Combination of the Silver-Ethylene Interaction and 3D Printing to

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Biological and Medical Applications of Materials and Interfaces

Combination of the Silver-Ethylene Interaction and 3D Printing to Develop Antibacterial Superporous Hydrogels for Wound Management Zhen Wu, and Youliang Hong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b14090 • Publication Date (Web): 22 Aug 2019 Downloaded from pubs.acs.org on August 24, 2019

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ACS Applied Materials & Interfaces

Combination of the Silver-Ethylene Interaction and 3D Printing to Develop Antibacterial Superporous Hydrogels for Wound Management Zhen Wu and Youliang Hong* National Engineering Research Centre for Biomaterials, Sichuan University, 610064, Chengdu, P. R. China KEYWORDS: wound dressings; superporous hydrogels; silver-ethylene interaction; 3D printing; silver nanoparticles

ABSTRACT: Due to insufficient biomedical functions of hydrogels for wound management, the exploitation of available methods to expand the biomedical functions of hydrogels always becomes the cutting-edge research. Here we report on the use of the silver-ethylene interaction and 3D printing technique to develop the antibacterial superporous polyacrylamide (PAM)/hydroxypropyl methylcellulose (HPMC) hydrogel dressings. Experiments demonstrated that the silver-ethylene interaction played significant roles in mediating the formation, dispersion and crosslinking of silver nanoparticles (AgNPs) in the hydrogel matrix, as well as the crosslinking of the PAM networks. At the same time, such organometallic complexes also controlled the release of AgNPs to balance the cytocompatibility and antibacterial activity of the AgNP-crosslinked hydrogels. On the other hand, the use of 3D printed templates and HPMC as

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the pore-making materials demonstrated could tailor hydrogels into 91.4% porosity and the formed pores into open channels, which endowing hydrogels with rapid water uptake rate and 14 times dead-weight of uptake capacity. Furthermore, experiments showed that the regular large pores arisen from 3D printed templates could buffer the swelling of superporous hydrogel dressings, thus decreasing the detachment risk of dressings from wounds. In vivo experiments demonstrated that the AgNP-crosslinked superporous hydrogel dressings could promote the healing of the infected wounds and restrain scar tissue formation.

INTRODUCTION The use of dressings to manage wounds has become an indispensable way in current clinic practice, and solid evidence has demonstrated that the performances of wound dressings can influence wound healing extremely.1-3 An excellent wound dressing even can double the rate of wound healing in comparison with traditional wound care.4 For example, a wound dressing with the performances of alleviating pain, maintaining wound moisture, absorbing and holding exudates, and impeding infection can be unquestionably beneficial to chronic wound management. Therefore, the improvement of the dressing performances put forward a direct and feasible way to favor wound management and accelerate wound healing. Hydrogels, which are cross-linked network polymers with high water contents and a variety of physical properties, have been claimed to be best dressings because their biomedical performances are closet to the requirements of ideal wound dressings.5,6 Still, the performances of current hydrogel dressings are insufficient and need be improved. For example, current commercially-available hydrogel dressings had low even no antibacterial activity. To endow hydrogels with antibacterial activity, the incorporation of various antibacterial reagents, i.e., Ag nanoparticles (AgNPs),7-9 zinc salts,10 and tannic acid11 has been attempted. Thereinto, AgNPs


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received most attention because AgNPs have broad-spectrum antibacterial activity and can aid in faster and aesthetic wound healing.12 However, current most universal approaches of introducing AgNPs into the hydrogel matrix were based on physical incorporation.13-17 Physical incorporation of Ag into hydrogels can give rise to rapid and continuous release of Ag to surrounding environment, leading to unwanted toxicity problems (The toxic effects of Ag has been well-documented).14 For example, Maneerung et al have reported that the AgNPs could be leaked rapidly from the freeze-dried AgNP-impregnated bacterial cellulose prepared by in situ AgNP reduction (~20% AgNPs could be released after 7 day immersion).15 As such, Abdelgawad et al have reported that the AgNPs physically-loaded in a kind of electrospun nanofibers could release rapidly from nanofibers at initial stage.16 Especially, Liu et al have demonstrated that the cytocompatibility of wound dressings was relative to the released dose of AgNPs and high dose-releasing of AgNPs resulted in the dressings with high cytotoxicity.17 To overcome such drawbacks, García-Astrain et al recently reported a strategy in which AgNPs were covalently immobilized into hydrogels, and thus the leakage of AgNPs to result in the toxicity problems was avoided.13 Still, the synthetic methods reported by García-Astrain is complex, and the used hydrogel material, chondroitin sulfate, is expensive, thus it raises the difficulty to translate the as-prepared hydrogels into the commercially-available dressings. In addition, extremely low AgNP leakage is also disadvantageous to inhibit bacterial growth. Low liquid absorption capacity (in general < 100%) is another defect of current commercially-available hydrogel dressings.18 Under the requirement of moist wound healing, especially, for chronic wound management, the dressings with high liquid-held capacity and water retention capability are beneficial to hold more exudate or prolong their use to prevent the superficial desiccation of wounds.19 To improve liquid absorption capacity of hydrogels, an


