Materials
Poly(ethylene oxide) (PEO) polymer with hexa-thiols (MW = 10 kDa) was purchased from Sunbio Inc. (Seoul, Korea). While the chemicals of carboxymethyl cellulose sodium salt (CMC) (MW = 90 kDa), adipic dihydrazide (ADH) (MW = 174 kDa), acrylic acid (MW = 72 Da), adipic acid dihydrazide (ADH), dimethyl sulfoxide (DMSO) and esterase solution from porcine liver (177 unit/mg) were purchased from Sigma-Aldrich Chemical Co. (MO, USA), N-(3-diethylpropyl)-N-ethylcarbodiimide hydrochloride (EDC) and 1-hydroxybenzotriazole hydrate (HOBt) were obtained from Fluka Chemie GmbH (Buchs, Switzerland). While DMEM-604 and penicillin-streptomycin were purchased from Lonza Korea (Switzerland), cell counting kit-8 (CCK-8) solution and live & dead viability/cytotoxicity kit for mammalian cells were bought from Dojindo Laboratories (Japan) and Invitrogen (USA), respectively. Fetal bovine serum (FBS) and in vitro toxicology assay kits such as bromodeoxyuridine (BrdU), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Neutral red were purchased from Sigma-Aldrich (MO, USA), Roche (Germany) and Gibco BRL (Australia), respectively. All chemicals were employed as received.
Synthesis of CMC-acrylate
CMC-acrylate was synthesized by sequential grafting of ADH and acrylic acid to CMC as below. 0.08 g ADH, 0.08 g HOBT and 0.10 mL EDC were separately added into 40 mL 5% CMC solution, and then chemical reaction proceeded for 1 hr (see overall schematics in Figure 1). After precipitating the resulting solution in ethanol, the products were dialyzed in distilled water with 50 g NaCl by employing cellulose dialysis membrane filter with molecular weight cut off of 6 ~ 8 kDa. CMC powder was obtained by lyophilizing for 2 d.
Fabrications of CMC-PEO hydrogel and porous gel film
The precursor solutions of both CMC-acrylates and PEO-thiols were in advance sterilized separately with a 3 mL syringe by filtering them by a syringe filter with poly(ether sulfone) membrane (Pall Corp., PN4612; USA). The CMC-PEO hydrogel was synthesized by mixing the sterilized precursor solutions in a 24 well plate overnight. Synthesis methods related have been reported in detail in our previous works [8,31].
Porous CMC-PEO gel films were fabricated by adding ammonium bicarbonate porogens into the precursor solutions of CMC-acrylate and PEO-thiols. The pore sizes of hydrogel films were controlled by employing different concentrations of porogens (12 and 18%) with different particle sizes ranging from 150–180 to 250–350 μm. The porous gel films were obtained by gas forming the porogen particles.
Attenuated total reflectance - Fourier transform infrared spectroscopy (ATR-FTIR)
After mixing 300 mg KBr and 1.0 mg CMC powders either with or without acrylated, a thin gel film (1.2 cm diameter and 1.0 mm thickness) was prepared by pressurizing 50 MPa into a sample holder containing the mixed powder under vacuum for 3 min. The gel film was chemically analyzed with an ATR-FTIR spectroscopy (FT/IR-62, Jasco; Japan). After establishing a standard curve with a polystyrene film, transmittance through the samples was scanned, ranging from 650 to 4000 cm−1.
1H nuclear magnetic resonance (1H-NMR)
1H-NMR spectra were obtained by employing an UI 500 MHz FT-NMR spectrometer (Varian, Japan) to observe an extent of grafting of acrylic acid to CMC. Chemical shift of the spectrum peak was measured for the samples of CMC and CMC-acrylate, by using 1% deuterium oxide (D2O) (w/v) as a solvent.
Morphologies of dehydrated CMC-PEO hydrogel
The morphologies of CMC-PEO gel films were visualized by a scanning electron microscopy (SEM; JEOL Ltd, Japan) after routine processing of dehydration and gold-sputter coating of the gel films as below. The swollen gel films (0.5 × 0.5 mm) were frozen in liquid nitrogen and then freeze-dried at −55°C overnight (FD-8508, Ilshin Bio Base; Korea). The dry samples were mounted on an aluminum stub with a double-sided tape, and then gold-coated for 1 min. The morphology of the dehydrated gold-coated CMC-PEO gel films was analyzed with SEM.
