 33342 (Sigma) and calcein AM (Invitrogen) considered to be viable. Random cell motility experiments were performed on 5 and 15% GelMA hydrogels using time-lapse microscopy on a spinning disc confocal microscope (Yokogawa CSU-X1) at 10x magnification for 24 hours (at 10 min time intervals). Cell motility was determined using the manual tracking plugin in Image J software. For comparison, random cell motility experiments

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were also performed on glass coverslips coated with 15% gelatin (w/v) and 15% (v/v) 2D collagen (rat tail collagen type I, Sigma), as well as on 3D collagen hydrogels (described above). 4. Synthesis and characterization of silver nanoparticles (AgNPs): AgNPs were synthesized using 2.0×10-6 mole/cm3 sodium borohydride (Sigma) as a primary reducing agent and 2.0×10-6 mole/cm3 tri-sodium citrate (Merck) as a secondary reducing and capping agent40. These reagents were mixed properly in deionized water at 60 oC for 30 min in the dark. Next, 1.17 ×10-6 mole/cm3 silver nitrate (Merck) was added dropwise and the reaction temperature was set to 90 oC. Upon reaching this temperature, pH was adjusted to 10.5 with 1 N NaOH (Thomas Baker) and solution was heated continuously for 20 minutes until the color changed to dark yellow. Solution was cooled at room temperature and centrifuged at 20,000 rpm for 1 hour followed by washing (3 times) with deionized water to remove unreacted chemicals. The obtained sample was stored at 4 o

C in the dark for further analysis.

To confirm AgNP synthesis, UV-Vis spectrophotometer (Jasco V-730 BIO) was used in absorbance mode (200-800 nm) with desired dilution of silver colloids in deionized water. Stability of silver colloidal solution was measured by using zeta potential analyzer (Zeta pals). The sample was sonicated for 10 minutes prior to use to prevent the aggregation of particles. Size and morphology of AgNPs were determined using a combination of DLS (Malvern) and TEM (FEI Technai 12 BioTwin 120 kV Cryo - Transmission Electron Microscope). For TEM, after sonication, AgNP solution was placed on a copper grid and kept for drying under an infra-red (IR) lamp. After drying, sample was placed inside the TEM instrument, voltage of 120 kV was applied and images were accquired. Concentration of AgNPs was determined by using Induced Coupled Plasma Atomic Emission Spectroscopy (ICP-AES). For ICP analysis, sample was prepared in 2% HNO3 solution. 5. Fabrication and characterization of AgNP embedded GelMA hydrogels: AgNP-embedded GelMA hydrogels were prepared by dissolving 15% (w/v) of lyophilized GelMA in deionized water at 60 °C, 1.5% (w/v) photo initiator (Irgacure 2959, Ciba Chemicals) and varying concentration of AgNPs. Sample preparation for Cryo mode was done as mentioned above. For imaging, 10 kV voltage was applied and images were acquired at 20,000 to 40,000x magnification using FEG-SEM (JSM-7600F) in backscattered mode.

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6. Release kinetics of AgNPs from GelMA gels: GelMA gels were prepared (1mm2) with different concentration of AgNPs (5, 10, 50, 150 µg/mL) in the presence of UV light (365 nm) for 60 sec. For release kinetic studies, gel samples (150 µl in volume) were incubated in 5 mL PBS solutions at 37 °C in different centrifuge tubes under shaking condition. After every 12 hours, 2 ml of solution was removed from each tube. Subsequently, each tube was replenished with 2 mL of fresh PBS so as to maintain a constant total volume of each tube. This process of removal of solution and replenishment with fresh PBS was continued till 72 hours, as the gels started fragmenting into smaller pieces making it obtain clear solutions for ICP analysis. AgNP concentration in the samples collected at different time-points was determined using ICP-AES. 7. Antibacterial activity of AgNPs and AgNP entrapped GelMA gels: To assess the antibacterial property of AgNPs, gram positive (Staphylococcus aureus MTCC 2043) and gram negative (Escherichia coli MTCC 2412) bacterial cells were grown in Luria broth (LB from Himedia) and optical density (O.D.) was recorded using a spectrophotometer41,42 For assessing antibacterial properties of AgNP entrapped GelMA gels, 10 µl/well of bacterial suspension (corresponding to 0.3 O.D.), 100 µl/well of LB and different concentrations of AgNPs (5, 10, 50, 100, 150, 200 µg/mL) embedded with and without 15% GelMA gels were added on to 96 well plates for 8 hours at 37 °C. Absorbance was recorded at 600 nm using plate reader (Thermo Multiskan Go) at 0 hour (for reference) and after 8 hours43. Minimum inhibitory concentration (MIC) was determined at the point when there was minimal turbidity and a sharp decline in absorption was observed44. The experiment was performed two independent times in duplicate. 8. Cytotoxicity of AgNPs and AgNP entrapped GelMA gels: 3T3 fibroblasts cultured in 24 well plates (10,000 cells/well) were incubated with different concentrations of AgNPs (0.5, 1, 2, 3, 4, 5, 10 µg/mL) for 24 hrs. Next day, cell cytotoxicity was assessed by staining cells with calcein AM (4 µg/mL) and Propidium iodide (PI) (3 µM, Himedia), respectively for 10 min. Images were acquired using a fluorescence microscope (Olympus IX 71) at 10x magnification. To assess cytotoxicity of AgNP entrapped GelMA hydrogels, GelMA gels were fabricated in 24 well plates with different concentrations of AgNPs (3, 5, 10, 50, 150 µg/mL). After culturing fibroblasts (10,000 cells/well) on these gels for 24 hours, cytotoxicity was assessed by staining (using for calcein AM and PI, as detailed above) as well as by Fluorescence-Activated Cell Sorting (FACS). For FACS analysis, cells were collected by trypsinizing the adherent cells, suspended in FACS

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buffer and stained with PI (3 µM) for 5 min followed by washing with FACS buffer. Samples were analysed using BD FACS Aria 1 instrument. 9. Wound healing on GelMA gels: For wound healing study, scratch assay was performed on 15% GelMA gels cast inside 24 well plates. For doing this, a polydimethylsiloxane (PDMS) device was used. The device was designed with 2 open areas separated by a central zone (800 m in width) (Fig. 6A). Cells were plated at a seeding density of 3×104 cells in these open areas. The PDMS device was firmly attached onto the GelMA gels so as to stop cells from migrating underneath the device. The seeding density was so chosen that within this time interval, the two zones are completely confluent. Removal of the PDMS device 12 hours after cell seeding exposed the cell-free gel area masked by the device thereby generating free space for cells to migrate. This free space mimics the formation of a scratch in a traditional scratch wound assay. However, unlike the scratch wound assay, this approach does not cause any cell death. Wound healing was tracked by imaging the gap created upon removal of the PDMS device. Images were acquired every 8 hours at 4x magnification in phase contrast mode (Nikon Eclipse Ti) up to a duration of 32 hours. To ensure that wound closure was a direct consequence of cell migration and not cell division, experiments were performed with media containing 2% serum only. Control experiments were performed on uncoated glass coverslips, glass coverslips coated with 15% gelatin (w/v), 15% GelMA (w/v) and 15% 2D collagen (v/v), as well as on 1.5 mg/ml 3D collagen hydrogels. 10. Statistical analysis: Statistical analysis was performed either by using two tail student’s t-test for three sets of experimental groups or by using one-way ANOVA with Fisher multiple comparison test for more than two sets of samples in an experiment. In case of statistically significant differences, the p (* ≤ 0.05, ** ≤ 0.01 and *** ≤ 0.001) values have been reported.

