Subscriber access provided by UB + Fachbibliothek Chemie | (FU-Bibliothekssystem)
Article
Cytocompatibility of Wood-derived Cellulose Nanofibril Hydrogels with Different Surface Chemistry Ahmad Rashad, Kamal Mustafa, Ellinor Bœvre Heggset, and Kristin Syverud Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01911 • Publication Date (Web): 06 Mar 2017 Downloaded from http://pubs.acs.org on March 8, 2017
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Biomacromolecules is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 32
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
Biomacromolecules
1
Cytocompatibility of Wood-derived Cellulose Nanofibril
2
Hydrogels with Different Surface Chemistry
3
Ahmad Rashad1, Kamal Mustafa1*, Ellinor Bævre Heggset2, Kristin Syverud2,3*
4
1
Department of Clinical Dentistry, University of Bergen, Norway
5
2
Paper and Fiber Research Institute, Trondheim, Norway
6
3
Norwegian University of Science and Technology (NTNU), Department of Chemical
7
Engineering, Trondheim, Norway
8
*
9
Kamal Mustafa, Ph.D.
Corresponding authors:
10
Department of Clinical Dentistry, University of Bergen, Norway
11
E-mail:
[email protected] 12
Phone: +4755586097
13
Kristin Syverud, Dr. ing.
14
Paper and Fiber Research Institute, Trondheim, Norway
15
E-mail:
[email protected] 16 17
KEYWORDS:
18
morphology.
TEMPO-mediated
oxidation,
Carboxymethylation,
Cytotoxicity,
Cell
1 ACS Paragon Plus Environment
Biomacromolecules
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
1
Page 2 of 32
Abstract
2
The current study aims to demonstrate the influence of the surface chemistry of wood-
3
derived cellulose nanofibril (CNF) hydrogels on fibroblasts for tissue engineering applications.
4
TEMPO-mediated oxidation or carboxymethylation pretreatments were employed to produce
5
hydrogels with different surface chemistry. This study demonstrates, firstly, the gelation of CNF
6
with cell culture medium and formation of stable hydrogels with improved rheological properties.
7
Secondly, the response of mouse fibroblasts cultured on the surface of the hydrogels or
8
sandwiched within the materials with respect to cytotoxicity, cell attachment, proliferation,
9
morphology and migration. Indirect cytotoxicity tests showed no toxic effect of either hydrogel.
10
The direct contact with the carboxymethylated hydrogel adversely influenced the morphology of
11
the cells and limited their spreading, while typical morphology and spreading of cells was
12
observed with the TEMPO-oxidized hydrogel. The porous fibrous structure may be a key to cell
13
proliferation and migration in the hydrogels.
14
2 ACS Paragon Plus Environment
Page 3 of 32
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
1
Biomacromolecules
Introduction
2
Native extracellular matrix (ECM) is a highly hydrated gel-like viscoelastic three
3
dimensional (3D) network of proteoglycans, multiscale protein fibers, and glycosaminoglycan
4
chains.1, 2 The hierarchical topographical features of the ECM range in size from nanometers to
5
micrometers.2 The nanotopography of the ECM plays a critical role in regulating cell behavior
6
since the cells interact with ECM via nanoscale proteins such as fibronectin and collagen fibrils.3-
7
5
8
the ECM to direct cell attachment, proliferation and differentiation to promote tissue formation.6,
9
7
In tissue engineering, the primary role of the scaffold is to provide a 3D environment similar to
Among different scaffolding biomaterials, hydrogels are promising for 3D cell cultures as they
10
may mimic the physicochemical, and biological properties of the tissues.8,
11
hydrogels, per se, might recapitulate the complex interaction between cells and their
12
microenvironments to guide tissue morphogenesis and function.10, 11 Polysaccharide biomaterials
13
such as alginate, chitosan, hyaluronic acid and cellulose can form hydrogels similar to ECM due
14
to their physicochemical structure, which places them in the frontline of tissue engineering
15
applications.12-14 Typically, most polysaccharides require separate cross-linking steps to form a
16
hydrogel network.15, 16 On the other hand, cellulose fibers from plants or bacteria form colloidal
17
dispersions in aqueous medium.17, 18 Cellulose is the most abundant natural polymer on earth
18
providing an unlimited source for environmentally friendly products.19,
19
biomedical applications, traditional cellulosic materials have been introduced for tissue
20
engineering uses by extracting cellulose at nanoscale.20, 21 These nanocellulosic materials include
21
wood-based cellulose nanofibril (CNF), bacterial nanocellulose (BNC) and cellulose nanocrystals
22
(CNC).21-24 As nanoscale materials, they have remarkable physicochemical properties different
23
from traditional cellulose including geometrical dimensions, morphology, high specific surface
24
area, crystallinity, orientation and good mechanical properties.25-27 BNC is a collagen-like
20
9
Nanostructured
After years of
3 ACS Paragon Plus Environment
Biomacromolecules
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
Page 4 of 32
1
material that has been extensively investigated for tissue engineering applications.24,
2
Nevertheless, a cost-efficient process for mass production of BNC is still not available.
