Characterization of cardiac fibroblast-extracellular matrix crosstalk across developmental ages provides insight into age-related changes in cardiac repair.

Heart failure afflicts an estimated 6.5 million people in the United States, driven largely by incidents of coronary heart disease (CHD). CHD leads to heart failure due to the inability of adult myocardial tissue to regenerate after myocardial infarction (MI). Instead, immune cells and resident cardiac fibroblasts (CFs), the cells responsible for the maintenance of the cardiac extracellular matrix (cECM), drive an inflammatory wound healing response, which leads to fibrotic scar tissue. However, fibrosis is reduced in fetal and early (<1-week-old) neonatal mammals, which exhibit a transient capability for regenerative tissue remodeling. Recent work by our laboratory and others suggests this is in part due to compositional differences in the cECM and functional differences in CFs with respect to developmental age. Specifically, fetal cECM and CFs appear to mitigate functional loss in MI models and engineered cardiac tissues, compared to adult CFs and cECM. We conducted 2D studies of CFs on solubilized fetal and adult cECM to investigate whether these age-specific functional differences are synergistic with respect to their impact on CF phenotype and, therefore, cardiac wound healing. We found that the CF migration rate and stiffness vary with respect to cell and cECM developmental age and that CF transition to a fibrotic phenotype can be partially attenuated in the fetal cECM. However, this effect was not observed when cells were treated with cytokine TGF-ß1, suggesting that inflammatory signaling factors are the dominant driver of the fibroblast phenotype. This information may be valuable for targeted therapies aimed at modifying the CF wound healing response and is broadly applicable to age-related studies of cardiac remodeling.

Extracellular matrix and cyclic stretch alter fetal cardiomyocyte proliferation and maturation in a rodent model of heart hypoplasia.

Introduction: Birth defects, particularly those that affect development of the heart, are a leading cause of morbidity and mortality in infants and young children. Babies born with heart hypoplasia (heart hypoplasia) disorders often have a poor prognosis. It remains unclear whether cardiomyocytes from hypoplastic hearts retain the potential to recover growth, although this knowledge would be beneficial for developing therapies for heart hypoplasia disorders. The objective of this study was to determine the proliferation and maturation potential of cardiomyocytes from hypoplastic hearts and whether these behaviors are influenced by biochemical signaling from the extracellular matrix (ECM) and cyclic mechanical stretch. Method: Congenital diaphragmatic hernia (CDH)-associated heart hypoplasia was induced in rat fetuses by maternal exposure to nitrofen. Hearts were isolated from embryonic day 21 nitrofen-treated fetuses positive for CDH (CDH+) and from fetuses without nitrofen administration during gestation. Results and discussion: CDH+ hearts were smaller and had decreased myocardial proliferation, along with evidence of decreased maturity compared to healthy hearts. In culture, CDH+ cardiomyocytes remained immature and demonstrated increased proliferative capacity compared to their healthy counterparts. Culture on ECM derived from CDH+ hearts led to a significant reduction in proliferation for both CDH+ and healthy cardiomyocytes. Healthy cardiomyocytes were dosed with exogenous nitrofen to examine whether nitrofen may have an aberrant effect on the proliferative ability of cardiomyocyte, yet no significant change in proliferation was observed. When subjected to stretch, CDH+ cardiomyocytes underwent lengthening of sarcomeres while healthy cardiomyocyte sarcomeres were unaffected. Taken together, our results suggest that alterations to environmental cues such as ECM and stretch may be important factors in the pathological progression of heart hypoplasia.

Evaluation of rapid transepithelial electrical resistance (TEER) measurement as a metric of kidney toxicity in a high-throughput microfluidic culture system.

Rapid non-invasive kidney-specific readouts are essential to maximizing the potential of microfluidic tissue culture platforms for drug-induced nephrotoxicity screening. Transepithelial electrical resistance (TEER) is a well-established technique, but it has yet to be evaluated as a metric of toxicity in a kidney proximal tubule (PT) model that recapitulates the high permeability of the native tissue and is also suitable for high-throughput screening. We utilized the PREDICT96 high-throughput microfluidic platform, which has rapid TEER measurement capability and multi-flow control, to evaluate the utility of TEER sensing for detecting cisplatin-induced toxicity in a human primary PT model under both mono- and co-culture conditions as well as two levels of fluid shear stress (FSS). Changes in TEER of PT-microvascular co-cultures followed a dose-dependent trend similar to that demonstrated by lactate dehydrogenase (LDH) cytotoxicity assays and were well-correlated with tight junction coverage after cisplatin exposure. Additionally, cisplatin-induced changes in TEER were detectable prior to increases in cell death in co-cultures. PT mono-cultures had a less differentiated phenotype and were not conducive to toxicity monitoring with TEER. The results of this study demonstrate that TEER has potential as a rapid, early, and label-free indicator of toxicity in microfluidic PT-microvascular co-culture models.

