This work was supported by the National Science Foundation through the UPenn MRSEC program (DMR-1720530) and graduate research fellowships (to V.G.M and M.E.P.) and the National Institutes of Health (R01AR077362 to J.A.B.).

The authors have no competing financial interests.

Herein, methods to fabricate granular hydrogels using extrusion fragmented microgels and packing by either centrifugation or vacuum-driven filtration are described. Compared to other microgel fabrication methods (i.e., microfluidics, batch emulsions, electrospraying, photolithography), extrusion fragmentation microgel fabrication is highly rapid, low-cost, easily scalable, and amenable to a wide variety of hydrogel systems. Further, this protocol is highly repeatable with minimal batch-to-batch variability, which was cha...

Granular hydrogels are fabricated through the packing of hydrogel particles (i.e., microgels) and are an exciting class of biomaterials with many advantageous properties for biomedical applications1,2,3. Due to their particulate structure, granular hydrogels are shear-thinning and self-healing, allowing for their use as extrusion printing (bio)inks, granular supports for embedded printing, and injectable therapeutics4,5,6,7,8,9. Additionally, the void space between microgels provides a microscale porosity for cell movement and molecular diffusion8,10,11. Further, multiple microgel populations can be combined into a single formulation to allow for enhanced tunability and material functionality8,10,12,13. These important properties have motivated the rapid expansion of granular hydrogel development in recent years.
There is a range of methods available to form microgels towards granular hydrogel fabrication, each with its own advantages and disadvantages. For example, microgels are often formed from water-in-oil emulsions using droplet microfluidics4,11,13,14,15,16,17, batch emulsions7,18,19,20,21,22, or electrospraying6,23,24,25. These methods yield spherical microgels with either uniform (microfluidics) or polydisperse (batch emulsions, electrospraying) diameters. There are some limitations to these water-in-oil emulsion fabrication methods, including potentially low-throughput production, the need for low-viscosity hydrogel precursor solutions, and the high cost and resources for setup. Additionally, these protocols may require harsh oils and surfactants that must be washed from the microgels using procedures that add processing steps, and may be difficult to translate to sterile conditions for biomedical applications in many labs. Removing the need for water-in-oil emulsions, (photo)lithography can also be used, where molds or photomasks are used to control the curing of microgels from hydrogel precursor solutions1,26,27. Like microfluidics, these methods may be limited in their production throughput, which is a major challenge when large volumes are needed.
As an alternative to these methods, mechanical fragmentation of bulk hydrogels has been used to fabricate microgels with irregular sizes19,28,29,30,31,32. For example, bulk hydrogels can be pre-formed and subsequently passed through meshes or sieves to form fragmented microgels, a process which has even been done in the presence of cells within microgel strands33,34. Bulk hydrogels have also been processed into microgels with mechanical disruption using techniques such as grinding with mortar and pestle or through the use of commercial blenders35,36,37. Others have also used mechanical agitation during hydrogel formation to fabricate fragmented microgels (i.e., fluid gels)31.
The methods herein expand on these mechanical fragmentation techniques and present a simple approach to fabricate microgels with extrusion fragmentation, using photocrosslinkable hyaluronic acid (HA) hydrogels as an example. Extrusion fragmentation uses only syringes and needles to fabricate fragmented microgels in a low-cost, high throughput, and easily scalable method that is appropriate for a wide range of hydrogels19,32. Further, methods to assemble these fragmented microgels into granular hydrogels are described using either centrifugation (low packing) or vacuum-driven filtration (high packing). Lastly, the application of these fragmented granular hydrogels is discussed for use as an extrusion printing ink. The goal of this protocol is to introduce simple methods that are adaptable to a wide variety of hydrogels and can be implemented in virtually any laboratory interested in granular hydrogels.

