Polysaccharide-Based Networks from Homogeneous Chitosan

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Polysaccharide-based networks from homogeneous chitosantripolyphosphate hydrogels: synthesis and characterization Pasquale Sacco, Massimiliano Borgogna, Andrea Travan, Eleonora Marsich, Sergio Paoletti, Fioretta Asaro, Mario Grassi, and Ivan Donati Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/bm500909n • Publication Date (Web): 18 Aug 2014 Downloaded from http://pubs.acs.org on August 23, 2014

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Polysaccharide-based networks from homogeneous

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chitosan-tripolyphosphate hydrogels: synthesis and

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characterization

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Pasquale Sacco 1, Massimiliano Borgogna1, Andrea Travan1, Eleonora Marsich2,

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Sergio Paoletti1, Fioretta Asaro 3, Mario Grassi4 and Ivan Donati1*.

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Department of Life Sciences, University of Trieste, Via Licio Giorgieri 5, I-34127 Trieste, Italy

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Department of Medical, Surgical and Health Sciences, University of Trieste, Piazza dell'Ospitale

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1, I-34129 Trieste, Italy 3

Department of Chemical and Pharmaceutical Sciences, University of Trieste, Via Licio Giorgieri

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1, I-34127 Trieste, Italy 4

Department of Engineering and Architecture, University of Trieste, Via Alfonso Valerio, 6/A

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I-34127 Trieste, Italy

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* Corresponding author

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Phone: +39-040-5588733

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Email: [email protected]

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Abstract

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Polysaccharide networks, in the form of hydrogels and dried membranes based on chitosan and on

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the cross-linker tripolyphosphate (TPP) were developed using a novel approach. TPP was

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incorporated into chitosan by slow diffusion to favor a controlled gelation. By varying chitosan,

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TPP and NaCl concentration, transition from inhomogeneous to homogeneous systems was

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achieved. Rheology and uniaxial compression tests enabled to identify the best performing hydrogel

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composition with respect to mechanical properties. FTIR,

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methods were used to investigate the interaction chitosan-TPP, the kinetics of phosphates diffusion

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during the dialysis and the amount of TPP in the hydrogel. A freeze-drying procedure enabled the

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preparation of soft pliable membranes. The lactate dehydrogenase assay demonstrated the

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biocompatibility of the membranes towards fibroblasts. Overall, we devised a novel approach to

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prepare homogeneous macroscopic chitosan/TPP-based biomaterials with tunable mechanical

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properties and good biocompatibility that show good potential as novel polysaccharide derivatives.

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P NMR and spectrophotometric

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Keywords. Chitosan, tripolyphosphate, ionotropic gelation, slow diffusion, hydrogel, membrane. ACS Paragon Plus Environment

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1. Introduction

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Tridimensional matrices play a pivotal role in the biomedical field, especially in tissue

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engineering, in which they are developed to support cells and to promote their differentiation and

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proliferation toward the formation of new tissues. 1 According to the definition given by Peppas,

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hydrogels are water-swollen, cross- linked polymeric structures containing either covalent bonds

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produced by the reaction of one or more co-monomers or physical cross-links.2

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Various polymers have been proposed for the development of hydrogels for biomedical use.

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Among them, natural polymers offer the considerable advantage of resembling biological

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macromolecules, both from chemical and physical point of view, and then well recognized by cell

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surface receptors influencing processes like adhesion, spreading and proliferation.3,4 Moreover, due

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to their similarity to the extracellular matrix, natural polymers may also avoid the stimulation of

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chronic inflammation or immunological reactions and toxicity, often detected when synthetic

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polymers are used. Among the vast class of biopolymers, chitosan, a polysaccharide consisting of β-

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1→4 linked 2-amino-2-deoxy-glucopyranose (GlcN) and 2-acetamido-2-deoxy-β-D-glucopyranose

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(GlcNAc) residues in variable relative amounts, shows very interesting features. It is produced

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commercially on a large scale by alkaline N-deacetylation of chitin isolated from the exoskeleton of

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crustaceans.5 It is widely employed in biomedical field owing to its biocompatibility,

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biodegradability, lack of pathological inflammatory response and antibacterial properties. 6-9

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Many chitosan-based matrices reported in the literature and proposed for biomedical

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applications are fabricated by three-dimensional hydrogels starting from chitosan solutions where

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the use of specific cross- linkers to form covalent or ionic bonds is a mandatory step.10-13 In addition,

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chitosan may form hydrogels without any cross- linker only by changing the pH or using

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hydroalcoholic media.14,15 Chitosan hydrogels can be further processed by employing several

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technologies (i.e. freeze-drying and air drying) in order to obtain novel matrices as well as scaffolds

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and membranes.16 The properties of cross-linked hydrogels depend mainly on their cross- linking

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density, i.e. the ratio of moles of cross- linker to moles of polymer repeating units. Moreo ver, a ACS Paragon Plus Environment

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critical number of cross-links per chain is required to allow the formation of a uniform polymeric

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network throughout the hydrogel defined as “wall- to-wall” system. The most common cross- linkers

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used to promote a covalent reticulation of chitosan are dialdehydes such as glyoxal and

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glutaraldehyde.17,18 The use of genipin represents an alternative solution.19,20 Nevertheless, most of

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the cross- linkers used to perform covalent cross- linking result toxic and then incompatible with

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biological environment.21 In order to overcome this drawback, ionic cross- linkers can be exploited

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because of their higher biocompatibility and milder conditions of use. Moreover, at variance with

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some covalent cross-linkers, the use of catalysts can be avoided.21

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Tripolyphosphate (TPP) is an ionic cross- linker already used to reticulate chitosan to obtain

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fibers, nanoparticles and micro/nano gels.22-25 Nevertheless, when an anionic tripolyphosphate

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solution is mixed with chitosan, TPP quickly interacts with the chitosan amino groups leading to the

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formation of inhomogeneous systems, as well defined nanoparticles or not-controlled aggregates

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depending on both chitosan and TPP concentration. 23,26 Inhomogeneity leads to hydrogels with

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uneven structure and marked anisotropy of the mechanical properties at the macroscopic scale, thus

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limiting their potential and final application.

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To the best of the authors’ knowledge, a process allowing the formation of homogeneous

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macroscopic chitosan-TPP hydrogels is still lacking. To this end, the aim of the present work is (i)

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to develop a novel slow diffusion-based technique to prepare homogeneous bulk chitosan-TPP

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hydrogels, (ii) to investigate their physical-chemical and mechanical properties, (iii) to prepare

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prototypes of membranes for biomedical applicatio ns by a freeze-dried technology16 and (iv) to

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evaluate the biocompatibility of the polysaccharide networks on eukaryotic cells.

