The authors would like to thank T. Pond, research support specialist at the Department of Chemical Engineering of The Pennsylvania State University (Penn State), the Nanofabrication Lab staff at Penn State, and Dr. J. de Rutte from Partillion Bioscience for the help and discussion regarding nanofabrication processes. A. Sheikhi acknowledges the support of the Materials Research Institute (MRI) and the College of Engineering Materials Matter at the Human Level seed grants, the Convergence Center for Living Multifunctional Material Systems (LiMC2) and the Cluster of Excellence Living, Adaptive and Energy-autonomous Materials Systems (livMatS) Living Multifunctional Materials Collaborative Research Seed Grant Program, and the startup fund from Penn State. Research reported in this publication was partially supported by the National Institute of Biomedical Imaging and Bioengineering (NIBIB) of the National Institutes of Health (NIH) under award number R56EB032672.

NOTE: See the Table of Materials for details related to all materials, instruments, and reagents used in this protocol.
1. GelMA synthesis
NOTE: GelMA synthesis should be conducted in a chemical fume hood, and proper personal protective equipment (PPE) should be used all the time.
<ol>
	<li>Add 200 mL of Dulbecco's phosphate buffered saline (DPBS, 1x) to an Erlenmeyer flask and heat the solution until it reaches 50 °C. Cover the flask with aluminum foil to prevent evaporation.</li>
	<li>Add 20 g of gelatin powder to the DPBS solution at 50 °C while stirring at 240 rpm until the powder is completely dissolved.</li>
	<li>Add 16, 2.5, or 0.5 mL of methacrylate anhydride (MA) to the gelatin solution dropwise via a glass Pasteur pipette to synthesize GelMA with a high, medium, or low degree of methacryloyl substitution, respectively. 
	​CAUTION: MA is a hazardous material. Proper PPE should be used when working with MA. MA is also light-sensitive, so protect the reaction from light by wrapping the flask with aluminum foil.</li>
	<li>After 2 h, add 400 mL of DPBS at 50 °C to stop the reaction. Allow stirring to continue at 50 °C for 10 min.</li>
	<li>Pour the solution into a dialysis membrane tubing with 12-14 kDa molecular weight cutoff,  and then place the tubing in a 5 L beaker filled with 40 °C ultrapure water. Stir the water at 240 rpm and 40 °C.</li>
	<li>Dialyze the solution against ultrapure water for 10 days and change the water 2x a day to remove unreacted methacrylate anhydride, byproducts, and other impurities.</li>
	<li>After 10 days, add 400 mL of ultrapure water at 40 °C to the GelMA solution. Stir the solution at 240 rpm for 15 min.</li>
	<li>Filter the solution twice using coffee filters, followed by vacuum filtration via a 0.2 µm vacuum filtration unit.</li>
	<li>Pour 25 mL of the filtered solution into 50 mL centrifuge tubes and freeze them at -80 °C, placing the tubes horizontally.</li>
	<li>After 2 days, remove the caps and cover the centrifuge tubes with laboratory wipes. Use tape or a rubber band to hold the wipes tightly.</li>
	<li>Lyophilize the frozen GelMA solution to yield white solid GelMA.</li>
	<li>To conduct proton nuclear magnetic resonance (1H NMR) spectroscopy, separately add 30 mg of gelatin powder (control) or lyophilized GelMA in 1 mL of deuterium oxide (D2O) and maintain the samples at 37 °C until the gelatin powder or GelMA is entirely dissolved.</li>
	<li>Obtain the 1H NMR spectra and determine the degree of methacryloyl substitution by integrating the aromatic acids and lysine methylene proton peaks at chemical shifts of ~6.5-7.5 and ~3.0 ppm, respectively. Use the aromatic acids peak as a reference and determine the degree of substitution (DS) using lysine methylene peaks based on equation (1): 
	DS (%) = [1 - (Area of lysine methylene in GelMA / Area of lysine methylene in gelatin)] × 100    (1)</li>
</ol>
<img alt="Gelatin methacryloyl synthesis diagram with ¹H NMR spectra analyzing gelatin and GelMA composition." class="xfigimg" src="/files/ftp_upload/64829/64829fig01.jpg" data-altupdatedat="1747929244000" data-alt="GelMA synthesis and ¹H NMR graph; gelatin to GelMA reaction, chemical shifts in ppm, peaks."> 
Figure 1: GelMA synthesis and characterization. (A) GelMA synthesis reaction. Gelatin is modified with methacrylic anhydride at 50 °C for 2 h. (B) The proton nuclear magnetic resonance (1H NMR) spectra of gelatin and GelMA: (a) the peak for aromatic acids, which is selected as the reference for calibration, (b) vinyl functional group peaks after the MA modification of gelatin, and (c) the peak for lysine proteins. In this example, the MA degree of substitution was 71% ± 3% (n = 3). This figure has been modified with permission from Ataie et al.11 Abbreviations: GelMA = gelatin methacryloyl; DPBS = Dulbecco's phosphate-buffered saline; MA = methacryloyl. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64829/64829fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. High-throughput GelMA microgel fabrication
<ol>
	<li>Device master mold microfabrication 
	NOTE: Master molds may be microfabricated via soft lithography using the KMPR 1000 negative photoresist series19.
	<ol>
		<li>Thaw KMPR 1025 and 1035 overnight. Avoid any light exposure.</li>
		<li>To coat the first layer on the wafer, add KMPR 1025 directly to the middle of the wafer to make an approximately 5 cm circle of photoresist. Run the spin coater at 3,000 rpm for 30 s.</li>
		<li>Soft bake for 12 min on a 100 °C hotplate. Then, cool down on the cooling plate for 5 min.</li>
		<li>Attach the first layer mask to the blank soda lime, then expose the coated wafer to UV light using a mask aligner for 645 mJ/cm2 of dosage.</li>
		<li>Post bake for 3 min on a 100 °C hotplate. Cool down on the cooling plate for 5 min. 
		NOTE: Do not develop after this step. Develop only once at the end of the process.</li>
		<li>Spin coat the second layer on the wafer using the KMPR 1035. Run the spin coater at 1,000 rpm for 30 s.</li>
		<li>Soft bake for 30 min on a 100 °C hotplate. Cool down on the cooling plate for 5 min.</li>
		<li>Attach the second layer mask to the blank soda lime and align the second mask using the aligner through standard alignment signs. Expose to UV light using a mask aligner up to 2,000 mJ/cm2.</li>
		<li>Post bake for 5 min on a 100 °C hotplate.</li>
		<li>Develop for &gt;6 min in the SU-8 developer. 
		NOTE: If the wafer looks milky, development should be continued for a longer time. Use fresh developer each time and in between for a better outcome.</li>
		<li>Spray with isopropanol. Make sure the wafer is clear, with no milky residues. Thoroughly dry the wafer using nitrogen (N2) gas.</li>
	</ol>
	</li>
	<li>Microfluidic device fabrication
	<ol>
		<li>Pour 50 g of polydimethylsiloxane (PDMS) base part into a transparent plastic cup. Then, add 5 g of the crosslinker to the plastic cup. Mix the base and crosslinker using a spatula vigorously until a creamy texture is obtained.</li>
		<li>Vacuum degas the mixture using a desiccator for 20 min until it becomes clear. Pour the mixture onto the master mold, which is placed inside and taped to a Petri dish. 