Results Fabrication and characterization of GelMA hydrogels: As a first step towards fabricating GelMA gels, we quantified the extent of methacrylation of gelatin using 1H-NMR. Modification of gelatin can be gleaned from the appearance of the distinctive peaks including, bond formation in double bond region at 5.3 and 5.5 ppm (due to -CH3 group) and at 1.8 ppm (due to –CH2 group of methacrylate anhydride) and a corresponding reduction in peak at 2.9 ppm (due to chemical bonding between lysine residue and acrylic proton of acrylate group)45 (Supp. Fig. S1). Using the

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formula

𝐴 (𝑙𝑦𝑠𝑖𝑛𝑒 𝑚𝑒𝑡ℎ𝑦𝑙𝑒𝑛𝑒 𝑜𝑓 𝐺𝑒𝑙𝑀𝐴)

% Methacrylation = 1 − 𝐴 (𝐿𝑦𝑠𝑖𝑛𝑒 𝑚𝑒𝑡ℎ𝑦𝑙𝑒𝑛𝑒 𝑜𝑓 𝑢𝑛𝑚𝑜𝑑𝑖𝑓𝑖𝑒𝑑 𝐺𝑒𝑙𝑀𝐴)46

the

degree

of

methacrylation was estimated to be ~70%.

Since our eventual aim is to use GelMA gels for in vivo applications, GelMA gels were fabricated with the duration of UV exposure limited to 1 minute to minimize UV induced damage to cells/tissues. Under these conditions, gelation was not observed for GelMA concentrations less than 5% GelMA. In addition, at concentrations beyond 15% GelMA, the solution was highly viscous leading to instantaneous but non-uniform gelation. To test the mechanical properties of 5 and 15% GelMA gels, rheological studies were performed. Frequency sweep measurements suggested that shear storage moduli (G’) of both 5 and 5% GelMA gels were nearly 10-fold higher than the loss moduli (G’’) and were constant upto 1 Hz frequency (Supp. Fig. S2A). However, 15% GelMA gels exhibited a stiffening response at higher frequencies. At 1 Hz frequency, shear storage modulus of 15% GelMA gels (~150 Pa) was nearly 3-fold of that of 5% GelMA gels (~35 Pa) (Supp. Fig. S2B). Strain sweep experiments at 1 Hz frequency revealed strain-dependent reduction of G’ values of 15% GelMA gels beyond 1% strain (Supp. Fig. S2C). However, no crossover in G’/G’’ values were observed. Together, these results suggest that both 5 and 15% GelMA gels are primarily elastic in nature, and are stable upto frequencies of 1 Hz.

Spreading, proliferation and migration of NIH 3T3 fibroblasts on GelMA gels: To next assess the effectiveness of GelMA gels for wound healing applications, NIH-3T3 fibroblasts were plated at identical seeding densities on 5 and 15% GelMA gels (w/v). After culturing on GelMA gels for 24 hours, cells were imaged for comparing their morphology and proliferation rate across these two gels. Though representative images of 3T3 fibroblasts cultured on 5 and 15% gels were indicative of comparable spreading across both these gels (Fig. 2A), quantification revealed higher extent of cell spreading on 15% GelMA gels (Fig. 2B). Additionally, comparable number of cells per frame on 5 and 15% gels at both 24 and 48 hours was indicative of similar proliferation rates across these two gels (Fig. 2C, D). When experiments were performed in 2% FBS, no cell proliferation was observed in either of these two gels as observed from the identical number of cells/frame at both 24 and 48 hours time points (Supp. Fig. S3).

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Since cell migration plays a key role during wound healing, to next compare cell motility of NIH3T3 fibroblasts on 5 and 15% GelMA gels, time lapse images were acquired for a duration of 12 hours at 10 min intervals. Analysis of single cell trajectories (Fig. 2E) revealed faster motility (~30%) of fibroblasts on 15% GelMA gels (Fig. 2F). Cell motility on 15% GelMA gels was also more efficient compared to that on glass coverslips coated with 15% gelatin/2D collagen, and that on 3D collagen hydrogels (Supp. Fig. S4). Taken together, the above results suggest that fibroblasts spread and migrate more extensively on 15% GelMA gels compared to 5% GelMA gels. Since, cells exhibited greater spreading and faster motility on 15% GelMA gels, for all subsequent experiments, we have chosen 15% GelMA gels.

Synthesis and characterization of silver nanoparticles (AgNPs): Preventing infection in chronic wounds plays a critical role for effective wound healing. With an aim to develop scaffolds with antibacterial property, we next synthesized AgNPs of sizes amenable to renal clearance. Synthesis of AgNPs was confirmed from the sharp peak in the UV-Vis spectrum observed at ~ 400 nm (Fig. 3A). While the synthesized nanoparticles were roughly spherical in shape (Fig. 3B), analysis of their size distribution by two separate techniques (TEM and DLS) yielded sizes in the range of 6-14 nm (Fig. 3C). Furthermore, a -22-mV zeta potential of these nanoparticles suggests that the synthesized AgNPs were stable and mono-disperse.

Antibacterial activity and release kinetics of AgNPs from GelMA gels: To next assess the antibacterial activity of the synthesized AgNPs, bacterial growth experiments were performed using both gram positive (S. aureus) and gram negative (E. coli) strains. In the absence of AgNPs, significant bacterial growth was observed for both the strains. Upon addition of different concentration of AgNPs (5, 10, 50, 100, 150 µg/mL) decrease in O.D. was observed in a dosedependent manner (Fig. 3D). While complete killing of E. coli was observed at ≥ 50 μg/mL AgNPs, this was higher for S. aureus (≥ 100 𝜇g/mL). Together, these experiments suggest that the synthesized AgNPs exhibit potent antibacterial activity towards both gram-positive and gramnegative bacterial strains.

To next probe the ability of the synthesized AgNPs to adsorb onto the GelMA gels, after incubation of AgNPs with 15% GelMA gels for 10 mins, the NP-laden gels were processed using Cryo-FEG-

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SEM in backscatter mode of operation (Fig. 4A). The presence of dot-like patterns on the AgNP laden GelMA gels in the zoomed-in images of the AgNP laden GelMA gels is indicative of the entrapment of AgNPs onto the gels. However, the presence of AgNPs did not alter the organization of the GelMA gels, as evidenced from the quantification of the pore sizes of GelMA gels with and without AgNPs is ~1µm2 (Fig. 4B). To next determine the timescale of release of AgNPs from the GelMA gels, gel samples containing AgNPs of varying concentrations were incubated in 5 mL PBS solution in different centrifuge tubes under shaking condition (Fig. 4C), and AgNP release tracked by monitoring AgNP concentration in the bulk solution over a period of 72 hours (Fig. 4D). In gels containing 10, 50 and 150 μg/mL AgNPs, the cumulative release profile is indicative of an initial burst release at 12 hours followed by gradual release.

To last test the antibacterial properties of AgNP entrapped GelMA gels, GelMA gels entrapped with different concentration of AgNPs (5, 10, 50, 100, 150, 200 µg/mL) were incubated with identical density of bacteria with (LB + bacteria) and (LB + bacteria + 15% GelMA gels) serving as positive controls, and only LB/(LB + 15% GelMA gels) serving as negative controls. Decrease in O.D. values corresponding to AgNP-induced bacterial death was observed in AgNP entrapped GelMA gels with MIC values of 100 μg/mL and 150 μg/mL for E. coli and S. aureus, respectively (Fig. 4E). This was roughly twice compared to the MIC values of AgNPs alone (Fig. 3D). In addition, complete killing was observed at concentrations of AgNP entrapped GelMA gels, higher than 150 μg/mL for both the bacterial strains. As expected, these concentrations were higher compared to AgNPs only. Collectively, our results suggest that AgNPs entrapped within GelMA gels are gradually released and exhibit potent antibacterial activity towards both gram-positive and gram-negative bacterial strains in a concentration-dependent manner.

