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Mieloch, et al.
the literature [11-13] . For the purpose of this study, two types 2.3. Material preparation
of CNT were selected: “bamboo-like” CNT (BNCT)
and multiwalled CNT (MWCNT). “Bamboo-like” CNT The PCL and CNT powders were mixed in 15 ml Falcon
(BCNT) resemble the cup-in-cup structure characteristic tubes by shaking until a visually homogeneous powder
was obtained. Subsequently, the powder was placed
of the bamboo stem, with a high presence of surface on a glass Petri dish and heated on a magnetic stirrer
defects. This type of CNT is comparatively inexpensive
as its imperfect morphology does not require stringent until melting occurred. The melt was cooled down
synthesis conditions. MWCNT are composed of multiple and folded several times to improve the homogeneity
single-walled CNT with diminishing diameters, arranged of the material. Finally, the melt was cut into pellet-
concentrically. Due to the diminished quantity of structural like pieces, which were suitable for the thermoplastic
defects in comparison to BCNT, MWNCT can be used printhead.
for tissue engineering applications requiring electrical 2.4. 3D printing
conductivity . In recent years, a lot of research has
[14]
been devoted to electrospun PCL/CNT nanocomposites 3D printing was performed with the Cellink BioX
for tissue engineering purposes, indicating a growing printer. In the process, the thermoplastic printhead
interest in biopolymers with CNT additives [15-19] . In was utilized. The 3D model of the grid was prepared
regard to PCL/CNT composites, a staggering amount by manual writing of a.gcode file. The printing was
of variables, such as CNT aspect ratios, purity, defects, performed with the following parameters: printhead
functionalizations, entanglement within a polymer nozzle diameter, 0.4 mm; printhead temperature,
matrix, and interfacial interactions reduce any predictive 180°C; printing speed, 4 mm/s; and extrusion pressure,
attempts of resulting properties to an educated guess. In 510 kPa.
addition, the potential cytotoxicity of a nanocomposite
material depends on the biodegradation rate and 2.5. Nanoindentation
subsequent gradual release of the nanofiller into the tissue The nanoindentation study was performed on a G200
environment. Therefore, despite a plethora of relevant (Agilent) nanoindenter equipped with a DCM head. Each
research, a specific application-driven design of CNT- sample was subjected to 12 indentations of 2000 nm in-
reinforced polymers still requires extensive laboratory depth, with a Berkovich-type probe, at room temperature.
work. This work aimed to evaluate mechanical and Analyses were performed at 500 – 1800 nm depth. Due
biological properties of PCL reinforced with BCNT and to the uneven topography of the surface, extreme results
MWCNT from a 3D bioprinting and tissue engineering were excluded from further analysis.
point of view.
2.6. Parallel plate rheometry
2. Materials and methods
The rheology study was performed with the Discovery
2.1. Materials Hybrid HR20 Rheometer (TA instruments). A 20 mm

CNT were purchased from NanoLab Inc. (USA, MA). aluminum parallel plate was used for the measurements.
MWCNT have a purity >85%, diameter 10 – 30 nm, and A temperature sweep analysis was performed in two
length 5 – 20 µm. BCNT have a purity >85%, diameter 10 ranges: 120 – 40°C and 50 – 80°C. For both ranges,
– 30 nm, and length 5 – 20 µm. PCL used in this work was the temp. step was set to 2°C, 1% strain, and angular
in powder (~50 000 MW, Polysciences Europe GmbH). frequency at 10 rad/s. The soak time was set to 30 s. A flow
LIVE/DEAD Viability/Cytotoxicity Kit for mammalian sweep analysis was performed at three temperatures:
cells (Invitrogen) was also used in this study. 180°C, 120°C, and 60°C. The range of shear rate was set
from 1 × 10 1/s to 500.0 1/s, at logarithmic step. Data
−3
2.2. Cell culture were analyzed using TA Instruments TRIOS Software
Human knee articular chondrocytes (NHAC-kn, Cat No: version 5.1.1.46572.
CC-2550, LONZA) were cultured in DMEM/F12 with 2.7. Differential scanning calorimetry (DSC)
L-glutamine (Corning) supplemented with 10% fetal
bovine serum (FBS), 50 µg/mL 2-phospho-L-ascorbic DSC analysis was performed on a DSC 8500 apparatus
acid, 50 U/ml penicillin, and 50 µg/ml streptomycin at (Perkin Elmer) in the temperature range of −90 – 180°C,
standard culture conditions. The medium was changed with nitrogen flow (20 ml/min). The temperature
every 3 days. Cells were subcultured at 80 – 90% change rate was set at 10°C/min. The thermal history
confluence with the TrypLE Express Enzyme (Gibco). of the raw material was erased before measurement.
Chondrocytes up to the ninth passage and with cell 3D-printed grids were not subjected to thermal history
viability above 95% were used for cell experiments. erasure.

International Journal of Bioprinting (2022)–Volume 8, Issue 2 71