3
Biomaterials without microbial or animal-derived components are preferred immunologically,
4
thus interest in wood-based CNF has grown recently.7, 14, 18 Functionalization of CNF utilizing
5
carboxymethylation or 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO-oxidation) as
6
chemical pretreatment to introduce different functional groups and charges to the surface of CNF
7
has been investigated.27, 29,
8
TEMPO oxidation may act as sites for covalent crosslinking using diamines and polyamines.31, 32
9
Furthermore, the carboxylate groups can facilitate gelation of CNF dispersion by introducing
10
30
28
Interestingly, the introduction of aldehyde groups formed during
ionic interactions with metal cations such as Ca2+ and Fe3+.33, 34
11
It is known that cell behavior on biomaterials is influenced by surface functional
12
groups.35-37 Although, there are many studies reporting the safety and biocompatibility of CNF
13
materials, cell-material interactions should be evaluated after any chemical modification or
14
crosslinking as it may affect cell behavior.21, 38-40 Cell proliferation and migration in hydrogels
15
are key steps in morphogenesis and regeneration of tissues and still present challenges in the field
16
of cell encapsulation and bioprinting.3, 9, 41, 42 Recently, it was reported that CNF hydrogel could
17
create 3D environment for proliferation and differentiation of human embryonic stem cells.7
18
Moreover, the behavior of liver cells cultured in CNF hydrogel was promoted without the
19
addition of bioactive components.14, 18 In light of the aforementioned characteristics of CNF, this
20
study aimed to produce two CNF hydrogels with different surface chemistry and study their
21
ability to form mechanically stable networks by ionic interactions with cell culture medium. The
22
CNF hydrogels were produced using i) TEMPO mediated oxidation or ii) carboxymethylation
23
pretreatment before fibrillation. The interactions of fibroblasts with the hydrogels were then
24
studied in terms of cell toxicity, attachment, proliferation, morphology and migration. 4 ACS Paragon Plus Environment
Page 5 of 32
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
Biomacromolecules
1
Materials and Methods
2
Chemicals
3
All chemicals used for hydrogel preparation were of laboratory grade purchased from
4
Sigma Aldrich. The cellulose pulp used as raw material was a fully bleached and never-dried
5
softwood kraft pulp that was kindly donated by Södra Cell (Växjö, Sweden). For the in vitro
6
cytotoxicity test, Dulbecco’s Modified Eagle Medium (DMEM) (low glucose, GlutaMAXTM) and
7
Dulbecco’s phosphate buffered saline (PBS), 4´,6-diamidino-2-phenylindole (DAPI), PicoGreen
8
and Live/Dead kit were purchased from Thermo Fisher Scientific. Lactate dehydrogenase (LDH)
9
in vitro toxicology kit was purchased from Abcam. Fetal bovine serum (FBS), dimethyl sulfoxide
10
(DMSO), ethanol, crystal violet, MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
11
bromide), glutaraldehyde and Phalloidin-Atto488 were purchased from Sigma Aldrich.
