Acta Biomaterialia 10 (2014) 2482–2494

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Arginine-based polyester amide/polysaccharide hydrogels and their biological response Mingyu He a, Alicia Potuck b, Yi Zhang a, Chih-Chang Chu a,c,⇑ a

Department of Fiber Science and Apparel Design, Cornell University, Ithaca, NY 14853-4401, USA Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA c Department of Biomedical Engineering, Cornell University, Ithaca, NY 14853, USA b

a r t i c l e

i n f o

Article history: Received 22 October 2013 Received in revised form 30 January 2014 Accepted 3 February 2014 Available online 12 February 2014 Keywords: Arginine Glycidyl methacrylate chitosan Inflammation Nitric oxide Arginase

a b s t r a c t An advanced family of biodegradable cationic hybrid hydrogels was designed and fabricated from two precursors via a UV photocrosslinking in an aqueous medium: unsaturated arginine (Arg)-based functional poly(ester amide) (Arg-UPEA) and glycidyl methacrylate chitosan (GMA-chitosan). These Arg-UPEA/GMA-chitosan hybrid hydrogels were characterized in terms of their chemical structure, equilibrium swelling ratio (Qeq), compressive modulus, interior morphology and biodegradation properties. Lysozyme effectively accelerated the biodegradation of the hybrid hydrogels. The mixture of both precursors in an aqueous solution showed near non-cytotoxicity toward porcine aortic valve smooth muscle cells at total concentrations up to 6 mg ml1. The live/dead assay data showed that 3T3 fibroblasts were able to attach and grow on the hybrid hydrogel and pure GMA-chitosan hydrogel well. Arg-UPEA/ GMA-chitosan hybrid hydrogels activated both TNF-a and NO production by RAW 264.7 macrophages, and the arginase activity was also elevated. The integration of the biodegradable Arg-UPEA into the GMA-chitosan can provide advantages in terms of elevated and balanced NO production and arginase activity that free Arg supplement could not achieve. The hybrid hydrogels may have potential application as a wound healing accelerator. Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction Wound healing is closely related to the metabolism of arginine (Arg), particularly in the inflammation and proliferation phases [1,2]. In many experimental wound model studies, activated macrophages avidly consumed or depleted arginine from culture medium via the action of inducible nitric oxide synthase (iNOS) and arginase [3,4]. The process of Arg metabolism generates several essential nitrogen-containing compounds, including creatine, polyamines, agmatine and nitric oxide (NO) [5]. Arg is the sole substrate for NO synthesis in biological systems, as NO is synthesized from Arg by the activity of nitric oxide synthase (NOS). In wound healing, NO production has an antibacterial function and is also critical to wound collagen accumulation for restoring mechanical strength [6]. Moreover, through the action of

⇑ Corresponding author at: Department of Fiber Science and Apparel Design, Cornell University, Ithaca, NY 14853-4401, USA. Tel.: +1 6072551938; fax: +1 6072551903. E-mail address: [email protected] (C.-C. Chu). http://dx.doi.org/10.1016/j.actbio.2014.02.011 1742-7061/Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

arginase, Arg is converted to urea and ornithine, generating polyamines, including putrescine, spermine and spermidine [2,7]. Ornithine is a precursor for proline, which serves as the substrate for collagen synthesis, whereas polyamines are involved in cell proliferation [8]. Because of the Arg’s role in wound healing, free Arg supplement to patients has been suggested and reported. For example, free Arg therapeutic treatment was applied to diabetic ulcer wounds, but the outcomes were not greatly improved [9–15]. Continuous supplemental arginine infusion produced significant and sustained increases in NO production in wound fluid, but no significant difference in concentrations of ornithine, citrulline or proline was found [9]. The lack of significant wound healing improvement may be attributed to such sustained NO production that may inhibit arginase function, thereby limiting polyamines and ornithine synthesis, which are also crucial to wound healing [9–17]. Moreover, free Arg through oral administration is easily dissolved in plasma and distributes systematically, instead of concentrating locally around the wound site. Wu et al., Song et al. and Pang et al. [18–24] reported the incorporation of Arg into the design and synthesis of a new family of

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synthetic biodegradable amino acid-based poly(ester amide)s (AA-PEA) biomaterials. Functional AA-PEAs have recently been reported to be a viable family of biodegradable biomaterials that have both protein and non-protein characteristics, and hence are referred to as ‘‘pseudo-proteins’’ [25,26]. Wu et al. reported that those Arg-based PEAs have some unique biological properties, such as improving cell attachment and proliferation [18–21], easy penetration through cell membranes, and the capability of capturing and delivering DNA as non-viral gene transfection vectors at a much lower level of cytotoxicity than Lipofectamine 2000Ò and SuperfectÒ [22,23]. Among the Arg-PEA biomaterials reported, one particular series of cationic Arg-based PEAs has photocrosslinkable double bonds on its polymer backbone (Arg-UPEA) [18,20]. These functional Arg-UPEAs were used as co-precursors to fabricate photocrosslinked hybrid hydrogels with synthetic functional pluronic acid diacrylate (F127) with a significant improvement in cellular adhesion and proliferation [18]. Like polyethylene glycol, F127 co-precursor, however, is a relatively biologically inert biomaterial, and in its hybrid with Arg-UPEA, F127 did not contribute any biological function, and therefore did not have a synergistic effect with Arg-UPEA. In addition, the Arg-UPEA/F127 hybrid hydrogels were relatively mechanically weak, with a compression modulus ranging from 0.77 to 4.35 kPa. In the present study, a modified but biologically active polysaccharide from chitosan was used as a co-precursor to fabricate an advanced generation of Arg-UPEA-based hybrid hydrogels for improving both the mechanical properties and the potential biological benefits of the resulting hybrids. Chitosan is a linear polymer of N-acetyl-D-glucosamine and a deacetylated glucosamine, which could be used as a wound-healing accelerator in clinical and veterinary medicine [27,28]. Shibata et al. [29] observed that the chitin oligosaccharides could activate the macrophage production of TNF-a and interleukin-12. The stimulation relies on the acetylated units. The mechanism of chitosan-induced macrophage activation involves mannose receptor-mediated phagocytosis. During inflammation, the mannose receptor is highly regulated on local macrophages, increasing the possibility to interact with the suitable ligands such as chitosan. Chitosan signaling through the up-regulated mannose and other possible receptors is one possible mechanism for the enhancement of the arginase pathway [28]. Glycidyl methacrylate modified chitosan (GMA-chitosan) was very recently synthesized by a new improved method, which could also achieve a much higher degree of GMA substitution with high yields [30]. GMA-chitosan is water soluble and biocompatible and, owing to the presence of the pendant photo-reactive vinyl group, GMAchitosan can be photocrosslinked to form biodegradable hydrogels by itself (i.e. without a co-precursor) [30]. The present paper reports on the feasibility of integrating this newly developed GMA-chitosan polysaccharide with Arg-UPEA, their characterization and some preliminary in vitro biological properties with a view to pursuing future in vivo study. The resulting hybrids could have the merits of both biologically active polysaccharides (i.e. chitosan) and pseudo-proteins (i.e. Arg-based PEAs) biomaterials. Such an integration could lead to an advanced family of biodegradable and biologically active biomaterials for a variety of biomedical applications, such as a potential treatment option to accelerate wound healing, which is difficult using a traditional free Arg supplement (dietary or infusion). Arg-UPEA/ GMA-chitosan-based hybrid hydrogels may have potential as model biomaterials in the study of wound healing, because they are able to provide an Arg-rich environment in situ. In addition, the hydrophilicity and soft, flexible nature of Arg-UPEA-based hybrid hydrogels may make this family of advanced biomaterials suitable for the treatment of wound healing.

