Four-dimensional (4D) printing is an emerging field in additive manufacturing, and the technology helps develop high-demand products in electronics, biomedicine , robotics and other fields. Currently, most reported 4D printing systems are based on the temporal shape transformation of printed objects. In addition to shape, the induction of temporal heterogeneity of functions can expand the scope of 4D printing. To this end, Professor Wang Jinye’s team from Shanghai Jiao Tong University reported a 4D printing method that uses a plant protein (zein) gel inspired by the amyloid fibril formation mechanism (Figure 1). In this study, zein gel was applied in additive manufacturing to produce scaffolds with controllable properties, while the gelation mechanism of the zein solution was investigated. The research results related to were published in "Bioact. Mater." on November 25, 2022 under the title "Water-responsive 4D printing based on self-assembly of hydrophobic protein "Zein" for the control of degradation rate and drug release".
Figure 1 Preparation and structural characterization of 4D printed
- zein gel based on the hydrophobic protein "zein"
The gelation mechanism of zein solution was inspired by amyloid fibrils, and hydrogen bonds drive the fibrils to form a gel network. The author used cryo-TEM, FTIR and ThT combined analysis for structural characterization. Zein formed spherical self-assemblies after dissolution in hydroalcoholic solvents (Fig. 2a), while in zein gels, fibrillar networks with a minimum diameter of 10 nm were observed (Fig. 2b). FTIR analysis showed a small shoulder −1 at 1625–1635 cm indicating the formation of β-sheets during gelation (Fig. 2c). Furthermore, a shift in β-sheet content was observed from the second derivative spectrum, indicating a new β-band (Fig. 2d). Positive ThT binding to the zein gel indicated that the gelation of the zein solution was caused by amyloid (Fig. 2f–h).
Figure 2 Preparation and structural characterization of zein gels
- Rheological properties and printability of zein gels
Next, the authors evaluated the rheological properties of the gels to establish a theoretical estimate of printability. The gel exhibits shear thinning behavior with low viscosity at higher shear rates (Figure 3a). The strain/stress scan curve showed that the zein gel was viscoelastic, with a higher elastic modulus value than the viscous modulus at low strains (Fig. 3b). For all zein gels, structural recovery ability was also observed after 100% strain, which resulted in the stability of the printed structures (Figure 3c). Overall, zein gels exhibit rheological properties suitable for direct printing. Furthermore, since zein gels are driven by hydrogen bonds, they can self-assemble after extrusion, resulting in continuous filamentous gel extrusion (Figure 3d–e). At the same time, the yield stress and structural recovery further support that the filaments do not collapse under gravity (Fig. 3f).
Figure 3 Printability evaluation of zein gels
- 4D printing of zein gels in a support bath
Hydrogen bonds exist between amyloid fibrils in zein gels, and upon water immersion, hydrophobic interactions dominate, causing the gel to solidify. Therefore, the authors performed a proof-of-concept and no size change of CB60 was observed within 12 hours, This may be due to balanced hydrogen and hydrophobic interactions between the zein gel and the support bath .
Figure 4 Mechanism of structural changes during printing of zein in Carbopol baths with different water concentrations (CB75 and CB40)
For the overall structural transformation, the authors proposed a mechanism, taking the dye-loaded butterfly structure in a double-layer Carbopol support bath (CB75/CB40) as an example (Figure 4) . In CB75, zein tends to self-assemble rapidly and hydrophobic attractions dominate, resulting in shrinkage of the structure/filaments. In CB40, due to the low water concentration, hydrogen bonds between the zein molecules and the solvent of the supporting medium are dominant, resulting in an increase in structure/filaments and the formation of a loose network.The video below further validates this and also illustrates the 4D printing of bath zein-supported gels. Degradation rate of printed constructs
The degradation rate of printed constructs (ductal) after drying was evaluated by calculating the percent weight loss in degradation medium containing protease XIV . As the water concentration in the support bath decreased from 75% to 40%, the degradation rate decreased significantly. The conduits printed in CB75 degraded by 88.2 ± 1.17% in 10 days, with the remaining structural integrity lost (collapse) and the wall thickness of the conduits also reduced during the degradation process (Figure 5a-b). Conduits printed in CB75 had higher porosity than conduits printed in CB40 (Fig. 5c). Higher porosity promotes penetration of the media, resulting in faster degradation, while lower porosity conduits (CB40) limit water penetration.