Right Ventricular Outflow Tract Surgical Resection in Young, Large Animal Model for the Study of Alternative Cardiovascular Patches.

Tetralogy of Fallot (ToF) is a severe congenital heart defect (CHD) that requires surgical reconstruction soon after birth. Reconstructive surgery involves the implantation of synthetic cardiovascular patches to widen the right ventricular outflow tract (RVOT) and repair defects in the septal wall. However, synthetic patches can cause complications for these patients later in life as they do not integrate or adapt in the tissue of a growing patient; a limitation that could be solved with the development of a patch fabricated from a degradable biomaterial. Unfortunately, the lack of appropriate pre-clinical models has hindered the development of novel patch materials. Currently, most studies use rodent models to study the efficacy of new patch materials; however, large animal models are necessary to develop realistically sized patches in a clinically relevant growing heart where gradients in diffusion and length scales for cell migration are more similar to the human. Here, we describe a novel method by which a Satinsky vascular clamp is used to isolate RVOT muscle for resection followed by implantation of a cardiovascular patch in an appropriately young, rapidly growing porcine model.

Self-Assembled Human Skin Equivalents Model Macrophage Activation of Cutaneous Fibrogenesis in Systemic Sclerosis.

OBJECTIVE: The development of precision therapeutics for systemic sclerosis (SSc) has been hindered by the lack of models that accurately mimic the disease in vitro. This study was undertaken to design and test a self-assembled skin equivalent (saSE) system that recapitulates the cross-talk between macrophages and fibroblasts in cutaneous fibrosis. METHODS: SSc-derived dermal fibroblasts (SScDFs) and normal dermal fibroblasts (NDFs) were cultured with CD14+ monocytes from SSc patients or healthy controls to allow de novo stroma formation. Monocyte donor-matched plasma was introduced at week 3 prior to seeding keratinocytes to produce saSE with a stratified epithelium. Tissue was characterized by immunohistochemical staining, atomic force microscopy, enzyme-linked immunosorbent assay, and quantitative reverse transcriptase-polymerase chain reaction. RESULTS: Stroma synthesized de novo from NDFs and SScDFs supported a fully stratified epithelium to form saSE. A thicker and stiffer dermis was generated by saSE with SScDFs, and more interleukin-6 and transforming growth factor ß (TGFß) was secreted by saSE with SScDFs compared to saSE with NDFs, regardless of the inclusion of monocytes. Tissue with SSc monocytes and plasma had amplified dermal thickness and stiffness relative to control tissue. Viable CD163+ macrophages were found within the stroma of saSE 5 weeks after seeding. Additionally, SSc saSE contained greater numbers of CD163+ and CD206+ macrophages compared to control saSE. TGFß blockade inhibited stromal stiffness to a greater extent in SSc saSE compared to control saSE. CONCLUSION: These data suggest reciprocal activation between macrophages and fibroblasts that increases tissue thickness and stiffness, which is dependent in part on TGFß activation. The saSE system may serve as a platform for preclinical therapeutic testing and for molecular characterization of SSc skin pathology through recapitulation of the interactions between macrophages and fibroblasts.

RNA sequencing indicates age-dependent shifts in the cardiac fibroblast transcriptome between fetal, neonatal, and adult developmental ages.

