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PVA hydrogels
Materials Science and Engineering C 29 (2009) 1574–1583
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Materials Science and Engineering C j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / m s e c
Cytocompatibility evaluation in cell-culture systems of chemically crosslinked chitosan/PVA hydrogels Herman S. Mansur a,⁎, Ezequiel de S. Costa Jr. a, Alexandra A.P. Mansur a, Edel Figueiredo Barbosa-Stancioli b a b
Department of Metallurgical Engineering and Materials, Federal University of Minas Gerais, Rua Espírito Santo, 35/206, Centro, 30.160-030, Belo Horizonte/MG, Brazil Department of Microbiology, Institute of Biological Science, UFMG, Brazil
a r t i c l e
i n f o
Article history: Received 30 July 2008 Received in revised form 21 November 2008 Accepted 10 December 2008 Available online 16 December 2008 Keywords: Cytocompatibility Cell culture Biomaterial Hydrogel Poly(vinyl alcohol) Chemical crosslinking
a b s t r a c t In the present study we report the preparation, characterization and cytocompatibility of novel polymeric systems based on blends of chitosan and poly(vinyl alcohol) (PVA) and chemically crosslinked by glutaraldehyde for biomedical applications. The structure of the hydrogels was characterized through Fourier Transform Infrared spectroscopy (FTIR) and their swelling behavior was investigated as preliminary in vitro test. Bioactivity, cytotoxicity and cell viability were assessed via MTT assay with 2 cell cultures and cell spreading-adhesion analysis. Moreover, the cell viability and potential biocompatibility were assessed by the secretion of nitric oxide by activated macrophages with gamma interferon (IFN-γ) cytokine and lipopolysaccharide (LPS). It was found that by increasing the chitosan to PVA ratio the swelling behavior was significantly altered. In addition, all tested hydrogels have clearly presented adequate cell viability, nontoxicity and suitable properties which can be tailored for prospective use in tissue engineering. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Chitosan is a copolymer of glucosamine and N-acetylglucosamine derived from the natural polymer chitin, which is commercially available. Chitosan can be considered as one of the most investigated materials in recent years. The non-toxicity, high biocompatibility, and antigenicity of chitosan have driven the interest of the scientific community due to its potential applications in biomedical field. It was reported that chitosan could regulate cell biology such as differentiation, proliferation, and cytokine production [1–4]. The biocompatibility of chitosan brings its utility as biomaterial, in tissue engineering, membranes and drug delivery systems [1–4]. But chitosan has some drawbacks, it is only soluble in aqueous medium in the presence of a small amount of acid such as acetic acid and its mechanical properties are not readily suitable for some biomedical applications. Also, it has been reported the toxicity of chitosan to some organisms associated with its bacteriostatic effect [4–7]. That has raised some concern about using chitosan as a fully biocompatible polymer. So, it is important to move beyond standard regular bioactivity methods in order to properly evaluate chitosan-derived hydrogels. As a result many researchers have tried to modify its properties without compromising the viability of future use in living organisms. One path to be pursuit is the utilization of natural or synthetic polymers, separately or blended, with grafted or crosslinked
⁎ Corresponding author. E-mail address: [email protected] (H.S. Mansur). 0928-4931/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2008.12.012
networks, in order to match the required properties. In other words, the polymer blended crosslinked system may present differential degradation behavior under physiological fluid conditions, where part of polymeric network may undergo to fast solvation and another portion may experience slow degradation by de-polymerization. Hence, chitosan joined to other polymers opened a window of research for altering or tailoring the property of interest [4–7]. Among organic polymers, poly(vinyl alcohol) (PVA) is one of the very few vinyl polymers soluble in water that has been studied intensively because of its attractive features for medical applications such as high hydrophilicity, good film forming and processability [7,8]. In addition, the published literature has indicated that no significant difference was found in quantitative or in qualitative cytotoxicity evaluation of PVA. It was also shown that the amount of PVA accumulated in organs was too small to affect the biological fate, which suggested that PVA is excreted to the same extent as for example PEG, supporting the cytocompatibility of PVA [9]. Focusing on modifying the polymeric network, glutaraldehyde has been broadly used as an active chemical crosslinker due to the formation of intra–interchain covalent bondings. Nevertheless, this synthetic crosslinking reagent has been reported as highly cytotoxic that may impair the biocompatibility of the crosslinked biomaterials [10]. That disadvantage can be overcome by assuring that all aldehyde functional groups are actually crosslinking the polymeric network or they are effectively blocked with molecules such as aminoacids and proteins which are widely present in living organism serum. MTT and cell adhesion have been used for preliminary evaluation of biocompatibility of biomaterials for quite some time. More recently, nitric oxide (NO) which is a potent
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signaling molecule secreted by healthy endothelial cells has been utilized to assess the activity of cells. NO is a highly reactive free radical involved in a number of physiological and pathological processes [11]. As a result, nitric oxide production by macrophages can be examined as indicator of cell activation. A prospective biomaterial has to undergo extensive in vitro and in vivo tests and should ensure non-toxicity to the biological hosting site where it will be utilized. Cell-based compatibility assays must be properly conducted as a reliable tool of predicting the biomaterial behavior in vivo and future clinical performance. Thus, in the present research we have developed a novel system by blending chitosan with PVA (low degree of hydrolysis), at different ratios followed by chemical crosslinking with glutaraldehyde, aiming for altering the polymeric network. These synthesized chitosan/PVA films were spectroscopically and structurally characterized via FTIR. Moreover, cell cultures were used for assessing cytocompatibility and cell viability cell behavior using MTT method, cell spreading-adhesion via SEM analysis and nitric oxide production by activated macrophages with gamma interferon (IFN-γ) cytokine and lipopolysaccharide (LPS). 2. Materials and methods 2.1. Materials All salts and reagents used were of analytical degree and Milli-Q water was used in all solutions (18.0 MΩ). Poly (vinyl alcohol-co-vinyl acetate) (PVA) supplied from Sigma-Aldrich with 80% degree of hydrolysis and molar weight MW = 9000–10,000 g/mol. Chitosan (Aldrich Chemical) powder, medium molecular weight, degree of deacetylation (DD) = 80%, was used without further purification. Glutaraldehyde (GA) or 1,5-pentane-dial (Aldrich Chemical) used as covalent chemical crosslinking reagent was purchased as a 25% (wt.%) aqueous solution. 2.2. Methods 2.2.1. Chitosan and PVA solution preparation Briefly, PVA hydrogels were prepared by fully dissolving 5.0 g of polymer powder without further purification in 100 mL of Milli-Q water, under magnetic stirring, at temperature of 75 °C± 2 °C, as previously reported by our group [7,8,12]. PVA 5% solution was cooled down to room temperature and the pH was corrected to (2.00 ± 0.05) with 1.0 M HCl (Sigma). Chitosan hydrogels (Chi) were produced in a similar procedure by fully dissolving 2.5 g in 250.0 mL of Milli-Q water with 2% of CH3COOH (Sigma), under constant magnetic stirring for 48 h. 2.2.2. Chitosan, PVA and blends films preparation Different quantities of PVA were added into the 1.0% chitosan solution in order to obtain chitosan/PVA mass ratios of (0:1), (1:3), (1:1), (3:1) and (1:0) and pH was corrected to (4.00 ± 0.05) with 1.0 M NaOH solution. The mixture was kept under stirring for 5 min until the PVA and chitosan completely formed a clear solution. Then, the crosslinker reagent (glutaraldehyde) was slowly added under constant stirring. The final concentration of glutaraldehyde in the gel solution precursors was 1% and 5% (wt.%). Further in the sequence, the solution was poured into plastic moulds (polyethylene, round-plate shape, diameter = 85 mm, height = 10 mm) and let drying for 72–120 h at room temperature, and finally dried at 40 °C for 24 h (constant weight). Chitosan/PVA samples chemically crosslinked were identified by (X:Y:Z) that is, X as chitosan content, Y as PVA content and Z as glutaraldehyde (wt.%). For instance, sample labeled as Chi/PVA/GA (1:3:1) represents the following proportion of reagents: 25% chitosan, 75% PVA and crosslinked with 1.0% GA (wt.%). The dried gel was stored in a desiccator before all subsequent characterization procedures. The average film thickness produced was assessed with a Mitotoyo (±10 µm) micrometer (4 measurements each sample).