Additionally, the authors evaluated the degradation rate of the CB75-CB40-CB75 three-segment catheter as a nerve conduit (15 mm) in the rat sciatic nerve injury model (Figure 5e) . The CB75-CB40-CB75 catheter showed faster degradation at the ends due to the presence of the highly porous CB75 part, while the middle part degraded slowly (CB40) (Figure 5f). The proximal portion of the catheter degrades faster than the distal portion because nerve regeneration occurs faster in the proximal portion. In contrast, in the CB40-CB75-CB40 catheter, the middle part (CB75) degraded faster than the end position (CB40) (Fig. 5g). The corresponding HE staining further proved this.
Figure 5 Evaluating the degradation rate of catheters
- Proof of concept for structural heterogeneity applications
To provide a proof of concept for structural heterogeneity applications, ciprofloxacin hydrochloride (CPFX) loaded catheters and doxorubicin (DOX) loaded butterfly constructs were printed in a bilayer support bath (CB75/CB40). CPFX is a commonly used antibiotic and DOX is used as an antitumor agent . The CPFX loading and drug release rate of catheters printed in CB75 were higher than those printed in CB40 (Fig. 6a–b). As with CPFX, higher rates of DOX loading and release were observed for structures printed in CB75 than those printed in CB40 (Fig. 6d–e). The construct was divided into two parts to evaluate the cytotoxicity of DOX-loaded butterflies, and higher cytotoxicity towards BEL7402 cells was found within the butterflies printed in CB75 (Figure 6f). In summary, the biphasic controlled release of may help achieve initial rapid release and long-term sustained release. Differential drug release dependent on the water concentration of the support bath can be explained by porosity. The higher porosity of the constructs printed in CB75 resulted in faster release of through easy penetration of the dissolving medium.
Figure 6 Demonstration of structural heterogeneity application
- Efficacy of catheters as ureteral stents in porcine models
Finally, The authors used a 4D printed zein catheter as a drug-eluting, biodegradable ureteral stent in a porcine model . In animal experiments, the authors designed experiments to evaluate the urinary excretion and antibacterial efficacy of CPFX-loaded catheters printed in CB75 or CB40 to verify differential drug release (Figure 7a). Before surgery, 3 min after intravenous injection of iohexol, renal pelvis , renal calyces, and ureters were observed, indicating normal secretion and excretion functions. The contrast medium in the renal pelvis and ureter disappeared 30 minutes after injection (Fig. 7b). Four days after surgery, the renal pelvis and ureter appeared 3 minutes after injection, and the renal pelvis and ureter were not dilated, indicating good secretion and excretion. The contrast agent passes through the stent implantation site. Sixty minutes after injection, the contrast agent disappeared from the renal pelvis and ureter (Fig. 7b). Furthermore, the CFUs counting test showed that scaffolds printed in CB75 had significantly higher CFUs than scaffolds printed in CB40 (Figure 7c). Overall, the zein scaffold showed urine drainage efficacy and antibacterial activity in animal models and can be used to prepare 4D printed drug-eluting biodegradable ureteral scaffolds .
Figure 7 Application of printed catheter as ureteral stent in porcine model
In summary, This paper applies zein gel to additive manufacturing to produce a stent with controllable properties. The authors found that hydrogen-bonded amyloid fibrils are responsible for zein gel formation under shear stress in and .Zein gels have water-induced self-assembly properties that were developed for 4D printing using a specialized support bath. hydrophobic attraction induces different self-assembly in support baths with different water concentrations. Hydrophobic self-assembly exhibits controllable printed structural properties, especially for biomedical purposes, and provides tunable drug loading, drug release, and degradation rates. Animal studies further validate the significant translational potential of this
Article source: https://doi.org/10.1016/j.bioactmat.2022.11.009
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