Highly Stretchable, Adhesive, Biocompatible, and Antibacterial Hydrogel Dressings for Wound Healing

Abstract Treatment of wounds in special areas is challenging due to inevitable movements and difficult fixation. Common cotton gauze suffers from incomplete joint surface coverage, confinement of joint movement, lack of antibacterial function, and frequent replacements. Hydrogels have been considered as good candidates for wound dressing because of their good flexibility and biocompatibility. Nevertheless, the adhesive, mechanical, and antibacterial properties of conventional hydrogels are not satisfactory. Herein, cationic polyelectrolyte brushes grafted from bacterial cellulose (BC) nanofibers are introduced into polydopamine/polyacrylamide hydrogels. The 1D polymer brushes have rigid BC backbones to enhance mechanical property of hydrogels, realizing high tensile strength (21–51 kPa), large tensile strain (899–1047%), and ideal compressive property. Positively charged quaternary ammonium groups of tethered polymer brushes provide long‐lasting antibacterial property to hydrogels and promote crawling and proliferation of negatively charged epidermis cells. Moreover, the hydrogels are rich in catechol groups and capable of adhering to various surfaces, meeting adhesive demand of large movement for special areas. With the above merits, the hydrogels demonstrate less inflammatory response and faster healing speed for in vivo wound healing on rats. Therefore, the multifunctional hydrogels show stable covering, little displacement, long‐lasting antibacteria, and fast wound healing, demonstrating promise in wound dressing.

4 accordance with laboratory rules and guidelines. All rat experiments were approved by the Animal Ethics Committee of South China Agricultural University. Histological analysis was tested by Wuhan Servicebio Technology Co.,Ltd (Wuhan, China).

Zeta potential measurements
Before analyzing the zeta potential of hydrogels, the lyophilized hydrogels were milled into powder form. [S1] The hydrogel powders were dispersed in water for 10 minutes, and filtered through filter screen to remove large size hydrogel. Finally, the zeta potential of filtrate was measured at 25 ℃ by using the Zetasizer Nano-ZS PN3702 system (Malvern Instruments, Worcestershire, England).

Self-healing tests
The hydrogels were cut in half along the middle with a clean blade, and then its cut surfaces were rejoined. The self-healing behaviors of the hydrogels were confirmed through tensile performance after 30 minutes and 2 hours.

Compressive tests
Compressive tests were performed on a universal mechanical testing machine (WD-5A, Guangzhou Experimental Instrument Factory, China) at the compressing speed of 5 mm min -1 . The tested 10‰BCD/PDA/PAM hydrogel was cylindrical of 12 mm in height and 16 mm in diameter. Compression resilience of the hydrogel was characterized by 5 cyclic loading-unloading compressive tests to a compressive strain of 60%. Every subsequent cycle was conducted after complete recovery of the hydrogel. Compressive strength of the hydrogel was determined by measuring the ratio of the maximum force 5 required to crush the hydrogel to the contact area.

Tensile tests
BCD/PDA/PAM hydrogels with different BCD contents were molded into dumbbell specimens (12 mm in length, 5 mm in width, and 1 mm in thickness) for tensile test.
Tensile property measurements of hydrogels were performed using a universal mechanical testing machine (WD-5A, Guangzhou Experimental Instrument Factory, China) with a 100 N load cell and an extension speed of 15 mm min −1 . The cyclic tensile test of 10‰BCD/PDA/PAM hydrogel was characterized by 4 cyclic loading-unloading tensile tests to a tensile strain of 400%.

Adhesive tests
The adhesion strengths of BCD/PDA/PAM hydrogels were evaluated by lap shear tests using the universal mechanical testing machine (WD-5A, Guangzhou Experimental Instrument Factory, China) equipped with a 100 N load cell and a 2 mm min -1 loading rate. [S2] Porcine skin which was chosen to mimic the adhesion on human tissue was cut into 20 mm×10 mm and glued to the glass. The tested hydrogels were cut to 10 mm× 10 mm and sandwiched between two pieces of porcine skin for lap shear tests. The adhesion tests were immediately conducted once the hydrogels were quickly attached on the porcine skin. The adhesion strengths were calculated by the maximum load divided by the initial bonded area. Cyclic adhesion tests of BCD/PDA/PAM hydrogels were evaluated. The tests were repeated with three parallel specimens.