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2.3. Characterization 2.3.1. Fourier transform infrared spectroscopy (FTIR) FTIR spectroscopy analysis was performed to characterize the presence of specific chemical groups in the hydrogels and also the effectiveness on the crosslinking process. Chitosan, PVA and their blends before and after crosslinking with GA (Chi/PVA/GA) were prepared as 100 to 200 µm thick films and analyzed by FTIR using ATR (attenuated total reflection) modes. FTIR spectra were collected with wavenumber ranging from 4000 to 650 cm− 1 during 64 scans, with 2 cm− 1 resolution (Paragon 1000, Perkin-Elmer, USA). The FTIR spectra were normalized and major vibration bands were identified and associated with the main chemical groups. 2.3.2. Simulated body fluid assay (SBF) Preliminary cytocompatibility was investigated in the present work, by using a protein-free acellular simulated body fluid medium (SBF or Kokubo solution) with pH (7.40) and ionic composition (Na+ 142.0, K+ 2− 2− 5.0, Ca2+ 2.5, Mg2+ 1.5, Cl− 147.8, HCO− 3 4.2, HPO4 1.0, SO4 0.5 mM) equal to those in blood plasma [12]. Hence, chitosan/PVA hydrogel samples were cut into 5.0 mm×5.0 mm square pieces and soaked in SBF at pH 7.4 for swelling assay. Samples were evaluated at 30 min, 2 h, 4 h, 24 h, 96 h and 192 h. At the end of each soaking period, the remaining solution excess on the gels was wiped with a lint-free tissue paper, and dried at 40 °C in an oven for 24 h. 2.3.2.1. Swelling assay. Fluid absorption studies are of paramount importance for preliminary analysis of biodegradable materials. For fluid-uptake measurements, all the specimens of the chitosan/PVA hydrogels with molar ratios of 0:1, 1:3, 1:1, 3:1 and 1:0 were prepared as described in the previous section, were weighed (W0) before being immersed in SBF at 37 °C. After immersion for different time periods, the samples were carefully removed from the medium and, after wiping off water excess on the surface with filter paper, they were weighed for the determination of the wet weight (Wf) as a function of the immersion time [12]. Swelling index (S) is given by the Eq. (1): S=
Wf − W0 × 100k W0
ð1Þ
Each SBF absorption experiment was repeated three times and the average value was taken to validate the results. 2.3.3. Cytocompatibility, cell viability and bioactivity assays on polymeric blends 2.3.3.1. Neutralization procedures. Phosphate buffered saline (PBS) was used in the procedure to neutralize remaining cytotoxic groups of non-reacted glutaraldehyde crosslinker. The chitosan/PVA films were immersed in polyethylene flasks with 75 mL PBS solution without cells and with an area/volume ratio ranging from 0.5 to 1.0 cm− 1. The flasks were placed in an incubator with controlled temperature of 37 °C for 2.5 h. Later the samples were washed in de-ionized water, and dried at 40 °C for 48 h. All the samples submitted to the cytotoxicity experiment have been previously sterilized by exposure to saturated steam of ethylene oxide. It is rather important to emphasize that despite of being reported cytotoxic, glutaraldehyde reacts under acid condition with both amine and hydroxyls groups from chitosan and PVA. Yet, any remaining aldehyde unreacted group is almost immediately blocked by aminoacids and proteins present in living organisms sera. 2.3.3.2. Bioviability of chitosan/PVA blends in VERO cell culture. As previously reported by our group [12,13], African green monkey kidney VERO cells, a fibroblastic cell line, were used for the experiments of cell biocompatibility MTT (3-[4,5-dimethyltriazol-2-y1]-2,5-diphenyl tretrazolium) and adhesion assays. It is worth to point out that a fully
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Fig. 1. FTIR spectra of (a) chitosan, (b) Chi/PVA/GA (1:3:0), (c) Chi/PVA/GA (1:1:0), (d) Chi/PVA/GA (3:1:0) and (e) PVA vibrational bands without chemical crosslinking.
detailed description of the MTT is beyond the major goal of this research as it has been extensively reported in the literature. Briefly, 5×104 cells were seeded on matrices samples within a 96-well plate. The cells were incubated at 37 °C in humidified atmosphere containing 5% CO2. After 24 h incubation, supernatant of each well was replaced with MTT diluted in serum free medium and the plates incubated at 37 °C for 4 h. After that, SDS 10%/HCl 0.04 N solution was added to supernatant and plates were re-incubated for more 24 h and after exhaustive pipetation, 200μL was transferred to a clean 96-well plate, where absorbance was measured at 595 nm using ASYS EXPERT PLUS spectrometric microplate reader. For analysis, all data were expressed as average±standard deviation for number of 4 replicates (n=4). Statistical significance was determined for all groups and P values were generated by ANOVA using the Dunnett Test for multiple comparisons to one control (Pb 0.05, n≥4 assays). This method relies on assumptions of normality and homogeneity of the variances of the distributions. 2.3.3.3. Bioviability of chitosan/PVA blends in culture of cementoblasts 2.3.3.3.1. Culture of cementoblasts. Cementoblast cells were chosen as a biological model for periodontal application of the chitosan/PVA polymer blend developed in this work. So, cementoblasts were iso-
lated from the molars extracted from Wistar male rats (8 week old, 220–250 g). The extracted molars were rinsed once in Dulbecco's phosphate-buffered saline without calcium and magnesium (PBS). Then the molars were immersed in a digestion solution, 20 mL of Dulbecco's Modified Eagle Medium containing 0.04 g collagenase and 0.5 mL trypsin at 37 °C for five consecutive digestions were performed and five cells populations were obtained. The two first populations were discarded. The other three digestions produced a suspension of cells periodontal ligament (cells PDL) in various differentiation stages including cementoblasts. The three suspensions of cells were centrifuged for 5 min at 1500 RPM. The cells in each population were then cultured in DMEM (Dulbecco's Modified Eagle's Medium) with 10% fetal bovine serum (FBS), penicillin G sodium (10 U/mL) and streptomycin sulfate (10 mg/mL) in a humidified atmosphere of 5% CO2 at 37 °C. The cells were used for experiments at the third passage. Cementoblasts cells were identified by RT-PCR (Reverse transcription polymerase chain reaction) F-Spondin cell marker. The cementoblasts viability was also evaluated via MTT assay. The chitosan/PVA blended films were prepared on the well surface of a 24-well plate cells culture. Every result was obtained by 12 replicates and average absorbance was calculated (n = 12). Wells with only cells culture were used as
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positive controls. Cementoblasts were seeded on chitosan films at density of 1.0 × 105 cells seeded and then incubated at 37 °C and 5% CO2. After that, morphology of cells spread onto the novel materials was observed under optical microscope (Olympus IX70). After 48 h of incubation the culture medium was changed to 210 µL and 170 µL of MTT (5 mg/mL) was added to each well. Four hours later, formazan salts were dissolved with isopropanol. Then 100 µL of solution from each well was aspirated and poured in 96-well plate for absorbance measurement at λ = 595 nm. The absorbance was directly proportional to cell viability. One way ANOVA was used to access statistical significance of results and were expressed as percentage of cell viability. 2.3.3.4. Bioactivity evaluation – cell adhesion-spreading assay. Cell viability was evaluated by spreading and attachment assays in order to examine their morphology, adhesion and spreading behavior. VERO cells were plated at 6 × 104 density on the hosting scaffold; Cell spreading was evaluated by scanning electron microscopy (SEM, JEOL/ Noran, JSM 6360LV) of the specimens after culturing for 2 h. Before microscopy analysis, specimens were fixed with 2% glutaraldehyde for 16 h and dehydrated by passing through a series of alcohol solutions (ethanol–water). Then, they were dried in nitrogen flowing reactor for 4 h and out-gassed in vacuum desiccator for 12 h. Before examination the samples were sputtered with a thin layer of gold using low
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deposition rate, and placed at the maximum distance from the target to prevent damaging them. The images were obtained using an accelerating voltage of 15 kV. 2.3.3.5. Cell activity – nitric oxide (NO) production assay. As far as biocompatibility is concerned it is of paramount importance to move beyond standard regular bioactivity methods in order to properly evaluate chitosan-derived hydrogels. Therefore, a novel approach is proposed in this research where nitric oxide secretion was stimulated by cytokine (IFN-γ) and other inductive moiety (LPS) to assess cell activity in contact with polymeric systems developed. Mice were euthanized by halothane inhalation (approved by university ethical committee), and then 7–10 mL ice-cold Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS) was injected into their peritoneal cavities. Lavage fluids were pooled from several mice and large, peritoneal macrophages cells (line C57BL/6J) were counted on a hemacytometer. Then, 200 µL of medium (RPMI 1640 with 10% FBS) with cells were seeded in 24-well tissue culture plates (Costar) at density at 1.0× 105 with chitosan/PVA samples previously placed (4 replicates of each). Negative controls were prepared by adding 200 µL of medium (RPMI 1640 with 10% FBS) without any samples (n = 4). Moreover, the macrophage cells were stimulated for NO production by addition of cytokine IFN-γ (120 U/mL) and lipopolysaccharide (LPS) (100 ng/mL) in the
Fig. 2. Schematic representation of chemical crosslinking by glutaraldehyde (A) PVA and (B) chitosan; Reaction of chitosan amino groups with glutaraldehyde (Scheme 1).
