The authors gratefully acknowledge support from the&#160;National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (No.2022R1F1A1074266).

The stimuli-responsive soft gripper was composed of three different types of hydrogels: non-stimuli-responsive acrylamide (AAm)-based hydrogel, thermally responsive N-isopropyl acrylamide (NIPAM)-based hydrogel, and magnetic responsive ferrogel (Figure 1). The three hydrogel inks were prepared by modifying previously published methods29,30,31. The data presented in this study are available on request from the corresponding author.
1. Preparation of hydrogel inks
<ol>
	<li>Non-stimuli-responsive AAm-based hydrogel inks (Figure 1A)
	<ol>
		<li>Dilute the acrylamide (AAm), the crosslinker N, N&#39;-methylenebisacrylamide (BIS) (see Table of Materials), and the photoinitiator 2-Hydroxy-4&#39;-(2-hydroxyethoxy)-2-methylpropiophenone (see Table of Materials) in distilled (DI) water using a magnetic stirrer for 24 h.</li>
		<li>Vortex the shear-thinning agent, laponite RD nanoclay, and fluorescein O-methacrylate dye (see Table of Materials) at 1,150 rpm for at least 6 h until they completely dilute.</li>
		<li>Prepare specific weights of AAm-based hydrogel ink per total 20 mL of solution base: 1.576 g of AAm, 0.332 g of BIS, 1.328 g of laponite RD, 0.166 g of photoinitiator, 0.1 mg of NaOH, 0.1 mg of fluorescein O-methacrylate (see Table of Materials), and 16.594 g of DI water.</li>
		<li>After total dilution, transfer the AAm-based hydrogel ink into an empty 3D printing cartridge (see Table of Materials) using a syringe.</li>
	</ol>
	</li>
	<li>Stimuli-responsive NIPAM-based hydrogel inks (Figure 1B)
	<ol>
		<li>Dilute N-isopropyl acrylamide (NIPAM), poly N-isopropyl acrylamide (PNIPAM), and the photoinitiator (see Table of Materials) in DI water using a magnetic stirrer for 24 h.</li>
		<li>Vortex the shear-thinning agent, laponite RD nanoclay, and fluorescein rhodamine 6G dye at 1,150 rpm for at least 6 h until they completely dilute.</li>
		<li>Prepare specific weights of NIPAM-based hydrogel ink per total 20 mL of solution base: 1.692 g of NIPAM, 0.02 g of pNIPAM, 1.354 g of laponite RD, 0.034 g of photoinitiator, 0.1 mg of rhodamine 6G (see Table of Materials), and 16.92 g of DI water.</li>
		<li>After complete dilution, transfer the NIPAM-based hydrogel ink into an empty 3D printing cartridge using a syringe.</li>
	</ol>
	</li>
	<li>Ferrogel inks (Figure 1C)
	<ol>
		<li>Prepare the A-solution: Dilute acrylamide (AAm) and crosslinker, N, N&#39;-methylenebisacrylamide (BIS), ferric oxide (Fe2O3), and N, N, N&#39;, N&#39;-tetramethylethylenediamine (TMEDA) (see Table of Materials) in DI water.</li>
		<li>Consider the specific weight percent (wt%) of the materials: 71% AAm, 3.5% BIS, and 25.5% Fe2O3 in 1.2 mL of DI water with 10 &#181;L of TMEDA accelerator.</li>
		<li>Prepare the B-solution: Dilute 0.8 g of ammonium persulphate (APS, see Table of Materials) in 10 mL of DI water.</li>
		<li>For polymerization, transfer 200 &#181;L of the A-solution and 5 &#181;L of the B-solution into a microcentrifuge tube.</li>
		<li>Vortex the microcentrifuge tube for 20 s.</li>
	</ol>
	</li>
</ol>
2. Optimization of the soft hybrid gripper design
NOTE: The elliptical soft hybrid gripper is composed of an AAm-based hydrogel outer layer, a NIPAM-based hydrogel inner layer, and a ferrogel upper layer (Figure 1D). The overall elliptical soft hybrid gripper was created using the AutoCAD software (see Table of Materials).
<ol>
	<li>Two-dimensional AAm-based hydrogel layer design
	<ol>
		<li>Draw an elliptical shape with a vertical axis of 24 mm and a horizontal axis of 20 mm at the outermost part.</li>
		<li>Draw another elliptical shape with a vertical axis of 20.8 mm and a horizontal axis of 16.8 mm with the same center point as the shape drawn in step 2.1.1.</li>
