Page 454 - IJB-10-3

International Journal of Bioprinting Biomimetic scaffolds for tendon healing

ramp type to logarithmic. The properties of the ink were Once the structure was printed, it was crosslinked using
determined from the graphical representation of modulus 1 mL of a mixture of CaCl (200 mM) and thrombin
2
vs. frequency. All the obtained rheological parameters of the (40 IU mL ), mixed at a 1:1 ratio, for 30 min at room
-1
ink are summarized in part (B) of the Supplementary File. temperature.
2.5. Printability For the 3D bioprinting process, the bioink containing
For the study of printability, a CELLINK BIO X printer tenocytes was utilized, using the same protocol and
(Gothenburg, Sweden) was used. In order to control the parameters as for the acellular scaffolds. After the
printing temperature, the printing process was performed crosslinking process, the tissue constructs were maintained
using a temperature-controlled printhead. The selected tip in Prigrow III culture medium supplemented with 10%
was a 22 G blue needle, and the speed was kept constant at (v/v) FBS and 1% (v/v) P/S at 37°C in a humid atmosphere
7 mm s . The printability of the ink was studied at different with 5% CO until analysis.
-1
2
pressures and temperatures. The extrudability of the ink at 2.7. Macroscopic imaging and cryo-SEM imaging
each temperature was tested. The photos of the obtained After printing, images of the scaffolds were captured to
filaments were used to identify the level of gelation (relevant examine their dimensions and to compare them with those
methodology is given in part (C) of the Supplementary of the original design. These images were also used to verify
File). To determine shape fidelity, also known as print the homogeneity of the obtained scaffolds. For analyzing
accuracy, a computer-aided design (CAD) was made with the morphology of the surface, cryo-scanning electron
horizontal and vertical lines at different distances in a single microscopy (cryo-SEM) technology was employed using a
Z plane, as described in part (C) of the Supplementary File TM4000Plus microscope (Hitachi, Japan).
(panel 4 in Figure S4 in the Supplementary File). For each
38
temperature, the ink was printed at different pressures. 2.8. Mechanical characterization
A photo of the obtained structure was taken and, using The mechanical properties of the acellular scaffolds and
ImageJ (National Institutes of Health, Bethesda, Maryland, tissue constructs were determined using a TA.XTplusC
USA), the area of the squares formed on the diagonal of texture analyzer and the exponent connect software
the printed structure (actual area) was determined. Finally, version 7.0.6.0 (Stable Micro Systems, United Kingdom).
the obtained actual area was compared with the theoretical The selected test was a static uniaxial compression test. A
area of the CAD design (Equation I). P/20 probe (20 mm diameter cylindrical aluminum probe)
Actualarea and a 5 kg loading cell were used. The initial position of
1 ,,
Shapefidelity TP A 1 (%) = Theoreticalarea 1 × 100 (I) the probe was 3 mm. The pre-test, test, and post-test speed
TP A 1
1 ,,
1
-1
1 ,,
T TP A 1 were established at 1 mm s . The target mode was the
1
The diameter of the filaments was used as a parameter strain, which was set to 80%. The trigger force was 5 g.
to determine the printability of the ink. In this case, the The mechanical properties of the two types of structures
strand printability was defined, analogously to the shape were analyzed at five different times. Four specimens were
fidelity, as follows (Equation II): tested at each time point. The force and displacement
values were measured by the machine. For the calculation
Experimental strand diameter
1 ,
Strand printability TP 1 (%) = T Theoreticalstranddiameter TP 1 × 100 (II) of the stress and strain values, the following equations
1 ,
1 ,
TP 1 were employed (Equations III and IV).
To calculate the strand printability, the average of
eight filaments was calculated for each condition (relevant Stress   N  = FN() 2 (III)
2 
(
methodology is given in part (C) of the Supplementary File).  mm  Amm )
2.6. Three-dimensional printing and bioprinting ∆ Hmm( )
Scaffolds were obtained using the Cellink BIO X Strain (%) = Hmm( ) ×100 (IV)
0
(Gothenburg, Sweden). To do this, the ink was transferred
to a cartridge, which was subsequently introduced into a The parameters obtained from the two
temperature-controlled printhead at 24°C. The selected representations (force vs. displacement, and stress vs.
tip was a 22 G blue needle. The speed was kept constant strain) included: (i) the maximum force value, (ii) the
at 7 mm s and the pressure at 120 kPa. The printbed stiffness, (iii) the maximum stress, (iv) the tangent
-1
temperature was established at 4°C. The designed scaffold elastic modulus, and (v) the work to break. The stiffness
was in a dimension of 1 cm (width) × 1 cm (length) × 1.44 cm was determined as the slope of the tangent line to the
(height), with 100% infill. This structure was used for all force–displacement curve at 50% of the ultimate force.
the assays described in this paper, unless otherwise stated. Similarly, the tangent elastic modulus was calculated
Volume 10 Issue 3 (2024) 446 doi: 10.36922/ijb.2632