3D bioprinted functional and contractile cardiac tissue constructs

Abstract

Bioengineering of a functional cardiac tissue composed of primary cardiomyocytes has great potential for myocardial regeneration and in vitro tissue modeling. However, its applications remain limited because the cardiac tissue is a highly organized structure with unique physiologic, biomechanical, and electrical properties. In this study, we undertook a proof-of-concept study to develop a contractile cardiac tissue with cellular organization, uniformity, and scalability by using three-dimensional (3D) bioprinting strategy. Primary cardiomyocytes were isolated from infant rat hearts and suspended in a fibrin-based bioink to determine the priting capability for cardiac tissue engineering. This cell-laden hydrogel was sequentially printed with a sacrificial hydrogel and a supporting polymeric frame through a 300-µm nozzle by pressured air. Bioprinted cardiac tissue constructs had a spontaneous synchronous contraction in culture, implying in vitro cardiac tissue development and maturation. Progressive cardiac tissue development was confirmed by immunostaining for α-actinin and connexin 43, indicating that cardiac tissues were formed with uniformly aligned, dense, and electromechanically coupled cardiac cells. These constructs exhibited physiologic responses to known cardiac drugs regarding beating frequency and contraction forces. In addition, Notch signaling blockade significantly accelerated development and maturation of bioprinted cardiac tissues. Our results demonstrated the feasibility of bioprinting functional cardiac tissues that could be used for tissue engineering applications and pharmaceutical purposes.

Statement of significance: Cardiovascular disease remains a leading cause of death in the United States and a major health-care burden. Myocardial infarction (MI) is a main cause of death in cardiovascular diseases. MI occurs as a consequence of sudden blocking of blood vessels supplying the heart. When occlusions in the coronary arteries occur, an immediate decrease in nutrient and oxygen supply to the cardiac muscle, resulting in permanent cardiac cell death. Eventually, scar tissue formed in the damaged cardiac muscle that cannot conduct electrical or mechanical stimuli thus leading to a reduction in the pumping efficiency of the heart. The therapeutic options available for end-stage heart failure is to undergo heart transplantation or the use of mechanical ventricular assist devices (VADs). However, many patients die while being on a waiting list, due to the organ shortage and limitation of VADs, such as surgical complications, infection, thrombogenesis, and failure of the electrical motor and hemolysis. Ultimately, 3D bioprinting strategy aims to create clinically applicable tissue constructs that can be immediately implanted in the body. To date, the focus on replicating complex and heterogeneous tissue constructs continues to increase as 3D bioprinting technologies advance. In this study, we demonstrated the feasibility of 3D bioprinting strategy to bioengineer the functional cardiac tissue that possesses a highly organized structure with unique physiological and biomechanical properties similar to native cardiac tissue. This bioprinting strategy has great potential to precisely generate functional cardiac tissues for use in pharmaceutical and regenerative medicine applications.

Keywords: Bioprinting; Body-on-a-chip; Cardiac tissue; Contractility; Heart failure; In vitro tissue model; Tissue engineering.

Fig. 1

Bioprinting of cardiac tissue constructs. (A) Basic motion program and (B,C) our customized ITOP system containing three components for bioprinting cardiac tissues. (D) Timelapse image sequence of cardiac tissue printing. Bioprinted cardiac tissue constructs: (E) string form and (F) patch form.

Fig. 2

(A) Gross appearance and cardiac beating measurement with time: (left) progress tissue development of the patch formed cardiac tissue was observed and (right) plotting of spontaneous beating based on bright field video with time. (B) Immunofluorescent images of bioprinted cardiac tissues: α-actinin at 4 days and 1 week in culture and α-actinin/connexin 43 at 3 weeks after bioprinting. Bioprinted cardiomyocytes expressed α-actinin (red), connexin (green) and cell nuclei (blue). (C) Quantification of cardiac tissue development at 4 days, 1, and 3 weeks in culture (n = 3). *P < 0.05 compared with control. **P < 0.05 compared with others. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Calcium imaging analysis of bioprinted cardiac tissues at 1 and 3 weeks in culture. (A) Immunofluorescence for α-actinin of bioprinted cardiac tissues at 1 and 3 weeks in culture, representing different levels on cellular organization and tissue development: α-actinin (red) and cell nuclei (blue). (B) Calcium imaging analysis for synchronization of bioprinted cardiac tissues at 1 and 3 weeks in culture (Supplementary Movie 1(b)). At 3 weeks, bioprinted cardiac tissue showed regular synchronous contractile behavior. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Cardiac drug responses. (A) Bioprinted cardiac tissue responded to epinephrine (200 nM) and carbachol (CCH, 10 μM) as confirmed by changes in beating frequency and amplitude (Supplementary Movie 1(c)). (B) Quantification of beating frequency (beats per minute, BPM) of bioprinted cardiac tissues, responding to cardiac drugs (n = 3). *,**P < 0.05 compared with control (baseline).

Fig. 5

Contractile force measurement of bioprinted cardiac tissues by micro-spring integration. (A) Printed micro-spring device used to measure contractile force generated by bioprinted cardiac tissues: (a) Illustration of micro-spring motion during diastolic and systolic phases of cardiac contraction cycle in bioprinted cardiac tissue. (b) Gross images of micro-spring for force measurement. (B) Motion (μm) derived from the contraction of bioprinted cardiac tissue was converted to force (mN). Dashed line shows 95% confidence range of linear aggregation. (C) Representative plots of the force generated by bioprinted cardiac tissues in response to different doses of epinephrine (Supplementary Movie 2). (D) Quantification of peak force generated by bioprinted cardiac tissues in response to different doses of epinephrine (n = 3). *P < 0.05 compared with control.

Fig. 6

Notch signaling blockade on bioprinted cardiac tissues. (A) Calcium images on synchronous contraction of bioprinted cardiac tissues with and without DAPT treatment at 1 week in culture, Notch signaling blockade (DAPT) resulted in early formation of synchronous contraction, while no synchronous contraction in control (non-treated). Scale bar = 100 μm. (B) Plotting of beating frequency of bioprinted cardiac tissues from Notch signaling blockade and control groups at 1 week in culture. (C) Immunofluorescent analyses of bioprinted cardiac tissues with and without DAPT treatment at 1 week in culture: α-actinin (red) and cell nuclei (blue). Scale bar = 100 μm. (D) Quantification of cardiac tissue development by measuring frequency of α-actinin positive cells, cardiomyocyte area, cardiac muscle cell alignment, and cardiomyocytes perimeter in Notch blockade and control groups (n = 3). **P < 0.05 compared with control. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)