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2. Materials and methods

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2.1. Materials. Highly deacetylated chitosan (residual acetylation degree approximately 16% as

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determined by means of 1 H-NMR), was purchased from Sigma-Aldrich (Chemical Co. USA) and

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purified by precipitation with isopropanol, followed by a d ialysis against deionized water.27 The ACS Paragon Plus Environment

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relative molar mass (“molecular weight”, MW) of chitosan determined by intrinsic viscosity measurements was found to be around 690 000.27 Pentasodium tripolyphosphate (TPP) (MW 367.86 g mol-1 ; technical grade 85% wt), sodium chloride (NaCl), glycerol (ReagentPlus® ≥ 99.0%) and Phosphate Buffered Saline PBS (0.01 M phosphate buffer, 0.0027 M KCl and 0.137 M NaCl) were purchased from Sigma-Aldrich (Chemical Co. USA). Ammonium vanadate and ammonium molybdate were purchased from Honeywell Specialty Chemicals Seelze GmbH (Germany) and Carlo Erba (Italy), respectively. LDH (lactate dehydrogenase)-based TOX-7 kit was purchased from Sigma Aldrich (Chemical Co. USA). All other chemicals and reagents were of the highest purity grade commercially available. 2.2. Chitosan-TPP hydrogel preparation. Chitosan- TPP (CH-TPP) hydrogels were fabricated by exploiting an ion diffusion technique (Figure 1). Chitosan solution (3.5% w/v) was prepared as a stock solution by dissolving chitosan powder (0.35 g) in 10 mL of 0.2 M acetic acid solution and diluted to reach the desired concentration. Glycerol (5% v/v) was used as plasticizer. Chitosan solutions were casted in a mold (diameter = 22 mm, height = 2.5 mm) closed by two dialysis membranes (average flat width 33 mm, Sigma Aldrich, Chemical Co. USA) and fixed by double circular stainless iron rings. The system was hermetically sealed and immersed into a TPP-NaClglycerol solution (gelling bath). The concentrations of both TPP and NaCl in the gelling bath have been varied over the range 1-3% w/v and 0-500 mM, respectively, in order to obtain a broad range of formulations. Conversely, glycerol was kept constant both for the inner chitosan solution and the outer TPP-NaCl solution for all experiments. Ion diffusion proceeded for 24 h under moderate stirring at room temperature. 2.3. Membrane preparation. CH- TPP hydrogels were washed twice at the end of dialysis (1 h for each washing) with 150 mM NaCl solution first and finally with deionized water in order to remove all residual traces of unbound tripolyphosphate. Hydrogels were thereafter cooled by immersion in a liquid cryostat (circulating bath 28L, VWR, Radnor, PA, USA). Ethylene Glycol in water (3:1) was


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used as refrigerant fluid. Temperature was decreased from 20 to -20 °C by 5 °C steps with 20 minutes intervals. Samples were then freeze-dried for 24 h. 2.4. Morphological analyses. Morphological analyses of hydrogels and membranes were performed by Scanning Electron Microscopy (Quanta250 SEM, FEI, Oregon, USA). In environmental conditions, hydrated specimens were mounted on aluminium stubs covered with double-sided conductive carbon adhesive tape. In the case of cross-section evaluation, samples were cut in parallel to the cylinder axis and then prepared as described above. The samples were then analysed in secondary electron detection mode. The working distance was adjusted in order to obtain the suitable magnification. Freeze-dried membranes were sectioned and directly visualized after sputter-coating with an ultrathin layer of gold and analysed as to both material surface and cross sections. The accelerating voltage was varied from 20 to 30 kV. 2.5. Mechanical spectroscopy. Rheological characterization of cylindrical chitosan hydrogels was performed by means of a controlled stress rheometer Haake Rheo-Stress RS150 operating at 25 °C using, as measuring device, a shagreened plate apparatus (HPP20 profiliert: diameter = 20 mm). To avoid water evaporation from the hydrogel, the measurements were led in a water-saturated environment formed by using a glass bell (solvent trap) containing a wet cloth. In addition, to prevent both wall-slippage28 and excessive gel squeezing (which could reflect in the alteration of polymeric network properties), the gap between plates was adjusted, for each sample, by executing a series of short stress sweep tests (f = 1 Hz; stress range 1 - 5 Pa; maximum deformation < 0.1%) characterized by a reducing gap. 29 The selected gap was that maximizing the value of the elastic modulus G' (used gaps ranged between 2.5 and 2.0 mm). For each hydrogel, the linear viscoelastic range was determined by means of a stress sweep test consisting in measuring elastic (G') and viscous (G'') moduli variation with increasing shear stress (1 Pa < τ < 103 Pa) at a frequency ν = 1 Hz (hence with ω = 2πν = 6.28 rad s-1 ). Mechanical spectrum of the hydrogel was determined by measuring the dependence of the elastic (G') and viscous (G'') moduli from the pulsation ω at


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constant shear stress τ = 3 Pa (well within the linear viscoelastic range which, for all samples, spans up to at least 30 Pa). 2.6. Uniaxial compression. Compressive properties of hydrogels were evaluated with a universal testing machine (Mecmesin Multitest 2.5- i) equipped with a 100 N load cell in controlled conditions (T = 25 °C, relative humidity = 50%). A compression speed of 6 mm min-1 was used. Toughness was calculated by measuring the area under the stress-strain curve at 70% of strain. All measurements were performed in triplicate. An unpaired Student's t-test was used to determine statistically significant differences for the toughness calculation. 2.7. Attenuated total reflectance fourie r transform infrared spectroscopy (FTIR-ATR). Fourier transform infrared spectra of chitosan, CH-TPP membrane (see membranes preparation, section 2.3.) and TPP powder were obtained using Spectrometer Nicolet 6700 (Thermo Electron Corporation, Madison WI, USA) with DTGS KBr detector. The following setup was used throughout the measurements: number of sample scans 16, resolution 6 cm-1 from 500 to 2 000 cm1

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2.8.

