Engineering Of Cell Mechanical Microenvironment Using Hydrogels And Their Applications For Muscle Tissue Regeneration

In vivo cells live in a complex microenvironment,which plays a vital role in maintaining cell functions.A variety of investigations reveal that in vivo cells experience and respond to various mechanical stimulus,which can regulate cell behavior and guide cell fate.Study of cell mechanical microenvironment aims to better understand development,healing,tissue remodelling and homeostasis attainment,which involves multidisciplinary fields including mechanics,materials science,chemistry and biology.Hydrogels find widespread applications in engineering cell microenvironment due to their hydrated environment and tunable properties(e.g.,mechanical,chemical,and biocompatible)similar to the native extracellular matrix.Thus,hydrogel can be selected as an appropriate matrix for mimicking cell mechanical microenvironment.Herein,we experimentally and numerically investigated the construction,characterization and application of cell mechanical microenvironment using hydrogels in vitro.In this study,the hydrogel has been selected as matrix for mimicking cell mechanical microenvironment in vitro.To test the mechanical properties of “soft” hydrogels,we developed an experimental model of a magnetic non-contact mechanical testing system.In this system,each hydrogel sample was composed of a magnetically-actuated layer,which can be stretched in an applied magnetic field.This platform can also apply mechanical stretching on such cell-laden hydrogels.To optimize the magnetic field strength and gradient,the finite element analysis was also used here.The mechanical properties of polyacrylamide and native tissues were characterized by magnetic-assisted testing system.To verify the accuracy and stability of our system,the experimental results were compared with results obtained from Bose mechanical testing platform.The mechanical properties of “soft” hydrogels,including poly(ethylene glycol)dimethacrylate and gelatin methacrylate,were then tested.The testing platform was also used for studying the effect of cell remodelling on mechanical properties of gelatin methacrylate hydrogels.The results indicated that the stiffness of hydrogels was enhanced by a large amount of ECM secreted by encapsulated cells.The developed testing platform can be a useful tool for construction and characterization of cell mechanical microenvironment.To construct stiffness microenvironment and stress/strain microenvironment,polyacrylamide and gelatin methacrylate hydrogels were used,respectively.We experimentally and numerically studied the deformation of hydrogels under magnetic field.Cell responses to matrix stiffness and stress/strain were both investigated.Our results indicated that cell viability,proliferation and adhesion was enhanced by increasing matrix stiffness(2 k Pa,10 k Pa,30 kpa and 60 kpa)in 2D microenvironment.Interestingly,cell functions were inhibited by increasing matrix stiffness(2 k Pa,6 k Pa,10 kpa and 20 kpa)in 3D microenvironment.On the contrary,tensile strain(ranging from 10%~100%)significantly promoted cell spreading,proliferation and alignment.Cell apoptosis was also evident in a large strain range(>120%).The stress/strain gradient microenvironment was also engineered in vitro by designing the shape of hydrogels.The stress/strain gradients on hydrogels were characterized.The effect of stress/strain gradients on cell alignment was also investigated.The results showed that cell aligned perpendicular to stretch direction under strain ranging from 17.0 ± 0.5%~19.7 ± 1.0%,whereas cell aligned parallel to stretch direction in strain ranging from 15.4 ± 0.4% ~13.5 ± 0.7%.To clarify the mechanism of mechanical stimulus on cell behavior,cardiac fibrosis models and functional muscle myofibers were both sucessfully fabricated through regulating cell mechanical microenvironment in vitro.For cardiac fibrosis models,the matrix stiffness will enhance cell secretion of ?-SMA by upregulating AT1 R and TGF-?1 markers,which is a key pathway for cardiac myofibroblast differentiation.In addition,the results demonstrated that hepatocyte growth factor secreted by human adipose-derived stem cells plays an essential role in attenuating cardiac fibrosis through downregulation of AT1 R and upregulation of Smad7.For regeneration of functional muscle myofibers,a simple,facile,and high-throughput technique was developed to fabricate 3D cell-laden hydrogel microfibers.Moreover,it is shown that these cell-laden microfibers can induce muscle myofiber formation by tensile stretching alone,which has also been verified in molecular level.This easily adaptable platform provides the experimental basis for regeneration of functional muscle tissues in vitro.