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medium (RPMI 1640 with 10% FBS). All plates were then kept in a 37 °C incubator supplemented with 5% CO2. Macrophages were allowed to adhere for 48 h, at which point the wells were washed three times with endotoxin-free, phosphate buffered saline (PBS) to rinse away most non-adherent cells. Production of nitric oxide was estimated by measuring nitrite levels in the supernatant with the Griess reagent (1:1 ratio of 0.1% of N-1-naphthylethylenediamine and 1% sulphanilamide in 5% phosphoric acid). Griess reagent was added to 100 µL of sample cell supernatant and was incubated at room temperature for 10 min. Absorbance was measured at wavelength = 550 nm. The absorbance values were converted to their corresponding concentration of nitric oxide produced using a standard curve for NaNO2. 3. Results and discussion 3.1. Qualitative assessment No heterogeneity was observed regarding to solubility, miscibility and phase segregation when the Chi/PVA blends with different proportions were visually inspected (not shown). Highly uniform yellowish optically transparent films were obtained with average thickness of 100 µm. It is needed to point out that as the crosslinker concentration was raised the chitosan/PVA films became relatively more brittle and fragile to be handled. 3.2. Fourier transform infrared spectroscopy (FTIR) FTIR spectroscopy was used to assess the polymer chemical groups (chitosan and PVA) and investigating the formation of crosslinked
networks from the blends with glutaraldehyde. Initially, Fig. 1 shows the FTIR spectra of samples prepared with chitosan and PVA with different ratios [Chi/PVA] without chemical crosslinking. Fig. 4a spectrum of pure chitosan shows peaks around 893 cm− 1 and 1156 cm− 1 corresponding to saccharide structure [7,12,14–17]. In spite of several peaks clustering in the amide II peak range from 1510 cm− 1 to 1570 cm− 1, there still were strong absorption peaks at 1658 cm− 1 and 1322 cm− 1, which are characteristic of chitosan and have been reported as amide I and III peaks, respectively. These peaks ranging from 1510 cm− 1 to 1570 cm− 1 may be attributed to partially deacetylated amino groups. In order words, there are contributions from both species, protonated amino (–NH) groups and acetyl groups (R–CfO) and confirms that the samples are not fully deacetylated (~80%). The broad peak at 1030 and 1080 cm− 1 indicates the C–O stretching vibration in chitosan. Another broad peak at 3447 cm− 1 is caused by amine N–H symmetrical vibration, which is used with 1650 cm− 1 for quantitative analysis of deacetylation of chitosan. Peaks at 2800 cm− 1 and 2900 cm− 1 are the typical C–H stretch vibrations [7,12,14–17]. The IR spectra of the Chi/PVA blended films (Fig. 1b, c and d) are different from that of the chitosan because of the ionization of the primary amino groups. There are two distinct peaks at 1408 cm− 1 and 1548–1560 cm− 1. Formation of the 1548–1560 cm− 1 peak is the symmetric deformation of –NH+ 3 resulting from ionization of primary amino groups in the acidic medium whereas the peak at 1408 cm− 1 indicates the presence of carboxylic acid in the polymers. The peaks at 1700–1725 cm− 1 are characteristic of the carboxylic acid dimmer. In the present study, the presence of carboxylic dimmer was due to the acetic acid used for dissolving the chitosan [7,12,14–17]. Hence, it can be clearly observed a significant reduction of intensities from main absorption bands related to chitosan, for instance amine
Fig. 3. Evaluation of chemical crosslinking of chitosan/PVA blend via FTIR spectroscopy with different concentration of GA. Left Y-axis amine band decrease (squares, solid line); Right Y-axis imine band increase (circles, dashed line). Schematic illustration of amine group (bottom left) and imine group (bottom right).
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Fig. 4. Evaluation of swelling degree of the Chi/PVA (1:3) blends with GA crosslinking content 0.0 wt.%, 1.0 wt.% and 5.0 wt.%. Legend: blank=Chi/PVA/GA (1:3:0); dashed=Chi/PVA/GA (1:3:1); solid=Chi/PVA/GA (3:1:5).
region (1500–1650 cm− 1), as its content was decreased from 100% (pure chitosan, Fig. 1a), 75% (Fig. 4b), 50% (Fig. 1c), 25% (Fig. 1d) and 0% (pure PVA, Fig. 1e). Fig. 1e shows the FTIR spectrum of PVA. All major peaks related to hydroxyl and acetate groups were observed. More specifically, the broad band observed between 3550 and 3200 cm− 1 is associated with the stretching O–H from the intermolecular and intramolecular hydrogen bonds. The vibrational band observed between 2840 and 3000 cm− 1 refers to the stretching C–H from alkyl groups and the peaks between 1750–1735 cm− 1 are due to the stretching CfO and C–O from acetate group remaining from PVA (saponification reaction of polyvinyl acetate) [15–17]. Furthermore, FTIR spectroscopy was used in order to investigate the evolution of chemical crosslinking associated with the addition of glutaraldehyde (GA). Fig. 2 presents a schematic representation of the chemical structures after reactions that may occur as covalent bonds are formed in PVA (Fig. 2A) and chitosan (Fig. 2B) as GA is added to the system. It is expected competitive and parallel reactions from aldehyde with both functional groups hydroxyls (–OH) and amines (–NH2), present in PVA and chitosan. Indeed, all results from chitosan-derived blends have shown a relative increase on their imine (–CfN–) bands and simultaneous drop on the amine (–NH2) vibrational band after chemical crosslinking by GA, as summarized in Fig. 3. The imine group was formed by the nucleophilic addition reaction of the amine from chitosan with the aldehyde giving a hemiaminal (–C(OH)(NHR)–) followed by an elimination of water (Schiff base). Schiff base (or azomethine), is a functional group that contains a carbon–nitrogen double bond with the nitrogen atom connected to an aryl or alkyl group, but not hydrogen. Schiff bases are of the general formula R1R2-C–N-R3 [12,14–17]. In
Fig. 3, the major IR vibrational bands associated with imine (right Y-axis), amine (left Y-axis) and reference were plotted which have given evidence of an increase on the network crosslinking as the glutaraldehyde concentration was gradually raised from 0% (without GA) up to 5%. Hence, it reasonable to assume that in fact, the crosslinking mechanism occurred via covalent bond formation, preferably with chitosan amine group (Fig. 2B, Scheme 1) reacting with bi-functional aldehyde (GA) as it is favored over the hydroxyl group (Fig. 2A). 3.3. Biocompatibility test in vitro – swelling test Swelling assays are widely used as preliminary in vitro test for prospective evaluation of biomaterials, particularly hydrogels. Therefore, swelling experiments were conducted with Chi/PVA blends, with different polymer proportions and crosslinked by GA. A typical swelling behavior is presented in Fig. 4 which was performed for Chi/ PVA blend [25:75] before and after chemical crosslinking with 1% and 5% of content. It was observed basically 2 stages, an initial rapid mass uptake, usually in approximately 30 min, intrinsically related to volume increase, followed by mass stabilization over a period of 192 h. These results have revealed a strong influence of the crosslinking on the swelling index, which dropped from approximately 700% in Chi/PVA (1:3) sample before chemically crosslinking to almost 400% and 200%, with 1.0% and 5% glutaraldehyde, respectively. That fact is attributed to a more rigid network formed by the inter-intra polymer chain reactions that have occurred, reducing the flexibility and number of hydrophilic groups of hydrogel which is unfavorable to the swelling behavior. So, these results are corresponding to the expected hydrogel mechanism, where hydrophilic moieties present
Fig. 5. Swelling degree of chitosan, PVA and Chi/PVA blends with GA crosslinking content 5.0 wt.% after swelling for 192.0 h.
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Fig. 6. Viability of VERO cell on different matrices. Biocompatibility assay for cell viability was carried out for VERO cells seeded on Chi/PVA/GA matrices (MTT test).
in polymer network will favor water absorption by free-energy stabilization. Before GA reaction, the PVA chains are physically entangled with the chitosan chains, forming a hydrogel network. In the sequence, when the GA content was increased the chemical crosslinking has occurred, forming covalent bonds among chains, fixing and reducing polymer mobility, which resulted in the lower swelling index, in this case, less than half of the same blend proportion without chemical crosslinking (arrow, Fig. 4). The effect of chitosan to PVA ratio was also analyzed and the results are presented in Fig. 5 for samples crosslinked at 5.0% GA. It was verified that the swelling behavior is especially influenced by the chitosan content in the blend, where the swollen mass reduced by increasing the chitosan concentration and reaching a minimum value at [Chi/PVA] = 50:50. The swelling degree reduced from 200% (PVA without chitosan) to 100% (50:50 Chi/PVA), then raised to about 140% at Chi/PVA ratios of 75:25 and 100% chitosan. These results are supported by understanding the crosslinking reaction which has occurred in the blended hydrogels, where the amine groups of chitosan are more reactive to glutaraldehyde than hydroxyls of PVA. The minimum value observed at 50:50 (Fig. 5) is probably related to the overall balance between amine and hydroxyl crosslinking which is caused by the formation of a rigid structure amongst the Chi/PVA chains, reducing drastically their possibility of solution uptake. Swelling method has to be considered as a relative assessment of hydrogel behavior under aqueous medium as it depends of several factors and broad variations have been reported [7,12]. Nevertheless, the swelling properties of PVA and chitosan found in our study are in good agreement with literature, where PVA has a swelling degree above 500% and chitosan of about 200%, depending of course of the solution medium, pH, temperature and so forth [7,12–14]. It is important to emphasize that some solvation and dissolution may have occurred while swelling assays were performed and they accounted for about 10–15% of mass change (mass loss) during the experiments. This value did not alter significantly the average trend that was observed as the results were usually one or two orders of magnitude higher than that (200–700%).
