Krista L. Sider

Influence of substrate stiffness on the phenotype of heart cells

Adult cardiomyocytes (CM) retain little capacity to regenerate, which motivates efforts to engineer heart tissues that can emulate the functional and mechanical properties of native myocardium. Although the effects of matrix stiffness on individual CM have been explored, less attention was devoted to studies at the monolayer and the tissue level. The purpose of this study was to characterize the influence of substrate mechanical stiffness on the heart cell phenotype and functional properties. Neonatal rat heart cells were seeded onto collagen‐coated polyacrylamide (PA) substrates with Youngs moduli of 3, 22, 50, and 144 kPa. Collagen‐coated glass coverslips without PA represented surfaces with effectively “infinite” stiffness. The local elastic modulus of native neonatal rat heart tissue was measured to range from 4.0 to 11.4 kPa (mean value of 6.8 kPa) and for native adult rat heart tissue from 11.9 to 46.2 kPa (mean value of 25.6 kPa), motivating our choice of the above PA gel stiffness. Overall, by 120 h of cultivation, the lowest stiffness PA substrates (3 kPa) exhibited the lowest excitation threshold (ET; 3.5 ± 0.3 V/cm), increased troponin I staining (52% positively stained area) but reduced cell density, force of contraction (0.18 ± 0.1 mN/mm2), and cell elongation (aspect ratio = 1.3–1.4). Higher stiffness (144 kPa) PA substrates exhibited reduced troponin I staining (30% positively stained area), increased fibroblast density (70% positively stained area), and poor electrical excitability. Intermediate stiffness PA substrates of stiffness comparable to the native adult rat myocardium (22–50 kPa) were found to be optimal for heart cell morphology and function, with superior elongation (aspect ratio > 4.3), reasonable ET (ranging from 3.95 ± 0.8 to 4.4 ± 0.7 V/cm), high contractile force development (ranging from 0.52 ± 0.2 to 1.60 ± 0.6 mN/mm2), and well‐developed striations, all consistent with a differentiated phenotype. Biotechnol. Bioeng. 2010;105: 1148–1160.

β-Catenin Mediates Mechanically Regulated, Transforming Growth Factor-β1–Induced Myofibroblast Differentiation of Aortic Valve Interstitial Cells

Objective—In calcific aortic valve disease, myofibroblasts and activation of the transforming growth factor-&bgr;1 (TGF-&bgr;1) and Wnt/&bgr;-catenin pathways are observed in the fibrosa, the stiffer layer of the leaflet, but their association is unknown. We elucidated the roles of &bgr;-catenin and extracellular matrix stiffness in TGF-&bgr;1-induced myofibroblast differentiation of valve interstitial cells (VICs). Methods and Results—TGF-&bgr;1 induced rapid &bgr;-catenin nuclear translocation in primary porcine aortic VICs in vitro through TGF-&bgr; receptor I kinase. Degrading &bgr;-catenin pharmacologically or silencing it with small interfering RNA inhibited TGF-&bgr;1-induced myofibroblast differentiation without altering Smad2/3 activity. Conversely, increasing &bgr;-catenin availability with Wnt3A alone did not induce differentiation. However, combining TGF-&bgr;1 and Wnt3A caused greater myofibroblast differentiation than TGF-&bgr;1 treatment alone. Notably, in VICs grown on collagen-coated PA gels with physiological stiffnesses, TGF-&bgr;1-induced &bgr;-catenin nuclear translocation and myofibroblast differentiation occurred only on matrices with fibrosa-like stiffness, but not ventricularis-like stiffness. In diseased aortic valves from pigs fed an atherogenic diet, myofibroblasts colocalized with increased protein expression of Wnt3A, &bgr;-catenin, TGF-&bgr;1, and phosphorylated Smad2/3 in the fibrosa. Conclusion—Myofibroblast differentiation of VICs involves matrix stiffness–dependent crosstalk between TGF-&bgr;1 and Wnt signaling pathways and may explain in part why the stiffer fibrosa is more susceptible to disease.

Measurement of layer-specific mechanical properties in multilayered biomaterials by micropipette aspiration.