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2. Materials and methods 2.1. Materials L-Arginine (L-Arg), fumaryl chloride, ethylene glycol, 1,4butanediol were purchased from Alfa Aesar (Ward Hill, MA). p-Toluene sulfonic acid monohydrate (TsOHH2O), p-nitrophenol (J.T. Baker, Philipsburg, NJ) and triethylamine (Avantor Performance Materials, Center Valley, PA) were used without further purification. Solvents including toluene, isopropyl alcohol, N,N-dimethylacetamide and DMSO were purchased from VWR Scientific (West Chester, PA); ethyl acetate and acetone were purchased from Mallinckrodt (St. Louis, MO). All solvents were ACS grade and used without further purification. Chitosan (77% deacetylated) of molecular weight (MW) 150 kg mol1 and bovine serum albumin (BSA) of molecular weight 66 kg mol1, polyethylene glycol diacrylate (PEGDA, Mn  750) and a-isonitrosopropiophenone were purchased from Sigma Chemical Company (St. Louis, MO). GMA (97%), 4-(N,N-dimethylamino) pyridine (DMAP, 99%), 0.05 M buffer solutions (pH 3, pH 7.4, pH 10), thiazolyl blue tetrazolium bromide and MnCl24H2O and lysozyme (from chicken egg) were purchased from VWR Scientific (West Chester, PA). Irgacure 2959 was donated by Ciba Specialty Chemicals Corp.

2.2. Synthesis of Arg-UPEA Two Arg-UPEA were synthesized by the same procedures reported before [18,20]. Briefly, the preparation steps could be divided into three major steps: the preparation of TsOH salts of Arg alkylene diester monomer (I); the preparation of di-p-nitrophenyl ester of dicarboxylic acid monomer (II), and the polymer synthesis of Arg-UPEA via solution polycondensation of the two monomers from (I) and (II) above. The detailed procedures are given elsewhere [18,20]. The Mn and Mw of 2-UArg-2-S synthesized by this method are 12.93 and 14.01 kg mol1, and those of 2-UArg-4-S are 15.71 and 17.49 kg mol1 [18]. 2.3. Synthesis of GMA-chitosan The GMA-chitosan was synthesized by the procedure described in a prior study [30]. The degree of substitution (DS; the amount of methacrylate (MA) groups per 100 chitosan repeat unit) of GMAchitosan was 37, which was determined by 1H NMR spectroscopy. 2.4. Fabrication of Arg-UPEA/GMA-chitosan hybrid hydrogel Although almost all Arg-UPEA have water solubility at room temperature, the methylene chain length (x) in the diol part between the two adjacent ester groups is one crucial material parameter of Arg-UPEA that can largely influence the polymer solubility in water [18]. Only Arg-UPEA with x = 2 and 4 were used in the fabrication of Arg-UPEA/GMA-chitosan hybrid hydrogel, because the water solubility of Arg-UPEA with x > 4 is too low to form a water-soluble hydrogel precursor solution with some other water-soluble polymer precursors, such as the GMA-chitosan. In a typical Arg-UPEA/GMA-chitosan hybrid hydrogel fabrication process, 0.3 g GMA-chitosan (DS 37) and desired amounts of Arg-UPEA (2-UArg-4-S or 2-UArg-2-S, 43–150 mg) were added to a glass vial and dissolved in deionized water (6.0 ml) to form a clear homogeneous solution with light yellow color. Then, 10 mg Irgacure 2959 photo-initiator was added to the precursors’ solution and dissolved completely. In order to keep the structure integrity of the hybrid hydrogels, the weight feed ratio of Arg-UPEA to GMA-chitosan could not be higher than 33/67.

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Every 400 ll of this aqueous solution mixture was transferred onto a custom-made 20-well Teflon mold (11 mm in diameter, 6 mm deep) using a micropipette and then irradiated by long wavelength UV light (100 W, 365 nm, mercury spot lamp, Blak-rayÒ) at room temperature for 30 min until disk-shaped solid hydrogels were formed. The resultant hydrogels were immersed in deionized water for 16 h at room temperature to leach the residues and to reach the swelling equilibrium. The swollen Arg-UPEA/GMA-chitosan hydrogel samples were then dehydrated on a Teflon plate in ambient air at room temperature until the dry weight was constant for further studies, such as Fourier transform infrared spectroscopy (FTIR) and equilibrium swelling ratio. Pure GMA-chitosan hydrogels were fabricated from 6 wt.% GMA-chitosan water solution [30]. In further study (swelling test, morphology, compressive mechanical study, enzymatic degradation, TNF-a and NO production and arginase activity), pure GMA-chitosan hydrogels served as the control samples. Sol fractions of the hybrid hydrogel and pure GMA-chitosan were tested by a gravimetric assay. Sol fraction = 100  (Wd/Wi)  100, where Wi is the initial weight of the dried sample and Wd is the weight of the dried insoluble part of the sample after extraction with water. 2.5. FTIR and elemental analysis of Arg-UPEA/GMA-chitosan hybrid hydrogel Dehydrated Arg-UPEA/GMA-chitosan hybrid hydrogels were analyzed by FTIR, and elemental analysis. FTIR spectra of ArgUPEA/GMA-chitosan hybrid hydrogel samples were recorded on a PerkinElmer (Madison, WI) Nicolet Magna 560 FTIR spectrophotometer with Omnic software for data acquisition and analysis. Elemental analysis of Arg-UPEA and Arg-UPEA/GMA-chitosan hybrid hydrogels was performed with a Thermo Scientific ConFlo III elemental analyzer by Stable Isotope Laboratory of Cornell University. 2.6. Equilibrium swelling ratio (Qeq) under different pH environment The Qeq of Arg-UPEA/GMA-chitosan hybrid hydrogel and the pure GMA-chitosan hydrogel (as control) were performed at room temperature (25 °C) by immersing dehydrated hydrogels individually in glass vials containing 15 ml 0.05 M buffers (pH 3, 7.4, 10). After 16 h, the Arg-UPEA/GMA-chitosan hybrid hydrogels reached their swelling equilibrium. The swollen hydrogels were then removed, the excess surface water was wiped, and the hydrogels were weighed until a constant weight was obtained. The swelling ratios of the hydrogels were calculated from the swollen and dry weights of the hydrogels according to the following equation

