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 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 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. 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 at low HMP packing density is demonstrated via the interfacial self-assembly of heterogeneously charged nanoparticles.
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 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 <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 °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 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.
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.
Figure 1: GelMA synthesis and characterization. (A) GelMA synthesis reaction. Gelatin is modified with methacrylic anhydride at 50 °C for 2 h. (B) The proton nuclear magnetic resonance (1H NMR) spectra of gelatin and GelMA: (a) the peak for aromatic acids, which is selected as the reference for calibration, (b) vinyl functional group peaks after the MA modification of gelatin, and (c) the peak for lysine proteins. In this example, the MA degree of substitution was 71% ± 3% (n = 3). This figure has been modified with permission from Ataie et al.11 Abbreviations: GelMA = gelatin methacryloyl; DPBS = Dulbecco's phosphate-buffered saline; MA = methacryloyl. Please click here to view a larger version of this figure.
2. High-throughput GelMA microgel fabrication
3. Converting microgels to resuspendable powder via the microengineered emulsion-to-powder (MEtoP) technology
NOTE: The MEtoP technology to convert the water-in oil emulsion-based HMPs to microparticle powder (micro-aerogels) with preserved properties, such as resuspendability, shape, size, and assembly, has been developed.
Figure 2: GelMA microparticle powder preparation via MEtoP technology. (A) Images of GelMA powder obtained from the MEtoP technology or conventional lyophilization of HMP. In MEtoP technology or conventional lyophilization, HMPs are suspended in oil-surfactant or aqueous media, respectively. The engineering fluid protects the dispersed phase (HMPs) from aggregation and preserves the physiochemical properties of GelMA microparticles during lyophilization. (B) Schematic illustration of dried HMPs prepared via the MEtoP compared with conventionally lyophilized HMP in an aqueous medium. (C) SEM images of dried GelMA microparticles prepared via the MEtoP compared with conventional lyophilization. Scale bars = 2 mm (left; A), 500 µm (right; A), 10 µm (left; C), and 200 µm (right; C). This figure was modified with permission from Sheikhi et al.26 Abbreviations: GelMA = gelatin methacryloyl; DPBS = Dulbecco's phosphate-buffered saline; MEtoP = microengineered emulsion-to-powder; HMP = hydrogel microparticle; SEM = scanning electron microscopy. Please click here to view a larger version of this figure.
4. GelMA GHS formation
NOTE: This protocol is for preparing 400 µL of microgel suspension. For larger quantities, scale-up is needed. To keep the GelMA HMPs physically crosslinked, all the steps should be performed at about 4 °C by placing the microgel containers in an ice-water bucket.
5. Nanoengineered granular bioinks (NGB) for the 3D bioprinting of GHS with preserved microporosity
Figure 3: Schematics of GelMA microgel and GHS formation. (A) Schematics of GelMA microgel separation from oil and NGB preparation. PFO (20% v/v in engineering fluid) was added to the GelMA microgel-oil emulsion at a 1:1 volumetric ratio, followed by vortexing and centrifugation at 300 × g for 15 s. To fabricate GelMA GHS, the PI solution (LAP 0.1% w/v in DPBS) was added to the GelMA HMPs, followed by vortexing and centrifugation at 3,000 × g for 15 s. For preparing the NGB, the PI solution (LAP 0.1% w/v in ultrapure water) and nanoplatelet dispersion (3% w/v in ultrapure water) were added to the GelMA HMP suspension, followed by vortexing and centrifugation at 3,000 × g for 15 s. Figure 3A was modified with permission from Ataie, Z. et al.11 (B) Exposing packed GelMA HMPs to light yields GHS. Figure 3B was modified with permission from Sheikhi et al.15 Abbreviations: GelMA = gelatin methacryloyl; GHS = granular hydrogel scaffold; NGB = nanoengineered granular bioink; PFO = 1H,1H-perfluoro-1-octanol; PI = photoinitiator; LAP = lithium phenyl-2,4,6-trimethylbenzoylphosphinate; HMP = hydrogel microparticle; DPBS = Dulbecco's phosphate-buffered saline. Please click here to view a larger version of this figure.