<table><tbody><tr><td>15 mL Plastic Conical Centrifuge Tube</td><td>Corning</td><td>430766</td><td></td></tr><tr><td>30 G NT Premium Series Dispensing Tip</td><td>Jensen Global</td><td>JG30-0.5HPX</td><td>Catalog Number listed here is for 30 G, 0.5&quot; needle. Various sizes are available.</td></tr><tr><td>BD Disposable Syringes with Luer-Lok Tips (3 mL)</td><td>Fisher Scientific</td><td>14-823-435</td><td>Catalog Number listed here is for 3 mL syringe. Various sizes are available (14-823-XXX).</td></tr><tr><td>Black folders</td><td>Various Vendors</td><td></td><td></td></tr><tr><td>Disposable Probe Needle For Use With Syringes and Dispensing Machines (18 G, 0.5&quot;)</td><td>Grainger</td><td>5FVH5</td><td>Catalog Number listed here is for 18 G, 0.5&quot; needle. Various sizes are available.</td></tr><tr><td>Disposable Probe Needle For Use With Syringes and Dispensing Machines (23 G, 0.5&quot;)</td><td>Grainger</td><td>5FVJ3</td><td></td></tr><tr><td>Disposable Probe Needle For Use With Syringes and Dispensing Machines (27 G, 1.5&quot;)</td><td>Grainger</td><td>5FVL0</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline</td><td>Fisher Scientific</td><td>14190-250</td><td>Catalog Number listed here is for a case of 10 x 500 mL bottles.</td></tr><tr><td>Durapore Membrane Filter, 0.22 &amp;micro;m</td><td>Millipore</td><td>GVWP04700</td><td></td></tr><tr><td>Epifluorescent or confocal microscope</td><td>Various Vendors</td><td></td><td>To visualize microgels and granular hydrogels</td></tr><tr><td>Eppendorf Snap-Cap Microcentrifuge Safe-Lock Tubes</td><td>Fisher Scientific</td><td>05-402-25</td><td></td></tr><tr><td>Extrusion printer</td><td>Custom-built</td><td></td><td>Other extrusion printers can be use,d such as commercially available BIOX.</td></tr><tr><td>Filter Adapters</td><td>Fisher Scientific</td><td>05-888-107</td><td>Catalog Number listed here is for a set of multiple sizes. Various sizes are available (05-888-XXX).</td></tr><tr><td>Filter Flask</td><td>Various Vendors</td><td></td><td></td></tr><tr><td>Fluorescein isothiocyanate-dextran (2 MDa)</td><td>Sigma-Aldrich</td><td>52471</td><td></td></tr><tr><td>Glass microscope slide</td><td>Various Vendors</td><td></td><td></td></tr><tr><td>ImageJ</td><td>National Institutes of Health</td><td></td><td>&quot;Analyze Particles&quot; information link: https://imagej.nih.gov/ij/docs/menus/analyze.html</td></tr><tr><td>Laptop</td><td>Various Vendors</td><td></td><td></td></tr><tr><td>Luer-Lock Tip Caps</td><td>Integrated Dispensin g Solutions</td><td>9991329</td><td></td></tr><tr><td>Metal spatula for scooping</td><td>Various Vendors</td><td></td><td></td></tr><tr><td>Microcentrifuge</td><td>Various Vendors</td><td></td><td>Capable of speed up to 18,000 x g</td></tr><tr><td>Microscoft Execl</td><td>Microsoft</td><td></td><td>Other programs can be used, such as Google Slides.</td></tr><tr><td>OmniCure S2000 Spot UV Curing System</td><td>Excelitas Technologies</td><td>S2000</td><td>Different light systems may be used to fabricate bulk hydrogels if desired.</td></tr><tr><td>Porcelain Buchner Funnel with Fixed Perforated Plate</td><td>Fisher Scientific</td><td>FB966C</td><td>Catalog Number listed here is for 56mm diameter plate. Various sizes are available.</td></tr><tr><td>Radiometer</td><td>Various Vendors</td><td></td><td></td></tr><tr><td>Repetier Host</td><td>Hot-World GmbH &amp;amp; Co. KG</td><td></td><td>3D printing software</td></tr><tr><td>Screw-based extrusion printer</td><td>Various Vendors</td><td></td><td>This study used a custom-modified 3D FDM printer (Velleman K8200). Many alternatives are available.</td></tr><tr><td>Solidworks/CAD software</td><td>Dassault Syst&amp;egrave;mes SolidWorks Corporation</td><td></td><td>Other programs can be used, such as Blender or TinkerCAD.</td></tr><tr><td>Tubing to Connect Filter Flask to Vacuum Line</td><td>Various Vendors</td><td></td><td></td></tr><tr><td>UV Eye Protection (i.e., safety glasses)</td><td>Various Vendors</td><td></td><td></td></tr></tbody></table>

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.

