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3D Printable PLA/BG Composite In Vitro Evaluation
Deutschland) before and after submersing the printed for 10 min and were immersed in a four-step ethanol
parts (n = 3) and removing of the supernatant. Afterward, gradient (50%, 75%, 96%, and 100%) for 5 min each.
the submersed and with agarose filled parts were weighed, After a short passage in 1,1,1,3,3,3-hexamethyldisilazane
too. In addition, these values were compared to calculated (Merck Schuchardt, Hohenbrunn, Germany) and
parameters within a CAD tool (Fusion 360, v2.0.12665, draining overnight, gold was deposited on the samples by
Autodesk, San Rafael USA). sputtering (5 × 60 s, Agar Sputter Coater; Agar Scientific
Ltd., Stansted, United Kingdom). Analysis was performed
2.6. Adhesion and metabolic activity of MSC using a SEM (Hitachi, Düsseldorf, Germany) and the
MSCs were seeded at a density of 1 × 10 cells/test specimen Digital Image Processing System 2.6 (Point Electronic,
4
and allocated in a well of a 12-well plate. For this purpose, Halle, Germany).
cells were suspended in 15 µL culture medium, transferred
in a droplet onto the test specimen, and incubated for 2.8. Determination of the osteogenicity of BG20
30 min at 100% humidity to allow the cells to adhere to Osteogenicity of the composite BG20 was analyzed in
the test material. The wells were then carefully filled with a functional test. For this purpose, 2 × 10 MSCs were
4
500 µL of complete culture medium each. seeded into individual wells of a 24-well plate. After
Fluorescence micrographs were taken to directly 24 h, the medium was changed and either 500 µL normal
detect adherent cells. For this purpose, MSCs on the test medium/well or 500 µL OD medium/well (PromoCell)
specimens were stained with Calcein-AM (BD Biosciences, were added in each well. In addition, a test specimen made
Heidelberg, Germany) 24 h after seeding. Specimens with of PLA or BG20 was coincubated contact-free through
cells were transferred into individual wells of a 24-well an insert (pore size 3 µm, Corning B.V. Life Sciences,
plate and 1 ml of pre-warmed culture medium containing Amsterdam, Netherlands) over a period of 14 days.
20 µM Calcein-AM was added to each well. Cells were The medium was changed twice a week. After 14 days,
incubated for 40 min at 37°C, washed carefully 3 times with calcium deposition of MSC was analyzed by alizarin red
phosphate-buffered saline (PBS), and stained with DAPI staining (Merck, Darmstadt, Germany) as described in
(1 µg/ml in PBS, Sigma-Aldrich, Taufkirchen, Germany). the previous study . For evaluation, the percentage of
[36]
After 10 min incubation, cells were again carefully washed alizarin red positive area per microscopic field of view
3 times with PBS. MSC adherence and distribution on was calculated using ImageJ software (https://imagej.nih.
the test specimen were then assessed by examining the gov/ij/). The experiment was performed 3 times with two
samples with fluorescence microscopy using a Zeiss Axio technical replicates each time.
Observer Z1 (Zeiss, Gottingen, Germany).
The metabolic activity of MSC adhering to the test 2.9. Assessment of OD and inflammatory
specimens was determined by 3-(4,5-dimethylthiazol- pathway gene expression
2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT)
assay (MTT, Roche Diagnostics, Mannheim, Germany). To evaluate the influence of the BG component on OD and
For the determination of the metabolic activity, test induction of inflammatory pathways, the expression of the
specimens were carefully moved 24 h, 72 h, and genes involved in osteogenesis (RUNX2 [transcription
168 h after seeding into a new 48-well plate to prevent factor], alkaline phosphatase [ALP], collagen-1α
the adherence of cells to the bottom of the wells [COL1A], and bone morphogenetic protein-2 [BMP2])
that could interfere with measurement results. MTT and inflammation mitogen-activated protein kinase
assay principle is based on the cleavage of the yellow ([MAPK]8 cJun-N-terminal kinase 1 [JNK-1], MAPK14
tetrazolium salt MTT (3-[4,5-dimethylthiazol-2-yl]-2,5- [p38α], and interleukin-6 [IL-6]) were measured.
diphenyltetrazoliumbromide) to purple formazan crystals Analogous to the previously described experiments,
by metabolically active cells. All experiments were 1 × 10 MSCs were seeded onto the different materials
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performed in duplicate. The MTT assay was performed and cultured in osteogenic medium for 24 h, 72 h, and
according to the manufacturer’s instructions. Briefly, 168 h. At the respective time points, the medium was
MTT reagent was incubated 2 h followed by overnight removed and total RNA was isolated using the RNeasy
lysis of the formazan crystals. The supernatants were kit, according to the manufacturer’s instructions (Qiagen,
collected and transferred into wells of a new 96-well plate Hilden, Germany). RNA concentration was measured
where its absorbance was measured at 570 nm using an photometrically using NanoDrop (ND-1000, NanoDrop
ELISA reader (Infinite M200, Tecan, Mainz, Germany). Technologies, Wilmington, Delaware, USA).
Each 1 µg of RNA was reversely transcribed using
2.7. Scanning electron microscope (SEM) an Affinity script QPCR-cDNA synthesis kit (Stratagene,
Scaffolds seeded with cells were analyzed using SEM. La Jolla, CA, USA) following the manufacturer’s
Samples were fixed with glutaraldehyde (2% in −/− PBS) instructions. Contaminated genomic DNA was removed

68 International Journal of Bioprinting (2022)–Volume 8, Issue 4