Q eq ð%Þ ¼ ðW t  W 0 Þ=W 0  100 where Wt is the weight of the hydrogel at swelling equilibrium, and W0 is the initial dry weight of the hydrogel before immersion. The reproducibility of the swelling profiles of a hydrogel was determined in triplicate. 2.7. Interior morphology of 2-UArg-4-S/GMA-chitosan hybrid hydrogel Scanning electron microscopy (SEM) was employed to analyze the interior microstructure of two hybrid hydrogels with different Arg-UPEA to GMA-chitosan feed ratios: 2-UArg-4-S/GMA-chitosan12.5/87.5 and 2-UArg-4-S/GMA-chitosan-33/67 hybrid hydrogels. A pure GMA-chitosan hydrogel was examined for comparison. Individual hydrogels were soaked in deionized water at room temperature to reach their swelling equilibrium. Then, the hydrogels were transferred to liquid nitrogen immediately to freeze and

retain the swollen structure. The samples were subsequently freeze-dried for 72 h in a Labconco (Kansas City, MO) Freezone 2.5 Freeze drier under vacuum at 50 °C, and finally glued onto aluminum stubs and coated with gold for 30 s for SEM observation using a Leica Microsystems GmbH (Wetzlar, Germany) 440 instrument. 2.8. Compressive mechanical properties of Arg-UPEA/GMA-chitosan hybrid hydrogel The mechanical testing of the GMA-chitosan and Arg-UPEA/ GMA-chitosan hybrid hydrogel was performed on a DMA Q800 Dynamic Mechanical Analyzer (TA Instrument Inc., New Castle, DE) in ‘‘controlled force’’ compression mode. A pure GMA-chitosan hydrogel was tested for comparison. The compressive mechanical properties of Arg-UPEA/GMA-chitosan hybrid hydrogels and GMA-chitosan hydrogel in circular disk shape after reaching their equilibrium swelling in deionized water were measured at room temperature (25 °C). The hydrogels were mounted between the movable compression probe (diameter 15 mm) and the fluid cup without any liquid media. A compression force from 0.01 to 4 N at a rate of 0.5 N min1 was applied to the swollen hydrogel samples at room temperature until a fragment of the hydrogels was produced. TA Universal Analysis software was used for mechanical data analysis. The initial compressive modulus and compressive strain at break were used to examine the hydrogel mechanical property. The initial compressive modulus was calculated from the slope of the initial linear portion of the curve. For each type of hydrogel, five samples were used, and their mean value was calculated with a standard deviation. 2.9. Enzymatic degradation of GMA-chitosan hydrogel and 2-UArg-4-S/GMA-chitosan-33/67 hybrid hydrogels The enzymatic biodegradation of the circular disk-shaped GMAchitosan and 2-UArg-4-S/GMA-chitosan-33/67 hybrid hydrogel was evaluated by their weight loss at 37 °C in 15 ml lysozyme (1 mg ml1) in 0.05 M pH 7.4 phosphate buffered saline (PBS) over a period of 10 days. A 15 ml PBS of pH 7.4 served as the control. The weight of each dry GMA-chitosan or 2-UArg-4-S/GMAchitosan-33/67 hydrogel was measured before immersion. At various immersion intervals, three GMA-chitosan hydrogel and 2-UArg-4-S/GMA-chitosan hybrid hydrogel samples were removed from the immersion solution and dried under vacuum at room temperature to constant weight. The weight loss was calculated according to the following equation

% Weight loss ¼ ðW o  W t Þ=W o  100% where Wo is the average initial (t = 0) dry weight of hydrogel in the biodegradation test, and Wt is the dry weight of the hydrogel tested after incubation at time t. The mean value of experimental data was calculated as the weight loss at time t with a standard deviation. 2.10. Cytotoxicity of hydrogel precursors and hybrid hydrogels 2.10.1. Cell culture Porcine aortic valve smooth muscle cells (PAVSMC) were maintained in minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) and 1% each of penicillin–streptomycin. The cells were incubated in CO2 incubator at 37 °C with 5% CO2. After reaching confluence, the cells were detached from the flask with Trypsin–EDTA (Invitrogen, Carlsbad, CA). The cell suspension was centrifuged at 3000 rpm for 3 min and then re-suspended in the growth medium for further study. PAVSMC were used between passages 4 and 7.

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2.10.2. MTT assay of hydrogel precursors The cytotoxicity of Arg-UPEA and GMA-chitosan precursors was performed using the MTT assay. A 20 ml Arg-UPEA and GMAchitosan (DS 37) at 33/67 weight ratio sample solution (total 3 wt.%) was obtained by dissolving 400 mg GMA-chitosan and 200 mg Arg-UPEA in PBS (0.05 M, pH 7.4). A total of six different concentrations of such Arg-UPEA and GMA-chitosan mixture solutions (0.1, 0.3, 0.6, 1, 3 and 6 mg ml1) were prepared by diluting 3 wt.% Arg-UPEA and GMA-chitosan stock solution with MEM media and sterilized after filtration through a 0.45 lm filter. PAVSMC at an appropriate cell density (2000 cells well1) were seeded onto 96-well plates and incubated overnight. After 24 h, the cells were treated with 100 ll freshly prepared Arg-UPEA and GMA-chitosan mixture in MEM media at six different concentrations. The cells treated only with normal cell culture media were used as the control. After 24 h treatment and incubation, 10 ll of MTT solution (5 mg ml1 thiazolyl blue tetrazolium bromide in deionized water after filtration by 0.22 lm filter) was added to each well and incubated for another 4 h at 37 °C under a 5% CO2 atmosphere to allow the formation of formazan crystals. After that, the cell culture medium including the Arg-UPEA and GMA-chitosan mixture was carefully removed, and 100 ll of acidic isopropyl alcohol (contains 10% Triton-X 100 with 0.1 M HCl) was added to each well and gently shaken at room temperature for 1 h to ensure that the purple crystals had been completely dissolved. The optical density of the solution was measured at wavelengths of 570 nm and 690 nm (Spectramax plus 384, Molecular Devices, USA). The cell viability (%) was calculated according to the following equation

Viability ð%Þ ¼ ½OD570 ðsampleÞ  OD690 ðsampleÞ=½OD570 ðcontrolÞ  OD690 ðcontrolÞ  100% where the OD570 (control) represents the measurement from the wells treated with medium only, and the OD570 (sample) from the wells treated with various polymers. Eight samples were analyzed for each experiment. 2.11. Live/dead assay of hybrid hydrogels (3T3 fibroblast) For the evaluation of the cellular response on the crosslinked hydrogels, Arg-UPEA/GMA-chitosan hybrid hydrogels (prepared as described in Section 2.4) were cut to 10 mm  5 mm (diameter  thickness) disks and transferred to 24-well plate. Pure GMA-chitosan hydrogel samples were prepared in the same way and used as the control. All samples were exposed to UV radiation for 30 min for sterilization. The hydrogels were then equilibrated in complete DMEM culture media for 24 h. NIH 3T3 Fibroblasts with a cell density of 2  105 cells ml1 were seeded on each sample. After 72 h incubation in 5% CO2 under 37 °C, live and dead cells were stained with the Live/Dead Viability/Cytotoxicity Assay Kit (Invitrogen, Carlsbad, CA), according to manufacturer’s protocol. The cell proliferation and distribution were observed under a Zeiss Axio Imager. M1 fluorescence microscope (Carl Zeiss Microscopy, Thornwood, NY). 2.12. Macrophage culture stimulation and assay RAW 264.7 macrophages (provided by Dr. Cynthia Leifer, Cornell Veterinary College) were grown at 37 °C and 5% CO2 in DMEM supplemented with 10% FBS (with 1:100 penicillin–streptomycin, Hepes, L-glutamine and sodium pyruvate). Photocrosslinked PEGDA hydrogel (10 ml 20 wt.% aqueous precursor with 2 mg ml1 Irgacure 2959 photo-initiator, 10 min, 100 W, 365 nm wavelength, UV radiation), GMA-chitosan hydrogel, and 2-UArg-4-S/GMAchitosan-33/67 hybrid hydrogels were purified by soaking in