		NOTE: Make sure the thickness (height) of the poured PDMS is ≤8 mm.</li>
		<li>Put the Petri dish into the desiccator and vacuum degas the PDMS mixture again for 20 min until all the bubbles are removed. Place the Petri dish in a 70 °C oven for 2 h until the PDMS is crosslinked. Take the Petri dish out of the oven and allow it to cool down.</li>
		<li>Cut the devices out of the mold using a scalpel. Detach the devices slowly from the master mold. Use the biopsy punch (1.5 mm diameter) to cut holes through the inlets and the outlet.</li>
		<li>Remove any dust from the PDMS devices and the glass slides using masking tape, and place the glass slides and the devices in a plasma cleaner chamber. Perform the plasma treatment for 45 s (starting when the chamber turns purple) with air pressure under 400 mTorr. Remove the slides and devices from the chamber, put the device on the glass slides, and apply slight pressure. Put the device in the 70 °C oven for 30 min to enhance the bonding.</li>
		<li>Fill the devices with trichloro(1H,1H,2H,2H-perfluorooctyl)silane (F-silane, 2% v/v) in the engineered fluid to render the channel surface fluorophilic. Inject the F-silane solution through the outlet and make sure all the devices are exposed. Wait for 5-10 min. 
		NOTE: F-silane should be prepared freshly. In addition, F-silane should not be exposed to air for a long time.</li>
		<li>Aspirate the F-silane solution out of the device through the aqueous solution inlet. Wash the device twice using the engineering fluid and aspirate again. Place the device in the 70 °C oven for 30 min to evaporate the remaining oil.</li>
	</ol>
	</li>
	<li>Droplet formation and GelMA microgel fabrication
	<ol>
		<li>Add 10 mg of lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) to 10 mL of DPBS to prepare a photoinitiator (PI) solution (0.1% w/v). Protect the solution from light by wrapping it in aluminum foil.</li>
		<li>Dissolve the desired amount of GelMA in the PI solution and place it in the 37 °C oven for 1 h until a clear solution is obtained. Protect the solution from light by wrapping in aluminum foil.</li>
		<li>To prepare the oil phase, make a 2% v/v of biocompatible surfactant solution in the engineering fluid.</li>
		<li>Insert the Tygon tubing in the inlets and outlet of the PDMS device. Insert a 25 G blunt needle in the other end of Tygon tubing for inlets. Use the minimum possible tubing length.</li>
		<li>Place the device under the microscope. Keep the environment warm (~40 °C) using a hair dryer and/or a space heater.</li>
		<li>Load the aqueous and oil solutions in separate syringes, connected to the device. Start the syringe pumps with flow rates of 160 and 80 µL/min for the oil (continuous) and aqueous (dispersed) phases, respectively. 
		NOTE: Start the oil phase first; make sure the oil fills the channel, then start the aqueous phase.</li>
		<li>Collect the droplets in a container and evaluate them in the imaging chamber via optical microscopy imaging.</li>
		<li>Place the droplets at 4 °C overnight while protecting them from light to initiate GelMA HMP physical crosslinking and convert the droplets to stable microgels at 4 °C.</li>
	</ol>
	</li>
</ol>
3. Converting microgels to resuspendable powder via the microengineered emulsion-to-powder (MEtoP) technology
NOTE: The MEtoP technology to convert the water-in oil emulsion-based HMPs to microparticle powder (micro-aerogels) with preserved properties, such as resuspendability, shape, size, and assembly, has been developed.
<ol>
	<li>To implement the MEtoP, collect the physically crosslinked HMPs in the engineering fluid using thermally durable microcentrifuge tubes or cryovials. Open the tube caps and seal them with a laboratory wipe and tape.</li>
	<li>Deep-freeze the physically crosslinked HMPs in liquid nitrogen (-196 °C) for 10 min.</li>
	<li>Transfer the flash-frozen tubes to a freeze-dryer instrument. Lyophilize the tubes at low pressure (e.g., 0.06 mbar) for at least 6 h to yield powder. 
	NOTE: When the lyophilization cycle is finished, break the pressure slowly so the powder is not lost.</li>
	<li>Add 1 mL of cooled PI solution (0.1% w/v, 4 °C) to the powder to make microgel suspensions. Vortex for 5 s, then centrifuge at 3,000 × g for 15 s. Discard the supernatant.</li>
	<li>Transfer the packed microgel suspension to a mold using a positive displacement pipette, followed by UV light exposure at 400 nm wavelength with an intensity of 15 mW/cm2 for 60 s to form GHS.</li>
</ol>
<img alt="MetoP vs. lyophilization micro-aerogels diagram; SEM comparison of particle structures and aggregates." class="xfigimg" src="/files/ftp_upload/64829/64829fig02.jpg" data-altupdatedat="1747929244000" data-alt="MetoP tech vs. conventional lyophilization, microstructures, SEM images, aerogel formation."> 
Figure 2: GelMA microparticle powder preparation via MEtoP technology. (A) Images of GelMA powder obtained from the MEtoP technology or conventional lyophilization of HMP. In MEtoP technology or conventional lyophilization, HMPs are suspended in oil-surfactant or aqueous media, respectively. The engineering fluid protects the dispersed phase (HMPs) from aggregation and preserves the physiochemical properties of GelMA microparticles during lyophilization. (B) Schematic illustration of dried HMPs prepared via the MEtoP compared with conventionally lyophilized HMP in an aqueous medium. (C) SEM images of dried GelMA microparticles prepared via the MEtoP compared with conventional lyophilization. Scale bars = 2 mm (left; A), 500 µm (right; A), 10 µm (left; C), and 200 µm (right; C). This figure was modified with permission from Sheikhi et al.26 Abbreviations: GelMA = gelatin methacryloyl; DPBS = Dulbecco's phosphate-buffered saline; MEtoP = microengineered emulsion-to-powder; HMP = hydrogel microparticle; SEM = scanning electron microscopy. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64829/64829fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. GelMA GHS formation
NOTE: This protocol is for preparing 400 µL of microgel suspension. For larger quantities, scale-up is needed. To keep the GelMA HMPs physically crosslinked, all the steps should be performed at about 4 °C by placing the microgel containers in an ice-water bucket.
<ol>
	<li>Add 400 µL of 1H,1H-perfluoro-1-octanol (PFO) solution in the engineering fluid (20% v/v) to the physically crosslinked GelMA HMPs. Then, vortex for 5 s and centrifuge for 15 s at 300 × g. 
	NOTE: The PFO solution in the engineering fluid should be prepared freshly and stored in an enclosed container to prevent evaporation.</li>
	<li>Remove the oil phase from the GelMA HMPs via pipetting.</li>
	<li>Add 400 µL of PI solution (0.1% w/v) at 4 °C to the microgel suspension. Then, vortex for 5 s and centrifuge at 300 × g for 15 s. Discard the oil afterward.</li>
	<li>Repeat the previous step but centrifuge at 3,000 × g. Remove the supernatant of packed GelMA HMPs via pipetting.</li>
	<li>Transfer the packed GelMA HMPs to a mold using a positive displacement pipette, followed by UV light exposure (wavelength = 400 nm, intensity = 15 mW/cm2, exposure time = 60 s).</li>
