Name: gelatin methacryloyl granular hydrogel scaffolds: high-throughput microgel fabrication, lyophilization, chemical assembly, and 3d bioprinting
Uploaded: 2022-12-09T15:00:00
Description: Watch this Scientific Journal Video about Gelatin Methacryloyl Granular Hydrogel Scaffolds: High-throughput Microgel Fabrication, Lyophilization, Chemical Assembly, and 3D Bioprinting at JoVE.com

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&#39;s phosphate buffered saline (DPBS, 1x) to an Erlenmeyer flask and heat the solution until it reaches 50 &#176;C. Co...

<ol>
	<li>Feng, Q., Li, D., Li, Q., Cao, X., Dong, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microgel+assembly:+Fabrication,+characteristics+and+application+in+tissue+engineering+and+regenerative+medicine.">Microgel assembly: Fabrication, characteristics and application in tissue engineering and regenerative medicine.</a> Bioactive Materials. 9, 105-119 (2022).</li><li>Daly, A. C., Riley, L., Segura, T., 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=Hydrogel+microparticles+for+biomedical+applications.">Hydrogel microparticles for biomedical applications.</a> Nature Reviews Materials. 5 (1), 20-43 (2020).</li><li>Griffin, D. R., 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=Activating+an+adaptive+immune+response+from+a+hydrogel+scaffold+imparts+regenerative+wound+healing.">Activating an adaptive immune response from a hydrogel scaffold imparts regenerative wound healing.</a> Nature Materials. 20 (4), 560-569 (2021).</li><li>Griffin, D. R., Weaver, W. M., Scumpia, P. O., Di Carlo, D., Segura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+wound+healing+by+injectable+microporous+gel+scaffolds+assembled+from+annealed+building+blocks.">Accelerated wound healing by injectable microporous gel scaffolds assembled from annealed building blocks.</a> Nature Materials. 14 (7), 737-744 (2015).</li><li>Ding, 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=Jammed+micro-flake+hydrogel+for+four-dimensional+living+cell+bioprinting.">Jammed micro-flake hydrogel for four-dimensional living cell bioprinting.</a> Advanced Materials. 34 (15), 2109394 (2022).</li><li>Muir, V. G., 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=Sticking+together:+injectable+granular+hydrogels+with+increased+functionality+via+dynamic+covalent+inter-particle+crosslinking.">Sticking together: injectable granular hydrogels with increased functionality via dynamic covalent inter-particle crosslinking.</a> Small. 18 (36), 2201115 (2022).</li><li>Sideris, E., 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=Particle+hydrogels+based+on+hyaluronic+acid+building+blocks.">Particle hydrogels based on hyaluronic acid building blocks.</a> ACS Biomaterials Science and Engineering. 2 (11), 2034-2041 (2016).</li><li>Molley, T. G., Hung, T., Kilian, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-laden+gradient+microgel+suspensions+for+spatial+control+of+differentiation+during+biofabrication.">Cell-laden gradient microgel suspensions for spatial control of differentiation during biofabrication.</a> Advanced Healthcare Materials. , 2201122 (2022).</li><li>Zoratto, N., 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=In+situ+forming+microporous+gelatin+methacryloyl+hydrogel+scaffolds+from+thermostable+microgels+for+tissue+engineering.">In situ forming microporous gelatin methacryloyl hydrogel scaffolds from thermostable microgels for tissue engineering.</a> Bioengineering and Translational. 5 (3), (2020).</li><li>Yuan, Z., 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=In+situ+fused+granular+hydrogels+with+ultrastretchability,+strong+adhesion,+and+mutli-bioactivities+for+efficient+chronic+wound+care.">In situ fused granular hydrogels with ultrastretchability, strong adhesion, and mutli-bioactivities for efficient chronic wound care.</a> Chemical Engineering Journal. 450, 138076 (2022).</li><li>Ataie, Z., 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=Nanoengineered+granular+hydrogel+bioinks+with+preserved+interconnected+microporosity+for+extrusion+bioprinting.">Nanoengineered granular hydrogel bioinks with preserved interconnected microporosity for extrusion bioprinting.</a> Small. 18 (37), 2202390 (2022).</li><li>Annabi, N., 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=25th+anniversary+article:+rational+design+and+applications+of+hydrogels+in+regenerative+medicine.">25th anniversary article: rational design and applications of hydrogels in regenerative medicine.</a> Advanced Materials. 26 (1), 85-124 (2014).</li><li>Rajabi, N., 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=Recent+advances+on+bioprinted+gelatin+methacrylate-based+hydrogels+for+tissue+repair.">Recent advances on bioprinted gelatin methacrylate-based hydrogels for tissue repair.</a> Tissue Engineering. 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J., 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=Three-dimensional+printing+of+complex+biological+structures+by+freeform+reversible+embedding+of+suspended+hydrogels.">Three-dimensional printing of complex biological structures by freeform reversible embedding of suspended hydrogels.</a> Science Advances. 1 (9), (2015).</li><li>Seymour, A. J., Shin, S., Heilshorn, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+printing+of+microgel+scaffolds+with+tunable+void+fraction+to+promote+cell+infiltration.">3D printing of microgel scaffolds with tunable void fraction to promote cell infiltration.</a> Advanced Healthcare Materials. 10 (18), 2100644 (2021).</li><li>Lee, 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=3D+bioprinting+of+collagen+to+rebuild+components+of+the+human+heart.">3D bioprinting of collagen to rebuild components of the human heart.</a> Science. 365 (6452), 482-487 (2019).</li><li>de Rutte, J. M., Koh, J., 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+high-throughput+production+of+modular+microgels+for+in+situ+assembly+of+microporous+tissue+scaffolds.">Scalable high-throughput production of modular microgels for in situ assembly of microporous tissue scaffolds.</a> Advanced Functional Materials. 29 (25), 1900071 (2019).</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=Modular+microporous+hydrogels+formed+from+microgel+beads+with+orthogonal+thermo-chemical+responsivity:+Microfluidic+fabrication+and+characterization.">Modular microporous hydrogels formed from microgel beads with orthogonal thermo-chemical responsivity: Microfluidic fabrication and characterization.</a> MethodsX. 6, 1747-1752 (2019).</li><li>Van Den Bulcke, A. 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>

