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Transition from bioinert to bioactive material by tailoring the biological cell response to carboxylated nanocellulose Kai Hua, Igor Rocha, Peng Zhang, Simon Gustafsson, Yi Ning, Maria Stromme, Albert Mihranyan, and Natalia Ferraz Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00053 • Publication Date (Web): 17 Feb 2016 Downloaded from http://pubs.acs.org on February 18, 2016
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Transition from bioinert to bioactive material by tailoring the biological cell response to carboxylated nanocellulose Kai Hua*, Igor Rocha*,§, Peng Zhang*, Simon Gustafsson*, Yi Ning*, Maria Strømme*, Albert Mihranyan*,‡ and Natalia Ferraz*,‡ *
Nanotechnology and Functional Materials, Department of Engineering Sciences, Uppsala University,
Box 534, 75121, Uppsala, Sweden.; §
CAPES Foundation, Ministry of Education of Brazil, Brasília - DF 70040-020, Brazil;
Abstract This work presents an insight into the relationship between cell response and physicochemical properties of Cladophora cellulose (CC) by investigating the effect of CC functional group density on the response of model cell lines. CC was carboxylated by electrochemical TEMPO-mediated oxidation. By varying the amount of charge passed through the electrolysis setup, CC materials with different degrees of oxidation were obtained. The effect of carboxyl group density on the material´s physicochemical properties was investigated together with the response of human dermal fibroblasts (hDF) and human osteoblastic cells (Saos-2) to the carboxylated CC films. The introduction of carboxyl groups resulted in CC films with decreased specific surface area and smaller total pore volume compared with the unmodified CC (u-CC). While u-CC films presented a porous network of randomly oriented fibers, a compact and aligned fiber pattern was depicted for the carboxylated-CC films. The decrease in surface area and total pore volume, and the orientation and aggregation of the fibers tended to augment parallel to the increase in the carboxyl group density. hDF and Saos-2 cells presented poor cell adhesion and spreading on u-CC, which gradually increased for the carboxylated CC as the degree of oxidation increased. It was found that a threshold value in carboxyl group density needs be reached to obtain a carboxylated-CC film with cytocompatibility comparable to commercial tissue culture material. Hence this study demonstrates that a normally bioinert nanomaterial can be rendered bioactive by carefully tuning the density of charged groups on the material
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surface, a finding that not only may contribute to the fundamental understanding of biointerface phenomena but also to the development of bioinert/bioactive materials.
Keywords: Biocompatibility, Cladophora nanocellulose, surface group density, surface topography, TEMPO oxidation
1. Introduction Biomaterials research is one of the main drivers of medical innovation and it has been transforming many aspects of preventive and therapeutic healthcare. Since biomaterials are intended to come in contact with living cells, they need to be either biocompatible, bioresorbable or biodurable. Furthermore, depending on the application biomaterials may be required to be bioactive, bioinert or bioadaptive. Bioactive materials interact with the surrounding biological systems, for example promoting the regeneration of natural tissue or specific cell responses, while the so called bioinert materials present a minimum interrelation with the surrounding tissues or cells and do not elicit foreign body response. Bioadaptive or bioresponsive materials respond and adapt to a specific biological stimuli/environment. To fulfill the criteria described above, several critical hinders related to developing novel biomaterials have been identified including: − lack of fundamental understanding of biointerface phenomena; − gaps in correlation between molecular structure and biofunctionality; − shortage of technologies to direct hierarchical self-assembly; − limitations in tissue and organ repair. One biomaterial that has been in focus of numerous studies is nanocellulose thanks to its mechanical strength, broad chemical modifying capacity and nanoscale features, such as high aspect ratio and large surface area.1,
2
It can be obtained from different sources such as
bacteria, algae, fungi, tunicates and higher plants. Although nanocellulose is generally described as biocompatible, the statement is mainly based on data from cellulose and therefore it needs to be further investigated to account for the nanocharacteristics of the
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material and its use in specific applications. Moreover, depending on the source and processing, the properties of nanocellulose can greatly vary, including fiber dimensions, degree of crystallinity, aspect ratio and surface charge,3-5 all properties with the potential to greatly affect the biocompatible profile of the nanomaterial.6 Bacterial cellulose is the member of the nanocellulose family that has been most widely studied for biomedical applications and therefore a broad range of biocompatibility studies can be found in the literature.7-9 Several authors have investigated the biocompatible profile of cellulose nanocrystals as reviewed by Dugan et al.7 Nanofibrillated cellulose (NFC) from wood and algae nanocellulose are relatively novel materials in the biomedical field. Some investigations of the interaction of NFC with biological systems have been carried out considering specific biomedical applications of the nanomaterial,10-17 including the potential use of NFC gels as 3D tissue scaffolds,11,