Cytotoxicity of AgNP entrapped GelMA gels: AgNPs are known to possess cytotoxic properties, particularly at higher concentrations. To assess the cytotoxicity of the above synthesized AgNPs, fibroblasts were cultured on glass substrates in the presence of increasing concentrations of AgNPs. Labelling of cells with calcein AM and propidium iodide (PI) after 24 hours allowed us to assess the extent of cytotoxicity of these AgNPs. As observed from the representative images, while cell death was minimal upto AgNP concentration of 5 µg/mL, there was dramatic increase in cell death at AgNP concentrations of 10 µg/mL and higher (Fig. 5A). Consistent with our

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qualitative observations, quantification of the %age of live cells revealed >75% viability for AgNP concentrations ≤5 µg/mL, with viability dropping to ~50% at AgNP concentration of 10 µg/mL (Fig. 5B). However, when AgNPs were entrapped inside the GelMA gels, majority of the cells were viable even at AgNP concentrations greater than 100 µg/mL (Fig. 5C). Quantification of acquired images revealed >95% cell viability up to 150 µg/mL concentration of AgNPs (Fig. 5D). To further confirm our imaging-based observations, FACS was used to quantitatively determine the fraction of viable cells on AgNP entrapped GelMA gels. Consistent with our imaging results, >95% viability was also observed in FACS measurements (Fig. 5E, F). Together, these results suggest that AgNP entrapped GelMA gels are non-toxic to cells even at very high concentrations of AgNPs.

Evaluation of wound healing on GelMA gels: To finally probe the effectiveness of GelMA gels in mediating wound healing, scratch assay was performed using 15% GelMA gels overlaid firmly with a PDMS device. The device was shaped as a ring with a central section 800 m in width such that upon seeding, cells attached and spread onto the exposed regions of the GelMA gels, but were excluded from the central zone (Fig. 6A). After 12 hours of culture, the device was removed thereby exposing the central cell-free zone which closely mimicked the wound. Subsequently, wound healing was tracked by imaging the cell-free zone after every 8 hours of time interval up to 32 hours. Apart from choosing GelMA gels with three different AgNP concentrations (0, 10 and 50 g/mL), control experiments were performed on uncoated glass coverslips, and glass coverslips coated with 15% gelatin, 15% GelMA and 2D collagen (15% v/v). In addition, experiments were also performed with 1.5 mg/ml collagen gels, which have pore sizes of (~ 2-5 μm2 )47 comparable to that of 15% GelMA gels. As seen from the representative wound healing images, in contrast to the different control substrates wherein wound closure (yellow dotted lines) was not complete even after 32 hours, wound closure was significantly faster on the GelMA gels (Fig. 6B). In line with our visual observations, quantification of %age wound healing, i.e., the wound area normalized to the initial area, revealed significantly faster healing on GelMA gels compared to different controls (Fig. 7A). Comparison of wound healing after 24 hrs revealed 1.5-2-fold faster rate of healing of GelMA gels compared to that on different control substrates, with no dependence on AgNP concentration (Fig. 7B). Collectively, these findings illustrate the potential of GelMA gels in mediating wound healing in a manner independent of AgNP concentration.

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Discussion Tissue adhesives have emerged as an attractive alternative to conventional wound closure devices such as sutures and staples48. Several natural tissue adhesives are already available in market such as Tisseel and Hemassel (Fibrin Sealants)49, Proceed (Fusion Medical Technologies, Mountain View, CA) and FloSeal (Sulzer Spine-tech Anaheim CA). However, these products suffer from several shortcomings. While Fibrin Glue has been reported as a blood-borne transmission agent and can also increase the risk of embolization9, collagen is very expensive and can cause antigenicity due to its structural variation from different sources18. To overcome the limitations of such adhesives, we have developed gelatin-based adhesives because gelatin is affordable, biocompatible and biodegradable, and does not cause antigenicity 10. Our in vitro results illustrate the suitability of AgNP-entrapped GelMA gels in driving wound healing by minimizing cell toxicity, supporting cell viability, enabling fast migration of fibroblasts, and concurrently, exhibiting antibacterial activity. Altogether, our results clearly show that NIH 3T3 fibroblasts spread, proliferate and migrate extensively on 15% GelMA gels, further testing of these gels in driving fibroblasts/keratinocyte migration and in animal models in vivo are required to fully assess the capability of these gels for wound healing applications.

Given the biocompatible, biodegradable, and bioadhesive properties of gelatin, chemical modifications of gelatin have been widely explored for wound healing applications50. Among the various modifications, methacrylation of gelatin using methacrylic anhydride represents an effective strategy for forming hydrogels via photopolymerization18. Though the extent of methacrylation was estimated to be ~70% in our case, in addition to formation of methacrylate groups, recent studies suggest that modification of proteins by methacrylic anhydride can also lead to the formation of meth-acrylamide groups51. While increase in UV exposure times is expected to induce greater crosslinking thereby leading to increased stiffness of GelMA gels, with an aim of using our GelMA gels for in vivo applications, UV exposure time was intentionally restricted to 1 minute for minimizing UV-induced damage to cells. This is consistent with past studies performed with variable UV exposure times that have shown that cell viability remains unchanged up to 200 seconds of exposure52. While administration of gel patches for wound healing does not place any constraint on the duration of UV exposure, but the patches may not fully conform to the shape of the wounds thereby leading to delayed healing, an aspect which has been experimentally

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observed 53. In this regard, in situ gel formation may enhance wound healing. For these cases, UV exposure times need to be optimized. Young’s modulus of native skin has been reported to lie in the range of 10 kPa to 50 MPa54. Stiffness of a biopolymer network can be tuned by varying concentration of the polymer, concentration of the crosslinker or both22. Though Young’s modulus of GelMA scaffolds can be made comparable to that of skin by changing the duration of UV exposure or by blending with other materials such as tropoelastin51, GelMA gels fabricated by us were only ~100s of Pa’s in storage modulus, and is consistent with our previous study55. However, the stiffness values reported by us are much lower than literature reported values56,57. In addition to GelMA concentration, stiffness of GelMA gels is collectively determined by multiple factors including power of the UV lamp, exposure time of UV, of thickness of the gels and also the distance between the UV lamp and the gelling solution58. The considerable low values of GelMA stiffness obtained by us may be partly attributed to ~2 cm gap between the UV lamp and the gelling solution used in our study, which is much larger compared to the ~750 µm gap used by Nichol et al. who reported compressive modulus values of ~20 kPa59. Substrate stiffness has been now established to play a crucial role in modulating cell spreading60 and cell motility in a wide variety of adherent cell types61,62, with limited spreading and motility observed on soft ~100-1000 Pa’s substrates. It is surprising how cells are able to spread and migrate so extensively on ~100 Pa GelMA hydrogels? We speculate that increased spreading on these gels may be a consequence of non-linear elasticity of gels63 which has been documented both in collagen gels64,65 as well as in crosslinked gelatin gels57,66. Indeed, non-linear stiffening was observed in 15% GelMA gels. In contrast to 2D culture conditions wherein increase in stiffness is associated with increased spreading and motility, in 3D culture, higher stiffness is associated with smaller pore size and decreased spreading and motility67. In line with this, 3T3 fibroblasts exhibit rounded morphology when embedded in high concentration and with higher stiffness of GelMA gels59. Thus, optimal cell infiltration into 3D GelMA gels may be achieved at moderate GelMA concentrations. However, since degradation rates of the gels are expected to be faster in vivo compared to the observed timescales in the presence PBS, a detailed in vivo study is required to

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identify the optimal combination of GelMA concentration and duration of crosslinking so that the gel remains stable over a longer duration but is still not limiting for cell migration.

