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., "microgels"). 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.
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.
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 adaptable to any photocrosslinkable hydrogel. See Discussion for more information.
Figure 1: Overview of fabricating bulk hydrogels inside a syringe using photocrosslinking. The figure depicts (A) removing the plunger from the syringe, (B) securing the tip cap to the syringe barrel, (C) adding hydrogel precursor to the syringe barrel, (D) returning the plunger to the syringe, (E) removing excess air and securing the tip cap, and (F) photocrosslinking bulk hydrogel inside of the syringe. Please click here to view a larger version of this figure.
2. Fabricating microgels using extrusion fragmentation
NOTE: An overview of microgel fabrication using extrusion fragmentation is shown in Figure 2.
Figure 2: Overview of microgel fabrication using extrusion fragmentation. The figure depicts (A) extruding bulk hydrogel into an empty syringe barrel and adding PBS, (B) securing a plunger in the syringe with fragmented hydrogel, (C) attaching an 18 G needle and extruding fragmented hydrogel suspension into an empty syringe barrel, and (D) repeating extrusion fragmentation steps with 23 G, 27 G, and 30 G needles, collecting fragmented hydrogel suspension in microcentrifuge tubes on final extrusion. Please click here to view a larger version of this figure.
3. Characterizing fragmented microgels using ImageJ
NOTE: An overview of characterizing the fragmented microgels using ImageJ is shown in Figure 3, as well as representative results for describing size distributions and shapes within a batch of fragmented microgels. Microgels should be fluorescently labeled prior to visualization. For example, high molecule weight FITC-dextran (2 MDa) can be encapsulated in the bulk hydrogel prior to fragmentation to create fluorescein-labeled microgels.
Figure 3: Overview of characterizing fragmented microgel particles using ImageJ. The figure depicts (A) creating a dilute suspension of fragmented microgel particles and using an epifluorescent or confocal microscope to image microgels in suspension (scale bar = 500 µm), (B) converting to a binary image in ImageJ and analyzing particles (count, shape descriptors, etc.), and (C) representative results. Error bars depict min and max with inner quartile ranges demarcated. A population size of n = 100 microgels is shown. Please click here to view a larger version of this figure.
4. Assembling fragmented microgels into granular hydrogels
NOTE: Two methods for the formulation of granular hydrogels from fragmented microgels are presented, using centrifugation and filtration. The method used will depend on the desired microgel packing (i.e., filtration packs particles more densely) and whether biological components are included (i.e., centrifugation will retain components between particles, whereas in filtration these may be lost). Prior work40 thoroughly describes comparative outcomes (i.e., mechanics, porosity) for granular hydrogels formed from either centrifuge or vacuum-driven filtration.
Figure 4: Overview of jamming microgels by vacuum-driven filtration to fabricate tightly-packed fragmented granular hydrogels. The figure depicts (A) placing a membrane filter on the vacuum filtration apparatus, (B) using a pipette to transfer fragmented microgel suspension onto the filter, (C) pulling the vacuum and waiting for microgels to jam and form a granular hydrogel, (D) turning off the vacuum and removing fragmented granular hydrogel using a metal spatula, and (E) using a metal spatula to transfer granular hydrogel to the syringe. Please click here to view a larger version of this figure.
5. Extrusion printing with granular hydrogel inks
NOTE: An overview of the extrusion printing process is shown in Figure 5, including a representative print of a star-shaped construct using fragmented granular hydrogels jammed with vacuum-driven filtration. The printing workflow consists of formulating an ink, planning the print design, and then printing the ink based on the desired design41. If desired, printed granular hydrogel constructs can be stabilized using photocrosslinking post-extrusion by adding excess DTT (5 mM) and I2959 (0.05 wt.%) to the fragmented microgel suspension prior to jamming. This will result in photocrosslinked covalent bonds formed between the microgels, leading to permanent stabilization of the granular hydrogel construct.