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P-nuclear magnetic resonance (31 P-NMR). All 31 P NMR spectra were recorded, at 25 °C,

on a JEOL Eclipse 400 (9.4 T) NMR spectrometer operating at 399.78 MHz for 1 H and 161.83 MHz for

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P. 512 scans were accumulated with a spectral width of 5.66 kHz over 8192 complex

data points. The employed pulse width was 45° and the total recycle time 2.5 s. The data were multiplied by a decaying exponential function (broadening factor 0.5 Hz) and zero filled twice prior to Fourier transform (FT). A coaxial insert filled with a solution of Triphenylphosphine (PPh3 ) in CDCl3 containing Tetramethylsilane (TMS), was employed for lock and reference purposes. The 31 P chemical shifts are referred to H3 PO 4 85%, considering it resonating at 40.5 (Ξ) at a field at which TMS protons resonate at exactly 100 MHz. 30,31 Chitosan (3.6 g L-1 ) and TPP (0.3% w/v) solutions

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in 0.2 M acetic acid-150 mM NaCl were used to record NMR spectra. Moreover, NMR analyses

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were performed to assess the amount of TPP and other phosphates diffusing into chitosan solution ACS Paragon Plus Environment

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during the dialysis. The gelling bath used in hydrogel preparation was collected at different time intervals (t = 0, 2, 4, 6, 8, 24 h) and subsequently analyzed. The decrease of the signals corresponding to phosphates was determined by normalizing the integrals with respect to the reference PPh3 . 2.9. Phosphate quantification. The amount of TPP present in the hydrogels was quantified by means of a protocol described elsewhere with slight modification. 22 Briefly, standard (TPP) or hydrogels were dissolved in hydrochloric acid 37% (Sigma Aldrich, Chemical Co. USA) at room temperature. 0.1 mL of the latter solution was added to 0.9 mL of deionized water. Finally, 0.2 mL of a solution containing ammonium molybdate (2.5% w/v) and ammonium vanadate (0.125% w/v) were added and mixed by shaking. After five minutes, the absorba nce of the yellowish solution was measured at 420 nm using Infinite M200 PRO NanoQuant, Tecan spectrophotometer. A blank solution was prepared with 1 mL of deionized water and 0.2 mL of the vanadium- molybdenum solution. A standard calibration curve for TPP was obtained with R2 > 0.99. All measurements were performed in triplicate. 2.10. Swelling studies. Swelling studies of hydrogels were carried out in PBS at room temperature. Samples disks (diameter = 22 mm, surface/volume ratio 4 cm-1 ) were placed in 10 mL of PBS under moderate stirring. After blotting the excess of water using filter paper, the hydrogels were weighed at different time intervals (t = 0, 1, 2, 3, 4, 5, 6, 7 and 24 h). The swelling ratio was calculated as the percentage of water loss with respect to the initial weight in agreement with the formula

where Wt is the weight of samples at different time intervals and W0 the dry weight of hydrogels at t = 0. All measurements were performed in triplicate. 2.11. Cell culture. Mouse fibroblast- like (NIH-3T3) cell line (ATCC® CRL-1658TM) was used for


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the in vitro experiments. Cells were cultured in Dulbecco’s Modified Eagle’s Medium high glucose (EuroClone, Italy), 10% heat- inactivated fetal bovine serum (Sigma Aldrich, Chemical Co. USA), 100 U mL-1 penicillin, 100 µg mL -1 streptomycin and 2 mM L-glutamine in a humidified atmosphere of 5% CO 2 at 37 °C. 2.12. Lactate dehydrogenase (LDH) assay. In vitro cytotoxicity of CH- TPP membranes was evaluated by using the lactate dehydrogenase assay on NIH-3T3 cells. UV-sterilized freeze-dried samples (30 minutes of irradiation for each side of sample) of approximately 6 mm in length and 2 mm in width were placed in Dulbecco’s modified Eagle’s medium, inactivated fetal bovine serum 10%, penicillin 100 U mL-1 , streptomycin 100 μg mL-1 and L-glutamine 2 mM for 72 h at 37 °C and 5% pCO 2 (extraction medium). After 72 h of incubation, the cytotoxicity test was performed by direct contact of the cells with the swollen membranes and by using the extraction medium, according to ISO 10993-5.32 In the case of the direct contact test, 25 000 cells were plated on 24well plates and, after complete adhesion, culture medium was changed with 300 μL of fresh medium. Tested materials were directly deposited on the cell layer. After 24 and 72 h, medium was collected and the LDH assay was performed according to the manufacture’s protocol. In the extraction test, extraction medium was added on cells seeded on 24-well plates (25 000 cells/well). The LDH assays were performed after 24 and 72 h as described above. As a positive control material, poly(urethane) films (PU) containing 0.25% zinc dibuthyldithiocarbamate (ZDBC) (6 mm disks) were used. As negative control material, plastic poly(styrene) sheets (PS) (6 mm disks) were used. Triton X-100 0.1% v/v was used as a positive control for extraction test. Each material test was performed in triplicate. Evaluation of cytotoxicity was calculated according to the formula

with A: LDH activity in the culture medium of membrane-treated or extraction medium- treated cells; B: LDH activity of culture medium from untreated cells and C: LDH activity after total cell


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lysis at 24 and 72 h.

3. Results and discussion 3.1. Preparation of chitosan-TPP networks. The present work aimed to prepare chitosan-based hydrogels and dried networks (membranes) starting from homogeneous hydrogels using tripolyphosphate as cross- linker. Typically, when a TPP solution is added to dilute chitosan solutions, the anions quickly cross- link the cationic chitosan chains leading to the formation of nanoparticles or aggregates.26 In accordance with literature, we observed that when chitosan concentration was increased up to 3% and TPP added in solution, a very fast reticulation took place causing the formation of polydisperse micro-aggregates. This leads to a highly inhomogeneous system with an unreacted liquid core and highly cross- linked surfaces. Using this experimental approach, it is then not possible to obtain a homogeneous macroscopic hydrogel with specific shape and regular structure. Therefore, it was necessary to control the reaction rate and the cross- linking density to tune the properties of the polymeric network. To this end, we devised an ion diffusionbased approach that enables TPP to diffuse slowly and gradually into the confined polymer solution from an external cross- linking bulk, aiming at a uniform reticulation throughout the chitosan matrix. The experimental setup is depicted in Figure 1.


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Figure 1. Experimental set up to develop chitosan-tripolyphosphate hydrogels by exploiting dialysis technique. (A) Enclosed viscous chitosan solution is placed into an outer TPP solution under stirring condition. During the dialysis TPP ions diffuse through semipermeable membranes and (B) after 24 h viscous solution has turned into a homogeneous hydrogel by ionotropic gelation.