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deionized water for 24 h, then soaking in PBS for another 24 h and sterilized by UV-exposure for 30 min before macrophage cell culture. RAW 264.7 macrophages were plated on top of the hydrogel samples in the 24-well format. Then 1  105 cells well1 were cultured on various hydrogels for 24 h in 24-well tissue culture plates with a total well volume of 500 ll. After 24 h, cell supernatants were collected, diluted and assayed for tumor necrosis factoralpha (TNF-a), NO production and arginase activity study. 2.13. Determination of TNF-a release TNF-a production by macrophage was measured by ELISA, according to the manufacturer’s instructions (Biolegend, San Diego, CA). The RAW 264.7 macrophages supernatant samples were diluted 1:2 (RAW 264.7 macrophages blank control), 1:4 (PEGDA hydrogel control), 1:20 (2-UArg-4-S/GMA-chitosan-33/67 hybrid hydrogel and GMA-chitosan hydrogel), 1:100 (lipopolysaccharide (LPS), total well concentration 100 ng ml1). All hydrogel samples were disk shaped and had the same dimensions: 11 mm diameter 6 mm thick. One hundred microliters pre-titrated diluted capture antibody (1:200) in coating buffer was added to each well of the 96 well plate (8.4 g NaHCO3, 3.56 g Na2CO3, add deionized H2O to 1 L, pH adjusted to 9.5) and allowed to incubate overnight at 4 °C. The next day, the plate was washed four times with wash buffer (0.5% Tween 20 in 1 PBS) and 200 ll of assay diluent (1% BSA in 1 PBS) added to block non-specific binding. After 1 h, the plate was washed four times, and 100 ll of sample (prediluted in assay diluent) was added to wells and allowed to incubate at room temperature for 2 h. Afterwards, the plate was again washed four times, followed by addition of 100 ll detection antibody to the diluted supernatants, and incubated at room temperature for 1 h, followed by washing an additional four times. After the plate was blotted dry, 100 ll avidin-horse radish peroxidase was added to each well (diluted 1:1000 in assay diluent) and incubated at room temperature for 30 min. A final plate washing was completed, with a 2 min soak between each of the five washings. The plate was blotted dry, and 100 ll of TMB solution (Sigma Aldrich) was added to each well and allowed to incubate at room temperature for 20 min in the dark. Finally, 100 ll of 2 N H2SO4 solution was added to each well, and the plate was read at 450 nm using a BioRad plate reader, and TNF-a concentrations were calculated using the standard curve generated using the recombinant murine TNF-a standard with twofold serial dilutions from 7.8 to 500 pg ml1. LPS served as the positive control. Three replicate wells were tested, and the average TNF-a release (corrected for volume and expressed as total release from the specified cell count) and standard error of the mean were determined. 2.14. NO production The 2-UArg-4-S/GMA-chitosan-33/67 hybrid hydrogels, GMAchitosan hydrogel, LPS and blank control were individually incubated for 24 h with 1  105 RAW 264.7 macrophages at 37 °C and 5% CO2. The nitrate was measured by Griess reagent system (Promega, Madison, WI). Then, 50 ll cell free culture supernatant samples were taken from the cell culture with the hydrogel samples and control groups, mixed with 50 ll sulfanilamide solution (1% sulfanilamide in 5% phosphoric acid) and incubated for 5–10 min at room temperature, protected from light. Then, 50 ll NED solution (0.1% N-1-napthylethylenediamine dihydrochloride in water) was dispensed to all wells, and incubated at room temperature for 5–10 min, protected from light. The absorbance was measured within 30 min in a plate reader at 540 nm. Nitrite concentration was calculated with a sodium nitrite standard curve

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generated for each experiment. Three samples of each hydrogel type and control groups were tested, and the mean value was calculated with a standard deviation. 2.15. Arginase activity test Individual samples (10 mg chitosan powder, 5 mg 2-UArg-4-S, 10 mg dehydrated GMA-chitosan hydrogel, 10 mg dehydrated 2-UArg-4-S/GMA-chitosan-33/67 hybrid hydrogel) were added to a 24-well plate. To study the effect of degradation products of 2-UArg-4-S on macrophage arginase activity, 5 mg 2-UArg-4-S or 10 mg 2-UArg-4-S/GMA-chitosan-33/67 hybrid hydrogel were hydrolyzed by 150 ll 0.1 M sodium hydroxide solution for 12 h in 20 ml vials and then neutralized by 0.1 M hydrochloric acid to pH 7. The degradation product solutions were also added to the 24-well plate. Free Arg (3 mg) and resident RAW 264.7 macrophages without treatment were used as positive and negative controls. Next, 2  105 RAW 264.7 macrophages in the 24-well plate were incubated with the samples individually, with a total well volume of 500 ll for 24 h. Then, the macrophages were lysed by 100 ll of 0.1% Triton X-100 containing 50 lg pepstatin, 50 lg aprotinin and 50 lg antipain as protease inhibitors for 30 min at room temperature in each well. Arginase activity was measured as described elsewhere [31]. After the cells were lysed, the lysate was mixed with 100 ll of 10 mM MnCl2 in 50 mM Tris–HCl (pH 7.5) filtered through a 0.45 lm filter, and the enzyme was activated for 10 min at 55 °C in the glass vials. Then, 800 ll of an acid mixture containing H2SO4/H3PO4/H2O (1/3/7) and 50 ll 9 wt.% a-isonitrosopropiophenone (dissolved in 100% ethanol) was added to the cell lysate in each vial, and the mixture solutions were heated at 95 °C for 45 min. After being cooled to room temperature, the absorbance of the mixture at 540 nm was determined for urea content by UV–Vis spectrophotometry. A urea calibration curve was prepared with increasing amounts of urea between 1.5 and 30 lg, using the procedure described above. One unit (U) of arginase activity was defined as the enzyme activity that catalyzes the production of 1 lM of urea per minute under the condition of the assay. Arginase activity was expressed as mU 105 cells. For each type of sample, three samples were used, and the mean value was calculated with a standard deviation. 3. Results and discussion 3.1. Arg-UPEA/GMA-chitosan hybrid hydrogel The 2-UArg-4-S/GMA-chitosan-33/67 hybrid hydrogel was used as a representative hybrid hydrogel sample for characterization and cell assays because, at this 33/67 Arg-UPEA to GMA-chitosan precursors’ feed ratio, the resulting hybrids have sufficient ArgUPEA content to show the Arg-UPEA component’s influence and yet maintain good mechanical strength. The optical images of 2-UArg-4-S/GMA-chitosan (DS 37)-33/67 hybrid hydrogel before and after swelling in deionized water (Fig. 1A) show that this hybrid hydrogel has good transparency, with a slight yellow tint from the 2-UArg-4-S component. A representative chemical network structure of the photocrosslinked Arg-UPEA/GMA-chitosan hybrid hydrogel is shown in Fig. 1B. The sol fractions of 2-UArg-4-S/ GMA-chitosan-33/67 hybrid hydrogel and pure GMA-chitosan hydrogel are 17 ± 3% and 11 ± 2%, respectively. Arg-UPEA is a polar polymer and is soluble in polar solvents, including DMSO, DMF methanol and water [18]. Owing to the presence of double bonds in the repeating unit of the Arg-UPEA backbone, Arg-UPEA is able to be photocrosslinked with another unsaturated polymeric precursor, such as PEGDA or Pluronic-DA