Figure 5: Overview of extrusion printing with fragmented granular hydrogels. The figure depicts (A) using a spatula to transfer fragmented granular hydrogel to a syringe barrel, (B) attaching a blunt-tip needle (18 G shown) and pushing the sample to the top, (C) a graphic representing the connection to computer software for printing, and (D) completing the printing of a star-shaped construct with fragmented granular hydrogel. Please click here to view a larger version of this figure.
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 µm (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 formed by the fragmentation process.
When packed together using either centrifugation or vacuum-driven filtration, the assembled granular hydrogel is shear-thinning and self-healing, as described in the previous work39. In addition, the fragmented granular hydrogel has high shape fidelity and mechanical integrity for an injectable hydrogel, as shown by the deposition of a hollow cylinder with a height of 2 cm being extrusion printed in Figure 6. Fragmented granular hydrogels fabricated with these simple and cost-effective methods are useful for many biomedical applications, including injectable therapeutics and 3D printing inks.
Figure 6: Protocol overview and representative results. The figure depicts (A) fragmentation, (B) microgels in suspension, (C) jamming by vacuum-driven filtration, and (D) jammed granular hydrogel being extruded through a needle and printed into a hollow cylinder. Please click here to view a larger version of this figure.
Supplemental File 1: Example .stl file Please click here to download this File.
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 characterized in the previous work39.
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, many hydrogel chemistries can be used to fabricate fragmented microgels using the methods described herein if a bulk hydrogel can be formed within the barrel of a syringe. It is also useful to understand the bulk hydrogel mechanical properties (e.g., compressive modulus). The bulk hydrogels used in this protocol have a bulk compressive modulus of about 30 kPa39. A bulk hydrogel with a higher compressive modulus will require more force to extrude during the fragmentation steps, which could lead to increased clogging or over-pressurization of the syringes; thus, it is recommended to use hydrogels with compressive moduli less than 80 kPa. Further, a bulk hydrogel with compressive moduli lower than 10 kPa may deform during the fragmentation steps, making it challenging to fragment.
This protocol is optimized for a UV spot cure lamp. As an alternative to the UV light source and UV-responsive photoinitiators, visible light sources can also be used along with visible light-responsive photoinitiators, such as water-soluble lithium phenyl-2,4,6-trimethylbenzoyl-phosphinate (LAP). Initiator concentration, light intensity, and sample volume will influence crosslinking times depending on the polymer and crosslinking system being used. Further, many lamp sources can be used as an alternative to spot cure systems.
The most critical step in the protocol is the serial extrusion through smaller and smaller needle gauges. In this procedure, it is suggested to use needle gauges from 18 G (838 µm inner diameter) down to 30 G (159 µm inner diameter). Adding PBS to the fragmented bulk hydrogel prior to extruding through needles is crucial to significantly reduce the force needed to extrude and fragment. No excessive force should be used to extrude the hydrogel, as excessive force can lead to back pressurization in the syringe and risk bursting the hydrogel out of the syringe back. Additional strategies to reduce the force required to extrude include using more needles in the series to reduce fragment size more gradually, as well as adding additional PBS between fragmenting steps.
When jamming the fragmented microgels using vacuum-driven filtration, there may be variability in the process. Some material systems may require more (or less) time to remove PBS and fully jam the microgels. It is suggested to record the time needed for individual material systems to ensure repeatability across experiments. The time to jam will also be dependent on the thickness and size of the sample added to the filter. Spreading the sample out evenly across the filter can help with uniform jamming.
The extrusion fragmentation microgel fabrication method can be adapted for many biomedical applications. For instance, therapeutics can be included in the hydrogel precursor solution and subsequently encapsulated within fragmented microgels to fabricate a shear-thinning, self-healing granular hydrogel for localized therapeutic delivery. In addition, fragmented microgels can be dried to allow for long-term storage and straightforward sterilization practices. However, one limitation to the extrusion fragmentation is the incorporation of cells within microgels. Due to the high shear rates during extrusion fragmentation, the method is likely not amenable to cell encapsulation within microgels, as the high shear may lead to significantly decreased cell viability. Still, cells and spheroids can easily be incorporated between microgels for in vitro culture and in vivo cell delivery.