First attempts with an initial chitosan concentration of 1.5% led to the formation of hydrogels characterized by highly reticulated rigid surfaces and a liquid core. This can be traced back to the fact that when the dialysis starts, TPP ions bind to chitosan charge groups at the boundary of the polymer- gelling bath, and polymer chain diffusion takes place towards the gelling zone causing the formation of a concentration gradient. Hence, chitosan progressively accumulates within the gelling zone, resulting into an inhomogeneous hydrogel with the highest concentration of polymer at the TPP-hydrogel interface. The inhomogeneity of the hydrogels decreased upon increasing the TPP concentration from 1% to 1.5%. However, even at a concentration of 3% of TPP a partial inhomogeneity was still detectable. At higher chitosan concentrations, a homogeneous hydrogel was obtained for all TPP concentrations explored. This phenomenon can be explained by taking


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into account the very high viscosity of the chitosan solution that hampered the diffusion of the polymer towards the gelling zone preventing the formation of the gradient. These experimental evidences are in line with what is known for alginate-Ca+2 systems.33 The addition of non-gelling ions (i.e. Na+) in the gelling bath is expected to slow down the gelation process by screening the strong electrostatic interactions between polymer and cross-linker. Recently, Huang et al. demonstrated that small amounts of sodium chloride affect the binding mechanism of chitosan and TPP by decreasing the fast kinetics of their self-assembly and by weakening the binding of TPP to the polysaccharide.34 Along this line, sodium chloride was added at two different concentrations (150 and 500 mM) into the gelling bath containing TPP. The presence of NaCl enabled to improve the homogeneity of the hydrogel for both NaCl concentrations. The outcome in the case of NaCl 150 mM was the hydrogel shown in Figure 2A, which appears as a homogenous wall-to-wall system, albeit not transparent. Environmental scanning electron microscopy (E-SEM) in the wet state revealed the presence of a uniform polymeric structure both on the hydrogel surface and in the inner core (Figure 2, B-C).


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Figure 2. CH-TPP hydrogels: (A) top view, (B) E-SEM micrograph of the hydrogel surface, (C) E-

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membrane (Figure 2, E-F) revealed a homogeneous polymeric mesh with a compact structure,

SEM micrograph of the hydrogel cross-section. CH-TPP membrane derived from the hydrogel depicted in (A): (D) top view, (E) SEM micrograph of the membrane surface, (F) SEM micrograph of the membrane cross-section.

No evidence of irregular gelling zones was found even at high magnifications. In the present CHTPP systems, the hydrogel formation may take place through a mechanism that involves primary particles formation which consequently aggregate until to reach a more homogeneous system, a similar phenomenon already demonstrated for the formation of CH-TPP particles and micro/nano gels35,23 , leading to a wall- to-wall isotropic reticulation. Table 1 summarizes the overall conditions used to fabricate CH-TPP hydrogels. A temperature-controlled freeze-drying procedure was applied to the hydrogels to obtain a soft pliable membrane as depicted in Figure 2D. SEM images of the


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which demonstrates the uniform reticulation occurred within the hydrogel phase.

Table 1. Experimental parameters investigated to fabricate CH-TPP hydrogels. : Non-continuous networks (inhomogeneous); ▲: Continuous networks (homogeneous). Both chitosan and tripolyphosphate are expressed as % w/v.

NaCl 0 mM

NaCl 150 mM

NaCl 500 mM

TPP 1%

TPP 1.5%

TPP 3%

TPP 1%

TPP 1.5%

TPP 3%

TPP 1%

TPP 1.5%

TPP 3%

Chitosan 1.5%







▲

▲

▲

▲

▲

▲

Chitosan 2.5%

▲

▲

▲

▲

▲

▲

▲

▲

▲

Chitosan 3%

▲

▲

▲

▲

▲

▲

▲

▲

▲

3.2. Mechanical behavior. The following characterizations were carried out on the homogenous systems screened according to Table 1. Hydrogels are systems exhibiting viscoelastic behavior when undergoing deformation depending on the frequency of the deformation. The ideal viscoelastic properties of a biomaterial depend on the intended use. For example, mechanical integrity is required for materials that are to be used as scaffolds for cell culture or substrates for patterning. In order to investigate mechanical properties of the chitosan-TPP hydrogels, rheological and uniaxial compression studies were performed. In detail, rheological analyses were carried out by measuring the mechanical spectrum of the hydrogels in oscillatory shear conditions. In all the cases considered, the storage (G') modulus is about ten times higher than the loss modulus (G'') and ACS Paragon Plus Environment

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basically independent on the stress frequency, thus describing the systems under consideration as “strong hydrogels”. The mechanical spectrum of the samples was interpreted in terms of the generalized Maxwell model.36 The storage and loss modulus can be modeled as function of oscillation frequency according to the following equations

where n is the number of Maxwell elements considered, Gi, ηi, and λi represent the spring constant, the dashpot viscosity, and the relaxation time of the ith Maxwell element, respectively. Ge is the spring constant of the last Maxwell element which is supposed to be purely elastic. 28 The influence of chitosan concentration on shear stress response was investigated first.


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Figure 3. (A) G' (full symbols) and G'' (open symbols) for CH-TPP hydrogels at a total polymer concentration of 3% (black squares), 2.5% (red triangles) and 1.5% (blue circles). (B) Dependence of the shear modulus - red solid line - (± SD) and Ge - black solid line - (± SD) versus Cp . (C) G' (full symbols) and G'' (open symbols) for CH-TPP hydrogels versus NaCl concentration of 0 mM (black squares), 150 mM (red triangles) and 500 mM (blue circles). (D) Dependence of the shear modulus - red solid line - (± SD) and Ge - black solid line - (± SD) versus NaCl concentration.

