Name: fragmenting bulk hydrogels and processing into granular hydrogels for biomedical applications
Uploaded: 2022-05-17T14:00:00
Description: Watch this Scientific Journal Video about Fragmenting Bulk Hydrogels and Processing into Granular Hydrogels for Biomedical Applications at JoVE.com

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.).

1. Fabricating bulk hydrogels inside of a syringe using photocrosslinking
NOTE: An overview of bulk hydrogel fabrication inside a syringe using photocrosslinking is shown in Figure 1. This protocol uses norbornene-modified hyaluronic acid (NorHA) to fabricate bulk hydrogels using a photo-mediated thiol-ene reaction. Detailed procedures for the synthesis of NorHA are described elsewhere38. However, this protocol is highly adap...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#34; 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&#34;)</td>
			<td>Grainger</td>
			<td>5FVH5</td>
			<td>Catalog Number listed here is for 18 G, 0.5&#34; needle. Various sizes are available.</td>
		</tr>
		<tr>
			<td>Disposable Probe Needle For Use With Syringes and Dispensing Machines (23 G, 0.5&#34;)</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&#34;)</td>
			<td>Grainger</td>
			<td>5FVL0</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;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 &#181;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>&#34;Analyze Particles&#34; 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 &#38; 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&#232;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 ...

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