polyaniline spongy-like hydrogels

Subscriber access provided by UNIV OF DURHAM

Tissue Engineering and Regenerative Medicine

ELECTROACTIVE GELLAN GUM/POLYANILINE SPONGY-LIKE HYDROGELS Pathomthat Srisuk, Fernanda V. Berti, Lucília Pereira da Silva, Alexandra P. Marques, Rui L. Reis, and Vitor M. Correlo ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00917 • Publication Date (Web): 12 Apr 2018 Downloaded from http://pubs.acs.org on April 13, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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

ACS Biomaterials Science & Engineering

Electroactive gellan gum/polyaniline spongy-like hydrogels Pathomthat Srisuk 1†‡⊥, Fernanda V. Berti 1†‡, Lucilia P. da Silva†‡, Alexandra P. Marques†‡, Rui L. Reis†‡§ , Vitor M. Correlo*†‡§ †

3B's Research Group, Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European

Institute of Excellence on Tissue Engineering and Regenerative Medicine, Avepark, 4806-909 Taipas, Guimarães, Portugal ‡

ICVS/3B's PT Government Associated Laboratory, Braga, Portugal

§

The Discoveries Centre for Regenerative and Precision Medicine, Headquarters at University of Minho, Avepark, 4805-

017 Barco, Guimarães, Portugal ⊥Division

of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Khon Kaen University, Mittraphap

Highway, Muang District, Khon Kaen 40002, Thailand

*corresponding author E-mail address: [email protected] 1

These authors contributed equally to this work.

ACS Paragon Plus Environment

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

Abstract The ability of electroactive materials to influence and modulate cell behavior has been revealing great interest specially in the field of skeletal muscle tissue engineering. Herein, we propose PANiGG electroactive spongy-like hydrogels as potential materials to modulate myoblast bioresponse. polyaniline (PANi) adds electroconductiviy to gellan gum (GG) spongy-like hydrogels that hold a high resemblance to the extracellular matrix (ECM), i.e. water content, mechanical properties and microarchitecture, and that can be further tuned to meet muscle tissue properties. For this purpose, PANi-GG spongy-like hydrogels were obtained by ionically crosslinking with calcium chloride (CaCl2) and further in situ aniline polymerization through oxidation with ammonium persulfate (APS) in a molar ratio of 1:1.05. The physicochemical characterization, surface morphology, electroconductivity, and mechanical performance were assessed by FTIR, SEM, four-point probe technique, and compression testing, respectively. The viability and proliferation of L929 was not compromised after direct contact of PANi-GG spongy-like hydrogels with L929 cells, as determined by MTS assay and DNA quantification, respectively. C2C12 myoblasts were entrapped within the electroactive materials and cells adhered and spread. Moreover, cells proliferated along the cell culture period showing myosin expression after 7 days of culture. These results highlight that PANi-GG spongy like hydrogels are attractive candidates to be used in skeleton muscle tissue engineering.

Keywords: Electroactive spongy-like hydrogels; gellan gum; polyaniline; oxidative polymerization


Page 2 of 34

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


1. Introduction One of the challenges in tissue engineering (TE) is to establish a physiological and functional structure for supporting cell growth, differentiation, and ultimately regeneration of a new tissue, which can only be achieved by understanding the cellular microenvironment1,2. Electrical stimulation (ES) has been posed as a key player in the regenerative processes of injured organs and tissues. Restoration of movements and improved ability to perform the activities of daily living are some of the clinical evidences 3. In fact, it has been demonstrated that the endogenous bioelectric current has a regulatory role in the healing process 4-6. The use of exogenous ES may mimic this natural endogenous current after neuromuscular electrical stimulation (NMES), transcutaneous electrical nerve stimulation (TENs), and functional electrical stimulation (FES) application in muscle atrophy, spinal cord injury, and muscle sclerosis. In fact, ES has also shown to modulate the behavior of cardiac, neural, and skeletal muscle cells 7-9. Hence, the ability of electroactive materials to influence and modulate cell behavior prompted the development of new and efficient electroactive materials to study cell bioresponse and ultimately trigger tissue regeneration. Moreover, the combination of composite/blend materials with electrical stimulation (electrical current) have showed promising results for instance with nerve cells 10-12, wound healing 13, and muscle cells 14. Conducting polymers (CPs) are bringing a novel focus on the research of biomaterials for TE applications that in addition to their physicochemical and biological properties also consider important the electrical property of biomaterials15, 16. CPs are attractive due to their electrical properties and easy synthesis. As an example, polypyrrole (PPy), polythiophene (PT), and polyaniline (PANi) have particularly inspired the development of non-resorbable CPs with inherent conductivity properties1,