</ol>
5. Nanoengineered granular bioinks (NGB) for the 3D bioprinting of GHS with preserved microporosity
<ol>
	<li>Add 100 mg of nanoplatelet powder to 3 mL of 4 °C ultrapure water to form a nanoparticle dispersion (3.33% w/v). Vortex the dispersion vigorously inside a 4 °C fridge for 15 min to exfoliate the otherwise aggregated nanoparticles. Properly exfoliated nanoparticles yield a clear dispersion.</li>
	<li>Dissolve 50 mg of LAP in 5 mL of 4 °C ultrapure water to prepare a stock PI solution (1% w/v).</li>
	<li>Add 333 µL of PI solution (1% w/v) to the exfoliated nanoparticle dispersion. Wrap in aluminum foil to protect against ambient light. Vortex for 1 min to mix the nanoparticle dispersion and PI. The final clay and PI concentrations are 3% w/v and 0.1% w/v, respectively.</li>
	<li>Add PFO 20% v/v in engineering fluid (4 °C) to the physically crosslinked GelMA HMPs at a 1:1 volume ratio. Vortex thoroughly for 5 s. Then, centrifuge at 300 × g for 15 s and discard the oil phase containing the surfactant.</li>
	<li>Add the LAP-supplemented nanoparticle dispersion (4 °C) to the washed GelMA HMPs. Vortex for 15 s, centrifuge at 3,000 × g for 15 s, and discard the remaining oil at the bottom as well as the supernatant dispersion.</li>
	<li>Store the suspension at 4 °C while protecting it from light using aluminum foil for 1 day. The product of this step is the GelMA NGB.</li>
	<li>Load the NGB into a 3 mL syringe, seal the loaded syringe with a cap and parafilm, and pulse centrifuge at 200 × g to remove the trapped air. Transfer the bioink to a 3 mL cartridge using a female-female Luer-Lok connector. Centrifuge the cartridge briefly at 200 × g again to remove the trapped air. Keep the NGB at 4 °C in a fridge before using.</li>
	<li>Before cell-laden bioink preparation, prepare a concentrated cell suspension (e.g., NIH/3T3 murine fibroblast cells), containing ~24 million cells in 100 µL of cell culture medium. Load the cell suspension into a 3 mL syringe, couple the NGB-loaded syringe and the cell-loaded syringe using a female-female Luer-Lok connector, and mix the cells and NGB gently by pushing back and forth 40x.</li>
	<li>Print the NGB or cell-laden NGB using a proper bioprinter with a standard conical nozzle. Load the nozzle into the 3 mL printhead. Keep the printing bed temperature below 10 °C. Optimize printing parameters such as speed and back pressure prior to printing.</li>
	<li>Select the substrate and nozzle type (pneumatic 3 mL syringe equipped with standard conical nozzle), calibrate the bioprinter using the device guidelines, select desirable gcode or STL file, and start printing. 
	NOTE: When performing cell-laden bioprinting, all the materials and devices should be maintained under the biological safety cabinet to minimize contamination.</li>
	<li>After the printing, expose the construct to UV light for photocrosslinking (wavelength = 400 nm, intensity = 15 mW/cm2, exposure time = 60 s).</li>
</ol>
<img alt="Synthesis and preparation of nanoengineered bioink using PFO, photoinitiator, and centrifugation process." class="xfigimg" src="/files/ftp_upload/64829/64829fig03.jpg" data-altupdatedat="1747929244000" data-alt="Bioink preparation diagram with PFO and GelMA; centrifuge steps for nanoengineered bioink."> 
Figure 3: Schematics of GelMA microgel and GHS formation. (A) Schematics of GelMA microgel separation from oil and NGB preparation. PFO (20% v/v in engineering fluid) was added to the GelMA microgel-oil emulsion at a 1:1 volumetric ratio, followed by vortexing and centrifugation at 300 × g for 15 s. To fabricate GelMA GHS, the PI solution (LAP 0.1% w/v in DPBS) was added to the GelMA HMPs, followed by vortexing and centrifugation at 3,000 × g for 15 s. For preparing the NGB, the PI solution (LAP 0.1% w/v in ultrapure water) and nanoplatelet dispersion (3% w/v in ultrapure water) were added to the GelMA HMP suspension, followed by vortexing and centrifugation at 3,000 × g for 15 s. Figure 3A was modified with permission from Ataie, Z. et al.11 (B) Exposing packed GelMA HMPs to light yields GHS. Figure 3B was modified with permission from Sheikhi et al.15 Abbreviations: GelMA = gelatin methacryloyl; GHS = granular hydrogel scaffold; NGB = nanoengineered granular bioink; PFO = 1H,1H-perfluoro-1-octanol; PI = photoinitiator; LAP = lithium phenyl-2,4,6-trimethylbenzoylphosphinate; HMP = hydrogel microparticle; DPBS = Dulbecco's phosphate-buffered saline. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64829/64829fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+rheological+properties+of+methacrylamide+modified+gelatin+hydrogels.">Structural and rheological properties of methacrylamide modified gelatin hydrogels.</a> Biomacromolecules. 1 (1), 31-38 (2000).</li><li>Qazi, T. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anisotropic+rod-shaped+particles+influence+injectable+granular+hydrogel+properties+and+cell+invasion.">Anisotropic rod-shaped particles influence injectable granular hydrogel properties and cell invasion.</a> Advanced Materials. 34 (12), 2109194 (2022).</li><li>Highley, C. B., Song, K. H., Daly, A. C., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Jammed+microgel+inks+for+3d+printing+applications.">Jammed microgel inks for 3d printing applications.</a> Advanced Science. 6 (1), 1801076 (2019).</li><li>Claa&#223;en, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+substitution+of+gelatin+methacryloyl:+best+practice+and+current+pitfalls.">Quantification of substitution of gelatin methacryloyl: best practice and current pitfalls.</a> Biomacromolecules. 19 (1), 42-52 (2018).</li><li>Sheikhi, A., Di Carlo, D., Khademhosseini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+converting+colloidal+systems+to+resuspendable/redispersable+powders+that+preserve+the+original+properties+of+the+colloids.">Methods for converting colloidal systems to resuspendable/redispersable powders that preserve the original properties of the colloids.</a> U.S. Patent Application. , (2022).</li><li>Sheikhi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microengineered+emulsion-to-powder+technology+for+the+high-fidelity+preservation+of+molecular,+colloidal,+and+bulk+properties+of+hydrogel+suspensions.">Microengineered emulsion-to-powder technology for the high-fidelity preservation of molecular, colloidal, and bulk properties of hydrogel suspensions.</a> ACS Applied Polymer Materials. 1 (8), 1935-1941 (2019).</li><li>Lee, S., de Rutte, J., Dimatteo, R., Koo, D., Di Carlo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scalable+fabrication+and+use+of+3d+structured+microparticles+spatially+functionalized+with+biomolecules.">Scalable fabrication and use of 3d structured microparticles spatially functionalized with biomolecules.</a> ACS Nano. 16 (1), 38-49 (2022).</li><li>Charlet, A., Bono, F., Amstad, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+reinforcement+of+granular+hydrogels.">Mechanical reinforcement of granular hydrogels.</a> Chemical Science. 13 (11), 3082-3093 (2022).</li></ol>