Representative results from these protocols are shown in Figure 3 and Figure 6. Extrusion fragmentation yields microgels with jagged, polygon shapes with diameters ranging from 10-300 &#181;m (Figure 3). Further, circularity ranges from 0.2 (not circular) to almost 1 (perfect circle), and the aspect ratio ranges from 1-3 (Figure 3). These parameters describe the irregular and jagged microgel shapes form...

Watch this Scientific Journal Video about Fragmenting Bulk Hydrogels and Processing into Granular Hydrogels for Biomedical Applications at JoVE.com

Fragmenting Bulk Hydrogels and Processing into Granular Hydrogels for 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 ...

fragmenting-bulk-hydrogels-processing-into-granular-hydrogels-for

Research

JoVE Journal

Bioengineering

5.4K Views.  University of Pennsylvania. Granulaire hydrogels zijn een spannende nieuwe klasse van biomaterialen met veel voordelige eigenschappen. Onze hoop is dat we door het delen van deze methoden de toegang en innovatie in granulaire hydrogels voor biomedische toepassingen kunnen vergroten. Deze techniek is eenvoudig, goedkoop, gemakkelijk aan te passen aan vele chemische stoffen en kan in vrijwel elk laboratorium worden geïmplementeerd.Laad een spuit van 3 milliliter met de hydrogel-precursoroplossing. Verwijder de zui...

Video: Fragmenting Bulk Hydrogels and Processing into Granular Hydrogels for Biomedical Applications

Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large variety of PEG molecular weights and configurations (arm number, arm length, and branching pattern) affords researchers tight control over resulting hydrogel structures and properties, including Young&#8217;s modulus and mesh size. This video will illustrate a rapid, efficient, solvent-free, microwave-assisted method to methacrylate PEG precursors into poly(ethylene glycol) dimethacrylate (PEGDM). This synthetic method provides much-needed starting materials for applications in drug delivery and regenerative medicine. The demonstrated method is superior to traditional methacrylation methods as it is significantly faster and simpler, as well as more economical and environmentally friendly, using smaller amounts of reagents and solvents. We will also demonstrate an adaptation of this technique for on-resin methacrylamide functionalization of peptides. This on-resin method allows the N-terminus of peptides to be functionalized with methacrylamide groups prior to deprotection and cleavage from resin. This allows for selective addition of methacrylamide groups to the N-termini of the peptides while amino acids with reactive side groups (e.g.&#160;primary amine of lysine, primary alcohol of serine, secondary alcohols of threonine, and phenol of tyrosine) remain protected, preventing functionalization at multiple sites. This article will detail common analytical methods (proton Nuclear Magnetic Resonance spectroscopy (;H-NMR) and Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-ToF)) to assess the efficiency of the functionalizations. Common pitfalls and suggested troubleshooting methods will be addressed, as will modifications of the technique which can be used to further tune macromer functionality and resulting hydrogel physical and chemical properties. Use of synthesized products for the formation of hydrogels for drug delivery and cell-material interaction studies will be demonstrated, with particular attention paid to modifying hydrogel composition to affect mesh size, controlling hydrogel stiffness and drug release.

Microwave-assisted Functionalization of Polyethylene glycol and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

This video will illustrate a rapid, efficient method to methacrylate poly(ethylene glycol), enabling chain polymerizations and hydrogel synthesis. It will demonstrate how to similarly introduce methacrylamide functionalities into peptides, detail common analytical methods to assess functionalization efficiency, provide suggestions for troubleshooting and advanced modifications, and demonstrate typical hydrogel characterization techniques.

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large ...