		<li>Draw a three-point arc passing through the points (&#8722;8.24, 2), (0, 6), and (8.24, 2) away from the center point of the ellipse.</li>
		<li>Trim the small upper part of the eclipse divided by the arc.</li>
	</ol>
	</li>
	<li>Two-dimensional NIPAM-based hydrogel layer design
	<ol>
		<li>Draw an oval with a vertical axis of 20.2 mm and a horizontal axis of 16.4 mm with the same center point as the shape drawn in step 2.1.1.</li>
		<li>Draw an ellipse with a vertical axis of 16.16 mm and a horizontal axis of 13.12 mm with the same center point as the shape drawn in step 2.1.1.</li>
		<li>Draw a three-point arc passing through the points (&#8722;7.86, 1.83), (0, 5.6), and (7.86, 1.83) away from the center point of the ellipse.</li>
		<li>Draw a three-point arc passing through the points (&#8722;5.47, 1.64), (0, 3.18), and (5.47, 1.64) away from the center point of the ellipse.</li>
		<li>Trimthe small upper part of the ellipses divided by the arcs.</li>
		<li>To make a pedestal, draw an arc with two points away from the center point at (&#8722;4.75, &#8722;2.71) and (4.75, &#8722;2.71) as both endpoints and one point away from the center point at (0, -4.59).</li>
	</ol>
	</li>
	<li>Two-dimensional ferrogel layer design
	<ol>
		<li>Draw a three-point arc passing through the points (&#8722;7, 4.92), (0, 9.2), and (7, 4.92) away from the center point of the ellipse.</li>
		<li>Draw a three-point arc passing through the points (&#8722;7, 4.92), (0, 7.6), and (7, 4.92) away from the center point of the ellipse.</li>
	</ol>
	</li>
	<li>Two-dimensional gripper tips design
	<ol>
		<li>To make the grasping part of the gripper, cut 0.8 mm from each side from the center line at the bottom of the ellipse.</li>
	</ol>
	</li>
	<li>Three-dimensional hybrid gripper design
	<ol>
		<li>To turn the overall 2D hybrid gripper design into 3D, extrude the pedestal of the responsive gel by 0.8 mm, and extrude the non-responsive gel, the cut oval of the responsive gel, and the ferrogel by 2.5 mm.</li>
	</ol>
	</li>
</ol>
3. Three-dimensional printing of the soft hybrid gripper
<ol>
	<li>Generate a G-code30 for each structure created in step 2 using Slic3r software (see Table of Materials) with a 0.4 mm layer height, a 10 mms&#8722;1 printing speed, and an infill density of 75%. Edit the G-code file using dual print heads.</li>
	<li>Save the G-code file on a secure digital (SD) card, and connect it to the 3D printer (see Table of Materials) to generate the printing paths of the soft gripper.</li>
	<li>Connect an air pump pressure control to the 3D printer.</li>
	<li>Choose nozzle tips with diameters of 0.25 mm and 0.41 mm for the NIPAM-based hydrogel and AAm-based hydrogel, respectively.</li>
	<li>Connect the AAm-based hydrogel cartridge to nozzle 1 and the NIPAM-based hydrogel cartridge to nozzle 2.</li>
	<li>Check if the two print heads of the cartridges are at the same position on the z-axis.</li>
	<li>Calibrate the X and Y coordinates precisely to avoid misalignments between the two nozzles.</li>
	<li>Set the printing pressure at 20-25 KPa for the AAm-based hydrogel and at 10-15 KPa for the NIPAM-based hydrogel.</li>
	<li>Repeat steps 3.5-3.8 when each sample is completely printed (Figure 2A).</li>
</ol>
4. UV photocuring of the soft hybrid gripper
<ol>
	<li>Before UV photocuring, inject the magnetic field-responsive ferrogel inks (prepared in step 1.3) into the targeted thin-hole area of the 3D-printed soft gripper using a syringe.</li>
	<li>After the injection of the ferrogel, place the gripper structure inside a UV source chamber with a 365 nm wavelength for 6 min. Fix the intensity of the UV light at 4.9 mJ/s.</li>
	<li>After UV photocuring, transfer the gripper structure to a DI water bath for at least 24 h until it reaches a fully swollen equilibrium state (Figure 2B-D).</li>
</ol>

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The authors declare no conflicts of interest.

