Ruogang Zhao

Calcification by Valve Interstitial Cells Is Regulated by the Stiffness of the Extracellular Matrix

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-&bgr;1 potentiated aggregate formation on stiff matrices by enhancing &agr;-smooth muscle actin expression and cellular contractility, but not on compliant matrices attributable to downregulation of TGF-&bgr; receptor I. Cell contraction by VICs inhibited Akt activation and enhanced apoptosis-dependent calcification on stiff matrices. Conclusions—Differentiation of VICs to pathological phenotypes in response to biochemical cues is modulated by matrix stiffness. Although osteogenic or myofibrogenic differentiation of VICs can result in calcification, the processes are distinct.

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.

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.

Decoupling Cell and Matrix Mechanics in Engineered Microtissues Using Magnetically Actuated Microcantilevers

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.

Comparison of analytical and inverse finite element approaches to estimate cell viscoelastic properties by micropipette aspiration

The viscoelastic properties of cells are important in predicting cell deformation under mechanical loading and may reflect cell phenotype or pathological transition. Previous studies have demonstrated that viscoelastic parameters estimated by finite element (FE) analyses of micropipette aspiration (MA) data differ from those estimated by the analytical half-space model. However, it is unclear whether these differences are statistically significant, as previous studies have been based on average cell properties or parametric analyses that do not reflect the inherent experimental and biological variability of real experimental data. To determine whether cell material parameters estimated by the half-space model are significantly different from those predicted by the FE method, we implemented an inverse FE method to estimate the viscoelastic parameters of a population of primary porcine aortic valve interstitial cells tested by MA. We found that inherent differences between the analytical and inverse FE estimation methods resulted in statistically significant differences in individual cell properties. However, in cases with small pipette to cell radius ratios and short loading periods, model-dependent differences were masked by experimental and cell-to-cell variability. Analytical models that account for finite cell-size and loading rate further relaxed the experimental conditions for which accurate cell material parameter estimates could be obtained. These data provide practical guidelines for analysis of MA data that account for the wide range of conditions encountered in typical experiments.

YAP and TAZ control peripheral myelination and the expression of laminin receptors in Schwann cells

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 and Taz are activated in Schwann cells by mechanical stimuli and regulate Schwann cell proliferation and transcription of basal lamina receptor genes, both necessary for 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.

An improved texture correlation algorithm to measure substrate–cytoskeletal network strain transfer under large compressive strain

Force-induced deformation of tissues is transduced to the cytoskeletal (CSK) network within cells via focal adhesions. Previous studies have characterized transfer of strains of less than 15% from the substrate to the cell, using mitochondria as surrogate markers for CSK deformation. However, it is unclear if intracellular strains determined from mitochondrial displacement accurately reflect CSK network deformation. Furthermore, previous studies have not characterized substrate-CSK network strain transfer for strain magnitudes exceeding 15%, as can occur in vivo and in cell culture studies. Here, we developed and characterized a texture correlation algorithm to address the image distortion problem caused by large strain. We then used this algorithm to characterize large compressive strain (-40%) transfer from the substrate to the CSK in living cells, using fluorescently tagged actin to perform the tracking and both fluorescently tagged actin and talin to make validation measurements. With this approach, we were able to demonstrate explicitly that substrate strain transfers directly to CSK deformation in living cells undergoing large compressive deformation, and that the strain transfer ratios are independent of cell alignment. The tools and approaches developed here enable improved characterization of cell-matrix interactions under large deformation and in doing so, may reveal new insights into mechanotransduction mechanisms in such circumstances.

NANOG reverses the Myogenic Differentiation Potential of Senescent Stem Cells by Restoring ACTIN Filamentous Organization and SRF‐Dependent Gene Expression

Cellular senescence as a result of organismal aging or progeroid diseases leads to stem cell pool exhaustion hindering tissue regeneration and contributing to the progression of age related disorders. Here we discovered that ectopic expression of the pluripotent factor NANOG in senescent or progeroid myogenic progenitors reversed cellular aging and restored completely the ability to generate contractile force. To elicit its effects, NANOG enabled reactivation of the ROCK and Transforming Growth Factor (TGF)‐β pathways—both of which were impaired in senescent cells—leading to ACTIN polymerization, MRTF‐A translocation into the nucleus and serum response factor (SRF)‐dependent myogenic gene expression. Collectively our data reveal that cellular senescence can be reversed and provide a novel strategy to regain the lost function of aged stem cells without reprogramming to the pluripotent state. Stem Cells 2017;35:207–221

Magnetic approaches to study collective three-dimensional cell mechanics in long-term cultures (invited)

Contractile forces generated by cells and the stiffness of the surrounding extracellular matrix are two central mechanical factors that regulate cell function. To characterize the dynamic evolution of these two mechanical parameters during tissue morphogenesis, we developed a magnetically actuated micro-mechanical testing system in which fibroblast-populated collagen microtissues formed spontaneously in arrays of microwells that each contains a pair of elastomeric microcantilevers. We characterized the magnetic actuation performance of this system and evaluated its capacity to support long-term cell culture. We showed that cells in the microtissues remained viable during prolonged culture periods of up to 15 days, and that the mechanical properties of the microtissues reached and maintained at a stable state after a fast initial increase stage. Together, these findings demonstrate the utility of this microfabricated bio-magneto-mechanical system in extended mechanobiological studies in a physiologically relevant 3D environment.

Semi-confined compression of microfabricated polymerized biomaterial constructs

Mechanical forces are critical parameters in engineering functional tissue because of their established influence on cellular behaviour. However, identifying ideal combinations of mechanical, biomaterial and chemical stimuli to obtain a desired cellular response requires high-throughput screening technologies, which may be realized through microfabricated systems. This paper reports on the development and characterization of a MEMS device for semi-confined biomaterial compression. An array of these devices would enable studies involving mechanical deformation of three-dimensional biomaterials, an important parameter in creating physiologically relevant microenvironments in vitro. The described device has the ability to simultaneously apply a range of compressive mechanical stimuli to multiple polymerized hydrogel microconstructs. Local micromechanical strains generated within the semi-confined hydrogel cylinders are characterized and compared with those produced in current micro- and macroscale technologies. In contrast to previous work generating unconfined compression in microfabricated devices, the semi-confined compression model used in this work generates uniform regions of strain within the central portion of each hydrogel, demonstrated here to range from 20% to 45% across the array. The uniform strains achieved simplify experimental analysis and improve the utility of the compression platform. Furthermore, the system is compatible with a wide variety of polymerizable biomaterials, enhancing device versatility and usability in tissue engineering and fundamental cell biology studies.