Page 507 - IJB-10-6

International Journal of Bioprinting Redefined collagen inks in cartilage printing

obtained excess heat capacity profiles were normalized to evaluated by the smallest pore that could be achieved.
the protein content. The tendency for filament collapse was observed by
printing over gaps with sizes of 1–16 mm. Finally, height
2.3. Collagen bioink formulation and printability maintenance was evaluated by printing a 10-layer “wall”
Neutral collagen bioinks were prepared by keeping the and following its height for 30 min at ambient conditions
solutions at a low temperature of around 5°C. First, the of 45% humidity and 20°C.
dry collagen was dissolved in cold dilute acetic acid.
Thereafter, a suitably selected concentration of phosphate- 2.4. Enzymatic hydrolysis of high-collagen
buffered saline (PBS) was added. Finally, the solution was concentration hydrogels
neutralized with NaOH solution. For instance, to prepare Stability against enzymatic digestion was established
a 5 mg/mL collagen solution, 25 mg dry collagen was in vitro by dipping in a 2 mL 0.1 M Tris-HCl buffer
dissolved in 3.5 mL 0.05 M acetic acid; 0.5 mL 10× PBS was containing 5 mM CaCl solution of collagenase from
2
then added; and finally, the solution was neutralized with Clostridium histolyticum, with an activity of 49 CDUs/
about 0.9 mL 0.2 M NaOH. The neutralization process was mL (collagen digestion units) at 37°C. Three different
closely monitored using the pH indicator phenol red. collagen concentrations were investigated, i.e., 1.0, 2.0,
and 4.0% (w/v). The neutralized hydrogels were thermally
The effect of various components of a typical collagen crosslinked at 37°C overnight before introducing them to
bioink on thermal gelation kinetics and complex modulus the collagenase solutions. Changes in the biopolymer mass
was investigated. In particular, 0.5% collagen solution were monitored for up to 8 h by washing the remaining
with neutral pH and with various concentrations of NaCl, gels with EDTA solution, freezing the hydrogels at −20°C,
Na SO , CaCl , phosphate buffer (Na HPO and KH PO in freeze-drying, and weighing the dry mass. This approach
2
2
2
4
2
4
4
a molar ratio 5.6:1), PBS (i.e., 1× PBS contains 1.37 M NaCl, eliminates the effect of mass increase due to hydrogel
0.027 M KCl, 0.1 M Na HPO , and 0.018 M KH PO ), and swelling, providing direct information on biopolymer
2
4
4
2
glucose were prepared and studied by rotational rheometry degradation kinetics.
in oscillation mode (Table 1).
Printability of a 2% collagen ink was evaluated on a 2.5. Characterization of elastic properties
newly developed mechanical extrusion bioprinter capable Rheological analysis of neutralized collagen solutions
of maintaining a cartridge temperature of ≤5°C indefinitely and inks was performed using Thermo Scientific HAAKE
and independent of the ambient temperature. Additionally, RHEOSTRESS 600 with parallel plate (20 mm diameter;
the bioprinter maintains a platform temperature of 37°C. 1 mm distance) geometry. Rotational controlled-shear-
stress oscillatory measurements at 1 Hz and amplitude of
The system uses 3 mL syringes with a Luer lock, which are 1 Pa were utilized to monitor thermal crosslinking and
widely available in sterile packaging.
viscoelastic properties. Thermal gelation was induced with
The printability of the formulated bioinks was two different temperature ramps at 1°C/min and 30°C/min.
characterized by their extrudability and capacity for
continuous uninterrupted extrusion. The resolution 2.6. Bioink functionalization with TGF-β1 and
was established by screening various extrusion factors, release studies
which effectively and directly determined the flow rate The release of two different concentrations of TGF-β1
until filament thinning resulted in the deposition of non- from thermally crosslinked collagen hydrogels with two
continuous filaments. The effect of filament spreading was different biopolymer concentrations was investigated at
37°C. Initially, 5 ng/mL GF was tested, as it is within the
range of typical concentrations employed in chondrogenic
Table 1. Tested concentrations of various components in the
collagen bioink. media. Simultaneously, for consistency with the bioprinting
Component Concentrations studies described below, the collagen concentration used
was 2%. In light of the repeatedly observed results, the
NaCl 75, 100, and 150 mM GF concentration was increased to 100 ng/mL, and the
Na SO 75, 100, and 150 mM collagen concentration was decreased to 1% to ensure
2 4
CaCl 2 75, 100, and 150 mM release within the sensitivity range of the ELISA kit.
Phosphate buffer 6, 12, and 18 mM Functionalization was performed in a laminar hood by
PBS 0.5×, 0.75×, and 1× adding TGF-β1 to a freshly neutralized collagen solution,
Glucose 5, 15, and 30 mM prepared aseptically, and homogenizing the solution with
Note: The concentrations of glucose were tested in 1× phosphate- a spatula while cooling on ice. The full volume of the
buffered saline (PBS); phosphate buffer was prepared with 0.1 M NaCl. functionalized collagen solution was then distributed into

Volume 10 Issue 6 (2024) 499 doi: 10.36922/ijb.4566