Page 453 - IJB-10-3

International Journal of Bioprinting Biomimetic scaffolds for tendon healing

2.4 Rheological characterization To carry out the amplitude sweep assay, the values of G’
The rheological characterization of the final ink was and G’’ of the ink were measured in a range of oscillation
carried out using an AR 1000 Rheometer (TA instrument, stress between 0.1 and 2000 Pa (type of test: oscillation
Waters Corporation, Milford, Massachusetts, USA) with amplitude). The frequency used was 1 Hz, and the angular
a 20 mm flat steel plate. The tests were performed with frequency was 6.28 rad s . A deviation of ±5% was used to
-1
a gap between the geometry and the Peltier plate of 600 determine the end of the LVER. The values of G’ and G’’
μm and at a temperature of 25°C, unless otherwise stated. were taken into account during the LVER to characterize
To ensure that the sample was at the test temperature the ink. The yield point (τ ), value of the shear stress at the
y
and that the sample loading process did not affect the limit of the LVER, and flow point (τ ), value of the shear
f
results, a sample-conditioning step was performed before stress at the crossover of the moduli, were established.
each assay with an equilibrium time of 3 min (but for In the gel-formation assay, the two parts of the ink,
the gel formation assay). The software used to carry out which were at 25°C, were mixed and loaded into the
the tests and the subsequent analysis was TRIOS 5.2 (TA rheometer (type of test: oscillation time). No conditioning
instrument, Waters Corporation, Milford, Massachusetts, step was performed in this test. The G’ and G’’ were
USA). Eight different rheological tests were performed. measured from time 0 min to 60 min using a frequency
In the shear rate sweep, the viscosity and shear stress of 0.1 Hz and a strain of 1%. The time-point where the
values were calculated for shear rate values from 0.1 to beginning of a gel-formation process takes place is referred
1000 s (type of test: flow sweep). The sampling rate was to as the gelation onset time (t ), and the time-point of
-1
CR
set to 10 points per decade, with a 5 sec delay before the sol/gel transition point is known as the gel point time
measurement and a measurement duration of 30 sec. The (t ). These values were determined from the modulus vs.
SG
zero-rate viscosity and the infinite-rate viscosity were time graph. The t was calculated as the onset of the G’
CR
determined from the representation of viscosity vs. shear values (5% difference from the initially recorded values).
rate by selecting the best mathematical fitting. The value The t was determined as the time at which the G’ and G’’
SG
of yield stress (σ ) was determined from the representation values intersect.
y
of shear stress vs. shear rate; the value with the best During the frequency sweep assay, the G’, G’’, and
mathematical fitting was selected. complex viscosity (η*) were determined over the frequency
In the recovery test, the viscosity values were recorded range of 0.1 to 100 Hz (type of test: oscillation frequency).
during five different steps (type of test: flow peak hold). In The oscillation amplitude was kept constant at a value of
steps 1, 3, and 5, high shear rate of 200 s was used, and 8 rad and the strain at 1%. The points per decade were set
-1
in steps 2 and 4, low shear rate of 0.1 s was used. Each to 10 and the ramp type to logarithmic. The properties of
-1
of the steps lasted for 2 min. There was no recovery time the ink were determined from the representation of the
between steps. The thixotropic character of the ink was modulus vs. the frequency.
established from representation of viscosity vs. time. The For the temperature sweep assay, three temperature
initial viscosity was calculated as the mean viscosity once ramps were performed (type of test: oscillation temperature
the parameter had stabilized (variation less than 5%). The sweep). In the first one, the values of G’ and G’’ were
initial viscosity, determined as the mean stabilized viscosity calculated while decreasing the temperature from 37°C to
with a variation of less than 5%, served as the baseline 4°C (cooling curve). In the second one, the temperature
and was assigned a reference value of 100%. The viscosity was increased from 4°C to 37°C (heating curve). In the last
values were measured at the conclusion of the 2-min, one, the temperature decreased again from 37°C to 4°C
high-shear-rate periods. They were then compared to the (cooling curve). The temperature ramp was established at
corresponding 100% baseline (initial resting viscosity). 1°C min , the strain percentage at 0.1%, and the frequency
-1
The strain sweep assay was used to determine the at 1.0 Hz. The gelation point (T ) determined from the
g
linear viscoelastic region (LVER) (type of test: oscillation cooling curve and the melting point (T ) obtained from the
m
amplitude). With a frequency of 1 Hz, the storage modulus heating curve were identified based on a change exceeding
(G’) and the loss modulus (G’’) were determined at 5% in the G’ value.
oscillation strains from 0.2% to 10000%. The points per Temperature frequency sweep (type of test: oscillation
decade were set to 6 and the ramp type to logarithmic. frequency) was the last assay performed. The complex
A deviation of ±5% in the value of the G’ was used to modulus (G*) was determined over the frequency range
determine the end of the LVER. The result of this assay of 0.1 to 100 Hz at different temperatures. The oscillation
served to determine the percentage of strain to use in the amplitude was kept constant at a value of 8 rad and the
rest of the performed oscillatory tests. strain at 1%. The points per decade were set to 10 and the

Volume 10 Issue 3 (2024) 445 doi: 10.36922/ijb.2632