Figure 3A compares the relaxation spectra of chitosan hydrogels with different chitosan concentration, TPP and NaCl concentrations of 1.5% and 500 mM, respectively. Lines represent the fitting of the experimental data according to equations (1) and (2). Experimental data were

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efficiently fitted by the generalized Maxwell model and supported by the statistical F test37 [F1.5%(4,

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concentration of 1.5% were fitted adequately by means of three Maxwell elements whereas hydrogels with concentrations of 2.5 and 3% required four spring-dashpot elements. The use of the generalized Maxwell model to describe chitosan systems enables the shear modulus, G, to be calculated as

By fitting data shown in Figure 3A according to equation (3), this modulus was found to scale (R2 > 0.99) with the total polymer concentration (C p ) according to the following power law G

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Cp3.1 (Figure 3B). Herein, red solid line represents the best fit of the experimental results. These findings are in agreement with those of Kong et al., who described a non- linear dependence of shear modulus versus polymer concentration for high MW alginate hydrogels in the range 2 to 4.5% w/v.38 As expected, these results indicate that mechanical performance of hydrogels versus shear stress is positively influenced by the increase of polymer concentration. A similar trend was identified for the purely elastic spring Ge who was found to scale in an analogue manner (Ge 206.5 Cp2.8 , R2 > 0.99). The effect of NaCl on mechanical behavior of hydrogels was then investigated. Figure 3C shows the mechanical spectra of chitosan hydrogels at different NaCl concentrations and constant chitosan and TPP concentrations (3 and 1.5%, respectively). Lines represent the fitting of the experimental data according to equations (1) and (2). In this case, shear modulus was not influenced significantly when small amount of NaCl (150 mM) was added to the system with respect to the salt free hydrogel (Figure 3D). However, shear modulus decreased when the sodium chloride concentration was increased further to 500 mM. A similar behavior was demonstrated for the G e parameter who was found to progressively diminish by augmenting NaCl concentration. This result is in a very good agreement with those of Huang et al. who demonstrated that at high concentration of NaCl (500 mM) the chitosan-TPP binding is weakened.34 This suggests that sodium chloride competes with the polymer-cross- linker binding in hydrogel formation.


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Similar experimental results were observed upon varying TPP concentration at constant polymer and NaCl concentrations (3% and 150 mM, respectively). By increasing cross- linking ion concentration (1, 1.5 and 3%) shear modulus was found to be 12.0 ± 0.5, 17.0 ± 0.5 and 9.3 ± 0.3 kPa, respectively. At the lowest concentration of tripolyphosphate (i.e. 1%), hydrogels showed the shear modulus higher than the hydrogels with 3% of TPP. This might be due to the very fast binding of the anion by the polysaccharide in the latter case that, despite the macroscopic continuous network formed (Table 1), induces anisotropy in polysaccharide distribution with the formation of a more liquid- like core. On the other hand, the best mechanical response was observed in hydrogels with 1.5% of TPP. Rheological data clearly suggested that the best mechanical behavior was observed only for a very narrow range of chemical concentrations explored, compared with those indicated in Table 1. To summarize, from rheological analyses we identified two optimum conditions for hydrogel formation: chitosan 3%, TPP 1.5%, NaCl 150 mM, glycerol 5% (F1) and chitosan 3%, TPP 1.5%, glycerol 5% (F2). The TPP-glucosamine molar ratio was the same in both cases. Since rheological tests could not point out significant differences between formulation F1 and F2, the mechanical behavior of both formulations was evaluated considering a different stress condition, i.e. uniaxial compression. These measurements were performed by applying a very slow compression stress to the material, which induces an elastic deformation of the polymeric network. The toughness at 70% of total strain was used as a parameter to compare the two formulations (Figure 4).

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Figure 4. Representative stress-strain curves of the hydrogels under uniaxial compression. Black line: F1 (with supporting salt NaCl); red line: F2 (without supporting salt NaCl). Dashed green line represents the 70% of total strain.

By integrating the areas under stress-strain curves, the toughness values were 20.5 ± 5.0 and 9.6 ± 1.5 kJ m-3 for F1 and F2, respectively. These data point out that, in the presence of supporting salt (F1) the hydrogels are able to accumulate significantly (p < 0.05, n = 3) more energy than hydrogels without NaCl (F2). These results clearly indicated that hydrogels F1 showed a better mechanical performance in compression compared to hydrogels F2. The interpretation of these results is somewhat unclear but it can be speculated that, although a continuous network is formed in both cases (Table 1), an anisotropic distribution of the polysaccharide component takes place which results in a stiffer structure. Given these results, hydrogel F1 was selected as the best candidate for further characterization studies and to optimize the preparation of the membranes.

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3.3. Chitosan-TPP interaction. In order to investigate the interaction between chitosan and TPP,

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FTIR-ATR analyses were performed. Figure 5 shows the spectra of dried chitosan, CH-TPP ACS Paragon Plus Environment

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membrane and TPP powder.

Figure 5. FTIR-ATR spectra of dried chitosan (black line), F1 membrane (blue line) and TPP powder (red line).

CH-TPP membrane was developed starting from the best candidate hydrogel F1. FTIR spectrum of dried chitosan (black line) showed two characteristic peaks at about 1645 and 1550 cm-1 which were attributed to C=O stretching in amide group (amide I vibration) and N-H bending in amide group (amide II vibration), respectively. 39 The peak at 1375 cm-1 was attributed to the distorting vibration of C–CH3 bond.39 Absorption bands at 1150 (anti-symmetric stretching of the C-O-C bridge), 1070 and 1025 cm-1 (skeletal vibrations involving the CO stretching) are characteristic peaks of the chitosan structure.40 TPP spectrum shows peaks at 1210 (P=O stretching), 1140 (symmetric and anti-symmetric stretching vibrations in PO 2 group) and 1093 cm-1 (symmetric and anti-symmetric stretching vibrations in PO 3 group).41 Spectrum referring to F1 membrane showed the appearance of the novel band at 1220 cm-1 , corresponding to anti-symmetric stretching


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vibrations of PO 2 groups and the significant shift of the peak at 1073 in dried chitosan to 1025 cm-1 in chitosan-TPP membrane. These peaks confirm the formation of ionic cross- links between NH3 + groups of chitosan and tripolyphosphate anions in agreement with the literature.41 The chitosan-TPP interaction was also investigated in diluted 0.2 M acetic acid-150 mM NaCl solutions by exploiting 31

P NMR. The latter analyses (Figure 6) showed the presence of characteristic peaks in TPP

spectrum referring to orthophosphate (0.266 ppm), pyrophosphate (-10.398 ppm) and triphosphate (-10.284 ppm: α terminal phosphorus; -22.679 ppm: β middle phosphorus atoms. JP, P = 19.5 Hz) species according to the literature.42,43

Figure 6.

31

P NMR spectrum of TPP (0.3% w/v) solution (red spectrum). The peak at -5.288 ppm

refers to PPh3 . Inset: magnification of the signal referring to PPi and α terminal phosphorus of TPP. Black spectrum: 31 P NMR of diluted solution composed by TPP (0.3% w/v), NaCl (150 mM) and chitosan (3.6 g L-1 ). All spectra were recorded at 25 °C.