Swelling of CMC-PEO hydrogel
After measuring the gel weight with a microbalance, swelling of the 5% CMC-PEO hydrogel was measured by immersing it in distilled water over time. Swelling behaviors of the CMC-PEO gel was also measured by immersing it in distilled water with different pHs at 4, 7 and 10. Adherent water was removed by blotting the wet CMC-PEO gel with a piece of Kimwipe paper before weighing on an electronic balance. The percentage of the gel swelling was calculated by a following formula over its soaking time in distilled water at room temperature.
$$ \mathrm{Swelling}\ \mathrm{of}\ \mathrm{the}\ \mathrm{hydrogel}\ \left(\%\right) = \frac{\left(\mathrm{W}\mathrm{s}-\mathrm{W}\mathrm{i}\right)}{\mathrm{Wi}}\times 100\left(\%\right) $$
Where W
s
and W
i
are the swollen weight of the CMC-PEO gel at time t and its weight after gelation, respectively.
Degradation of CMC-PEO hydrogel by esterase
Degradation of 5% CMC-PEO gel (1:1) was tried as below by addition of 2 mL esterase solution from porcine liver in distilled water. After swelling 200 μL hydrogel in 2 mL DW, fresh esterase solution with either 2, 10 or 30 units was sprayed over the hydrogel at every 2 d (n = 3). The weight percentage of the remained hydrogel was calculated by employing flowing equation.
$$ \mathrm{Gel}\ \mathrm{weight}\ \mathrm{loss}\ \mathrm{b}\mathrm{y}\ \mathrm{esterase}\ \left(\%\right) = \left(1-{\boldsymbol{W}}_{\boldsymbol{e}}/{\boldsymbol{W}}_{\boldsymbol{c}}\right)\times 100\ \left(\%\right) $$
$$ {W}_e-\mathrm{weight}\ \mathrm{of}\ \mathrm{C}\mathrm{M}\mathrm{C}-\mathrm{P}\mathrm{E}\mathrm{O}\ \mathrm{gel}\ \mathrm{treated}\ \mathrm{with}\ \mathrm{esterase}\ \mathrm{at}\ \mathrm{time}\ t, $$
$$ {W}_c-\mathrm{weight}\ \mathrm{of}\ \mathrm{C}\mathrm{M}\mathrm{C}-\mathrm{P}\mathrm{E}\mathrm{O}\ \mathrm{gel}\ \mathrm{in}\ \mathrm{water}\ \mathrm{without}\ \mathrm{esterase}\ \mathrm{at}\ \mathrm{time}\ t. $$
Evaluation of biocompatibility
In vitro cell behaviors on/in CMC-PEO hydrogel film
Porcine aorta smooth muscle cells (passage 8) were in vitro cultured in DMEM-640 media (Lonza, Switzerland) containing both 10% fetal bovine serum (Lonza, Switzerland) and penicillin-streptomycin (100 IU/mL) (Lonza, Switzerland) in an in vitro incubator with 5% CO2 at 37°C. Cell culture was performed by initial seeding of smooth muscle cells either inside or on the surface of 200 μL CMC-PEO gel correspondingly at the densities of either 100,000 or 10,000 cells/ea for 7 d. Cell culture plate with 24 wells was employed for loading of hydrogels. In vitro tissue regeneration was performed on the porous gel films by seeding 200,000 cells/scaffolds for 35 d.
Cell adhesion and proliferation were evaluated with the assays of both cell counting kit-8 (CCK-8, Dojindo, Japan) and live & dead after seeding smooth muscle cells at a density of 10,000 cells per sample. The cell number was counted as below with the CCK-8 assay by using a microplate reader (Tecan, Australia), where the cell culture medium was measured as a background with the OD value of 1.2 to 1.4. 100 μL CCK-8 solution was inserted into the 900 μL DMEM-640 medium and then the cell culture plate with samples was inserted in the in vitro CO2 incubator. After 2 hr, 100 μL medium with CCK-8 solution was aliquoted into a 96 well plate and an optical density of the CCK-8 loaded medium was measured at the wavelength of 450 nm by the microplate reader.
