Microfluidics-Enabled Multimaterial Maskless Stereolithographic Bioprinting

Abstract

A stereolithography-based bioprinting platform for multimaterial fabrication of heterogeneous hydrogel constructs is presented. Dynamic patterning by a digital micromirror device, synchronized by a moving stage and a microfluidic device containing four on/off pneumatic valves, is used to create 3D constructs. The novel microfluidic device is capable of fast switching between different (cell-loaded) hydrogel bioinks, to achieve layer-by-layer multimaterial bioprinting. Compared to conventional stereolithography-based bioprinters, the system provides the unique advantage of multimaterial fabrication capability at high spatial resolution. To demonstrate the multimaterial capacity of this system, a variety of hydrogel constructs are generated, including those based on poly(ethylene glycol) diacrylate (PEGDA) and gelatin methacryloyl (GelMA). The biocompatibility of this system is validated by introducing cell-laden GelMA into the microfluidic device and fabricating cellularized constructs. A pattern of a PEGDA frame and three different concentrations of GelMA, loaded with vascular endothelial growth factor, are further assessed for its neovascularization potential in a rat model. The proposed system provides a robust platform for bioprinting of high-fidelity multimaterial microstructures on demand for applications in tissue engineering, regenerative medicine, and biosensing, which are otherwise not readily achievable at high speed with conventional stereolithographic biofabrication platforms.

Keywords: bioprinting; digital light prototyping; digital micromirror devices; microfluidics; multimaterials.

Figure 1.

a) Planar schematics showing the setup of the bioprinter, including a UV lamp (385 nm), optical lenses and objectives, a DMD chip, and a microfluidic device. b) Schematic showing the actual setup of the entire optical platform. c) Schematic showing an open-chamber microfluidic chamber used to create single-material printouts.

Figure 2.

a) Schematic showing the assembly of the microfluidic chip having four inlets and one common outlet. b) The operation of the microfluidic device for consecutive injection of different bioinks and the washes in between the injections. c) The defined CFD model and the velocity profile (m/s) of PEGDA (with a density 1.06 kg/m³ and a viscosity 1×10⁻⁵ Pa s) in the closed chamber under sinusoidal fluid flow. d-f) The role of mixing and washing observed by flow streamlines in GelMA solution (15% w/v) mixed by food dye in the microfluidic chip for a star pattern, two rectangular patterns (made of PMMA molds), and no pattern, respectively.

Figure 3.

a) Schematic showing the microfluidic chip containing a moving part at the center of the bottom chamber. b) Computational domain of the finite element analysis built for an isotropic, incompressible, hyperelastic membrane supported on the rigid piston. c) Simulation result showing principle strain and stress values of the PDMS membrane at 4-mm displacement. d) Schematics showing the four-step bioprinting process inside the microfluidic chip for fabricating 3D objects. e-g) Examples of multi-component bioprinted constructs: e) a two-component GelMA-7% construct filled by fluorescent dyes (left) and a three-component pattern of colored PEGDA-50% (right); f) a 3D fluidic mixer made by three different colors (white, orange, blue) printed from PEGDA-50%; and g) a single-component (green) and a three-component (white, blue, purple) star-shaped pyramid of PEGDA-50%.

Figure 4.

a) A tumor angiogenesis model: i) schematic showing the tumor angiogenesis; ii) schematic of the mask for printing; iii) bioprinted microvasculature in PEGDA; iv) bioprinted MCF7 cell (red)-laden microvascular bed of GelMA further seeded with HUVECs (green) in the channels. b) A skeletal muscle model: i) schematic showing the skeletal muscle tissue; ii) schematic of the mask for printing; iii) bioprinted structure of GelMA containing patterned C2C12 cells (red) and fibroblasts (blue) after 48 h of culture; iv) Presto blue measurements of cell proliferation in the bioprinted structures. c) A tendon-to-bone insertion model: i) schematic of the tendon-to-bone insertion site; ii) schematic of the mask for printing; iii) bright-field optical micrograph showing a bioprinted dye-laden GelMA structure; iv) bioprinted structure of GelMA containing patterned osteoblasts (blue), MSCs (red), and fibroblasts (green).

Figure 5.

a) A concentration-gradient model generated by multi-material DMD bioprinting: i) schematic of the construct showing the PEGDA (35% v/v) frame and three GelMA strips of 5, 10, and 15% w/v mass concentrations with a uniform thickness of 1 mm; ii) a bioprinted model where the GelMA strips contained green fluorescent beads; iii) the rat subcutaneous model used to assess the bioprinted constructs. b) Photographs showing the retrieved implants at Day 10 and Day 30, along with confocal images of the retrieved constructs at Day 30 stained for nuclei (blue) and for CD31 (red), where the bright-field views were pseudo-colored in green. c) Immunostaining of the retrieved implants for CD31 (red), for different GelMA concentrations (5, 10, and 15%), in the absence and presence of VEGF; the nuclei were counterstained with DAPI (blue). d) H&E staining of the retrieved implants for different GelMA concentrations, in the absence and presence of VEGF.