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CNT/PCL Nanocomposite for Tissue Engineering
2.8. LIVE/DEAD viability/cytotoxicity kit for by a 50 – 80°C range sweep. Subsequently, flow sweep
mammalian cells analysis was performed at three temperatures: 180°C,
120°C, and 60°C. Exemplary results of a pure PCL are
3D-printed grids were cut and sterilized by ultraviolet (UV) presented in Figure 1.
light (30 min on each side). This experiment was carried The rest can be found in Supplementary File
out on 24-well Ultra-Low Attachment plates (Corning) (Figures S2-S4). From the temperature sweep analysis, a
in three biological replications. The chondrocytes were point of modulus cross-over was obtained. The modulus
seeded on the grids at a density of 2 × 10 cells per well. cross-over signifies a temperature at which a change of
4
The culture medium was changed every three days. The the dominant modulus occurs. If the storage modulus
assay was carried out after 2 weeks of culture under (G’) dominates, a material presents more elastic (solid-
standard conditions. The final concentrations of calcein- like) behavior. Conversely, if the loss modulus (G’’)
AM and EthD-1 were 2 and 4 µM, respectively. The dominates, the material presents a more viscous (liquid-
labeled cells were visualized under confocal microscopy like) behavior. From the flow sweep analysis, zero-rate
(Olympus XI83).
viscosity was calculated. The results are presented in
2.9. Cell Titer-Glo 2.0 cell viability assay Table 1.
The addition of CNT resulted in elevated temperature
To investigate the potential toxicity of the materials, of modulus cross-over during cooling, regardless of
3D-printed PCL grids were cut and sterilized with UV CNT type. The highest increase was observed for 0.2%
light (30 min on each side). This experiment was carried addition of BCNT and 0.2% MWCNT: 6.3°C and
out on 24-well plates in three biological replications. 5.2°C increase, respectively, in comparison to pure
Chondrocytes were seeded at a density of 2 × 10 cells per PCL. The temperature of modulus cross-over during
4
well. Positive control was cells seeded without the grid. heating was not pronouncedly affected by CNT addition.
After 3 and 6 days, the CellTiter-Glo 2.0 cell viability assay These observations indicate that CNT influence the
was carried out according to the manufacturer’s manual solidification of PCL, but not melting. This led us to the
with slight modifications. Briefly, 500 µl of fresh medium suspicion that CNT may affect the crystallization of PCL
was added to each well, equilibrated to room temperature, either by acting as nucleation centers or by facilitating
followed by the addition of the CellTiter reagent. The heat transfer through the polymer. Such phenomena
plates were mixed for 2 min and incubated for 10 min have been described previously [20-22] . Regarding zero-
at room temperature. Then, 200 µl of the solution was rate viscosity, 0.01% addition of BCNT and MWCNT
transferred to 96-well opaque-walled plates in at least two increased the polymer’s viscosity. Interestingly, at the
technical repeats. The luminescence was recorded using highest concentrations, a decrease in zero-rate viscosity
the Infinite 200 PRO plate reader (TECAN). was observed instead. Congruous data were obtained

2.10. Statistical analysis for all three temperatures. This result could be attributed
to a gradual shift in dominant interactions between the
Statistical analysis was performed using GraphPad Prism CNT and PCL chains. At 0.01%, PCL/CNT interactions
ver. 8.0.1. may predominate over PCL/PCL and CNT/CNT. With
increasing CNT concentrations, the balance shifts toward
3. Results CNT/CNT interactions, resulting in a decrease in zero-

3.1. Scanning electron microscopy (SEM) rate viscosity. The best fit flow analysis of viscosity vs.
imaging strain curve confirmed the pseudoplastic behavior of all
tested materials. In most cases, the Carreau-Yasuda model
SEM imaging of the printed constructs revealed that was attributed to the experimental data [23,24] . Regardless of
the addition of CNT, regardless of their type and CNT addition, all samples showed similarity in viscosity
concentration, resulted in a much smoother surface in vs. strain curve (Figure S4). At 60°C, the Newtonian
comparison to the pure PCL (Figure S1). The initial plateau persisted until the shear rate of c.a. 6 1/s, where
assumption was that the decrease in surface roughness the transition region occurred. Due to limitations of the
will result in diminished cell attachment. apparatus, further measurements beyond this point were
unattainable. At 120°C, a prolonged Newtonian plateau
3.2. Parallel plate rheometry was observed, followed by a well-pronounced transition
Rheological properties are crucial from the standpoint of region starting at c.a. 25 1/s, and a precipitous drop in
3D printing and polymer manufacturing. It also provides viscosity, signifying the power-law region. At 180°C,
valuable insight into the supramolecular interactions the Newtonian plateau extended to the shear viscosity
present within a material. The materials were subjected to of c.a. 100 1/s. The transition region and the power-law
the temperature sweep in the 120 – 40°C range, followed were, in general, more flattened in comparison to 120°C.

72 International Journal of Bioprinting (2022)–Volume 8, Issue 2