The authors declare no conflicts of interest.

Gelatin and its derivatives are the most commonly used protein-based biomaterials for HMP fabrication. The challenge of throughput versus particle size monodispersity trade-off can be overcome using step-emulsification microfluidic devices. These devices are capable of forming more than 40 million droplets per hour, with a coefficient of variation less than 5%27. In this article, we discussed the microfabrication of droplets containing GelMA solutions, followed by converting them to GelMA HMPs, powder, GHS, and NGB.
The thermoresponsivity of GelMA enables facile microfluidic-enabled HMP fabrication and stabilization. At a temperature higher than the UCST (e.g., 37 &#176;C), GelMA dissolves in an aqueous solution, yielding a suitable aqueous fluid for water-in-oil emulsion formation in step-emulsification devices. Decreasing temperature (e.g., 4 &#176;C) enables GelMA HMP formation via physical crosslinking after droplet formation. GelMA HMPs may be used as building blocks for GHS fabrication through various approaches. Thermally stable HMPs may be photoassembled to form a mechanically robust GHS, attaining one of the highest reported compression moduli among granular scaffolds, excluding the interpenetrating percolating networks28. In the photoassembly method, all the procedures should be performed at low temperature (e.g., 4 &#176;C) to avoid GelMA HMP melting.
The 3D (bio)printing of HMPs enables the fabrication of geometrically well-defined GHS; however, this has been performed using tightly packed HMPs, compromising the microporosity of additively manufactured granular constructs. To address this challenge, we show how the reversible self-assembly of heterogeneously charged nanoparticles adsorbed to HMP surfaces renders loosely packed HMPs shear-yielding and 3D printable (NGB) with preserved microporosity.

Assembling HMP building blocks to form tissue engineering scaffolds has gained tremendous attention in the past few years1. GHS, fabricated via HMP assembly, have unique properties compared with their bulk counterparts, including cell-scale microporosity originating from the void spaces among the discrete building blocks. Additional properties, such as injectability, modularity, and decoupled stiffness from porosity, render GHS&#160;a promising platform to enhance tissue repair and regeneration2. Different biomaterials have been used for GHS fabrication, including synthetic PEG-based polymers3,4 and polysaccharides, such as alginate5 and hyaluronic acid6,7. Among naturally derived polymers, the most common protein-based biopolymer for GHS fabrication is GelMA8,9,10,11, a crosslinkable, biocompatible, bioadhesive, and biodegradable biomaterial12,13.
HMPs may be fabricated via batch emulsification8, flow-focusing14,15 or step-emulsification9,11 microfluidic devices, blending16, or complex coacervation17,18. Usually, there is a trade-off between the fabrication throughput and HMP monodispersity. For instance, the blending technique yields irregularly shaped and highly polydispersed HMPs. Batch emulsification or complex coacervation enables the production of large volumes of polydispersed spherical HMPs. Flow-focusing microfluidic devices have been used to fabricate highly monodispersed droplets with a coefficient of variation of &#60;5%, however the throughput is significantly low. In step-emulsification microfluidic devices, the highly parallelized steps enable the high-throughput fabrication of monodispersed HMPs19.
Methacryloyl-modified gelatin (GelMA) HMP building blocks are thermoresponsive and (photo)chemically crosslinkable, enabling facile GHS fabrication20. Upon cooling below the upper critical solution temperature (UCST)21 (e.g., at 4 &#176;C), droplets containing a GelMA solution are converted to physically crosslinked HMPs. These HMP building blocks are then packed using external forces (e.g., via centrifugation) to yield jammed microgel suspensions. Interparticle linkages are established between adjacent HMPs via (photo)chemical crosslinking to form mechanically robust GHS14. One of the most important properties of GHS&#160;is microporosity, enabling facile cell penetration in vitro11 and enhanced tissue ingrowth in vivo22. Three-dimensional (3D) bioprinting of HMPs is conventionally performed using tightly packed microgel suspensions, compromising microporosity23.
We have recently developed a novel class of granular bioinks based on the interfacial nanoengineering of GelMA microgels via the adsorption of heterogeneously charged nanoparticles, followed by nanoparticle reversible self-assembly. This strategy renders loosely packed microgels shear-yielding and extrusion 3D bioprintable, which preserves the microscale porosity of additively manufactured GHS11. This article presents the methods for high-throughput GelMA droplet fabrication, converting these droplets to physically crosslinked HMPs, fabricating GelMA HMPs using resuspendable powder, GelMA GHS formation, GelMA nanoengineered granular bioink (NGB) preparation, and 3D bioprinting.

