3D bioprinting in cardiac tissue engineering

Abstract

Heart disease is the main cause of death worldwide. Because death of the myocardium is irreversible, it remains a significant clinical challenge to rescue myocardial deficiency. Cardiac tissue engineering (CTE) is a promising strategy for repairing heart defects and offers platforms for studying cardiac tissue. Numerous achievements have been made in CTE in the past decades based on various advanced engineering approaches. 3D bioprinting has attracted much attention due to its ability to integrate multiple cells within printed scaffolds with complex 3D structures, and many advancements in bioprinted CTE have been reported recently. Herein, we review the recent progress in 3D bioprinting for CTE. After a brief overview of CTE with conventional methods, the current 3D printing strategies are discussed. Bioink formulations based on various biomaterials are introduced, and strategies utilizing composite bioinks are further discussed. Moreover, several applications including heart patches, tissue-engineered cardiac muscle, and other bionic structures created via 3D bioprinting are summarized. Finally, we discuss several crucial challenges and present our perspective on 3D bioprinting techniques in the field of CTE.

Keywords: 3D bioprinting; bioinks; cardiac muscle; printed biomaterials; tissue engineering.

Figure 1

Adult rat hearts were harvested, sliced, and stained for cells and collagen to analyze variations in the transmural orientation. (A) Masson's trichrome staining of a transmural block cut from the ventricular wall showing the macroscopic variation in fiber orientation across the wall. (B) Analysis of collagen fiber orientation revealed that the degree of alignment from the epicardial side to the endocardial side had a 100° shift. Adapted with permission from . Copyright 2017, National Academy of Sciences.

Figure 2

Traditional fabrication methods in CTE. (A-C) Examples of micropatterned scaffolds fabricated from bioelastomers. (A) Grid-patterned scaffold for myocardial repair prepared on a polydimethylsiloxane (PDMS) mold. Adapted with permission from . Copyright 2017, Springer Nature. (B) Accordion-like honeycomb CTE scaffolds fabricated by excimer laser microablation. Adapted with permission from . Copyright 2008, Springer Nature. (C) Multi-layered micropatterned elastic CTE scaffold fabricated using a microelectromechanical technique and packaging approach. Adapted with permission from . Copyright 2013, John Wiley & Sons. (D-E) Examples of electrospun nanofibrous scaffolds for CTE applications. (D) Conductive electrospun nanofibrous sheet based on poly(L-lactic acid)/polyaniline. The scaffold promoted the maturation and spontaneous beating of primary CMs. Adapted with permission from . Copyright 2017, Elsevier. (E) Random or aligned electrospun nanofibrous mats based on PLLA/chitosan. These scaffolds showed promise as platforms for regenerating myocardia and drug screening applications. Adapted with permission from . Copyright 2017, Elsevier.

Figure 3

Examples of 3D CTE scaffolds with multi-layered structures (A) Multi-layer 3D CTE scaffold fabricated via layer-by-layered deposition of cardiac cells and graphene oxide (GO)-coated poly-L-lysine (PLL) films. Adapted with permission from . Copyright 2014, John Wiley & Sons. (B) Multi-layered NFYs/hydrogel core-shell scaffold within a transition in the 3D orientation of CMs. Adapted with permission from . Copyright 2017, American Chemical Society. (C) Grooved electrospun scaffolds stacked with a slight angular shift for guiding the orientation of multi-layered CMs. Adapted with permission from . Copyright 2017, National Academy of Sciences.

Figure 4

Schematic illustrations of various 3DBP technologies. (A) Inkjet printing: thermal or piezoelectric actuators are applied to form droplets of bioink-cell hybrids. (B) Digital light processing printing: ultraviolet or visible light is used to cure a photopolymer in a vat for layer-by-layer manufacturing of a 3D model. (C) Extrusion printing: pneumatic, piston, or screw forces are applied to extrude continuous beads of bioink. (D) Freeform reversible embedding printing: bioink is extruded into a reversible support bath for fabrication of support-free structures.

Figure 5

Examples of FRE printing strategies. (A) Schematic of the printing and crosslinking process of hydrogel (green) within a gelatin slurry support bath (FRESH v1.0). A whole neonatal-scale human heart was printed via FRESH v1.0. Adapted with permission from . Copyright 2015, American Association for the Advancement of Science. (B) Gelatin microspheres with smaller diameter were used as the support bath in FRESH v2.0, resulting in printed structures with higher resolution. Adapted with permission from . Copyright 2019, American Association for the Advancement of Science. (C) A xanthan gum support bath was shown to support complicated structures (hollow sphere, small hand). Adapted with permission from . Copyright 2019, John Wiley & Sons. (D) Helical and tubular structures printed in Carbopol support gel by freeform extrusion. Adapted with permission from . Copyright 2016, American Chemical Society. (E) A continuous knot of aqueous fluorescent microspheres in Carbopol support gel written without the simultaneous building of any other support structure. Adapted with permission from . Copyright 2015, American Association for the Advancement of Science.

