Gelatin Methacryloyl Granular Hydrogel Scaffolds: High-throughput Microgel Fabrication, Lyophilization, Chemical Assembly, and 3D Bioprinting

Summary

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.

Abstract

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.

Introduction

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 polymers^3<....

Protocol

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.

1. Add 200 mL of Dulbecco's phosphate buffered saline (DPBS, 1x) to an Erlenmeyer flask and heat the solution until it reaches 50 °C. Cover the flask with aluminum foil to prevent evaporation.
2. Add 20 g of gelatin powder to the DPBS solution at 50 °C while stirring at 240....

Results

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 gelat.......

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
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