<table><tbody><tr><td>1H,1H-perfluoro-1-octanol</td><td>Alfa Aesar, MA, USA</td><td>B20156-18</td><td>98% purity</td></tr><tr><td>Biopsy punch</td><td>Integra Miltex, NY, USA</td><td>33-31A-P/25</td><td>1.5 mm Biopsy Punch with Plunger System</td></tr><tr><td>Blunt needle</td><td>SANANTS</td><td>30-002-25</td><td>25 G</td></tr><tr><td>Bruker Avance NEO 400 MHz</td><td>400 MHz Bruker NEO, MA, USA</td><td></td><td>NMR device</td></tr><tr><td>Centrifuge</td><td>Eppendorf, Germany</td><td>5415 C</td><td></td></tr><tr><td>Centrifuge tube</td><td>Celltreat, MA ,USA</td><td>229423</td><td></td></tr><tr><td>Coffee filters</td><td>BUNN, IL, USA</td><td>20104.0006</td><td>BUNN 8-12 Cup Coffee Filters, 6 each, 100 ct</td></tr><tr><td>Desiccator</td><td>Thermo Scientific</td><td>5311-0250</td><td>Nalgene Vacuum Desiccator, PC Cover and Body, 280 mm OD</td></tr><tr><td>Deuterium oxide</td><td>Sigma, MA, USA</td><td>151882</td><td></td></tr><tr><td>Dialysis membrane (12-14 kDa)</td><td>Spectrum Laboratories, NJ, USA</td><td>08-667E</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate buffered saline (DPBS, 1x)</td><td>Sigma, MA, USA</td><td>56064C-10L</td><td>dry powder, without calcium, without magnesium, suitable for cell culture</td></tr><tr><td>Erlenmeyer flask</td><td>Corning, NY, USA</td><td>4980</td><td>Corning PYREX&amp;nbsp;</td></tr><tr><td>Ethanol</td><td>VWR, PA, USA</td><td>89125-188</td><td>Koptec 200 proof</td></tr><tr><td>External thread cryogenic vials (cryovials)</td><td>Corning, NY, USA</td><td>430659</td><td></td></tr><tr><td>Freeze dryer</td><td>Labconco, MO, USA</td><td>71042000</td><td>Equipped with vacuum pump (Catalog# 7587000)</td></tr><tr><td>Gelatin powder</td><td>Sigma, MA, USA</td><td>G1890-5100G</td><td>Type A from porcine skin, gel strength ~300 g Bloom</td></tr><tr><td>Glass microscope slides</td><td>VWR, PA, USA</td><td>82027-788</td><td></td></tr><tr><td>Hotplate</td><td>FOUR E&#039;S SCIENTIFIC</td><td>MI0102003</td><td>5 inch Magnetic Hotplate Stirrer Max Temp 280 &amp;deg;C/536 &amp;deg;F&amp;nbsp;</td></tr><tr><td>Kimwipes</td><td>Fischer scientific, MA, USA</td><td>06-666</td><td></td></tr><tr><td>KMPR 1000 negative photoresist series</td><td>Kayaku Advanced Materials, MA, USA</td><td>121619</td><td>KMPR1025 and KMP1035 are included</td></tr><tr><td>LAPONITE XLG</td><td>BYK USA Inc., CT, USA</td><td>2344265</td><td></td></tr><tr><td>Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP)</td><td>Sigma, MA, USA</td><td>900889-1G</td><td>&gt;95%</td></tr><tr><td>Luer-Lok connector</td><td>BD, NJ, USA</td><td>BD 302995&amp;nbsp;</td><td></td></tr><tr><td>MA/BA Gen4-Serie Mask- und Bond-Aligner</td><td>S&amp;Uuml;SS MicroTeck, German</td><td></td><td>Nanofabrication device</td></tr><tr><td>Methacylate anhydride</td><td>Sigma, MA, USA</td><td>276685-100ML</td><td>contains 2,000&amp;nbsp;ppm topanol A as inhibitor, 94%</td></tr><tr><td>Milli-Q water</td><td>Millipore Corporation, MA, USA</td><td>ZRQSVR5WW</td><td>electrical resistivity &amp;asymp; 18 M&amp;Omega; at 25 &amp;deg;C, Direct-Q 5 UV Remote Water Purification System</td></tr><tr><td>Novec 7500 engineering fluid</td><td>3M, MN, USA</td><td></td><td>3M ID&amp;nbsp;7100003723</td></tr><tr><td>Oven</td><td>VWR, PA, USA</td><td>VWR-1410</td><td>1410 Vacuum Oven</td></tr><tr><td>Parafilm</td><td>Fischer scientific, MA, USA</td><td>HS234526C</td><td></td></tr><tr><td>Pasteur pipette</td><td>VWR, PA, USA</td><td>14673-010</td><td></td></tr><tr><td>Petri dish</td><td>VWR, PA, USA</td><td>25384-092</td><td>polystyrene</td></tr><tr><td>Pico-Surf</td><td>Sphere Fluidics, UK</td><td>C022</td><td>(5% (w/w) in Novec 7500)</td></tr><tr><td>Pipette</td><td>VWR, PA, USA</td><td>89079-970</td><td></td></tr><tr><td>Pipette tips</td><td>VWR, PA, USA</td><td>87006-060</td><td></td></tr><tr><td>Plasma cleaner chamber</td><td>Harrick Plasma, NY, USA</td><td>PDC-001-HP&amp;nbsp;</td><td></td></tr><tr><td>Polydimethylsiloxane</td><td>Dow Corning, MI, USA</td><td>&amp;nbsp;2065623</td><td>SYLGARD 184 Silicone Elastomer Kit</td></tr><tr><td>Positive displacement pipette</td><td>Microman E M100E, Gilson, OH, USA</td><td>M100E</td><td></td></tr><tr><td>Silicon wafers</td><td>UniversityWafer, MA, USA</td><td>452/1196</td><td>4-inch mechanical grade</td></tr><tr><td>Spatula</td><td>VWR, PA, USA</td><td>231-0104</td><td>Disposable</td></tr><tr><td>SU-8&amp;nbsp;</td><td>Kayaku Advanced Materials, MA, USA</td><td></td><td></td></tr><tr><td>Syringe pump</td><td>Harvard Apparatus, MA, USA</td><td>70-2001</td><td>PHD 2000</td></tr><tr><td>Trichloro(1H,1H,2H,2H-perfluorooctyl)silane</td><td>Millipore Sigma, MA, USA</td><td>448931-10G</td><td>97%</td></tr><tr><td>Tygon tubings</td><td>Saint-globain, PA, USA</td><td>AAD04103&amp;nbsp;</td><td></td></tr><tr><td>UV light&amp;nbsp;</td><td>QUANS</td><td></td><td>Voltage: 85 V-265 V AC / Power: 20 W</td></tr><tr><td>Vacuum filtration unit</td><td>VWR, PA, USA</td><td>10040-460</td><td>0.20 &amp;micro;m</td></tr><tr><td>Vortex</td><td>Fischer scientific, USA</td><td>14-955-151</td><td>Mini Vortex Mixer</td></tr></tbody></table>