Though AgNPs have been widely used in biomedical applications due to their antibacterial properties, they are not biodegradable and may cause cell toxicity68. In vivo, bio-distribution and clearance of AgNPs is dictated by their size, shape and charge69. While AgNPs ≤5 nm in size are capable of crossing the blood brain barrier, AgNPs 10-20 nm in size get eliminated through renal clearance70. In contrast, AgNPs ≥20 nm in size are not amenable to renal clearance and get accumulated inside the body thereby causing cell toxicity. Since the size of synthesized AgNPs ≈ 10 nm, these are expected to be cleared renally. Though AgNPs were found to be cytotoxic at concentrations greater than 10 µg/mL concentrations, when entrapped inside the gels, the gels were non-toxic even at AgNP concentrations greater than 150 µg/mL, which corresponds to the AgNP concentration required to achieve complete killing of both the bacterial strains. This can be attributed to the slow release of AgNPs from the GelMA gels such that at any point of time, the concentration of released AgNPs was lesser than the cytotoxic limit. Thus, the GelMA gels offer a wide window for tuning the AgNP concentration without impacting cell viability. Hence, our results suggest that synthesized AgNPs adsorb onto GelMA gels, get released gradually and also exhibit antibacterial property.

For hydrogel-mediated healing of deep dermal wounds, dermal fibroblasts must first attach, and migrate into the hydrogels, proliferate, and subsequently remodel the gels via a combination of hydrogel degradation and deposition of native extracellular matrix proteins, namely collagen. While we had observed identical fibroblast proliferation rates on 5 and 15% GelMA gels, cells migrated faster on 15% GelMA gels. Though wound healing was also faster on GelMA gels compared to control conditions, the extent of cell infiltration into GelMA gels under 3D conditions remains to be probed. However, in a recent study, compared to electrospun gelatin and PLGA scaffolds, electrospun GelMA scaffolds have been shown to support greater extent of fibroblast infiltration as well as expression of Collagen I and II57. Given the sustained release of AgNPs from our GelMA gels, coating of AgNPs with fibroblast growth factor (FGF) may be used as a strategy to further increase the efficiency of fibroblast infiltration.

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Conclusion: In conclusion, this work demonstrates the potential of AgNP entrapped soft GelMA gels for wound healing applications. First, using 5 and 15% GelMA gels, we have shown that 15% GelMA gels are more effective in supporting cell motility. Second, we show that AgNPs entrapped within the GelMA gels exhibit antibacterial activity at concentrations beyond 50 µg/mL for both gram positive and gram negative bacterial strains without compromising cell viability. This antibacterial activity is provided by the sustained release of AgNPs from the GelMA gels. Finally, using a PDMS-based device, we show that wound healing is efficient on GelMA gels, with the incorporated AgNPs not influencing cell motility. While these findings are promising, future in vivo experiments are required to fully demonstrate the efficacy of these gels in driving wound healing. Blending of GelMA with other materials, and encapsulation of growth factors for inducing directed migration can be explored as future directions to further improve the wound healing properties of these hydrogels.

Acknowledgements Authors thank IIT Bombay for providing Cryo FEG-SEM, TEM, ICP-AES, NMR and FACS facilities. SS acknowledges financial support from ICMR (Grant # 5/3/8/336/2017-ITR). NS was supported by the Inspire Fellowship from the Department of Science and Technology (Govt. of India).

Figure Legends Figure 1: Schematic depicting proposed mode of action of silver nanoparticle (AgNP) entrapped methacrylated gelatin (GelMA) gels. Precursor solution containing methacrylated gelatin, AgNPs and photoinitiator are poured at the site of wound and subjected to 1 min UV exposure to form GelMA gels. Fibroblasts surrounding the wound site migrate into the GelMA gel, proliferate inside the gel, and gradually remodel the GelMA gel thereby healing the wound. In addition, AgNPs entrapped inside the GelMA gels provide antibacterial activity during the wound healing.

Figure 2: Spreading proliferation and motility of NIH 3T3 fibroblasts on GelMA gels. (A) Representative phase contrast images of fibroblasts cultured on 5 and 15% GelMA gels for 24

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hours. Scale Bar = 20 µm. (B) Quantification of cell spreading after 24 hours of culture on 5 and 15% GelMA gels. (n = 170-175 cells per condition across 3 independent experiments). ∗∗ 𝑝 < 0.01; statistical significance was determined by student’s t-test. Error bars represent standard errors of mean (± SEM). (C) Representative images of fibroblasts cultured on 5 and 15% GelMA gels for 24 and 48 hours and stained for calcein AM and Hoechst 33342. Scale Bar = 100 µm. (D) Cell proliferation was characterized by quantifying the average number of cells per frame positive for calcein AM at 24 and 48 hours (n = 490-500 cells per condition across 3 independent experiments). ∗∗ 𝑝 < 0.01; statistical significance was determined by student’s t-test. Error bars represent standard errors of mean (± SEM). (E) Representative random cell migration trajectories of fibroblasts cultured on 5 and 15% GelMA gels. (F) Quantitative analysis of cell speed of fibroblasts cultured on 5 and 15% GelMA gels (n = 95-100 cells per condition across 3 independent experiments). ∗ 𝑝 < 0.05. Statistical significance was determined by student’s t-test. Error bars represent standard error of mean (± SEM).

Figure 3: Characterization of AgNPs and their antibacterial activity. (A) Representative absorption spectra of AgNPs in distilled water. Synthesis of AgNPs was confirmed from the sharp peak in the UV-Vis spectrum at ~400 nm. (B) Representative TEM image of synthesized AgNPs Scale Bar = 10 nm. (C) Size distribution of AgNPs analyzed by TEM and DLS. Both the techniques yielded AgNP size of ~10 nm (n = 200-250 NPs per condition across 3 independent experiments). Statistical significance was determined by student’s t-test (ns: not significant). Error bars represent standard error of mean (± SEM). (D) Antibacterial activity of synthesized AgNPs against E. coli and S. aureus characterized by quantifying the change in O.D. ( O.D.) upon incubation of bacterial strains with varying concentrations of AgNPs. Experiment was repeated twice in duplicates. Statistical significance was determined by one-way ANOVA/Fisher test (∗ 𝑝 < 0.05, ∗∗ 𝑝 < 0.01, ∗∗∗ 𝑝 < 0.001 ). Error bars represent standard error of mean (± SEM).

