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Osteosarcoma growth on trabecular bone mimicking structures manufactured via laser direct write

wards) and pressure was controlled by a needle valve chanical rocker for 24 hours to ensure complete dis-
(Edwards LV10K). The flow rate of acrylic acid mo- solving of formazan crystals prior to the removal of
3
nomer was established through the chamber at 2.4 cm ∙ 200 μL of MTT solution (triplicate readings) from
−1
min . The electromagnetic field was generated by each sample. The absorbance of the solution was
radiofrequency generator (Coaxial power systems li- measured using a Biotek absorbance reader (Model
mited) through a coil wrapped around the chamber. Elx800) at a single wavelength of 562 nm. Statistical
The power to this coil was set to 15 W and the plasma analysis of the results was carried out using the
was left on for 20 minutes. Graphpad Prism program. The significance between
the control and test values was compared using the
2.3 In Vitro Biocompatibility two tailed t-test with an assumption of equal variance.
In vitro biocompatibility of PolyHIPE materials in the The levels of significance are indicated in the graphs.
form of both EHA80 disks and woodpile structures 2.4 Cell Imaging: Scanning Electron Microscopy,
was investigated using the human osteosarcoma cell Confocal Microscopy
line MG-63. MG-63s were cultivated in Dulbecco’s
modified Eagle’s medium (DMEM) supplemented The morphology of PolyHIPE structures and MG63
with 10% fetal calf serum, 1% penicillin and strepto- cells was investigated using Philips XL-20 scanning
mycin, 1% L-glutamine and 0.25% amphotericin B in electron microscope operating at 10.0 kV. The Poly-
a humidified 5% CO 2 atmosphere at 37°C. Cells were HIPE disks were washed in PBS three times and fixed
seeded on PolyHIPE disks and woodpile scaffold in 2.5% glutaraldehyde for 1 hour. The samples were
structures and incubated for 7 days in vitro. PolyHIPE further washed in PBS, and then soaked in distilled
scaffolds and disks were placed in 12-well plates and water for a further 5 minutes. Finally, the disks were
secured using marine grade steel rings (2 cm outer dehydrated for 15 minutes in a series of ethanol solu-
diameter, 1 cm inner diameter). The disks were seeded tions at 35%, 60%, 80%, 90% and 100% concentra-
at a density of 20,000 cells per disk (n = 6 of each tion. The disks were finally treated with HDMS/EtOH
type per day) and the woodpile scaffolds were seeded (1:1 EtOH + HDMS) for 1 hour following a rinsing in
at a density of 100,000 cells per scaffold. The required 100% HDMS for 5 minutes. The samples were air
numbers of cells were seeded on the samples in 10 μL dried prior to sputter coating with gold and prior to be
of DMEM cell suspension placed in the centre of the attached by adhesive carbon tabs onto aluminium stubs.
samples and was left in an incubator for 50 minutes at SEM images were taken from different sections of the
37°C. A further 990 μL of DMEM was added to each same PolyHIPE structure and random selection of 25
well and were left in the incubator for the duration of voids from each SEM image was made and statistical
the experiment (1, 3 and 7 days). Media was changed correction factor was applied to the average void di-
[1]
every two days. The controls (n = 6) involved seeding ameter . The average void diameter of the structures
cells on tissue culture plastics containing the standard was quantified using the software ImageJ 1.48.
medium. All the disks were incubated at 37°C in 5% The cell seeded woodpile structures were used for
CO 2 for 1, 3 and 7 days. SEM. Single plane images (1024 × 1024 pixels) were
MTT assay is a quantitative indicator of metaboli- obtained using a Zeiss LSM Meta upright confocal
cally active cells, which is widely used as an indicator microscope. Z-stack images (512 × 512 pixels) were
to analyse cell proliferation as well as cell viability. obtained using the same settings as single plane im-
The MTT [3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyl- ages but repeated images were obtained of the same
tetrazolium bromide] solution was prepared in ad- area, translated 11 µm in the z direction after each
-1
vance at a concentration of 0.5 mg∙mL . The samples capture. After fixation with 3.7% formaldehyde (ap-
were washed in PBS and 1 mL of MTT solution was proximately 30 minutes) at room temperature, the
added to each disk and was left in the incubator for 50 woodpile structures were permeabilised with Triton-
minutes. The disks were washed slowly with PBS to X100 (1%) for approximately 3 minutes. Cells were
minimise any risks involved in the accidental removal washed further with PBS three times. Finally, the cells
of produced formazan salts. Ethoxyethanol reagent were treated with 0.1% nuclear staining DAPI and
plus (700 μL) solvent (Sigma, UK) was added to each 0.1% Phalloiding-FITC. DAPI was excited via a two-
sample to dissolve the formazan crystals resulting photon 800 nm laser (11% transmission) and the
from MTT reduction. The samples were left on a me- emission detected between 435 and 485 nm. FITC-

70 International Journal of Bioprinting (2016)–Volume 2, Issue 2