Cardiac fibroblasts are responsible for extracellular matrix turnover and repair in the cardiac environment and serve to help facilitate immune responses. However, it is well established that they have a significant phenotypic heterogeneity with respect to location, physiological conditions, and developmental age. The goal of this study was to provide an in-depth transcriptomic profile of cardiac fibroblasts derived from rat hearts at fetal, neonatal, and adult developmental ages to ascertain variations in gene expression that may drive functional differences in these cells at these specific stages of development. We performed RNA sequencing (RNA-seq) of cardiac fibroblasts isolated from fetal, neonatal, and adult rats and compared with the rat genome. Principal component analysis of RNA-seq data suggested that data variance was predominantly due to developmental age. Differential expression and gene set enrichment analysis against Gene Ontology and Kyoto Encyclopedia of Genes and Genomes datasets indicated an array of differences across developmental ages, including significant decreases in cardiac development and cardiac function-associated genes with age and a significant increase in immune- and inflammatory-associated functions, particularly immune cell signaling and cytokine and chemokine production, with respect to increasing developmental age. These results reinforce established evidence of diverse phenotypic heterogeneity of fibroblasts with respect to developmental age. Furthermore, based on our analysis of gene expression, age-specific alterations in cardiac fibroblasts may play a crucial role in observed differences in cardiac inflammation and immune response observed across developmental ages.

The effects of membrane potential and extracellular matrix composition on vascular differentiation of cardiac progenitor cells.

Historically, the field of tissue engineering has been adept at modulating the chemical and physical microenvironment. This approach has yielded significant progress, but it is imperative to further integrate our understanding of other fundamental cell signaling paradigms into tissue engineering methods. Bioelectric signaling has been demonstrated to be a vital part of tissue development, regeneration, and function across organ systems and the extracellular matrix is known to alter the bioelectric properties of cells. Thus, there is a need to bolster our understanding of how matrix and bioelectric signals interact to drive cell phenotype. We examine how cardiac progenitor cell differentiation is altered by simultaneous changes in both resting membrane potential and extracellular matrix composition. Pediatric c-kit+ cardiac progenitor cells were differentiated on fetal or adult cardiac extracellular matrix while being treated with drugs that alter resting membrane potential. Smooth muscle gene expression was increased with depolarization and decreased with hyperpolarization while endothelial and cardiac expression were unchanged. Early smooth muscle protein expression is modified by matrix developmental age, with fetal ECM appearing to amplify the effects of resting membrane potential. Thus, combining matrix composition and bioelectric signaling represents a potential alternative for guiding cell behavior in tissue engineering and regenerative medicine.

Stimuli-responsive composite biopolymer actuators with selective spatial deformation behavior.

Bioinspired actuators with stimuli-responsive and deformable properties are being pursued in fields such as artificial tissues, medical devices and diagnostics, and intelligent biosensors. These applications require that actuator systems have biocompatibility, controlled deformability, biodegradability, mechanical durability, and stable reversibility. Herein, we report a bionic actuator system consisting of stimuli-responsive genetically engineered silk-elastin-like protein (SELP) hydrogels and wood-derived cellulose nanofibers (CNFs), which respond to temperature and ionic strength underwater by ecofriendly methods. Programmed site-selective actuation can be predicted and folded into three-dimensional (3D) origami-like shapes. The reversible deformation performance of the SELP/CNF actuators was quantified, and complex spatial transformations of multilayer actuators were demonstrated, including a biomimetic flower design with selective petal movements. Such actuators consisting entirely of biocompatible and biodegradable materials will offer an option toward constructing stimuli-responsive systems for in vivo biomedicine soft robotics and bionic research.

Heart-on-a-Chip Model with Integrated Extra- and Intracellular Bioelectronics for Monitoring Cardiac Electrophysiology under Acute Hypoxia.

We demonstrated a bioelectronic heart-on-a-chip model for studying the effects of acute hypoxia on cardiac function. A microfluidic channel enabled rapid modulation of medium oxygenation, which mimicked the regimes induced by a temporary coronary occlusion and reversibly activated hypoxia-related transduction pathways in HL-1 cardiac model cells. Extracellular bioelectronics provided continuous readouts demonstrating that hypoxic cells experienced an initial period of tachycardia followed by a reduction in beat rate and eventually arrhythmia. Intracellular bioelectronics consisting of Pt nanopillars temporarily entered the cytosol following electroporation, yielding action potential (AP)-like readouts. We found that APs narrowed during hypoxia, consistent with proposed mechanisms by which oxygen deficits activate ATP-dependent K+ channels that promote membrane repolarization. Significantly, both extra- and intracellular devices could be multiplexed, enabling mapping capabilities unachievable by other electrophysiological tools. Our platform represents a significant advance toward understanding electrophysiological responses to hypoxia and could be applicable to disease modeling and drug development.

Systemic Sclerosis Dermal Fibroblasts Induce Cutaneous Fibrosis Through Lysyl Oxidase-like 4: New Evidence From Three-Dimensional Skin-like Tissues.