Figure 4: Characterization of AgNP entrapped GelMA gels. (A) Representative Cryo FEGSEM images of 15 % GelMA gels in the absence and presence of 50 µg/ml AgNPs. White arrows in zoomed-in image show AgNPs physically adsorbed onto the gels. Scale Bar = 0.5 µm. (B) Quantification of pore size of GelMA gels in the absence and presence of 50 µg/ml AgNPs (n = 100-120 pores per condition across 3 independent experiments). Statistical significance was

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determined by student’s t-test (ns: not significant). Error bars represent standard error of mean (± SEM). (C) Experimental setup for quantifying kinetics of AgNP release from GelMA gels. GelMA gels entrapped with varying concentration of AgNPs were incubated in 5 ml of PBS. 2 ml of PBS was removed every 12 hours to assess quantity of AgNP in solution. 2 ml of fresh PBS was added to maintain volume constant (n = 6 gels per condition across 2 independent experiments). (D) Cumulative release of AgNPs from 15% GelMA gels entrapped with different initial concentration of AgNPs. Error bars represent standard error of mean (± SEM). (E) Antibacterial activity of AgNP entrapped 15% GelMA gels against E. coli and S. aureus characterized by quantifying the change in O.D. ( O.D.) upon incubation of bacterial strains with GelMA gels entrapped with varying concentrations of AgNPs. Experiment was repeated twice in duplicates. Statistical significance was determined by one-way ANOVA/Fisher test (∗ 𝑝 < 0.05, ∗∗ 𝑝 < 0.01, ∗∗∗ 𝑝 < 0.001). Error bars represent standard error of mean (± SEM).

Figure 5: Cytotoxicity of AgNPs and AgNP entrapped GelMA gels. (A) Representative images of cells cultured on glass coverslips for 24 hours in the presence of different concentration of AgNPs. Scale Bar = 20 µm. Cells were labeled with Calcein AM and propidium iodide (PI) for visualizing live and dead cells, respectively. (B) Quantification of %age of live cells after 24 hours of culture in the presence of different concentration of AgNPs (n = 400-500 cells per condition across 3 independent experiments). Statistical significance was determined by two tailed student’s t-test (∗ 𝑝 < 0.05). Error bars represent standard error of mean (± SEM). (C) Representative images of cells cultured for 24 hours on GelMA gels entrapped with different initial concentration of AgNPs. Scale Bar = 20 µm. Cells were labeled with Calcein AM and propidium iodide (PI). (D) Quantification of the %age of live cells after 24 hours of culture on GelMA gels entrapped with different initial concentration of AgNPs hours (n = 90-100 cells per condition across 3 independent experiments). Statistical significance was determined by two tailed student’s t -test. Error bars represent standard error of mean (± SEM). (E) Representative images showing gating strategy of FACS to distinguish live and dead cells. Effect of AgNP entrapped GelMA gels on cell viability. (F) Quantification of %age of live cells after 24 hours of culture on GelMA gels entrapped with different initial concentration of AgNPs (n = 6 gels per condition across 3 independent experiments). Statistical significance was determined by two tailed student’s t-test. Error bars represent standard error of mean (± SEM).

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Figure 6: Wound healing on GelMA gels using a PDMS device: Schematic representation of in vitro scratch assay on GelMA gels/glass coverslips using PDMS device. Control experiments were performed on uncoated glass coverslips, glass coverslips coated with 15% gelatin (w/v), 15% GelMA (w/v) and 15% collagen (v/v), as well as on 1.5 mg/ml collagen gels. (A) After placing the PDMS device firmly on the gels/glass coverslips, cells were seeded on the exposed areas. After 12 hours culture, the device was removed leaving ~800 m gap in the center of the gel. Sensing the gap, cells migrate inward thereby closing the gap. This process closely resembles wound healing without causing cell death. (B) Representative images show temporal dynamics of wound healing on glass coverslips, collagen gels and GelMA gels entrapped with varying concentrations of AgNPs. Scale Bar = 400 µm.

Figure 7: Quantification of wound healing: (A) Quantification of the %age of wound closure as a function of time (n = 6 samples per condition across 3 independent experiments). (B) Quantification of %age of wound healing after 24 hours. Statistical significance was determined by one-way ANOVA/Fisher’s test (∗ 𝑝 < 0.05, ∗∗ 𝑝 < 0.01). Error bars represent standard error of mean (± SEM).

Supporting Information: Figure S1: 1H NMR spectrum of gelatin and modified gelatin with methacrylic anhydride Figure S2: Storage and loss modulus of GelMA gels Figure S3: Proliferation of NIH 3T3 fibroblasts on GelMA gels in 2% FBS Figure S4: Random motility of NIH 3T3 fibroblasts

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Fig.2

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References

(1)

Annabi, N.; Rana, D.; Shirzaei Sani, E.; Portillo-Lara, R.; Gifford, J. L.; Fares, M. M.; Mithieux, S. M.; Weiss, A. S. Engineering a Sprayable and Elastic Hydrogel Adhesive with Antimicrobial Properties for Wound Healing. Biomaterials 2017, 139, 229–243. https://doi.org/10.1016/j.biomaterials.2017.05.011.

(2)

Bruns, T. B.; Worthington, J. M. Using Tissue Adhesive for Wound Repair: A Practical Guide to Dermabond. Am. Fam. Physician 2000, 61 (5), 1383–1388.

(3)

Chan Choi, Y.; Choi, J. S.; Jung, Y. J.; Cho, Y. W. Human Gelatin Tissue-Adhesive Hydrogels Prepared by Enzyme-Mediated Biosynthesis of DOPA and Fe Crosslinking.

J.

Mater.

Chem.

B

2014,

2

(2),

3+

Ion

201–209.

https://doi.org/10.1039/C3TB20696C. (4)

Berry, M.; Stanek, J. J. Fibrin Tissue Adhesive for Face- and Necklift. J. Plast. Reconstr. Aesthetic Surg. 2015, 68 (10), 1325–1331. https://doi.org/10.1016/j.bjps.2015.06.021.

(5)

Ryu, J. H.; Lee, Y.; Kong, W. H.; Kim, T. G.; Park, T. G.; Lee, H. AC51-CatecholFunctionalized Chitosan/Pluronic Hydrogels for Tissue Adhesives and Hemostatic Materials.

Biomacromolecules

2011,

12

(7),

2653–2659.

https://doi.org/10.1021/bm200464x. (6)

Sekine, T.; Nakamura, T.; Shimizu, Y.; Ueda, H.; Matsumoto, K.; Takimoto, Y.; Kiyotani, T. A New Type of Surgical Adhesive Made from Porcine Collagen and Polyglutamic Acid. J. Biomed. Mater. Res. 2001, 54 (2), 305–310. https://doi.org/10.1002/10974636(200102)54:23.0.CO;2-B.

(7)

Fan, C.; Fu, J.; Zhu, W.; Wang, D. A. A Mussel-Inspired Double-Crosslinked Tissue Adhesive Intended for Internal Medical Use. Acta Biomater. 2016, 33, 51–63. https://doi.org/10.1016/j.actbio.2016.02.003.

(8)

Wang, T.; Nie, J.; Yang, D. Dextran and Gelatin Based Photocrosslinkable Tissue Adhesive. Carbohydr.

Polym.

2012,

90

(4),

1428–1436.

https://doi.org/10.1016/j.carbpol.2012.07.011. (9)

Petersen, B.; Barkun, A.; Carpenter, S.; Chotiprasidhi, P.; Chuttani, R.; Silverman, W.; Hussain, N.; Liu, J.; Taitelbaum, G.; Ginsberg, G. G. Tissue Adhesives and Fibrin Glues.

ACS Paragon Plus Environment

ACS Applied Bio Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Gastrointest.

Endosc.

2004,

60

(3),

327–333.

Page 30 of 36

https://doi.org/10.1016/S0016-

5107(04)01564-0. (10)

Yue, K.; Trujillo-De Santiago, G.; Mois Es Alvarez, M.; Tamayol, A.; Annabi, N.; Khademhosseini, A. Synthesis, Properties, and Biomedical Applications of Gelatin Methacryloyl

(GelMA)

Hydrogels.