gelatin methacryloyl granular hydrogel scaffolds: high-throughput microgel fabrication, lyophilization, chemical assembly, and 3d bioprinting

The emergence of granular hydrogel scaffolds (GHS), fabricated via assembling hydrogel microparticles (HMPs), has enabled microporous scaffold formation in situ. Unlike conventional bulk hydrogels, interconnected microscale pores in GHS facilitate degradation-independent cell infiltration as well as oxygen, nutrient, and cellular byproduct transfer. Methacryloyl-modified gelatin (GelMA), a (photo)chemically crosslinkable, protein-based biopolymer containing cell adhesive and biodegradable moieties, has widely been used as a cell-responsive/instructive biomaterial. Converting bulk GelMA to GHS&#160;may open a plethora of opportunities for tissue engineering and regeneration. In this article, we demonstrate the procedures of high-throughput GelMA microgel fabrication, conversion to resuspendable dry microgels (micro-aerogels), GHS formation via the chemical assembly of microgels, and granular bioink fabrication for extrusion bioprinting. We show how a sequential physicochemical treatment via cooling and photocrosslinking enables the formation of mechanically robust GHS.&#160;When light is inaccessible (e.g., during deep tissue injection), individually crosslinked GelMA HMPs may be bioorthogonally assembled via enzymatic crosslinking using transglutaminases. Finally, three-dimensional (3D) bioprinting of microporous GHS&#160;at low HMP packing density is demonstrated via the interfacial self-assembly of heterogeneously charged nanoparticles.

GelMA was synthesized through the reaction of gelatin with MA, as presented in Figure 1A. By tailoring the reaction conditions, such as MA concentration, different degrees of MA substitution were obtained. To quantify the degree of MA substitution, GelMA was assessed via 1H NMR spectroscopy (Figure 1B). Vinyl functional groups with representative peaks at the chemical shifts of ~5-6 ppm confirmed the successful GelMA synthesis from gelatin. The reaction yield after dialysis and sterile filtration was &gt;70% (mg of GelMA/mg of gelatin). The droplet/microgel fabrication yield was ~100%. Different methods can be used to quantify the degree of substitution24. We assessed the decrease of lysine amino acid (primary amine), normalized using the unaffected aromatic acid proton based on equation (1).
HMPs are commonly suspended in aqueous media, such as DPBS or cell culture media. The hydrated state of HMPs may introduce several challenges in sterilization, shipping, storage, and long-term stability. MEtoP is a novel method to convert HMPs to dried powder without affecting their original molecular and colloidal properties25. The MEtoP technology yields resuspendable, dried HMPs (micro-aerogels) via low-pressure freeze-drying, while protecting the HMPs from aggregation and severe deformation using a volatile oil, rather than an aqueous medium (Figure 2A). Using this technique, the microgels are individually dried without aggregation (Figure 2B), thus retaining their spherical shape after lyophilization (Figure 2C). These microparticles readily recover their initial properties upon rehydration, yielding HMP suspensions that are ready to form GHS upon assembly.
Step-emulsification microfluidic devices yield high-throughput monodispersed GelMA droplets, independent of the aqueous/oil phase flow rates. Since GelMA is a thermosensitive biopolymer, droplets are converted to thermally crosslinked HMPs by reducing the temperature to ~4 °C. The stable HMPs may be rinsed to remove the oil and surfactant using PFO (20% v/v). After oil/surfactant removal, the GelMA HMPs can be mixed with a PI for chemical assembly or with nanoparticles for interfacial self-assembly (Figure 3A). GHS formation is initiated via light (wavelength = 400 nm, intensity = 15 mW/cm2, exposure time = 60 s)-mediated free-radical polymerization, resulting in microgel-microgel bonding (Figure 3B).
Microporosity is one of the major characteristics of GHS, enabling facile oxygen and cellular waste exchange, cell infiltration, migration, and proliferation. To assess the microporosity, a high molecular weight fluorescence dye is used to visualize the void spaces among the HMPs. Figure 4A shows the top and 3D views of GHS scaffolds, with the green area showing the interconnected microporosity. Figure 4B presents a fluorescence image, assessed using a custom-written MATLAB script to detect the pore area. Void fraction (Figure 4C) and median pore equivalent diameter (Figure 4D) measurements show no significant difference between GHS and 3D printed NGB scaffolds, which attest to the availability and interconnectivity of microscale pores of NGB.
<img alt="3D bioprinting pore analysis; fluorescence microscopy diagrams; charts of void fraction and pore size." class="xfigimg" src="/files/ftp_upload/64829/64829fig04.jpg" data-altupdatedat="1747929244000" data-alt="3D bioprinted hydrogel structure, fluorescence images, pore analysis, void fraction comparison chart."> 
Figure 4: Pore characterization of GelMA GHS and NGB. (A) Top and 3D orthographic views of GHS and NGB scaffolds. Increments are 100 µm. (B) The fluorescence image of void area and pore detection via a custom-written MATLAB code for photocrosslinked GelMA GHS and NGB. Scale bar = 200 µm. (C) The void fraction of GelMA GHS and NGB. (D) The median equivalent pore diameter of GelMA GHS and NGB. This figure was modified with permission from Ataie et al.11 Abbreviations: GelMA = gelatin methacryloyl; GHS = granular hydrogel scaffold; NGB = nanoengineered granular bioink; NS = non-significant. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64829/64829fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
NGB is designed as a printable HMP suspension with preserved microporosity. To demonstrate the extrudability and printability of NGB, we performed extrusion-based 3D printing, as presented in Figure 5. PSU letters were printed using the NGB, followed by light exposure (Figure 5A). To assess the superiority of NGB to tightly packed and loosely packed GelMA HMPs, the hanging filament length (Lf) was measured (Figure 5B). The NGB had the highest Lf compared with the packed HMPs. The loosely packed HMPs did not yield filaments. Additionally, a hollow cylinder was 3D printed, and the whole construct was exposed to light for photocrosslinking (Figure 5D) and physically held (Figure 5E) to demonstrate the 3D printability and shape fidelity of photocrosslinked GelMA NGB, respectively.
<img alt="3D printing diagram with light exposure, material properties, and structural comparison chart." class="xfigimg" src="/files/ftp_upload/64829/64829fig05.jpg" data-altupdatedat="1747929244000" data-alt="Photopolymerization technique, syringe-based extrusion, and structural stability; diagram and chart."> 
Figure 5: Printability of the NGB followed by UV-light mediated photocrosslinking (wavelength = 395-405 nm, intensity = 15 mW/cm2, exposure time = 60 s). (A) Schematic of the printing process, showing PSU letters being 3D printed using the fluorescently labeled NGB. Scale bar = 3 mm. (B) Visual comparison of filament extrusion using the NGB, tightly packed GelMA HMPs, and loosely packed GelMA HMPs. Scale bar = 10 mm. (C) Hanging filament length of NGB, tightly packed, and loosely packed granular hydrogels. The NGB forms longer hanging filaments (filament length Lf = 45.0 ± 5.0 mm, n = 10) than tightly packed microgels (Lf = 19.3 ± 0.7 mm, n = 10). The loosely packed microgels yield droplet-size (Lf = 5.7 ± 0.7 mm, n = 10) filaments. (D) The NGB was used for the 3D printing of hollow cylinders with a diameter of 5 mm and a height of 10 mm. (E) The whole construct (hollow cylinder with d = 5 and h = 10 mm) was printed, then exposed to UV light. Layer-by-layer photocrosslinking might increase shape fidelity but decreases structural integrity as the layers are not crosslinked together. The hollow printed cylinder was held using tweezers, showing mechanical robustness. Scale bar = 1 cm. This figure was modified from Ataie et al.11 Abbreviations: NGB = nanoengineered granular bioink; GelMA = gelatin methacryloyl; HMP = hydrogel microparticle. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64829/64829fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Gelatin Methacryloyl Granular Hydrogel Scaffolds: High-throughput Microgel Fabrication, Lyophilization, Chemical Assembly, and 3D Bioprinting at JoVE.com