Chemistry

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field of 3D templates is rising due to the potential resemblance of those materials to the natural extracellular matrix. Protein-based hydrogels are particularly promising because they can easily be functionalized and can achieve defined structures with adjustable physicochemical properties. However, the production of macroporous 3D templates for cell culture applications using natural materials is often limited by their weaker mechanical properties compared to those of synthetic materials. Here, different methods were evaluated to produce macroporous bovine serum albumin (BSA)-based hydrogel systems, with adjustable pore sizes in the range of 10 to 70 &#181;m in radius. Furthermore, a method to generate channels in this protein-based material that are several hundred microns long was established. The different methods to produce pores, as well as the influence of pore size on material properties such as swelling ratio, pH, temperature stability, and enzymatic degradation behavior, were analyzed. Pore sizes were investigated in the native, swollen state of the hydrogels using confocal laser scanning microscopy. The feasibility for cell culture applications was evaluated using a cell-adhesive RGD peptide modification of the protein system and two model cell lines: human breast cancer cells (A549) and adenocarcinomic human alveolar basal epithelial cells (MCF7).

Different methods to manipulate three-dimensional architecture in protein-based hydrogels are evaluated here with respect to material properties. The macroporous networks are functionalized with a cell-adhesive peptide, and their feasibility in cell culture is evaluated using two different model cell lines.

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

Biochemistry

The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and functionalize polymer chains with compounds that are able to improve biocompatibility, mechanical properties, or cell viability allows the design of novel materials to meet new challenges in the biomedical field. With the polymer functionalization strategies, click chemistry is a powerful tool to improve cell-compatibility and drug delivery properties of polymeric devices. Similarly, the fundamental need of biomedicine to use sterile tools to avoid potential adverse-side effects, such as toxicity or contamination of the biological environment, gives rise to increasing interest in the microwave-assisted strategy.
The combination of click chemistry and the microwave-assisted method is suitable to produce biocompatible hydrogels with desired functionalities and improved performances in biomedical applications. This work aims to synthesize RGD-functionalized hydrogels. RGD (arginylglycylaspartic acid) is a tripeptide that can mimic cell adhesion proteins and bind to cell-surface receptors, creating a hospitable microenvironment for cells within the 3D polymeric network of the hydrogels. RGD functionalization occurs through Huisgen 1,3-dipolar cycloaddition. Some PAA carboxyl groups are modified with an alkyne moiety, whereas RGD is functionalized with azido acid as the terminal residue of the peptide sequence. Finally, both products are used in a copper catalyzed click reaction to permanently link the peptide to PAA. This modified polymer is used with carbomer, agarose and polyethylene glycol (PEG) to synthesize a hydrogel matrix. The 3D structure is formed due to an esterification reaction involving carboxyl groups from PAA and carbomer and hydroxyl groups from agarose and PEG through microwave-assisted polycondensation. The efficiency of the gelation mechanism ensures a high degree of RGD functionalization. In addition, the procedure to load therapeutic compounds or biological tools within this functionalized network is very simple and reproducible.

We present a protocol for the synthesis of RGD-functionalized hydrogels as devices for cell and drug delivery. The procedure involves copper catalyzed alkyne-azide cycloaddition (CuAAC) between alkyne-modified polyacrylic acid (PAA) and a RGD-azide derivative. The hydrogels are formed using microwave-assisted polycondensation and their physicochemical properties are investigated.

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and ...

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

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

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

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

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 ...

An Open Source Technology Platform to Manufacture Hydrogel-Based 3D Culture Models in an Automated and Standardized Fashion

