Cardiac Tissue Engineering: A Journey from Scaffold Fabrication to In Vitro Characterization

Abstract

Cardiac tissue engineering has been rapidly evolving with diverse applications, ranging from the repair of fibrotic tissue caused by "adverse remodeling," to the replacement of specific segments of heart tissue, and ultimately to the creation of a whole heart. The repair or replacement of cardiac tissue often involves the development of tissue scaffolds or constructs and the subsequent assessment of their performance and functionality. For this, the design and/or selection of biomaterials, and cell types, scaffold fabrication, and in vitro characterizations are the first starting points, yet critical, to ensure success in subsequent implantation in vivo. This highlights the importance of scaffold fabrication and in vitro experiments/characterization with protocols for cardiac tissue engineering. Yet, a comprehensive and critical review of these has not been established and documented. As inspired, herein, the latest development and advances in scaffold fabrication and in vitro characterization for cardiac tissue engineering are critically reviewed, with focus on biomaterials, cell types, additive manufacturing techniques for scaffold fabrication, and common in vitro characterization techniques or methods. This article would be of benefit to the ones who are working on cardiac tissue engineering by providing insights into the scaffold fabrication and in vitro investigations.

Keywords: 3D‐printing; biomaterials; cell types; electrospinning; manufacturing techniques.

Figure 1

Different approaches for 3D‐printing tissue scaffolds: A) cell‐free scaffolds: materials without cells are 3D‐printed, and the 3D‐printed scaffolds are either used for in vivo studies or first cultured in vitro to grow cells onto/into the scaffold before implantation; B) cell‐laden constructs: materials with cells are 3D‐printed, and then they are either used directly for in vivo studies or they are cultured first to form a 3D tissue construct prior to in vivo studies; and C) pre‐vascularized constructs: cell‐free or cell‐laden scaffolds are 3D‐printed, cultured in vitro to form neovascularization, and then are transplanted into the animal.

Figure 2

Histological images of engineered cardiac tissues captured by light microscopy. A) H&E staining to confirm the penetration of human cardiac stem cells inside the scaffolds composed of (a1–a2) ECM with polysaccharides at a ratio of 75:25 (E75/P25) and (a3–a4) ECM only. Reproduced with permission.^{[ 70 ]} Copyright 2020, Elsevier. B) H&E staining to confirm the biocompatibility of CMs loaded Antheraea mylitta silk fibroin scaffolds on (b1) day 8 and (b2) day 20 (scale bar: 50 μm). Reproduced with permission.^{[ 69 ]} Copyright 2012, Elsevier. C) H&E staining (c1) and Masson trichrome (c2) of bioprinted cardiac patches to confirm presence of the cells and fibrous tissue in the matrix (scale bar: 100 μm). Reproduced with permission.^{[ 71 ]} Copyright 2017, Nature. (license: http://creativecommons.org/licenses/by/4.0/.).

Figure 3

Immunofluorescence staining of different engineered cardiac tissues. A) 3D‐printed constructs seeded with human embryonic cell‐derived cardiomyocytes (hiPSC‐CMs) after 10 days of in vitro culture stained with α‐actinin (a1 and a2) and cardiac troponin T (cTNT, a3 and a4), and DAPI (a5 and a6). (a7) and (a8) Represent the merged images, indicating a promoted striation pattern of the engineered tissues with regularly arranged myofibrils along one long axis. Reproduced with permission.^{[ 27 ]} Copyright 2023, John Wiley and Sons. (licence: https://creativecommons.org/licenses/). B) Scaffold‐free 3D‐printed model for cardiac cells embedded in fibrin‐based biomaterial after 7 days of in vitro culture stained with Cx43 antibody and Hoechst (b1), single plane images corresponding to (b1): F‐actin alone (b2), Cx43 alone (b3), and merge of all channel (b4). The staining shows the cardiac cellular morphology that mimic native cardiac cell culture as well as overexpression of CX43 due to enhanced functional organization of cardiac cells within the engineered cardiac tissue. Reproduced with permission.^{[ 48 ]} Copyright 2022, Elsevier. C) Electrospun scaffolds 6 days after in vitro incubation in differentiation media stained with MYH2 (green) and DAPI (blue) composed of (c1) PLA, (c2) PLA/PANI1.5, and (c3) PLA/PANI3. The images demonstrate the impact of conductivity on the maturity of myotubes, revealing that more mature myotubes were formed on PLA/PANI1.5 and PLA/PANI3 compared to PLA nanofibrous sheets. Reproduced with permission.^{[ 78 ]} Copyright 2017, Elsevier.

Figure 4

Calcium transients imaging on A) PLA, PLA/PANI1.5, and PLA/PANI3 nanofibrous sheets. ROIs were randomly assigned in a) PLA, b) PLA/PANI1.5, and c) PLA/PANI3 groups. (d–f) Spontaneous calcium transients are shown through line scanning across each ROI (scale bar:10 s). (g–i) F/F0 representing the normalized fluorescence intensity of each ROI. Reproduced with permission.^{[ 78 ]} Copyright 2017, Elsevier. B) Nanoengineered peptide‐based conductive biomaterial. (a–d) Calcium transient imaging analysis for seven assigned ROI. (e–h)Calcium transient imaging analysis for each ROI in the presence of an electrical stimulation. (a,d) F/F0 for each ROI; (b,f) Calcium flux delay between different ROI; (c,g) The seven separate sites representing each ROI; (d,h) Heat map of propagation of Ca2+ signal. Reproduced with permission.^{[ 87 ]} Copyright 2021, Wiley & Sons.