Page 325 - IJB-9-3

International Journal of Bioprinting New fibrillar collagen for 3D printing and bioprinting

are expressed as O.D. after subtracting the absorbance region when the viscoelastic parameters of the material
provided by the controls. are strain-independent below a critical strain. That is,
For each bioink, both the cell viability and proliferation the response of the material will only be dependent on
were assessed at 0, 2, 4, 6, and 12 days after 3D bioprinting. its structure. The end of the LVR is sometimes difficult
To minimize the influence of cells growing over the to define, since it does not correspond to the crossover
petri dish (outside of the scaffold), the constructs were point visible in the amplitude sweep test, but to a previous
moved to new petri dishes before Live/Dead and CCK-8 point in which the storage modulus (Gʹ, also called
experiments. All the scaffolds were maintained in static “elastic modulus”) and/or the loss modulus (G˝, “plastic
culture conditions (5% CO and 95% relative humidity). modulus”) varies around 5% from the plateau value. From
2
this point, the collagen bioink losses the elastic behavior
2.6.4. Mechanical properties of the bioprinted and starts behaving like a viscous fluid. The oscillatory
scaffolds strain amplitude sweeps of ColA and ColN inks are shown
To study the influence of the bioprinting process, the culture in Figure 2A and Figure S1. It is clear that the end of the
conditions and the presence of cells within the constructs LVR depends on the collagen concentration, needing
over the mechanical performance of the constructs, the higher amplitude values to finish the LVR.
textural properties were assessed at different time points. In fact, the higher is the collagen concentration (up
To do so, a 2% (w/w) and 3% (w/w) of ColN L929-laden to 5%), the wider is the LVR (Figure 2A and Figure S1).
bioinks were bioprinted, covered with culture medium and Likewise, the values of Gʹ and G˝ within the LVR grow
maintained at 37°C in a cell culture chamber for 24 h before proportionally to the collagen content in the ink. This means
the first compression measurement. Moreover, another that the ink is generally stiffer as the collagen concentration
batch of constructs were maintained in vitro for 12 days grows, disregarding the pH of the formulation (Figure 2A).
and subsequently subjected to uniaxial compression. Particularly, for the amplitude sweep profiles of the neutral
The corresponding counterparts without cells were also inks, the modulus (Gʹ and G˝) are significantly reduced
printed and monitored as controls. Before the compression in comparison with the acidic inks (pH 3), which can be
analysis, the culture medium was withdrawn and each explained by the amphoteric nature of the biopolymer.
scaffold was gently dried with tissue paper to eliminate Rising the pH up to values closer to neutrality, the swelling
excessive amount of liquid. capacity of the collagen is reduced, the protein reaches

At this point, the 3D-bioprinted constructs were the lowest viscosity and maximum turbidity due to
evaluated by means of uniaxial compression tests fiber aggregation . This fiber aggregation is also driven
[26]
performed with a TA.XTplusC texture analyzer (Stable by hydrophobic interactions and swelling reduction
Micro Systems) equipped with a P/50 cylinder probe. (water withdrawal). Since the collagen ink maintains the
The 3D constructs (n = 6) were compressed at 1 mm·seg protein’s native structure, the viscosity drops right after
-1
until 80% of strain. Compression data were collected neutralization, and so do the elastic and viscous modulus.
and analyzed through Exponent Connect software. The Despite these lower values, the mechanical properties of
compressive Young’s moduli (Pa) values were obtained the printed scaffold are high and a self-standing structure
from the stress–strain curves, which were obtained during is obtained without the need of jellification or further
the uniaxial compression tests. addition of crosslinkers.
2.7. Statistical analysis The temperature sweep of the acidic and neutral collagen
Statistical differences were determined by one-way inks (Figure 2B) shows no elastic modulus variation (Gʹ)
analysis of variance (ANOVA, after confirming the normal from 4°C to 37°C and from 37°C to 4°C. The present
distribution of the samples (Shaphiro–Wilk, p > 0.05, results are contradictory to those previously reported by Li
n < 50 in all cases). SPSS software was used to carry out et al., which found that type I collagen viscosity increased
[27]
the statistical analysis, and differences were considered with increase in temperature . A feasible explanation for
significant at p < 0.05. Only significant differences this difference may be the sample concentration: Li et al.
are reported. used highly diluted collagen solutions in acetic acid. In
these conditions, collagen is soluble (molecular collagen,
3. Results and discussion tropocolagen) and no collagen fibers are present (limpid
dissolution). Under these circumstances, environmental
3.1. Ink characterization changes such as temperature can trigger the fibrillogenesis,
3.1.1. Rheology measurements and so, the fiber formation and viscosity increase. The same
The oscillatory strain amplitude sweep allows to identify results were reported for “Viscoll collagen bioinks” ,
[28]
the linear viscoelasticity region (LVR), which defines the
working with porcine, soluble collagen type I; again,

Volume 9 Issue 3 (2023) 317 https://doi.org/10.18063/ijb.712