in an aqueous medium, even though the Arg-UPEA alone is not able to be crosslinked by itself [18]. The GMA-chitosan co-precursor was synthesized by an improved procedure with DMAP as a catalyst [30]. In a previous study, GMA-chitosan (DS 37) showed a cationic nature (zeta potential +14.15 to +18.04 mV in aqueous solution at 0.5 mg ml1), good biocompatibility and biodegradability. Its aqueous precursor solution was able to be photocrosslinked to fabricate robust, transparent hydrogels. Using the GMA-chitosan as a co-precursor to fabricate Arg-UPEA/GMA-chitosan hybrid hydrogel provides an easy means to integrate the merits of both biologically active polysaccharides (i.e. chitosan) and pseudo-protein (i.e. Arg-UPEA) into a single polymeric biomaterial and hence enhance the performance and expand the utility beyond individual polysaccharide or pseudo-proteins. Fig. 2 shows the FTIR spectra of the 2-UArg-4-S and GMAchitosan precursors and their hybrid hydrogels. The carbonyl bands of the ester bonds at 1738–1742 cm1 are shown on 2-UArg-4-S [20,32]. The ester bond of GMA on GMA-chitosan is 1725 cm1. The characteristic absorption bands of the unsaturated C@C bond (C@C< bonds during the photo-crosslinking reaction [32,33]. The 2-UArg-4-S and 2-UArg-4-S/GMA-chitosan also show amide (I) and amide (II) bands at 1648–1650 cm1 and 1538–1542 cm1, respectively (Fig. 2A and B). The C–O–C structure of GMA-chitosan repeat unit absorption at 1150 cm1 is also shown in the 2-UArg-4-S/GMA-chitosan hybrid hydrogel spectra (Fig. 2B and C) which is absent at 2-UArg-4-S spectra (Fig. 2A). Carbon and nitrogen elemental analysis was also used to characterize the chemical composition of GMA-chitosan (DS 37) and Arg-UPEA (Supporting data, Table S1). The theoretical data were calculated with the assumption that 100% of the two precursors reacted together. Since the Arg-UPEA had much higher nitrogen contents (13.5–14%) than GMA-chitosan (6.4%), the nitrogen contents of the resulting Arg-UPEA/GMA-chitosan hybrid hydrogels synthesized at different ratios of the two precursors ranged from 6.4% to 7.8%, which was between the upper (from a pure Arg-UPEA) and the lower (from a pure GMA-chitosan) limits. These element data indicate that Arg-UPEA was covalently bonded to the GMA-chitosan in the hybrid hydrogels. For all ArgUPEA/GMA-chitosan hybrid hydrogels, the nitrogen contents acquired in the test increased with an increasing Arg-UPEA to GMA-chitosan feed ratio, and were lower than theoretically calculated data (Table S1). For example, 2-UArg-4-S/GMA-chitosan-33/ 67 had a higher nitrogen content (7.5%) than the same precursors, but at a lower Arg-UPEA to GMA-chitosan feed ratio of 20/80 (6.4%). The slightly higher theoretical nitrogen contents in the hybrid hydrogels than the corresponding experimental nitrogen contents indicates that there was a small portion of the Arg-UPEA precursor that was not covalently linked to the GMA-chitosan matrix. This water-soluble Arg-UPEA residue could be easily removed from the hybrid hydrogels during the purification process in deionized water. 3.2. Equilibrium swelling ratio (Qeq) of Arg-UPEA/GMA-chitosan hybrid hydrogels Fig. 3 shows the effect of the pH environment on the swelling behavior of Arg-UPEA/GMA-chitosan hybrid hydrogels at the same ionic strength (0.05 M). The swelling results demonstrated that the Arg-UPEA/GMA-chitosan hybrid hydrogels and pure GMA-chitosan hydrogel changed their ability to swell significantly when the environmental pH was altered, i.e. a lower swelling at a higher pH. Apparently, the swelling ratio decreased as the pH increased from 3 to 10 for all GMA-chitosan hydrogels and

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A

B

Fig. 1. Optical images of 2-UArg-4-S/GMA-chitosan-33/67 hybrid hydrogel: (A) 2-UArg-4-S/GMA-chitosan-33/67 hybrid hydrogels, before and after swelling in water: (B) representative chemical structure of Arg-UPEA/GMA-chitosan hybrid hydrogel.

Arg-UPEA/GMA-chitosan hybrid hydrogels. It is known that the swelling ratios of polymer network structures in various pH environments depend on the available free volume of the expanded polymer matrix, polymer chain relaxation and availability of ionizable functional groups, such as amine groups and guanidine groups. A reduction in swelling ratios from pH 3 to 10 in the Arg-UPEA/GMA-chitosan hybrid hydrogels is because the pKa of guanidine groups in the Arg-UPEA is 12.5 [22], and the pKa of the amine groups of GMA-chitosan is 6.5 [34]. Both the primary amine groups and guanidine groups of hydrogels tended to be ionized at an acid pH. The osmotic pressure inside the hydrogels decreases with pH increasing from 3 to 10, which led to swelling decreasing. The pH environment has the most significant influence on hybrid hydrogel swelling, while the contents of Arg-UPEA’s effect on swelling only appear at neutral pH. The Qeq of the hybrid hydrogels at 7.4 generally decreased with an increase in the Arg-UPEA contents. The reason is probably related to the difference in the molecular weight (MW) of these two polymers. The MW of

chitosan used in the synthesis of GMA-chitosan is 150 kg mol1, and after grafting the MA groups, the MW of GMA-chitosan molecules was 200 kg mol1. While the MW of Arg-UPEA synthesized by polycondensation in solution ranged from 12.93 to 15.71 kg mol1 [18]. This suggests that the polymer chains of GMA-chitosan precursor were much longer than those of Arg-UPEA precursor. Thus, an increase in the Arg-UPEA contents in the hybrid hydrogels would make the resulting hydrogel network structure tighter, i.e. more compact and less swelling. When at the acidic/basic environment, i.e. pH 3 or pH 10, the swelling difference caused by content of Arg-UPEA/GMA-chitosan or the type of Arg-UPEA can be neglected, because the electrostatic interaction between the ionizable groups of the hydrogels and the buffer solution plays the dominant role. The swelling data in Fig. 3A and B also indicate that the methylene chain length (x) in the Arg alkylene diester repeating unit of the Arg-UPEA precursor did not show much influence on the Qeq of hybrid hydrogels at all pH environments. The pH effect on the swelling property of other chitosan-based hybrid hydrogels

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Amide (I) and amide (II)

C=C

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Fig. 2. FTIR of (A) 2-UArg-4-S, (B) 2-UArg-4-S/GMA-chitosan-33/67 and (C) GMA-chitosan.

