Objective-Extensive remodeling of the valve ECM in calcific aortic valve sclerosis alters its mechanical properties, but little is known about the impact of matrix mechanics on the cells within the valve interstitium. In this study, the influence of matrix stiffness in modulating calcification by valve interstitial cells (VICs), and their differentiation to pathological phenotypes was assessed. Methods and Results-Primary porcine aortic VICs were cultured in standard media or calcifying media on constrained type I fibrillar collagen gels. Matrix stiffness was altered by changing only the thickness of the gels. Calcification did not occur in standard media, regardless of matrix stiffness. However, when VICs were grown in calcifying media on relatively compliant matrices with stiffness similar to that of normal tissue, they readily formed calcified aggregates of viable cells that expressed osteoblast-related transcripts and proteins. In contrast, VICs cultured in calcifying media on stiffer matrices (similar to stenotic tissue) differentiated to myofibroblasts and formed calcified aggregates that contained apoptotic cells. Actin depolymerization reduced aggregation on stiff, but not compliant, matrices. TGF-␤1 potentiated aggregate formation on stiff matrices by enhancing ␣-smooth muscle actin expression and cellular contractility, but not on compliant matrices attributable to downregulation of TGF-␤ receptor I. Cell contraction by VICs inhibited Akt activation and enhanced apoptosis-dependent calcification on stiff matrices. Key Words: aortic valve Ⅲ matrix mechanics Ⅲ mechanobiology Ⅲ collagen Ⅲ sclerosis D ysregulation of normal cellular processes 1,2 leads to aortic valve sclerosis (AS), a common disease 3 that involves chronic inflammation, fibrosis, and calcification. 4,5 The consequences of AS are serious, as even minor valve calcification increases the risk of other cardiovascular disorders by 50%, and the prognosis with progression to sclerosis is poor. 6 Treatment is limited to surgical replacement of the stenotic valves, as effective medical therapies do not exist. Conclusions-Differentiation of VICs to pathological phenotypes in response toThe progression of sclerosis and calcification is mediated primarily by valve interstitial cells (VICs) that populate the interstitial matrix. 1,7 As in the vasculature, 8 calcification of the aortic valve occurs through multiple mechanisms, 9 including apoptosis-related calcification typically associated with myofibrogenic activation of VICs, 10,11 calcium deposition associated with necrotic cells, 12 and bone formation by resident VICs 13 or bone marrow-derived cells. 10 However, details of the cellular mechanisms by which VICs contribute to calcification are not well understood, largely because of the limited number of studies in vitro and difficulties with their interpretation. For example, when VICs are induced to form calcified multicellular aggregates in vitro, the aggregates are associated with the expression of bone-related transcripts and proteins, the expr...

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 Young's 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/mm(2)), 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/mm(2)), and well-developed striations, all consistent with a differentiated phenotype.

Myelination is essential for nervous system function. Schwann cells interact with neurons and the basal lamina to myelinate axons, using known receptors, signals and transcription factors. In contrast, the transcriptional control of axonal sorting and the role of mechanotransduction in myelination are largely unknown. Yap and Taz are effectors of the Hippo pathway that integrate chemical and mechanical signals in cells. We describe a previously unknown role for the Hippo pathway in myelination. Using conditional mutagenesis in mice we show that Taz is required in Schwann cells for radial sorting and myelination, and that Yap is redundant with Taz. Yap/Taz are activated in Schwann cells by mechanical stimuli, and regulate Schwann cell proliferation and transcription of basal lamina receptor genes, both necessary for proper radial sorting of axons and subsequent myelination. These data link transcriptional effectors of the Hippo pathway and of mechanotransduction to myelin formation in Schwann cells.

A novel bio‐magnetomechanical microtissue system is described for magnetic actuation of arrays of 3D microtissues using microcantilevers. This system enables both in situ measurements of fundamental mechanical properties of engineered tissue, such as contractility and stiffness, as well as dynamic stimulation of the microtissues. Using this system, cell and extracellular matrix contributions to the tissue mechanical properties are decoupled for the first time under both static and dynamic loading conditions.

Fibrosis is a severe health problem characterized by progressive stiffening of tissues which causes organ malfunction and failure. A major bottleneck in developing new anti-fibrosis therapies is the lack of in vitro models that recapitulate dynamic changes in tissue mechanics during fibrogenesis. Here we create membranous human lung microtissues to model key biomechanical events occurred during lung fibrogenesis including progressive stiffening and contraction of alveolar tissue, decline in alveolar tissue compliance and traction force-induced bronchial dilation. With these capabilities, we provide proof of principle for using this fibrotic tissue array for multi-parameter, phenotypic analysis of the therapeutic efficacy of two anti-fibrosis drugs recently approved by the FDA. Preventative treatments with Pirfenidone and Nintedanib reduce tissue contractility and prevent tissue stiffening and decline in tissue compliance. In a therapeutic treatment regimen, both drugs restore tissue compliance. These results highlight the pathophysiologically relevant modeling capability of our novel fibrotic microtissue system.