(Fig. 6). Although it is possible to observe in Fig. 6 that the cell viability numbers varying from approximately 78% to 97% (comparing to VERO cell control as 100%), this difference was not statistically significant (ANOVA, p b 0.05; n ≥ 4). MTT method has been widely used as a suitable biocompatibility assay for in vitro evaluation of biomaterials. Usually, cytotoxicity tests using cell cultures have been accepted as the first step in identifying active compounds and for biosafety testing [7,12,15,18,19]. As a consequence, from MTT results it can be inferred that all the hydrogel matrices produced in the present research shows potential to be tested in vivo assays. 3.4.2. Cementoblast cell culture In order to move a step forward on estimating biocompatibility of chitosan-PVA matrices a novel test using cementoblast cell culture was conducted. Cementoblasts are derived from cells of the fibrous and cellular connective tissue that fills the space between the tooth and its bony socket and mediates tooth attachment to bone. They were used as biological model for potential application of the polymeric network on repairing periodontal tissues in the future [20]. So, as it can be observed in Fig. 7, the result of typical chitosan/PVA [1:3] blend before and after crosslinking concentration have shown strong evidence to be fully biocompatible to the culture under investigation. Despite a slight increase of compatibility as the glutaraldehyde content
3.4. Chitosan/PVA cytocompatibility assays with cell cultures 3.4.1. VERO cell culture Initially, cell viability was measured using MTT assay and that represents the active mitochondrial enzymes present in a cell capable of reducing MTT. In this study the viability test was measured at 24 h interval after cell seeding. The ability of the CHI/PVA matrices (crosslinked or not) to support all viability and proliferation shown that these samples evaluated exhibited comparable biocompatibility
Fig. 7. Viability of cementoblast cells seeded on Chi/PVA (1:3) with different network crosslinking densities (without GA, 1.0% and 5.0% GA) via MTT biocompatibility assay; Cellular control (CC) was used as reference.
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Fig. 8. Adhesion and spreading of VERO cells for biocompatibility assay. VERO cells seeded on Chi/PVA/GA matrices. (A) Cartoon of cell-interaction process: adhesion, spreading, proliferation; (B), (C) and (D) SEM images illustrating typical morphology of cells seeded on Chi/PVA/GA(1:3:1) blend.
was raised, all samples have presented over 80% (within the statistical variation) when compared to the cellular control reference. The results of samples with different chitosan to PVA ratio have indicated very similar trend. These results have endorsed our previous MTT results as far as cytotoxicity of chitosan-PVA is concerned. 3.4.3. Chitosan/PVA biocompatibility and spreading assay From MTT assays, the ability of the Chi/PVA matrices (crosslinked or not) to support cell viability was verified, where the whole set of hydrogel samples evaluated exhibited comparable biocompatibility. Beyond that, cell proliferation and growth was assessed via adhesion and spreading test, where general morphology was observed by SEM (Fig. 8). It can be noted that the VERO cells seeded on matrix with good adhesion and spreading morphology regular for this fibroblastic lineage. Since cellular attachment, adhesion, and spreading belong to the first phase of cell/material interactions, the quality of this phase will influence proliferation and differentiation of cells on biomaterials surfaces. Based on the SEM results obtained in our work, one may attribute the VERO cell spreading and adhesion verified on the chitosan/PVA blends to be a reliable proof of biocompatibility and non-cytotoxicity of samples. According to the literature [19–25], cell spreading are usually divided into three main interaction levels: (a) not spread: cells were still spherical in appearance, protrusions or lamellipodia were not yet produced; (b) partially spread: at this stage, cells began to spread laterally at one or more sides, but the extensions of plasma membrane were not completely confluent; and (c) fully spread. The last model (c) would represent the best result for material cell hosting. A schematic drawing of the cell-substrate interacting
process with its major steps is shown in Fig. 8A. Additionally, the cell line (VERO) used as a model in this research plays a role in producing many of the components essential to connective tissue, for example extracellular components such as glycosaminoglycans and, in fibrous tissue, collagen. Promoting the attachment of fibroblast cells would aid in integrating soft connective tissue to the implant, improving vascularity at the implant surface and decreasing the chance of fibrous encapsulation. In summary, cell adhesion and spreading are of vital importance in living biology processes and are involved in various natural phenomena such as embryogenesis, maintenance of tissue structure, wound healing, immune response, metastasis, and tissue integration of biomaterials [7,13,19–25]. 3.4.4. Cell activity – nitric oxide (NO) secretion assay Before going into reporting the results it is required some background on foreign body reaction from living organism induced by biomaterials. In summary, the interactions at the cell–substrate interface are mediated by extracellular signals such as cytokines or growth factors that induce changes in the cell substrate adhesion and the extracellular matrix. A prospective biomaterial has to undergo extensive in vitro and in vivo tests and should ensure non-toxicity to the biological hosting site where it will be utilized. Moreover, it should not elicit appreciable macrophage and cytokine response. Macrophages are involved in the host defense and play an important role in immune regulation. They induce generation of nitric oxide, a high level of which could lead to the rejection of a biomaterial during in vivo studies. Nitric oxide is a potent signaling molecule secreted by healthy endothelial cells that is capable of inhibiting the activation
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Fig. 9. Evaluation of nitric oxide secretion in macrophages before (blank columns) and after cytokine (IFN-γ) + LPS stimulation (solid black column) of chitosan-PVA blends. (The nitrite was measured by the Griess reagent method, absorbance reading at wavelength = 550 nm; data presented as average ± standard deviation of 4 replicates).
and adhesion of platelets, preventing inflammation and inducing vasodilation. Nitric oxide is the product of the oxidation of L-arginine to L-citrulline by nitric oxide synthase. Cytokines and endotoxin, for instance interferon (IFN)-γ and lipopolysaccharide (LPS), respectively, induce the expression of one isoform of nitric oxide synthase, which generates high levels of nitric oxide that is cytotoxic or cytostatic to target cells [26–29]. Consequently, it is essential to assess the NO production to reliably investigate biocompatibility. One should be aware that is beyond the scope of this study to present an in-depth description of the complete macrophage response to cytokines, as they have a rather complex pathway that it not yet understood, but the application of the concept on evaluating the hydrogels based on chitosan-PVA blends. Using this assay, it was discovered that chitosanPVA blends have presented satisfactory response regarding to nitric oxide secretion, as summarized in Fig. 9. It can be observed that all blends have given nitric oxide production values ranging from about 50% to 100% relatively compared to the value of control group of cells under cytokine stimulation (IFN-γ + LPS). Nevertheless, more than absolute values, some samples for instance Chi/PVA/GA (1:0:1), Chi/PVA/GA (1:3:1) and Chi/PVA/GA (0:1:5) (Fig. 9) have given results above 75% compared to control group even though this difference was not statistically significant (ANOVA, p b 0.05; n ≥ 4). It should be emphasized at this point that chemically crosslinked samples, Chi/PVA/GA (1:3:1) and Chi/PVA/GA (0:1:5), with 1% and 5% of glutaraldehyde, respectively, with NO secretion above 85%, have provided strong evidence that they are undoubtedly biocompatible. On the other hand, samples Chi/PVA/GA(1:3:0), Chi/PVA/GA(1:3:5) have shown lower NO response compared to control group with significant statistical difference (ANOVA, p b 0.05; n ≥ 4). Another important aspect to be addressed is concerned to the PVA samples exhibiting suitable nitric oxide secretion which is of paramount importance once several reports have been published where PVA was used as a standard control for inducing inflammation response in vivo. Again, the present study clearly shows that the biocompatibility response of biomaterials is much more complex than just running a specific in vitro assay and accepting the result as
generally and widely applicable to a whole class of materials. In fact, the most recent researchers have found that these systems should be analyzed according to the dynamical process that takes place in the living organism focusing on the inflammatory and wound healing responses following implantation of a medical device, prosthesis, or biomaterial as it gets in contact with the hosting tissue. That means, the correct approach should be the early-stage of inflammatory response followed by a cascade of molecules signaling and a end-stage of wound healing and tissue repairing. Therefore, it becomes essential to characterize the material factors determining the host response and to design new strategies for minimizing both acute and chronic inflammatory responses against implanted biomaterials. 4. Conclusion Chitosan/PVA blends were synthesized and chemically crosslinked with bi-functional aldehyde. The results have shown that by altering the proportion of chitosan to PVA, associated with different crosslinker concentration, some important properties from hydrogels can be modified. A significant reduction on the swelling behavior was verified as the chitosan content was increased and also as the concentration of crosslinking reagent (GA) was raised. Moreover, cytocompatibility, cell viability and preliminary bioactivity assays have given important evidence that all systems evaluated are non-toxic, biotolerant and potentially biocompatible. In summary, these novel functional hydrogels based on chitosan/PVA blends have broadened the number of choices of biomaterials to be potentially used in biomedical applications such as biomaterial, drug delivery vehicles and skin tissue engineering. Acknowledgements The authors acknowledge FAPEMIG-PPM/CAPES for financial support on this work. Authors also thank Mr. Mateus Laguardia and DDS Sandhra Carvalho for conducting MTT laboratory protocols.