GelMA was synthesized through the reaction of gelatin with MA, as presented in Figure 1A. By tailoring the reaction conditions, such as MA concentration, different degrees of MA substitution were obtained. To quantify the degree of MA substitution, GelMA was assessed via 1H NMR spectroscopy (Figure 1B). Vinyl functional groups with representative peaks at the chemical shifts of ~5-6 ppm confirmed the successful GelMA synthesis from gelatin. The reaction yield after dialysis and sterile filtration was >70% (mg of GelMA/mg of gelatin). The droplet/microgel fabrication yield was ~100%. Different methods can be used to quantify the degree of substitution24. We assessed the decrease of lysine amino acid (primary amine), normalized using the unaffected aromatic acid proton based on equation (1).
HMPs are commonly suspended in aqueous media, such as DPBS or cell culture media. The hydrated state of HMPs may introduce several challenges in sterilization, shipping, storage, and long-term stability. MEtoP is a novel method to convert HMPs to dried powder without affecting their original molecular and colloidal properties25. The MEtoP technology yields resuspendable, dried HMPs (micro-aerogels) via low-pressure freeze-drying, while protecting the HMPs from aggregation and severe deformation using a volatile oil, rather than an aqueous medium (Figure 2A). Using this technique, the microgels are individually dried without aggregation (Figure 2B), thus retaining their spherical shape after lyophilization (Figure 2C). These microparticles readily recover their initial properties upon rehydration, yielding HMP suspensions that are ready to form GHS upon assembly.
Step-emulsification microfluidic devices yield high-throughput monodispersed GelMA droplets, independent of the aqueous/oil phase flow rates. Since GelMA is a thermosensitive biopolymer, droplets are converted to thermally crosslinked HMPs by reducing the temperature to ~4 °C. The stable HMPs may be rinsed to remove the oil and surfactant using PFO (20% v/v). After oil/surfactant removal, the GelMA HMPs can be mixed with a PI for chemical assembly or with nanoparticles for interfacial self-assembly (Figure 3A). GHS formation is initiated via light (wavelength = 400 nm, intensity = 15 mW/cm2, exposure time = 60 s)-mediated free-radical polymerization, resulting in microgel-microgel bonding (Figure 3B).
Microporosity is one of the major characteristics of GHS, enabling facile oxygen and cellular waste exchange, cell infiltration, migration, and proliferation. To assess the microporosity, a high molecular weight fluorescence dye is used to visualize the void spaces among the HMPs. Figure 4A shows the top and 3D views of GHS scaffolds, with the green area showing the interconnected microporosity. Figure 4B presents a fluorescence image, assessed using a custom-written MATLAB script to detect the pore area. Void fraction (Figure 4C) and median pore equivalent diameter (Figure 4D) measurements show no significant difference between GHS and 3D printed NGB scaffolds, which attest to the availability and interconnectivity of microscale pores of NGB.
Figure 4: Pore characterization of GelMA GHS and NGB. (A) Top and 3D orthographic views of GHS and NGB scaffolds. Increments are 100 µm. (B) The fluorescence image of void area and pore detection via a custom-written MATLAB code for photocrosslinked GelMA GHS and NGB. Scale bar = 200 µm. (C) The void fraction of GelMA GHS and NGB. (D) The median equivalent pore diameter of GelMA GHS and NGB. This figure was modified with permission from Ataie et al.11 Abbreviations: GelMA = gelatin methacryloyl; GHS = granular hydrogel scaffold; NGB = nanoengineered granular bioink; NS = non-significant. Please click here to view a larger version of this figure.
NGB is designed as a printable HMP suspension with preserved microporosity. To demonstrate the extrudability and printability of NGB, we performed extrusion-based 3D printing, as presented in Figure 5. PSU letters were printed using the NGB, followed by light exposure (Figure 5A). To assess the superiority of NGB to tightly packed and loosely packed GelMA HMPs, the hanging filament length (Lf) was measured (Figure 5B). The NGB had the highest Lf compared with the packed HMPs. The loosely packed HMPs did not yield filaments. Additionally, a hollow cylinder was 3D printed, and the whole construct was exposed to light for photocrosslinking (Figure 5D) and physically held (Figure 5E) to demonstrate the 3D printability and shape fidelity of photocrosslinked GelMA NGB, respectively.