OBJECTIVE: Systemic sclerosis (SSc) is a clinically heterogeneous disease characterized by increased collagen accumulation and skin stiffness. Our previous work has demonstrated that transforming growth factor ß (TGFß) induces extracellular matrix (ECM) modifications through lysyl oxidase-like 4 (LOXL-4), a collagen crosslinking enzyme, in bioengineered human skin equivalents (HSEs) and self-assembled stromal tissues (SAS). We undertook this study to investigate cutaneous fibrosis and the role of LOXL-4 in SSc pathogenesis using HSEs and SAS. METHODS: SSc-derived dermal fibroblasts (SScDFs; n = 8) and normal dermal fibroblasts (NDFs; n = 6) were incorporated into HSEs and SAS. These 3-dimensional skin-like microenvironments were used to study the effects of dysregulated LOXL-4 on ECM remodeling, fibroblast activation, and response to TGFß stimulation. RESULTS: SScDF-containing SAS showed increased stromal thickness, collagen deposition, and interleukin-6 secretion compared to NDF-containing SAS (P < 0.05). In HSE, SScDFs altered collagen as seen by a more mature and aligned fibrillar structure (P < 0.05). With SScDFs, enhanced stromal rigidity with increased collagen crosslinking (P < 0.05), up-regulation of LOXL4 expression (P < 0.01), and innate immune signaling genes were observed in both tissue models. Conversely, knockdown of LOXL4 suppressed rigidity, contraction, and α-smooth muscle actin expression in SScDFs in HSE, and TGFß-induced ECM aggregation and collagen crosslinking in SAS. CONCLUSION: A limitation to the development of effective therapeutics in SSc is the lack of in vitro human model systems that replicate human skin. Our findings demonstrate that SAS and HSE can serve as complementary in vitro skin-like models for investigation of the mechanisms and mediators that drive fibrosis in SSc and implicate a pivotal role for LOXL-4 in SSc pathogenesis.

Functional maturation of human neural stem cells in a 3D bioengineered brain model enriched with fetal brain-derived matrix.

Brain extracellular matrix (ECM) is often overlooked in vitro brain tissue models, despite its instructive roles during development. Using developmental stage-sourced brain ECM in reproducible 3D bioengineered culture systems, we demonstrate enhanced functional differentiation of human induced neural stem cells (hiNSCs) into healthy neurons and astrocytes. Particularly, fetal brain tissue-derived ECM supported long-term maintenance of differentiated neurons, demonstrated by morphology, gene expression and secretome profiling. Astrocytes were evident within the second month of differentiation, and reactive astrogliosis was inhibited in brain ECM-enriched cultures when compared to unsupplemented cultures. Functional maturation of the differentiated hiNSCs within fetal ECM-enriched cultures was confirmed by calcium signaling and spectral/cluster analysis. Additionally, the study identified native biochemical cues in decellularized ECM with notable comparisons between fetal and adult brain-derived ECMs. The development of novel brain-specific biomaterials for generating mature in vitro brain models provides an important path forward for interrogation of neuron-glia interactions.

3D extracellular matrix microenvironment in bioengineered tissue models of primary pediatric and adult brain tumors.

Dynamic alterations in the unique brain extracellular matrix (ECM) are involved in malignant brain tumors. Yet studies of brain ECM roles in tumor cell behavior have been difficult due to lack of access to the human brain. We present a tunable 3D bioengineered brain tissue platform by integrating microenvironmental cues of native brain-derived ECMs and live imaging to systematically evaluate patient-derived brain tumor responses. Using pediatric ependymoma and adult glioblastoma as examples, the 3D brain ECM-containing microenvironment with a balance of cell-cell and cell-matrix interactions supports distinctive phenotypes associated with tumor type-specific and ECM-dependent patterns in the tumor cells' transcriptomic and release profiles. Label-free metabolic imaging of the composite model structure identifies metabolically distinct sub-populations within a tumor type and captures extracellular lipid-containing droplets with potential implications in drug response. The versatile bioengineered 3D tumor tissue system sets the stage for mechanistic studies deciphering microenvironmental role in brain tumor progression.

Knockdown of CD44 expression decreases valve interstitial cell calcification in vitro.