12, 17-22

. PANi, in particular, has well-known chemical and electrical

properties; is easily synthesized by chemical in situ reactions, is highly doped by protonic acids, and presents high unique electrical conductivity (≈ 4.4 ± 1.7 S cm-1) 23. When blended with natural gelatin (≈0.01-0.021 S/cm) 24 and poly(aniline-co-3-aminobenzoic acid)- poly (lactic acid) (≈0.9-8.1



mS/cm-1) 25. PANi has been shown to support cell attachment and promote cell proliferation in the presence of ES influence. Gellan gum (GG) is a linear anionic polysaccharide that we have been explored for a wide range of applications. In particular, GG has specially been processed as spongy-like hydrogels that are structures that retain the features of hydrogels relevant for TE applications, i.e. depict improved mechanical features and more importantly intrinsic cell-adhesive ability 26. Aiming to overcome the limited conductivity of GG spongy-like hydrogels, we propose the combination of GG with PANi, and its processing as spongy-like hydrogels to further advance the functionalities of these structures 27-28

.

In this context, electroactive spongy-like hydrogels obtained by the combination of GG and PANi were produced by in situ chemical reaction, and the physicochemical and electrical properties of PANi-GG spongy-like hydrogels were studied. Additionally, we evaluated the cytotoxicity of PANiGG electroactive spongy-like hydrogels using L929 standard cell line, and studied the ability of C2C12 myoblasts to adhere, proliferate and differentiate within PANi-GG spongy-like hydrogels. 2. Experimental 2.1 Materials and methods Low acyl gellan gum (Gelzan™ CM-Gelite® Fw= 1000000); aniline, C6H7N (ACS reagent, ≥ 99.5%); ammonium persulfate, H8N2O8S2 (ACS reagent, ≥98%) were obtained from Sigma-Aldrich. Aniline was purified by distillation under vacuum before use. Gelzan™ and H8N2O8S2 were prepared using Milli-Q water (Millipore, Milli-Q Direct 16) and HCl solution subsequently. 2.2 Fabrication of GG and PANi-GG spongy-like hydrogels As shown in Figure 1, a solution of 1.25% wt. gellan gum (GG) was prepared by dissolving Gelzan™ in Milli-Q water. The solution was heated up to 90OC with vigorous stirring for 20 min.


Page 4 of 34

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


Subsequently, when the temperature of GG solution reached 55-60OC the crosslinking agent (0.016 mol/L calcium chloride (CaCl2), 1:5 (v/v)), was added. The mixture was then poured into a plastic petri dish and covered with PBS for 1h to stabilize the cross-linking. After that, the GG hydrogels were cut with a 5 mm diameter flat-ended cylindrical metallic punch. The 3D cylindrical GG hydrogels obtained were submersed for 1h in a solution of 1 M aniline monomer doped in 1 M of HCl. After removal of the aniline solution, ammonium persulfate dissolved in 1 M of HCl was added to start the in situ oxidative reaction. The oxidative reaction was taken place at 4°C for 18h. The three dimensional PANi-GG hydrogels were washed several times with PBS solution for 1h each to remove residues of the oxidative reaction. GG and PANi-GG hydrogels were then frozen at -80°C (48h) and freeze dried during 72h to obtain GG and PANi-GG dried polymeric networks that form the spongy-like hydrogels after hydration[28]. Samples were sterilized with ethylene oxide. GG hydrogels were produced following the procedure previously described by da Silva, L.P. et al. (2014).

Figure 1. Schematic representation of GG and PANi-GG hydrogels preparation and oxidation



Page 6 of 34

2.3 FTIR spectroscopy Chemical characterization of GG and PANi-GG dried polymeric networks were performed by Fourier Transform Infrared (FTIR) (IR Prestige 21 spectrometer, Shimadzu, Japan) in attenuated total reflectance (ATR) mode. Samples scans were recorded at 4 cm-1 spectral resolution with 70 scans and recorded in ranging from 4400 to 700 cm-1. The experimental study was independently performed for 3 repeating samples per each condition (n=3) and a representative spectrum of each sample was interpreted. 2.4 Differential scanning calorimetry Differential scanning calorimetry (DSC) measurements were performed by using T.A. Instrument DSC Q100 (UK). The empty aluminum pan and lid was used as the reference for all measurements. Nitrogen gas was used as a purge gas at a rate of ≈ 40 mL.min-1. GG and PANi-GG dried polymeric networks (~3.0 mg) were loaded into the aluminum pan and hermetically sealed. The analysis was carried out from -20OC to 300OC at heating rates of 10OC.min-1. 2.5 Electrical conductivity analysis The electrical resistivity measurements from a four-point-probe technique were employed. The current (I) that passed through the outer probe, measured voltages (V) between the inner probes, thickness (W) and a correction factor (CF) for the geometry were assumed. The conductivity of the electroactive GG and PANi-GG dried polymeric networks (4 × 2 mm2) were considered. The following equations were used to calculate the electrical conductivity:

= × × .