Antibacterial property of BCD/PDA/PAM hydrogels
Bacterial growth curves 6 To investigate the antibacterial activity of BCD/PDA/PAM hydrogels, S. aureus and E. coli were used for the tests. [S3] All the hydrogels were taken under UV exposure for 24 hours before bacterial culture. The original bacterium fluid of S. aureus and E. coli was inoculated in Luria-Bertani (LB) growth medium for 24 hours at 37 ℃ with constant shaking. The typical colony was taken out by an inoculation ring to 50 mL of nutrient broth at 37 ℃ for 12 hours. The concentration of bacteria was 10 7 colonies forming units (CFU)/mL. The resulting S. aureus and E. coli suspensions were stored in a sterile medical bottle. The solution was further diluted 100 times to 10 5 CFU/mL. S. aureus or E. coli suspensions (4 mL) were added to the small glass bottles containing BCD/PDA/PAM hydrogels (size: 15 mm ×15 mm×1 mm). In this assay, a bacterial solution without any treatment was used as the control group. Each sample was repeated for three times. The bottles were incubated at 37 ℃ for 24-96 hours with 180 rpm.
During the incubation, the optical density (OD 600 ) value of the above bacterial solutions was measured at different times. Meanwhile, the turbidity change of bacterial liquids at different times was observed.

CFU test and live/dead bacteria assay
Briefly, after incubation at 37 ℃ for 24 hours with 180 rpm, 15 μL of the bacterial solution was uniformly spread on an agar plate. After incubating on agar plate at 37 °C for another 24 hours, the bacterial colony forming units were photographed. Meanwhile, the hydrogels were taken out and washed with PBS for 2-3 times. Subsequently, the hydrogels were soaked in the solution containing 1.5 μL SYTO9 dye, 1.5 μL PI dye and 7 Finally, the hydrogels were washed three times with PBS and then visualized using a fluorescence microscope (Olympus IX73, Japan).

In vitro cytotoxicity
The mouse bone marrow-derived mesenchymal stem cells (BMSCs, SCSP-405) were resuspended in a complete culture medium consisting of mesenchymal stem cell medium (MSCM) with 5% w/v fetal bovine serum (FBS), 0.5 mL mesenchymal stem cell growth additive and 0.5 mL penicillin/streptomycin solution, and then were cultured in a carbon dioxide cell incubator (37 °C, 5% CO2). The complete medium was replaced every two days to achieve the purpose of cell proliferation. Cellular cytotoxicity and proliferation were measured using cell counting kit-8 reagent (CCK-8, Domino, Japan). Specifically, the hydrogels were cut to an appropriate size and placed in the bottom of a 96-well plate. After soaking in a mixture of absolute ethanol and PBS (v/v = 75%) for 12 hours for sterilization, the hydrogels were washed with PBS for 5 times to remove ethanol. Subsequently, BMSCs were seeded into hydrogels as a cell density of 2×10 4 /well, placed in carbon dioxide cell incubator (37 °C, 5% CO2) and continued to culture until the cells returned to the normal adherent state. 10% CCK-8 reagent was added to the corresponding wells at different time points (Day 1, 3, and 5), and the incubation was continued for 2 hours. Cells seeded into culture without hydrogel served as the control group. The obtained supernatants were transferred to another 96-well plate. Then, the optical density at 450 nm (OD 450 ) was measured by a microplate reader, and the cell viability was calculated.
Effect of BCD/PDA/PAM hydrogels on the cell viability was observed by 8 fluorescence staining. Briefly, BMSCs were seeded onto the hydrogels and cultured for 3 days. Subsequently, all cell substrates were fixed on ice with 4% paraformaldehyde for 15 minutes, followed by washing three times with PBS. Then permeabilization of the cells was done with 0.1% Triton-PBS for 10 minutes, followed by washing three times with PBS. After that, DAPI (Beyotime, China) was used to visualize the cell nucleus, while Actin-Tracker Green (Beyotime, China) was utilized to show cytoskeleton. After staining in the dark, the fluorescence images were taken using a fluorescence microscope (Olympus IX73, Japan). The cells were seeded at a density of 1×10 4 cells/cm 2 . Briefly, 500 μL of cell suspension was gently added to the hydrogels along the edge of culture plate. After 24-hour culture, the above cells were fixed with 2.5% glutaraldehyde aqueous solution for 15 minutes, and washed with PBS three times to remove residual glutaraldehyde. Subsequently, the cell morphology and attachment were observed by a fluorescence microscope (Olympus IX73, Japan).

In vivo wound healing
Briefly, 6 male Sprague Dawley (SD) rats weighing 200-240 g were used. After being 9 anesthetized with pentobarbital (2 wt.%, 1.8 mL kg -1 ), the dorsal area of rats was totally depilated and 3 full-thickness circular wounds (10 mm in diameter) were created on the upper back of each rat by a disposable 10 mm skin biopsy punch. [S4,S5] On each rat, a wound without hydrogel dressing treatment was used as control. After dropping 10 μL of S. aureus (10 5 CFU/mL) into the wound, a tailored hydrogel was placed, and a 3M Tegaderm TM (Neuss, Germany) was covered. Then, the healing of the wound was observed and photographed every day. During the observation period, a rat was executed on day 5, 10, and 15, respectively. The wound site from the executed rat was harvested in full layer with scissors in conjunction with surrounding tissues, and then soaked in 10% formalin solution. Paraffin embedding was performed as soon as possible for subsequent histological analysis.