In vitro cell viability was also observed with smooth muscle cells both inside and on the surface of the 200 μL 5% CMC-PEO gel. Live & dead viability/cytotoxicity kit for mammalian cells was prepared according to the protocol suggested by the vendor (Invitrogen, USA) by adding the solutions of both 1.2 μL ethidium homodimer-1 (EthD-1) (2 mM) and 4 mM calcein AM (0.3 μL) into 600 μL PBS. After performing the reaction with the prepared agents for 30 min in the in vitro CO2 incubator, cell viability on the hydrogel was observed by a fluorescence microscope (Leica DMLB, Germany).
In vitro direct contact of CMC-PEO gel with cells on tissue culture plate
Effects of direct contact of the 5% CMC-PEO gel on smooth muscle cells have been evaluated by covering the hydrogels on the cells in a 12 well plate. For the assay, smooth muscle cells were initially seeded at a density of 2×104 cell/well on a 12 well plate and then its surface was completely covered with either 200 μL hydrogel, Teflon or Latex (1 × 1 cm). After locating the samples carefully on the cells for 24 hr, both the cell growth characteristics and any signs of morphological change by cytotoxicity were observed by an inverted light microscope.
Bromodeoxyuridine (BrdU) assay
After loading 3 kinds of extract solution (1 mL) of the 5% CMC-PEO gel, Latex and Teflon (1 × 1 cm2), smooth muscle cells at a density of 1×104 cells were in vitro cultured in a 96 well plate for 24 hr. Cell culture lasted for another 2 hr with addition of 10 μL BrdU labeling solution. Subsequent to removal of labeling medium, solutions of both 200 μL FixDenat and 100 μL anti-BrdU peroxidase-labeled anti-BrdU antibody per well were treated, as suggested by the manufacturer. 25 μL 1 M H2SO4 solution was added into each well after washing. Optical density of the samples was measured at an absorbance wavelength of 450 nm by the microplate reader by referencing that of 690 nm.
Thiazolyl blue tetrazolium bromide (MTT) assay
After seeding smooth muscle cells in a 96 well plate at a density of 1×104 cells, cell culture lasted in a 5% CO2 incubator at 37°C for 24 hr, and then 1 mL extracts of the 5% CMC-PEO gel, Teflon and Latex (1 × 1 cm) were added into the cell culture media for another 24 hr. Cell culture lasted for additional 4 hr after insertion of 20 μL MTT solution (2 mg/ml in PBS) in the culture medium, and then sequential removal of the culture medium and addition of 100 μL dimethyl sulfoxide followed. The optical density of the final solution was measured by the microplate reader at a wavelength of 570 nm.
Neutral red assay
Extracts from the 5% CMC-PEO gel was obtained after loading the gel in the cell culture medium (1 mL) for 72 hr. Cell culture lasted in 5% CO2 incubator at 37°C for 24 hr after seeding smooth muscle cells on the hydrogel at a density of 1×104 cells/well. The medium was removed from the well, and then cell culture lasted for another 24 hr after addition of the extract solution. Subsequent to addition of the culture medium and 0.33% Neutral red solution at a ratio of 9:1 in the in vitro incubator, cell culture lasted for another 2 hr. After washing with the fixation solution of the assay and then proceeding of reaction for 10 min, 100 μL solubilization solution was added per well. Optical density was measured at an absorbance wavelength of 550 nm by the microplate reader by referencing that of 690 nm.
Histological staining of the in vitro tissue-regenerated porous CMC-PEO gel film
The porous CMC-PEO gel films in vitro cell cultured were visualized by light microscopy after staining with hematoxylin and eosin (H&E) and Masson’s trichrome (MT) as below. H&E stain was processed after cross-linking the cell-cultured porous CMC-PEO gel films with 4% paraformaldehyde for 1 d and then stored in a refrigerator in phosphate buffered solution. After paraffin embedding of the samples (Tissue Embedding; Leica: Germany), micro-sectioning were performed with a microtome (RM2245; Leica: Germany). The sections were treated with xylene and graded ethanols (Thermo Scientific; USA), and then stained with H&E and Masson’s trichrome. The stained sections were visualized with a light microscopy (CK40-F200, Olympus: Japan) and the images were stored by an image processor (UTHSCSA Image Tool 3.0, Samwoo Science Co.; Seoul, Korea).
Statistical analysis
Data were expressed as mean ± standard deviations. Statistical significance was assessed with one-way and multi-way ANOVA by employing the SPSS 12.0 program (ver. 18.0, SPSS Inc.; IL, USA). The comparisons between two groups were carried out using a t-test. The samples were considered as significantly different when p< 0.05.