Equation 1

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


= 4.532 !" ($) =

& '()*)+*,*+-

Equation 2

Three samples were analyzed. 2.6 Scanning electron microscopy (SEM) and Micro-computed tomography (µ-CT) analysis The microstructure of three dimensional GG and PANi-GG dried polymeric networks were observed by SEM (SEM, Jeol JSM-6010LV, Japan). Cross section of GG and PANi-GG samples were obtained after freezing in nitrogen liquid (2 min). All samples (n=3) were sputter-coated with a thin gold layer under vacuum using a sputter coater (Cressington model 108auto, TED PELLA, INC, USA) prior examination by SEM (10kV). GG and PANi-GG dried polymeric networks (approximately 2 × 2 mm3) were also analyzed using XRay Microtomography (MICRO-CT) system (Model 1072, SKYSCAN) at room temperature. Three specimens per sample were used for MICRO-CT analysis and each sample was mounted vertically on a metal support and rotated 180O around the long axis (z-axis) of the sample. The X-ray radiograph image was collected at 41 kV/234 µA, 8.70 µm pixel every 0.45O angle of rotation (0O180O rotation) and exposure time of 1.2 seconds with 1.0 of gain. These projection radiographs were used in standard cone beam reconstruction NRecon software to generate a series of 417 axial slices to the within slide pixel spacing. The resulting 3D data sets were isotropic with the entire spatial range. 3D reconstruction of the internal pore morphology was carried out using these axial binary images and the porosity, pore wall thickness and pore size were measured and analyzed using CTAn software. 2.7 Compressive modulus and recovery capability analysis GG and PANi-GG dried polymeric networks and also spongy-like hydrogels were submitted to compression tests using a Universal Mechanical Testing machine (model 5543, INSTRON) and then



Page 8 of 34

the recovery capability was measured. GG and PANi-GG dried polymeric networks were submerged in Dulbecco’s modified Eagle medium (DMEM) (pH 7.4, room temperature) up to 10 min to obtain spongy-like hydrogels and prior to starting of the compression. Measurements were conducted at 1 mm.min-1 using a step mode with compression of 60% of their three dimensional microstructures. The recovery capacity was evaluated in DMEM bathing solution after compression of 60% by measuring, using a caliper, the specimen height before (Hi), immediately (HDef) and after 10 min, 1h and 24h, according to the following Equation 3 and 4:

./0 = 0.4 × .

Recovery (%) =

Equation 3

(9:;9) × 9

100

Equation 4

For the calculations, deformation of 60 % was set as 0 % of recovery. Six samples were analyzed. 2.8 Swelling capability The swelling capability of GG and PANi-GG spongy-like hydrogels was determined by submerging dried polymeric networks in DMEM, at 37OC up to 7 days. The swelling capacity of GG and PANiGG spongy-like hydrogels was calculated from the initial weight of dried polymeric networks (Wi) and the weight of swollen spongy-like hydrogels, at different time point (Wt), in sixplicate (Equation 5). Swelling capacity =

I+;J* I*

× 100

Equation 5

2.9 Weight loss For weigh loss analysis upon their degradation, all samples were immersed in PBS solution at pH of 7.4 with 0.02% of sodium azide and 37oC under orbital agitation (60 rpm) up to 60 days. GG and


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


PANi-GG dried polymeric networks were weighted at the beginning. At each time point, the samples were removed from the PBS solution, frozen at -80OC (48h) and lyophilized for 72h. After lyophilization, the samples were weighted (Wf). All data were obtained in sixplicate. The percentage of weight loss was calculated as Equation 6; % Weight loss =