12
Preparation of Hydrogels
13
TEMPO oxidized CNF (sample TO-CNF) was produced according to the method
14
described by Saito et al.45 using 2,2,6,6-tetramethylpiperidinyl-1-oxyl (TEMPO) and sodium
15
bromide (NaBr) to catalyze the oxidation of the alcohol group in the C6 position to aldehyde and
16
carboxylic acid. Sodium hypochlorite was used in the oxidation. The pH was kept constant at
17
10.5 by titration of sodium hydroxide (NaOH) during the reaction. The reaction was finalized
18
when pH stopped decreasing, and pH was adjusted to 7 by addition of hydrochloric acid (HCl).
19
The pulp was washed by filtration using deionized water until the filtrate had conductivity below
20
5µS/cm.
21
Carboxymethylated CNF (sample CM-CNF) was produced according to Wagberg et al.27
22
The pulp was disintegrated and the solvent was exchanged from water to ethanol. The fibers were
23
impregnated in a solution of 2% monochloroacetic acid in 500 mL isopropanol for 30 min. This
24
was transferred to a 5 L reaction vessel equipped with reflux and containing a heated solution of 5 ACS Paragon Plus Environment
Biomacromolecules
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
Page 6 of 32
1
16.2 g NaOH in a mixture of 500 mL methanol and 2 L isopropanol. After one hour, the pulp was
2
washed with 20 L deionized water and 2 L 0.1 M acetic acid followed by additional 10 L
3
deionized water. The carboxyl groups were converted to sodium form by soaking the pulp in a
4
4% NaHCO3 solution for 60 min. The pulp was finally filtered and washed with deionized water
5
until the conductivity was below 5 µS/cm.
6
Fibrillation of the pretreated samples was done using a Rannie 15 type 12.56X
7
homogenizer (APV, SPX Flow Technology, Silkeborg, Denmark) using two passes, the first at
8
600 and the second at 1000 bar pressure.
9
To evaluate the influence of cell culture medium, prepared CNF hydrogels were stirred
10
for 30 min, transferred to polystyrene containers and covered with DMEM for 24 h at 37°C.
11
Characterization of Hydrogels
12
Determination of Surface Groups
13
The content of carboxyl and aldehyde groups was determined by conductometric titration,
14
as described by Saito et al.43 About 55 mL water and 5 mL 0.01M NaCl were added to 0.3 g dry
15
sample. The pH was adjusted to approximately 2.5 by the addition of 0.1M HCl. Titration was
16
performed with 0.04 M NaOH solution added at a rate of 0.1 mL/min up to pH of approximately
17
11. The conductivity of the sample was automatically measured at increments of 0.01 mL using a
18
Metrohm 856 Conductivity Module, and the data was recorded by Tiamo® Titration Software.
19
The carboxylate content was calculated from the titration curve. This analysis was also done after
20
oxidation of aldehyde groups to carboxyl groups with NaClO2. The difference in carboxylate
21
content before and after the NaClO2 oxidation yields the aldehyde content.
22
Structural Characterization
23
To evaluate the structure of the CNF hydrogels, thin films were prepared by drying CNF
24
dispersion on coverslips at room temperature. Films were analyzed by an optical microscope 6 ACS Paragon Plus Environment
Page 7 of 32
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
Biomacromolecules
1
(Nikon Eclipse 80i, Tokyo, Japan) and a JEOL JSM-7400F field emission scanning electron
2
microscope. For the nanostructures, the films were imaged using an atomic force microscope
3
(diMultiMode V AFM, Bruker, USA).
4
FTIR Characterization
5
The infrared spectra of dried samples were collected on a Bio-Rad Excalibur series FTS
6
3000 spectrophotometer. The spectra were acquired in transmission mode on the films at a
7
spectral range of 4000−500 cm−1. The films were made from the hydrogels before and after
8
exposure to DMEM and had a basis weight of 20 g/m2 of CNF.
