Endothelial extracellular vesicles contain protective proteins and rescue ischemia-reperfusion injury in a human heart-on-chip.

Extracellular vesicles (EVs) derived from various stem cell sources induce cardioprotective effects during ischemia-reperfusion injury (IRI). These have been attributed mainly to the antiapoptotic, proangiogenic, microRNA (miRNA) cargo within the stem cell-derived EVs. However, the mechanisms of EV-mediated endothelial signaling to cardiomyocytes, as well as their therapeutic potential toward ischemic myocardial injury, are not clear. EV content beyond miRNA that may contribute to cardioprotection has not been fully illuminated. This study characterized the protein cargo of human vascular endothelial EVs (EEVs) to identify lead cardioactive proteins and assessed the effect of EEVs on human laminar cardiac tissues (hlCTs) exposed to IRI. We mapped the protein content of human vascular EEVs and identified proteins that were previously associated with cellular metabolism, redox state, and calcium handling, among other processes. Analysis of the protein landscape of human cardiomyocytes revealed corresponding modifications induced by EEV treatment. To assess their human-specific cardioprotection in vitro, we developed a human heart-on-a-chip IRI assay using human stem cell-derived, engineered cardiac tissues. We found that EEVs alleviated cardiac cell death as well as the loss in contractile capacity during and after simulated IRI in an uptake- and dose-dependent manner. Moreover, we found that EEVs increased the respiratory capacity of normoxic cardiomyocytes. These results suggest that vascular EEVs rescue hlCTs exposed to IRI possibly by supplementing injured myocytes with cargo that supports multiple metabolic and salvage pathways and therefore may serve as a multitargeted therapy for IRI.

Biomimetic and estrogenic fibers promote tissue repair in mice and human skin via estrogen receptor ß.

The dynamic changes in estrogen levels throughout aging and during the menstrual cycle influence wound healing. Elevated estrogen levels during the pre-ovulation phase accelerate tissue repair, whereas reduced estrogen levels in post-menopausal women lead to slow healing. Although previous reports have shown that estrogen may potentiate healing by triggering the estrogen receptor (ER)-ß signaling pathway, its binding to ER-α has been associated with severe collateral effects and has therefore limited its use as a therapeutic agent. To this end, soy phytoestrogens, which preferentially bind to the ER-ß, are currently being explored as a safer therapeutic alternative to estrogen. However, the development and evaluation of phytoestrogen-based materials as local ER-ß modulators remains largely unexplored. Here, we engineered biomimetic and estrogenic nanofiber wound dressings built from soy protein isolate (SPI) and hyaluronic acid (HA) using immersion rotary jet spinning. These engineered scaffolds were shown to successfully recapitulate the native dermal architecture, while delivering an ER-ß-triggering phytoestrogen (genistein). When tested in ovariectomized mouse and ex vivo human skin tissues, HA/SPI scaffolds outperformed controls (no treatment or HA only scaffolds) towards promoting cutaneous tissue repair. These improved healing outcomes were prevented when the ER-ß pathway was genetically or chemically inhibited. Our findings suggest that estrogenic fibrous scaffolds facilitate skin repair by ER-ß activation.

Alfalfa Nanofibers for Dermal Wound Healing.

Engineering bioscaffolds for improved cutaneous tissue regeneration remains a healthcare challenge because of the increasing number of patients suffering from acute and chronic wounds. To help address this problem, we propose to utilize alfalfa, an ancient medicinal plant that contains antibacterial/oxygenating chlorophylls and bioactive phytoestrogens, as a building block for regenerative wound dressings. Alfalfa carries genistein, which is a major phytoestrogen known to accelerate skin repair. The scaffolds presented herein were built from composite alfalfa and polycaprolactone (PCL) nanofibers with hydrophilic surface and mechanical stiffness that recapitulate the physiological microenvironments of skin. This composite scaffold was engineered to have aligned nanofibrous architecture to accelerate directional cell migration. As a result, alfalfa-based composite nanofibers were found to enhance the cellular proliferation of dermal fibroblasts and epidermal keratinocytes in vitro. Finally, these nanofibers exhibited reproducible regenerative functionality by promoting re-epithelialization and granulation tissue formation in both mouse and human skin, without requiring additional proteins, growth factors, or cells. Overall, these findings demonstrate the potential of alfalfa-based nanofibers as a regenerative platform toward accelerating cutaneous tissue repair.

Acute pergolide exposure stiffens engineered valve interstitial cell tissues and reduces contractility in vitro.

