Samuel E. Senyo

Inflammation is a key driver of cardiac disease progression, including heart failure and myocardial infarction, with macrophage‐cardiomyocyte interactions playing a central role in shaping the local inflammatory microenvironment. Prolonged exposure to pro‐inflammatory cytokines, many produced by macrophages, induces cardiomyocyte stress, dysfunction, and pathological tissue remodeling. However, the spatial and temporal dynamics of this crosstalk remain poorly understood, limiting the development of targeted therapies. To address this gap, we developed an in vitro cardiac inflammation model that enables controlled investigation of macrophage‐cardiomyocyte interactions. Cardiomyocytes were transduced with an NF‐κB‐responsive fluorescent reporter to allow real‐time monitoring of inflammatory signaling. MATLAB‐based simulations were used to optimize medium delivery and cell densities by modeling TNFα diffusion. Using a multiplex microfluidic co‐culture chip, we identified the TNFα threshold of 50 ng mL ^{− 1} with a 6‐h exposure as sufficient to activate cardiomyocyte NF‐κB signaling. Both macrophage‐conditioned medium and direct co‐culture significantly increased NF‐κB activation, indicating enhanced cardiomyocyte inflammatory stress. Dexamethasone markedly suppressed NF‐κB activation when administered at onset of co‐culture to both cell types, whereas IL‐10 showed limited efficacy. Overall, this cardiac inflammation model provides a versatile platform for dissecting immune‐cardiac crosstalk and supports future studies of multicellular crosstalk, spatiotemporal signaling dynamics, and therapeutic screening.

Microfluidic platforms offer controlled microenvironments for studying cell migration such as haptotaxis. In many gradient-based assays, protein gradients are first visualized using higher concentrations of fluorescent labels, since gradients formed at biologically relevant ligand densities often fall below the detection limits of conventional imaging methods. In this study, we demonstrate the feasibility of characterizing fibronectin gradients using a more sensitive, high-resolution approach with Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS). Our methods increase analytical sensitivity to fibronectin gradients formed on commonly used synthetic surfaces to better elucidate physiological mechanisms and ensure experimental reproducibility. We utilized a microfluidic chip with a silicone housing placed on an optically clear plastic microscope slide designed for live-cell microscopy. Slides were coated with fibronectin incorporating surrogate labels for ToF-SIMS analysis to investigate spatial distribution. To enhance signal detection, fibronectin was labeled or conjugated with copper, bromine, and fluorine-containing BODIPY as surrogate elements. Among the tested labels, BODIPY-fibronectin (BODIPY-FN) provided the lowest background signal, enabling fluorescence-based detection at concentrations of 10 µg mL⁻¹ or higher, whereas ToF-SIMS demonstrated greater sensitivity, detecting fibronectin gradients at concentrations of 1 µg mL⁻¹ or higher. Semi-quantitative measurements using imaging mass spectrometry confirmed a graded distribution of fibronectin within the physiologically relevant “haptotaxis zone”, with detection sensitivity exceeding the limits of standard fluorescent microscopy. These results establish ToF-SIMS as a valid method for detecting surrogate-labeled ligand gradients, providing a reliable approach for future quantitative ligand measurements.

Therapeutic tissue regeneration remains a significant unmet need in heart failure and cardiovascular disease treatment, which are among the leading causes of death globally. Decellularized heart matrix (DHM) offer promising advantages for tissue engineering, including low immunogenicity and seamless integration into biological processes, facilitating biocompatibility. However, DHM is challenged by weak mechanical properties that limit its utility to biomedical applications like tissue engineering. To address this limitation, we functionalized DHM with methacryloyl functional groups (DHMMA) that support UV-induced crosslinking to enhance mechanical properties. By modulating the degree of methacryloyl substitution, a broad range of stiffness was achieved while maintaining cell viability on crosslinked DHMMA. Additionally, we show that increasing UV exposure time and pH increases DHMMA stiffness. Furthermore, topographical features transferred on DHMMA via soft lithography facilitated physical orientation of cells in culture. We demonstrate DHMMA as a scaffold with tunable stiffness and matrix-degradation properties suitable for cell survival and microfabrication for cardiac tissue engineering applications.

Skull morphogenesis is a complex, dynamic process involving two different germ layers and progressing to the coordinated, directional growth of individual bones. The mechanisms underlying directional growth toward the apex are not completely understood. Here, a microfluidic chip‐based approach is utilized to test whether calvarial osteoblast progenitors undergo haptotaxis on a gradient of Fibronectin1 (FN1) via lamellipodia. Mimicking the embryonic cranial mesenchyme's FN1 pattern, FN1 gradients is established in the chip using computer modeling and fluorescent labeling. Primary mouse calvarial osteoblast progenitors are plated in the chip along an array of segmented gradients of adsorbed FN1. The study performs single‐cell tracking and measures protrusive activity. Haptotaxis is observed at an intermediate FN1 concentration, with an average directional migration index (yFMI) of 0.07, showing a significant increase compared to the control average yFMI of ‐0.01. A significant increase in protrusive activity is observed during haptotaxis. Haptotaxis is an Arp2/3‐dependent, lamellipodia‐mediated process. Calvarial osteoblast progenitors treated with the Arp2/3 (Actin Related Protein 2/3 complex) inhibitor CK666 show significantly diminished haptotaxis, with an average yFMI of 0.01. Together, these results demonstrate haptotaxis on an FN1 gradient as a new mechanism in the apical expansion of calvarial osteoblast progenitors during development and shed light on the etiology of calvarial defects.