9
Rheological Characterization
10
To measure the mechanical properties of the hydrogels before and after soaking in
11
DMEM, rheological assessments of the CNF hydrogels were done by using a shear Anton Paar
12
Physica rheometer (MCR 301). Cone-plate geometry and roughened plates were used for all
13
measurements (cone = 2.01° gradient, gap = 57 µm at center, diameter = 39.96 mm). The
14
temperature was kept constant at 25°C. Preshearing at 100 s-1 for one minute followed by resting
15
for two minutes was done before each measurement. Strain sweeps were done in order to
16
determine the linear viscoelastic regime of CNF dispersions. The strain was increased from 0.01
17
to 100% while keeping the frequency constant at 0.01 Hz. Based on the results from the strain
18
sweeps, a strain of 1% was chosen for frequency sweep. Frequency sweeps were done for
19
determination of the storage and loss moduli (G′ and G′′) respectively. The frequency was varied
20
from 0.01 to 10 Hz. Shear viscosity as a function of shear rate, was obtained for shear rates from
21
0.1 to 1000 s-1.
22
Cell Culture
7 ACS Paragon Plus Environment
Biomacromolecules
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
Page 8 of 32
1
Mouse fibroblasts (L929 cells, American Type Culture Collection CCL-1) were cultured
2
in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37°C in 5% CO2
3
humidified atmosphere.
4
Indirect Cytotoxicity
5
CNF hydrogels were first sterilized by autoclave and then incubated in DMEM (1 g/5 mL)
6
without serum at 37°C with constant shaking (60 rpm) for 24 h (CNF-24h extract) and 72 h
7
(CNF-72h extract) and then the extracts were filtered (0.2 µm pore size). As control samples,
8
DMEM without hydrogels was kept at the same extraction conditions. Cells were cultured in
9
proper cell culture plates and incubated in complete medium for 24 h to attach and then the media
10
were replaced with extracts supplemented with 10% FBS. After 1, 4 and 7 days, cell
11
proliferation, morphology and cytotoxicity were assessed utilizing crystal violet, MTT and LDH
12
assays. For the crystal violet assay, cells (1×104 cells/well) were cultured in 24-well plates and
13
treated with the hydrogel extracts (1 mL/well). At predetermined time points, the medium was
14
discarded and cells were washed with PBS, fixed with paraformaldehyde, stained with 0.05%
15
crystal violet for 30 min and then analyzed using an inverted microscope (Nikon Eclipse Ti,
16
Tokyo, Japan). Afterwards, the crystal violet was solubilized in 500 µL of 95% ethanol and
17
absorbance was measured at wavelength 595 nm using a microplate reader (FLUOstar OPTIMA,
18
BMG LABTECH, Germany).
19
For mitochondrial activity, cells (5×103/well) were cultured in 96-well plates and treated
20
with the extracts. At predetermined time points, the MTT solution (200 µL of 0.5 g/L in medium)
21
was then added to the culture wells. After incubating for 4 h at 37°C in a 5% CO2, the MTT
22
solution was removed, and DMSO containing 6.25% (v/v) 0.1M NaOH was added to dissolve
23
formazan crystals. The optical density was measured at wavelength 570 nm using a microplate
24
reader. 8 ACS Paragon Plus Environment
Page 9 of 32
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
Biomacromolecules
1
To evaluate plasma membrane integrity, lactate dehydrogenase (LDH) was measured
2
(LDH Assay Kit, abcam). Cells (1×105/well) in 12-well plates were treated with extracts for 1
3
and 4 days. Following the manufacturer’s instructions, cells were washed with PBS,
4
homogenized in the assay buffer and centrifuged at 10,000 × g for 15 min at 4°C. The
5
supernatant was collected and mixed with the LDH reaction solution and incubated for 30 min.
6
The LDH activity was measured in a microplate reader at 450 nm.