Fragmented granular hydrogels are a promising biomaterial for biomedical applications. In recent years, granular hydrogels made from various fragmentation methods (i.e., mortar and pestle, blenders, and mesh graters) have been used as cell-laden 3D printing inks48, therapeutic delivery vehicles29, injectable tissue repair scaffolds30, and spheroid-culture platforms39. Of the fragmentation methods previously reported, the extrusion fragmentation method described herein is one of the most simple and cost-effective methods with numerous advantages. Sharing the methods herein will increase accessibility to granular hydrogel fabrication and lead to significant advances in the growing field of granular hydrogel biomaterials, allowing more researchers to engineer innovative biomedical solutions with fragmented granular hydrogels.
The authors have nothing to disclose.
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.).
15 mL Plastic Conical Centrifuge Tube | Corning | 430766 | |
30 G NT Premium Series Dispensing Tip | Jensen Global | JG30-0.5HPX | Catalog Number listed here is for 30 G, 0.5" needle. Various sizes are available. |
BD Disposable Syringes with Luer-Lok Tips (3 mL) | Fisher Scientific | 14-823-435 | Catalog Number listed here is for 3 mL syringe. Various sizes are available (14-823-XXX). |
Black folders | Various Vendors | ||
Disposable Probe Needle For Use With Syringes and Dispensing Machines (18 G, 0.5") | Grainger | 5FVH5 | Catalog Number listed here is for 18 G, 0.5" needle. Various sizes are available. |
Disposable Probe Needle For Use With Syringes and Dispensing Machines (23 G, 0.5") | Grainger | 5FVJ3 | |
Disposable Probe Needle For Use With Syringes and Dispensing Machines (27 G, 1.5") | Grainger | 5FVL0 | |
Dulbecco's Phosphate Buffered Saline | Fisher Scientific | 14190-250 | Catalog Number listed here is for a case of 10 x 500 mL bottles. |
Durapore Membrane Filter, 0.22 µm | Millipore | GVWP04700 | |
Epifluorescent or confocal microscope | Various Vendors | To visualize microgels and granular hydrogels | |
Eppendorf Snap-Cap Microcentrifuge Safe-Lock Tubes | Fisher Scientific | 05-402-25 | |
Extrusion printer | Custom-built | Other extrusion printers can be use,d such as commercially available BIOX. | |
Filter Adapters | Fisher Scientific | 05-888-107 | Catalog Number listed here is for a set of multiple sizes. Various sizes are available (05-888-XXX). |
Filter Flask | Various Vendors | ||
Fluorescein isothiocyanate-dextran (2 MDa) | Sigma-Aldrich | 52471 | |
Glass microscope slide | Various Vendors | ||
ImageJ | National Institutes of Health | "Analyze Particles" information link: https://imagej.nih.gov/ij/docs/menus/analyze.html | |
Laptop | Various Vendors | ||
Luer-Lock Tip Caps | Integrated Dispensin g Solutions | 9991329 | |
Metal spatula for scooping | Various Vendors | ||
Microcentrifuge | Various Vendors | Capable of speed up to 18,000 x g | |
Microscoft Execl | Microsoft | Other programs can be used, such as Google Slides. | |
OmniCure S2000 Spot UV Curing System | Excelitas Technologies | S2000 | Different light systems may be used to fabricate bulk hydrogels if desired. |
Porcelain Buchner Funnel with Fixed Perforated Plate | Fisher Scientific | FB966C | Catalog Number listed here is for 56mm diameter plate. Various sizes are available. |
Radiometer | Various Vendors | ||
Repetier Host | Hot-World GmbH & Co. KG | 3D printing software | |
Screw-based extrusion printer | Various Vendors | This study used a custom-modified 3D FDM printer (Velleman K8200). Many alternatives are available. | |
Solidworks/CAD software | Dassault Systèmes SolidWorks Corporation | Other programs can be used, such as Blender or TinkerCAD. | |
Tubing to Connect Filter Flask to Vacuum Line | Various Vendors | ||
UV Eye Protection (i.e., safety glasses) | Various Vendors |