Human-Recombinant-Elastin-Based Bioinks for 3D Bioprinting of Vascularized Soft Tissues

Abstract

Bioprinting has emerged as an advanced method for fabricating complex 3D tissues. Despite the tremendous potential of 3D bioprinting, there are several drawbacks of current bioinks and printing methodologies that limit the ability to print elastic and highly vascularized tissues. In particular, fabrication of complex biomimetic structure that are entirely based on 3D bioprinting is still challenging primarily due to the lack of suitable bioinks with high printability, biocompatibility, biomimicry, and proper mechanical properties. To address these shortcomings, in this work the use of recombinant human tropoelastin as a highly biocompatible and elastic bioink for 3D printing of complex soft tissues is demonstrated. As proof of the concept, vascularized cardiac constructs are bioprinted and their functions are assessed in vitro and in vivo. The printed constructs demonstrate endothelium barrier function and spontaneous beating of cardiac muscle cells, which are important functions of cardiac tissue in vivo. Furthermore, the printed construct elicits minimal inflammatory responses, and is shown to be efficiently biodegraded in vivo when implanted subcutaneously in rats. Taken together, these results demonstrate the potential of the elastic bioink for printing 3D functional cardiac tissues, which can eventually be used for cardiac tissue replacement.

Keywords: GelMA; MeTro; bioprinting; cardiac tissue; elastic bioinks; elasticity; vascularized tissue.

Figure 1.. Synthesis and mechanical characterization of GelMA/MeTro composite hydrogel.

(A) Domain map of human tropoelastin. Methacrylation of (B) tropoelastin and (C) gelatin. (D) A schematic to describe the formation of MeTro/GelMA hydrogels. GelMA and MeTro polymers are covalently crosslinked upon exposure to visible light in the presence of LAP photoinitiator to form a highly elastic hydrogel network. Mechanical properties of MeTro, GelMA and MeTro/GelMA composite hydrogels, showing (E) representative tensile stress-strain curves; (F) tensile moduli; (G) extensibility; (H) representative compressive stress-strain curves; (I) compressive moduli; and (J) energy loss. (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001)

Figure 2.. Optimization of 3D printing parameters.

(A) Bioink formulation for 3D bioprinting. (B) Viscosity of MeTro/GelMA/gelatin, gelatin, and MeTro/GelMA as a function of temperature at shear rate 50 s⁻¹. (C) Shear stress of MeTro/GelMA bioink measured as a function of shear rate. The diamond points indicate the actual shear stresses on the cells encapsulated in the bioink exiting the nozzle under the extrusion pressures 5, 10, 15, 20, and 25 kPa, respectively (from left to right). (D) Optimization of the printing speed and extrusion pressure: (i) a schematic to illustrate printing speed and extrusion pressure; (ii) MeTro/GelMA bioink filaments printed into the support bath at different printing pressures and speeds; (iii) qualitative evaluation of the MeTro/GelMA bioink printability at different printing pressures and speeds. (E) Optimized printing parameters for MeTro/GelMA bioink. (F) A lattice-shaped construct printed up to 16 layers to form constructs with a linear relationship between the number of layers and the height of the construct. (G) Various 3D constructs printed with MeTro/GelMA bioink (from left to right: heart slice, lattice cube and cat toy).

Figure 3.. 3D bioprinting of cell-laden elastic constructs using MeTro/GelMA bioink.

(A) A schematic illustration of 3D bioprinting of lattice scaffolds using HUVECs- and CMs/CFs-laden MeTro/GelMA bioinks. Green and red food colors were used to distinguish the HUVECs- and CMs/CFs-laden inks, respectively, only for imaging experiments. (B) Immunostaining of the lattice structure against sarcomeric α-actinin (red), CD31 (green), and DAPI (blue) at day 7 post bioprinting. Printed CMs/CFs and HUVECs are marked with red and green boxes, respectively. (C) A schematic to describe 3D bioprinting vascularized cardiac constructs with HUVECs-laden MeTro/GelMA bioink and CMs/CFs/HUVECs-laden GelMA bioink. (D) A vascularized cardiac construct in the support bath right after printing process and (E) the construct after the photocrosslinking and washing steps. Green and red food colors were used to distinguish the MeTro/GelMA and GelMA bioinks, respectively, only for imaging experiments. (F) A cross-sectional fluorescence image of the vascularized cardiac construct. Fluorescein and rhodamine dyes were added to the MeTro/GelMA and GelMA bioinks, respectively. (G) Viability of HUVECs (in MeTro/GelMA bioink) and CMs/CFs/HUVECs (in GelMA bioink) within vascularized cardiac tissue constructs. (H) Live/dead staining of the vascularized cardiac construct at day 10 post bioprinting. (I) Immunostaining of the vascularized cardiac construct against sarcomeric α-actinin (red), CD31 (green), and DAPI (blue) at day 10 post bioprinting. HUVECs (in MeTro/GelMA bioink) and CMs/CFs/HUVECs (in GelMA bioink) are marked with green and red boxes, respectively.

Figure 4.. Evaluation of in vitro function and in vivo biocompatibility of the 3D printed vascularized cardiac constructs.

(A) Representative images of the cardiac constructs with acellular and HUVECs-laden channel 0, 4, and 12 after infusion of FITC-Dex to quantify the barrier properties imparted by HUVECs in the printed construct. (B) FITC-Dex distribution within the constructs plotted as a function of distance from vasculature. (C) Diffusional permeability of FITC-Dex in acellular and HUVECs-laden channels. (D) A representative image of the vascularized cardiac construct at day 15 post printing. Beating CMs were identified and marked with red boxes as region of interests (ROIs). (E) Beating quantification of the CMs in vascularized cardiac construct at day 15 post bioprinting. In the representative plot, beating was recorded as change in pixel intensity within three different ROIs. (F) Beat rate and (G) degree of coordination measured for the beating CMs. (H) A schematic of the 3D printed implant structure. (I) In vivo biodegradation rate of the 3D printed constructs. (J) H&E staining of the 3D printed constructs explanted at day 21 post implantation. (K) H&E staining images showing the interface between implanted hydrogel constructs and tissues at day 7 and 21 post implantation at 10 × (top) and 40 × (bottom) magnifications. (L) IHC staining against CD3, (top, green) and CD68 markers (bottom, green) and DAPI (blue) in the explanted 3D printed constructs at day 7 and 21 post implantation. Inflammatory response was quantified by calculating fluorescence intensity from IHC images. (* p<0.05, ** p<0.01, *** p<0.001)