Current mixing steps of viscous materials rely on repetitive and time-consuming tasks which are performed mainly manually in a low throughput mode. These issues represent drawbacks in workflows that can ultimately result in irreproducibility of research findings. Manual-based workflows are further limiting the advancement and widespread adoption of viscous materials, such as hydrogels used for biomedical applications. These challenges can be overcome by using automated workflows with standardized mixing processes to increase reproducibility. In this study, we present step-by-step instructions to use an open source protocol designer, to operate an open source workstation, and to identify reproducible mixtures. Specifically, the open source protocol designer guides the user through the experimental parameter selection and generates a ready-to-use protocol code to operate the workstation. This workstation is optimized for pipetting of viscous materials to enable automated and highly reliable handling by the integration of temperature docks for thermoresponsive materials, positive displacement pipettes for viscous materials, and an optional tip touch dock to remove excess material from the pipette tip. The validation and verification of mixtures are performed by a fast and inexpensive absorbance measurement of Orange G. This protocol presents results to obtain 80% (v/v) glycerol mixtures, a dilution series for gelatin methacryloyl (GelMA), and double network hydrogels of 5% (w/v) GelMA and 2% (w/v) alginate. A troubleshooting guide is included to support users with protocol adoption. The described workflow can be broadly applied to a number of viscous materials to generate user-defined concentrations in an automated fashion.

This protocol serves as a comprehensive tutorial for standardized and reproducible mixing of viscous materials with a novel open source automation technology. Detailed instructions are provided on the operation of a newly developed open source workstation, the usage of an open source protocol designer, and the validation and verification to identify reproducible mixtures.

Current mixing steps of viscous materials rely on repetitive and time-consuming tasks which are performed mainly manually in a low throughput mode. These ...

Controlling Particle Fraction in Microporous Annealed Particle Scaffolds for 3D Cell Culture

Microgels are the building blocks of microporous annealed particle (MAP) scaffolds, which serve as a platform for both in vitro cell culture and in vivo tissue repair. In these granular scaffolds, the innate porosity generated by the void space between microgels enables cell infiltration and migration. Controlling the void fraction and particle fraction is critical for MAP scaffold design, as porosity is a bioactive cue for cells. Spherical microgels can be generated on a microfluidic device for controlled size and shape and subsequently freeze-dried using methods that prevent fracturing of the polymer network. Upon rehydration, the lyophilized microgels lead to controlled particle fractions in MAP scaffolds. The implementation of these methods for microgel lyophilization has led to reproducible studies showing the effect of particle fraction on macromolecule diffusion and cell spreading. The following protocol will cover the fabrication, lyophilization, and rehydration of microgels for controlling particle fraction in MAP scaffolds, as well as annealing the microgels through bio-orthogonal crosslinking for 3D cell culture in vitro.

Minimizing the variability in the particle fraction within granular scaffolds facilitates reproducible experimentation. This work describes methods for generating granular scaffolds with controlled particle fractions for in vitro tissue engineering applications.

Microgels are the building blocks of microporous annealed particle (MAP) scaffolds, which serve as a platform for both in vitro cell culture and in vivo ...

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.

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 ...

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 ...

Bioprinting Bacteria in Hydrogel Matrices for the Study of Motility and Growth

3D Printing Bacteria to Study Motility and Growth in Complex 3D Porous Media

Bacteria are ubiquitous in complex three-dimensional (3D) porous environments, such as biological tissues and gels, and subsurface soils and sediments. However, the majority of previous work has focused on studies of cells in bulk liquids or at flat surfaces, which do not fully recapitulate the complexity of many natural bacterial habitats. Here, this gap in knowledge is addressed by describing the development of a method to 3D-print dense colonies of bacteria into jammed granular hydrogel matrices. These matrices have tunable pore sizes and mechanical properties; they physically confine the cells, thus supporting them in 3D. They are optically transparent, allowing for direct visualization of bacterial spreading through their surroundings using imaging. As a proof of this principle, here, the capability of this protocol is demonstrated by 3D printing and imaging non-motile and motile Vibro cholerae, as well as non-motile Escherichia coli, in jammed granular hydrogel matrices with varying interstitial pore sizes.

Author Spotlight: Studying Bacterial Growth in 3D Hydrogel Matrices

This protocol describes a procedure for three-dimensional (3D) printing of bacterial colonies to study their motility and growth in complex 3D porous hydrogel matrices that are more akin to their natural habitats than conventional liquid cultures or Petri dishes.

Bacteria are ubiquitous in complex three-dimensional (3D) porous environments, such as biological tissues and gels, and subsurface soils and sediments. ...

Fragmenting Bulk Hydrogels and Processing into Granular Hydrogels for Biomedical Applications

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