Biomaterials

2015,

73,

254–271.

https://doi.org/10.1016/j.biomaterials.2015.08.045. (11)

Galis, Z. S.; Khatri, J. J. Matrix Metalloproteinases in Vascular Remodeling and Atherogenesis: The Good, the Bad, and the Ugly. Circ. Res. 2002, 90 (3), 251–262. https://doi.org/10.1161/hh0302.105345.

(12)

Liu, Y.; Chan-Park, M. B. A Biomimetic Hydrogel Based on Methacrylated Dextran-GraftLysine and Gelatin for 3D Smooth Muscle Cell Culture. Biomaterials 2010, 31 (6), 1158– 1170. https://doi.org/10.1016/j.biomaterials.2009.10.040.

(13)

Fukaya, C.; Nakayama, Y.; Murayama, Y.; Omata, S.; Ishikawa, A.; Hosaka, Y.; Nakagawa, T. Improvement of Hydrogelation Abilities and Handling of Photocurable Gelatin-Based Crosslinking Materials. J. Biomed. Mater. Res. - Part B Appl. Biomater. 2009, 91 (1), 329– 336. https://doi.org/10.1002/jbm.b.31406.

(14)

Marois, Y.; Chakfé, N.; Deng, X.; Marois, M.; How, T.; King, M. W.; Guidoin, R. Carbodiimide Cross-Linked Gelatin: A New Coating for Porous Polyester Arterial Prostheses. Biomaterials 1995, 16 (15), 1131–1139. https://doi.org/10.1016/01429612(95)93576-Y.

(15)

Nickerson, M. T.; Patel, J.; Heyd, D. V.; Rousseau, D.; Paulson, A. T. Kinetic and Mechanistic Considerations in the Gelation of Genipin-Crosslinked Gelatin. Int. J. Biol. Macromol. 2006, 39 (4–5), 298–302. https://doi.org/10.1016/j.ijbiomac.2006.04.010.

(16)

Lee, Y.; Bae, J. W.; Oh, D. H.; Park, K. M.; Chun, Y. W.; Sung, H. J.; Park, K. D. In Situ Forming Gelatin-Based Tissue Adhesives and Their Phenolic Content-Driven Properties. J. Mater. Chem. B 2013, 1 (18), 2407–2414. https://doi.org/10.1039/c3tb00578j.

(17)

Nguyen, A. H.; McKinney, J.; Miller, T.; Bongiorno, T.; McDevitt, T. C. Gelatin Methacrylate Microspheres for Controlled Growth Factor Release. Acta Biomater. 2015, 13, 101–110. https://doi.org/10.1016/j.actbio.2014.11.028.

(18)

Van Den Bulcke, I.; Bogdanov, B.; De Rooze, N.; Schacht, E. H.; Cornelissen, M.; Berghmans, H. Structural and Rheological Properties of Methacrylamide Modified Gelatin

ACS Paragon Plus Environment

Page 31 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Bio Materials

Hydrogels. Biomacromolecules 2000, 1 (1), 31–38. https://doi.org/10.1021/bm990017d. (19)

Klotz, B. J.; Gawlitta, D.; Rosenberg, A. J. W. P.; Malda, J.; Melchels, F. P. W. GelatinMethacryloyl Hydrogels: Towards Biofabrication-Based Tissue Repair. Trends Biotechnol. 2016, 34 (5), 394–407. https://doi.org/10.1016/j.tibtech.2016.01.002.

(20)

Zhao, X.; Sun, X.; Yildirimer, L.; Lang, Q.; Lin, Z. Y. W; Zheng, R.; Zhang, Y.; Cui, W.; Annabi, N.; Khademhosseini, A. Cell Infiltrative Hydrogel Fibrous Scaffolds for Accelerated

Wound

Healing.

Acta

Biomater.

2017,

49,

66–77.

https://doi.org/10.1016/j.actbio.2016.11.017. (21)

Nichol, J. W.; Koshy, S. T.; Bae, H.; Hwang, C. M.; Yamanlar, S.; Khademhosseini, A. Cell-Laden Microengineered Gelatin Methacrylate Hydrogels. Biomaterials 2010, 31 (21), 5536–5544. https://doi.org/10.1016/j.biomaterials.2010.03.064.

(22)

Schuurman, W.; Levett, P. A.; Pot, M. W.; Van W, P. R.; Dhert, W. J. A.; Hutmacher, D. W.; Melchels, F. P. W.; Klein, T. J.; Malda, J. Gelatin-Methacrylamide Hydrogels as Potential Biomaterials for Fabrication of Tissue-Engineered Cartilage Constructs. Macromol. Biosci. 2013, 13 (5), 551–561. https://doi.org/10.1002/mabi.201200471.

(23)

Shin, S. R.; Aghaei, G. B. B.; Dang, T. T.; Topkaya, S. N.; Gao, X.; Yang, S. Y.; Jung, S. M.; Oh, J. H.; Dokmeci, M. R.; Tang, X.; Khademhosseini, A. Cell-Laden Microengineered and Mechanically Tunable Hybrid Hydrogels of Gelatin and Graphene Oxide. Adv. Mater. 2013, 25 (44), 6385–6391. https://doi.org/10.1002/adma.201301082.

(24)

Benton, J. A.; DeForest, C. A.; Vivekanandan, V.; Anseth, K. S. Photocrosslinking of Gelatin Macromers to Synthesize Porous Hydrogels That Promote Valvular Interstitial Cell Function.

Tissue

Eng.

Part

A

2009,

15

(11),

3221–3230.

https://doi.org/10.1089/ten.tea.2008.0545. (25)

Cencetti, C.; Bellini, D.; Pavesio, A.; Senigaglia, D.; Passariello, C.; Virga, A.; Matricardi, P. Preparation and Characterization of Antimicrobial Wound Dressings Based on Silver, Gellan,

PVA

and

Borax.

Carbohydr.

Polym.

2012,

90

(3),

1362–1370.

https://doi.org/10.1016/j.carbpol.2012.07.005. (26)

Gunasekaran, T.; Nigusse, T.; Dhanaraju, M. D. Silver Nanoparticles as Real Topical Bullets for Wound Healing. J. Am. Coll. Clin. Wound Spec. 2011, 3 (4), 82–96. https://doi.org/10.1016/j.jcws.2012.05.001.

(27)

Hiro, M. E.; Pierpont, Y. N.; Ko, F.; Wright, T. E.; Robson, M. C.; Payne, W. G.

ACS Paragon Plus Environment

ACS Applied Bio Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 36

Comparative Evaluation of Silver-Containing Antimicrobial Dressings on in Vitro and in Vivo Processes of Wound Healing. Eplasty 2012, 12, e48. (28)

Hussain, S.; Ferguson, C. Best Evidence Topic Report. Silver Sulphadiazine Cream in Burns. Emerg. Med. J. 2006, 23 (12), 929–932. https://doi.org/10.1136/emj.2006.043059.

(29)

Chen, X.; Schluesener, H. J. Nanosilver: A Nanoproduct in Medical Application. Toxicol. Lett. 2008, 176 (1), 1–12. https://doi.org/10.1016/j.toxlet.2007.10.004.

(30)

Galandáková, A.; Franková, J.; Ambrožová, N.; Habartová, K.; Pivodová, V.; Zálešák, B.; Šafářová, K.; Smékalová, M.; Ulrichová, J. Effects of Silver Nanoparticles on Human Dermal Fibroblasts and Epidermal Keratinocytes. Hum. Exp. Toxicol. 2016, 35 (9), 946– 957. https://doi.org/10.1177/0960327115611969.

