Rapid Continuous Multimaterial Extrusion Bioprinting

Abstract

The development of a multimaterial extrusion bioprinting platform is reported. This platform is capable of depositing multiple coded bioinks in a continuous manner with fast and smooth switching among different reservoirs for rapid fabrication of complex constructs, through digitally controlled extrusion of bioinks from a single printhead consisting of bundled capillaries synergized with programmed movement of the motorized stage.

Keywords: bioinks; bioprinting; hydrogels; multimaterial; tissue engineering.

Figure 1. Design of the digitally tunable continuous multi-material extrusion bioprinter

(A and B) Schematics showing the design of the 7-channel printhead connected to reservoirs that are individually actuated by programmable pneumatic valves. (C and D), Photographs showing the setup of the Festo valves and printhead. (E) Schematic of a sample code for continuous bioprinting of a single serpentine microfiber consisting of 1–7 bioinks. (F) Photograph showing the printed microfiber. (G) Side view of the end of the microfiber indicating the 3D volume containing 7 individually segmented bioinks. Printing conditions: 5% nanosilicate aqueous suspension dyed in 7 different colors, printhead moving speed = 400 mm min−1, pneumatic pressure = 50 psi.

Figure 2. Multi-material bioprinting of 3D constructs

(A and B) Bioprinting of dual- and triple-layered cuboid blocks. (C–E) Bioprinting of blood vessel-like structures (transverse plane) containing dual, triple, and quadruple materials. (F) Bioprinting of a pyramid containing 7 layers of different bioinks. (G and H) Bioprinting of 3 and 10-layered blocks with continuous segments of 7 different bioinks. (I) Bioprinting of human organ-like constructs from multiple bioinks, including brain, lung, heart, liver, kidneys, pancreas, stomach, small/large intestines, bladder, and prostate. The organ-like constructs were individually printed, photographed, and stitched together in the same image at relative locations as those in the human body. (J–N) Side view of selected organ-like constructs indicating their 3D nature: (J) brain, (K) lung vasculature, (L) kidney, (M) left atrium of heart, (N) bladder/prostate. The organ-like structures were not printed to scale to each other. (A–N) printing conditions: 5% nanosilicate aqueous suspension dyed in 7 different colors, printhead moving speed = 400 mm min⁻¹, pneumatic pressure = 50 psi. (O–Q) embedded bioprinting of free-form (O) coils (printing conditions: 20% PEGDA, 2% alginate, and 0.5% PI extruded in 23% Pluronic aqueous solution; printhead moving speed = 100 mm min⁻¹, pneumatic pressue = 30 psi), (P) dual-layer hollow tube, and (Q) DNA helix in front and side views. (P and Q) printing conditions: 2% alginate dyed in different colors extruded in 23% Pluronic aqueous solution containing 0.05% CaCl₂; printhead moving speed = 100 mm min⁻¹, pneumatic pressue = 30 psi.

Figure 3. Multi-material bioprinting of cell-laden structures

(A) Fluorescence image showing a printed multi-component heart-like structure. Fluorescent microbeads were used to aid the macroscopic visualization. The inset shows a schematic of the design. (B–E) Fluorescence micrographs of different junction regions showing the co-existence of HDFs stained with different cell trackers in the printed cell-embedding construct. (F) Schematic showing the design of a vascularized tissue construct containing the four types of cells stained with different cell trackers. (G–I) Fluorescence micrographs of different junction regions showing the co-existence of desired cell types in the printed construct. (J) Quantification of viability of the four cell types immediately, 1 and 7 days post bioprinting. (K) Proliferation of the cells over a course of 3 days. Printing conditions: 5% GelMA and 1% alginate encapsulating different cells, printhead moving speed = 400 mm min⁻¹, pneumatic pressure = 3 psi.

Figure 4. Extended applications of the continuous multi-material bioprinting platform

(A) Printed concentric hydrogel structures containing inward-out gradient of HAp. (B) Alizarin Red staining of the same construct revealing the gradient in HAp contents. (C) Quantification of the Alizarin Red staining intensities for each ring. (D) Printed bone-like structure with desired bioinks containing different concentrations of HAp post staining with Alizarin Red. (E) Quantification of the Alizarin Red staining intensities at selected locations. (F and G) Fluorescence micrograph showing the spreading of preosteoblasts on printed hydrogel blocks with gradients of (F) HAp and (G) nanosilicates. (H and I) Quantifications of area coverages by the cells on the two substrates with HAp and nanosilicate gradients, respectively. Printing conditions: (A to I) 5% GelMA, 2.67% alginate, and 0.5% PI containning various concentrations of HAp and nanosilicates, printhead moving speed = 400 mm min⁻¹, pneumatic pressure = 3 psi. (J) Schematic showing the design of the bioelectronic circuit composed of conductive bioinks with a series concentrations of CNTs. (K) Photograph of a printed circuit. (L) Photograph of a completed circuit where the LEDs showed a series of differential luminescence intensities. (M and N) Quantifications of the resistance and LED luminescence for bioinks containing different concentrations of CNTs. Printing conditions: 2% alginate containning various concentrations of CNTs, printhead moving speed = 400 mm min⁻¹, pneumatic pressure = 3 psi.