14, 17
as wound healing dressings10,
12
and in
composites for ligament and tendons substitution.15, 16 Nevertheless, the understanding on how biological systems respond to changes in nanocellulose´s physicochemical and structural properties is still limited. With the aim of increasing such knowledge we have been investigating the influence of surface modifications to nanocellulose on its physicochemical properties and on its biocompatibility. In our previous study involving NFC from wood and highly crystalline nanocellulose from Cladophora algae, it was observed that surface modification of nanocellulose could be directly translated into a different biological response in contact with living cells.18 In particular, changes in nanocellulose surface topography, chemistry and charge had a measurable effect on proliferation and morphology of human dermal fibroblasts (hDF). For instance, carboxylated negatively charged Cladophora cellulose (a-CC) could promote hDF adhesion and spreading, comparable to the cells cultured with Thermanox® (TMX, a standard cell culture substrate), while the positively charged hydroxypropyltrimethylammonium modified CC (c-CC) or unmodified CC (u-CC) were unsuitable for fibroblast adhesion and spreading. Moreover, when studying the inflammatory response of monocytes/macrophages to the functionalized CC films, it was found that under pro-inflammatory stimuli a-CC films caused a more pronounced reduction in the levels of the pro-inflammatory cytokine TNF-α compared with the c-CC films.19 Interestingly, the a-CC films further showed higher degree of 3 ACS Paragon Plus Environment
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unidirectional nanofiber alignment, which was not observed in c-CC and u-CC films. In another study, we also found that surface chemical groups on the NFC have the ability to direct THP-1 monocytes activity. Monocytes cultured on carboxymethylated-NFC tended to produce higher level of TNF-α than cells on hydroxypropyltrimethylammonium-modified NFC films.20 Although these studies are indicative of the possibility of eliciting specific biological response by tailoring the surface properties of nanocellulose, more thorough studies are required to be able to attribute the observed effect to a single most important parameter. Thus, the present work is the continuation of our previous translational studies between the biological cell response and structural characteristics of nanocellulose, especially CC. Highly crystalline CC features several unique attributes such as entangled fibrous web structure, large specific surface area as well as excellent mechanical and rheological properties.21-23 Due to these unique features, many studies have been carried out to explore the potential applications of CC materials in the biomedical field, such as hemodialysis membranes,24-28 drug delivery vehicles,29, 30 virus removal filters31-33 and electrochemically controlled DNA extraction34-36. Previously, in order to produce a-CC, 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO) mediated oxidation of cellulose primary alcohols to carboxyls was used.18 TEMPO is a mild oxidant, which can be regenerated by auxiliary co-oxidants, e.g. NaClO or NaBrO. The TEMPO/NaBr/NaClO at pH ≥ 9 and TEMPO/NaClO/NaClO2 at pH ~ 5 have been extensively used for surface-limited cellulose carboxylation.37-43 An alternative to the use of auxiliary co-oxidants is to regenerate the reduced TEMPO molecules by electrochemical energy, the so called TEMPO electro-mediated reaction.44 This electrochemical approach has been previously reported as a green chemical process for selective oxidation of primary hydroxyls of water-soluble mono-, di- and polysaccharides45-47 as well as native cellulose44, 48. Recently, the electrochemically controlled TEMPO oxidation method was described to produce a-CC.49 The major benefit of this method lies in the possibility of precise control of the degree of CC oxidation during a TEMPO-mediated process since the density of carboxylate groups is directly proportional to the amount of charge passed through the electrolysis setup. Thus, the oxidation can be halted at a distinct point, and a range of 4 ACS Paragon Plus Environment
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materials with varying density of surface carboxylate groups can be obtained. Then, by studying the biological cell response to these materials fundamental understanding of the relation between surface charge density and functionality can be derived. Therefore, the goal of this study is to investigate the effect of CC functional groups density, varied through electrochemical TEMPO-mediated oxidation, on the behavior of adherent model cell lines, i.e. human dermal fibroblasts and human osteoblastic Saos-2 cells.
2. Materials and Methods 2.1 Chemicals Dulbecco's Modified Eagle Medium (DMEM-F12), Dulbecco’s phosphate buffered saline (PBS), dimethyl sulfoxide (DMSO, CAS No. 67-68-5), ethanol (CAS No. 64-17-5), hexamethyldisilazane (HMDS, CAS No. 999-97-3), glutaraldehyde (CAS No. 111-30-8), sodium chloride (NaCl, CAS No. 7647-14-5), sodium hydrogen carbonate (NaHCO3, CAS No.
