3D bioprinting for cardiovascular regeneration and pharmacology

Abstract

Cardiovascular disease (CVD) is a major cause of morbidity and mortality worldwide. Compared to traditional therapeutic strategies, three-dimensional (3D) bioprinting is one of the most advanced techniques for creating complicated cardiovascular implants with biomimetic features, which are capable of recapitulating both the native physiochemical and biomechanical characteristics of the cardiovascular system. The present review provides an overview of the cardiovascular system, as well as describes the principles of, and recent advances in, 3D bioprinting cardiovascular tissues and models. Moreover, this review will focus on the applications of 3D bioprinting technology in cardiovascular repair/regeneration and pharmacological modeling, further discussing current challenges and perspectives.

Keywords: 3D bioprinting; Cardiac tissue; Cardiovascular; Myocardium; Pharmacology; Regeneration; Tissue model; Valves; Vasculature.

Fig. 1.

Schematic diagram outlined in this review, including the techniques of 3D cardiovascular bioprinting, bioengineering methods, and bio-applications in regeneration and pharmacology.

Fig. 2.

Schematic diagram of cardiovascular system structure. Micro-physiological models of the 3D bioprinted heart have focused mostly on generating the myocardium, valve, and vessels.

Fig. 3.

(A) Schematic bioprinting setup based on LIFT [187]. (B) Arrangement of transferred cells by LIFT observed after 24h: Human MSC were pre-stained with PKH26 and patches were stained with polyclonal goat anti-Pecam1 24h after LIFT to separate grid patterned HUVEC [187]. (C) Patch implantation in vivo: After LAD-ligation rats received the cardiac patch sutured onto the area of blanched myocardium [187]. (D) Seven rows of Hy-Stem/CMPCs mixture were printed at a distance of 2.5mm from each other, both horizontally and vertically [189]. (E) Live-dead assay performed 2h after printing showed the vast majority of CMPCs to be alive (green) and only a few were dead (red), scale bar 1000mm [189]. (F) In vivo CMPC survival. Assessment of cell survival by bioluminescent imaging showed no significant decrease in luciferase positive signal over 1month [189]. Transplanted scaffolds were visible in all treated mice as a patch on the infarcted area of the ventricular wall both at 1 (G) and 4 (I) weeks [189]. The presence of human transplanted cells was confirmed by immunofluorescence analysis for human-specific b-integrin (H) and human Lamin A/C (J), which reveled robust presence of CMPCs inside the matrix at all time points [189]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4.

(A) Optical and microscopic images of native and decellularized heart tissue (scale bar, 100mm) [190]. (B) Heart tissue construct was printed with heart dECM (hdECM) [190]. (C) Representative microscopic images of hdECM construct (scale bar, 400mm), Structural maturation of myoblasts in (D) COL and (E) hdECM construct showing Myh7 (red) and cell nuclei (DAPI, blue) (scale bar, 200mm) [190]. (F) Illustration of pre-vascularized stem cell patch including multiple cell-laden bioinks and supporting PCL polymer [191]. (G) Fabricated patch including the two types of cell-laden bioink and PCL supporting layer (Scale bar (left top), 1mm; Scale bar (bottom), 200mm) [191]. (H) Optical image of pre-vascularized stem cell patch implanted into post-MI rat [191]. (I) Vessel formation in pattern C/M patch and migration phenomenon from patch to injured myocardium at week 8 (scale bar, 50mm; S represents the strut of the PCL mesh; BV represents the newly formed blood vessels) [191]. (J) Bioprinting of heart valve conduit with encapsulation of HAVIC within the leaflets [200]. (K) representative image of immunohistochemical staining for α-SMA (green) and vimentin (red), and Draq 5 counterstaining for cell nuclei (blue) [200]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5.

(A) Confocal fluorescence micrograph showing the cross-sectional view of a three-layer scaffold at Day 14, indicating the formation of the endothelium by the HUVECs [192]. (B) Confocal fluorescence micrograph showing the GFP-HUVECs in a single fiber for CD31, GFP, and nuclei [192]. (C) High-resolution confocal fluorescence micrograph showing the distribution of the HUVECs in a single microfiber at Day 14. Left: projection view; Right: 3D rendering of the tubular structure at the position of the dotted line [192]. (D) Immunofluorescence staining of sarcomeric α-actinin (red) and connexin-43 (Cx-43, green) of cardiomyocytes seeded on bioprinted microfibrous scaffolds, showing the sarcomeric banding [192]. (E) Schematic showing a native myocardium containing blood vessels embedded in a matrix of cardiomyocytes [192]. (F) Schematic and high-resolution confocal fluorescence micrograph showing an endothelialized myocardial tissue formed by seeding neonatal rat cardiomyocytes onto the bioprinted endothelialized microfibrous scaffold after 15days of pre-endothelialization [192]. (G, H) Relative beating of the endothelialized myocardial tissues and the levels of vWF expression by the endothelial cells, respectively, upon treatment with different dosages of doxorubicin [192]. (I) The fully printed final device. Insert 1: Confocal microscopy image of immunostained laminar NRVM cardiac tissue on the cantilever surface. Blue, DAPI nuclei stain. White, α-actinin. Scale bar, 10μm. Insert 2: Still images of a cantilever deflecting upon tissue contraction. Insert 3: Example resistance signal [208]. (J) Left, 1: Modified device cantilever containing micro-pin and micro-well to support thicker laminar NRVM tissue. 2: Detail of cantilever with micro-pins. 3: Still images of a cantilever deflecting upon tissue contraction. Right, 1: z-projection of immunostained thicker laminar tissue on the cantilever surface with micro-pin (scale bar, 30μm). 2: x–z line scan of thicker laminar tissue in grooves (scale bar, 10μm). Blue, DAPI nuclei stain. White, α-actinin. Red, actin [208]. (K, L) Dose–response curves for thicker laminar NRVM tissues. Stress values for thicker tissues assumed linear proportional to relative resistance change: σ~ΔR/R0. Stress normalized between maximal and minimal values [208]. (K) Dose–response curve for verapamil (n=4). Error bars are s.e.m. Individual data points included (circles), tissue paced at 1.5Hz. Apparent half maximal effective concentration (EC₅₀) 7.90×10⁻⁷ M [208]. (L) Dose–response curve for isoproterenol (n=3). Error bars are s.e.m. Individual data points included (circles), tissue paced at 1.5Hz. Apparent EC₅₀ 1.16×10⁻⁹M [208]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)