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available method is to endow hydrogels with porosities. Up to now, a variety of methods have been reported to prepare porous hydrogels, such as the porogenation technique,20 phase separation technique,21 freeze-drying,22 and microemulsion formation.23 Thereinto, superporous hydrogels (also are called as the interpenetrating polymer network hydrogels) prepared through a combination reaction of ethylene monomers (e.g., acrylamide, N-isopropyl acrylamide, acrylic acid, vinyl pyrrolidone) with a kind of polymers (mainly natural polysaccharides or hydroxyl (or carboxyl) group-contained polymers) are particularly attractive because these hydrogels had high porosities24,25 and were nonfouling for cell/protein adhesion.26 These features of superporous hydrogels are favorable to manage chronic wounds. Still, these hydrogels have two defects: lack of antibacterial activity and high swelling once liquid uptake. To overcome above defects, herein we report on the combination of the silver-ethylene interaction and 3D printing technique to prepare high absorbent capacity of antibacterial superporous hydrogel dressings, as shown in Scheme 1. To our knowledge, this is the first report on the use of the silver-ethylene interaction to develop the Ag nanoparticle-crosslinked nanocomposite hydrogels as wound dressings. The silver-ethylene interaction was employed because (i) the precursors of superporous hydrogels involve double-ethylene N,N’-methylene bisacrylamide (MBAM) monomers, which provide the sites to fix Ag. Therefore the addition of other components to fix Ag can be avoided, and at the same time the synthetic process is simplified; (ii) the silver-ethylene interaction is a supramolecular synthon.27 Before superporous hydrogel polymerization, MBAM monomers interact with Ag ions through the silver-ethylene interaction to form the organometallic complexes, which subsequently were polymerized and reduced into the AgNP-loaded nanocomposite hydrogels, thus at the same time of endowing the hydrogel dressings with antibacterial activity, decreasing the toxicity. The use of 3D printed


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porous templates can enhance pore volume and more importantly, can achieve open pores, which allow liquid to enter open channels by the capillary effects and reach the swelling equilibrium rapidly.28 It should be noted that although up to now many 3D printed hydrogels have been reported,29-31 little attention has been paid for their applications as wound dressings. In addition, in this work hydroxypropyl methylcellulose (HPMC) was selected to incorporate into the goal hydrogels. HPMC was taken into account based on two factors: (i) HPMC possesses the characteristics of high water uptake and retention, which have been widely used for artificial tears and lubricants of artificial eyes;32 (ii) HPMC is a kind of high swelling polymers.

Scheme 1. The preparation procedure of the AgNP-crosslinked superporous hydrogel dressing. EXPERIMENTAL SECTION Materials. Absolute alcohol, silver nitrate (AgNO3), sodium borohydride (NaBH4), dichloromethane, acrylamide (AM), N,N’-methylene bisacrylamide (MBAM), and ammonium persulfate (APS) all are analytical reagent (AR) and were purchased from Chengdu Kelong Reagent Co., China. Hydroxypropyl methyl cellulose (HPMC) was purchased from Shangdong Weifang Lite Composite Materials Co., China. Sodium Alginate (viscosity: 20±20 mpa·s) and


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chitosan (deacetylation degree ≥ 95%, viscosity: 100-200 mpa·s) was purchased from Aladdin Industrial Co., China. Polylactic acid (PLA) 3D printing filaments (diameter: 1.75 mm) was purchased from Dongguan Hengli Dejian Plastic Electronic Product Factory. The used water was deionized water (DI water). Preparation of Superporous Hydrogel Dressings. Preparation of Porous PLA Templates. A fused deposition modeling (FDM) 3D printer (JGAURORA, Shenzhen Aurora Technology Co.) was used to print the porous PLA scaffolds according to the designed STL format files as the hydrogel templates. The scaffold prototypes (15×15×3 mm) with various pore diameters were designed using the 3D design software, Solidworks. Synthesis of the AgNP-Loaded Superporous Hydrogel Dressings. The illustrative synthesis process is shown in Scheme 1. At first, the silver-ethylene interaction was carried out by dissolving MBAM (0.034g) and AgNO3 (The quantity of AgNO3 changed in light of specific experiments) into 3.0g DI water. After silver ions chelated fully with MBAM (2 h), AM (0.2g), APS (0.015g), and HPMC (0.1g) were added with agitation into the Ag-MBAM solution, which subsequently was cast into the molds or a printed porous PLA templates and reacted for 12 h to form hydrogels. The as-prepared hydrogels were soaked into a NaBH4 aqueous solution for 2 h to reduce silver ions into nanoparticles. After washed using DI water for twice, the prepared AgNP-loaded hydrogels were dried by soaking into absolute alcohol overnight and then soaked into dichloromethane to remove the PLA templates to achieve superporous dressings. The AgNP-loaded hydrogels prepared by above method were named as the AgNP-crosslinked hydrogels. For comparison, the hydrogels without Ag were prepared by repeating above experimental process but no AgNO3 was added and no process of Ag ion reduction was performed.


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Furthermore, such hydrogels were used to prepare another kind of the AgNP-loaded hydrogels. The detailed process was as follows. The hydrogels firstly were soaked in dark into the AgNO3 solution (0.035 mol/l) for 2 h. After dried in dark and at room temperature overnight, the hydrogels absorbing Ag ions were soaked into a NaBH4 aqueous solution for 2 h to reduce silver ions into nanoparticles. Then the AgNP-loaded hydrogels were washed using DI water for twice and dried using by absolute alcohol soaking overnight. The AgNP-loaded hydrogels prepared by above process were named as the AgNP-absorbed hydrogels. In addition, HPMC was replaced by alginate and chitosan to prepare the chitosan-contained and alginate-contained hydrogels (i.e., the PAM/chitosan and PAM/alginate hydrogels). Physicochemical Characterization. FTIR Analysis. The as-prepared hydrogels were analyzed using a Fourier Transform Infrared Spectroscopy (FTIR, PerkinElmer Autoimage). Before measurement, all samples were dried and processed into thin slices by a potassium bromide tableting technique. The FTIR spectra of all samples were measured in the wavenumber range 4000-400 cm-1. AgNPs Release. The AgNP-crosslinked and AgNP-absorbed rectangular hydrogel blocks were soaked into respective DI water with shaking for 7 days at room temperature. The soaked DI water then were analyzed using an inductive coupled plasma atomic emission spectrometry (ICP-AES) (Perkin-Elmer Optima, 7000DV). Transmission Electron Microscope (TEM). The dried AgNP-crosslinked hydrogels were tested using a TEM (Tecnai G2 F20 S-TWIN, FEI). Before measurement, the hydrogels were embedded through methyl methacrylate resin and cut into thin slices (thickness: 5 μm). A piece of thin slice then was loaded onto the 300 mesh copper grid. The sample was observed at acceleration voltage of 200 kV.