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By integrating the area under the aforementioned peaks, it was revealed that the percentage of orthophosphate and pyrophosphate was 1 and 17% w/w, respectively, and that of TPP was 82% w/w. This is in line with the 85% w/w purity of tripolyphosphate reported by the producer. The pKa values reported in the literature are: orthophosphoric acid pKa 1 2.17, pKa2 7.31, pKa3 12.36; pyrophosphoric acid pKa1 very small, pKa2 2.64, pKa3 6.76, pKa4 9.42; tripolyphosphoric acid pKa1 and pKa2 very small, pKa3 2.30, pKa4 6.50, pKa5 9.24.44 Thus, in the present medium containing 0.2 M acetic acid - the most abundant species should bear one negative charge in the case of orthophosphate, two charges in the case of the pyrophosphate, and three charges in the case of tripolyphosphate, one on each monophosphate unit in the latter two cases. When chitosan was added to the cross- linking ion solution, the signals of TPP and pyrophosphate disappear, being broadened beyond detection limit by the strong interaction with the polymeric chain, extremely slowly reorienting. Also the orthophosphate signal seems to disappear likely indicating that it binds to chitosan, although the very low signal prevents a clear interpretation. 3.4. Phos phate quantification. In order to assess the amount of tripolyphosphate embedded within the best candidate hydrogel, a spectrophotometric method was exploited based on an acid hydrolysis of TPP by means of hydrochloric acid followed by a colorimetric reaction with a mixture of vanadium- molybdenum salts. The reaction rate of TPP hydrolysis is equal for all samples analysed, provided pH and time treatment are equal. Thus, the comparison of the absorbance of the sample and those of solutions containing known amounts of tripolyphosphate provides a direct evaluation on the TPP concentration. Considering that the concentration of TPP in the gelling bath is equal to 1.5% w/v, the TPP concentration entered in the hydrogel was equal to 0.2% w/v, calculated on the basis of the amount of TPP measured in hydrogel by the spectrophotometric method. Thus, only a limited amount of TPP (estimated to be about 13%) was uptaken. To better understand how the concentration of TPP and other phosphates present in the gelling bath decreased upon time,

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P NMR spectra were performed on the gelling solution. The chemical shifts of the


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signals did not change with time, at variance with their integrated intensities (data not shown) that allowed to calculate the concentration which results 1.3% w/v after 24 h in agreement with the spectrophotometric method. In addition, NMR spectroscopy enables to distinguish the signals of the phosphate species. The P, PPi and TPP concentrations slowly decreased over time as depicted in Figure 7, in accordance with an anion uptake by the chitosan solution.

Figure 7. Concentration decrease of P (black line), PPi (red line) and TPP (blue line) species (mM) with time (h) in the gelling solution as calculated from the integrals of 31 P NMR spectra recorded at 25 °C.

The orthophosphate (P) concentration was very small and did not significantly vary, whereas the tripolyphosphate (TPP) and pyrophosphate (PPi) concentrations (mM) decreased appreciably in 24

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hours (from 32.80 at t = 0 h to 29.20 at t = 24 h and from 9.28 at t = 0 h to 6.79 at t = 24 h,

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respectively). The PPi present as minor component in the commercial TPP sample used did not ACS Paragon Plus Environment

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hamper the hydrogel formation. Indeed, it was reported that it is able to ionically cross-link polyelectrolytes, chitosan included.45,46 The molar ratio of the latter two species (TPP/PPi) increased from 3.53 (t = 0 h) to 4.30 (t = 24 h). This might be due either to a faster diffusion of PPi through the dialysis membrane or to a more effective binding of PPi than TPP to chitosan. This represents a very interesting finding which will be investigated further in forthcoming works. The decrease of TPP and PPi concentrations due to diffusion into chitosan solution during the dialysis was 3.6 and 2.4 mM, respectively. Therefore, the contribution of PPi to the gelation process cannot to be ruled out. Indeed, the spectrum of chitosan-TPP mixture depicted in Figure 6 showed the disappearance of peaks at -10.398, -10.284 and -22.679 ppm, respectively, indicating that both TPP and PPi are implicated in binding with the polymer. 3.5. Swelling studies. The swelling capacity of ionically cross- linked hydrogels plays an important role to evaluate their potential use as biomaterials for biomedical applications. Swelling is mainly influenced by ionic interactions between chitosan chains, depending in turn on the cross- linking density.47,48 With the aim of investigating the swelling behavior of F1 hydrogels in physiological pH conditions, the water loss kinetics of samples was followed in PBS at room temperature for 24 h and the results are reported in Figure 8. Samples were characterized by a fast de-swelling that took place during the first 7 hours. Further, the loss of solvent was basically stable up to 24 h (from 19.32 ± 4.97 to -20.28 ± 5.89%). A parallel investigation was performed on hydrogels with the same chitosan and NaCl concentration but with a different amount of TPP (1%) compared to F1. A similar trend was observed to that of depicted in Figure 8 (data not shown) albeit a greater experimental error was identified likely due to a higher instability of hydrogels. To explain the loss of solvent experienced by the hydrogels, it should be noted that during the hydrogel formation the dialysis takes place at a pH of 4.3 for F1. In this case, chitosan amino groups are almost completely protonated.5 When hydrogels are placed in PBS with pH of 7.4, chitosan amino groups are in the form of -NH2 , which makes it insoluble and leads to a shrinkage of the network with a loss of


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solvent. The same effect was observed by Lopez-Leon and co-workers who demonstrated that the diameter of CH-TPP nanoparticles diminished appreciably when pH was varied from 4 to 7.49 In this case, the lack of repulsive electrostatic forces at pH 7 between nanoparticles influenced intermolecular interactions, thus the uncharged nanospheres started to aggregate.

Figure 8. Hydrogel de-swelling (%) versus time (h) of F1 hydrogels. The data are expressed as mean (± SD) of three samples.

3.6. Cytotoxicity evaluation. The cytotoxicity characterization has been carried out on the prototype membranes obtained by freeze-drying. Pati et al. reported that TPP at concentrations higher than 4 mM can be toxic for the 3T3 cell line.50 In order to exclude the release of toxic concentrations of TPP from the material, LDH assay was performed on mouse fibroblasts using the freeze-dried sample F1. As reported in Figure 9A, CH- TPP specimens did not exert any cytotoxic

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effect both when the material was placed directly in contact with cells and when the extract medium

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was added on the cell layer. Indeed, there was no significant difference in the release of lactate ACS Paragon Plus Environment

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dehydrogenase between sample-treated cells and negative control groups (indicated as Ps and Ad, respectively) after 24 and 72 h. To support this conclusion, optical microscopic analyses (Figure 9B) were performed and confirmed lack of cytotoxicity of the biomaterial. Indeed, cells showed a normal fibroblast-like morphology, good spreading and absence of suffering signals such as cytoplasmic vacuoles, chromatin aggregation or formation of cellular debris, even after 72 h of treatment.