Figure 6

Examples of natural and synthetic biomaterials applied as bioinks. (A) Schematic diagram of the process for preparing dECM bioink. Adapted with permission from . Copyright 2018, John Wiley & Sons. (B) Schematic diagram of alginate calcium-induced crosslinking. (C) Schematic diagram of GelMA formation and UV light-induced crosslinking. Adapted with permission from . Copyright 2018, Mary Ann Liebert. (D) Schematic diagram of MeCol formation and UV light-induced cross-linking. (E) Chemical structures of synthesized polymers commonly used in 3DBP. PLA, polylactic acid; PCL, polycaprolactone; PEG, poly(ethylene glycol); PEGDA, poly(ethylene glycol) diacrylate. (F) Schematic diagram of acrylated PCL-PEG-PCL triblock polymer synthesis, and simple gelation into single-network hydrogel using visible light. Adapted with permission from . Copyright 2018, American Chemical Society.

Figure 7

Preparation of hybrid bioinks for 3DBP in CTE. (A) Schematic diagram showing the two-step crosslinking process for alginate-GelMA hybrid bioinks, The alginate component was first physically crosslinked by CaCl₂ then GelMA was chemically crosslinked via UV illumination to stabilize the 3D printed scaffolds. Adapted with permission from . Copyright 2016, Elsevier. (B) Schematic diagram illustrating how salt particles can be used as a temporary mechanical support and to facilitate thermoplastic processes, including fused deposition modeling (FDM) 3D printing. Adapted with permission from . Copyright 2019, John Wiley & Sons. (C) Schematic diagram of a microfluidic system used to flow two bioinks (containing red and green fluorescent beads) that exited the device through a single extruder. This device opened new routes for the creation of complex and heterogeneous tissue fibers on demand. Adapted with permission from . Copyright 2015, John Wiley and Sons. (D) Schematic diagram of a multi-printer system used to fabricate pre-vascularized stem cell patches from multiple cell-laden bioinks and supporting PCL polymer. The printed dual stem cell structure improved cell-to-cell interactions and cell differentiation and promoted functionality for tissue regeneration. Adapted with permission from . Copyright 2017, Elsevier.

Figure 8

Various applications of 3D-bioprinted constructs for CTE. (A) Left: Schematic illustration of vascularized heart patches printed from iPSC-CM-laden bioink and HUVEC-laden bioink. Right: Representative images of patches after 7 days of culture showing expression of troponin I (red) and connexin 43 (green) in CMs and von Willebrand factor (green) in HUVECs, indicating that a well-developed vascular network was formed in the printed structures. Adapted with permission from . Copyright 2018, Springer Nature. (B) Schematic illustration of the perfusable, multifunctional epicardial device (PerMed) consisting of a biodegradable elastic patch, permeable hierarchical microchannel networks and a system to enable delivery of therapeutic agents from a subcutaneously implanted pump (top). The process of PerMed implantation in pigs via laparoscopic surgery (bottom). Adapted with permission from . Copyright 2021, Springer Nature. (C) Top: Schematic illustration and electron microscopy images of bioprinted microchanneled hydrogels with variable spacing. Bottom: Fluorescence microscopy images assessing the effect of the hydrogels on the alignment, elongation, and differentiation of hMSCs. Adapted with permission from . Copyright 2018, IOP Publishing. (D) Design of a cardiac muscle chip with a stress sensor to monitor muscle contraction for applications in drug screening. Adapted with permission from . Copyright 2016, Springer Nature. (E) Images of an organ-scale tri-leaflet heart valve (top left), a neonatal-scale human heart (bottom left), and a human cardiac ventricle model (right) printed by FRE, showing the capability of this strategy for precise deposition of bioink. Adapted with permission from . Copyright 2019, American Association for the Advancement of Science. (F) Schematic illustration (top left) and photograph (top right) of a heart printed within a support bath and 3D confocal images (bottom) showing its robust structure and perfusable. Adapted with permission from . Copyright 2019, John Wiley & Sons.

Figure 9

Examples of 3DBP strategies combined with other biofabrication techniques. (A) A microfluidic printing head guaranteeing high-resolution bioprinting generated heterogeneous constructs composed of iPSC-derived CMs and HUVECs with different spatial distributions, enabling fabrication of 3D cardiac tissue models enriched with a vascular network. Adapted with permission from . Copyright 2018, Springer Nature. (B) 3DBP was combined with electrospinning to create patient-specific nanofiber tissue-engineered vascular grafts, which were effective in repairing inferior vena cava. Adapted with permission from . Copyright 2016, Elsevier. (C) A custom-built melt electrospinning writing device enabled high-resolution (~10 µm) deposition of bioink (left) to print rectangular patterns that promoted CPC alignment (right). Adapted with permission from . Copyright 2017, John Wiley & Sons.