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Owen, et al.
2.3 Scanning electron microscopy (SEM) added to the well, just covering the top scaffold
surface. The seeding suspension was gently pulsed
Samples were sputter coated with gold (SC500, by manual pipetting every 45 min to improve
Emscope) to improve conductivity before imaging seeding distribution. After 2 h, scaffolds were
(XL-20 SEM, Philips). transferred to a 48 well plate so that only adhered
2.4 Mechanical testing cells remained. Cell viability (PrestoBlue) was
quantified to determine baseline cell numbers,
Compressive testing was performed on a BOSE then scaffolds were maintained in supplemented
ElectroForce 3200 with a 450 N load cell at a rate media (SM) consisting of BM with 5 mM beta-
of 0.01 mm/s to a maximum displacement of 4 mm. glycerolphosphate and 50 μg/mL ascorbic acid
The samples were placed centrally on parallel 2-phosphate. Media were changed every 2 – 3 days.
compression plates and a preload of 1 N applied
before test initiation. The compressive modulus 2.7 Cell viability
was calculated from the force-displacement curves. Viability was quantified on days 0, 7, and 14 by
2.5 Plasma modification PrestoBlue which measures metabolic activity.
PrestoBlue reagent was diluted 1:10 in Hank’s
To increase hydrophilicity for cell culture, Balanced Salt Solution. 1 mL was added to each
polyHIPE samples were air plasma treated well and incubated for 1 h. 200 μL of the reduced
(Zepto W6 Plasma System, Diener Electronic). solution was then transferred in triplicate to a black
The samples were placed uniformly on a flat 96 well plate and the fluorescence measured on a
aluminum foil wrapped stage and placed centrally plate reader (Tecan infinite 200-pro, λ 540 nm,
ex
in the plasma chamber. A range of parameters λ 590 nm). Scaffolds were rinsed in PBS before
em
were tested as charring readily occurred in adding fresh SM.
the particulate leached polyHIPEs. The final
parameters used were 15 W at an initial pressure 2.8 Mineralized matrix deposition
of 0.4 mbar for 1 min. Calcium and collagen deposition were quantified
2.6 Cell culture on days 7 and 14 by alizarin red S (ARS) and
direct red 80 (DR80), respectively, as previously
MLO-A5 murine post-osteoblasts (kindly donated reported Owen et al. Briefly, the samples were
[48]
by Dr. Lynda Bonewald, University of Missouri) rinsed twice in PBS then fixed by immersion in
were used for all experiments. Cells were passaged 3.7% formaldehyde for 30 min. Scaffolds were
in gelatin-coated flasks in basal media (BM) rinsed twice in diH O, and then submerged in
2
consisting of alpha-minimum essential medium 1 w/v% ARS in diH O for 30 min to stain for
2
(alpha-MEM) (Lonza, UK) with 10% fetal bovine calcium. Stained samples were washed repeatedly
serum (FBS, Labtech, UK), 2 mM L-glutamine, in diH O until wash water remained clear, then
2
100 U/mL penicillin, and 100 μg/mL streptomycin. air dried and photographed. To quantify, scaffolds
To sterilize, scaffolds were placed in 70% were submerged in 1 mL 5% perchloric acid and
ethanol and put under vacuum to remove all orbitally shaken for 15 min at 100 rpm. 150 μL
air. After 90 min, ethanol was exchanged for was then transferred in triplicate to a 96 well
phosphate-buffered saline (PBS) and orbitally plate and read at an absorbance of 405 nm (Tecan
shaken at 150 rpm for 15 min. The washing stage infinite 200-pro). The concentration of ARS was
was repeated a further 2 times, then PBS was determined from a standard curve. Scaffolds were
exchanged for BM for 1 h. Media were removed then washed 3 times in diH O before immersing
2
from the scaffolds for seeding. in 1 w/v% DR80 in saturated picric acid for
To seed, scaffolds were placed in a 96 well 1 h to stain for collagen. Stained samples were
plate. 350,000 MLO-A5 in 100 μL BM were washed repeatedly in diH O until wash water
2
International Journal of Bioprinting (2020)–Volume 6, Issue 2 103