In terms of material selection for the soft hybrid gripper, a multi-responsive material system composed of a non-stimuli-responsive AAm-based hydrogel, a thermally responsive NIPAM-based hydrogel, and a magnetic-responsive ferrogel was first prepared to allow the soft hybrid gripper to exhibit programmable locomotion and shape transformation. Owing to their thermally responsive swelling-deswelling properties, NIPAM-based hydrogels exhibit bending, folding, or wrinkling when fabricated as bilayer or bi-strip structures wi...

The development of stimuli-responsive soft robots is important from both technical and intellectual perspectives. The term stimuli-responsive soft robots generally refers to devices/systems composed of hydrogels, polymers, elastomers, or hybrids that exhibit shape changes in response to external cues, such as heat, pH, and light1,2,3,4. Among the many stimuli-responsive soft robots, N-isopropylacrylamide (NIPAM) hydrogel-based soft robots perform the desired tasks or interactions using spontaneous shape transformation5,6,7,8. Generally, the NIPAM-based hydrogels exhibit a low critical solution temperature (LCST), and swelling (hydrophilicity below the LCST) and deswelling (hydrophobicity above the LCST) property changes occur inside the hydrogel system near physiological temperatures between 32 &#176;C and 36 &#176;C9,10. This reversible swelling-deswelling mechanism near the sharp critical transition point of the LCST can generate the shape transformation of NIPAM-based hydrogel soft robots2. As a result, thermally responsive NIPAM-based hydrogel soft robots have improved operations, such as walking, gripping, crawling, and sensing, which are important in multifunctional manipulators, healthcare systems, and smart sensors2,3,4,11,12,13,14,15,16,17,18,19,20,21.
In the fabrication of stimuli-responsive soft robots, three-dimensional (3D) printing approaches have been widely employed using a direct layer-by-layer additive process22. A variety of materials, such as plastics and soft hydrogels, can be printed with 3D printing23,24. Recently, 4D printing has been extensively highlighted as an innovative technique for creating shape-programmable soft robots25,26,27,28. This 4D printing is based on 3D printing, and the key characteristic of 4D printing is that the 3D structures can change their shapes and properties over time.&#160;The combination of 4D printing and stimuli-responsive hydrogels has provided another innovative route to create smart 3D devices that change shape over time when exposed to appropriate external stimulus triggers, such as heat, pH, light, and magnetic and electric fields25,26,27,28. The development of this 4D printing technique using diverse stimuli-responsive hydrogels has provided an opportunity for the emergence of shape-transformable soft robots that display multifunctionality with improved response speeds and feedback sensitivity.
This study describes the creation of a 3D printing-driven thermally responsive soft gripper that displays shape transformation and locomotion. Notably, the specific procedure described can be utilized to fabricate various multifunctional soft robots with overall sizes ranging from the millimeter to centimeter length scales. Finally, it is expected that this protocol can be applied in several fields, including soft robots (e.g., smart actuators and locomotion robots), flexible electronics (e.g., optoelectrical sensors and lab-on-a-chip), and healthcare systems (e.g., drug delivery capsules, biopsy tools, and surgical devices).