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Scanning Electron Microscopy (SEM). The porous structure and size of the as-prepared hydrogel dressings were observed by a SEM (Hitachi S-4800). Before observation, the hydrogel samples were coated by a thin layer of gold through gold sputter coating for 30 s. By comparison, pure PAM hydrogels were prepared by repeating above hydrogel synthesis process but no HPMC was added. Before measurement, all samples were soaked into DI water for 24 h to swell fully and then dried by soaking into absolute alcohol overnight. Mechanical Testing. The mechanical properties of dried superporous hydrogel dressings were measured with a Universal Mechanical Tester (Shimadzu AG-IC, Japan). The samples (50×15×1 mm) were strained to failure at a rate of 0.1 mm/min. UV-vis DRS Analysis. The samples were analyzed using a solid ultraviolet-visible diffuse reflection spectroscopy (UV-vis DRS, Hitachi U-2900, Japan), and before measurement, all samples were prepared into dried thin films (0.5 mm) and fixed at a quartz plate. Water Uptake Capacity. The dried hydrogels with specific volume were weighted (Wd) and subsequently soaked into DI water (50 mL) at 37 °C. The soaked hydrogels were gotten out and weighed (Wm) over and over again at specific time intervals. The water uptake capacity (Cw) is calculated as Cw = [(Wm-Wd)/Wd]×100%. Swelling Observation. The dried solid and superporous AgNP-PAM/HPMC hydrogel dressings with same size were prepared and then soaked into DI water for 24 h to observe the change of volume. Cytotoxicity Assessment. Cell viability was tested by a quantitative CCK8 cytotoxicity assay. Because PAM hydrogel was nonfouling for cell adhesion,26 a method of extracting the hydrogel leakage to assess the cytotoxicity of the as-prepared samples was used.33 In detail, the L929 cells suspension (cell density 1×104) was transferred by 100 μL/well into 96-well plates at first. The


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cell-seeded plates then were incubated at 37 °C in an incubator with 5% CO2. After L929 cells have fully adhered to the well bottom to form a cell monolayer (24 h), the hydrogel extract was used instead of common cell culture medium to incubate the seeded-cells. After the cells were cultured by the hydrogel extract for 24 h, 48 h, or 72 h, the hydrogel extract was replaced by CCk8 reagent (50 μL/well) and the cells were incubated for another 2 h in darkness. The cell-incubated CCK8 reagent then was measured at 450 nm using a microplate reader (Bio-Rad model 550, USA). The relative cell viability was calculated in light of the formula: Vcell = (ODS-ODB)/(ODc-ODB)×100%, where Vcell denotes the cell viability, ODs the absorbance value of sample, ODB the absorbance value of blank, ODC the absorbance value of control (i.e., the cells were always incubated by common cell medium). For each hydrogel extract three same samples were tested. Furthermore, the live-dead analysis was carried out. In detail, the cell-attached plates were washed using sterile PBS and stained using propidium iodide (PI) for dead cells and fluorescein diacetate (FDA) for live cells. A confocal laser scanning microscopy (CLSM, Olympus IX 95) was used to observe the FDA-PI stained L929 cells. In Vitro Antibacterial Activity Assessment. Gram-positive bacteria Staphylococcus aureus (S. aureus, ATCC 29213) and Gram-negative bacteria Escherichia coli (E. coli, ATCC 25922) were used to evaluate the antibacterial activity of the hydrogel dressings. Before the samples were assessed, the S. aureus/E. coli solutions with 1×106 CFU/mL were cultured in tryptic soy broth (TSB, BD). The dressings (10×10×1.5 mm) and the S. aureus or E. coli suspensions were introduced respectively into 24-well culture plates, which subsequently were incubated at 37 °C in an incubator for 24 h. The optical density (OD) of the S. aureus or E. coli suspensions was measured at 600 nm using the microplate reader (Bio-Rad model 550, USA). The antibacterial


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activity of sample was calculated in light of the formula: AD = (ODC-ODS)/(ODC-ODB)×100%, where AD denotes the antibacterial activity of sample, ODC the absorbance value of the bacterial suspension without samples, ODS the absorbance value of the bacterial suspension with sample, and ODB the absorbance value of culture medium. For each sample, five same samples were assessed. Antibacterial activities of the hydrogel dressings were further assessed by a disc diffusion approach.17 In brief, 100μL of the S. aureus or E. coli suspensions with 1×106 CFU/mL were plated on the 1.5% LB agar plates, and then the superporous hydrogel dressings were place on the plates against the S. aureus or E. coli. After the plates with S. aureus or E. coli and dressings were incubated at 37 °C in an incubator for 24 h, the inhibition zones resulted from samples were observed. In Vivo Wound Management. Sprague-Dawley (SD) rats (250±50g) were purchased from Experimental Animal Center of West China College of Pharmacy, Sichuan University. The rats were housed in laboratory, in which the temperature was kept at 22-24 °C and the humidity was kept at 40-70%, and the light-dark cycle was 12 h. The rats were provided water and food ad libitum. The in vivo experiments were conducted strictly in light of state regulations and laws and Standing Committee on Ethics in China34 on the use and care of laboratory animals. At the same time, these experiments were done with the guidelines established by Institute for Experimental Animals of Sichuan University and were approved by the Sichuan University Committee on Animal Care and Use. After one week of adaptation, eligible rats were used for experiments. The SD rats were anesthetized through intraperitoneal injection of 0.3ml/100g chloral hydrate and operations were performed under sterile conditions. The hair of the dorsal skin then was removed using depilatory paste before skin excision, and the surgical area was disinfected with