The lack of pharmaceutical targets available to treat patients with calcific aortic valve disease (CAVD) necessitates further research into the specific mechanisms of the disease. The significant changes that occur to the aortic valves extracellular matrix (ECM) during the progression of CAVD suggests that these proteins may play an important role in calcification. Exploring the relationship between valve interstitial cells (VICs) and the ECM may lead to a better understand of CAVD mechanisms and potential pharmaceutical targets. In this study, we look at the effect of two ECM components, collagen and hyaluronic acid (HA), on the mineralization of VICs within the context of a two-dimensional, polyacrylamide (PAAM) model system. Using a novel, nondestructive imaging technique, we were able to track calcific nodule development in culture systems over a 3-wk time frame. We saw a significant increase in the size of the nodules grown on HA PAAM gels as compared with collagen PAAM gels, suggesting that HA has a direct effect on mineralization. Directly looking at the two known receptors of HA, CD44 and receptor for HA-mediated motility (RHAMM), and using siRNA knockdown revealed that a decrease in CD44 expression resulted in a reduction of calcification. A decrease in CD44, through siRNA knockdown, reduces mineralization on HA PAAM gels, suggesting a potential new target for CAVD treatment. NEW & NOTEWORTHY Our in vitro model of calcific aortic valve disease shows an interaction between the hyaluronic acid binding protein CD44 with the osteogenic factor OPN as a potential mechanism of aortic valve calcification. Using siRNA knockdown of CD44, we show an upregulation of OPN expression with a decrease in overall mineralization.

Bioengineered in Vitro Tissue Model of Fibroblast Activation for Modeling Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a complex disease of unknown etiology with no current curative treatment. Modeling pulmonary fibrotic (PF) tissue has the potential to improve our understanding of IPF disease progression and treatment. Rodent animal models do not replicate human fibroblastic foci (Hum-FF) pathology, and current iterations of in vitro model systems (e.g., collagen hydrogels, polyacrylamide hydrogels, and fibrosis-on-chip systems) are unable to replicate the three-dimensional (3D) complexity and biochemical composition of human PF tissue. Herein, we fabricated a 3D bioengineered pulmonary fibrotic (Eng-PF) tissue utilizing cell laden silk collagen type I dityrosine cross-linked hydrogels and Flexcell bioreactors. We show that silk collagen type I hydrogels have superior stability and mechanical tunability compared to other hydrogel systems. Using customized Flexcell bioreactors, we reproduced Hum-FF-like pathology with airway epithelial and microvascular endothelial cells. Eng-PF tissues can model myofibroblast differentiation and permit evaluation of antifibrotic drug treatments. Further, Eng-PF tissues could be used to model different facets of IPF disease, including epithelial injury with the addition of bleomycin and cellular recruitment by perfusion of cells through the hydrogel microchannel.

Applications of Cardiac Extracellular Matrix in Tissue Engineering and Regenerative Medicine.

The role of the cardiac extracellular matrix (cECM) in providing biophysical and biochemical cues to the cells housed within during disease and development has become increasingly apparent. These signals have been shown to influence many fundamental cardiac cell behaviors including contractility, proliferation, migration, and differentiation. Consequently, alterations to cell phenotype result in directed remodeling of the cECM. This bidirectional communication means that the cECM can be envisioned as a medium for information storage. As a result, the reprogramming of the cECM is increasingly being employed in tissue engineering and regenerative medicine as a method with which to treat disease. In this chapter, an overview of the composition and structure of the cECM as well as its role in cardiac development and disease will be provided. Additionally, therapeutic modulation of cECM for cardiac regeneration as well as bottom-up and top-down approaches to ECM-based cardiac tissue engineering is discussed. Finally, lingering questions regarding the role of cECM in tissue engineering and regenerative medicine are offered as a catalyst for future research.

Conditional deletion of RB1 in the Tie2 lineage leads to aortic valve regurgitation.

OBJECTIVE: Aortic valve disease is a complex process characterized by valve interstitial cell activation, disruption of the extracellular matrix culminating in valve mineralization occurring over many years. We explored the function of the retinoblastoma protein (pRb) in aortic valve disease, given its critical role in mesenchymal cell differentiation including bone development and mineralization. APPROACH AND RESULTS: We generated a mouse model of conditional pRb knockout (cKO) in the aortic valve regulated by Tie2-Cre-mediated excision of floxed RB1 alleles. Aged pRb cKO animals showed significantly more aortic valve regurgitation by echocardiography compared to pRb het control animals. The pRb cKO aortic valves had increased leaflet thickness without increased cellular proliferation. Histologic studies demonstrated intense α-SMA expression in pRb cKO leaflets associated with disorganized extracellular matrix and increased leaflet stiffness. The pRb cKO mice also showed increased circulating cytokine levels. CONCLUSIONS: Our studies demonstrate that pRb loss in the Tie2-lineage that includes aortic valve interstitial cells is sufficient to cause age-dependent aortic valve dysfunction.