Gelatin Methacryloyl Granular Hydrogel Scaffolds: High-throughput Microgel Fabrication, Lyophilization, Chemical Assembly, and 3D Bioprinting

This article describes protocols for high-throughput gelatin methacryloyl microgel fabrication using microfluidic devices, converting microgels to resuspendable powder (micro-aerogels), the chemical assembly of microgels to form granular hydrogel scaffolds, and developing granular hydrogel bioinks with preserved microporosity for 3D bioprinting.

The emergence of granular hydrogel scaffolds (GHS), fabricated via assembling hydrogel microparticles (HMPs), has enabled microporous scaffold formation ...

gelatin-methacryloyl-granular-hydrogel-scaffolds-high-throughput

Nanyang Technological University

Research

JoVE Journal

Bioengineering

6.6K Views.  The Pennsylvania State University. Extracellulaire matrix of ECM numerieke hydrogel microdeeltjes kunnen worden gebruikt als de bouwstenen voor steigerfabricage. Dit protocol demonstreert de fabricage, zuivering, lyofilisatie, fotoassemblage en 3D-bioprinting van gelatinemethacryloyl of GelMA-hydrogelmicrodeeltjes. GelMA is een fotochemisch, crosslinkable, op eiwitten gebaseerd biopolymeer dat cellijm en biologisch afbreekbare stoffen bevat en dat op grote schaal is gebruikt als een celresponsief en leerzaam biomateriaal.

Video: Gelatin Methacryloyl Granular Hydrogel Scaffolds: High-throughput Microgel Fabrication, Lyophilization, Chemical Assembly, and 3D Bioprinting

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

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

Advancing 3D Bioprinting with &#954;-Carrageenan Sub-Microgel Medium

Embedded Bioprinting of Tissue-like Structures Using &#954;-Carrageenan Sub-Microgel Medium

Embedded three-dimensional (3D) bioprinting utilizing a granular hydrogel supporting bath has emerged as a critical technique for creating biomimetic scaffolds. However, engineering a suitable gel suspension medium that balances precise bioink deposition with cell viability and function presents multiple challenges, particularly in achieving the desired viscoelastic properties. Here, a novel &#954;-carrageenan gel supporting bath is fabricated through an easy-to-operate mechanical grinding process, producing homogeneous sub-microscale particles. These sub-microgels exhibit typical Bingham flow behavior with small yield stress and rapid shear-thinning properties, which facilitate the smooth deposition of bioinks. Moreover, the reversible gel-sol transition and self-healing capabilities of the &#954;-carrageenan microgel network ensure the structural integrity of printed constructs, enabling the creation of complex, multi-layered tissue structures with defined architectural features. Post-printing, the &#954;-carrageenan sub-microgels can be easily removed by a simple phosphate-buffered saline wash. Further bioprinting with cell-laden bioinks demonstrates that cells within the biomimetic constructs have a high viability of 92% and quickly extend pseudopodia, as well as maintain robust proliferation, indicating the potential of this bioprinting strategy for tissue and organ fabrication. In summary, this novel &#954;-carrageenan sub-microgel medium emerges as a promising avenue for embedded bioprinting of exceptional quality, bearing profound implications for the in vitro development of engineered tissues and organs.

Author Spotlight: Development of Homogeneous &#954;-Carrageenan Sub-Microgel Baths for High-Resolution 3D Bioprinting

This study introduces a novel &#954;-carrageenan sub-microgel suspension bath, displaying remarkable reversible jamming-unjamming transition properties. These attributes contribute to the construction of biomimetic tissues and organs in embedded 3D bioprinting. The successful printing of heart/esophageal-like tissues with high resolution and cell growth demonstrates high-quality bioprinting and tissue engineering applications.

Embedded three-dimensional (3D) bioprinting utilizing a granular hydrogel supporting bath has emerged as a critical technique for creating biomimetic ...

Engineering

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 ...

