The neurovascular unit (NVU) regulates metabolic homeostasis as well as drug pharmacokinetics and pharmacodynamics in the central nervous system. Metabolic fluxes and conversions over the NVU rely on ...interactions between brain microvascular endothelium, perivascular pericytes, astrocytes and neurons, making it difficult to identify the contributions of each cell type. Here we model the human NVU using microfluidic organ chips, allowing analysis of the roles of individual cell types in NVU functions. Three coupled chips model influx across the blood-brain barrier (BBB), the brain parenchymal compartment and efflux across the BBB. We used this linked system to mimic the effect of intravascular administration of the psychoactive drug methamphetamine and to identify previously unknown metabolic coupling between the BBB and neurons. Thus, the NVU system offers an in vitro approach for probing transport, efficacy, mechanism of action and toxicity of neuroactive drugs.
Cardiac tissue development and pathology have been shown to depend sensitively on microenvironmental mechanical factors, such as extracellular matrix stiffness, in both in vivo and in vitro systems. ...We present a novel quantitative approach to assess cardiac structure and function by extending the classical traction force microscopy technique to tissue-level preparations. Using this system, we investigated the relationship between contractile proficiency and metabolism in neonate rat ventricular myocytes (NRVM) cultured on gels with stiffness mimicking soft immature (1 kPa), normal healthy (13 kPa), and stiff diseased (90 kPa) cardiac microenvironments. We found that tissues engineered on the softest gels generated the least amount of stress and had the smallest work output. Conversely, cardiomyocytes in tissues engineered on healthy- and disease-mimicking gels generated significantly higher stresses, with the maximal contractile work measured in NRVM engineered on gels of normal stiffness. Interestingly, although tissues on soft gels exhibited poor stress generation and work production, their basal metabolic respiration rate was significantly more elevated than in other groups, suggesting a highly ineffective coupling between energy production and contractile work output. Our novel platform can thus be utilized to quantitatively assess the mechanotransduction pathways that initiate tissue-level structural and functional remodeling in response to substrate stiffness.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The lack of a robust pipeline of medical therapeutic agents for the treatment of heart disease may be partially attributed to the lack of in vitro models that recapitulate the essential ...structure–function relationships of healthy and diseased myocardium. We designed and built a system to mimic mechanical overload in vitro by applying cyclic stretch to engineered laminar ventricular tissue on a stretchable chip. To test our model, we quantified changes in gene expression, myocyte architecture, calcium handling, and contractile function and compared our results vs. several decades of animal studies and clinical observations. Cyclic stretch activated gene expression profiles characteristic of pathological remodeling, including decreased α- to β-myosin heavy chain ratios, and induced maladaptive changes to myocyte shape and sarcomere alignment. In stretched tissues, calcium transients resembled those reported in failing myocytes and peak systolic stress was significantly reduced. Our results suggest that failing myocardium, as defined genetically, structurally, and functionally, can be replicated in an in vitro microsystem by faithfully recapitulating the structural and mechanical microenvironment of the diseased heart.
Abstract The heart is a muscular organ with a wrapping, laminar structure embedded with neural and vascular networks, collagen fibrils, fibroblasts, and cardiac myocytes that facilitate contraction. ...We hypothesized that these non-muscle components may have functional benefit, serving as important structural alignment cues in inter- and intra-cellular organization of cardiac myocytes. Previous studies have demonstrated that alignment of engineered myocardium enhances calcium handling, but how this impacts actual force generation remains unclear. Quantitative assays are needed to determine the effect of alignment on contractile function and muscle physiology. To test this, micropatterned surfaces were used to build 2-dimensional myocardium from neonatal rat ventricular myocytes with distinct architectures: confluent isotropic (serving as the unaligned control), confluent anisotropic, and 20 μm spaced, parallel arrays of multicellular myocardial fibers. We combined image analysis of sarcomere orientation with muscular thin film contractile force assays in order to calculate the peak sarcomere-generated stress as a function of tissue architecture. Here we report that increasing peak systolic stress in engineered cardiac tissues corresponds with increasing sarcomere alignment. This change is larger than would be anticipated from enhanced calcium handling and increased uniaxial alignment alone. These results suggest that boundary conditions (heterogeneities) encoded in the extracellular space can regulate muscle tissue function, and that structural organization and cytoskeletal alignment are critically important for maximizing peak force generation.