Many biomaterials and tissues are complex multilayered structures in which the individual layers have distinct mechanical properties that influence the mechanical behavior and define the local cellular microenvironment. Characterization of the mechanical properties of individual layers in intact tissues is technically challenging. Micropipette aspiration (MA) is a proven method for the analysis of local mechanical properties of soft single-layer biomaterials, but its applicability for multilayer structures has not been demonstrated. We sought to determine and validate MA experimental parameters that would permit measurement of the mechanical properties of only the top layer of an intact multilayer biomaterial or tissue. To do so, we performed parametric nonlinear finite-element (FE) analyses and validation experiments using a multilayer gelatin system. The parametric FE analyses demonstrated that measurement of the properties of only the top layer of a multilayer structure is sensitive to the ratio of the pipette inner diameter (D) to top layer thickness (ttop), and that accurate measurement of the top layer modulus requires D/ttop<1. These predictions were confirmed experimentally by MA of the gelatin system. Using this approach and an inverse FE method, the mean effective modulus of the fibrosa layer of intact porcine aortic valve leaflets was determined to be greater than that of the ventricularis layer (P<0.01), consistent with data obtained by tensile testing of dissected layers. This study provides practical guidelines for the use of MA to measure the mechanical properties of single layers in intact multilayer biomaterials and tissues.

Animal Models of Calcific Aortic Valve Disease

Calcific aortic valve disease (CAVD), once thought to be a degenerative disease, is now recognized to be an active pathobiological process, with chronic inflammation emerging as a predominant, and possibly driving, factor. However, many details of the pathobiological mechanisms of CAVD remain to be described, and new approaches to treat CAVD need to be identified. Animal models are emerging as vital tools to this end, facilitated by the advent of new models and improved understanding of the utility of existing models. In this paper, we summarize and critically appraise current small and large animal models of CAVD, discuss the utility of animal models for priority CAVD research areas, and provide recommendations for future animal model studies of CAVD.

Hydrogels modified with QHREDGS peptide support cardiomyocyte survival in vitro and after sub-cutaneous implantation

Myocardial cell injection and tissue engineering could provide novel treatment options for heart diseases; however both approaches are limited by the loss of the transplanted myogenic cells. We hypothesized that novel hydrogels could promote cardiomyocyte survival and remedy this critical limitation. The hydrogel described here is based on a photocrosslinked form of chitosan, Az-chitosan, which was covalently bound to the QHREDGS peptide to promote cell survival. The QHREDGS amino acid sequence is thought to be the integrin binding site in angiopoietin-1, a growth factor which has cardioprotective properties. Covalent immobilization was performed using 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide chemistry. Elastic moduli of the Az-chitosan hydrogel were within the lower physiological range for the neonatal rat heart (1.9 ± 0.2 kPa for 10 mg/ml and 3.5 ± 0.6 kPa for 20 mg/ml). After 6 days of cultivation of neonatal rat heart cells encapsulated with the hydrogels, cell viability and elongation was significantly higher in the peptide modified groups compared to the Az-chitosan control. No significant differences were found in the ability of RGDS and QHREDGS hydrogels to support contractile function in vitro. After subcutaneous implantation of cardiomyocyte hydrogel-peptide constructs in Lewis rats for 7 days, both QHREDGS and RGDS similarly recruited endothelial cells. However, Az-chitosan-QHREDGS gel had a higher percentage of smooth muscle actin (SMA)-positive myofibroblasts. The QHREDGS peptide gel promoted cardiomyocyte elongation and assembly of contractile apparatus and reduced cardiomyocyte apoptosis significantly better than the RGDS peptide. The new Az-chitosan-QHREDGS hydrogel may markedly improve cardiac regeneration by cell therapy.

Evaluation of a porcine model of early aortic valve sclerosis

BACKGROUND Calcific aortic valve disease (CAVD) is associated with significant cardiovascular morbidity. While late-stage CAVD is well-described, early pathobiological processes are poorly understood due to the lack of animal models that faithfully replicate early human disease. Here we evaluated a hypercholesterolemic porcine model of early diet-induced aortic valve sclerosis. METHODS Yorkshire swine were fed either a standard or high-fat/high-cholesterol diet for 2 or 5 months. Right coronary aortic valve leaflets were excised and analyzed (immuno)histochemically. RESULTS Early human-like proteoglycan-rich onlays formed between the endothelial layer and elastic lamina in the fibrosa layer of valve leaflets, with accelerated formation associated with hypercholesterolemia (P<.05). Lipid deposition was more abundant in hypercholesterolemic swine (P<.001), but was present in a minority (28%) of onlays. No myofibroblasts, MAC387-positive macrophages, or fascin-positive dendritic cells were detected in 2-month onlays, with only scarce myofibroblasts present at 5 months. Cells that expressed osteochondral markers Sox9 and Msx2 were preferentially found in dense proteoglycan-rich onlays (P<.05) and with hypercholesterolemia (P<.05). Features of more advanced human CAVD, including calcification, were not observed in this necessarily short study. CONCLUSIONS Early aortic valve sclerosis in hypercholesterolemic swine is characterized by the formation of proteoglycan-rich onlays in the fibrosa, which can occur prior to significant lipid accumulation, inflammatory cell infiltration, or myofibroblast activation. These characteristics mimic those of early human aortic valve disease, and thus the porcine model has utility for the study of early valve sclerosis.