7
Cell- Hydrogel Interactions
8
Prepared CNF hydrogels were autoclaved, transferred to 24-well plates (400 µL/ well)
9
and covered with DMEM overnight at 37°C in 5% CO2 humidified atmosphere. Then cells
10
(1×105/well) were seeded on the surface of the hydrogels and incubated for 2 h to attach. In
11
addition, a sandwich-seeding method was tested by embedding cells between two layers of
12
hydrogels (200 µL). The cells were seeded on the first layer and incubated for 2 h to attach, then
13
a second layer was added and medium was added to give a total volume of 1 mL. Cell responses
14
in terms of attachment, proliferation, toxicity, viability and morphology were examined. Cells
15
cultured in tissue culture plates (TCP) were used as controls.
16
Cell Attachment
17
Cells were seeded onto the hydrogels and allowed to attach for 2 h and then covered with
18
1 mL of medium. After two additional hours, the culture medium was collected and unattached
19
cells were quantified using an automated cell counter (CountnessTM, Invitrogen Carlsbad, CA,
20
USA). In addition, hydrogels were rinsed with PBS, fixed with 4% paraformaldehyde for 30
21
minutes, then stained with DAPI or crystal violet and examined with optical and fluorescence
22
microscopes. Cell attachment was quantified as described below: ℎ (%) =
( − ) 100 9
ACS Paragon Plus Environment
Biomacromolecules
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
1
Page 10 of 32
Cell Proliferation
2
Cell proliferation was evaluated by dsDNA assay (Quant-iTTM PicoGreen®, Invitrogen).
3
At predetermined time points, medium was removed from the wells and CNF hydrogels were
4
washed three times with PBS. Then 400 µL of 0.05% Triton-X/PBS was used to cover the
5
hydrogel before freezing and storing at −80°C. Samples were then subjected to freezing-thawing
6
cycles and sonicated for 30 seconds. Solution (50 µL) was then transferred to 96-well plates and
7
150 µL PicoGreen® working solution was added to each well. The plate was read on microplate
8
reader (excitation wavelength: 480 nm, emission wavelength: 520 nm).
9
Cell Toxicity by LDH
10
The medium corresponding to the PicoGreen® proliferation assay was collected after 1
11
and 4 days and centrifuged at 10,000 × g at 4°C. The enzymatic assay was performed in the
12
supernatant following the manufacturer’s instructions.
13
Cell Viability by Live/Dead Assay
14
The viability of cells was evaluated with Live/Dead assay using calcein-AM and ethidium
15
homodimer. The cell seeded hydrogels were rinsed with PBS and incubated in 1 mL Live/Dead
16
solution at room temperature for 40 min. The samples were sectioned, transferred to glass slides
17
and imaged with a fluorescence microscope.
18
Cell Morphology
19
The seeded hydrogels were rinsed with PBS and fixed with paraformaldehyde 4% at room
20
temperature for 30 min. The samples were sectioned, stained with crystal violet, squeezed
21
between glass slides and cover slips and imaged with an optical microscope.
22
Cell Cytoskeleton in 2D Culture
23
To evaluate the effect of the surface groups of the hydrogels on the cytoskeleton of the cells,
24
6-well plates were coated with a thin layer of diluted CNF hydrogels (1g of hydrogel in 3 mL of 10 ACS Paragon Plus Environment
Page 11 of 32
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
Biomacromolecules
1
water) and then incubated at 37°C overnight to evaporate the water. Two wells without coating
2
were used as controls. Cells were then grown on the wells and incubated for 72 h. After fixation
3
with 4% paraformaldehyde, Phalloidin-Atto488 in PBS was added to the cells for 40 min in the
4
dark. The cells were then examined with an inverted fluorescent microscope. To calculate the cell
5
number and cell surface area, eight images were taken from each group (4 images/ well) and
6
analyzed utilizing ImageJ software (1.46r).