Brain in vitro models are critically important to developing our understanding of basic nervous system cellular physiology, potential neurotoxic effects of chemicals, and specific cellular mechanisms ...of many disease states. In this study, we sought to address key shortcomings of current brain in vitro models: the scarcity of comparative data for cells originating from distinct brain regions and the lack of multiregional brain in vitro models. We demonstrated that rat neurons from different brain regions exhibit unique profiles regarding their cell composition, protein expression, metabolism, and electrical activity in vitro. In vivo, the brain is unique in its structural and functional organization, and the interactions and communication between different brain areas are essential components of proper brain function. This fact and the observation that neurons from different areas of the brain exhibit unique behaviors in vitro underline the importance of establishing multiregional brain in vitro models. Therefore, we here developed a multiregional brain-on-a-chip and observed a reduction of overall firing activity, as well as altered amounts of astrocytes and specific neuronal cell types compared with separately cultured neurons. Furthermore, this multiregional model was used to study the effects of phencyclidine, a drug known to induce schizophrenia-like symptoms in vivo, on individual brain areas separately while monitoring downstream effects on interconnected regions. Overall, this work provides a comparison of cells from different brain regions in vitro and introduces a multiregional brain-on-a-chip that enables the development of unique disease models incorporating essential in vivo features.
Due to the scarcity of comparative data for cells from different brain regions in vitro, we demonstrated that neurons isolated from distinct brain areas exhibit unique behaviors in vitro. Moreover, in vivo proper brain function is dependent on the connection and communication of several brain regions, underlining the importance of developing multiregional brain in vitro models. We introduced a novel brain-on-a-chip model, implementing essential in vivo features, such as different brain areas and their functional connections.
Wounds in the fetus can heal without scarring. Consequently, biomaterials that attempt to recapitulate the biophysical and biochemical properties of fetal skin have emerged as promising ...pro-regenerative strategies. The extracellular matrix (ECM) protein fibronectin (Fn) in particular is believed to play a crucial role in directing this regenerative phenotype. Accordingly, Fn has been implicated in numerous wound healing studies, yet remains untested in its fibrillar conformation as found in fetal skin. Here, we show that high extensional (∼1.2 ×105 s−1) and shear (∼3 ×105 s−1) strain rates in rotary jet spinning (RJS) can drive high throughput Fn fibrillogenesis (∼10 mL/min), thus producing nanofiber scaffolds that are used to effectively enhance wound healing. When tested on a full-thickness wound mouse model, Fn nanofiber dressings not only accelerated wound closure, but also significantly improved tissue restoration, recovering dermal and epidermal structures as well as skin appendages and adipose tissue. Together, these results suggest that bioprotein nanofiber fabrication via RJS could set a new paradigm for enhancing wound healing and may thus find use in a variety of regenerative medicine applications.
Abstract In vitro cardiovascular disease models need to recapitulate tissue-scale function in order to provide in vivo relevance. We have developed a new method for measuring the contractility of ...engineered cardiovascular smooth and striated muscle in vitro during electrical and pharmacological stimulation. We present a growth theory-based finite elasticity analysis for calculating the contractile stresses of a 2D anisotropic muscle tissue cultured on a flexible synthetic polymer thin film. Cardiac muscle engineered with neonatal rat ventricular myocytes and paced at 0.5 Hz generated stresses of 9.2 ± 3.5 kPa at peak systole, similar to measurements of the contractility of papillary muscle from adult rats. Vascular tissue engineered with human umbilical arterial smooth muscle cells maintained a basal contractile tone of 13.1 ± 2.1 kPa and generated another 5.1 ± 0.8 kPa when stimulated with endothelin-1. These data suggest that this method may be useful in assessing the efficacy and safety of pharmacological agents on cardiovascular tissue.