A new bone vascular perfusion compound for the simultaneous analysis of bone and vasculature.

Bone is a highly vascular tissue, which plays an important role in bone development and healing. The ability to analyze both the bone and vasculature simultaneously can enhance the understanding of wound healing, development, and disease in bone. At present, analysis methods are limited in their ability to allow for this simultaneous analysis of bone and bone vasculature in three dimensions, without using the most recent dual‐energy computed tomography (CT) techniques. In this study, we present a new barium sulfate (BaSO4) radiopaque vascular perfusion compound for performing postmortem microangiography with single‐beam microcomputed tomography (microCT), which allows for such simultaneous analysis. This compound differs from currently available contrast mediums due to (1) the high weight‐to‐volume ratio of BaSO4 achieved, (2) small BaSO4 aggregate size (<5 μm), (3) minimal additives, and (4) its miscibility with blood and saline. Most notably, it achieves a radiodensity of 2.4× that of cortical bone, with high perfusion of both the arterial and venous systems and the intervening capillary bed, resulting in an in vivo radiodensity that ranges from that of bone to titanium. Our results, verified using a rat femoral gap‐healing model, show that the compound is uniquely suited to high‐contrast imaging of the vasculature in the presence of undecalcified bone, with a versatility to be used in other tissues. Microsc. Res. Tech., 2010.

Microdevice arrays with strain sensors for 3D mechanical stimulation and monitoring of engineered tissues

Native and engineered tissue development are regulated by the integrative effects of multiple microenvironmental stimuli. Microfabricated bioreactor array platforms can efficiently dissect cue-response networks, and have recently integrated critical 2D and 3D mechanical stimulation for greater physiological relevance. However, a limitation of these approaches is that assessment of tissue functional properties is typically limited to end-point analyses. Here we report a new deformable membrane platform with integrated strain sensors that enables mechanical stretching or compression of 3D cell-hydrogel arrays and simultaneous measurement of hydrogel construct stiffness in situ. We tested the ability of the integrated strain sensors to measure the evolution of the stiffness of cell-hydrogel constructs for two cases. First, we demonstrated in situ stiffness monitoring of degradable poly (ethylene glycol)-norbornene (PEG-NB) hydrogels embedded with mesenchymal stromal cells (MSCs) and cultured with or without cyclic tensile stimulation for up to 15 days. Whereas statically-cultured hydrogels degraded and softened throughout the culture period, mechanically-stimulated gels initially softened and then recovered their stiffness corresponding to extensive cell network and collagen production. Second, we demonstrated in situ measurement of compressive stiffening of MSC-seeded PEG-NB gels cultured statically under osteogenic conditions, corresponding to increased mineralization and cellularization. This measurement technique can be generalized to other relevant bioreactor and organ-on-a-chip platforms to facilitate online, non-invasive, and high-throughput functional analysis, and to provide insights into the dynamics of engineered tissue development that are otherwise not available.

Comparison of Analytical and Finite Element Implementation of Exponential Constitutive Models for Valve Tissue Under Micropipette Aspiration

Micropipette aspiration (MA) has been widely used to measure the biomechanical properties of cells and biomaterials [1]. Typically a linear elastic half-space model is used to fit the experimental load-deformation data [1]. However, load-deformation relationships for most biological tissues are highly nonlinear, suggesting alternative constitutive models are necessary. In the case of aortic heart valve tissue, exponential-type constitutive models have been found to fit the biaxial stress-strain behavior well [2]. Based on these studies, Butcher et al. used an exponential constitutive model to characterize the response of chicken embryonic valve (atrioventricular cushion) under MA [3]. To do so, they implemented an analytical exponential constitutive model [2] and directly related the stress and strain to the experimentally measured pressure and aspiration length. This allowed the authors to fit the tissue MA data without accounting for the complexities of the boundary conditions and multicomponent strain field inherent in MA. However, it is unclear whether the material parameters estimated using this approach are different from those estimated by solving the more complex boundary value problem, which presumably more faithfully simulates the physical process of tissue aspiration.Copyright