Page 188 - IJB-10-1

International Journal of Bioprinting Nanoclay biopolymer inks for 3D printing

DMA Q800 TA Instruments (New Castle, DE, USA) effort to achieve improved fidelity and stability, different
was used to evaluate the mechanical behavior of the weight fractions of alginate and salecan were investigated
obtained 3D-printed hydrogel samples. Using a modified and their inks were used in additive manufacturing.
compression clamp, the cylindrical 3D-printed samples These bi-component hydrogels were further doped
with ~12 mm in diameter and ~3 mm in thickness were with different concentrations of clay nanoparticles and
employed for the analyses, which were carried out in then used to print 3D nanocomposite structures. The
dynamic frequency sweeps mode at 25°C to avoid the resulted polysaccharides-based 3D constructions were
effects of water evaporation. Dynamic frequency sweeps further submerged in a CaCl bath to allow alginate –
2
were carried out over a frequency range of 0.1–5 Hz with COO functional groups to react with Ca ions. Alginate
–
2+
a constant strain of 0.1% (in the linear viscoelastic area) to networks became intertwined with salecan chains, creating
record the storage (G’) and loss (G”) moduli of the swollen semi-interpenetrating networks which were stabilized
3D-printed samples. To confirm reproducibility, frequency through physical interactions by alginate ionic crosslinking
tests were conducted three times, and the G’ and G” were and also H–bonding interactions.
plotted against frequency. The frequency sweep modulus A schematic representation of the crosslinking
was analyzed using TA Universal Analysis. mechanism of alginate–salecan networks in the presence

Nanoindentation tests were performed using a TI of clay nanofiller is proposed in Scheme 1.
Premier System (Hysitron Inc., Minneapolis, MN, USA)
equipped with a three-side pyramidal Berkovich tip (total 3.1. 3D printing of polysaccharide-based
angles of 142.35° and radius of curvature of 150 nm). A hydrogel inks
normal load of 100 µN was applied using the trapezoidal To reach the main goal of this research, i.e., obtaining
load function (5 s loading, 2 s hold, 5 s unloading) to appropriate inks for printing 3D constructs with
determine the values of reduced modulus (E), calculated excellent fidelity and stability, eight formulations were
using the Oliver–Pharr method. investigated. The 3D printing process was carried out at
room temperature, employing the 3D printer’s extrusion
2.8. Preliminary biological studies dispenser. Different types of needles (plastic or metallic
Human dermal fibroblasts were used to test biocompatibility with a diameter of 0.41 mm), printing pressures ranging
of the 3D-printed biomaterials. Cells were cultured in from 150 to 620 kPa, and speeds ranging from 4 to
Dulbecco’s Modified Eagle Medium with 10% fetal bovine 12 mm/s were examined as a function of ink composition
serum at a density of 10 cells per well. For 24 h, the 3D as presented in Table 1.
5
structures were placed on top of the cells after being UV-
sterilized. Following the manufacturer’s instructions, Initially, the alginate hydrogel was prepared after
cytotoxicity was assessed using the LDH (lactate tests involving 3D printing were performed on it. The
dehydrogenase) Cytotoxicity kit (Sigma). With the use of low viscosity of the ink caused instability throughout the
a NanoQuant Infinite M200 Pro equipment, absorbance printing process; also, the layers collapsed as they came
was measured at 490 nm. Utilizing the Live/Dead test (cat. into contact with the glass slides, and the printed shape
no. L3224), the viability of the cells was evaluated. Using displayed apparent distortion due to the weight of the
a Zeiss fluorescent microscope, imaging was done at λ = added layers.
494/517 (living cells) and λ = 517/617 (dead cells). Cell In order to improve the rheological properties of
proliferation was quantified using the CyQUANT™ MTT the weak alginate hydrogel and to obtain 3D-printed
(3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium constructs with improved shape fidelity, salecan was added
bromide) Cell Viability Assay (thermos Scientific) in as a second biopolymer network. Thus, three compositions
accordance with the manufacturer recommendations. of salecan and alginate in different weight ratios were
created: salecan-alginate 1.687:3.620 (AV1), 2.653:2.653
2.9. Statistical analyses (AV2), and 3.62:1.687 (AV3). 3D printing tests were
The data are expressed as mean and standard deviation. To performed using speeds in the range of 4–10 mm/s and
evaluate the significance of differences, one-way analysis of pressures in the range of 180–500 kPa. The results showed
variance (ANOVA) was performed. The significance was that raising the salecan concentration enhanced the ink
assessed if the p value was less than 0.05. stability during the printing process when compared with
neat alginate ink, and the resulted 3D-printed platforms
3. Results and discussion maintained greater fidelity of up to seven layers (Figure 1).

Our present study investigated in the first step the Based on the 3D printing behavior cumulated with
possibility to obtain alginate–salecan hydrogels. In an the results obtained from salecan retention in the alginate

Volume 10 Issue 1 (2024) 180 https://doi.org/10.36922/ijb.0967