Medications based on ergoline-derived dopamine and serotonin agonists are associated with off-target toxicities that include valvular heart disease (VHD). Reports of drug-induced VHD resulted in the withdrawal of appetite suppressants containing fenfluramine and phentermine from the US market in 1997 and pergolide, a Parkinson's disease medication, in 2007. Recent evidence suggests that serotonin receptor activity affected by these medications modulates cardiac valve interstitial cell activation and subsequent valvular remodeling, which can lead to cardiac valve fibrosis and dysfunction similar to that seen in carcinoid heart disease. Failure to identify these risks prior to market and continued use of similar drugs reaffirm the need to improve preclinical evaluation of drug-induced VHD. Here, we present two complimentary assays to measure stiffness and contractile stresses generated by engineered valvular tissues in vitro. As a case study, we measured the effects of acute (24 h) pergolide exposure to engineered porcine aortic valve interstitial cell (AVIC) tissues. Pergolide exposure led to increased tissue stiffness, but it decreased both basal and active contractile tone stresses generated by AVIC tissues. Pergolide exposure also disrupted AVIC tissue organization (i.e., tissue anisotropy), suggesting that the mechanical properties and contractile functionality of these tissues are governed by their ability to maintain their structure. We expect further use of these assays to identify off-target drug effects that alter the phenotypic balance of AVICs, disrupt their ability to maintain mechanical homeostasis, and lead to VHD.

Human airway musculature on a chip: an in vitro model of allergic asthmatic bronchoconstriction and bronchodilation.

Many potential new asthma therapies that show promise in the pre-clinical stage of drug development do not demonstrate efficacy during clinical trials. One factor contributing to this problem is the lack of human-relevant models of the airway that recapitulate the tissue-level structural and functional phenotypes of asthma. Hence, we sought to build a model of a human airway musculature on a chip that simulates healthy and asthmatic bronchoconstriction and bronchodilation in vitro by engineering anisotropic, laminar bronchial smooth muscle tissue on elastomeric thin films. In response to a cholinergic agonist, the muscle layer contracts and induces thin film bending, which serves as an in vitro analogue for bronchoconstriction. To mimic asthmatic inflammation, we exposed the engineered tissues to interleukin-13, which resulted in hypercontractility and altered relaxation in response to cholinergic challenge, similar to responses observed clinically in asthmatic patients as well as in studies with animal tissue. Moreover, we reversed asthmatic hypercontraction using a muscarinic antagonist and a ß-agonist which are used clinically to relax constricted airways. Importantly, we demonstrated that targeting RhoA-mediated contraction using HA1077 decreased basal tone, prevented hypercontraction, and improved relaxation of the engineered tissues exposed to IL-13. These data suggest that we can recapitulate the structural and functional hallmarks of human asthmatic musculature on a chip, including responses to drug treatments for evaluation of safety and efficacy of new drugs. Further, our airway musculature on a chip provides an important tool for enabling mechanism-based search for new therapeutic targets through the ability to evaluate engineered muscle at the levels of protein expression, tissue structure, and tissue function.

A tissue-engineered jellyfish with biomimetic propulsion.

Reverse engineering of biological form and function requires hierarchical design over several orders of space and time. Recent advances in the mechanistic understanding of biosynthetic compound materials, computer-aided design approaches in molecular synthetic biology 4,5 and traditional soft robotics, and increasing aptitude in generating structural and chemical micro environments that promote cellular self-organization have enhanced the ability to recapitulate such hierarchical architecture in engineered biological systems. Here we combined these capabilities in a systematic design strategy to reverse engineer a muscular pump. We report the construction of a freely swimming jellyfish from chemically dissociated rat tissue and silicone polymer as a proof of concept. The constructs, termed 'medusoids', were designed with computer simulations and experiments to match key determinants of jellyfish propulsion and feeding performance by quantitatively mimicking structural design, stroke kinematics and animal-fluid interactions. The combination of the engineering design algorithm with quantitative benchmarks of physiological performance suggests that our strategy is broadly applicable to reverse engineering of muscular organs or simple life forms that pump to survive.

Muscle on a chip: in vitro contractility assays for smooth and striated muscle.

INTRODUCTION: To evaluate the viability of a muscle tissue, it is essential to measure the tissue's contractile performance as well as to control its structure. Accurate contractility data can aid in development of more effective and safer drugs. This can be accomplished with a robust in vitro contractility assay applicable to various types of muscle tissue. METHODS: The devices developed in this work were based on the muscular thin film (MTF) technology, in which an elastic film is manufactured with a 2D engineered muscle tissue on one side. The tissue template is made by patterning extracellular matrix with microcontact printing. When muscle cells are seeded on the film, they self-organize with respect to the geometric cues in the matrix to form a tissue. RESULTS: Several assays based on the "MTF on a chip" technology are demonstrated. One such assay incorporates the contractility assay with striated muscle into a fluidic channel. Another assay platform incorporates the MTFs in a multi-well plate, which is compatible with automated data collection and analysis. Finally, we demonstrate the possibility of analyzing contractility of both striated and smooth muscle simultaneously on the same chip. DISCUSSION: In this work, we assembled an ensemble of contractility assays for striated and smooth muscle based on muscular thin films. Our results suggest an improvement over current methods and an alternative to isolated tissue preparations. Our technology is amenable to both primary harvests cells and cell lines, as well as both human and animal tissues.