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Materials Science and Engineering C j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / m s e c
Cytocompatibility evaluation in cell-culture systems of chemically crosslinked chitosan/PVA hydrogels Herman S. Mansur a,⁎, Ezequiel de S. Costa Jr. a, Alexandra A.P. Mansur a, Edel Figueiredo Barbosa-Stancioli b a b
Department of Metallurgical Engineering and Materials, Federal University of Minas Gerais, Rua Espírito Santo, 35/206, Centro, 30.160-030, Belo Horizonte/MG, Brazil Department of Microbiology, Institute of Biological Science, UFMG, Brazil
a r t i c l e
i n f o
Article history: Received 30 July 2008 Received in revised form 21 November 2008 Accepted 10 December 2008 Available online 16 December 2008 Keywords: Cytocompatibility Cell culture Biomaterial Hydrogel Poly(vinyl alcohol) Chemical crosslinking
a b s t r a c t In the present study we report the preparation, characterization and cytocompatibility of novel polymeric systems based on blends of chitosan and poly(vinyl alcohol) (PVA) and chemically crosslinked by glutaraldehyde for biomedical applications. The structure of the hydrogels was characterized through Fourier Transform Infrared spectroscopy (FTIR) and their swelling behavior was investigated as preliminary in vitro test. Bioactivity, cytotoxicity and cell viability were assessed via MTT assay with 2 cell cultures and cell spreading-adhesion analysis. Moreover, the cell viability and potential biocompatibility were assessed by the secretion of nitric oxide by activated macrophages with gamma interferon (IFN-γ) cytokine and lipopolysaccharide (LPS). It was found that by increasing the chitosan to PVA ratio the swelling behavior was significantly altered. In addition, all tested hydrogels have clearly presented adequate cell viability, nontoxicity and suitable properties which can be tailored for prospective use in tissue engineering. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Chitosan is a copolymer of glucosamine and N-acetylglucosamine derived from the natural polymer chitin, which is commercially available. Chitosan can be considered as one of the most investigated materials in recent years. The non-toxicity, high biocompatibility, and antigenicity of chitosan have driven the interest of the scientific community due to its potential applications in biomedical field. It was reported that chitosan could regulate cell biology such as differentiation, proliferation, and cytokine production [1–4]. The biocompatibility of chitosan brings its utility as biomaterial, in tissue engineering, membranes and drug delivery systems [1–4]. But chitosan has some drawbacks, it is only soluble in aqueous medium in the presence of a small amount of acid such as acetic acid and its mechanical properties are not readily suitable for some biomedical applications. Also, it has been reported the toxicity of chitosan to some organisms associated with its bacteriostatic effect [4–7]. That has raised some concern about using chitosan as a fully biocompatible polymer. So, it is important to move beyond standard regular bioactivity methods in order to properly evaluate chitosan-derived hydrogels. As a result many researchers have tried to modify its properties without compromising the viability of future use in living organisms. One path to be pursuit is the utilization of natural or synthetic polymers, separately or blended, with grafted or crosslinked
⁎ Corresponding author. E-mail address: [email protected] (H.S. Mansur). 0928-4931/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2008.12.012
networks, in order to match the required properties. In other words, the polymer blended crosslinked system may present differential degradation behavior under physiological fluid conditions, where part of polymeric network may undergo to fast solvation and another portion may experience slow degradation by de-polymerization. Hence, chitosan joined to other polymers opened a window of research for altering or tailoring the property of interest [4–7]. Among organic polymers, poly(vinyl alcohol) (PVA) is one of the very few vinyl polymers soluble in water that has been studied intensively because of its attractive features for medical applications such as high hydrophilicity, good film forming and processability [7,8]. In addition, the published literature has indicated that no significant difference was found in quantitative or in qualitative cytotoxicity evaluation of PVA. It was also shown that the amount of PVA accumulated in organs was too small to affect the biological fate, which suggested that PVA is excreted to the same extent as for example PEG, supporting the cytocompatibility of PVA [9]. Focusing on modifying the polymeric network, glutaraldehyde has been broadly used as an active chemical crosslinker due to the formation of intra–interchain covalent bondings. Nevertheless, this synthetic crosslinking reagent has been reported as highly cytotoxic that may impair the biocompatibility of the crosslinked biomaterials [10]. That disadvantage can be overcome by assuring that all aldehyde functional groups are actually crosslinking the polymeric network or they are effectively blocked with molecules such as aminoacids and proteins which are widely present in living organism serum. MTT and cell adhesion have been used for preliminary evaluation of biocompatibility of biomaterials for quite some time. More recently, nitric oxide (NO) which is a potent
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signaling molecule secreted by healthy endothelial cells has been utilized to assess the activity of cells. NO is a highly reactive free radical involved in a number of physiological and pathological processes [11]. As a result, nitric oxide production by macrophages can be examined as indicator of cell activation. A prospective biomaterial has to undergo extensive in vitro and in vivo tests and should ensure non-toxicity to the biological hosting site where it will be utilized. Cell-based compatibility assays must be properly conducted as a reliable tool of predicting the biomaterial behavior in vivo and future clinical performance. Thus, in the present research we have developed a novel system by blending chitosan with PVA (low degree of hydrolysis), at different ratios followed by chemical crosslinking with glutaraldehyde, aiming for altering the polymeric network. These synthesized chitosan/PVA films were spectroscopically and structurally characterized via FTIR. Moreover, cell cultures were used for assessing cytocompatibility and cell viability cell behavior using MTT method, cell spreading-adhesion via SEM analysis and nitric oxide production by activated macrophages with gamma interferon (IFN-γ) cytokine and lipopolysaccharide (LPS). 2. Materials and methods 2.1. Materials All salts and reagents used were of analytical degree and Milli-Q water was used in all solutions (18.0 MΩ). Poly (vinyl alcohol-co-vinyl acetate) (PVA) supplied from Sigma-Aldrich with 80% degree of hydrolysis and molar weight MW = 9000–10,000 g/mol. Chitosan (Aldrich Chemical) powder, medium molecular weight, degree of deacetylation (DD) = 80%, was used without further purification. Glutaraldehyde (GA) or 1,5-pentane-dial (Aldrich Chemical) used as covalent chemical crosslinking reagent was purchased as a 25% (wt.%) aqueous solution. 2.2. Methods 2.2.1. Chitosan and PVA solution preparation Briefly, PVA hydrogels were prepared by fully dissolving 5.0 g of polymer powder without further purification in 100 mL of Milli-Q water, under magnetic stirring, at temperature of 75 °C± 2 °C, as previously reported by our group [7,8,12]. PVA 5% solution was cooled down to room temperature and the pH was corrected to (2.00 ± 0.05) with 1.0 M HCl (Sigma). Chitosan hydrogels (Chi) were produced in a similar procedure by fully dissolving 2.5 g in 250.0 mL of Milli-Q water with 2% of CH3COOH (Sigma), under constant magnetic stirring for 48 h. 2.2.2. Chitosan, PVA and blends films preparation Different quantities of PVA were added into the 1.0% chitosan solution in order to obtain chitosan/PVA mass ratios of (0:1), (1:3), (1:1), (3:1) and (1:0) and pH was corrected to (4.00 ± 0.05) with 1.0 M NaOH solution. The mixture was kept under stirring for 5 min until the PVA and chitosan completely formed a clear solution. Then, the crosslinker reagent (glutaraldehyde) was slowly added under constant stirring. The final concentration of glutaraldehyde in the gel solution precursors was 1% and 5% (wt.%). Further in the sequence, the solution was poured into plastic moulds (polyethylene, round-plate shape, diameter = 85 mm, height = 10 mm) and let drying for 72–120 h at room temperature, and finally dried at 40 °C for 24 h (constant weight). Chitosan/PVA samples chemically crosslinked were identified by (X:Y:Z) that is, X as chitosan content, Y as PVA content and Z as glutaraldehyde (wt.%). For instance, sample labeled as Chi/PVA/GA (1:3:1) represents the following proportion of reagents: 25% chitosan, 75% PVA and crosslinked with 1.0% GA (wt.%). The dried gel was stored in a desiccator before all subsequent characterization procedures. The average film thickness produced was assessed with a Mitotoyo (±10 µm) micrometer (4 measurements each sample).