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70% ethanol. Full-thickness wounds (1×1 cm in diameter) were established on both dorsal sides to the depth of loose subcutaneous tissue. Then the infected wounds were created

by dropping

2 drops of S. aureus (1×108 CFU/20 mL) suspension into the wounds. The rats were divided into four groups and each group contains 3 rats). Group 1 (Blank): The wounds always were exposed at air. Group 2: The wounds were covered by the PAM/chitosan superporous hydrogel dressings. Group 3: The wounds were covered by the PAM/HPMC superporous hydrogel dressings. Group 4: The wounds were covered by the AgNP-PAM/HPMC superporous hydrogel dressings. The size of all hydrogel dressings was 15×15×1.5 mm. After covered by the hydrogel dressings, the wounds were further fixed with film dressings (TegadermTM, 3M, USA). The rats were caged individually following identification and the hydrogel dressings were renewed every 2 days. The wounds were observed and photographed to assess the reduction of wound size. The wound areas were measured in light of photographs by the Image-Pro Plus V.6.3 (Media Cybernetics, USA). The wound measurement was presented as the reduction of wound size (%) and was calculated as Wound size reduction (%) = [(Ai-Am)/Ai]×100, where Ai denotes initial wound area, Am the wound area measured at specific time. Histologic Analysis. The rats used for histological assay were sacrificed at day 14 and biopsies were fixed with 10% formalin and embedded in paraffin. The embedded skin tissues were sectioned (5 μm) by a high-speed precision microtome (LEICA SP1600, Germany) and then stained using the hematoxylin and eosin (H&E) and Masson’s trichrome in light of the protocol. The stained samples were observed by an optical microscope.


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Statistical Analysis. For all experiments, the data were reported as mean ± standard deviations (s.d.). Data were analyzed by an unpaired Student t-test. Confidence levels of > 95% (p < 0.05) were considered to be statistically significant. RESULTS AND DISCUSSION Synthesis of the AgNP-Crosslinked PAM/HPMC Hydrogels. It has well-known that silver can interact with ethylene to form the organometallic complexes.27,35 In this work, we investigated the interaction of Ag with MBAM at first by mixing excessive AgNO3 with MBAM (The mole ratio of Ag/MBAM was 1). The FTIR results (Figure 1a) show that the bands at 993 and 910 cm-1, which were assigned as ethylene bends of MBAM, disappeared after AgNO3 was reacted MBAM, and a new band at 949 cm-1, assigned as the silver-ethylene bond, emerged.36 Besides the ethylene groups, MBAM also contains amide (C=N) and carbonyl (C=O) groups, which possibly interact with Ag.37 However, the FTIR curves show that a wide peak at 1535-1547 cm-1, which assigned to the amide II stretch bands, did not change essentially after AgNO3 was added into the MBAM solution. This result demonstrated that no interaction between Ag and amide. As such, the peak of carbonyl at 1633 cm-1 did not shift, demonstrating no interaction between Ag and carbonyl. The FTIR results demonstrated that the silver-ethylene bond is main chemical interaction between Ag and MBAM, as shown in Figure 1(b).


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Figure 1. (a) FTIR of Ag-MBAM interaction: (i) MBAM, (ii) Ag-MBAM (mole ratio of Ag/MBAM was 1:2). (b) The suggested molecular structures of the Ag-MBAM. Then, the Ag-MBAM complexes with the mole ratio of Ag/MBAM 1:2 was prepared and added into the HPMC contained PAM precursors to synthesize the Ag-contained hydrogels, which further were soaked into a NaBH4 solution for 1 h to form the AgNP-crosslinked PAM/HPMC (or named as AgNP-APM/HPMC) hydrogels. At the same time, pure PAM, PAM/HPMC, and AgNP-absorbed PAM/HPMC hydrogels also were prepared for comparison. Figure 2(a) shows the FTIR spectra of HPMC and above prepared samples. In HPMC, the peaks at 3443 and 1650 cm-1 were assigned to the O-H stretching, the weak peaks at 1468 and 1381 cm-1 the OH deformation, and the wide peaks at 1210-1000 cm-1 the C-O-C stretching. The PAM hydrogel showed bands at 3415 and 3194 cm-1, corresponding to a stretching vibration of N-H, and at 1665 cm-1 for C=O stretching. The bands at 1610 cm-1 (N-H deformation for primary amine), 1457 cm-1 (CH2 in-plane scissoring), 1416 cm-1 (C-N stretching for primary amide), 1351 cm-1 (C-H deformation), and 1118 cm-1 (NH2 in-plane rocking), and 1200 cm-1 (C-N stretching of secondary amine) were also detected.38 In the PAM/HPMC hydrogel, the FTIR curve showed that except the peaks at 1060 cm-1 that assigned to the C-O-C stretching of HPMC and the strength reduction of the peak at 3194 cm-1, all other peaks and their peak strength could well correspond to PAM, and no new peaks formed. The disappearance of the peaks at 1650, 468 and 1381 cm-1, assigned to -OH groups in HPMC, presumably arisen from the remained water in HPMC. Such result suggested during PAM polymerization, no interaction occurred between


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HPMC and PAM, and thus suggested that the PAM/HPMC hydrogel is the semi-interpenetrating polymer network structure.39,40 In the AgNP-crosslinked PAM/HPMC hydrogel, the intensities of N-H peak at 1610 cm-1 and C-N peak at 1416 cm-1 decreased. It demonstrated that AgNPs strongly attached with amine (-NH and -NH2 groups). In addition, the peak at 949 cm-1, assigned to silver-ethylene bond formed. This result shows that AgNPs were bound both with -NH2 groups and with ethylene groups in the PAM/HPMC hydrogels. In light of the FTIR result, the interaction of the formed AgNPs with hydrogels can be suggested in Figure 2(b) (It should be noted that AgNPs did not bound with the NH groups in MBAM, as demonstrated in Figure 1). In the AgNP-absorbed PAM/HPMC hydrogel, however, as can be seen from Figure 2(a), the FTIR curve was completely same with the PAM/HPMC hydrogels, demonstrating no interaction between AgNPs with the PAM/HPMC hydrogel.

