Page 520 - IJB-9-5

International Journal of Bioprinting 3D printability and biochemical analysis of orange peel waste

ARE-250, Thinky Corporation, Tokyo, Japan) for 5 min lens. Surface profiling and color map overlay were done
at 2000 rpm at 25°C. using the original software developed for the microscope.
2.3. Rheological characterization 2.6. Extraction and liquid chromatography-mass
The oscillatory rheometer (Discovery Hybrid Rheometer spectrometry profiling of 3D-printed samples
DHR-2, TA Instruments, Delaware, USA) was used to About 0.1 g of orange peel powder and 0.5 g of ink and
measure the rheological properties of OPW inks. Stainless printed samples were weighed and extracted using
steel parallel plates with a diameter of 20 mm and a methanol extraction [31,32] . Briefly, 15 mL of methanol was
truncation gap of 500 mm were used for all measurements. added into each tube and placed in a sonicator with a
Viscosity tests were conducted by applying a stepwise shear water bath, and subjected to sonication at high frequency
rate ramp from 0.01 to 1000 s . To assess the viscoelastic for 30 min at 40°C [31,32] . Extracts were subsequently filtered
-1
properties of the samples, stress sweep measurements and used for subsequent experiments through a 0.45-µm
were performed with a logarithmically increasing shear filter and deposited in vials for compound analysis in the
stress at a constant frequency of 1 Hz over the range of auto-sampling rack [31,32] . Liquid chromatography-mass
0.1–4000 Pa. Excess material outside the parallel plates was spectrometry (LC/MS; Zorbax SB-C18 3.5 microns, 2.1 ×
removed before measurements to prevent the edge effect. 50 mm; Agilent, USA) was used to measure the level of
All rheological measurements were conducted at room flavonoids detected in the 3D-printed samples. The column
temperature on triplicates. temperature was kept at 40°C, and the total flow rate was
fixed at 1.0 mL/min . The injection volume was 10 µL,
[32]
2.4. DIW 3D printing
and it was separated concurrently in Nexera X-2 HPLC
A pneumatic extrusion-based DIW printer (SHOTmini (Shimadzu, Japan) and LCMS-8050 LC-MS (Shimadzu,
200 Sx, Musashi Engineering, Inc., Tokyo, Japan) was Japan) . A mobile phase containing 0.1% formic acid in
[30]
used to print 3D models. MuCAD V software (Musashi water and acetonitrile was used in the gradient elution [31,32] .
Engineering, Inc., Tokyo, Japan) was used to control the The gradient parameter was calculated in a negative mode
speed and printing path of the DIW printer. Solidworks (M-H-) using the following conditions: 0.10 min at 10%,
(Dassault Systèmes, Waltham, MA, USA), a computer-aided 15.00 min at 100%, 15.10 min at 10%, and 25.10 min at
design (CAD) software, was used to create a 3D model. The 10%; this condition was used to re-equilibrate the column
created 3D model was then exported from Solidworks as a to its starting settings . The electrospray ionization mass
[32]
standard tessellation language (STL) file, which was then spectrometry (ESI) was run with a 2.8 L/min nebulizing
loaded into Slic3r, open-source software that divides the gas flow, a 300°C interface temperature, and a 9.0 L/min
model into layers and creates G-code for 3D printers. In heating and drying gas flow [31,32] . Prior to getting an average
Slic3r, the infill level was selected. A home-made Python reading for the three samples, the measured peak intensity
programming script was used to convert the resulting for each sample was normalized to achieve a consistent
G-code to MuCAD V code, which was then put onto the sum [31,32] .
DIW printer. All samples were loaded into a 50-mL Luer
lock dispensing syringe (V-S liquid control equipment, 2.7. Antioxidant capacity assays
China) fitted with a 20-G nozzle (Birmingham Gauge) The 1,1-diphenyl-2-picrylhydrazyl (DPPH) and azinobis-
(V-S liquid control equipment, China). All substrates used 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assays
in this work were pre-cleaned glass substrates (Matsunami were deployed in assessing the antioxidant capacity of the
Glass Ind., Ltd, Osaka, Japan). Before printing, the standoff extracts obtained from 3D-printed samples. The principle
distance between the substrate and nozzle was calibrated of both assays allowed us to determine the antioxidant
to the layer thickness (set at 0.40 mm) using a height feeler properties of the extracts by quantifying their ability to
gauge (QST Express-01, China). Throughout the printing scavenge free radicals [31,33,34] . In the DPPH assay, the blank
process, the printing speed and dispensing pressure were (200 µL of methanol), and 100 µL of extracts from orange
30 mm/s and 0.160 MPa, respectively. All printings were peel powder, ink, and printed samples were transferred
conducted at room temperature in a chamber to maintain into the 96-well plate. Consequently, excluding the blank,
a sterile environment. The process is illustrated and 100 µL of 100 µM DPPH solution were added into each
[32]
with the photograph of the experimental printer setup well for incubation at 25°C for 30 min . All experiments
(Figure 1). were performed in triplicates. Ascorbic acid (10 mM) was
used as the positive control for antioxidant scavenging
2.5. Microscope imaging activity . For the ABTS assay, 88 µL of 140 mM potassium
[32]
The height of the two-layer grid patterns was evaluated persulfate solution was added to 7 mM ABTS solution and
using an optical microscope (VHX-7000N, Keyence, Osaka, the mixture was stored in the dark for 16 h . This produced
[31]
Japan). The images were taken with a 40× magnification a dark purple-colored solution, consisting of ABTS•+,

Volume 9 Issue 5 (2023) 512 https://doi.org/10.18063/ijb.776