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2.3. Characterization 2.3.1. Fourier transform infrared spectroscopy (FTIR) FTIR spectroscopy analysis was performed to characterize the presence of specific chemical groups in the hydrogels and also the effectiveness on the crosslinking process. Chitosan, PVA and their blends before and after crosslinking with GA (Chi/PVA/GA) were prepared as 100 to 200 µm thick films and analyzed by FTIR using ATR (attenuated total reflection) modes. FTIR spectra were collected with wavenumber ranging from 4000 to 650 cm− 1 during 64 scans, with 2 cm− 1 resolution (Paragon 1000, Perkin-Elmer, USA). The FTIR spectra were normalized and major vibration bands were identified and associated with the main chemical groups. 2.3.2. Simulated body fluid assay (SBF) Preliminary cytocompatibility was investigated in the present work, by using a protein-free acellular simulated body fluid medium (SBF or Kokubo solution) with pH (7.40) and ionic composition (Na+ 142.0, K+ 2− 2− 5.0, Ca2+ 2.5, Mg2+ 1.5, Cl− 147.8, HCO− 3 4.2, HPO4 1.0, SO4 0.5 mM) equal to those in blood plasma [12]. Hence, chitosan/PVA hydrogel samples were cut into 5.0 mm×5.0 mm square pieces and soaked in SBF at pH 7.4 for swelling assay. Samples were evaluated at 30 min, 2 h, 4 h, 24 h, 96 h and 192 h. At the end of each soaking period, the remaining solution excess on the gels was wiped with a lint-free tissue paper, and dried at 40 °C in an oven for 24 h. 2.3.2.1. Swelling assay. Fluid absorption studies are of paramount importance for preliminary analysis of biodegradable materials. For fluid-uptake measurements, all the specimens of the chitosan/PVA hydrogels with molar ratios of 0:1, 1:3, 1:1, 3:1 and 1:0 were prepared as described in the previous section, were weighed (W0) before being immersed in SBF at 37 °C. After immersion for different time periods, the samples were carefully removed from the medium and, after wiping off water excess on the surface with filter paper, they were weighed for the determination of the wet weight (Wf) as a function of the immersion time [12]. Swelling index (S) is given by the Eq. (1): S=
Wf − W0 × 100k W0
ð1Þ
Each SBF absorption experiment was repeated three times and the average value was taken to validate the results. 2.3.3. Cytocompatibility, cell viability and bioactivity assays on polymeric blends 2.3.3.1. Neutralization procedures. Phosphate buffered saline (PBS) was used in the procedure to neutralize remaining cytotoxic groups of non-reacted glutaraldehyde crosslinker. The chitosan/PVA films were immersed in polyethylene flasks with 75 mL PBS solution without cells and with an area/volume ratio ranging from 0.5 to 1.0 cm− 1. The flasks were placed in an incubator with controlled temperature of 37 °C for 2.5 h. Later the samples were washed in de-ionized water, and dried at 40 °C for 48 h. All the samples submitted to the cytotoxicity experiment have been previously sterilized by exposure to saturated steam of ethylene oxide. It is rather important to emphasize that despite of being reported cytotoxic, glutaraldehyde reacts under acid condition with both amine and hydroxyls groups from chitosan and PVA. Yet, any remaining aldehyde unreacted group is almost immediately blocked by aminoacids and proteins present in living organisms sera. 2.3.3.2. Bioviability of chitosan/PVA blends in VERO cell culture. As previously reported by our group [12,13], African green monkey kidney VERO cells, a fibroblastic cell line, were used for the experiments of cell biocompatibility MTT (3-[4,5-dimethyltriazol-2-y1]-2,5-diphenyl tretrazolium) and adhesion assays. It is worth to point out that a fully
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Fig. 1. FTIR spectra of (a) chitosan, (b) Chi/PVA/GA (1:3:0), (c) Chi/PVA/GA (1:1:0), (d) Chi/PVA/GA (3:1:0) and (e) PVA vibrational bands without chemical crosslinking.
detailed description of the MTT is beyond the major goal of this research as it has been extensively reported in the literature. Briefly, 5×104 cells were seeded on matrices samples within a 96-well plate. The cells were incubated at 37 °C in humidified atmosphere containing 5% CO2. After 24 h incubation, supernatant of each well was replaced with MTT diluted in serum free medium and the plates incubated at 37 °C for 4 h. After that, SDS 10%/HCl 0.04 N solution was added to supernatant and plates were re-incubated for more 24 h and after exhaustive pipetation, 200μL was transferred to a clean 96-well plate, where absorbance was measured at 595 nm using ASYS EXPERT PLUS spectrometric microplate reader. For analysis, all data were expressed as average±standard deviation for number of 4 replicates (n=4). Statistical significance was determined for all groups and P values were generated by ANOVA using the Dunnett Test for multiple comparisons to one control (Pb 0.05, n≥4 assays). This method relies on assumptions of normality and homogeneity of the variances of the distributions. 2.3.3.3. Bioviability of chitosan/PVA blends in culture of cementoblasts 2.3.3.3.1. Culture of cementoblasts. Cementoblast cells were chosen as a biological model for periodontal application of the chitosan/PVA polymer blend developed in this work. So, cementoblasts were iso-
lated from the molars extracted from Wistar male rats (8 week old, 220–250 g). The extracted molars were rinsed once in Dulbecco's phosphate-buffered saline without calcium and magnesium (PBS). Then the molars were immersed in a digestion solution, 20 mL of Dulbecco's Modified Eagle Medium containing 0.04 g collagenase and 0.5 mL trypsin at 37 °C for five consecutive digestions were performed and five cells populations were obtained. The two first populations were discarded. The other three digestions produced a suspension of cells periodontal ligament (cells PDL) in various differentiation stages including cementoblasts. The three suspensions of cells were centrifuged for 5 min at 1500 RPM. The cells in each population were then cultured in DMEM (Dulbecco's Modified Eagle's Medium) with 10% fetal bovine serum (FBS), penicillin G sodium (10 U/mL) and streptomycin sulfate (10 mg/mL) in a humidified atmosphere of 5% CO2 at 37 °C. The cells were used for experiments at the third passage. Cementoblasts cells were identified by RT-PCR (Reverse transcription polymerase chain reaction) F-Spondin cell marker. The cementoblasts viability was also evaluated via MTT assay. The chitosan/PVA blended films were prepared on the well surface of a 24-well plate cells culture. Every result was obtained by 12 replicates and average absorbance was calculated (n = 12). Wells with only cells culture were used as
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positive controls. Cementoblasts were seeded on chitosan films at density of 1.0 × 105 cells seeded and then incubated at 37 °C and 5% CO2. After that, morphology of cells spread onto the novel materials was observed under optical microscope (Olympus IX70). After 48 h of incubation the culture medium was changed to 210 µL and 170 µL of MTT (5 mg/mL) was added to each well. Four hours later, formazan salts were dissolved with isopropanol. Then 100 µL of solution from each well was aspirated and poured in 96-well plate for absorbance measurement at λ = 595 nm. The absorbance was directly proportional to cell viability. One way ANOVA was used to access statistical significance of results and were expressed as percentage of cell viability. 2.3.3.4. Bioactivity evaluation – cell adhesion-spreading assay. Cell viability was evaluated by spreading and attachment assays in order to examine their morphology, adhesion and spreading behavior. VERO cells were plated at 6 × 104 density on the hosting scaffold; Cell spreading was evaluated by scanning electron microscopy (SEM, JEOL/ Noran, JSM 6360LV) of the specimens after culturing for 2 h. Before microscopy analysis, specimens were fixed with 2% glutaraldehyde for 16 h and dehydrated by passing through a series of alcohol solutions (ethanol–water). Then, they were dried in nitrogen flowing reactor for 4 h and out-gassed in vacuum desiccator for 12 h. Before examination the samples were sputtered with a thin layer of gold using low
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deposition rate, and placed at the maximum distance from the target to prevent damaging them. The images were obtained using an accelerating voltage of 15 kV. 2.3.3.5. Cell activity – nitric oxide (NO) production assay. As far as biocompatibility is concerned it is of paramount importance to move beyond standard regular bioactivity methods in order to properly evaluate chitosan-derived hydrogels. Therefore, a novel approach is proposed in this research where nitric oxide secretion was stimulated by cytokine (IFN-γ) and other inductive moiety (LPS) to assess cell activity in contact with polymeric systems developed. Mice were euthanized by halothane inhalation (approved by university ethical committee), and then 7–10 mL ice-cold Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS) was injected into their peritoneal cavities. Lavage fluids were pooled from several mice and large, peritoneal macrophages cells (line C57BL/6J) were counted on a hemacytometer. Then, 200 µL of medium (RPMI 1640 with 10% FBS) with cells were seeded in 24-well tissue culture plates (Costar) at density at 1.0× 105 with chitosan/PVA samples previously placed (4 replicates of each). Negative controls were prepared by adding 200 µL of medium (RPMI 1640 with 10% FBS) without any samples (n = 4). Moreover, the macrophage cells were stimulated for NO production by addition of cytokine IFN-γ (120 U/mL) and lipopolysaccharide (LPS) (100 ng/mL) in the
Fig. 2. Schematic representation of chemical crosslinking by glutaraldehyde (A) PVA and (B) chitosan; Reaction of chitosan amino groups with glutaraldehyde (Scheme 1).