Figure 2. (a) FTIR spectra of HPMC, PAM, PAM/HPMC, and AgNPs-crosslinked/absorbed PAM/HPMC. (b) Chemical structures of the suggested AgNPs-crosslinked PAM.


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The formed AgNPs were further investigated using a TEM observation and a UV-vis DRS measurement. TEM images and the diameter statistic result (Figure 3a-b) show the AgNPs with spherical shape and with the diameter of 1-15 nm formed and disperse well within the hydrogel matrix, and more importantly no AgNP aggregation was observed. Such result showed that the preparation of AgNPs by in situ reduction is favorable to impede the AgNP aggregation. The UV-vis DRS measurement (Figure 3c) shows that the PAM/HPMC hydrogels had not absorption at 200-800 nm. In the Ag-PAM/HPMC hydrogels, a peak at 302 nm, which can assigned to the silver ions or silver oxide, demonstrating Ag was incorporated into hydrogel. In the AgNP-PAM/HPMC hydrogels, however, the peak at 302 nm disappeared, and was taken place of two new peaks, at 200-290 nm and 300-500 nm, respectively. These peaks could be attributed to different diameters of AgNPs, where the strong peak at long wavelength correlates to large AgNPs and the weak and wide peak at short wavelength small AgNPs.41 The UV-vis DRS results well demonstrated the TEM observation.


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Figure 3. (a) TEM images of the AgNP-PAM/HPMC. (b) Diameter distribution of AgNPs. (C) UV-vis DRS of the PAM/HPMC, Ag-PAM/HPMC, and AgNP-PAM/HPMC hydrogels. Pore Structure, Porosity and Mechanical Properties of Hydrogels. The pore structure of the dried hydrogels was observed at first. SEM images (Figure 4a) shows that abundant irregular pores with average diameter 100 μm formed in the PAM, PAM/HPMC, and AgNP-PAM/HPMC hydrogels. Still, the pores in the PAM hydrogel were close but those in the PAM/HPMC and AgNP-PAM/HPMC hydrogels were open and interconnecting. It shows that the incorporation of HPMC into PAM was very important to form the open pores. The porosity of the dried AgNP-PAM/HPMC hydrogels was further evaluated using an alcohol displacement method.42 Figure 4(b) shows that with the increase of the AgNPs content in hydrogels, the porosity of hydrogels also increased. Such result suggested that the incorporation of AgNPs into hydrogels reduced the crosslinking points of the PAM networks, and thereby enhancing the swelling ratio of hydrogels. Indeed, due to the Ag-MBAM interaction, a considerable amount of MBAM has changed into MBAM-AgNP-MBAM, therefore reducing the amount of single MBAM molecules to crosslink the PAM chains. In addition, although the FTIR result (Figure 2a) showed that AgNPs also interacted with NH2 groups of PAM, the increase of porosity suggested that the AgNP-amine interaction hardly acted as the crosslinking points in hydrogels. Furthermore, the mechanical properties of the fully-swollen hydrogels were further evaluated. Figure 4(c) shows that the AgNP-PAM/HPMC hydrogels had smaller stress and strain than the PAM/HPMC hydrogels. The Young's moduli of the hydrogels with and without AgNPs measured were 2.27 MPa and 1.74 MPa, respectively. This result further demonstrated that the AgNP-crosslinked hydrogels had fewer crosslinking points and higher extension degree of


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polymer chains than the PAM/HPMC hydrogels, and the AgNP-amine interaction hardly acted as the crosslinking points. The decrease of the crosslinking points decreased the hydrogel stress and the PAM chain coiling, thus presenting higher Young’s modulus.

Figure 4. (a) SEM images of the PAM, PAM/HPMC, and AgNP-PAM/HPMC (Ag/MBAM: 1:2, Ag content: 2 wt%) hydrogels. (b) Ag content as a function of porosity of the AgNP-PAM/HPMC hydrogels (n=3). (c) Mechanical properties of the PAM/HPMC and AgNP-PAM/HPMC (Ag/MBAM: 1:2, Ag content: 2 wt%) hydrogels. Ag Release of the AgNPs-Loaded PAM/HPMC Hydrogels. To check the bonding degree of AgNPs with the hydrogel networks, the AgNP-PAM/HPMC hydrogels with weight 100 mg were soaked in deionized water under shaking for 7 days, and the soaked aqueous solutions were tested by an ICP-AES. At the same time, the same weight of the AgNP-absorbed PAM/HPMC hydrogels also was used as a control (i.e., such hydrogels were prepared by soaking the


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PAM/HPMC hydrogel into the AgNO3 solution, which was reduced by NaBH4. See the experiment section). Figure 5 shows that after 7 day immersion, the aqueous solution soaking the Ag-absorbed hydrogels changed into yellow, and the ICP showed that 7.3±0.5 ppm Ag was released into aqueous solution. In contrast, the aqueous solution soaking the AgNP-crosslinked hydrogels was almost colorless, and the ICP showed that the Ag release concentration was 3.5±0.3 ppm. Such results demonstrated that using the silver-ethylene interaction, the AgNPs could be well crosslinked within the hydrogel networks. In contrast, the physical loading of AgNPs in the hydrogels was weak (FTIR spectra shown in Figure 2 showed that no interaction between AgNPs with the PAM/HPMC hydrogel), which had difficult to impede the leakage of AgNPs.