<table><tbody><tr><td>2-Hydroxy-4&#039;-(2-hydroxyethoxy)-2-methylpropiophenone</td><td>Sigma Aldrich</td><td>410896-50G</td><td>Irgacure 2959, photoinitiator</td></tr><tr><td>3D WOX 2X</td><td>sindoh</td><td>n/a</td><td>3D printer for fabricating a maze</td></tr><tr><td>Acrylamide</td><td>Sigma-Aldrich</td><td>29-007</td><td>&amp;ge;99%</td></tr><tr><td>Airbrush compressor</td><td>WilTec</td><td>AF18-2</td><td></td></tr><tr><td>Ammonium persulfate</td><td>Sigma Aldrich</td><td>A4418</td><td></td></tr><tr><td>Auto CAD</td><td>Autodesk</td><td>n/a</td><td>software for computer-aided-design file</td></tr><tr><td>BLX UV crosslinker</td><td>BIO-LINK</td><td>U01-133-565</td><td></td></tr><tr><td>Cartridge</td><td>CELLINK</td><td>CSC010300102</td><td></td></tr><tr><td>Digital stirring Hot Plates</td><td>Corning</td><td>6798-420D</td><td></td></tr><tr><td>Fluorescein O-methacrylate</td><td>Sigma Aldrich</td><td>568864</td><td>dye of AAm gel</td></tr><tr><td>INKREDIBLE+ bioprinter</td><td>CELLINK</td><td>n/a</td><td></td></tr><tr><td>Iron(III) Oxide red</td><td>DUKSAN general science</td><td>I0231</td><td></td></tr><tr><td>Laponite RD</td><td>BYK</td><td>n/a</td><td>nanoclay</td></tr><tr><td>Microcentrifuge tube</td><td>SPL</td><td>60615</td><td></td></tr><tr><td>Micro stirrer bar</td><td>Cowie</td><td>27-00360-08</td><td></td></tr><tr><td>N, N, N&#039;, N&#039;-tetramethylethylenediamine</td><td>Sigma Aldrich</td><td>T7024-100ML</td><td></td></tr><tr><td>N, N&#039;-methylenebisacrylamide</td><td>Sigma Aldrich</td><td>M7279</td><td>&amp;ge;99.5%</td></tr><tr><td>N-isopropylacrylamide</td><td>Sigma-Aldrich</td><td>415324-50G</td><td></td></tr><tr><td>Poly(N-isopropylacrylamide)</td><td>Sigma-Aldrich</td><td>535311</td><td></td></tr><tr><td>Rhodamine 6G</td><td>Sigma Aldrich</td><td>R4127</td><td>dye of NIPAM gel</td></tr><tr><td>Slic3r software (v1.2.9)</td><td>Slic3r</td><td>n/a</td><td>open-source software to convert .stl file to gcode</td></tr><tr><td>Sodium hydroxide beads</td><td>Sigma Aldrich</td><td>S5881</td><td></td></tr><tr><td>Sterile high-precision conical bioprinting nozzles</td><td>CELLINK</td><td>NZ3270005001</td><td>22 G, 25 G</td></tr><tr><td>Syringe</td><td>Korea vaccine</td><td>K07415389</td><td>10 CC 21 G (1-1/4 INCH)</td></tr><tr><td>Vortex mixer</td><td>DAIHAN</td><td>DH.WVM00030</td><td></td></tr></tbody></table>

four-dimensional printing of stimuli-responsive hydrogel-based soft robots

The present protocol describes the creation of four-dimensional (4D), time-dependent, shape-changeable, stimuli-responsive soft robots using a three-dimensional (3D) bio-printing method. Recently, 4D printing techniques have been extensively proposed as innovative new methods for developing shape-transformable soft robots. In particular, 4D time-dependent shape transformation is an essential factor in soft robotics because it allows effective functions to occur at the right time and place when triggered by external cues, such as heat, pH, and light. In line with this perspective, stimuli-responsive materials, including hydrogels, polymers, and hybrids, can be printed to realize smart shape-transformable soft robotic systems. The current protocol can be used to fabricate thermally responsive soft grippers composed of N-isopropylacrylamide (NIPAM)-based hydrogels, with overall sizes ranging from millimeters to centimeters in length. It is expected that this study will provide new directions for realizing intelligent soft robotic systems for various applications in smart manipulators (e.g., grippers, actuators, and pick-and-place machines), healthcare systems (e.g., drug capsules, biopsy tools, and microsurgeries), and electronics (e.g., wearable sensors and fluidics).

The NIPAM-based hydrogel was primarily considered when designing the thermally responsive soft gripper owing to its sharp LCST, which causes it to exhibit significant swelling-deswelling properties9,10. In addition, the AAm-based hydrogel was considered as a non-stimuli-responsive system to maximize the shape transformation of the soft hybrid gripper while reducing the delamination of the interface during multiple heating and cooling processes. In addition, ferro...

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Four-Dimensional Printing of Stimuli-Responsive Hydrogel-Based Soft Robots

This manuscript describes a 4D printing strategy for fabricating intelligent stimuli-responsive soft robots. This approach can provide the groundwork to facilitate the realization of intelligent shape-transformable soft robotic systems, including smart manipulators, electronics, and healthcare systems.