Cardiac fibroblasts play a pivotal role in maintaining heart homeostasis by depositing extracellular matrix (ECM) to provide structural support for the myocardium, vasculature, and neuronal network and by contributing to essential physiological processes. In response to injury such as myocardial infarction or pressure overload, fibroblasts become activated, leading to increased ECM production that can ultimately drive left ventricular remodeling and progress to heart failure. Recently, the American Journal of Physiology-Heart and Circulatory Physiology issued a call for papers on cardiac fibroblasts that yielded articles with topics spanning fibroblast physiology, technical considerations, signaling pathways, and interactions with other cell types. This mini-review summarizes those articles and places the new findings in the context of what is currently known for cardiac fibroblasts and what future directions remain.

Paracrine signaling is challenging to study in vitro, as conventional culture tools dilute soluble factors and offer little to no spatiotemporal control over signaling. Microfluidic chips offer potential to address both of these issues. However, few solutions offer both control over onset and duration of cell–cell communication, and high throughput. We have developed a microfluidic chip designed to culture cells in adjacent chambers, separated by valves to selectively allow or prevent exchange of paracrine signals. The chip features 16 fluidic inputs and 128 individually-addressable chambers arranged in 32 sets of 4 chambers. Media can be continuously perfused or delivered by diffusion, which we model under different culture conditions to ensure normal cell viability. Immunocytochemistry assays can be performed in the chip, which we modeled and fine-tuned to reduce total assay time to 1 h. Finally, we validate the use of the chip for co-culture studies by showing that HEK293Ta cells respond to signals secreted by RAW 264.7 immune cells in adjacent chambers, only when the valve between the chambers is opened.

Paracrine signaling is challenging to study in vitro, as conventional culture tools dilute soluble factors and offer little to no spatiotemporal control over signaling. Microfluidic chips offer potential to address both of these issues. However, few solutions offer both control over onset and duration of cell-cell communication, and high throughput. We have developed a microfluidic chip designed to culture cells in adjacent chambers, separated by valves to selectively allow or prevent exchange of paracrine signals. The chip features 16 fluidic inputs and 128 individuallyaddressable chambers arranged in 32 sets of 4 chambers. Media can be continuously perfused or delivered by diffusion, which we model under different culture conditions to ensure normal cell viability. Immunocytochemistry assays can be performed in the chip, which we modeled and fine-tuned to reduce total assay time to 1h. Finally, we validate the use of the chip for co-culture studies by showing that HEK293Ta cells respond to signals secreted by RAW 264.7 immune cells in adjacent chambers, only when the valve between the chambers is opened.

Ischemic heart injury causes permanent cardiomyocyte loss and fibrosis impairing cardiac function. Tissue derived biomaterials have shown promise as an injectable treatment for the post‐ischemic heart. Specifically, decellularized extracellular matrix (dECM) is a protein rich suspension that forms a therapeutic hydrogel once injected and improves the heart post‐injury response in rodents and pig models. Current dECM‐derived biomaterials are delivered to the heart as a liquid dECM hydrogel precursor or colloidal suspension, which gels over several minutes. To increase the functionality of the dECM therapy, an injectable solid dECM microparticle formulation derived from heart tissue to control sizing and extend stability in aqueous conditions is developed. When delivered into the infarcted mouse heart, these dECM microparticles protect cardiac function promote vessel density and reduce left ventricular remodeling by sustained delivery of biomolecules. Longer retention, higher stiffness, and slower protein release of dECM microparticles are noted compared to liquid dECM hydrogel precursor. In addition, the dECM microparticle can be developed as a platform for macromolecule delivery. Together, the results suggest that dECM microparticles can be developed as a modular therapy for heart injury.

The cardiogenesis of the fetal heart is absent in juveniles and adults. Cross-transplantation of decellularized extracellular matrix (dECM) can stimulate regeneration in myocardial infarct (MI) models. We have previously shown that dECM and tissue stiffness have cooperative regulation of heart regeneration in transiently regenerative day 1 neonatal mice. To investigate underlying mechanisms of mechano-signaling and dECM, we pharmacologically altered heart stiffness and administered dECM hydrogels in non-regenerative mice after MI. The dECM combined with softening exhibits preserved cardiac function, LV geometry, increased cardiomyocyte mitosis and lowered fibrosis while stiffening further aggravated ischemic damage. Transcriptome analysis identified a protein in cardiomyocytes, CLCA2, confirmed to be upregulated after MI and downregulated by dECM in a mechanosensitive manner. Synthetic knock-down of CLCA2 expression induced mitosis in primary rat cardiomyocytes in the dish. Together, our results indicate that therapeutic efficacy of extracellular molecules for heart regeneration can be modulated by heart microenvironment stiffness in vivo.