Figure 5: Printability of the NGB followed by UV-light mediated photocrosslinking (wavelength = 395-405 nm, intensity = 15 mW/cm2, exposure time = 60 s). (A) Schematic of the printing process, showing PSU letters being 3D printed using the fluorescently labeled NGB. Scale bar = 3 mm. (B) Visual comparison of filament extrusion using the NGB, tightly packed GelMA HMPs, and loosely packed GelMA HMPs. Scale bar = 10 mm. (C) Hanging filament length of NGB, tightly packed, and loosely packed granular hydrogels. The NGB forms longer hanging filaments (filament length Lf = 45.0 ± 5.0 mm, n = 10) than tightly packed microgels (Lf = 19.3 ± 0.7 mm, n = 10). The loosely packed microgels yield droplet-size (Lf = 5.7 ± 0.7 mm, n = 10) filaments. (D) The NGB was used for the 3D printing of hollow cylinders with a diameter of 5 mm and a height of 10 mm. (E) The whole construct (hollow cylinder with d = 5 and h = 10 mm) was printed, then exposed to UV light. Layer-by-layer photocrosslinking might increase shape fidelity but decreases structural integrity as the layers are not crosslinked together. The hollow printed cylinder was held using tweezers, showing mechanical robustness. Scale bar = 1 cm. This figure was modified from Ataie et al.11 Abbreviations: NGB = nanoengineered granular bioink; GelMA = gelatin methacryloyl; HMP = hydrogel microparticle. Please click here to view a larger version of this figure.
Gelatin and its derivatives are the most commonly used protein-based biomaterials for HMP fabrication. The challenge of throughput versus particle size monodispersity trade-off can be overcome using step-emulsification microfluidic devices. These devices are capable of forming more than 40 million droplets per hour, with a coefficient of variation less than 5%27. In this article, we discussed the microfabrication of droplets containing GelMA solutions, followed by converting them to GelMA HMPs, powder, GHS, and NGB.
The thermoresponsivity of GelMA enables facile microfluidic-enabled HMP fabrication and stabilization. At a temperature higher than the UCST (e.g., 37 °C), GelMA dissolves in an aqueous solution, yielding a suitable aqueous fluid for water-in-oil emulsion formation in step-emulsification devices. Decreasing temperature (e.g., 4 °C) enables GelMA HMP formation via physical crosslinking after droplet formation. GelMA HMPs may be used as building blocks for GHS fabrication through various approaches. Thermally stable HMPs may be photoassembled to form a mechanically robust GHS, attaining one of the highest reported compression moduli among granular scaffolds, excluding the interpenetrating percolating networks28. In the photoassembly method, all the procedures should be performed at low temperature (e.g., 4 °C) to avoid GelMA HMP melting.
The 3D (bio)printing of HMPs enables the fabrication of geometrically well-defined GHS; however, this has been performed using tightly packed HMPs, compromising the microporosity of additively manufactured granular constructs. To address this challenge, we show how the reversible self-assembly of heterogeneously charged nanoparticles adsorbed to HMP surfaces renders loosely packed HMPs shear-yielding and 3D printable (NGB) with preserved microporosity.
The authors have nothing to disclose.
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.