The present protocol describes the creation of four-dimensional (4D), time-dependent, shape-changeable, stimuli-responsive soft robots using a ...

four-dimensional-printing-stimuli-responsive-hydrogel-based-soft

Research

JoVE Journal

Engineering

2.8K Views.  Sookmyung Women's University. Het gepresenteerde protocol zal naar verwachting nieuwe richtingen bieden voor het realiseren van intelligente, vorm transformeerbare, zachte robotsystemen voor verschillende toepassingen. Het 4D-tijdsafhankelijke printproces kan diverse prikkelgevoelige zachte robots creëren, met een breed formaatbereik, van millimeters tot centimeters. Deze 4D-bioprinttechniek kan worden uitgebreid naar gerichte medicijnafgifte, microchirurgie en minder invasieve biopsie in de gezondheidszorg.Alleen...

Video: Four-Dimensional Printing of Stimuli-Responsive Hydrogel-Based Soft Robots

Micro 3D Printing Using a Digital Projector and its Application in the Study of Soft Materials Mechanics

Buckling is a classical topic in mechanics. While buckling has long been studied as one of the major structural failure modes1, it has recently drawn new attention as a unique mechanism for pattern transformation. Nature is full of such examples where a wealth of exotic patterns are formed through mechanical instability2-5. Inspired by this elegant mechanism, many studies have demonstrated creation and transformation of patterns using soft materials such as elastomers and hydrogels6-11. Swelling gels are of particular interest because they can spontaneously trigger mechanical instability to create various patterns without the need of external force6-10. Recently, we have reported demonstration of full control over buckling pattern of micro-scaled tubular gels using projection micro-stereolithography (P&mu;SL), a three-dimensional (3D) manufacturing technology capable of rapidly converting computer generated 3D models into physical objects at high resolution12,13. Here we present a simple method to build up a simplified P&mu;SL system using a commercially available digital data projector to study swelling-induced buckling instability for controlled pattern transformation.
A simple desktop 3D printer is built using an off-the-shelf digital data projector and simple optical components such as a convex lens and a mirror14. Cross-sectional images extracted from a 3D solid model is projected on the photosensitive resin surface in sequence, polymerizing liquid resin into a desired 3D solid structure in a layer-by-layer fashion. Even with this simple configuration and easy process, arbitrary 3D objects can be readily fabricated with sub-100 &mu;m resolution.
This desktop 3D printer holds potential in the study of soft material mechanics by offering a great opportunity to explore various 3D geometries. We use this system to fabricate tubular shaped hydrogel structure with different dimensions. Fixed on the bottom to the substrate, the tubular gel develops inhomogeneous stress during swelling, which gives rise to buckling instability. Various wavy patterns appear along the circumference of the tube when the gel structures undergo buckling. Experiment shows that circumferential buckling of desired mode can be created in a controlled manner. Pattern transformation of three-dimensionally structured tubular gels has significant implication not only in mechanics and material science, but also in many other emerging fields such as tunable matamaterials.

We demonstrate controlled pattern transformation of swelling gel tubes by elastic instability. A simple projection micro stereo-lithography setup is built using an off-the-shelf digital data projector to fabricate three-dimensional polymeric structures in a layer-by-layer fashion. Swelling hydrogel tubes under mechanical constraint display various circumferential buckling modes depending on dimension.

Buckling is a classical topic in mechanics. While buckling has long been studied as one of the major structural failure modes1, it has recently drawn new ...