(31)

Guzmán, M. G. M.; Dille, J.; Godet, S. Synthesis of Silver Nanoparticles by Chemical Reduction Method and Their Antibacterial Activity. Proc. World Acad. Sci. Eng. Technol. 2009, 45, 357–364. https://doi.org/10.1007/s11814-010-0067-0.

(32)

Franková, J.; Pivodová, V.; Vágnerová, H.; Juránová, J.; Ulrichová, J. Effects of Silver Nanoparticles on Primary Cell Cultures of Fibroblasts and Keratinocytes in a WoundHealing

Model.

J.

Appl.

Biomater.

Funct.

Mater.

2016,

14

(2),

e1–e6.

https://doi.org/10.5301/jabfm.5000268. (33)

Rath, G.; Hussain, T.; Chauhan, G.; Garg, T.; Goyal, A. K. Collagen Nanofiber Containing Silver Nanoparticles for Improved Wound-Healing Applications. J. Drug Target. 2016, 24 (6), 520–529. https://doi.org/10.3109/1061186X.2015.1095922.

(34)

Jeong, L.; Park, W. H. Preparation and Characterization of Gelatin Nanofibers Containing Silver

Nanoparticles.

Int.

J.

Mol.

Sci.

2014,

15

(4),

6857–6879.

https://doi.org/10.3390/ijms15046857. (35)

You, C.; Li, Q.; Wang, X.; Wu, P.; Ho, J. K.; Jin, R.; Zhang, L.; Shao, H.; Han, C. Silver Nanoparticle Loaded Collagen/Chitosan Scaffolds Promote Wound Healing via Regulating Fibroblast Migration and Macrophage Activation. Sci. Rep. 2017, 7 (1), 1–11. https://doi.org/10.1038/s41598-017-10481-0.

(36)

Neibert, K.; Gopishetty, V.; Grigoryev, A.; Tokarev, I.; Al-Hajaj, N.; Vorstenbosch, J.; Philip, A.; Minko, S.; Maysinger, D. Wound-Healing with Mechanically Robust and Biodegradable Hydrogel Fibers Loaded with Silver Nanoparticles. Adv. Healthc. Mater. 2012, 1 (5), 621–630. https://doi.org/10.1002/adhm.201200075.

ACS Paragon Plus Environment

Page 33 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Bio Materials

(37)

García, A, C.; Chen, C.; Burón, M.; Palomares, T.; Eceiza, A.; Fruk, L.; Corcuera, M. Á.; Gabilondo, N. Biocompatible Hydrogel Nanocomposite with Covalently Embedded Silver Nanoparticles.

Biomacromolecules

2015,

16

(4),

1301–1310.

https://doi.org/10.1021/acs.biomac.5b00101. (38)

Resmi, R.; Unnikrishnan, S.; Krishnan, L. K.; Kalliyana Krishnan, V. Synthesis and Characterization of Silver Nanoparticle Incorporated Gelatin-Hydroxypropyl Methacrylate Hydrogels for Wound Dressing Applications. J. Appl. Polym. Sci. 2017, 134 (10), 1–9. https://doi.org/10.1002/app.44529.

(39)

Das, A.; Monteiro, M.; Barai, A.; Kumar, S.; Sen, S. MMP Proteolytic Activity Regulates Cancer Invasiveness by Modulating Integrins. Sci. Rep. 2017, 7 (1), 1–13. https://doi.org/10.1038/s41598-017-14340-w.

(40)

Agnihotri, S.; Mukherji, S.; Mukherji, S. Size-Controlled Silver Nanoparticles Synthesized over the Range 5–100 Nm Using the Same Protocol and Their Antibacterial Efficacy. RSC Adv. 2014, 4 (8), 3974–3983. https://doi.org/10.1039/C3RA44507K.

(41)

Haase, H.; Jordan, L.; Keitel, L.; Keil, C.; Mahltig, B. Comparison of Methods for Determining the Effectiveness of Antibacterial Functionalized Textiles. PLoS One 2017, 12 (11), 1–16. https://doi.org/10.1371/journal.pone.0188304.

(42)

Wahab, R.; Khan, S. T.; Dwivedi, S.; Ahamed, M.; Musarrat, J.; Al-Khedhairy, A. A. Effective Inhibition of Bacterial Respiration and Growth by CuO Microspheres Composed of Thin Nanosheets. Colloids Surfaces B: Biointerfaces 2013, 111, 211–217. https://doi.org/10.1016/j.colsurfb.2013.06.003.

(43)

Lee, H.; Ryu, D.; Choi, S.; Lee, D. Antibacterial Activity of Silver-Nanoparticles Against Staphylococcus Aureus and Escherichia Coli. Korean J. Microbiol. Biotechnol. 2011, 39 (1), 77–85.

(44)

Banjara, R. A.; Jadhav, S. K.; Bhoite, S. A. MIC for Determination of Antibacterial Activity of Di-2-Ethylaniline Phosphate. J. Chem. Pharm. Res. 2012, 4 (1), 648–652.

(45)

Shin, H.; Olsen, B. D.; Khademhosseini, A. The Mechanical Properties and Cytotoxicity of Cell-Laden Double-Network Hydrogels Based on Photocrosslinkable Gelatin and Gellan Gum

Biomacromolecules.

Biomaterials

2012,

33

(11),

3143–3152.

https://doi.org/10.1016/j.biomaterials.2011.12.050. (46)

Brinkman, W. T.; Nagapudi, K.; Thomas, B. S.; Chaikof, E. L. Photo-Cross-Linking of

ACS Paragon Plus Environment

ACS Applied Bio Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 36

Type I Collagen Gels in the Presence of Smooth Muscle Cells: Mechanical Properties, Cell Viability,

and

Function.

Biomacromolecules

2003,

4

(4),

890–895.

https://doi.org/10.1021/bm0257412. (47)

Das, A.; Barai, A.; Monteiro, M.; Kumar, S.; Sen, S. Nuclear Softening Is Essential for Protease-Independent

Migration.

Matrix

Biol.

2019.

https://doi.org/10.1016/j.matbio.2019.01.001. (48)

Pascual, G.; Rodríguez, M.; Mesa-Ciller, C.; Pérez-Köhler, B.; Fernández-Gutiérrez, M.; San Román, J.; Bellón, J. M. Sutures versus New Cyanoacrylates in Prosthetic Abdominal Wall Repair: A Preclinical Long-Term Study. J. Surg. Res. 2017, 220, 30–39. https://doi.org/10.1016/j.jss.2017.06.074.

(49)

Ryou, M.; Thompson, C. C. Tissue Adhesives: A Review. Tech. Gastrointest. Endosc. 2006, 8 (1), 33–37. https://doi.org/10.1016/j.tgie.2005.12.007.

(50)

Soucy, J. R.; Shirzaei Sani, E.; Portillo Lara, R.; Diaz, D.; Dias, F.; Weiss, A. S.; Koppes, A. N.; Koppes, R. A.; Annabi, N. Photocrosslinkable Gelatin/Tropoelastin Hydrogel Adhesives for Peripheral Nerve Repair. Tissue Eng. Part A 2018, 24 (17–18), 1393–1405. https://doi.org/10.1089/ten.tea.2017.0502.

(51)

Yue, K.; Li, X.; Schrobback, K.; Sheikhi, A. Biomaterials Structural Analysis of Photocrosslinkable Methacryloyl-Modi Fi Ed Protein Derivatives. Biomaterials 2017, 139, 163–171. https://doi.org/10.1016/j.biomaterials.2017.04.050.