has also been reported [35]. In the Zhong et al. study of maleic-chitosan /PEGDA hydrogels, they also discovered pH-dependent swelling; but contrary to the findings of the current Arg-UPEA/GMA-chitosan hybrid system, the maleic-chitosan/PEGDA hybrid hydrogel achieved a higher swelling ratio in an alkaline pH than in an acidic condition [35]. This is because the ionizable groups in maleic-chitosan/PEGDA are mainly carboxyl group (pKa 1.8–2.4) that are ionized more easily (deprotonized) in an alkaline condition. The high water content and pH-responsive swelling of the hybrid hydrogel system can help better transport of cell signaling molecules and proteins in the milieu of the wound bed. 3.3. Interior morphology of 2-UArg-4-S/GMA-chitosan hybrid hydrogels The cross-sectional interior morphology of the 2-UArg-4-S/ GMA-chitosan hybrid hydrogels at two different precursor weight feed ratios (i.e. 12.5/87.5 and 33/67 of 2-UArg-4-S to GMAchitosan) were examined and are shown in Fig. 4. No obvious phase separation of the 2-UArg-4-S/GMA-chitosan hybrid hydrogel was observed, probably because of the good miscibility between the two components. Compared with a pure GMA-chitosan hydrogel (Fig. 4A), 2-UArg-4-S/GMA-chitosan-12.5/87.5 hybrid hydrogel (Fig. 4B) and 2-UArg-4-S/GMA-chitosan-33/67 hybrid hydrogel (Fig. 4C) had three-dimensional (3-D) porous network structures, and an increase in the 2-UArg-4-S to GMA-chitosan feed ratio led to a smaller pore size and compact network structure (Fig. 4B vs. Fig. 4C). For example, GMA-chitosan hydrogel and 2-UArg-4-S/ GMA-chitosan-12.5/87.5 hybrid hydrogel showed the most pores with a diameter of 20–40 lm, whereas 2-UArg-4-S/GMAchitosan-33/67 had relatively smaller 10–18 lm pores. This micro-morphological change was also reflected in the swelling data in which, as the 2-UArg-4-S to GMA-chitosan feed ratio increased, the Qeq of the hybrid hydrogel became lower at neutral pH (Fig. 3A). The 3-D microporous interior structure of a hydrogel along with its high swelling in an aqueous environment would

provide a better culture environment for cells than commercial two-dimensional tissue culture plates. 3.4. Compressive mechanical properties of Arg-UPEA/GMA-chitosan hybrid hydrogel The compressive moduli of the Arg-UPEA/GMA-chitosan hybrid hydrogels and the pure GMA-chitosan hydrogel are shown in Table S2 (Supporting data). Arg-UPEA/GMA-chitosan hybrid hydrogels showed compressive initial moduli similar to a pure GMAchitosan hydrogel, but smaller compressive strain at break than a pure GMA-chitosan hydrogel. Increasing the contents of Arg-UPEA slightly increased the rigidity of the hybrid hydrogels. The data in Table S2 also show that the molecular structure of Arg-UPEA (x value) did not significantly influence the compressive modulus and strain. For example, at a fixed feed weight ratio of Arg-UPEA to GMA-chitosan of 33/67, 2-UArg-2-S/GMA-chitosan and 2-UArg-4-S/GMA-chitosan hydrogels had the same compressive strain at break. The compressive moduli of these two hybrid hydrogels were also very close. These compressive modulus data of the 2-UArg-2-S/GMAchitosan hybrid hydrogels are significantly higher than the Arg-UPEA/F127-DA hybrid hydrogel system reported by Wu et al. [18]. This Arg-UPEA/GMA-chitosan hybrid hydrogel system offered mechanically stronger tissue engineering scaffolds than the Arg-UPEA/F127-DA hybrid hydrogel system reported earlier. The improved mechanical properties in the Arg-UPEA/GMA-chitosan hybrid hydrogel system may also be a better biomaterial choice for treating wounds. 3.5. Enzymatic biodegradation of 2-UArg-4-S/GMA-chitosan hybrid hydrogels The biodegradation behavior of 2-UArg-4-S/GMA-chitosan-33/ 67 hybrid hydrogels was evaluated in terms of their weight loss in both pure PBS buffer control and PBS lysozyme solution of pH

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Fig. 3. The influence of pH environment on the equilibrium swelling ratio Qeq of Arg-UPEA/GMA-chitosan hybrid hydrogels of different Arg-UPEA to GMA-chitosan feed ratio in an ionic strength 0.05 M aqueous buffer at room temperature: (A) 2-UArg-4-S/GMA-chitosan hybrid hydrogel; (B) 2-UArg-2-S/GMA-chitosan hybrid hydrogel.

7.4 at 37 °C over a period of 10 days. Fig. 5 shows that lysozyme accelerated the biodegradation in both the pure GMA-chitosan and Arg-UPEA/GMA-chitosan hybrids. And 2-UArg-4-S/GMAchitosan-33/67 hybrid hydrogel was degraded faster than a pure GMA-chitosan hydrogel in the presence of either 1 mg ml1 lysozyme or PBS buffer only. It is also noted that, most of the weight loss of the pure GMAchitosan hydrogels occurred during the first 4–6 days, and after that the weight loss rates of pure GMA-chitosan hydrogel in both PBS and lysozyme solution became near zero, i.e. no change in weight with time. The weight loss profiles of the Arg-UPEA/GMAchitosan hybrid hydrogels are quite different from the pure GMA-chitosan, faster at the late stage (after 4–6 days). Lysozyme is an enzyme usually present in plasma, serum, saliva and wound exudation fluid in vivo. The lysozyme in wound exuda-

tion fluid with inflammation could accelerate the degradation of the chitosan-derived hydrogels [36]. The biodegradation mechanism of enzyme-mediated polymer hydrogel is likely to be complex. Lysozyme can biodegrade many natural and synthetic polymers, including chitosan [37], dextran [38], poly-(HEMA) [39] and polyester [40]. The degradation of a pure GMA-chitosan hydrogel can be attributed to two reasons: (1) the ester bonds of MA group can be degraded by pure hydrolysis; and (2) lysozyme is able to cleave GMA-chitosan backbone structure at the b(1,4) linked glucosamine unit and the N-acetyl-D-glucosamine unit [37]. The observed accelerated degradation of a pure GMA-chitosan in the first 4 days in the presence of lysozyme compared with PBS is due to the cleavage of the chitosan backbone chain by the lysozyme. However, the weight loss data in Fig. 5 show no meaningful weight loss after the initial 4–6 days in either lysozyme or

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GMA-chitosan in PBS GMA-chitosan in 1mg/mL lysozyme PBS solution 2-UArg-4-S/GMA-chitosan-33/67 in PBS 2-UArg-4-S/GMA-chitosan-33/67 in 1mg/mL lysozyme PBS solution 60

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Incubation time (day) Fig. 5. In vitro enzymatic degradation of GMA-chitosan hydrogel and 2-UArg-4-S / GMA-chitosan-33/67 hybrid hydrogel at 37 °C, pH 7.4, 0.05 M PBS, containing lysozyme: solid symbol, degradation of hydrogels in the presence of 1 mg ml1 lysozyme; open symbol, degradation of hydrogel control samples in PBS.

hybrid hydrogel, both the diester bonds in the Arg-UPEA moiety and the ester bond in the MA groups of GMA-chitosan can be hydrolyzed, which would create more open space for lysozyme to approach the N-acetyl-D-glucosamine unit of the GMA-chitosan macromolecular chains for additional biodegradation. This may be one of the reasons for the hybrid hydrogels showing more continued weight loss after the initial 4–6 days than a pure GMAchitosan hydrogel, which showed very little weight loss after that initial period. Another possible reason for the faster degradation of 2-UArg-4-S/GMA-chitosan hybrid hydrogel than a pure GMAchitosan hydrogel is that the hybrid hydrogel combined two polymer compositions with different molecular weight and different degradable building blocks. The 2-UArg-4-S segment hydrolyzed faster than GMA-chitosan in terms of weight loss, because the Arg-UPEA has a much lower MW than GMA-chitosan, and hence would take a shorter time to be hydrolyzed, to an extent that the water-soluble degradation-product can be eliminated from the hydrogel network to create more open space for lysozyme to diffuse into the network for additional biodegradation. Fig. 4. SEM images at 500 showing the interior morphology of: (A) GMA-chitosan hydrogel; (B) 2-UArg-4-S/GMA-chitosan-12.5/87.5 hybrid hydrogel; (C) 2-UArg-4S/GMA-chitosan-33/67 hybrid hydrogel.