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

Bioengineering

Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact bioprinting1-4 (extrusion, dip pen and soft lithography), contactless bioprinting5-7 (laser forward transfer, ink-jet deposition) and laser based techniques such as two photon photopolymerization8. It can be used for many applications such as tissue engineering9-13, biosensor microfabrication14-16 and as a tool to answer basic biological questions such as influences of co-culturing of different cell types17. Unlike common photolithographic or soft-lithographic methods, extrusion bioprinting has the advantage that it does not require a separate mask or stamp. Using CAD software, the design of the structure can quickly be changed and adjusted according to the requirements of the operator. This makes bioprinting more flexible than lithography-based approaches.
Here we demonstrate the printing of a sacrificial mold to create a multi-material 3D structure using an array of pillars within a hydrogel as an example. These pillars could represent hollow structures for a vascular network or the tubes within a nerve guide conduit. The material chosen for the sacrificial mold was poloxamer 407, a thermoresponsive polymer with excellent printing properties which is liquid at 4 &deg;C and a solid above its gelation temperature ~20 &deg;C for 24.5% w/v solutions18. This property allows the poloxamer-based sacrificial mold to be eluted on demand and has advantages over the slow dissolution of a solid material especially for narrow geometries. Poloxamer was printed on microscope glass slides to create the sacrificial mold. Agarose was pipetted into the mold and cooled until gelation. After elution of the poloxamer in ice cold water, the voids in the agarose mold were filled with alginate methacrylate spiked with FITC labeled fibrinogen. The filled voids were then cross-linked with UV and the construct was imaged with an epi-fluorescence microscope.

A bioprinter was used to create patterned hydrogels based on a sacrificial mold. The poloxamer mold was backfilled with a second hydrogel and then eluted, leaving voids which were filled with a third hydrogel. This method uses fast elution and good printability of poloxamer to generate complex architectures from biopolymers.

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact ...

Immunology and Infection

Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper composition, targeted modulus, and well-defined architectural features. Biomaterials that recapitulate the intrinsic architecture of in vivo tissue are vital for studying diseases as well as to facilitate the regeneration of lost and malformed soft tissue. A novel biofabrication technique was developed which combines state of the art imaging, three-dimensional (3D) printing, and selective enzymatic activity to create a new generation of biomaterials for research and clinical application. The developed material, Bovine Serum Albumin rubber, is reaction injected into a mold that upholds specific geometrical features. This sacrificial material allows the adequate transfer of architectural features to a natural scaffold material. The prototype consists of a 3D collagen scaffold with 4 and 3 mm channels that represent a branched architecture. This paper emphasizes the use of this biofabrication technique for the generation of natural constructs. This protocol utilizes a computer-aided software (CAD) to manufacture a solid mold which will be reaction injected with BSA rubber followed by the enzymatic digestion of the rubber, leaving its architectural features within the scaffold material.

An innovative biofabrication technique was developed to engineer three-dimensional constructs that resemble the architectural features, components, and mechanical properties of in vivo tissue. This technique features a newly developed sacrificial material, BSA rubber, which transfers detailed spatial features, reproducing the in vivo architectures of a wide variety of tissues.

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper ...

Fabricating Degradable Thermoresponsive Hydrogels on Multiple Length Scales via Reactive Extrusion, Microfluidics, Self-assembly, and Electrospinning

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), their ultimate clinical use has been hampered by the lack of biologically-relevant degradation observed for most smart materials. This is particularly true for temperature-responsive hydrogels, which are almost uniformly based on polymers that are functionally non-degradable (e.g., poly(N-isopropylacrylamide) (PNIPAM) or poly(oligoethylene glycol methacrylate) (POEGMA)). As such, to effectively translate the potential of thermoresponsive hydrogels to the challenges of remote-controlled or metabolism-regulated drug delivery, cell scaffolds with tunable cell-material interactions, theranostic materials with the potential for both imaging and drug delivery, and other such applications, a method is required to render the hydrogels (if not fully degradable) at least capable of renal clearance following the required lifetime of the material. To that end, this protocol describes the preparation of hydrolytically-degradable hydrazone-crosslinked hydrogels on multiple length scales based on the reaction between hydrazide and aldehyde-functionalized PNIPAM or POEGMA oligomers with molecular weights below the renal filtration limit. Specifically, methods to fabricate degradable thermoresponsive bulk hydrogels (using a double barrel syringe technique), hydrogel particles (on both the microscale through the use of a microfluidics platform facilitating simultaneous mixing and emulsification of the precursor polymers and the nanoscale through the use of a thermally-driven self-assembly and cross-linking method), and hydrogel nanofibers (using a reactive electrospinning strategy) are described. In each case, hydrogels with temperature-responsive properties similar to those achieved via conventional free radical cross-linking processes can be achieved, but the hydrazone cross-linked network can be degraded over time to re-form the oligomeric precursor polymers and enable clearance. As such, we anticipate these methods (which may be generically applied to any synthetic water-soluble polymer, not just smart materials) will enable easier translation of synthetic smart materials to clinical applications.