Figure 5. The ICP-AES analysis of the Ag release from the hydrogels in which Ag was loaded by physical absorption or chemical crosslinking (The as-prepared hydrogels have soaked in DI water for 7 days) (n=3). The inserted picture: (a) the hydrogels that have soaked in DI water for 7 days, (b) the 7 day-soaked water.


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Preparation of the AgNP-PAM/HPMC Superporous Hydrogel Dressings. In light of the preparing procedure of the superporous hydrogel dressings shown in Scheme 1, 3D porous PLA templates were printed at first. The porous PLA templates can be prepared conveniently and cost-effectively by the prototype design and subsequent printing by a fused deposition modeling. Figure 6(a-b) shows the designed prototype and corresponding printed PLA templates (15×15×3 mm; pore diameter: 800 μm; template diameter: 400 um). Because the matrix and pore diameters of the PLA template could be adjusted conveniently by changing the prototype design, the porosities of subsequent hydrogel dressings can be controlled easily. Then the hydrogel precursors were cast in porous PLA templates and subsequently above synthesis process was repeated to prepare the AgNP-PAM/HPMC hydrogel dressings. After the PLA template-contained hydrogels were soaked into absolute alcohol overnight, the templates were removed through dichloromethane immersion to achieve superporous hydrogel dressings, as shown in Figure 6(c). SEM images (Figure 6d) show that besides the regular pores resulted from the PLA templates, abundant irregular pores with average diameter 100 μm formed in the dressing matrixes. The porosity of superporous hydrogel dressings measured using the alcohol displacement method was 91.4±2.3%, which was 37.7% higher than the solid hydrogels (did not use the 3D printed PLA templates), as can be seen from Figure 6(e). Furthermore, the mechanical properties of superporous hydrogel dressings were measured. Figure 6(f) shows that the mechanical strength of both superporous dressings decreased markedly but their strain increased largely in comparison with solid hydrogels (Figure 4c). Furthermore, Figure 6(f) shows that after superporous hydrogel dressings began to break, whole dressings did not break completely, but did gradually. Such change of the hydrogels’ mechanical properties obviously can be attributed to large pores resulted from the 3D printed PLA templates.


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The increase of large pores in the hydrogels makes hydrogels the softer materials, which is of benefit to wound management.

Figure 6. (a) The designed prototype and (b) the printed PLA template (15×15×3 mm, the pore diameter: 1.2×1.2×0.8 mm, the diameter of the printed PLA wires: 0.8×0.8×0.8 mm) in light to the designed prototype. (c) The superporous AgNP-PAM/HPMC hydrogel dressings. (d) SEM image of the as-prepared superporous hydrogel dressing. (e) Porosity of solid and superporous


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AgNP-PAM/HPMC hydrogels (n=3). (f) Mechanical properties of superporous PAM/HPMC and AgNP-PAM/HPMC hydrogel dressings. Water Uptake of Superporous Hydrogel Dressings. The water uptake profiles of superporous hydrogel dressings were measured and the results were shown in Figure 7(a). In comparison the PAM/HPMC dressings, the AgNPs-crosslinked dressings displayed faster water uptake ratio and higher water uptake capacity, and the water uptake capacity of the AgNP-PAM/HPMC dressings arrived at 14 times dead-weight (Figure 7a-b). Such result further demonstrated that the AgNP-PAM/HPMC superporous dressings had lower crosslinking degree than the PAM/HPMC ones, thereby allowing the AgNP-PAM/HPMC superporous dressings to absorb more water. Furthermore, alginate and chitosan were used instead of HPMC to prepare the superporous hydrogel dressings. Figure 7(b) shows that these non-HPMC contained hydrogel dressings had significantly lower water uptake capacity than the HPMC-contained hydrogel dressings, demonstrating that HPMC is better exudate uptake materials for wound management.

Figure 7. (a) Water uptake profiles of the AgNP-PAM/HPMC and PAM/HPMC superporous hydrogel dressings. (b) Water uptake capacity of the AgNP-PAM/HPMC, PAM/HPMC, PAM/alginate, and PAM/chitosan superporous hydrogel dressings (n=3).


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Swelling of Superporous Hydrogel Dressings. It has well-reported that the PAM-based interpenetrating polymer network hydrogels will swell once these hydrogels absorbed water,43-45 and some reports even took advantage of the swelling feature of the PAM-based interpenetrating polymer network hydrogels for drug delivery.46,47 However, the swelling feature is disadvantageous for wound management because the swelling easily results in bad attachment of dressings with wounds even detachment of dressings from wounds. Nevertheless, our dressings with the regular porous structures overcome such defect to a large extent. As shown in Figure 8(a), in the superporous hydrogel dressing, after the dried dressing had soaked into water for 24 h, the dressing had very small increase in volume (19×19×4 mm) in comparison with the dried one (15×15×3.2 mm). In contrast, the dried solid hydrogel which had same size as the superporous dressing displayed far higher swelling ratio (23.5×23.5×4 mm) than the superporous hydrogel dressing. The underlying mechanism of superporous hydrogel dressing with low swelling ratio can be explained by a schematic illustration shown in Figure 8(b). Due to the existence of regular large pores, the superporous hydrogel dressing can extend inwards during swelling to buffer the swelling of dressing. In fact, as can be seen from Figure 8(a), in the superporous hydrogel dressing, the regular large pores almost disappeared and are filled fully by the swollen hydrogel after swelling. In contrast, during swelling the solid hydrogel only can expand outwards due to the lack of large pores. Therefore the superporous hydrogel dressings with large pores are desirable for wound management.