(52)

Lin, R.-Z.; Chen, Y.-C.; Moreno-Luna, R.; Khademhosseini, A.; Melero-Martin, J. M. Transdermal Regulation of Vascular Network Bioengineering Using a Photopolymerizable Methacrylated

Gelatin

Hydrogel.

Biomaterials

2013,

34

(28),

6785–6796.

https://doi.org/10.1016/j.biomaterials.2013.05.060. (53)

Yeo, Y.; Highley, C. B.; Bellas, E.; Ito, T.; Marini, R.; Langer, R.; Kohane, D. S. In Situ Cross-Linkable Hyaluronic Acid Hydrogels Prevent Post-Operative Abdominal Adhesions in

a

Rabbit

Model.

Biomaterials

2006,

27

(27),

4698–4705.

https://doi.org/10.1016/j.biomaterials.2006.04.043. (54)

Liang, X.; Boppart, S. A. Biomechanical Properties of in Vivo Human Skin from Dynamic Optical Coherence Elastography. IEEE Trans. Biomed. Eng. 2010, 57 (4), 953–959. https://doi.org/10.1109/TBME.2009.2033464.

(55)

George, E.; Barai, A.; Shirke, P.; Majumder, A.; Sen, S. Engineering Interfacial Migration

ACS Paragon Plus Environment

Page 35 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Bio Materials

by Collective Tuning of Adhesion Anisotropy and Stiffness. Acta Biomater. 2018, 72, 82– 93. https://doi.org/10.1016/j.actbio.2018.03.016. (56)

Liu, B.; Wang, Y.; Miao, Y.; Zhang, X.; Fan, Z.; Singh, G.; Zhang, X.; Xu, K.; Li, B.; Hu, Z.; Xing, M. Hydrogen Bonds Autonomously Powered Gelatin Methacrylate Hydrogels with Super-Elasticity, Self-Heal and Underwater Self-Adhesion for Sutureless Skin and Stomach

Surgery

and

E-Skin.

Biomaterials

2018,

171,

83–96.

https://doi.org/10.1016/j.biomaterials.2018.04.023. (57)

Zhao, X.; Lang, Q.; Yildirimer, L.; Lin, Z. Y.; Cui, W.; Annabi, N.; Ng, K. W.; Dokmeci, M. R.; Ghaemmaghami, A. M.; Khademhosseini, A. Photocrosslinkable Gelatin Hydrogel for Epidermal Tissue Engineering. Adv. Healthc. Mater. 2016, 5 (1), 108–118. https://doi.org/10.1002/adhm.201500005.

(58)

O’Connell, C. D.; Zhang, B.; Onofrillo, C.; Duchi, S.; Blanchard, R.; Quigley, A.; Bourke, J.; Gambhir, S.; Kapsa, R.; Di Bella, C.; Choong, P.; Wallace.G.G. Tailoring the Mechanical Properties

of

Gelatin

Photocrosslinking

Methacryloyl

Conditions.

Soft

Hydrogels Matter

through 2018,

Manipulation 14

(11),

of

the

2142–2151.

https://doi.org/10.1039/C7SM02187A. (59)

Nichol, J. W.; Koshy, S. T.; Bae, H.; Hwang, C. M.; Yamanlar, S.; Khademhosseini, A. Cell-Laden Microengineered Gelatin Methacrylate Hydrogels. Biomaterials 2010, 31 (21), 5536–5544. https://doi.org/10.1016/j.biomaterials.2010.03.064.

(60)

Engler, A.; Bacakova, L.; Newman, C.; Hategan, A.; Griffin, M.; Discher, D. Substrate Compliance versus Ligand Density in Cell on Gel Responses. Biophys. J. 2004, 86 (1), 617– 628. https://doi.org/10.1016/S0006-3495(04)74140-5.

(61)

Peyton, S. R.; Putnam, A. J. Extracellular Matrix Rigidity Governs Smooth Muscle Cell Motility in a Biphasic Fashion. J. Cell. Physiol. 2005, 204 (1), 198–209. https://doi.org/10.1002/jcp.20274.

(62)

Solon, J.; Levental, I.; Sengupta, K.; Georges, P. C.; Janmey, P. A. Fibroblast Adaptation and Stiffness Matching to Soft Elastic Substrates. Biophys. J. 2007, 93 (12), 4453–4461. https://doi.org/10.1529/biophysj.106.101386.

(63)

Winer, J. P.; Oake, S.; Janmey, P. A. Non-Linear Elasticity of Extracellular Matrices Enables Contractile Cells to Communicate Local Position and Orientation. PLoS One 2009, 4 (7), 1–13. https://doi.org/10.1371/journal.pone.0006382.

ACS Paragon Plus Environment

ACS Applied Bio Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(64)

Page 36 of 36

Mohammadi, H.; Arora, P. D.; Simmons, C. A.; Janmey, P. A.; McCulloch, C. A. Inelastic Behaviour of Collagen Networks in Cell-Matrix Interactions and Mechanosensation. J. R. Soc.

Interface

2014,

12

(102),

20141074–20141074.

https://doi.org/10.1098/rsif.2014.1074. (65)

Oosten, V. A. S. G.; Vahabi, M.; Licup, A. J.; Sharma, A.; Galie, P. A.; MacKintosh, F. C.; Janmey, P. A. Uncoupling Shear and Uniaxial Elastic Moduli of Semiflexible Biopolymer Networks: Compression-Softening and Stretch-Stiffening. Sci. Rep. 2016, 6 (1), 19270. https://doi.org/10.1038/srep19270.

(66)

Yang, Z.; Hemar, Y.; Hilliou, L.; Gilbert, E. P.; McGillivray, D. J.; Williams, M. A. K.; Chaieb, S. Nonlinear Behavior of Gelatin Networks Reveals a Hierarchical Structure. Biomacromolecules 2016, 17 (2), 590–600. https://doi.org/10.1021/acs.biomac.5b01538.

(67)

Ehrbar, M.; Sala, A.; Lienemann, P.; Ranga, A.; Mosiewicz, K.; Bittermann, A.; Rizzi, S. C.; Weber, F. E.; Lutolf, M. P. Elucidating the Role of Matrix Stiffness in 3D Cell Migration and

Remodeling.

Biophys.

J.

2011,

100

(2),

284–293.

https://doi.org/10.1016/j.bpj.2010.11.082. (68)

Braakhuis, H. M.; Gosens, I.; Krystek, P.; Boere, J. A. F.; Cassee, F. R.; Fokkens, P. H. B.; Post, J. A.; van Loveren, H.; Park, M. V. D. Z. Particle Size Dependent Deposition and Pulmonary Inflammation after Short-Term Inhalation of Silver Nanoparticles. Part. Fibre Toxicol. 2014, 11, 49. https://doi.org/10.1186/s12989-014-0049-1.

(69)

Patchin, E. S.; Anderson, D. S.; Silva, R. M.; Uyeminami, D. L.; Scott, G. M.; Guo, T.; Van Winkle, L. S.; Pinkerton, K. E. Size-Dependent Deposition, Translocation, and Microglial Activation of Inhaled Silver Nanoparticles in the Rodent Nose and Brain. Environ. Health Perspect. 2016, 124 (12), 1870–1875. https://doi.org/10.1289/EHP234.

(70)

Choi, H. S.; Liu, W.; Misra, P.; Tanaka, E.; Zimmer, J. P.; Itty Ipe, B.; Bawendi, M. G.; Frangioni, J. V. Renal Clearance of Nanoparticles. Nat. Biotechnol. 2007, 25 (10), 1165– 1170. https://doi.org/10.1038/nbt1340.Renal.

ACS Paragon Plus Environment