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Söhling, et al.
2. Materials and methods OD was detected by Alizarin Red staining , adipogenic
[36]
differentiation was detected by Oil Red O staining ,
[35]
2.1. Ethics and chondrogenic differentiation of a MSC pellet
A MSC pool was obtained from residual bone marrow was detected by dimethylmethylene blue staining as
[36]
samples from five healthy donors. The use for research described .
purposes is covered by an ethics vote (329/10 of the
Department of Medicine of the Goethe University). For 2.3. Filament fabrication
the whole-blood stimulation assay, the analysis of blood Pre-dried (12 h; 40°C) PLA granules (mean granule size
samples from volunteers is covered by ethics vote 89/19 of 2-5 mm) (PLA-Filament Kristall Natur, 3dk.berlin,
of the same department. All donors signed informed Berlin, Germany) were mixed with pre-dried S53P4-BG
consent. The study was conducted according to the (mean granule size 25-42 µm; BG composition: 53% SiO ,
2
guidelines of the Declaration of Helsinki and approved 23% Na O, 20% CaO, and 4% P O (S53P4; BonAlive
2
2
5
by the Ethics Committee of University Hospital Frankfurt Biomaterials Ltd., Turku, Finland) according to weight
am Main, Goethe University (project no. 89/19). with 0%, 5%, 10%, and 20%. Using a filament extruder
2.2. Establishment and characterization of the (NEXT 1.0 Advanced, 3devo, Utrecht, Netherlands), the
MSC pool resulting granulate mixture was heated within the heating
zone, extruded at 4 rpm and then cooled down using a fan.
Pooled MSCs were used in this study to minimize the Automatic extrusion speed ensured a constant diameter
influence of individual differences between individuals. of 1.75 mm for the subsequent printing process. This
Mononuclear cells were isolated by Ficoll density gradient took place on a commercially available fused filament
centrifugation from EDTA-anti-coagulated bone marrow fabrication (FFF) 3D printer (i3 MK3S, Prusa Research,
samples (approximately 1-2 ml volume) as previously Prague, Czech Republic), whereby the slicing process
described . Mononuclear cells were seeded at a density was first carried out using the Cura Ultimaker software
[34]
of 1 × 10 cells/cm using MesenCult+supplements (V4.6, Ultimaker, Utrecht, Netherlands). The detailed
5
2
(hereinafter referred to as “complete medium,” Stemcell filament manufacturing process is described in Schätzlein
Technologies, Cologne, Germany) and cultured in et al. [37]
75 cm culture flasks for two additional passages after
2
reaching confluence. Cells were then harvested and 1 × 2.4. Test specimens and scaffold fabrication
10 MSC each per 1ml of freezing medium consisting The prepared PLA/BG composite was now used to print
6
of 90% FCS and 10% DMSO was stored in liquid two-layer test specimens (diameter 5 mm; height 0.3 mm)
nitrogen until the target number of donors was reached. for the in vitro studies on a 3D printer (i3 MK3S, Prusa
To create the pool, one vial of MSC from each donor was Research, Prague, Czech Republic) with a nozzle diameter
thawed and cultured in complete medium over another of 0.4 mm. Design was performed with computer-aided
passage. Cells were then enzymatically detached and design software NX 12 (Siemens NX 12, Siemens AG,
counted. 1 × 10 cells from each donor were pooled, then Berlin and Munich, Germany) and preprocessed with
6
centrifuged, resuspended in freezing medium, and stored Cura Ultimaker v.4.6. (Ultimaker, Utrecht, Netherlands).
in liquid nitrogen until use. Pooled MSCs were cultured To verify whether complex 3D structures are also
after thawing for one further passage in complete medium
and used either for characterization or experiments. printable with the prepared composites, cylindrical porous
scaffolds (height 6 mm; diameter 5 mm; macroscopic
Expression of MSC markers CD73 (CD73-PE, clone pores 700 µm, microscopic pores 150 µm, wall thickness
AD2, BD Biosciences, Heidelberg, Germany), CD90 1.5 mm) were printed under same printing conditions
(CD90-APC, clone 5E10, BD Biosciences), CD105
(CD105-FITC, clone 266, BD-Biosciences, Heidelberg, and examined by light microscopy (Axio Observer Z1,
Germany), and absence of hematopoietic markers CD34 Carl Zeiss, Göttingen, Germany). Filaments from 1.65
(CD34-FITC, clone 581, BD Biosciences, Heidelberg, to 1.85 mm diameter were used. The detailed printing
[37]
Germany) and CD45 (CD45-PerCP, clone 2D1, BD process is described in Schätzlein et al.
Biosciences, Heidelberg, Germany) were analyzed 2.5. Porosity
by flow cytometry. Fluorescence conjugated, isotype
identical, and unspecific antibodies served as control (all Porosity for the printed columns was estimated using
BD Biosciences) as previously described . In addition, slightly modified Archimedes principle as shown in
[35]
trilineage differentiation of MSC was performed using various other works [38,39] . A printed box with predetermined
osteogenic, adipogenic, and chondrogenic differentiation specific volume was first filled with agarose (A9539-
media (PromoCell, Heidelberg, Germany) and 500G, SIGMA, St. Louis, USA) and weighed with a high
differentiation protocols supplied by the manufacturer. precision scale (S-234, Denver Instrument, Göttingen,

International Journal of Bioprinting (2022)–Volume 8, Issue 4 67