PBS control media. The possible reasons could be attributed to the unique chitosan backbone structure. Chitosan is the partially deacetylated derivative of chitin, and has two major units in its backbone: N-acetyl-D-glucosamine and D-glucosamine. In the current study, the chitosan used was 77% deacetylated. Since the Nacetyl-Dglucosamine unit is the one to be targeted by lysozyme, the weight loss of GMA-chitosan during the first 4–6 days in Fig. 5 may already reflect the complete or near complete fragmentation of the N-acetyl-D-glucosamine units in the GMA-chitosan hydrogels. The grafted MA could not be biodegraded by lysozyme, and thus, no meaningful weight loss of the pure GMA-chitosan hydrogel was observed beyond the first 4 days in lysozyme medium. In a study of enzyme-catalyzed biodegradation of chitosan, Hirano et al. [37] suggested that the accessibility of the enzyme to polysaccharide-based hydrogel is one key factor influencing the biodegradation rate. In the case of 2-UArg-4-S/GMA-chitosan

3.6. Cytotoxicity of 2-UArg-4-S/GMA-chitosan-33/67 precursors and hybrid hydrogel Fig. 6 shows PAVSMC MTT assay data after 24 h treatment in six different concentrations (0.1, 0.3, 0.6, 1, 3, 6 mg ml1) of the aqueous mixture of 2-UArg-4-S and GMA-chitosan precursors at the feed ratio of 33/67 in MEM media. PAVSMC cultured on the 24-well cell culture plate without 2-UArg-4-S and GMA-chitosan treatment were used as the blank control. The MTT data indicated no significant difference in cytotoxicity in all the concentrations of 2-UArg4-S and GMA-chitosan precursor mixed aqueous solution compared with a blank control. It is evident from the results that at least 85% of PAVSMC were viable when these cells were incubated with up to 6 mg ml1 2-UArg-4-S and GMA-chitosan aqueous mixture at a 33/67 mixed ratio for 24 h. These results indicate that the 2-UArg-4-S and GMA-chitosan mixed solutions up to 6 mg ml1 at 33/67 feed ratio had virtually no cytotoxicity to PAVSMC cells. Arg-UPEA was reported to be non-cytotoxic to rat vascular smooth muscle cells, Detroit 539 human fibroblast cells and bovine aortic endothelial cells [18,41]. Arg-UPEA also had a special func-

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as control (Fig. 7B). After 3 days culture, most of the 3T3 fibroblasts on the Arg-UPEA/GMA-chitosan hybrid hydrogels and pure GMAchitosan hydrogel were viable and healthy (stained in green). The combination of 2-UArg-4-S and GMA-chitosan moieties into a hybrid hydrogel could provide a cell-friendly environment that may make the Arg-UPEA/GMA-chitosan hybrid hydrogels suitable for cell seeding in various biomedical applications.

3.7. Macrophage-induced TNF-a and NO production

Fig. 6. PAVSMC viability test by MTT assay of 2-UArg-4-S/GMA-chitosan-33/67 mixed solution at 0.1, 0.3, 0.6, 1, 3, 6 mg ml1 total concentration: 24 h culture and culture medium without the polymers as the control.

A TNF-Alpha (pg/mL)

tion to improve the cell attachment, proliferation and cell viability in the Arg-UPEA/pluronic acid (F127) hybrid hydrogels, probably owing to its cationic property [18]. In the current study, the aqueous solution mixture of Arg-UPEA and a new chitosan-based derivative (GMA-chitosan) also showed good biocompatibility with PAVSMC. The lack of cytotoxicity of hybrid hydrogels was also demonstrated in the live–dead fibroblast assay. Fig. 7 shows the 3T3 fibroblast cell viability cultured on the 2-UArg-4-S/GMA-chitosan-33/ 67 hybrid hydrogel (Fig. 7A) with a pure GMA-chitosan hydrogel

The RAW 264.7 macrophages cultured on Arg-UPEA/GMAchitosan hybrid hydrogel and pure GMA-chitosan hydrogel released high levels of TNF-a (5293 and 5020 pg ml1, respectively) at 24 h, shown in Fig. 8A, compared with the level from LPS (1008 pg ml1). The Arg-UPEA composition of the hybrid hydrogel shows no significant influence on TNF-a production compared with the pure GMA-chitosan hydrogel, because cells cultured with Arg-UPEA have a low level of inflammation, i.e. inducing low TNF-a production, which is a characteristic of the amino acid–based polyester amides [25]. The blank control and the PEGDA hydrogel have only 25 pg ml1 and 39 pg ml1 TNF-a, respectively. The PEGDA hydrogel sample is used as a control to measure the inflammation response of macrophages, which may be induced by the physical stimuli when macrophages were cultured on a soft and hydrophilic polymer surface. TNF-α secretion 8000 7000 6000 5000 4000 3000 2000 1000 0

B Nitrate Concentration (μM)

NO production 8 7 6 5 4 3 2 1 0

Fig. 7. Fluorescence image (2.5) of 3T3 fibroblasts on (A) 2-UArg-4-S/GMAchitosan-33/67 hybrid hydrogel and (B) GMA-Chitosan hydrogel cultivated by 72 h live/dead assay (merged images: green, live cells; red, dead/unhealthy cells).

Fig. 8. TNF-a secretion and NO production from RAW 264.7 macrophages cultured on the 2-UArg-4-S/GMA-chitosan hydrogel and GMA-chitosan hydrogel for 24 h. RAW macrophage serves as the blank control: (A) TNF-a secretion; (B) NO production.