Protocols are described for the fabrication of degradable thermoresponsive hydrogels based on hydrazone cross-linking of polymeric oligomers on the bulk scale, microscale, and nanoscale, the latter for preparation of both gel nanoparticles and nanofibers.

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), ...

An Additive Manufacturing Technique for the Facile and Rapid Fabrication of Hydrogel-based Micromachines with Magnetically Responsive Components

Polyethylene glycol (PEG)-based hydrogels are biocompatible hydrogels that have been approved for use in humans by the FDA. Typical PEG-based hydrogels have simple monolithic architectures and often function as scaffolding materials for tissue engineering applications. More sophisticated structures typically take a long time to fabricate and do not contain moving components. This protocol describes a photolithography method that allows for facile and rapid microfabrication of PEG structures and devices. This strategy involves an in-house developed fabrication stage that allows for the rapid fabrication of 3D structures by building upwards in a layer-by-layer fashion. Independent moving components can also be aligned and assembled onto support structures to form integrated devices. These independent components are doped with superparamagnetic iron oxide nanoparticles that are sensitive to magnetic actuation. In this manner, the fabricated devices can be actuated using external magnets to yield movement of the components within. Hence, this technique allows for the fabrication of sophisticated MEMS-like devices (micromachines) that are composed entirely out of a biocompatible hydrogel, able to function without an onboard power source, and respond to a contact-less method of actuation. This manuscript describes the fabrication of both the fabrication set-up as well as the step-by-step method for the microfabrication of these hydrogels-based MEMS-like devices.

An additive manufacturing strategy for processing UV-crosslinkable hydrogels has been developed. This strategy allows for the layer-by-layer assembly of microfabricated hydrogel structures as well as the assembly of independent components, yielding integrated devices containing moving components that are responsive to magnetic actuation.

Polyethylene glycol (PEG)-based hydrogels are biocompatible hydrogels that have been approved for use in humans by the FDA. Typical PEG-based hydrogels ...

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed from mammal collagen. Thus, the arginine-glycine-aspartic acid (RGD) sequences and target motifs of matrix metalloproteinase (MMP) remain on the molecular chains, which help achieve cell attachment and degradation. Furthermore, formation properties of GelMA are versatile. The methacrylamide groups allow a material to become rapidly crosslinked under light irradiation in the presence of a photoinitiator. Therefore, it makes great sense to establish suitable methods for synthesizing three-dimensional (3D) structures with this promising material. However, its low viscosity restricts GelMA's printability. Presented here are methods to carry out 3D bioprinting of GelMA hydrogels, namely the fabrication of GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. The resulting structures and biocompatibility of the materials as well as the printing methods are discussed. It is believed that this protocol may serve as a bridge between previously applied biomaterials and GelMA as well as contribute to the establishment of GelMA-based 3D architectures for biomedical applications.

Presented here is a method for the 3D bioprinting of gelatin methacryloyl.

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...

Bioinspired Soft Robot with Incorporated Microelectrodes

Bioinspired soft robotic systems that mimic living organisms using engineered muscle tissue and biomaterials are revolutionizing the current biorobotics paradigm, especially in biomedical research. Recreating artificial life-like actuation dynamics is crucial for a soft-robotic system. However, the precise control and tuning of actuation behavior still represents one of the main challenges of modern soft robotic systems. This method describes a low-cost, highly scalable, and easy-to-use procedure to fabricate an electrically controllable soft robot with life-like movements that is activated and controlled by the contraction of cardiac muscle tissue on a micropatterned sting ray-like hydrogel scaffold. The use of soft photolithography methods makes it possible to successfully integrate multiple components in the soft robotic system, including micropatterned hydrogel-based scaffolds with carbon nanotubes (CNTs) embedded gelatin methacryloyl (CNT-GelMA), poly(ethylene glycol) diacrylate (PEGDA), flexible gold (Au) microelectrodes, and cardiac muscle tissue. In particular, the hydrogels alignment and micropattern are designed to mimic the muscle and cartilage structure of the sting ray. The electrically conductive CNT-GelMA hydrogel acts as a cell scaffold that improves the maturation and contraction behavior of cardiomyocytes, while the mechanically robust PEGDA hydrogel provides structural cartilage-like support to the whole soft robot. To overcome the hard and brittle nature of metal-based microelectrodes, we designed a serpentine pattern that has high flexibility and can avoid hampering the beating dynamics of cardiomyocytes. The incorporated flexible Au microelectrodes provide electrical stimulation across the soft robot, making it easier to control the contraction behavior of cardiac tissue.

