Page 153 - IJB-9-2

International Journal of Bioprinting Enhanced osteogenesis in gelatin releasing bioink

salt hydrate (Tokyo Chemical Industry Co., Japan) and 2.7. Cell viability
10 mM β-glycerophosphate disodium salt, pentahydrate The viability of the cells encapsulated in the hydrogels was
(EMD Millipore, USA). determined using a live/dead viability/cytotoxicity kit for
mammalian cells (Invitrogen, USA) in accordance with the
2.5. Characterization of hydrogel manufacturer’s protocol. The samples were stained with
All hydrogel characterizations were completed using cell- fluorescent dyes (calcein-AM and EthD-III) after 3 weeks
free hydrogel disks (1 mm height, 8 mm diameter). The of culturing.
water content was calculated from the weight of the water
equilibrated (W ) and dried (W ) hydrogel. In brief, each To quantitate the DNA within the cells encapsulated
w
d
hydrogel disk (MA-alginate/gelatin ratios of 10:0, 1:9, 3:7, in the hydrogels, after 1, 2, and 3 weeks, 1 mL of cell
and 5:5) was immersed in 1 mL cell culture media at 37°C lysis buffer (CelLyticTM M, Sigma, USA) was added to
for 0 h (right after fabrication) and 3 weeks. At the specific each sample, and the samples were homogenized. DNA
time point, the water-soaked hydrogel disks were removed was detected using a picogreen assay kit (Thermo Fisher
from the media and their surfaces were gently wiped. Scientific, USA). For the external cells which cultured in
Each disk was weighed until an equilibrium weight was 24-well plates, after 1, 4, and 7 days, 1 mL of cell lysis buffer
reached. Then, they were freeze-dried and reweighed. The was added and DNA was detected using the same protocol
% swelling was calculated using the following equation: as described in the above.

Waterof wetsample ( ) −W w Water of driedsample( ) 2.8. Quantitative analysis of alkaline phosphatase
W
( )
d
Swelling % ( ) = ×100 % activity
Watterofdried sample W ( )
d
At the predetermined time points (1, 2, and 3 weeks),
After the hydrogel disks had been incubated in cell hydrogel disks containing cells from each group and external
culture media for different times, the compressive modulus cell cultured in 24-well plates were transferred to test tubes,
of the hydrogel disks was measured using a rheometer and 1 mL of cell lysis buffer was added to each tube. The
(DHR-1, TA instruments, USA) and a flat parallel plate hydrogel disks were broken down with a homogenizer (T10
(8 mm, crosshatched) at room temperature. Each disk was basic, IKA, Germany). To determine alkaline phosphatase
subjected to a compressive load at a constant linear rate of (ALP) activity, p-nitrophenyl phosphate (pNPP) was used
5.0 μm/s. The load-displacement data were transformed to as the substrate. The absorbance was measured using a
stress–strain plots. The slope of a linear fit for the strain range spectrophotometer at 405 nm.
of 0.1–0.3 was used as a measure of the compressive modulus.
2.9. Osteogenic marker gene expression
2.6. Gelatin releasing test After 3 weeks of culturing the cell-loaded hydrogels in the
Cell-free hydrogel disks were used in the gelatin release osteogenic differentiation media and 1 week of cultured
test. Fluorescein dye was attached to gelatin and used to external cells, quantitative real-time polymerase chain
assess gelatin release. Gelatin was dissolved in MES buffer reaction (qPCR) analysis was observed to evaluate the
solution consisting of 0.3 M NaCl (pH 6.5) to produce expression of relevant osteogenic markers. To extract
a 1% (w/v) solution. Then, 0.01 g of sulfo-fluorescein RNA and reverse-transcribe it into complementary
(Thermo Fisher Scientific, USA) and 0.08 g of EDC DNA (cDNA), the RNAiso extraction reagent (Takara,
were added and reacted overnight at 37°C. The resulting Japan) was used. According to the product manual,
solutions were filtered, freeze-dried, and stored at 4°C until 20 μL of the solutions were incubated in a PCR thermal
use. Fluorescein-gelatin was mixed with MA-alginate using cycler (C1000; Bio-Rad, USA) initially at 25°C for 5 min,
the same method, which is mentioned in section 2.4, and then at 45°C for 1 h, and finally at 95°C for 5 min. Gene
hydrogel disks with different ratios of the components were expression was quantitatively measured using RT-PCR
fabricated. The fabricated disks were soaked in cell culture equipment (TP900, Takara, Japan) and TB Green qPCR
media at 37°C with 5% CO , which are the same conditions Mix (Takara, Japan). The cycling conditions were as
2
used for cell culturing. At each time point (0 h, 1, 7, 14, follows: initial denaturation cycle at 95°C for 30 s, followed
and 21 days), 200 μL of buffer was removed from each by 40 cycles at 95°C for 5 s, and 60°C of annealing and
sample, and the relative fluorescence units were measured extension for 10 s. The cycle threshold for every transcript
using a spectrophotometer at an excitation wavelength of expression was exported from the Takara dice real-
480 nm and an emission wavelength of 520 nm to detect the time software. The comparative 2 -∆∆Ct method was used
fluorescein-gelatin released from the hydrogel disks. The to calculate the relative abundance of mRNA. Primers
remaining gelatin in each hydrogel disk was measured at the used included: 5’-CCATCCAATCGGTAGTAGCG-3’
same time points using a fluorescent imaging and analysis (sense) and 5’-GTAACCCGTTGAACCCCATT-3’ (anti-
system (Visque inVivo smart, Vieworks, South Korea). sense) for glyceraldehyde 3-phosphate dehydrogenase

Volume 9 Issue 2 (2023) 145 https://doi.org/10.18063/ijb.v9i2.660