Engineered cell and tissue models of pulmonary fibrosis.

Pulmonary fibrosis includes several lung disorders characterized by scar formation and Idiopathic Pulmonary Fibrosis (IPF) is a particularly severe form of pulmonary fibrosis of unknown etiology with a mean life expectancy of 3years' post-diagnosis. Treatments for IPF are limited to two FDA approved drugs, pirfenidone and nintedanib. Most lead candidate drugs that are identified in pre-clinical animal studies fail in human clinical trials. Thus, there is a need for advanced humanized in vitro models of the lung to improve candidate treatments prior to moving to human clinical trials. The development of 3D tissue models has created systems capable of emulating human lung structure, function, and cell and matrix interactions. The specific models accomplish these features and preliminary studies conducted using some of these systems have shown potential for in vitro anti-fibrotic drug testing. Further characterization and improvements will enable these tissue models to extend their utility for in vitro drug testing, to help identify signaling pathways and mechanisms for new drug targets, and potentially reduce animal models as standard pre-clinical models of study. In the current review, we contrast different in vitro models based on increasing dimensionality (2D, 2.5D and 3D), with added focus on contemporary 3D pulmonary models of fibrosis.

Bioengineering Human Lung Grafts on Porcine Matrix.

OBJECTIVE: Bioengineering of viable, functional, and implantable human lung grafts on porcine matrix. SUMMARY BACKGROUND DATA: Implantable bioartificial organ grafts could revolutionize transplant surgery. To date, several milestones toward that goal have been achieved in rodent models. To make bioengineered organ grafts clinically relevant, scaling to human cells and graft size are the next steps. METHODS: We seeded porcine decellularized lung scaffolds with human airway epithelial progenitor cells derived from rejected donor lungs, and banked human umbilical vein endothelial cells. We subsequently enabled tissue formation in whole organ culture. The resulting grafts were then either analyzed in vitro (n = 15) or transplanted into porcine recipients in vivo (n = 3). RESULTS: By repopulating porcine extracellular matrix scaffolds with human endothelial cells, we generated pulmonary vasculature with mature endothelial lining and sufficient anti-thrombotic function to enable blood perfusion. By repopulating the epithelial surface with human epithelial progenitor cells, we created a living, functioning gas exchange graft. After surgical implantation, the bioengineered lung grafts were able to withstand physiological blood flow from the recipient's pulmonary circulation, and exchanged gases upon ventilation during the 1-hour observation. CONCLUSIONS: Engineering and transplantation of viable lung grafts based on decellularized porcine lung scaffolds and human endothelial and epithelial cells is technically feasible. Further graft maturation will be necessary to enable higher-level functions such as mucociliary clearance, and ventilation-perfusion matching.

In vitro and in vivo analysis of visible light crosslinkable gelatin methacryloyl (GelMA) hydrogels.

Photocrosslinkable materials have been frequently used for constructing soft and biomimetic hydrogels for tissue engineering. Although ultraviolet (UV) light is commonly used for photocrosslinking such materials, its use has been associated with several biosafety concerns such as DNA damage, accelerated aging of tissues, and cancer. Here we report an injectable visible light crosslinked gelatin-based hydrogel for myocardium regeneration. Mechanical characterization revealed that the compressive moduli of the engineered hydrogels could be tuned in the range of 5-56 kPa by changing the concentrations of the initiator, co-initiator and co-monomer in the precursor formulation. In addition, the average pore sizes (26-103 µm) and swelling ratios (7-13%) were also shown to be tunable by varying the hydrogel formulation. In vitro studies showed that visible light crosslinked GelMA hydrogels supported the growth and function of primary cardiomyocytes (CMs). In addition, the engineered materials were shown to be biocompatible in vivo, and could be successfully delivered to the heart after myocardial infarction in an animal model to promote tissue healing. The developed visible light crosslinked hydrogel could be used for the repair of various soft tissues such as the myocardium and for the treatment of cardiovascular diseases with enhanced therapeutic functionality.