IN;I* I*

× 100

Equation 6

2.10 Cytotoxicity assay L929 cells were used to evaluate the cytotoxicity of PANi-GG spongy-like hydrogels by direct contact following ISO 10993-5. To perform direct contact assay, L929 cells were plated at a density of 25,000 cells/well on tissue culture plates (TCPS, 24 wells). After 24h, PANi-GG and GG dried polymeric networks were placed in direct contact with the adhered L929 cells becoming spongy-like hydrogels. The spongy-like hydrogels were kept in contact with L929 cells for 7 days considering that the metabolic activity of L929 cells was quantified by MTS assay (Promega Corporation, USA) and Quant-iTTM PicoGreen dsDNA Assay kit quantification kit (Life Technologies, U.S). C2C12 cells (ATCC) were grown in DMEM supplemented with 10% of FBS (Life Technologies, U.S.), 1% of antimycotic agent (Life Technologies, U.S.), and maintained at 37ºC, in a humidified tissue culture incubator with a 5% CO2 atmosphere. 2.10.1 MTS Assay The effect of GG and PANi-GG spongy-like hydrogels on L929 metabolic activity was evaluated by MTS (Cell Titer 96 AQueous One Solution Cell Proliferation Assay, Promega) assay at day 1, 3, and 7 of culture. After each time point, the culture medium was removed, the constructs were washed with PBS solution and 600 µL/well of MTS solution (1:6 in culture medium without phenol red) was added. The plate was incubated at 37°C for 3h. After the incubation time, MTS solution was homogenized by pipetting and 100 µL of MTS was transferred to wells of a 96-well plate. The absorbance (490 nm) was finally measured using a microplate reader (Synergy HT, Bio-TeK, USA).



2.10.2 Quant-iTTM PicoGreen  dsDNA Assay To measure the cytotoxic effect of GG and PANi-GG spongy-like hydrogels leached chemicals on the proliferation profile of L929 cells, we quantified Cell DNA using the Quant-iTTM PicoGreen dsDNA Assay kit (InvitrogenTM). After 1, 3, and 7 days of culture, the materials and the culture medium were removed, cells were washed with PBS solution and 1 mL of ultrapure water was added to each well. After added water, the plates were incubated at 37°C for 1h to provide DNA exposure by the osmotic balance and then frozen at -80°C. To analyze the proliferative profile of L929 cells, lambda DNA-standard provided by PicoGreen kit was used to construct the calibration curve. dsDNA of L929 cells were quantified following the guidelines of PicoGreen kit. Fluorescence spectroscopy was used to quantify dsDNA using 485/20 nm for excitation and 528/20 nm for emission. Assays for metabolic activity and proliferation of L929 were performed without changing the culture medium during all the experimental time; the interference levels of leached chemicals from GG and PANi-GG spongy-like hydrogels on the proliferation of L929 cells were quantified by the dsDNA assay mentioned here. 2.11 Cell adhesion assay and immunocytochemistry To evaluate the adhesiveness and morphology of C2C12 cultured on PANi-GG and GG spongy-like hydrogels, 25,000 cells/mm3 were entrapped within spongy-like hydrogels using DMEM supplemented with 2% of horse serum and 1% of antimycotic agent (differentiation medium). C2C12 cells were cultured on PANi-GG and GG spongy-like hydrogels for 96 hours at atmosphere of 37°C. PANi-GG and GG dried polymeric networks were not soaked with culture medium prior seeding. C2C12 cells were used from the passage (P) 15 to 17. After 24, 48 and 96h, cells were fixed with formalin (10%, Bio-Optica Milano S.p.a., Italy) and the cytoskeleton of the cells stained with Phalloidin-TRITC (1:500, Sigma-Aldrich, USA). Nuclei were


Page 10 of 34

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


stained with 4', 6-diamidino-2-phenylindole (DAPI, 1:1000, Sigma-Aldrich, USA). Samples were observed in an Axio Imager Z1m fluorescence microscopy (Zeiss, Germany) and images were acquired with the ZEN 2.1 blue edition software.

2.12 Immunocytochemistry After 7 days, cells were fixed with formalin (10%, Bio-Optica Milano S.p.a., Italy), permeabilized with 0.2% v/v of cold Triton-X 100 and blocked with 2.5 % w/v of horse serum (Vector Laboratories, USA) for 30-45 min. Cell-laden spongy-like hydrogels were incubated with primary antibody myosin (1:50, EBioscience) overnight at 4ºC and with the secondary antibody Alexa Fluor 488 for 2h at RT, according to the host of the primary antibody. Between antibodies incubations, cells were washed with PBS for 2h by replenishing the PBS every 10/20 min. Cells were also incubated with DAPI and phalloidin-TRITC, as previously stated. Cells were observed using a Leica TCS SP8 confocal microscope (Leica, Germany). 2.13 Statistic and data analysis Quantitative data were statically analyzed using Statistica. Significant differences were evaluated by Breakdown & one-way ANOVA pairwise comparisons were performed using a Tukey’s adjustment. P-values

polyaniline spongy-like hydrogels

Recommend Documents