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Fig. 8B shows that Arg-UPEA/GMA-chitosan hybrid hydrogel and pure GMA-chitosan hydrogel have 8–12-fold the NO production (3.9 lM and 5.8 lM) of the blank control (0.47 lM), and also much higher than the LPS-treated macrophages (0.58 lM). It is noteworthy that the 2-UArg-4-S/GMA-chitosan-33/67 hybrid hydrogel stimulated a lower NO level, 2/3 of a pure GMAchitosan hydrogel. It is well-known that macrophages can respond to LPS by releasing inflammatory mediators, including TNF-a and IL-8 [42]. TNF-a has regulatory effects on phagocytic cells, including augmented adhesion of neutrophils to endothelial surfaces, enhanced production of oxidative radicals, increased degranulation, increased receptor expression and increased toxicity for parasitic infections [43]. It has also been reported that macrophage has 20–30% higher NO production within 12 h incubation with LPS stimulation [42,44]. Chitin/chitosan derivatives are able to up-regulated macrophage TNF-a, IL-1 and colony-stimulating factor production and induce immunologic adjuvant effects [45,46]. Also, the treatment of resident macrophage with chitosan increased the levels of NO production by 30% compared with a control at 0.1% w/v [28]. In the present study, GMA-chitosan-based hydrogel showed significantly higher TNF-a and NO production than the blank control group and even LPS-treated macrophage positive control. Macrophages appear to express the receptors for mannose and N-acetyl-D-glucosamine-glycoproteins, which mediate the uptake of glycoproteins into macrophages and hence activate the release of biological mediators [28,47]. Because GMA-chitosan-based hydrogel was highly swollen in the cell culture media (i.e. creating macroporous space), the macrophages seeded on the surface of the hydrogel were able to access the N-actetyl-D-glucosamine moiety of chitosan and be activated, and thus increased TNF-a and NO production. This phenomenon is similar to the TNF-a and NO data from the reported studies of water-soluble chitosan derivatives or chitosan/chitin particles [42,48,49]. Porporatto et al. reported that chitosan activated the NOS pathway and enhanced the arginase activity of macrophages compared with the untreated cells [28]. It is well known that the relationship of NOS and arginase metabolism pathways of Arg are reciprocal, and the activity of one or the other pathway could be altered by many factors, such as proteose peptone, LPS (which increases NOS activity and reduce arginase activity) and IL-4, IL-10 (having the opposite effect) [50]. The reason for Arg-UPEA/GMA-chitosan hybrid hydrogel having comparatively lower NO production than the pure GMA-chitosan hydrogel is probably that GMA-chitosan and Arg-UPEA components in the hybrid hydrogel increased the flux of Arg through arginase pathway more than NOS pathway. It was reported that the arginase I expression is elevated by the low molecular weight chitosan with slightly enhanced expression of iNOS. The arginase can decrease the pool of Arg in the cells and thus limit the production of NO [28]. 3.8. Arginase activity test The arginase activity assay was done by incubating RAW 264.7 macrophages with raw chitosan powder, 2-UArg-4-S, GMA-chitosan hydrogel, 2-UArg-4-S/GMA-chitosan-33/67 hybrid hydrogels, hydrolyzed 2-UArg-4-S and hydrolyzed 2-UArg-4-S/GMAchitosan-33/67 hybrid hydrogels (shown in Fig. 9). Arginase is one cytosolic enzyme, so the arginase activity was determined in the macrophages only after cell lysis [17]. Arg-containing materials, including Arg-UPEA and the Arg-UPEA/GMA-chitosan hybrid hydrogel, were able to improve the arginase activity over the blank control. The free Arg control had the highest arginase activity. Thus, arginase activity depends mainly on the availability of Arg to macrophages. Arg-UPEA/GMA-chitosan hybrid hydrogel showed

Fig. 9. Arginase activity of 2-UArg-4-S (5 mg), GMA-chitosan hydrogel (10 mg) and 2-UArg-4-S/GMA-chitosan-33/67 hybrid hydrogel (10 mg) in RAW 264.7 macrophages. Significant differences between Arg-UPEA/GMA-chitosan hybrid hydrogel and GMA-chitosan hydrogel are indicated (⁄P < 0.05; two-way t-test).

a significantly higher arginase activity than the pure GMA-chitosan hydrogel at equal weight. Through the arginase activity, the Arg contents in the Arg-based PEA are converted to ornithine and urea, while the degradation products of Arg-UPEA can be used more efficiently because the Arg contents are readily available. In the absence of the Arg-containing biomaterials to provide Arg, chitosan powder did not show the enhanced arginase activity. Macrophages cultured on GMA-chitosan hydrogel showed moderately elevated arginase activity. As shown in Fig. 8B, the NO production of the Arg-UPEA/GMAchitosan hybrid hydrogel is much higher than the control groups, but is lower than that of a pure GMA-chitosan hydrogel (33% reduction). These data suggest that GMA-chitosan can improve both pathways of Arg metabolism (arginase and NOS), but also has some influence to induce more Arg metabolism through the arginase pathway than the NOS pathway. Porporatto et al. reported that chitosan (1 kg mol1) was able to induce the NO production and enhance arginase activity [28]. Chitosan signaling through the up-regulated receptor (mannose receptor) could be used to explain the enhancement of the arginase pathway in chitosan-treated macrophages [28]. Unlike the raw chitosan solution (Mn 1 kg mol1 and 50 kg mol1) used in the Porporatto et al. study [28], the GMAchitosan macromolecule used in the current study has a larger MW (150 kg mol1). Nevertheless, the GMA-chitosan hydrogel structure is very hydrophilic and efficiently expanded in an aqueous environment that can create enough interactions between chitosan and macrophage to up-regulate the arginase metabolism pathway. For the chitosan powder sample in the present study, the interaction between the chitosan molecules and macrophages was not sufficient to change the arginase activity, owing to the poor solubility of chitosan in cell culture media. In an inflammatory milieu, Arg is the only amino acid whose concentration decreases over time [28,50]. The supplement of Arg-containing biomaterial such as Arg-UPEA could provide an extra source for ornithine and NO production. NOS and arginase pathways have opposing biological effects. The NO resulting from the NOS pathway is cytotoxic to microbes, parasites and tumors, and is mainly anti-proliferative. The ornithine and polyamines produced through the arginase pathways appear to create an environment that could favor cell growth and proliferation [50]. Simple supplemental arginine infusion produced significant and sustained

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increases in wound fluid NO production with no significant difference in concentrations of ornithine, citrulline or proline. Sustained NO production maybe inhibit arginase function, thereby limiting polyamines and ornithine synthesis, with consequent reductions in angiogenesis and granulation tissue formation, which are also crucial to wound healing [9,11]. Therefore, when applied to wound healing, the GMA-chitosan component in the hybrid hydrogel is expected to be able to activate the macrophage and help the Arg metabolism to achieve a better balance. While the Arg-UPEA component in the hybrid hydrogel is able to compensate the consumed Arg, the degradation process of Arg-UPEA also prevents the drawback brought by free Arg supplement. Future in vivo animal trials will be initiated to confirm the in vitro data observed. 4. Conclusion An advanced family of cationic biodegradable Arg-UPEA/GMAchitosan hybrid hydrogels was designed and fabricated in an aqueous medium via a long wavelength UV photocrosslinking of both Arg-UPEA and GMA-chitosan precursors. By varying the feed ratio of Arg-UPEA to GMA-chitosan precursors and the methylene group length (x) of the diol building block of Arg-UPEA, the swelling mechanical and morphological properties of this hybrid hydrogels could be controlled. The Arg-UPEA and GMA-chitosan precursors’ mixed aqueous solutions, and their hybrid hydrogels had no cytotoxicity and can effectively activate the RAW 264.7 macrophages. Both TNF-a and NO production by the macrophage were improved when it was cultured on the hybrid hydrogel. The arginase activity in macrophages cultured on the hybrid hydrogel was also elevated compared with the pure GMA-chitosan hydrogels and blank control. This Arg-UPEA/GMA-chitosan hybrid hydrogel family offers advantages in terms of high water content, 3-D microporous structure, biocompatibility, enzymatic biodegradability, and biological functionality of GMA-chitosan and Arg contents, which regulate the Arg metabolism in macrophages. This new hybrid hydrogel family has the potential to be used as the model biomaterial in terms of wound healing study and has promising applications as a wound healing accelerator. Acknowledgements The authors would like to thank the support of Vincent V.C. Woo Fellowship to Mingyu He, and the Rebecca Q. Morgan Foundation for its fellowship to Alicia Potuck and partial funding support to this study. The authors also wish to thank Dr. Leifer, who provided mouse Macrophage 264.7 macrophages and cell culture facility. Appendix A. Figures with essential color discrimination Certain figures in this article, particularly Figs. 1, 3 and 5–9, are difficult to interpret in black and white. The full color images can be found in the on-line version, at http://dx.doi.org/10.1016/ j.actbio.2014.02.011. Appendix B. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actbio.2014. 02.011. References [1] Young A, McNaught CE. The physiology of wound healing. Surgery 2011;29(10):475–9.

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