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, po...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#39;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&#160;</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&#39;S SCIENTIFIC</td>
			<td>MI0102003</td>
			<td>5 inch Magnetic Hotplate Stirrer Max Temp 280 &#176;C/536 &#176;F&#160;</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>&#62;95%</td>
		</tr>
		<tr>
			<td>Luer-Lok connector</td>
			<td>BD, NJ, USA</td>
			<td>BD 302995&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>MA/BA Gen4-Serie Mask- und Bond-Aligner</td>
			<td>S&#220;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&#160;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 &#8776; 18 M&#937; at 25 &#176;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&#160;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&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane</td>
			<td>Dow Corning, MI, USA</td>
			<td>&#160;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&#160;</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&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>UV light&#160;</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 &#181;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&#160;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 gelat...

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 ...

Extracellular matrix or ECM numeric hydrogel microparticles may be used as the building blocks for scaffold fabrication. This protocol demonstrates the fabrication, purification, lyophilization, photo assembly, and 3D bioprinting of gelatin methacryloyl or GelMA hydrogel microparticles. GelMA is a photochemically, crosslinkable, protein-based biopolymer containing cell adhesive and biodegradable moieties which has been widely used as a cell-responsive and instructive biomaterial.In comparison to conventional bulk hydrogel, GelMA granular hydrogel scaffolds are microporous, enabling rapid cell infiltration that can be tailored by the building block size. GelMA granular hydrogel scaffolds may open new opportunities for the 3D bioprinting of thick tissues, organs, and diseases on cheap platforms and regenerative engineering. To begin, add 10 milligrams of LAP to 10 milliliters of DPBS to prepare a 0.1%photoinitiator solution.Protect the solution from light by wrapping it with aluminum foil, and label it. Next, dissolve the desired amount of GelMA in the photoinitiator solution. Place the aluminum-foil-wrapped solution in the 37-degrees-Celsius oven for one hour to get a clear solution.To prepare the oil phase, make a 2%biocompatible surfactant solution in the engineering fluid. Insert the Tygon tubing in the inlets and outlet of the PDMS device, then insert a 25-gauge blunt needle in the other end of Tygon tubing for inlets. Place the device under the microscope, and keep the environment warm at approximately 40 degrees Celsius using a hairdryer or a space heater.Load the aqueous and oil solutions in separate syringes connected to the device. Start the syringe pump for the oil phase with a flow rate of 160 microliters per minute. After oil fills the channel, start the aqueous phase at 80 microliters per minute.Check the droplet formation under the microscope. Collect the droplets in a container, and evaluate them in the imaging chamber by optical microscopy imaging. Keep the droplets at four degrees Celsius overnight while protecting them from light to initiate GelMA microgel's physical crosslinking and convert the droplets to stable microgels.To implement the microengineered emulsion-to-powder, MEtoP, method, collect the physically crosslinked microgels in the engineering fluid using thermally durable microcentrifuge tubes or cryovials. Seal the open tube with a laboratory wipe and tape. Deep-freeze the physically crosslinked microgels in liquid nitrogen for 10 minutes to proceed with lyophilization.After lyophilization, a solid powder will be obtained. Add one milliliter of cooled, 0.1%photoinitiator solution at four degrees Celsius to the lyophilized powder to make microgel suspensions. Centrifuge the vortexed mixture at 3, 000g for 15 seconds.After discarding the supernatant, transfer the packed microgel suspension to a mold using a positive displacement pipette, and expose it to light at 400 nanometers wavelength with an intensity of 15 milliwatts per square centimeter for 60 seconds to form granular hydrogel scaffolds or GHS. For 400 microliters of microgel suspension, add an equal amount of ice-cold PFO solution, 20%in engineering fluid to the physically crosslinked