144-55-8),
sodium
hydroxide
(NaOH,
CAS
No.
1310-73-2)
and
2,2,6,6,-tetramethylpiperidine-1-oxyl radical (TEMPO, CAS No. 2564-83-2) were purchased from Sigma Aldrich (Germany) and used as received. Alamar blue cell viability reagent (CAS No. 62758-13-8) was purchased from Invitrogen (USA).
2.2 Preparation of materials CC was obtained from FMC Biopolymers (USA) and purified by treating it with 0.1 mM NaOH aqueous solution for 1.5 h, which is referred to as unmodified CC (u-CC). Carboxylated CC [referred to as anionic-CC (a-CC)] was prepared by co-oxidant free, electrochemically controlled TEMPO oxidation (Figure 1), as previously described by Carlsson et al.49 The quantity of charge passed through the electrolysis setup was externally controlled during the reaction. Charge quantity corresponding to 20, 90, 250 and 430 C was used to produce a-CC containing different amount of carboxyl groups herein referred to as a-CC (Q20), a-CC (Q90), a-CC (Q250) and a-CC (Q430), respectively. In order to produce CC films, 300 mg of CC were dispersed in 50 mL deionized water using high-energy ultrasonic treatment (Vibracell 600 W, 20 kHz, USA), followed by vacuum filtration over a nylon filter membrane (0.1 µm mesh size). After air-drying overnight at room temperature and 5 ACS Paragon Plus Environment
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50% relative humidity, the CC films with a thickness of 0.2-0.3 mm were obtained and punched into 13 mm diameter discs. The CC discs, sterilized by autoclaving at 1.5 MPa for 15 min, were used in the cell studies.
Figure 1. Schematic representation of electrochemical TEMPO-mediated oxidation of nanocellulose
2.3 Characterization 2.3.1 Fourier transform infrared (FTIR) Spectroscopy Fourier transform infrared-attenuated total reflection (FTIR-ATR) spectra were recorded on a Spectrum One FTIR spectrometer equipped with a Diamond/ZnSe crystal (PerkinElmer, U.S.). The resolution was set to 4 cm−1, and 40 scans were averaged. Duplicates of each sample were analyzed at three different points in time, and peak positions were determined by the Spectrum software (PerkinElmer, U.S.).
2.3.2 Surface functional group quantification The amount of carboxyl groups in u-CC and a-CC was determined by conductometric titration. About 100 mg of material were dispersed in 60 mL NaCl (0.01 M) through high-energy ultrasonication (Vibracell 600 W, 20 kHz, USA) and the pH was adjusted to 2.8 by adding 6 ACS Paragon Plus Environment
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HCl (1 M) to ensure that all carboxyl groups on the nanocellulose surface were protonated. The dispersion was purged with nitrogen for 20 min prior to titrations in order to remove dissolved gases that can influence the pH of the medium. The titration was made with NaOH (0.05 M) in a Mettler Toledo T70 titrator, according to the procedure described by Carlsson et al.49
2.3.3 Scanning electron microscopy (SEM) SEM micrographs were taken with a Leo 1550 SEM instrument (Zeiss, Germany). Samples were mounted on aluminium stubs using a double-sided adhesive carbon tape and sputtered with Au/Pd with a plasma current of 30 mA for 30 s. The thickness of Au/Pd layer was approx. 10 nm.
2.3.4 Atomic force microscopy (AFM) A Bruker Dimension FastScan AFM system using a Bruker silicon nitride SCANASYST-AIR probe was used to obtain the images. The probe had a symmetric pyramidal geometry with a nominal tip radius of 2 nm. The samples were mounted on a magnetic holder using a double adhesive tape. The images were acquired in the peak-force tapping mode, using the manufacturer’s ScanAsyst automatic optimization algorithm. Post processing of the pictures was done in Bruker NanoScope Analysis v 1.6.
2.3.5 ζ-potential measurements Dispersions of 0.001% (w/w) a-CC and u-CC in NaCl (10 mM) were prepared through high-energy ultrasonication (Vibracell 600 W, 20 kHz, USA) and pH was adjusted to 7 with NaOH 0.05M. The electrophoretic mobility of the samples was measured using a universal dip cell, a ZetaSizer Nano instrument and a ZetaSizer Properties Software, all purchased from Malvern Instruments, UK.
