Wet-Spun Polycaprolactone Scaffolds Provide Customizable Anisotropic Viscoelastic Mechanics for Engineered Cardiac Tissues.

Myocardial infarction is a leading cause of death worldwide and has severe consequences including irreversible damage to the myocardium, which can lead to heart failure. Cardiac tissue engineering aims to re-engineer the infarcted myocardium using tissues made from human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to regenerate heart muscle and restore contractile function via an implantable epicardial patch. The current limitations of this technology include both biomanufacturing challenges in maintaining tissue integrity during implantation and biological challenges in inducing cell alignment, maturation, and coordinated electromechanical function, which, when overcome, may be able to prevent adverse cardiac remodeling through mechanical support in the injured heart to facilitate regeneration. Polymer scaffolds serve to mechanically reinforce both engineered and host tissues. Here, we introduce a novel biodegradable, customizable scaffold composed of wet-spun polycaprolactone (PCL) microfibers to strengthen engineered tissues and provide an anisotropic mechanical environment to promote engineered tissue formation. We developed a wet-spinning process to produce consistent fibers which are then collected on an automated mandrel that precisely controls the angle of intersection of fibers and their spacing to generate mechanically anisotropic scaffolds. Through optimization of the wet-spinning process, we tuned the fiber diameter to 339 ± 31 µm and 105 ± 9 µm and achieved a high degree of fidelity in the fiber structure within the scaffold (fiber angle within 1.8° of prediction). Through degradation and mechanical testing, we demonstrate the ability to maintain scaffold mechanical integrity as well as tune the mechanical environment of the scaffold through structure (Young's modulus of 120.8 ± 1.90 MPa for 0° scaffolds, 60.34 ± 11.41 MPa for 30° scaffolds, 73.59 ± 3.167 MPa for 60° scaffolds, and 49.31 ± 6.90 MPa for 90° scaffolds), while observing decreased hysteresis in angled vs. parallel scaffolds. Further, we embedded the fibrous PCL scaffolds in a collagen hydrogel mixed with hiPSC-CMs to form engineered cardiac tissue with high cell survival, tissue compaction, and active contractility of the hiPSC-CMs. Through this work, we develop and optimize a versatile biomanufacturing process to generate customizable PCL fibrous scaffolds which can be readily utilized to guide engineered tissue formation and function.

Stimulating Calcium Handling in hiPSC-Derived Engineered Cardiac Tissues Enhances Force Production.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have profound utility in generating functional human engineered cardiac tissues (ECT) for heart repair. However, the field at large is concerned about the relative immaturity of these hiPSC-CMs as we aim to develop clinically relevant models for regenerative therapy and drug testing. Herein, we develop a novel calcium (Ca2+) conditioning protocol that maintains ECTs in a physiological range of Ca2+ and assesses contractility in increasing calcium environments. Lactate-based selection served as a method to purify and shift the metabolic profile of hiPSC-CMs to evaluate the role of metabolism on Ca2+ sensitivity. After 2 weeks, we observe 2-fold greater peak twitch stress in high-Ca2+ conditioned ECTs, despite having lower stiffness and no change in Ca2+ sensitivity of twitch force. Interestingly, the force-calcium relationship reveals higher Ca2+ sensitivity in lactate conditioned tissues, suggesting that metabolic maturation alters mitochondrial Ca2+ buffering and regulation. Ca2+ sensitivity and force amplitude are not coupled, as lactate conditioned tissues produce force comparable to that of controls in high calcium environments. An upregulation of calcium handling protein gene expression likely contributes to the greater Ca2+ sensitivity in lactate conditioned hiPSC-CMs. Our findings support the use of physiological Ca2+ to enhance the functional maturation of excitation-contraction coupling in hiPSC-CMs and demonstrate that metabolic changes induced by lactate conditioning alter cardiomyocyte sensitivity to external Ca2+. These conditioning methods may be used to advance the development of engineered human cardiac tissue for translational applications in vitro and in vivo as a regenerative therapy.

Engineering a collagen matrix for cell-instructive regenerative angiogenesis.

Engineering an angiogenic material for regenerative medicine requires knowledge of native extracellular matrix remodeling by cellular processes in angiogenesis. Vascularization remains a key challenge in the field of tissue engineering, one that can be mitigated by developing platforms conducive to guiding dynamic cell-matrix interactions required for new vessel formation. In this review, we highlight nuanced processes of angiogenesis and demonstrate how materials engineering is being used to interface with dynamic type I collagen remodeling, Notch and VEGF signaling, cell migration, and tissue morphogenesis. Because α1(I)-collagen is secreted by endothelial tip cells during sprouting angiogenesis and required for migration, collagen is a very useful natural biomaterial and its angiogenic modifications are described. The balance between collagen types I and IV via secretion and degradation is tightly controlled by proteinases and other cell types that are capable of internalizing collagen to maintain tissue integrity. Thus, we provide examples in skin and cardiac tissue engineering of collagen tailoring in diverse cellular microenvironments for tissue regeneration. As our understanding of how to drive collagen remodeling and cellular phenotype through angiogenic pathways grows, our capabilities to model and manipulate material systems must continue to expand to develop novel applications for wound healing, angiogenic therapy, and regenerative medicine.

Optimizing Blended Collagen-Fibrin Hydrogels for Cardiac Tissue Engineering with Human iPSC-derived Cardiomyocytes.

Natural polymer hydrogels are used ubiquitously as scaffold materials for cardiac tissue engineering as well as for soft tissue engineering more broadly because of FDA approval, minimal immunogenicity, and well-defined physiological clearance pathways. However, the relationships between natural polymer hydrogels and resident cell populations in directing the development of engineered tissues are poorly defined. This interaction is of particular concern for tissues prepared with iPSC-derived cell populations, in which population purity and batch-to-batch variability become additional critical factors to consider. Herein, the design space for a blended fibrin and collagen scaffold is characterized for applications in creating engineered myocardium with human iPSC-derived cardiomyocytes. Stiffness values of the acellular hydrogel formulations approach those of native myocardium in compression, but deviate significantly in tension when compared to rat myocardium in both transverse and longitudinal fiber orientations. A response surface methodology approach to understanding the relationship between collagen concentration, fibrin concentration, seeding density, and cardiac purity found a statistically significant predictive model across three repeated studies that confirms that all of these factors contribute to tissue compaction. In these constructs, increased fibrin concentration and seeding density were each associated with increased compaction, while increased collagen concentration was associated with decreased compaction. Both the lowest (24.4% cTnT+) and highest (60.2% cTnT+) cardiomyocyte purities evaluated were associated with decreased compaction, whereas the greatest compaction was predicted to occur in constructs prepared with a 40-50% cTnT+ population. Constructs prepared with purified cardiomyocytes (≥75.5% cTnT+) compacted and formed syncytia well, although increased fibrin concentration in these groups was associated with decreased compaction, a reversal of the trend observed in unpurified cardiomyocytes. This study demonstrates an analytical approach to understanding cell-scaffold interactions in engineered tissues and provides a foundation for the development of more sophisticated and customized scaffold platforms for human cardiac tissue engineering.