GelMA microgels. Then centrifuge the vortexed mixture at 300g for 15 seconds, and remove the oil phase from the GelMA microgels by pipetting.Then add 400 microliters of ice-cold, 0.1%photoinitiator solution to the microgel suspension. After centrifuging the vortexed solution at 300g for 15 seconds, discard the oil. Repeat the steps of photoinitiator solution addition and centrifugation, but centrifuge at 3, 000g this time and discard the supernatant of packed GelMA microgels.Transfer the packed GelMA microgels to a mold, followed by light exposure to form the GHS. Add 100 milligrams of nanoplatelet powder to three milliliters of ice-cold, ultra-pure water to form a 3.33%nanoparticle dispersion. Vortex the dispersion vigorously inside a four-degrees-Celsius refrigerator for 15 minutes to exfoliate the aggregated nanoparticles.Properly exfoliated nanoparticles yield a clear dispersion. Dissolve 50 milligrams of LAP in five milliliters of ice-cold, ultra-pure water to prepare a 1%photoinitiator stock solution. Then add 333 microliters of 1%photoinitiator solution to the exfoliated nanoparticle dispersion, and wrap it in aluminum foil to protect against the ambient light.Vortex the mixture for one minute to mix the nanoparticle dispersion and the photoinitiator. Add ice-cold PFO, 20%in engineering fluid to the physically crosslinked GelMA microgels at a one-to-one volume ratio. After centrifuging the vortexed mixture at 300g for 15 seconds, discard the oil phase containing the surfactant.Add the ice-cold, LAP-supplemented nanoparticle dispersion to the washed GelMA microgels. After centrifuging at 3, 000g for 15 seconds, discard the remaining oil at the bottom and the supernatant dispersion. Store the suspension at four degrees Celsius while protecting it from light using aluminum foil for one day to yield the product GelMA nanoengineered granular bioink or NGB.The following day, load the NGB into a three-milliliter syringe. Seal the loaded syringe with a cap and parafilm, and do a pulse centrifugation at 200g to remove the trapped air. Transfer the bioink to a three-milliliter cartridge using a female-female Luer lock connector.Centrifuge the cartridge briefly at 200g again to remove the trapped air, and keep the NGB at four degrees Celsius before use. Next, load the prepared murine fibroblast cell suspension into a three-milliliter syringe. Couple the NGB and the cell-loaded syringes using a female-female Luer lock connector, and mix the contents gently by pushing back and forth 40 times.Print the NGB or cell-laden NGB using a proper bioprinter with a standard conical nozzle by loading the nozzle into the three-milliliter printhead. Keep the printing bed temperature below 10 degrees Celsius, and optimize parameters such as speed and back pressure. Select the desirable gcode or STL file.Then select the substrate and the nozzle type for a pneumatic three-milliliter syringe equipped with a standard conical nozzle. Calibrate the bioprinter using the device guidelines, and start printing to obtain the 3D structures. The MEtoP technology yielded resuspendable dried microgel powder by low-pressure freeze-drying while protecting microgels from aggregation and severe deformation.The SEM images showed that the dried GelMA microparticles using this technology retain their spherical shape after lyophilization compared with the aggregation observed with conventional lyophilization. The micropore characterization of GelMA, GHS, and NGB with a high molecular weight fluorescence dye showed the interconnected microscale void spaces. Also, the fluorescence image assessed using a custom-written MATLAB script detected the pores.The void fraction and median pore equivalent diameter measurements showed no significant difference attesting to the availability and interconnectivity of microscale pores in the 3D-printed scaffolds using the NGB. The superiority of NGB scaffolds to tightly and loosely packed GelMA microgels by the hanging filament length measurement revealed that NGB had a greater length than the packed microgels. The loosely packed microgels did not yield filaments.A hollow cylinder was 3D-printed, and the whole construct was exposed to light for photocrosslinking. The printed structure was physically held to show the shape, fidelity, and structural integrity. If the scaffold is used for biological assessment, for example, in cell-laden bioprinting, remember to perform all the steps under certain conditions, under the biological safety cabinet.We hope to democratize the use of granular hydrogel scaffolds as a newly emerged biomaterial platform in biomedicine.

