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Arguchinskaya, et al.
of additional support to improve the quality and stability of of the material were previously described . Briefly,
[12]
the scaffold both during and after biofabrication processes. the material is a soluble collagen fraction obtained by
Synthetic polymers such as poly(lactic acid) (PLA) or acidic extraction from porcine tendons, purified with a
polycaprolactone (PCL) are widely used [5,10,11] . These few salt precipitation and ion-exchange chromatography,
components provide a scaffold with necessary stability but sterilized by filtration, and stored in lyophilized condition.
do not fully correspond to hydrogel printing conditions Collagen gel was prepared by reconstitution of 10 mM
(e.g., a temperature). Thus, its application with hydrogels acetic acid to a concentration of 80 mg/ml.
for biofabrication using bioprinting is limited. Another Gelatin derived from porcine skin (80–120 g
approach related to the use of temporary hydrogel support Bloom, Type A, Sigma-Aldrich, cat. no. G6144) was
(e.g., gelatin) still could be applied. In accordance with the used as temporal material. It was dissolved in distilled
technique described in the study, a temporary component is water and sterilized by autoclaving (120°С, 1 bar).
located only in places where the main material needs some Gelatin concentration was 12%. At room temperature, the
prop. Each layer of biomaterial is laid on the previous one material was in gel state .
[13]
consisting either of the same biomaterial or the supporting
material. It guarantees stability of the structure during the 2.3. 3D-printing procedure
printing process and formation of required scaffold geometry The scaffold was created using extrusion-based print-
in the result of biofabrication. heads of Rokit in vivo 3D-bioprinter (South Korea).
Non-neutralized collagen at the stock concentration Slicing of the model was conducted in NewCreatorK
was applied as the main cartilage scaffold component. 1.57.63. The scaffold and the support were printed
It helped to validate the support performance since no with two 10 ml syringe dispensers. The materials
gelation (that increases elasticity of the material) occurred. were supplied through 21G needles. The printing was
At the same time, biocompatibility of neutralized collagen performed on a glass attached to the printing table with
was also assessed. The supporting part of the scaffold magnets. Temperature (23°C) was maintained within the
consists of gelatin, which was a temporary element. The chamber, on the printing table, as well as in the syringes.
aim of the study was to develop an approach for human The main printing options are presented in Table 1 and
thyroid cartilage scaffold temporal support formation, were the same for both materials. Immediately after the
which is applicable in the case of extrusion-based printing was completed, the object was placed in a cold
bioprinting with hydrogels. buffer (80 mM Tris-HCl, PanEco, Russia).
Due to certain mobility of plastic syringes and
2. Materials and methods needles, the centers of printing heads had to be adjusted.
2.1. 3D-model preparation For this purpose, the outlines of a 10 mm side square
were printed. Previously, one of the outlines had been
The thyroid cartilage model was based on CT images slightly displaced to improve misalignment visualization.
obtained as a part of routing diagnostic procedures. Informed The difference was evaluated by microscopy (Biomed 3,
consent was obtained from all patients before the study. Russia) and ImageJ 1.52.
Acquisition of CT data was conducted by a multi-detector
CT-scanner (Siemens Somatom Emotion 6, Germany). Neck 2.4. CT-verification
scanning was performed with a slice thickness of 1.25 mm, The scaffold geometry was verified using CT. The study
0 gantry tilt, and image resolution of 512 × 512 pixels. The was performed on Optima CT660 (GE Medical Systems,
acquisition data were stored in DICOM files. USA) with 120 kV and 340 mA. The acquisition protocol
Reconstruction of DICOM images was performed included the use of 64 detectors, 0.625 mm cut thickness,
by 3D Slicer 4.10.2 and MeshLab 2016.12. The integrity 0 gantry tilt, and 1.0 s rotation time. Post-processing of
of a mesh forming the model was checked using CT-acquisition was performed in Advantage Workstation
Autodesk Meshmixer 3.5.474. The same software was 4.6 (GE Medical Systems, USA). Further procedures were
used to estimate the wall thickness of the supporting carried out in NewCreatorK 1.57.63 and FreeCAD 0.17.
part of the scaffold. Further processing of the model and
its modification for bioprinting was carried out using Table 1. Printing parameters
FreeCAD 0.17. The volume of obtained models was
calculated by NewCreatorK 1.57.63. Veusz 3.0.1 was Parameter Value
used to visualize the results. Layer height, µm 386
Input flow, % 150
2.2. Hydrogels Fill density, % 66

Porcine atelocollagen type I (Viscoll, Imtek Ltd, Russia) Infill pattern concentric
was used as the main scaffold material. The properties Print speed, mm/s 5.0

International Journal of Bioprinting (2021)–Volume 7, Issue 2 105