title: Collective cell traction force analysis on aligned smooth muscle cell sheet between three-dimensional microwalls.
abstract: During the past two decades, novel biomaterial scaffold for cell attachment and culture has been developed for applications in tissue engineering, biosensing and regeneration medicine. Tissue engineering of blood vessels remains a challenge owing to the complex three-layer histology involved. In order to engineer functional blood vessels, it is essential to recapitulate the characteristics of vascular smooth muscle cells (SMCs) inside the tunica media, which is known to be critical for vasoconstriction and vasodilation of the circulatory system. Until now, there has been a lack of understanding on the mechanotransduction of the SMC layer during the transformation from viable synthetic to quiescent contractile phenotypes. In this study, microfabricated arrays of discontinuous microwalls coated with fluorescence microbeads were developed to probe the mechanotransduction of the SMC layer. First, the system was exploited for stimulating the formation of a highly aligned orientation of SMCs in native tunica medium. Second, atomic force microscopy in combination with regression analysis was applied to measure the elastic modulus of a polyacrylamide gel layer coated on the discontinuous microwall arrays. Third, the conventional traction force assay for single cell measurement was extended for applications in three-dimensional cell aggregates. Then, the biophysical effects of discontinuous microwalls on the mechanotransduction of the SMC layer undergoing cell alignment were probed. Generally, the cooperative multiple cell-cell and cell-microwall interactions were accessed quantitatively by the newly developed assay with the aid of finite-element modelling. The results show that the traction forces of highly aligned cells lying in the middle region between two opposing microwalls were significantly lower than those lying adjacent to the microwalls. Moreover, the spatial distributions of Von Mises stress during the cell alignment process were dependent on the collective cell layer orientation. Immunostaining of the SMC sheet further demonstrated that the collective mechanotransduction induced by three-dimensional topographic cues was correlated with the reduction of actin and vinculin expression. In addition, the online two-dimensional LC-MS/MS analysis verified the modulation of focal adhesion formation under the influence of microwalls through the regulation in the expression of three key cytoskeletal proteins.
authors.affiliation: Center of Biotechnology, School of Chemical and Biomedical Engineering , Nanyang Technological University , 639798 Singapore , Singapore.
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Center of Biotechnology, School of Chemical and Biomedical Engineering , Nanyang Technological University , 639798 Singapore , Singapore.
authors.forename: Soon Seng
authors.initials: SS
authors.lastname: Ng
authors.affiliation: Center of Biotechnology, School of Chemical and Biomedical Engineering , Nanyang Technological University , 639798 Singapore , Singapore.
authors.forename: Yilei
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Center of Biotechnology, School of Chemical and Biomedical Engineering , Nanyang Technological University , 639798 Singapore , Singapore.
authors.forename: Huixing
authors.initials: H
authors.lastname: Feng
authors.affiliation: Center of Biotechnology, School of Chemical and Biomedical Engineering , Nanyang Technological University , 639798 Singapore , Singapore.
authors.forename: Wei Ning
authors.initials: WN
authors.lastname: Chen
authors.affiliation: Center of Biotechnology, School of Chemical and Biomedical Engineering , Nanyang Technological University , 639798 Singapore , Singapore.
authors.forename: Mary B
authors.initials: MB
authors.lastname: Chan-Park
authors.affiliation: Department of Biomedical Engineering , National Yang Ming University , Taipei 11221 , Taiwan , Republic of China ; Department of Mechanical Engineering , National Central University , Jhongli, Taoyuan 32001 , Taiwan, Republic of China.
authors.forename: Chuan
authors.initials: C
authors.lastname: Li
authors.affiliation: Center of Biotechnology, School of Chemical and Biomedical Engineering , Nanyang Technological University , 639798 Singapore , Singapore.
authors.forename: Vincent
authors.initials: V
authors.lastname: Chan
keywords: aligned smooth muscle cell sheet; cell traction force microscopy; mechanical stress; micropatterned scaffold
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