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medium (RPMI 1640 with 10% FBS). All plates were then kept in a 37 °C incubator supplemented with 5% CO2. Macrophages were allowed to adhere for 48 h, at which point the wells were washed three times with endotoxin-free, phosphate buffered saline (PBS) to rinse away most non-adherent cells. Production of nitric oxide was estimated by measuring nitrite levels in the supernatant with the Griess reagent (1:1 ratio of 0.1% of N-1-naphthylethylenediamine and 1% sulphanilamide in 5% phosphoric acid). Griess reagent was added to 100 µL of sample cell supernatant and was incubated at room temperature for 10 min. Absorbance was measured at wavelength = 550 nm. The absorbance values were converted to their corresponding concentration of nitric oxide produced using a standard curve for NaNO2. 3. Results and discussion 3.1. Qualitative assessment No heterogeneity was observed regarding to solubility, miscibility and phase segregation when the Chi/PVA blends with different proportions were visually inspected (not shown). Highly uniform yellowish optically transparent films were obtained with average thickness of 100 µm. It is needed to point out that as the crosslinker concentration was raised the chitosan/PVA films became relatively more brittle and fragile to be handled. 3.2. Fourier transform infrared spectroscopy (FTIR) FTIR spectroscopy was used to assess the polymer chemical groups (chitosan and PVA) and investigating the formation of crosslinked
networks from the blends with glutaraldehyde. Initially, Fig. 1 shows the FTIR spectra of samples prepared with chitosan and PVA with different ratios [Chi/PVA] without chemical crosslinking. Fig. 4a spectrum of pure chitosan shows peaks around 893 cm− 1 and 1156 cm− 1 corresponding to saccharide structure [7,12,14–17]. In spite of several peaks clustering in the amide II peak range from 1510 cm− 1 to 1570 cm− 1, there still were strong absorption peaks at 1658 cm− 1 and 1322 cm− 1, which are characteristic of chitosan and have been reported as amide I and III peaks, respectively. These peaks ranging from 1510 cm− 1 to 1570 cm− 1 may be attributed to partially deacetylated amino groups. In order words, there are contributions from both species, protonated amino (–NH) groups and acetyl groups (R–CfO) and confirms that the samples are not fully deacetylated (~80%). The broad peak at 1030 and 1080 cm− 1 indicates the C–O stretching vibration in chitosan. Another broad peak at 3447 cm− 1 is caused by amine N–H symmetrical vibration, which is used with 1650 cm− 1 for quantitative analysis of deacetylation of chitosan. Peaks at 2800 cm− 1 and 2900 cm− 1 are the typical C–H stretch vibrations [7,12,14–17]. The IR spectra of the Chi/PVA blended films (Fig. 1b, c and d) are different from that of the chitosan because of the ionization of the primary amino groups. There are two distinct peaks at 1408 cm− 1 and 1548–1560 cm− 1. Formation of the 1548–1560 cm− 1 peak is the symmetric deformation of –NH+ 3 resulting from ionization of primary amino groups in the acidic medium whereas the peak at 1408 cm− 1 indicates the presence of carboxylic acid in the polymers. The peaks at 1700–1725 cm− 1 are characteristic of the carboxylic acid dimmer. In the present study, the presence of carboxylic dimmer was due to the acetic acid used for dissolving the chitosan [7,12,14–17]. Hence, it can be clearly observed a significant reduction of intensities from main absorption bands related to chitosan, for instance amine
Fig. 3. Evaluation of chemical crosslinking of chitosan/PVA blend via FTIR spectroscopy with different concentration of GA. Left Y-axis amine band decrease (squares, solid line); Right Y-axis imine band increase (circles, dashed line). Schematic illustration of amine group (bottom left) and imine group (bottom right).
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Fig. 4. Evaluation of swelling degree of the Chi/PVA (1:3) blends with GA crosslinking content 0.0 wt.%, 1.0 wt.% and 5.0 wt.%. Legend: blank=Chi/PVA/GA (1:3:0); dashed=Chi/PVA/GA (1:3:1); solid=Chi/PVA/GA (3:1:5).
region (1500–1650 cm− 1), as its content was decreased from 100% (pure chitosan, Fig. 1a), 75% (Fig. 4b), 50% (Fig. 1c), 25% (Fig. 1d) and 0% (pure PVA, Fig. 1e). Fig. 1e shows the FTIR spectrum of PVA. All major peaks related to hydroxyl and acetate groups were observed. More specifically, the broad band observed between 3550 and 3200 cm− 1 is associated with the stretching O–H from the intermolecular and intramolecular hydrogen bonds. The vibrational band observed between 2840 and 3000 cm− 1 refers to the stretching C–H from alkyl groups and the peaks between 1750–1735 cm− 1 are due to the stretching CfO and C–O from acetate group remaining from PVA (saponification reaction of polyvinyl acetate) [15–17]. Furthermore, FTIR spectroscopy was used in order to investigate the evolution of chemical crosslinking associated with the addition of glutaraldehyde (GA). Fig. 2 presents a schematic representation of the chemical structures after reactions that may occur as covalent bonds are formed in PVA (Fig. 2A) and chitosan (Fig. 2B) as GA is added to the system. It is expected competitive and parallel reactions from aldehyde with both functional groups hydroxyls (–OH) and amines (–NH2), present in PVA and chitosan. Indeed, all results from chitosan-derived blends have shown a relative increase on their imine (–CfN–) bands and simultaneous drop on the amine (–NH2) vibrational band after chemical crosslinking by GA, as summarized in Fig. 3. The imine group was formed by the nucleophilic addition reaction of the amine from chitosan with the aldehyde giving a hemiaminal (–C(OH)(NHR)–) followed by an elimination of water (Schiff base). Schiff base (or azomethine), is a functional group that contains a carbon–nitrogen double bond with the nitrogen atom connected to an aryl or alkyl group, but not hydrogen. Schiff bases are of the general formula R1R2-C–N-R3 [12,14–17]. In
Fig. 3, the major IR vibrational bands associated with imine (right Y-axis), amine (left Y-axis) and reference were plotted which have given evidence of an increase on the network crosslinking as the glutaraldehyde concentration was gradually raised from 0% (without GA) up to 5%. Hence, it reasonable to assume that in fact, the crosslinking mechanism occurred via covalent bond formation, preferably with chitosan amine group (Fig. 2B, Scheme 1) reacting with bi-functional aldehyde (GA) as it is favored over the hydroxyl group (Fig. 2A). 3.3. Biocompatibility test in vitro – swelling test Swelling assays are widely used as preliminary in vitro test for prospective evaluation of biomaterials, particularly hydrogels. Therefore, swelling experiments were conducted with Chi/PVA blends, with different polymer proportions and crosslinked by GA. A typical swelling behavior is presented in Fig. 4 which was performed for Chi/ PVA blend [25:75] before and after chemical crosslinking with 1% and 5% of content. It was observed basically 2 stages, an initial rapid mass uptake, usually in approximately 30 min, intrinsically related to volume increase, followed by mass stabilization over a period of 192 h. These results have revealed a strong influence of the crosslinking on the swelling index, which dropped from approximately 700% in Chi/PVA (1:3) sample before chemically crosslinking to almost 400% and 200%, with 1.0% and 5% glutaraldehyde, respectively. That fact is attributed to a more rigid network formed by the inter-intra polymer chain reactions that have occurred, reducing the flexibility and number of hydrophilic groups of hydrogel which is unfavorable to the swelling behavior. So, these results are corresponding to the expected hydrogel mechanism, where hydrophilic moieties present
Fig. 5. Swelling degree of chitosan, PVA and Chi/PVA blends with GA crosslinking content 5.0 wt.% after swelling for 192.0 h.
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Fig. 6. Viability of VERO cell on different matrices. Biocompatibility assay for cell viability was carried out for VERO cells seeded on Chi/PVA/GA matrices (MTT test).
in polymer network will favor water absorption by free-energy stabilization. Before GA reaction, the PVA chains are physically entangled with the chitosan chains, forming a hydrogel network. In the sequence, when the GA content was increased the chemical crosslinking has occurred, forming covalent bonds among chains, fixing and reducing polymer mobility, which resulted in the lower swelling index, in this case, less than half of the same blend proportion without chemical crosslinking (arrow, Fig. 4). The effect of chitosan to PVA ratio was also analyzed and the results are presented in Fig. 5 for samples crosslinked at 5.0% GA. It was verified that the swelling behavior is especially influenced by the chitosan content in the blend, where the swollen mass reduced by increasing the chitosan concentration and reaching a minimum value at [Chi/PVA] = 50:50. The swelling degree reduced from 200% (PVA without chitosan) to 100% (50:50 Chi/PVA), then raised to about 140% at Chi/PVA ratios of 75:25 and 100% chitosan. These results are supported by understanding the crosslinking reaction which has occurred in the blended hydrogels, where the amine groups of chitosan are more reactive to glutaraldehyde than hydroxyls of PVA. The minimum value observed at 50:50 (Fig. 5) is probably related to the overall balance between amine and hydroxyl crosslinking which is caused by the formation of a rigid structure amongst the Chi/PVA chains, reducing drastically their possibility of solution uptake. Swelling method has to be considered as a relative assessment of hydrogel behavior under aqueous medium as it depends of several factors and broad variations have been reported [7,12]. Nevertheless, the swelling properties of PVA and chitosan found in our study are in good agreement with literature, where PVA has a swelling degree above 500% and chitosan of about 200%, depending of course of the solution medium, pH, temperature and so forth [7,12–14]. It is important to emphasize that some solvation and dissolution may have occurred while swelling assays were performed and they accounted for about 10–15% of mass change (mass loss) during the experiments. This value did not alter significantly the average trend that was observed as the results were usually one or two orders of magnitude higher than that (200–700%).