2.3.6 X-ray diffraction (XRD)
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An X-ray diffractometer with Bragg-Brentano geometry and Cu Kα Radiation (λ = 1.54 Å) was used (Diffractometer D5000, Siemens, Germany). The crystallinity index (CrI) was calculated in accordance with Segal et al.50 CrI =
I 22o − I18o I 22o
× 100 ,
where I22° is the overall intensity of the peak at 2θ = 22° and I18° is the intensity of the baseline at 2θ = 18°.
2.3.7 Specific surface area (SSA) and pore size distribution analysis The nitrogen adsorption–desorption experiments were performed at 77K using a Micrometrics ASAP2020 surface area analyzer (Micromeritics, Norcross GA, USA). The samples were degassed at high vacuum (1×10-4 Pa) at 70°C for 24 h. The SSA was calculated using the Brunauer–Emmett–Teller (BET) method51 on the adsorption branch of the isotherm at P/P0 between
0.05
and
0.3.
The
pore
size
distribution
was
calculated
using
the
Barrett-Joyner-Halenda (BJH) method52 based on the desorption branch of the isotherm.
2.4 Cell culture Human dermal fibroblast (hDF) and human osteoblastic Saos-2 cells were cultured in DMEM-F12 medium supplemented with 10% (v/v) fetal bovine serum (FBS), 100 IU mL-1 penicillin, 100 µg mL-1 streptomycin in a humidified atmosphere of 95% air and 5% CO2 at 37oC. Cells were harvested using trypsin-EDTA treatment and counted using a haemocytometer. Cell viability was assessed through trypan blue staining (95-99% viable cells).
2.5 Cell response to the CC films The discs of CC films (u-CC, a-CC (Q20), a-CC (Q90), a-CC (Q250) and a-CC (Q430)), were placed in 24-well tissue culture plates and pre-soaked with PBS. Then, hDF or Saos-2 cells at a density of 50 000 cells per well were seeded onto the CC films and cultured at 37oC, 5% CO2 in a humidified atmosphere for 24±2 h. Cells cultured on TMX discs in the absence and
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presence of 5% DMSO served as the positive and negative controls, respectively. Samples were run in triplicate for each experiment and the experiments were repeated three times.
2.5.1 Alamar blue assay The number of viable cells adhered to the CC films was determined by the alamar blue assay. After 24 ± 2 h culture, the CC films were transferred to a new 24-well plate. 500 µL of alamar blue solution (1:10 in PBS) was added to the wells and incubated at 37oC, 5% CO2 in a humidified atmosphere for 90 min. Aliquots of 100 µL were transferred from each well to a 96-well plate. The fluorescence intensity was read at 560 nm of excitation wavelength and 590 nm of emission wavelength by using a spectrofluorometer (Tecan infinite® M200). The results are expressed as arbitrary units and reported as mean value ± standard error of the mean for n = 3. We have previously reported no interactions between the alamar blue reagent and the CC films.18
2.5.2 Scanning electron microscopy The CC films with adherent cells were moved to another 24-well plate after 24 ± 2 h of cell culture, rinsed with PBS and fixed with 2.5% (v/v) glutaraldehyde in PBS. Samples were dehydrated through a series of ethanol concentrations [10, 30, 50, 70, 90, 100% (v/v)], followed by incubations with HMDS solutions (HMDS 1:2 ethanol, HMDS 2:1 ethanol and 100% HMDS). After dehydration, samples were air-dried. Cell number and morphology were evaluated by using Leo 1550 SEM instrument (Zeiss, Germany). 2.6 Statistical analysis IBM SPSS statistics v.19 was used to perform statistical analysis with one way ANOVA (LSD and Tamhane post hoc test). Normal distribution was evaluated by Shapiro-Wilk test, and equal variances were evaluated by Levenes’s homogeneity of variance test. p value < 0.05 was considered to be statistically significant. 3. Results
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The electrochemical TEMPO-mediated modification of CC produced samples with different degrees of oxidation controlled by the charge used during the electrolysis. Figure 2 shows FTIR-ATR spectra of the different samples where it is possible to observe the increase in intensity of the peak centered at 1732 cm-1 referent to the C=O stretching of the carboxyl groups in oxidized CC chains49 (Full FTIR-ATR spectra can be found in the supporting information file).
0.00
Intensity (a.u.)