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

Research

JoVE Journal

Bioengineering

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as organ replacements for implantation in patients, and also, when created on a smaller size scale as model "organoids" that can be used in in vitro systems for drug and toxicology screening.
Despite development of a wide variety of bioprinting devices, application of bioprinting technology can be limited by the availability of materials that both expedite bioprinting procedures and support cell viability and function by providing tissue-specific cues. Here we describe a versatile hyaluronic acid (HA) and gelatin-based hydrogel system comprised of a multi-crosslinker, 2-stage crosslinking protocol, which can provide tissue specific biochemical signals and mimic the mechanical properties of in vivo tissues. 
Biochemical factors are provided by incorporating tissue-derived extracellular matrix materials, which include potent growth factors. Tissue mechanical properties are controlled combinations of PEG-based crosslinkers with varying molecular weights, geometries (linear or multi-arm), and functional groups to yield extrudable bioinks and final construct shear stiffness values over a wide range (100 Pa to 20 kPa). Using these parameters, hydrogel bioinks were used to bioprint primary liver spheroids in a liver-specific bioink to create in vitro liver constructs with high cell viability and measurable functional albumin and urea output. This methodology provides a general framework that can be adapted for future customization of hydrogels for biofabrication of a wide range of tissue construct types.

We describe a set of protocols that together provide a tissue-mimicking hydrogel bioink with which functional and viable 3-D tissue constructs can be bioprinted for use in in vitro screening applications.

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell lines using conventional two-dimensional (2D) cell culture. These challenges can be overcome by using bioprinting techniques to build heterogeneous three-dimensional (3D) tumor models whereby different types of cells are embedded. Alginate and gelatin are two of the most common biomaterials employed in bioprinting due to their biocompatibility, biomimicry, and mechanical properties. By combining the two polymers, we achieved a bioprintable composite hydrogel with similarities to the microscopic architecture of a native tumor stroma. We studied the printability of the composite hydrogel via rheology and obtained the optimal printing window. Breast cancer cells and fibroblasts were embedded in the hydrogels and printed to form a 3D model mimicking the in vivo microenvironment. The bioprinted heterogeneous model achieves a high viability for long-term cell culture (&#62; 30 days) and promotes the self-assembly of breast cancer cells into multicellular tumor spheroids (MCTS). We observed the migration and interaction of the cancer-associated fibroblast cells (CAFs) with the MCTS in this model. By using bioprinted cell culture platforms as co-culture systems, it offers a unique tool to study the dependence of tumorigenesis on the stroma composition. This technique features a high-throughput, low cost, and high reproducibility, and it can also provide an alternative model to conventional cell monolayer cultures and animal tumor models to study cancer biology.