7
Cell Migration
8
The migration of the cells was investigated using a transwell migration assay (BD Falcon,
9
8µm, USA). Hydrogels (100 µL) were added into the upper chamber of the wells and incubated
10
with DMEM overnight at 37°C in 5% CO2 humidified atmosphere. The medium was then
11
discarded and cells (1×104/well) in 50µL serum-free DMEM were then placed onto the center of
12
the hydrogels and allowed to attach for 4 h before covering with 250 µL serum-free DMEM.
13
Cells were allowed to migrate along a gradient of medium with 10% FBS. After 72 h, cells on the
14
lower surface of the insert membrane were fixed and stained with crystal violet for light
15
microscope imaging. Next, crystal violet was solubilized in ethanol and the absorbance was
16
measured using a microplate reader. Additionally, hydrogels of 10 mm thickness were seeded
17
with cells for 72 h then fixed with paraformaldehyde, sectioned longitudinally and stained with
18
DAPI for fluorescence microscope investigation.
19
Statistical Analysis
20
Data (n ≥3) are presented as means ± standard error of the mean (SEM).The means were
21
compared using one-way ANOVA followed by a post hoc test for multiple comparisons using
22
IBM SPSS Statistics 21. A p value ≤ 0.05 was considered to be statistically significant.
23
Results and Discussion
24
Characterization of Hydrogels 11 ACS Paragon Plus Environment
Biomacromolecules
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
Page 12 of 32
1
Several functional groups can be introduced onto surface of CNF using different
2
pretreatment processes.29 In the present study, CNF hydrogels with different surface groups were
3
produced by TEMPO mediated oxidation or carboxymethylation pretreatment (Table 1). The
4
TEMPO oxidized CNF sample had aldehyde (211 ± 60 µmol/g) and carboxyl (764 ± 60 µmol/g)
5
groups which produced more negatively charged surface than the carboxymethylated CNF
6
sample had, with carboxymethyl (346 ± 26 µmol/g) and carboxyl (58 ± 1 µmol/g) groups. The
7
TO-CNF and CM-CNF hydrogels had low solid content of 1.06 ± 0.01 and 1.07 ± 0.01%
8
respectively. This low solid content was enough to form a weak hydrogel-like material as shown
9
in Figure 1A. This weak gelation could be attributed to the coupling of nanofibrils with hydrogen
10
bonding and van der Waals interactions.17 Structural investigation by optical microscopy, SEM
11
and AFM revealed that the hydrogels have a multiscale fibrous structure ranging from several
12
micrometers (microfibers) (Figure 1B, C) to nanometers (nanofibrils) (Figure 1D). Such
13
hierarchical fibrous structure mimics the features of the natural ECM providing mechanical
14
strength and suitable 3D environment for cell attachment and migration.44-46
15
Table 1. Surface groups on CNF hydrogels CNF
Aldehyde
Carboxyl
Carboxymethyl
(µmol/g)
(µmol/g)
(µmol/g)
211 ± 60
764 ± 60
−
−
58 ± 1
346 ± 26
Functional groups
Hydroxyl, carboxyl TO-CNF aldehyde, Hydroxyl, carboxyl CM-CNF carboxymethyl 16
12 ACS Paragon Plus Environment
Page 13 of 32
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
Biomacromolecules
1 2 3 4
Figure 1. Multiscale fibrous structure of the prepared CNF hydrogels: (A) Macroscopic image of the prepared hydrogel-like materials. (B) Optical microscope images (C) SEM images showing the microscale fibers (D) AFM images showing the nanoscale fibrils.
5
Mechanically, prepared CNF hydrogels showed typical shear-thinning behavior. At low
6
stress level a solid-like behavior of the CNF hydrogels was observed, while with high shear stress
7
conditions (such as stress during injection) the hydrogels exhibit viscous flow (Figure 2A, B).