Figure 9. (A) Effect of the freeze-dried sample F1 on NIH-3T3 cell line. The cytotoxicity test was performed both with the extract from the sample (CH-TPP-E) and with the sample in direct contact with cell layer (CH-TPP-C). Control cells cultured in adhesion in complete DMEM medium (Ad). Polyurethane sheets (Pu) were used as contact positive control, plastic poly(styrene) sheets (Ps) were used as contact negative control and Triton X-100 was used as extract positive control. The data are expressed as mean (± SD) of three independent experiments. (B) Optical microscopy of mouse fibroblast NIH-3T3 cells after 72 h of treatment with membrane.

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Conclusions Homogeneous hydrogels and membranes based on chitosan and TPP were developed by exploiting an innovative approach through an ionotropic gelation process. A diffusion technique was selected in order to avoid the local saturation of chitosan binding sites, thus obtaining a macroscopic uniform hydrogel structure. The influence of the concentrations of chitosan, TPP and NaCl was studied in order to tune the homogeneity of gelation. In addition, it was revealed that not only TPP but also PPi, albeit present as minor component in TPP powder, contributes to the gelation of these chitosanbased hydrogels. Soft and pliable membranes have been prepared through a temperature-controlled freeze-drying procedure. Morphological analyses of both hydrogels and freeze-dried membranes pointed out a uniform polymeric network with considerable mechanical properties. The possibility of properly tune the mechanical performance of the biopolymeric network combined with their demonstrated lack of cytotoxicity suggests the use of these hydrophilic biomaterials based on the unique biological properties of chitosan for several biomedical applications such as in the fields of wound dressing (topical applications, in particular in combination with antibacterial drugs or silver nanoparticles), tissue engineering (articular cartilage repair) and drug delivery.51-53

Acknowledge ment. This study was supported by the Italian Ministry of University and Research MIUR (PRIN 2012, project “Spinal injury: towards the development of cell- instructive scaffolds for nerve tissue repair) and by the Friuli-Venezia Giulia Regional Government (Project: “Nuovi biomateriali per terapie innovative nel trattamento delle ferite difficili”- LR 47/78). The financial support to P.S. (PhD position) by the Friuli-Venezia Giulia Regional Government and by the European Social Fund (S.H.A.R.M. project-Supporting human assets in research and mobility) is gratefully acknowledged. We gratefully thank Dr. Gianluca Turco and Dr. Michela Abrami for technical support in SEM and rheological analyses.


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References and Notes (1)

Langer, R.; Vacanti, J. P. Science 1993, 260, 920–6.

(2)

Peppas, N. A.; Bures, P.; Leobandung, W.; Ichikawa, H. Eur. J. Pharm. Biopharm. 2000, 50, 27–46.

(3)

Marsich, E.; Mozetic, P.; Ortolani, F.; Contin, M.; Marchini, M.; Vetere, A.; Pacor, S.; Semeraro, S.; Vittur, F.; Paoletti, S. Matrix Biol. 2008, 27, 513–25.

(4)

Marsich, E.; Borgogna, M.; Donati, I.; Mozetic, P.; Strand, B. L.; Salvador, S. G.; Vittur, F.; Paoletti, S. J. Biomed. Mater. Res. A 2008, 84, 364–76.

(5)

Tømmeraas, K.; Köping-Höggård, M.; Vårum, K. M.; Christensen, B. E.; Artursson, P.; Smidsrød, O. Carbohydr. Res. 2002, 337, 2455–62.

(6)

Mao, S.; Sun, W.; Kissel, T. Adv. Drug Deliv. Rev. 2010, 62, 12–27.

(7)

Kean, T.; Thanou, M. Adv. Drug Deliv. Rev. 2010, 62, 3–11.

(8)

Molinaro, G.; Leroux, J.-C.; Damas, J.; Adam, A. Biomaterials 2002, 23, 2717–2722.

(9)

Ferrero, F.; Periolatto, M.; Vineis, C.; Varesano, A. Carbohydr. Polym. 2014, 103, 207–212.

(10)

Garnica-Palafox, I. M.; Sánchez-Arévalo, F. M.; Velasquillo, C.; García-Carvajal, Z. Y.; García-López, J.; Ortega-Sánchez, C.; Ibarra, C.; Luna-Bárcenas, G.; Solís-Arrieta, L. J. Biomater. Sci. Polym. Ed. 2014, 25, 32–50.

(11)

Grolik, M.; Szczubiałka, K.; Wowra, B.; Dobrowolski, D.; Orzechowska-Wylęgała, B.; Wylęgała, E.; Nowakowska, M. J. Mater. Sci. Mater. Med. 2012, 23, 1991–2000.

(12)

Kangjian, W.; Nianhua, D.; Shiwei, X.; Yichun, Y.; Weihua, D. J. Membr. Biol. 2012, 245, 707–16.

(13)

Silva, S. S.; Caridade, S. G.; Mano, J. F.; Reis, R. L. Carbohydr. Polym. 2013, 98, 581–8.

(14)

Montembault, A.; Viton, C.; Domard, A. Biomacromolecules 2005, 6, 653–62.

(15)

Boucard, N.; Viton, C.; Domard, A. Biomacromolecules 2005, 6, 3227–37.

(16)

Risbud, M. V; Hardikar, A. A.; Bhat, S. V; Bhonde, R. R. J. Control. Release 2000, 68, 23– 30.

(17)

Minet, E. P.; O’Carroll, C.; Rooney, D.; Breslin, C.; McCarthy, C. P.; Gallagher, L.; Richards, K. G. Chemosphere 2013, 93, 2854–8.

(18)

Ji, C.; Khademhosseini, A.; Dehghani, F. Biomaterials 2011, 32, 9719–29.

(19)

Feng, H.; Zhang, L.; Zhu, C. Colloids Surf. B. Biointerfaces 2013, 103, 530–7.


28

Page 29 of 31

1 2 3 566 4 5 6 567 7 568 8 9 569 10 11 12 570 13 571 14 15 572 16 573 17 18 19 574 20 575 21 22 576 23 24 577 25 26 578 27 579 28 29 580 30 31 581 32 33 582 34 583 35 36 37 584 38 39 585 40 586 41 42 43 587 44 588 45 46 589 47 48 590 49 50 51 591 52 53 592 54 593 55 56 57 594 58 595 59 596 60

597

Biomacromolecules

(20)

Cui, L.; Jia, J.; Guo, Y.; Liu, Y.; Zhu, P. Carbohydr. Polym. 2014, 99, 31–8.