The clinical onset of type 1 diabetes is characterized by the destruction of the insulin-producing β cells of the pancreas and is caused by autoantigen-induced inflammation (insulitis) of the islets ...of Langerhans. The current standard of care for type 1 diabetes mellitus patients allows for management of the disease with exogenous insulin, but patients eventually succumb to many chronic complications such as limb amputation, blindness, and kidney failure. New therapeutic approaches now on the horizon are looking beyond glycemic management and are evaluating new strategies from protecting and regenerating endogenous islets to treating the underlying autoimmunity through selective modulation of key immune cell populations. Currently, there are no effective treatments for the autoimmunity that causes the disease, and strategies that aim to delay or prevent the onset of the disease will play an important role in the future of diabetes research. In this review, we summarize many of the key efforts underway that utilize molecular approaches to selectively modulate this disease and look at new therapeutic paradigms that can transform clinical treatment.
Extracellular matrix (ECM) structure and biochemistry provide cell-instructive cues that promote and regulate tissue growth, function, and repair. From a structural perspective, the ECM is a scaffold ...that guides the self-assembly of cells into distinct functional tissues. The ECM promotes the interaction between individual cells and between different cell types, and increases the strength and resilience of the tissue in mechanically dynamic environments. From a biochemical perspective, factors regulating cell–ECM adhesion have been described and diverse aspects of cell–ECM interactions in health and disease continue to be clarified. Natural ECMs therefore provide excellent design rules for tissue engineering scaffolds. The design of regenerative three-dimensional (3D) engineered scaffolds is informed by the target ECM structure, chemistry, and mechanics, to encourage cell infiltration and tissue genesis. This can be achieved using nanofibrous scaffolds composed of polymers that simultaneously recapitulate 3D ECM architecture, high-fidelity nanoscale topography, and bio-activity. Their high porosity, structural anisotropy, and bio-activity present unique advantages for engineering 3D anisotropic tissues. Here, we use the heart as a case study and examine the potential of ECM-inspired nanofibrous scaffolds for cardiac tissue engineering. We asked: Do we know enough to build a heart? To answer this question, we tabulated structural and functional properties of myocardial and valvular tissues for use as design criteria, reviewed nanofiber manufacturing platforms and assessed their capabilities to produce scaffolds that meet our design criteria. Our knowledge of the anatomy and physiology of the heart, as well as our ability to create synthetic ECM scaffolds have advanced to the point that valve replacement with nanofibrous scaffolds may be achieved in the short term, while myocardial repair requires further study in vitro and in vivo.
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Myocardial development is regulated by an elegantly choreographed ensemble of signaling events mediated by a multitude of intermediates that take a variety of forms. Cellular differentiation and ...maturation are a subset of vertically integrated processes that extend over several spatial and temporal scales to create a well-defined collective of cells that are able to function cooperatively and reliably at the organ level. Early efforts to understand the molecular mechanisms of cardiomyocyte fate determination focused primarily on genetic and chemical mediators of this process. However, increasing evidence suggests that mechanical interactions between the extracellular matrix (ECM) and cell surface receptors as well as physical interactions between neighboring cells play important roles in regulating the signaling pathways controlling the developmental processes of the heart. Interdisciplinary efforts have made it apparent that the influence of the ECM on cellular behavior occurs through a multitude of physical mechanisms, such as ECM boundary conditions, elasticity, and the propagation of mechanical signals to intracellular compartments, such as the nucleus. In addition to experimental studies, a number of mathematical models have been developed that attempt to capture the interplay between cells and their local microenvironment and the influence these interactions have on cellular self-assembly and functional behavior. Nevertheless, many questions remain unanswered concerning the mechanism through which physical interactions between cardiomyocytes and their environment are translated into biochemical cellular responses and how these signaling modalities can be utilized in vitro to fabricate myocardial tissue constructs from stem cell-derived cardiomyocytes that more faithfully represent their in vivo counterpart. These studies represent a broad effort to characterize biological form as a conduit for information transfer that spans the nanometer length scale of proteins to the meter length scale of the patient and may yield new insights into the contribution of mechanotransduction into heart development and disease.
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