1H,1H-perfluoro-1-octanol | Alfa Aesar, MA, USA | B20156-18 | 98% purity |
Biopsy punch | Integra Miltex, NY, USA | 33-31A-P/25 | 1.5 mm Biopsy Punch with Plunger System |
Blunt needle | SANANTS | 30-002-25 | 25 G |
Bruker Avance NEO 400 MHz | 400 MHz Bruker NEO, MA, USA | NMR device | |
Centrifuge | Eppendorf, Germany | 5415 C | |
Centrifuge tube | Celltreat, MA ,USA | 229423 | |
Coffee filters | BUNN, IL, USA | 20104.0006 | BUNN 8-12 Cup Coffee Filters, 6 each, 100 ct |
Desiccator | Thermo Scientific | 5311-0250 | Nalgene Vacuum Desiccator, PC Cover and Body, 280 mm OD |
Deuterium oxide | Sigma, MA, USA | 151882 | |
Dialysis membrane (12-14 kDa) | Spectrum Laboratories, NJ, USA | 08-667E | |
Dulbecco's phosphate buffered saline (DPBS, 1x) | Sigma, MA, USA | 56064C-10L | dry powder, without calcium, without magnesium, suitable for cell culture |
Erlenmeyer flask | Corning, NY, USA | 4980 | Corning PYREX |
Ethanol | VWR, PA, USA | 89125-188 | Koptec 200 proof |
External thread cryogenic vials (cryovials) | Corning, NY, USA | 430659 | |
Freeze dryer | Labconco, MO, USA | 71042000 | Equipped with vacuum pump (Catalog# 7587000) |
Gelatin powder | Sigma, MA, USA | G1890-5100G | Type A from porcine skin, gel strength ~300 g Bloom |
Glass microscope slides | VWR, PA, USA | 82027-788 | |
Hotplate | FOUR E'S SCIENTIFIC | MI0102003 | 5 inch Magnetic Hotplate Stirrer Max Temp 280 °C/536 °F |
Kimwipes | Fischer scientific, MA, USA | 06-666 | |
KMPR 1000 negative photoresist series | Kayaku Advanced Materials, MA, USA | 121619 | KMPR1025 and KMP1035 are included |
LAPONITE XLG | BYK USA Inc., CT, USA | 2344265 | |
Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) | Sigma, MA, USA | 900889-1G | >95% |
Luer-Lok connector | BD, NJ, USA | BD 302995 | |
MA/BA Gen4-Serie Mask- und Bond-Aligner | SÜSS MicroTeck, German | Nanofabrication device | |
Methacylate anhydride | Sigma, MA, USA | 276685-100ML | contains 2,000 ppm topanol A as inhibitor, 94% |
Milli-Q water | Millipore Corporation, MA, USA | ZRQSVR5WW | electrical resistivity ≈ 18 MΩ at 25 °C, Direct-Q 5 UV Remote Water Purification System |
Novec 7500 engineering fluid | 3M, MN, USA | 3M ID 7100003723 | |
Oven | VWR, PA, USA | VWR-1410 | 1410 Vacuum Oven |
Parafilm | Fischer scientific, MA, USA | HS234526C | |
Pasteur pipette | VWR, PA, USA | 14673-010 | |
Petri dish | VWR, PA, USA | 25384-092 | polystyrene |
Pico-Surf | Sphere Fluidics, UK | C022 | (5% (w/w) in Novec 7500) |
Pipette | VWR, PA, USA | 89079-970 | |
Pipette tips | VWR, PA, USA | 87006-060 | |
Plasma cleaner chamber | Harrick Plasma, NY, USA | PDC-001-HP | |
Polydimethylsiloxane | Dow Corning, MI, USA | 2065623 | SYLGARD 184 Silicone Elastomer Kit |
Positive displacement pipette | Microman E M100E, Gilson, OH, USA | M100E | |
Silicon wafers | UniversityWafer, MA, USA | 452/1196 | 4-inch mechanical grade |
Spatula | VWR, PA, USA | 231-0104 | Disposable |
SU-8 | Kayaku Advanced Materials, MA, USA | ||
Syringe pump | Harvard Apparatus, MA, USA | 70-2001 | PHD 2000 |
Trichloro(1H,1H,2H,2H-perfluorooctyl)silane | Millipore Sigma, MA, USA | 448931-10G | 97% |
Tygon tubings | Saint-globain, PA, USA | AAD04103 | |
UV light | QUANS | Voltage: 85 V-265 V AC / Power: 20 W | |
Vacuum filtration unit | VWR, PA, USA | 10040-460 | 0.20 µm |
Vortex | Fischer scientific, USA | 14-955-151 | Mini Vortex Mixer |