8
Generally, shear-thinning behavior of hydrogels is important for cell encapsulation and
9
bioprinting applications.18,
47
This behavior may allow incorporation of cells and/or bioactive
10
molecules into the hydrogel and site-specific delivery from needle tips and nozzles.18, 47, 48 For
11
cell encapsulation and bioprinting, alginate has been extensively used due to its easy crosslinking
12
by divalent cations, such as Ca2+.47-49 Gelation of CNF aqueous dispersions by divalent or
13
trivalent cations has also been reported in many studies.33,
14
hydrogels from CNF that were ionically crosslinked using metal salts.33 Dong et al. showed that
15
addition of 50 mM salt solution of divalent cations could initiate gelation of TEMPO oxidized
16
CNF dispersions and create interconnected porous nanofibril networks.34
34, 50
Zander et al. prepared stable
13 ACS Paragon Plus Environment
Biomacromolecules
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
Page 14 of 32
1
Since DMEM culture medium contains cations such as Na+, Ca2+ and Fe3+ in varying
2
concentrations, ionic interactions with the negative charged fibrils can occur.51 In the present
3
study, after soaking in DMEM, the prepared hydrogels held their shape and became mechanically
4
strong enough to be cut and transferred to multiwell plates without affecting their injectability
5
(Figure 2B). FTIR was used to investigate the interactions between CNF hydrogels and DMEM
6
(Figure 2C). In the spectrum of CNF hydrogels before exposure to DMEM, a strong band
7
corresponding to the hydrogen-bonded hydroxyl group was observed at 3350-3345 cm−1. The
8
spectrum also demonstrated an asymmetric −OCO− stretching band at 1614-1609 cm−1 and a
9
symmetric stretching band at 1435-1424 cm−1, resulting from carboxylate anions. The band
10
around 1038 cm−1 resulted from CO stretching. DMEM-induced cross-linking of the CNF
11
hydrogels resulted in a slight difference in the location and absorption intensity of the –OCO–
12
stretching bands. This may be caused by the formation of ionic bonds between the cations and the
13
carboxylate groups.34, 50
14
To confirm the ability of the DMEM to ionically crosslink the prepared CNF hydrogels,
15
dynamic rheological properties before and after soaking in DMEM were investigated as a
16
function of frequency (Figure 2D, E, F). Generally, the storage (elastic) modulus (G′) was greater
17
than the loss (viscous) modulus (G″) in all conditions which is a typical characteristic of
18
hydrogels. The G′ values of the CNF hydrogels after soaking in DMEM were much larger than
19
the corresponding G′ of the original CNF hydrogels indicating the interaction between DMEM
20
and CNF hydrogels. Before exposure to DMEM, the average G′ values over the frequency range
21
of the TO-CNF and CM-CNF hydrogels were 380.8 ± 25.9 and 779.2 ± 11 Pa respectively. These
22
values increased to 4428.2 ± 389.4 and 7502.9 ± 1137 Pa respectively after soaking in DMEM.
23
Dong et al. demonstrated that the G′ values of CNF crosslinked with divalent cations ranged
24
from 3390 to 11700 Pa at 0.01 rad/s frequency.34 TEMPO oxidation or carboxymethylation 14 ACS Paragon Plus Environment
Page 15 of 32
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
Biomacromolecules
1
pretreatments produce negatively charged fibrils.39 These negative charges create interfibril
2
repulsion which may be screened in the presence of cations in the cell culture media and
3
dominate hydrogel properties through ionic crosslinking.37 Based on these findings we propose
4
that DMEM may induce gelation of CNF, thus improving their mechanical properties. The ability
5
to be cross-linked by exposure to cell culture media could enhance the biocompatibility of CNF
6
hydrogels by avoiding chemical crosslinking reagents that may reduce cell viability.40
7
8 9 10 11 12 13
Figure 2. Interactions between CNF hydrogels and DMEM culture medium: (A) Flow curves of the CNF hydrogels as prepared. (B) Stable free-standing and injectable hydrogel after exposure to DMEM. (C) FTIR spectra of the hydrogels before and after exposure to DMEM. (D−F) Change in G″ and G′ of CNF hydrogels before and after exposure to DMEM. Statistical significant difference: **P
