Page 72 - IJB-7-1

Using Plant Proteins to Develop Composite Scaffolds
PCL scaffolds. Although the composite inks we discussed and no significant viscosity difference was observed
are supplied to EHDP system for scaffold building, the between these inks.
application of such inks can also be implemented on
several different kinds of 3D printing systems, such as 2.3. EHDP system setup and fabrication process
extrusion-based printing and electrospinning. monitoring

2. Materials and methods Figure 1A shows a schematic diagram of a solution-
based EHDP setup, which includes an ink feeding system,
Natural extracellular matrix (ECM) creates complex a high voltage power supply (0 – 10 kV, Dongwen Co.
physical and chemical environment to support cell/ Ltd., China) and a three-axis precision motorized stage
tissue functions. To introduce such complexities to from Aerotech Company (Pittsburgh, PA, USA). The
ECM-mimicking fibrous scaffolds, EHDP has been solution feeding system consists of a micro-syringe
implemented to fabricate scaffolds with aligned fibers to pump, a disposable syringe (5 mL), a flexible plastic
create controllable microstructures using single polymer- hose, and a stainless steel needle (G20). The voltage
based material system. To enhance such EHDP scaffolds’ output from the high-power supply was applied between
performance, our group has developed plant protein-based the nozzle and the substrate to trigger and maintain EHDP
composite inks which could distinctly improve fiber jetting process. The precision stage has a travel range of
surface biocompatibility during cell culture studies. 150 mm/s with 3 μm accuracy on x and y axes. A polished
silicon wafer placed on x-y stage was used as the substrate
2.1. Materials for fiber deposition. The stage moving speed along x and
The gliadin power was purchased from Tokyo Chemical y directions is set between 100 mm/s and 300 mm/s. The
Industrial Ltd. (Japan). 3-(4,5-dimethylthiazol-2-yl)- ejected fiber could continuously deposit on the stage
2,5-diphenyltetrazolium bromide (MTT) cytotoxicity with the mechanical drawing force which was generated
assay and CellTiter 96 AQueous One Solution Cell with the stage moving along x and y axes. As shown in
®
Proliferation Assay were obtained from Abcam Ltd. Figure 1B, this deposited fiber stacked up gradually and
(China) and Promega Co. Ltd. (USA), respectively. The formed a scaffold by following predesigned moving path.
rest of the chemicals and reagents are similar to those
reported previously . 2.4. EHDP fabrication process monitoring
[23]
As the EHDP’s printing resolution can approach submicron
2.2. Preparation of PCL/zein and PCL/gliadin to nanoscale, any slight fluctuations of the environmental
biomaterial inks factors, such as the variations of temperature, humidity,

PCL, PCL/zein, and PCL/gliadin biomaterial inks were air flow, and the printing platform vibration due to stage
[12]
prepared for EHDP scaffold fabrication. PCL ink (70 w/v% movement, might affect printing accuracy . It also took
in glacial acetic acid [AcOH], g/mL) was prepared by some time to achieve and maintain a steady flow rate
dissolving PCL pellets (3.5 g) in acetic acid (5 mL) with and stage speed, especially for high viscous biopolymer
stirring for 1 h to allow complete dissolution. Both zein solutions. Besides, corona discharge phenomena are
and gliadin are soluble in acetic acid, and we used two quite common when using PCL/gliadin and PCL/zein
weight ratios, that is, 10% w/v and 20% w/v to prepare inks. This is ascribed to residual charges remained in the
both PCL/zein and PCL/gliadin inks in this study. Zein peptide chains of gliadin or zein protein, which alters
or gliadin powder were first dissolved in glacial AcOH the electrical properties of the composite inks. When the
to obtain a clear solution. Subsequently, PCL pellets accumulated charges on the droplet surface at the nozzle
were added to either solution under ultrasonic condition tip exceeded a critical value, the corona discharge could
at 50°C and the mixture was stirred 30 min to produce be observed. If this discharge lasts for a longer time, the
homogenous PCL/gliadin or PCL/zein biomaterial inks. EHDP fabrication system can be damaged.
Two types of PCL/zein biomaterial inks were To achieve a reliable fabrication process, it is
prepared: PCL/zein-10 (60% w/v PCL, 10% w/v zein in essential to develop a system to monitor and identify the
AcOH) and PCL/zein-20 (50% w/v PCL, 20% w/v zein status of triggered jet and cone. In the developed system,
in AcOH). Similarly, two PCL/gliadin biomaterial inks the jet and cone images were recorded using digital
were prepared: PCL/gliadin-10 (60% w/v PCL, 10% w/v microscope (Supereyes B011 digital camera with 1 – 500
gliadin in AcOH) and PCL/gliadin-20 (50% w/v PCL, magnifications and 30 frames/s) to observe the details
20% w/v gliadin in AcOH). The viscosity of zein and of Taylor cone and jet region. To capture images, the
gliadin solution (in acetic acid) is low. Thus, the viscosity camera position, focal length, and shooting angle should
of such biomaterial inks is mainly determined by the be calibrated by comparing the overlapping area of the
viscosity of PCL solution due to its high concentration, grayscale nozzle image with a predefined position.

68 International Journal of Bioprinting (2021)–Volume 7, Issue 1