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

We developed a heterogeneous breast cancer model consisting of immortalized tumor and fibroblast cells embedded in a bioprintable alginate/gelatin bioink. The model recapitulates the in vivo tumor microenvironment and facilitates the formation of multicellular tumor spheroids, yielding insight into the mechanisms driving tumorigenesis.

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell ...

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 ...

Preparation and Characterization of Graphene-Based 3D Biohybrid Hydrogel Bioink for Peripheral Neuroengineering

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global problem that occurs in 1.5%-5% of emergency patients and may lead to significant job losses. Today, tissue engineering-based approaches, consisting of scaffolds, appropriate cell lines, and biosignals, have become more applicable with the development of three-dimensional (3D) bioprinting technologies. The combination of various hydrogel biomaterials with stem cells, exosomes, or bio-signaling molecules is frequently studied to overcome the existing problems in peripheral nerve regeneration. Accordingly, the production of injectable systems, such as hydrogels, or implantable conduit structures formed by various bioprinting methods has gained importance in peripheral neuro-engineering. Under normal conditions, stem cells are the regenerative cells of the body, and their number and functions do not decrease with time to protect their populations; these are not specialized cells but can differentiate upon appropriate stimulation in response to injury. The stem cell system is under the influence of its microenvironment, called the stem cell niche. In peripheral nerve injuries, especially in neurotmesis, this microenvironment cannot be fully rescued even after surgically binding severed nerve endings together. The composite biomaterials and combined cellular therapies approach increases the functionality and applicability of materials in terms of various properties such as biodegradability, biocompatibility, and processability. Accordingly, this study aims to demonstrate the preparation and use of graphene-based biohybrid hydrogel patterning and to examine the differentiation efficiency of stem cells into nerve cells, which can be an effective solution in nerve regeneration.

In this manuscript, we demonstrate the preparation of a biohybrid hydrogel bioink containing graphene for use in peripheral tissue engineering. Using this 3D biohybrid material, the neural differentiation protocol of stem cells is performed. This can be an important step in bringing similar biomaterials to the clinic.

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global ...

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 ...

Synthesis of Strong Adhesive Hydrogel, Gelatin O-Nitrosobenzaldehyde

Adhesive materials have become popular biomaterials in the field of biomedical and tissue engineering. In our previous work, we presented a new material - gelatin o-nitrosobenzaldehyde (gelatin-NB) - which is mainly used for tissue regeneration and has been validated in animal models of corneal injury and inflammatory bowel disease. This is a novel hydrogel formed by modifying biological gelatin with o-nitrosobenzaldehyde (NB). Gelatin-NB was synthesized by activating the carboxyl group of NB-COOH and reacting with gelatin through 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The obtained compound was purified to generate the final product, which can be stably stored for at least 18 months. NB has a strong adhesion to -NH2 on the tissue, which can form many C = N bonds, thus increasing the adhesion of gelatin-NB to the tissue interface. The preparation process comprises steps for the synthesis of the NB-COOH group, modification of the group, synthesis of gelatin-NB, and purification of the compound. The goal is to describe the specific synthesis process of gelatin-NB in detail and to demonstrate the application of gelatin-NB to damage repair. Moreover, the protocol is presented to further strengthen and expand the nature of the material produced by the scientific community for more applicable scenarios.

The protocol presented here shows the synthesis of a strong adhesive hydrogel gelatin o-nitrosobenzaldehyde (gelatin-NB). Gelatin-NB has rapid and efficient tissue adhesion ability, which can form a strong physical barrier to protect wound surfaces, so it is expected to be applied to the field of injury repair biotechnology.

Adhesive materials have become popular biomaterials in the field of biomedical and tissue engineering. In our previous work, we presented a new material - ...

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 ...