Page 322 - IJB-9-3

International Journal of Bioprinting New fibrillar collagen for 3D printing and bioprinting

collagen bioink, 3 g of ColA was loaded in one syringe, and technique has been chosen in view of already reported
2 g of water was loaded in another syringe, connected and studies stating that cryo-SEM enables better preservation
homogenized. of the collagen microarchitecture . ColN collagen were
[25]
observed in a table-top TM4000Plus SEM (Hitachi)
2.2.2. Neutral ink preparation equipped with a coolstage (Deben UK Ltd) for cryo-
Starting from ColN ink (Viscofan S.A., Spain) with 5% of SEM analysis. Samples were prepared as follows: Acid
collagen content, the mass was neutralized by adding TRIS- collagen sample was diluted to 0.5% (w/w) in milliQ water.
HCl (1.5 M, pH 7.4) in a 3:2 and 2:3 volume ratio, thus Regarding the neutral samples, acid collagen was first
diluting the collagen concentration to 3% (w/w) and 2% neutralized with TRIS-HCl (1.5M, pH 7.5–7.6) until 2%
(w/w), respectively. Tris(hydroxymethyl)aminomethane (w/w). Then, the sample was further diluted with milliQ
(VWR, UK) was dissolved to a concentration of 1.5 M water until reaching 0.5% (w/w) concentration. Cryo-
by magnetic stirring. To perform the neutralization, the SEM analysis was performed by positioning a sample of
fibrillar collagen ink was introduced in a 10-mL syringe the aforementioned dilutions in a cryo-SEM sample holder
with Luer lock, and the necessary amount of TRIS-HCl in a and rapidly freezing it to -50°C once inside the microscope
different syringe, with eccentric Luer lip. The syringes were chamber. Secondary electron (SE) images were obtained at
connected through a Luer female-to-female connector 5 kV and medium vacuum conditions.
(Fisher, USA), and mixed 40 times.
2.3. 3D printing
2.2.3. Rheology measurements The acidic ColA and the neutral ColN neutral inks were
The rheology measurements were performed with a Haake prepared at different concentrations and printed with the
Mars 40 rheometer (Thermo Scientific) equipped with pneumatic extrusion-based bioprinter BioX (Cellink).
a plate–plate geometry (20 mm ∅) and stainless steel Squared 20 × 20 mm digital design with a 20% gyroid infill
smooth surface, working with a 0.8 mm gap at 25°C in all and two layers height (Figure 1A) were printed at different
experiments, unless otherwise stated. collagen concentrations in an attempt to determine the
To perform the rheological measurements of the ColA optimal printing conditions of each formulation. All the
acidic ink, a 2-cm layer of collagen was prepared between scaffolds were printed at room temperature with a 20-G
two Teflon sheets to avoid moisture loss. After a stabilization nozzle (inner diameter 0.61 mm, Cellink). The speed and
of the mass of 30 minutes at 4°C, a sample of 20 mm of the pressure were adjusted for each collagen formulation.
diameter was punched out and placed in the bottom plate Additionally, a 27-layer scaffold (L × W × H: 20 × 20 ×
of the rheometer. When the measuring gap was reached, 20 mm) was obtained with ColA working at 300 kPa and
the acidic bioink was equilibrated for 10 minutes between 5 mm/s.
the plates. The neutral ink (ColN) was prepared following
the “neutral ink preparation” protocol (see section 2.2.2), 2.4. Cell cultures
and it was extruded in the bottom plate just after the Mouse fibroblast cell line (L929) was used for the in vitro
neutralization. Once again, the sample was equilibrated for cytotoxicity assays. These cells were cultured in DMEM
10 minutes between the plates prior to analysis. supplemented with 1% (v/v) of penicillin/streptomycin
and 10% (v/v) of FCS.
Oscillatory strain amplitude sweep tests were obtained
Regarding the 3D bioprinting and the production
by subjecting the samples to 1 Pa to 15,000 Pa in a logarithmic of cell-laden collagen bioinks, two types of cells were
ramp with 6 points per order of magnitude, working used: L929 and pluripotent D1 mesenchymal stem cells
at constant frequency (1 Hz) at 25°C. The temperature (MSC-D1). MSC-D1 cells were also cultured in DMEM
sweep oscillatory tests (0.1% amplitude, 1 Hz) were 30-2002 (Gibco, Thermo-Fisher Scientific) supplemented
performed from 4°C to 37°C at a heating rate of 1 K/min. with 1% (v/v) of penicillin/streptomycin and 10% (v/v)
Subsequently, the same sample was cooled from 37°C to of FBS. In all cases, Tissue Culture Flasks (Corning®
4°C working under the same conditions.
Costar®) were used for the expansion and maintenance
Finally, the flow curves were obtained by subjecting the of both MSC-D1 and L929 cells. Culture conditions were
sample from 0.01 to 1000 1/s of shear rate (0.166 1/s steps), controlled by a cell culture incubator set at 37°C, with 5%
obtaining 6 points per order of magnitude. CO and 95% relative humidity.
2
2.2.4. Scanning electron microscopy 2.5. In vitro cytotoxicity
The micro-architectural properties of ColN collagen gels The biocompatibility profile of ColA and ColN ink was
(before and after neutralization) were analyzed through assessed by means of the WST-1 assay kit, based on the
cryogenic scanning electron microscopy (SEM). This

Volume 9 Issue 3 (2023) 314 https://doi.org/10.18063/ijb.712