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Figure 8. (a) Photos of the fully-swollen solid and superporous hydrogels (The inserted photos were the dried hydrogels). (b) Schematic illustration of the swelling of solid and superporous hydrogels. Cytotoxicity of Superporous Hydrogel Dressings. L929 cells were used in this study to evaluate the cytocompatibility of the as-prepared superporous hydrogel dressings. L929 cells were selected because these cells are fibroblasts, which play essential roles in wound healing.48 In the experiments, cell viability was tested by contacting the extracts of hydrogel dressings. The underlying reason of using the hydrogel extracts is because: (i) our pilot experiments have confirmed that L929 cells did not attached onto PAM-contained superporous hydrogel dressings; (ii) perhaps the substances released from dressings will influence cell viability. Figure 9(a) shows that a quantitative CCK8 cytotoxicity assay, in which no significant difference was indicated among three kinds of hydrogel extracts and in comparison with normal cell culture medium during 3 days of cell culture. The live-dead staining (Figure 9b) shows that only at day 1


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a few dead cells were observed in all media. Above results demonstrated that all dressings were cytocompatibility for L929 cells, and low dose of Ag releasing in the AgNP-crosslinked dressing did not induce the cytotoxicity.


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Figure 9. Cytocompatibility of various hydrogel dressings. (a) The quantitative CCK8 cytotoxicity assay (The normal culture medium was used as the control (n=3); (b) The live-dead staining. Antibacterial Activity of Superporous Hydrogel Dressings. The in vitro antibacterial activity of the AgNP-loaded superporous hydrogel dressings was assessed against Gram-positive S. aureus and Gram-negative E. coli using an disc diffusion method (i.e., the inhibitory effect was measured based on the clear zone surrounding the circular-shaped sample).17 At the same time, the HPMC-contained and chitosan-contained hydrogel dressings were used as the control. Figure 10(a) shows that no inhibition zones surrounded the HPMC-/chitosan-contained hydrogel dressings in both the S. aureus and E. coli colonies, and the removal of the dressings showed that the sterile zones were same as the dressing areas or a lot of bacteria even grew within the dressing-removed zones. In contrast, the AgNP-crosslinked dressings caused clear inhibition zones both bacterial colonies, in the S. aureus colony and in the E. coli colony, and the diameter of the inhibition zone in the S. aureus colony was 1.25 mm and in the E. coli colony 1.12 mm, respectively. Quantitative analysis of the samples’ antibacterial activity was further performed using the bacterial suspension assay.49 Figure 10(b) shows that after 24 h co-culture, the antibacterial rate of the AgNP-crosslinked dressings against S. aureus and E. coli reached 86.5±3.2%

and

75.8±2.7%,

respectively.

In

contrast,

the

HPMC-contained

and

chitosan-contained dressings presented low antibacterial rate against S. aureus (the HPMC-contained dressings were 27.3±3.2% and the chitosan-contained ones 28.1±2.7%), even promoted the growth of E. coli.


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Figure 10. Antibacterial activity of three kinds of hydrogel dressings against S. aureus and E. coli. (a) The disc diffusion method and (b) the bacterial suspension assay (n=3). Taken together, above results demonstrated that the HPMC-contained and chitosan-contained hydrogel dressings lacked antibacterial activity, and the incorporation of AgNPs into dressings


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endowed hydrogel dressings with good antibacterial activity. Although much literature has reported that chitosan possessed considerable antibacterial activity, chitosan did not exhibit such biological effect in our experiments. A possible explanation is that the compound of chitosan with PAM weakens the antibacterial activity of chitosan. It has well-known that the antibacterial activity of AgNPs is relative to the release of Ag0.50 In our samples, as shown in Figure 5, the AgNP-crosslinked hydrogels could release certain concentration of Ag, which surely inhibited the growth of bacteria. In Vivo Wound Management. To assess the wound healing ability of the AgNP-PAM/HPMC superporous hydrogel dressings, full-thickness skin wounds (1.0×1.0 cm in diameter) with the S. aureus infected model were established on the dorsal side of SD rats, as shown in Figure 11(a-b). Due to the existence of S. aureus in wounds, white pus formed 24 h later in all wounds (Arrows in Figure 11aii&b), suggesting that all wounds had been festering. After cleaned by physiological saline, the infected wounds, except the control group, were covered with different superporous hydrogel dressings (Figure 11a). The photographs (Figure 11b) showed the healing process of the infected wounds managed by different hydrogel dressings or exposed at air over time. Although all wounds have closed completely at day 14, statistical data (Figure 11c) showed that their closure rates were significant difference. The wounds covered by the AgNP-crosslinked dressings had faster healing rate than other wounds, and at the same time, the wounds had smoother surface during healing and smallest scarring morphology after 14 day healing than other wounds. In addition, the wounds exposed at air formed dried scab during healing and showed slowest closure rate in comparison with those covered by the hydrogel dressings.


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Figure 11. Photographs of an in vivo wound healing study. (a) The establishment and treatment of the S. aureus-infected wounds. (i) The established fresh wounds on the dorsal side of SD rats; (ii) a S. aureus-infected wound (24 h); (iii-v) different superporous hydrogel dressings were covered on the infected wounds. (b) The healing process of wounds treated by different methods over time (Dm denotes the wounds were established just now, D0 the S. aureus-infected wound (24 h), and D4-D16 the dressing-managed wounds). (c) The closure degree of wounds over time (n=3, * p < 0.05). Further histological analyses were carried out to the healed wounds (at day 14). The H&E staining (Figure 12a) showed that the wounds treated by the AgNP-crosslinked hydrogel dressings presented thickest newborn granulation tissue with smallest scar width. The Masson’s trichrome staining (Figure 12b) showed that thickest neo-epidermis and collagen deposition with some rete-pegs (Asterisks in Figure 12b) formed in the wounds treated by the AgNP-crosslinked dressings. In contrast, the thickness of neo-epidermis and collagen deposition in the wounds treated by other methods was significantly lower and no rete-pegs were observed. These results further demonstrated that the existence of AgNPs in dressings played a dominant role in