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0.02 0.04
1732 cm-1 0.06 0.08 0.10 0.12 1800
a-CC Q20 a-CC Q90 a-CC Q250 a-CC Q430
1750
1700
1650
1600
-1
Wavenumber (cm ) Figure 2. FTIR-ATR spectra of a-CC samples produced with different electrolysis charges. The spectra were normalized with respect to the C-H stretching vibration at 2897 cm-1. Note the increase in intensity of the peak centered at 1732 cm-1 that corresponds to the C=O stretching of the carboxyl groups in oxidized CC chains. The peak centered at 1640 cm-1 corresponds to the stretch of the OH groups in the water molecules present in the samples.53
The physicochemical properties of the samples are summarized in Table 1 which shows that conductometric titration and ζ-potential analyses corroborated FTIR spectroscopy results. The amount of carboxyl groups progressively increased from 32 µmol g-1 to 423 µmol g-1 with increasing amount of charge passed during the electrolysis. The corresponding ζ-potential values became increasingly more negative ranging from - 16 to - 41 mV, respectively.
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Table 1. Physicochemical properties of the CC materials under study Amount of
ζ-potential
Specific
Total pore
Degree of
carboxyl groups
pH 7 (mV)
surface area
volume
crystallinity
(m2 g-1)
(cm3 g-1)
(%)
(µmol g-1) u-CC
32±4
-16±1
106.7
0.441
92.6
Q20
77±7
-20±1
71.6
0.265
93.9
Q90
112±13
-30±2
79.6
0.300
93.6
Q250
259±10
-36±1
57.3
0.212
94.1
Q430
423±14
-41±3
64.4
0.188
93.9
Furthermore, while surface charge and composition were progressively altered during electrolysis, no significant difference in the degree of crystallinity was detected, which slightly varied between 92.6 and 94.1 %. According to BET gas adsorption surface analysis, u-CC has the highest SSA (106.7 m2 g-1). As the amount of the charge passing through the electrolysis setup is increased, the SSA values tended to decrease. Furthermore, the increased degree of surface oxidation of cellulose was generally paralleled with a decreasing total pore volume, suggesting a more compact structure for highly carboxylated samples. For instance, the total pore volume of a-CC (Q430) sample was more than halved as compared to u-CC. BJH pore size distribution analysis, derived from the desorption branch of the isotherm, reveals that the pore diameter ranges between approximately 2 and 30 nm with the average pore width at around 18 nm (Figure 3). A marked shift to smaller pore size was observed for a-CC (Q430) compared with the other materials.
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Q20 Q90 Q250 Q430 u-CC
2.0
3
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
dV/dlog(D) Pore Volume (cm g )
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1.5
1.0
0.5
0.0 1
10
100
Pore Width (nm) Figure 3. Pore size distribution of the CC materials
SEM images of the different CC materials (Figure 4) indicate that at higher degrees of oxidation there is an increase in the unidirectional fiber alignment. Compared with u-CC, which shows a relatively random fibril orientation, the a-CC (Q430) film shows highly aligned surface texture with much more compact arrangement, consistent with the nitrogen gas adsorption results. The compact and aligned pattern is further verified in high magnification AFM images as shown in Figure 5, suggesting that fiber aggregation tends to augment as the carboxyl group density is increased, in accordance with SSA and total pore volume data.
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a
1 µm
b
c
1 µm
d
1 µm
e
1 µm
1 µm
Figure 4. SEM images of the different CC films: a) u-CC, b) a-CC (Q20), c) a-CC (Q90), d) a-CC (Q250), e) a-CC (Q430). Note the random fiber orientation in the u-CC films and the increase in unidirectional fiber alignment for the films with higher degree of oxidation. Fiber aggregation seemed to augment as the carboxyl group density increased.
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a
b
c
d
e
Figure 5. AFM images of the different CC films: a) u-CC, b) a-CC (Q20), c) a-CC (Q90), d) a-CC (Q250), e) a-CC (Q430). The color scale represents the height difference of the sample surfaces.
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Cell response to CC with different degrees of oxidation The results in Figure 6 show that hDF cultured on u-CC had poor viability, even significantly lower than the value found for the negative control (5% DMSO). However, the viability of the adherent cells was improved when the carboxyl groups were incorporated onto the nanocellulose fibrils (the values found for all studied a-CC films were statistically significant higher than that of the negative control). The hDF on the a-CC (Q430) film showed the highest viability, comparable to the values found for the positive control, followed by cells cultured on a-CC (Q250) which was significantly higher than cells seeded on a-CC (Q20) and a-CC (Q90). No significant difference was observed between a-CC (Q20) and a-CC (Q90).
Viable adherent cells (AU)
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25000
*
20000
*
15000
*
*
10000
* 5000
0
u-CC
a-CC Q20 a-CC Q90 a-CC Q250 a-CC Q430
TMX
5% DMSO
Figure 6. Cell viability of hDF cultured on u-CC and a-CC films of varying functional group density. TMX served as positive control, while cells cultured on TMX in the presence of 5% DMSO were the negative control. *denotes a statistically significant difference versus the positive control (p