(21)

Berger, J.; Reist, M.; Mayer, J. M.; Felt, O.; Peppas, N. A.; Gurny, R. Eur. J. Pharm. Biopharm. 2004, 57, 19–34.

(22)

Pati, F.; Adhikari, B.; Dhara, S. Carbohydr. Res. 2011, 346, 2582–8.

(23)

Rampino, A.; Borgogna, M.; Blasi, P.; Bellich, B.; Cesàro, A. Int. J. Pharm. 2013, 455, 219– 228.

(24)

Zhang, H.; Mardyani, S.; Chan, W. C. W.; Kumacheva, E. Biomacromolecules 2006, 7, 1568–72.

(25)

Schütz, C. A.; Juillerat-Jeanneret, L.; Käuper, P.; Wandrey, C. Biomacromolecules 2011, 12, 4153–61.

(26)

Calvo, P.; Remunan-Lopez, C.; Vila-Jato, J. L.; Alonso, M. J. J. Appl. Polym. Sci. 1997, 63, 125–132.

(27)

Donati, I.; Stredanska, S.; Silvestrini, G.; Vetere, A.; Marcon, P.; Marsich, E.; Mozetic, P.; Gamini, A.; Paoletti, S.; Vittur, F. Biomaterials 2005, 26, 987–98.

(28)

Lapasin, R.; Pricl, S. Rheology of industrial polysaccharides: theory and applications; Chapman and Hall: London, Ed.; 1995; p. 620.

(29)

Meyvis, T. K. L.; De Smedt, S. C.; Demeester, J.; Hennink, W. E. J. Rheol. (N. Y. N. Y). 1999, 43, 933.

(30)

Edzes, H. T. Magn. Reson. Chem. 1992, 30, 850–854.

(31)

Harris, R. K.; Becker, E. D.; Cabral de Menezes, S. M.; Goodfellow, R.; Granger, P. In Magnetic Resonance in Chemistry; 2002; Vol. 40, pp. 489–505.

(32)

ISO 10993-5: Biological evaluation of medical devices - Part 5: Tests for in vitro cytotoxicity; 2nd ed.; 1999.

(33)

Skjåk-Bræk, G.; Grasdalen, H.; Smidsrød, O. Carbohydr. Polym. 1989, 10, 31–54.

(34)

Huang, Y.; Lapitsky, Y. Langmuir 2011, 27, 10392–9.

(35)

Huang, Y.; Lapitsky, Y. Biomacromolecules 2012, 13, 3868–76.

(36)

Turco, G.; Donati, I.; Grassi, M.; Marchioli, G.; Lapasin, R.; Paoletti, S. Biomacromolecules 2011, 12, 1272–82.

(37)

Grassi, M.; Grassi, G.; Lapasin, R.; Colombo, I. Understanding Drug Release and Absorption Mechanisms: A Physical and Mathematical Approach; CRC Press: Boca Raton (FL, USA), 2007; p. 627.

(38)

Kong, H.-J.; Lee, K. Y.; Mooney, D. J. Polymer. 2002, 43, 6239–6246.


29

Biomacromolecules

1 2 3 598 4 5 6 599 7 8 600 9 601 10 602 11 12 13 603 14 15 604 16 17 18 605 19 606 20 21 607 22 23 24 608 25 26 609 27 610 28 29 611 30 31 612 32 33 613 34 614 35 36 37 615 38 39 616 40 617 41 42 43 618 44 45 619 46 620 47 48 621 49 50 51 622 52 53 623 54 55 56 624 57 58 59 625 60

Page 30 of 31

(39)

Wang, Q.; Dong, Z.; Du, Y.; Kennedy, J. F. Carbohydr. Polym. 2007, 69, 336–343.

(40)

Pawlak, A.; Mucha, M. Thermochim. Acta 2003, 396, 153–166.

(41)

Ostrowska-Czubenko, M.; Gierszewska-Drużyńska, J. In Progress on chemistry and application of chitin and its derivative, Volume XV; M.M. Jaworska, Ed.; Polish Chitin Society: Łódź, 2010; pp. 25–32.

(42)

James G. Colson, D. H. M. Anal. Chem. 1973, 45, 370–371.

(43)

Hrynczyszyn, P.; Jastrzebska, A.; Szłyk, E. Anal. Chim. Acta 2010, 673, 73–8.

(44)

Manahan, S. E. Environmental Chemistry; Seventh ed.; CRC Press LLC: Boca Raton, 2010; p. 912.

(45)

Huang, Y.; Lapitsky, Y. J. Phys. Chem. B 2013, 117, 9548–57.

(46)

Cai, Y.; Lapitsky, Y. Colloids Surf. B. Biointerfaces 2014, 115, 100–8.

(47)

Mi, F.-L.; Chen, C.-T.; Tseng, Y.-C.; Kuan, C.-Y.; Shyu, S.-S. J. Control. Release 1997, 44, 19–32.

(48)

Mi, F.-L.; Shyu, S.-S.; Wong, T.-B.; Jang, S.-F.; Lee, S.-T.; Lu, K.-T. J. Appl. Polym. Sci. 1999, 74, 1093–1107.

(49)

López-León, T.; Carvalho, E. L. S.; Seijo, B.; Ortega-Vinuesa, J. L.; Bastos-González, D. J. Colloid Interface Sci. 2005, 283, 344–51.

(50)

Pati, F.; Adhikari, B.; Dhara, S. J. Mater. Sci. Mater. Med. 2012, 23, 1085–96.

(51)

Travan, A.; Pelillo, C.; Donati, I.; Marsich, E.; Benincasa, M.; Scarpa, T.; Semeraro, S.; Turco, G.; Gennaro, R.; Paoletti, S. Biomacromolecules 2009, 10, 1429–35.

(52)

Van Vlierberghe, S.; Dubruel, P.; Schacht, E. Biomacromolecules 2011, 12, 1387–408.

(53)

Wang, J. J.; Zeng, Z. W.; Xiao, R. Z.; Xie, T.; Zhou, G. L.; Zhan, X. R.; Wang, S. L. Int. J. Nanomedicine 2011, 6, 765–74.

626


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Polysaccharide-Based Networks from Homogeneous Chitosan

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