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Bioink for Reconstruction of Rigid-living Systems
2.5. Image processing for 3D printing lyophilized. Bruker Topspin 3.5pl7 software (Bruker

The similarity structural index measurement (SSIM) was BioSpin, Rheinstetten, Germany) and MestReNova
(Mestrelab Research, Spain) were used for data collection
measured by comparing pixels between images (Figure S2); and analysis. In addition, Solution-State NMR, the NMR
it can be visually seen how the comparison is made between spectra (1H and 13C) of biopolymer-base were recorded
two fixed figures to get this numerical value . The response using Bruker Avance 400 MHz spectrometer (Bruker,
[14]
surface methodology (RSM) was applied to evaluate the USA) at room temperature. The sample was prepared to
effect of enhancing the developed ink with hydroxyapatite dissolve 5 mg powder in 500 µl of d6-DMSO (Cambridge
and calcium carbonate over the structural similarity index Isotope Laboratories, USA) and then transferred into 5 mm
from the printed structure. According to the statistical NMR tubes. Bruker Topspin 3.5pl7 and MestReNova
modeling reported in recent studies [15,16] , the elaboration software were used for data collection and processing,
of the RSM was performed with the MATLAB software, respectively, of NMR of H-NMR, C-NMR for the solid
®
and the model’s constants were obtained with the Minitab and liquid state, the PI was not added as it behaves similar
18 software (Minitab® LLC, USA). The general model is to paramagnetic species; therefore, the equipment will not
presented in Equation 1, and the simplified resulting model detect any significant signal. A complete sample of a printed
is shown in Equation 2.
coral was ground for X-ray diffraction compared with
bioceramics spectra. For thermochemical characterization,
γ = δ ± δα ± δβ ± δα ± δβ ± ( ) + ε (1)
δαβ
2
2
0 1 2 3 4 5 ijk both thermogravimetric analysis (TGA) and differential
scanning calorimetry (DSC) (TA Instruments), the final
Equation 1. A general model for the effect of printed inks were ground and analyzed by both instruments.
hydroxyapatite and calcium carbonate over the structural Around 20 mg of material were used for each sample. The
similarity index of the printed structure; where Y is the ranges of temperatures used, were 25-850°C for TGA and
response and α,β are the factors of the model, δ 0,1,2,3,4,5 25-400°C for DSC.
represent the constants of the model, and ε is the total
ijk
error. 2.8. Viscoelastic characterization
The mechanical properties of non-crosslinked ink were
γ = δ + δα − δβ δα− 2 + δβ ; ∂γ = ϕ� i (2) analyzed using TA Ares-G2 Rheometer equipped with
2
∂i Advanced Peltier System. A freshly prepared ink was
2
1
0
4
3
measured using an 8 mm parallel plate with a 1.8 mm
Equation 2. A simplified model for the effect of gap at 25°C. The stiffness was analyzed through a time-
hydroxyapatite and calcium carbonate over the structural sweep test for 5 min with angular frequency and one
similarity index of the printed structure; where Y is the rad/s and 0.1% strain, respectively. A temperature sweep
response and α,β are the factors of the model, δ 0,1,2,3,4 the was subsequently performed on the sample by applying
constants of the model, i represents any of the two factors, a gradual temperature increase from 25°C to 50°C with
and φ the solved values from the partial derivatives. similar angular frequency and strain.
i
2.6. Morphological imaging The viscosity of the ink before crosslinking was
determined using 25 mm parallel plate geometry with
The scanning electron microscope FEI Magellan extreme- a 0.5 mm gap at 25°C. Three replicate samples were
high-resolution imaging was applied to a grid of the 3D measured using a 25 mm parallel plate geometry with
printed formulation, crosslinked, and dried overnight, a gap of 0.5 mm at 25°C. The flow experiment was set
with an accelerating voltage of 3 kV. The dried peptides up by starting the shear rate from 0.001 to 300 s for a
−1
were sputter-coated with 5 nm Ir before imaging. An 600-s duration. The value of the shear rate that we choose
optical microscope was used to obtain the macrography for our printing system was calculated using the equation
with a source of light in the upper side from the sample. below [17,18] :
8Q
2.7. Chemical characterization γ = (3)
πd 3
For Fourier-transform infrared (FTIR) spectroscopy, a
−1
Thermo Nicolet iS10 FTIR Spectrometer (Thermofisher) Equation 3. γ: shear rate (s ); Q: flow rate (2 µL/s);
was used; the samples were prepared and crosslinked by d: diameter of needle (0.84 mm).
two different sources individually compared to control 2.9. Biological assessments
with exposure at room conditions. For Solid-State NMR,
the 13C Magic Angle Spinning (MAS) NMR spectra were Undifferentiated mesenchymal stem cells (MSCs) were
recorded using Bruker Avance 400 MHz spectrometer seeded at a density of 15.5E3 cells/cm and incubated
2
(Bruker, USA) at room temperature. The sample was for 7 days (5% CO , 37°C) in supplemented DMEM-F12
2
68 International Journal of Bioprinting (2021)–Volume 7, Issue 4