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Söhling, et al.
by digestion with the RNase-free DNase Kit following free sealable tubes at a ratio of 1:4 with Dulbecco’s
the manufacturer’s protocol (Qiagen). Modified Eagle Medium (DMEM, Invitrogen, Bleiswijk,
Real-time RT-PCR was performed using primer Netherlands) and three test specimens (PLA or BG20)
assays (all obtained from Qiagen, first: gene accession were added to each mixture under sterile conditions. Only
number, 2. order number for primer set) for RUNX2 normal medium was used as negative control, and the
(NM_001015051, PPH01897B), ALP (NM_000478, positive control was stimulated with lipopolysaccharide
PPH01311F), COL1A (NM_000088, PPH01299F), (LPS) (5 µg/mL, Sigma-Aldrich). After 24 h incubation
BMP2 (NM_001200, PPH00549C), MAPK8 at 37°C, 5% CO , the cell supernatant was separated by
2
(NM_002750, PPH00720B), MAPK14 (NM_001315, centrifugation (15 min, 1100 ×g, 4°C), aliquoted, and
PPH00750B), IL6 (NM_000600, PPH00560C), and stored at −80°C until analysis.
GAPDH (NM_002046.3, PPH00150F) as housekeeping Analysis of secreted mediators in the supernatant
gene. Quantitative RT-PCR over 40 cycles (94°C: 15 s, was performed semi-quantitatively using the Proteome
70°C: 30 s) was performed on a Stratagene MX3005p Profiler Human XL Cytokine Array Kit (RnD-Systems,
qPCR system (Stratagene, La Jolla, CA, USA). A melting Minneapolis, Minnesota, USA), according to the
curve analysis was applied to ensure the specificity of manufacturer’s instructions. Complete parameter list
the PCR reaction. Relative quantification of the mRNA is available at https://www.rndsystems.com/products/
levels of the target genes was determined using the Livak proteome-profiler-human-xl-cytokine-array-kit_ary022b,
method (2 −ΔΔCT ) normalizing gene expression to GAPDH (accessed on 09/24/2021). Membranes were photographed
and MSC cultured under normal conditions . immediately after the addition of the chemiluminescent
[40]
substrate using a Fusion FX7 gel scanner (Vilber
2.10. Effect of calcium ions released from the BG Lourmat, Eberhardzell, Germany). The exposure time
component was 5 min for each membrane. The images were saved
as uncompressed 16-bit TIFF files. The densitometric
In this experiment, it was investigated whether calcium analysis of the spots was performed using ImageJ. For
ions released from the BG component led to the observed this, the product of spot area (pixel) and mean gray value
changes in gene expression. The expression of the IL-6 was calculated for each spot. Subsequently, the results
gene marker was selected for this purpose, since its were normalized to the reference spots (n = 6/membrane).
expression was strongly attenuated by incubation with
BG20. From preliminary experiments, it is known that 2.12. Statistics
within 72 h, an average of 0.35 nmol/µL calcium is
released from a test sample BG20 . Here, 200 µL of Statistical comparisons were made using the
[37]
normal medium was conditioned over 72 h with one nonparametric Kruskal–Wallis test with Bonferroni-
BG20 or PLA specimen as control. Released calcium Holm corrected post hoc analysis according to Dunn.
P < 0.05 indicates statistically significant differences.
ions were bound by the addition of the specific calcium P values between 0.05 and 0.1 were considered statistical
chelator EGTA (ethylene glycol tetraacetic acid, Sigma- trends. Statistical analyses were performed using Bias
Aldrich). In respective assays, EGTA was added
equimolar to the putative released calcium in a volume of 11.12 software (Epsilon Verlag, Darmstadt, Germany).
Unless otherwise indicated, results are presented as box
2 µL. In each case, 1 × 10 MSCs/well were incubated for plots of the median and interquartile ranges (median 50%,
4
72 h in a 96-well plate with the conditioned media with quartile 25%, and quartile 75%) in diagrams.
or without EGTA. This was followed by analysis of IL-6
gene expression as described in the previous section. The 3. Results
experiment was conducted 3 times with two technical
replicates each. 3.1. Filament, test specimen, and scaffold
properties
2.11. Immunostimulatory potential of the
composite Filaments with a BG content of up to 20% can be produced.
The BG is distributed homogeneously in the filaments
Whole-blood stimulation assay was used to investigate and can be found both in the center of the strand and at the
the immunostimulatory potential of BG20. The assay interfaces (Figure 1B, black arrows). The entire strand
was performed as previously described with the is interspersed with BG particles (Figure 1B and C).
following modifications . Blood was collected using A more detailed characterization of the filament can be
[41]
lithium heparin-coated Monovette (Sarstedt, Nümbrecht, found in Schätzlein et al. (under submission).
Germany) from three healthy volunteers (N.S., S.A., and With all four filaments, high-resolution 3D printing
D.H., one female and two males, mean age 39 ± 14 years). is possible on a standard FFF printer. Thereby, the strand
In each case, 500 µL blood were mixed in endotoxin- thickness in the body seems to be independent of the BG

International Journal of Bioprinting (2022)–Volume 8, Issue 4 69