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accelerating wound healing. In addition, the wounds treated by the chitosan-contained and the HPMC-contained hydrogel dressings presented similar results, suggesting that chitosan did not indicate distinct biological effects in improving wound healing. A possible explanation is that chitosan has similarly weak antibacterial activity as HPMC and thereby cannot interfere with the infected wounds (See Figure 10). The wounds exposed at air presented lowest thickness of neo-epidermis and highest white colored empty spaces (The emergence of white colored empty spaces means the lack of collagen), further demonstrating that dry wound environment was not conducive to wound closure and healing. Furthermore, the Masson’s trichrome staining shows that all wounds presented capillary vessels (An important marker of scar tissue formation)51,52 in newborn granulation tissues. Presumably the newborn capillary vessels resulted from S. aureus, which recruited excessive inflammatory cells (e.g., neutrophils and macrophages)53 into wounds to express a great deal of vascular endothelial growth factor (VEGF).54 Nevertheless, Figure 12(b) shows that the wounds treated by the AgNP-crosslinked dressing had farther smaller numbers of capillary vessels, suggesting that inflammatory cells were significantly fewer than those in other wounds. Previously, some reports have claimed that AgNPs could facilitate the aesthetic wound healing 55,56.

However, there are no histological results to support such conclusion. Herein, by using the

S. aureus-infected wound model we definitely demonstrated that the existence of AgNPs in dressings could restrain scar tissue formation.


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Figure 12. Histological analyses of the 14 day-healing wounds. (a) The H&E-stained wound sections: (i) representative photomicrographs (Yellow line and blue dotted line indicate granulation tissue and scar width, respectively); (ii) newborn granulation tissue; (iii) scar width. (b) The Masson’s trichrome stained wound sections: (i) representative photomicrographs (arrows, triangles, asterisks, and prismatic indicate capillary vessels, epidermis, rete-pegs, and granulation tissues, respectively); (ii) thickness of neo-epidermis; (iii) the number of newborn capillary vessels. (n=3, * p < 0.05) CONCLUSIONS In summary, we have successfully prepared superporous AgNP-crosslinked PAM/HPMC semi-interpenetrating polymer network hydrogel dressings by combing the silver-ethylene interaction and the pore-making materials consisted of 3D printed porous templates and HPMC. Experiments demonstrated that by the aid of the Ag-MBAM interaction, dispersive AgNPs with diameter 1-15 nm could in situ form and crosslink in the hydrogel matrix. At the same time, the Ag-MBAM bonding also decreased the crosslinking of PAM network, thus increasing the swelling rate of the formed hydrogels. Furthermore, the AgNP-MBAM complexes also decreased the leakage of AgNPs from the as-prepared hydrogels. In vitro experiments demonstrated that such dose release of AgNPs from hydrogels was desirable because it balanced well the cytocompatibility and antibacterial activity of hydrogel dressings. The combination of 3D printed porous templates and HPMC as the pore-making materials was favorable to prepare the semi- interpenetrating polymer network hydrogels with superporous structure. The combination of two pore-making materials could tailor hydrogels into 91.4% porosity and the formed pores into open channels. The hydrogels with such superporous structure were endowed with rapid water uptake rate and 14 times dead-weight of uptake capacity. More


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interestingly, our experiments demonstrated that the regular large pores arisen from 3D printed templates could buffer the swelling of superporous hydrogel dressings during water uptake. Such feature of our hydrogel dressings was advantageous for wound management because it decreased the detachment risk of dressings from wounds. In vivo experiments demonstrate that in comparison with other dressings, the AgNP-crosslinked superporous hydrogel dressings presented faster wound healing rate and the healed wounds had smoother surface and smallest scarring morphology. Histological analyses showed that the AgNP-crosslinked hydrogel dressings could restrain scar tissue formation. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Tel/Fax: +86-28-85412848. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources This study was supported by the National Natural Science Foundation of China (Grant 31570977) and by the Innovation Huohua Project of Sichuan University (2019SCUH0018). Notes The authors declare no competing financial interest. ACKNOWLEDGMENT


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We deeply acknowledge the financial support provided by the National Natural Science Foundation of China (Grant 31570977) and by the Innovation Huohua Project of Sichuan University (2019SCUH0018). ABBREVIATIONS 3D, three dimensional; PAM, polyacrylamide; HPMC, hydroxypropyl methylcellulose; AgNPs, Ag nanoparticles; AM, acrylamide; MBAM, N,N’-methylene bisacrylamide; APS, ammonium persulfate; AR, analytical reagent; PLA, polylactic acid; DI water, deionized water; FDM, fused deposition modeling; FTIR, Fourier Transform Infrared; ICP-AES, inductive coupled plasma atomic emission spectrometry;

TEM, Transmission Electron Microscope; SEM, Scanning

Electron Microscopy; UV-vis DRS, ultraviolet-visible diffuse reflection spectroscopy; PBS, phosphate buffered saline; FDA, fluorescein diacetate; PI, propidium iodide; CLSM, confocal laser scanning microscopy; S. aureus, Staphylococcus aureus; E. coli, Escherichia coli. REFERENCES (1) Vowden, K.; Vowden, P. Wound Dressings: Principles and Practice. Surgery (Oxford) 2017, 35, 489-494. (2) Abdelrahman, T.; Newton, H. Wound Dressings: Principles and Practice. Surgery (Oxford) 2011, 29, 491-495. (3) Watson, N. F. S.; Hodgkin, W. Wound Dressings. Surgery (Oxford) 2005, 23, 52-55. (4) Hampton, S. Selecting Wound Dressings for Optimum Healing. Nurs. Times. 2015, 111(49-50): 20-3.


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Combination of the Silver-Ethylene Interaction and 3D Printing to

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