A bioinspired scaffold is fabricated by a soft photolithography technique using mechanically robust and electrically conductive hydrogels. The micropatterned hydrogels provide directional cardiomyocyte cell alignment, resulting in a tailored direction of actuation. Flexible microelectrodes are also integrated into the scaffold to bring electrical controllability for a self-actuating cardiac tissue.

Bioinspired soft robotic systems that mimic living organisms using engineered muscle tissue and biomaterials are revolutionizing the current biorobotics ...

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized in vitro stretching methods. Various available systems use 2D substrate-based stretchers, while the accessibility of 3D techniques to strain soft hydrogels, is more restricted. Here, we describe a method that allows external stretching of soft hydrogels from their circumference, using an elastic silicone strip as the sample carrier. The stretching system utilized in this protocol is constructed from 3D-printed parts and low-cost electronics, making it simple and easy to replicate in other labs. The experimental process begins with polymerizing thick (&#62;100 &#956;m) soft fibrin hydrogels (Elastic Modulus of ~100 Pa) in a cut-out at the center of a silicone strip. Silicone-gel constructs are then attached to the printed-stretching device and placed on the confocal microscope stage. Under live microscopy the stretching device is activated, and the gels are imaged at various stretch magnitudes. Image processing is then used to quantify the resulting gel deformations, demonstrating relatively homogenous strains and fiber alignment throughout the gel&#8217;s 3D thickness (Z-axis). Advantages of this method include the ability to strain extremely soft hydrogels in 3D while executing in situ microscopy, and the freedom to manipulate the geometry and size of the sample according to the user&#8217;s needs. Additionally, with proper adaptation, this method can be used to stretch other types of hydrogels (e.g., collagen, polyacrylamide or polyethylene glycol) and can allow for analysis of cells and tissue response to external forces under more biomimetic 3D conditions.

The presented method involves uniaxial stretching of 3D soft hydrogels embedded in silicone rubber while allowing live confocal microscopy. Characterization of the external and internal hydrogel strains as well as fiber alignment are demonstrated. The device and protocol developed can assess the response of cells to various strain regimes.

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...

Microgel-Extracellular Matrix Composite Support for the Embedded 3D Printing of Human Neural Constructs

The embedded 3D printing of cells inside a granular support medium has emerged in the past decade as a powerful approach for the freeform biofabrication of soft tissue constructs. However, granular gel formulations have been restricted to a limited number of biomaterials that allow for the cost-effective generation of large amounts of hydrogel microparticles. Therefore, granular gel support media have generally lacked the cell-adhesive and cell-instructive functions found in the native extracellular matrix (ECM).
To address this, a methodology has been developed for the generation of self-healing annealable particle-extracellular matrix (SHAPE) composites. SHAPE composites consist of a granular phase (microgels) and a continuous phase (viscous ECM solution) that, together, allow for both programmable high-fidelity printing and an adjustable biofunctional extracellular environment. This work describes how the developed methodology can be utilized for the precise biofabrication of human neural constructs.
First, alginate microparticles, which serve as the granular component in the SHAPE composites, are fabricated and combined with a collagen-based continuous component. Then, human neural stem cells are printed inside the support material, followed by the annealing of the support. The printed constructs can be maintained for weeks to allow the differentiation of the printed cells into neurons. Simultaneously, the collagen continuous phase allows for axonal outgrowth and the interconnection of regions. Finally, this works provides information on how to perform live-cell fluorescence imaging and immunocytochemistry to characterize the 3D-printed human neural constructs.

This work describes a protocol for the freeform embedded 3D printing of neural stem cells inside self-healing annealable particle-extracellular matrix composites. The protocol enables the programmable patterning of interconnected human neural tissue constructs with high fidelity.

The embedded 3D printing of cells inside a granular support medium has emerged in the past decade as a powerful approach for the freeform biofabrication ...

Four-Dimensional Printing of Stimuli-Responsive Hydrogel-Based Soft Robots

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