(Fig. 6). Although it is possible to observe in Fig. 6 that the cell viability numbers varying from approximately 78% to 97% (comparing to VERO cell control as 100%), this difference was not statistically significant (ANOVA, p b 0.05; n ≥ 4). MTT method has been widely used as a suitable biocompatibility assay for in vitro evaluation of biomaterials. Usually, cytotoxicity tests using cell cultures have been accepted as the first step in identifying active compounds and for biosafety testing [7,12,15,18,19]. As a consequence, from MTT results it can be inferred that all the hydrogel matrices produced in the present research shows potential to be tested in vivo assays. 3.4.2. Cementoblast cell culture In order to move a step forward on estimating biocompatibility of chitosan-PVA matrices a novel test using cementoblast cell culture was conducted. Cementoblasts are derived from cells of the fibrous and cellular connective tissue that fills the space between the tooth and its bony socket and mediates tooth attachment to bone. They were used as biological model for potential application of the polymeric network on repairing periodontal tissues in the future [20]. So, as it can be observed in Fig. 7, the result of typical chitosan/PVA [1:3] blend before and after crosslinking concentration have shown strong evidence to be fully biocompatible to the culture under investigation. Despite a slight increase of compatibility as the glutaraldehyde content
3.4. Chitosan/PVA cytocompatibility assays with cell cultures 3.4.1. VERO cell culture Initially, cell viability was measured using MTT assay and that represents the active mitochondrial enzymes present in a cell capable of reducing MTT. In this study the viability test was measured at 24 h interval after cell seeding. The ability of the CHI/PVA matrices (crosslinked or not) to support all viability and proliferation shown that these samples evaluated exhibited comparable biocompatibility
Fig. 7. Viability of cementoblast cells seeded on Chi/PVA (1:3) with different network crosslinking densities (without GA, 1.0% and 5.0% GA) via MTT biocompatibility assay; Cellular control (CC) was used as reference.
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Fig. 8. Adhesion and spreading of VERO cells for biocompatibility assay. VERO cells seeded on Chi/PVA/GA matrices. (A) Cartoon of cell-interaction process: adhesion, spreading, proliferation; (B), (C) and (D) SEM images illustrating typical morphology of cells seeded on Chi/PVA/GA(1:3:1) blend.
was raised, all samples have presented over 80% (within the statistical variation) when compared to the cellular control reference. The results of samples with different chitosan to PVA ratio have indicated very similar trend. These results have endorsed our previous MTT results as far as cytotoxicity of chitosan-PVA is concerned. 3.4.3. Chitosan/PVA biocompatibility and spreading assay From MTT assays, the ability of the Chi/PVA matrices (crosslinked or not) to support cell viability was verified, where the whole set of hydrogel samples evaluated exhibited comparable biocompatibility. Beyond that, cell proliferation and growth was assessed via adhesion and spreading test, where general morphology was observed by SEM (Fig. 8). It can be noted that the VERO cells seeded on matrix with good adhesion and spreading morphology regular for this fibroblastic lineage. Since cellular attachment, adhesion, and spreading belong to the first phase of cell/material interactions, the quality of this phase will influence proliferation and differentiation of cells on biomaterials surfaces. Based on the SEM results obtained in our work, one may attribute the VERO cell spreading and adhesion verified on the chitosan/PVA blends to be a reliable proof of biocompatibility and non-cytotoxicity of samples. According to the literature [19–25], cell spreading are usually divided into three main interaction levels: (a) not spread: cells were still spherical in appearance, protrusions or lamellipodia were not yet produced; (b) partially spread: at this stage, cells began to spread laterally at one or more sides, but the extensions of plasma membrane were not completely confluent; and (c) fully spread. The last model (c) would represent the best result for material cell hosting. A schematic drawing of the cell-substrate interacting
process with its major steps is shown in Fig. 8A. Additionally, the cell line (VERO) used as a model in this research plays a role in producing many of the components essential to connective tissue, for example extracellular components such as glycosaminoglycans and, in fibrous tissue, collagen. Promoting the attachment of fibroblast cells would aid in integrating soft connective tissue to the implant, improving vascularity at the implant surface and decreasing the chance of fibrous encapsulation. In summary, cell adhesion and spreading are of vital importance in living biology processes and are involved in various natural phenomena such as embryogenesis, maintenance of tissue structure, wound healing, immune response, metastasis, and tissue integration of biomaterials [7,13,19–25]. 3.4.4. Cell activity – nitric oxide (NO) secretion assay Before going into reporting the results it is required some background on foreign body reaction from living organism induced by biomaterials. In summary, the interactions at the cell–substrate interface are mediated by extracellular signals such as cytokines or growth factors that induce changes in the cell substrate adhesion and the extracellular matrix. A prospective biomaterial has to undergo extensive in vitro and in vivo tests and should ensure non-toxicity to the biological hosting site where it will be utilized. Moreover, it should not elicit appreciable macrophage and cytokine response. Macrophages are involved in the host defense and play an important role in immune regulation. They induce generation of nitric oxide, a high level of which could lead to the rejection of a biomaterial during in vivo studies. Nitric oxide is a potent signaling molecule secreted by healthy endothelial cells that is capable of inhibiting the activation
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Fig. 9. Evaluation of nitric oxide secretion in macrophages before (blank columns) and after cytokine (IFN-γ) + LPS stimulation (solid black column) of chitosan-PVA blends. (The nitrite was measured by the Griess reagent method, absorbance reading at wavelength = 550 nm; data presented as average ± standard deviation of 4 replicates).
and adhesion of platelets, preventing inflammation and inducing vasodilation. Nitric oxide is the product of the oxidation of L-arginine to L-citrulline by nitric oxide synthase. Cytokines and endotoxin, for instance interferon (IFN)-γ and lipopolysaccharide (LPS), respectively, induce the expression of one isoform of nitric oxide synthase, which generates high levels of nitric oxide that is cytotoxic or cytostatic to target cells [26–29]. Consequently, it is essential to assess the NO production to reliably investigate biocompatibility. One should be aware that is beyond the scope of this study to present an in-depth description of the complete macrophage response to cytokines, as they have a rather complex pathway that it not yet understood, but the application of the concept on evaluating the hydrogels based on chitosan-PVA blends. Using this assay, it was discovered that chitosanPVA blends have presented satisfactory response regarding to nitric oxide secretion, as summarized in Fig. 9. It can be observed that all blends have given nitric oxide production values ranging from about 50% to 100% relatively compared to the value of control group of cells under cytokine stimulation (IFN-γ + LPS). Nevertheless, more than absolute values, some samples for instance Chi/PVA/GA (1:0:1), Chi/PVA/GA (1:3:1) and Chi/PVA/GA (0:1:5) (Fig. 9) have given results above 75% compared to control group even though this difference was not statistically significant (ANOVA, p b 0.05; n ≥ 4). It should be emphasized at this point that chemically crosslinked samples, Chi/PVA/GA (1:3:1) and Chi/PVA/GA (0:1:5), with 1% and 5% of glutaraldehyde, respectively, with NO secretion above 85%, have provided strong evidence that they are undoubtedly biocompatible. On the other hand, samples Chi/PVA/GA(1:3:0), Chi/PVA/GA(1:3:5) have shown lower NO response compared to control group with significant statistical difference (ANOVA, p b 0.05; n ≥ 4). Another important aspect to be addressed is concerned to the PVA samples exhibiting suitable nitric oxide secretion which is of paramount importance once several reports have been published where PVA was used as a standard control for inducing inflammation response in vivo. Again, the present study clearly shows that the biocompatibility response of biomaterials is much more complex than just running a specific in vitro assay and accepting the result as
generally and widely applicable to a whole class of materials. In fact, the most recent researchers have found that these systems should be analyzed according to the dynamical process that takes place in the living organism focusing on the inflammatory and wound healing responses following implantation of a medical device, prosthesis, or biomaterial as it gets in contact with the hosting tissue. That means, the correct approach should be the early-stage of inflammatory response followed by a cascade of molecules signaling and a end-stage of wound healing and tissue repairing. Therefore, it becomes essential to characterize the material factors determining the host response and to design new strategies for minimizing both acute and chronic inflammatory responses against implanted biomaterials. 4. Conclusion Chitosan/PVA blends were synthesized and chemically crosslinked with bi-functional aldehyde. The results have shown that by altering the proportion of chitosan to PVA, associated with different crosslinker concentration, some important properties from hydrogels can be modified. A significant reduction on the swelling behavior was verified as the chitosan content was increased and also as the concentration of crosslinking reagent (GA) was raised. Moreover, cytocompatibility, cell viability and preliminary bioactivity assays have given important evidence that all systems evaluated are non-toxic, biotolerant and potentially biocompatible. In summary, these novel functional hydrogels based on chitosan/PVA blends have broadened the number of choices of biomaterials to be potentially used in biomedical applications such as biomaterial, drug delivery vehicles and skin tissue engineering. Acknowledgements The authors acknowledge FAPEMIG-PPM/CAPES for financial support on this work. Authors also thank Mr. Mateus Laguardia and DDS Sandhra Carvalho for conducting MTT laboratory protocols.
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