3D Printed Porous Cellulose Nanocomposite Hydrogel Scaffolds

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with controlled pore structure and mechanical properties. Cellulose nanocrystals (CNCs, 69.62 wt%) based hydrogel ink with matrix (sodium alginate and gelatin) was developed and 3D printed into scaffolds with uniform and gradient pore structure (110-1,100 &#181;m). The scaffolds showed compression modulus in the range of 0.20-0.45 MPa when tested in simulated in vivo conditions (in distilled water at 37 &#176;C). The pore sizes and the compression modulus of the 3D scaffolds matched with the requirements needed for cartilage regeneration applications. This work demonstrates that the consistency of the ink can be controlled by the concentration of the precursors and porosity can be controlled by the 3D printing process and both of these factors in return defines the mechanical properties of the 3D printed porous hydrogel scaffold. This process method can therefore be used to fabricate structurally and compositionally customized scaffolds according to the specific needs of patients.&#160;

The three critical steps of this protocol are i) developing the right composition and consistency of the cellulose hydrogel ink, ii) 3D printing of scaffolds into various pore structures with good shape fidelity and dimensions and iii) demonstration of the mechanical properties in simulated body conditions for cartilage regeneration.

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with ...

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 ...

Fragmenting Bulk Hydrogels and Processing into Granular Hydrogels for Biomedical Applications

Granular hydrogels are jammed assemblies of hydrogel microparticles (i.e., &#34;microgels&#34;). In the field of biomaterials, granular hydrogels have many advantageous properties, including injectability, microscale porosity, and tunability by mixing multiple microgel populations. Methods to fabricate microgels often rely on water-in-oil emulsions (e.g., microfluidics, batch emulsions, electrospraying) or photolithography, which may present high demands in terms of resources and costs, and may not be compatible with many hydrogels. This work details simple yet highly effective methods to fabricate microgels using extrusion fragmentation and to process them into granular hydrogels useful for biomedical applications (e.g., 3D printing inks). First, bulk hydrogels (using photocrosslinkable hyaluronic acid (HA) as an example) are extruded through a series of needles with sequentially smaller diameters to form fragmented microgels. This microgel fabrication technique is rapid, low-cost, and highly scalable. Methods to jam microgels into granular hydrogels by centrifugation and vacuum-driven filtration are described, with optional post-crosslinking for hydrogel stabilization. Lastly, granular hydrogels fabricated from fragmented microgels are demonstrated as extrusion printing inks. While the examples described herein use photocrosslinkable HA for 3D printing, the methods are easily adaptable for a wide variety of hydrogel types and biomedical applications.

This work describes straightforward, adaptable, and low-cost methods to fabricate microgels with extrusion fragmentation, process the microgels into injectable granular hydrogels, and apply the granular hydrogels as extrusion printing inks for biomedical applications.

Granular hydrogels are jammed assemblies of hydrogel microparticles (i.e., &#34;microgels&#34;). In the field of biomaterials, granular hydrogels have ...

Interlinked Macroporous 3D Scaffolds from Microgel Rods

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions without further additives. Continuous photoinitiated on-chip gelation enables variation of the microgel aspect ratio, which determines the building block properties for the obtained constructs. Glycidyl methacrylate (GMA) or 2-aminoethyl methacrylate (AMA) monomers are copolymerized into the microgel network based on polyethylene glycol (PEG) star-polymers to achieve either epoxy or amine functionality. A focusing oil flow is introduced into the microfluidic outlet structure to ensure continuous collection of the functionalized microgel rods. Based on a recent publication, microgel rod-based constructs result in larger pores of several hundred micrometers and, at the same time, lead to overall higher scaffold stability in comparison to a spherical-based model. In this way, it is possible to produce higher-volume constructs with more free volume while reducing the amount of material required. The interlinked macroporous scaffolds can be picked up and transported without damage or disintegration. Amine and epoxy groups not involved in interlinking remain active and can be used independently for post-modification. This protocol describes an optimized method for the fabrication of microgel rods to form macroporous interlinked scaffolds that can be utilized for subsequent cell experiments.

Microgel rods with complementary reactive groups are produced via microfluidics with the ability to interlink in aqueous solution. The anisometric microgels jam and interlink into stable constructs with larger pores compared to spherical-based systems. Microgels modified with GRGDS-PC form macroporous 3D constructs that can be used for cell culture.

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions ...

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 ...

Agarose Fluid Gels Formed by Shear Processing During Gelation for Suspended 3D Bioprinting

The use of granular matrices to support parts during the bioprinting process was first reported by Bhattacharjee et al. in 2015, and since then, several approaches have been developed for the preparation and use of supporting gel beds in 3D bioprinting. This paper describes a process to manufacture microgel suspensions using agarose (known as fluid gels), wherein particle formation is governed by the application of shear during gelation. Such processing produces carefully defined microstructures, with subsequent material properties that impart distinct advantages as embedding print media, both chemically and mechanically. These include behaving as viscoelastic solid-like materials at zero shear, limiting long-range diffusion, and demonstrating the characteristic shear-thinning behavior of flocculated systems. 
On the removal of shear stress, however, fluid gels have the capacity to rapidly recover their elastic properties. This lack of hysteresis is directly linked to the defined microstructures previously alluded to; because of the processing, reactive, non-gelled polymer chains at the particle interface facilitate interparticle interactions-similar to a Velcro effect. This rapid recovery of elastic properties enables bioprinting high-resolution parts from low-viscosity biomaterials, as rapid reformation of the support bed traps the bioink in situ, maintaining its shape. Furthermore, an advantage of agarose fluid gels is the asymmetric gelling/melting transitions (gelation temperature of ~30 &#176;C and melting temperature of ~90 &#176;C). This thermal hysteresis of agarose makes it possible to print and culture the bioprinted part in situ without the supporting fluid gel melting. This protocol shows how to manufacture agarose fluid gels and demonstrates their use to support the production of a range of complex hydrogel parts within suspended-layer additive manufacture (SLAM).

Shear processing during hydrogel formation results in the production of microgel suspensions that shear-thin but rapidly restructure following the removal of shear forces. Such materials have been used as a supporting matrix for bioprinting complex, cell-laden structures. Here, methods used to manufacture the supporting bed and compatible bioinks are described.

The use of granular matrices to support parts during the bioprinting process was first reported by Bhattacharjee et al. in 2015, and since then, several ...

Gelatin Methacryloyl Granular Hydrogel Scaffolds: High-throughput Microgel Fabrication, Lyophilization, Chemical Assembly, and 3D Bioprinting

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