Mechanical microenvironments of living cells: a critical frontier in mechanobiology

The fields of biomechanics and mechanobiology have long been predicated on the premise that mechanics governs cell behavior. However, over the past few years, a growing body of evidence has suggested that the mechanical environment very close to cells–the cell microenvironment–plays the most important role in determining what a cell feels and how it responds to tissue-level stimuli. To complicate matters further, cells can actively manipulate their microenvironments through pathways of recursive mechanobiological feedback. Harnessing this recursive behavior to understand and control cell physiology and pathophysiology is a critical frontier in the field of mechanobiology. Recent results suggest that the key to opening this scientific frontier to investigation and engineering application is understanding a different frontier: the physical frontier that cells face when probing their mechanical microenvironments.

The Matrix Stiffness and Physical Confinement of Hydrogel Microchannel Jointly Induce the Mesenchymal-Amoeboid Transition for Cancer Cell Migration

The migration mode transition of cancer cell enhances its invasive capability and the drug resistance,where physical confinement of cell microenvironment has been revealed to induce the mesenchymal-amoeboid transition(MAT).However,most existing studies are performed in PDMS microchannels,of which the stiffness is much higher than that of most mammalian tissues.Therefore,the amoeboid migration transition observed in these studies is actually induced by the synergistic effect of matrix stiffness and confinement.Since the stiffness of cell microenvironment has been reported to influence the cell migration in 2D substrate,the decoupling of stiffness and confinement effects is thus in need for elucidating the underlying mechanism of MAT.However,it is technically challenging to construct microchannels with physiologically relevant stiffness and channel size,where existing microchannel platforms with physiological relevance stiffness are all with>10μm channel width.Such size is too wide to mimic the physical confinement that migrating cancer cells confront in vivo,and also larger than the width of PDMS channel,in which the MAT of cancer cell was observed.Therefore,an in vitro cell migration platform,which could mimic both stiffness and confinement of the native physical microenvironment during cancer metastasis,could profoundly contribute to researches on cancer cell migration and cellular mechanotransduction.In this paper,we overcome the limitations of engineering soft materials in microscale by combining the collagen-alginate hydrogel with photolithography.This enables us to improve the accuracy of molded microchannel,and thus successfully construct a 3D microchannel platform,which matches the stiffness and width ranges of native environmental confinement that migrating cancer cells confront in vivo.The stiffness(0.3~20 kPa),confinement(channel width:3.5~14μm)and the adhesion ligand density of the microchannel can be tuned independently.Interestingly,using this platform,we observed that the migration speed of cancer cell is influenced by the synergistic effect of channel stiffness and width,and the increasing stiffness reverses the effect of channel width on the migration speed of cancer cells.In addition,MAT has a strong correlation with the channel stiffness.These findings make us reconsider the widely accepted hypothesis:physical confinement can induce MAT.Actually,this transition can only occur in stiff confined microenvironment not in soft one.For soft microchannels,the compliance of the channel walls could cause little cell/nucleus deformation,and the MAT could not be induced.To further investigate the mechanism of MAT,we developed a computational model to simulate the effect of nucleus deformation on MAT.With the model,we found that deforming the cell nuclear by decreasing the nucleus stiffness will reduce the cellmigration speed.This implies that nuclear stiffness plays an important role in the regulation of cancer migration speed and thus MAT in microchannels.The effect of channel stiffness on MAT and migration speed as observed in our experiment could partially explain previous findings reported in the literature,where the increasing matrix stiffness of tumor microenvironment promotes cancer metastasis.Our observations thus highlight the critical role of cell nuclear deformation not only in MAT,but also in regulating cellular mechanotransduction and cell-ECM interactions.This developed platform is capable of mimicking the native physical microenvironment during metastasis,providing a powerful tool for high-throughput screening applications and investigating the interaction between cancer migration and biophysical microenvironment.

Effect of viscoelasticity on skin pain sensation

Pain sensation may appear under long-lasting mechanical stimulation. Although people have the experience that pain sensation generally decreases with time while the stimulation remains, the underlying mechanism remains elusive. We experimentally studied the thermal and strain rate- dependent viscoelastic behavior of skin in uniaxial stretch and numerically investigated the effects of temperature and strain rate on pain sensation. The results indicate that the viscosity of skin tissue decreases with increasing temperature and reducing strain rate, which subsequently decreases the discharge frequency of skin nociceptor and thus relieves the pain sensation. The results would contribute to the understanding of pain relief mechanism and optimizing for mechanical treatment.

A Mechanoelectrical Coupling Model of Neurons under Stretching

Introduction Neurons are situated in a microenvironment composed of various biochemical and biophysical cues,where stretching is thought to have a major impact on neurons.For instance,during a moderate traumatic brain impact,the injury region in axons exhibits significant longitudinal strain;and in a rat model of spinal cord injury,the most severe axonal injury is located in the largest strain region.Stretching may result in microstructural changes in neural tissue and further leading to abnormal electrophysiological function.Hence,it is of great importance to understand the coupled mechanoelectricalbehaviors of neurons under stretching.In spite of significant experimental efforts,the underlying mechanism remains elusive,more works are needed to provide a detailed description of the process that leads to the observed phenomena.Mathematical modeling is a powerful tool that offers a quantitative description of the underlying mechanism of an observed biological phenomenon,including mechanical and electrophysiological behaviors of neurons.Thus,we developed a mechanoelectrical coupling model of neurons under stretching in this study.Mathematical model The mathematical model consists of three submodels,i.e.,the mechanical submodel,the mechanoelectrical coupling submodel and the electrophysiological submodel.The mechanical submodel deals with the relationship between stretching and the deformation of axons,which has specially considered the plastic deformation of axons.The electrophysiological submodel characterizes the feature of neuronal action potential(AP),which is based on the classical H-H model and the cable theory.The mechanoelectrical coupling submodel links the mechanical and electrophysiological submodels through strain-induced equivalent circuit parameter alteration and ion channel injury.Besides,we have discussed a more general deformation condition,where an expanded model coupling the axonal deformation and electrophysiology alteration was explored.As the most essential parameters in an electrophysiological assessment,the amplitude of the AP,the neuronal firing frequency and the electrophysiological signal conduction velocity,which could be affected by stretching,were used as outputs of the model.Results&discussion To understand the mechanoelectrical coupling of neurons under stretching,we developed a mechanoelectrical coupling model.To verify the model,we simulated a slow stretching on an axon following the experimental study in the literature,we observed that as the strain increases,the peak AP declines faster,which is consistent with the experimental data.Moreover,the reduced AP cannot be restored to the original peak,implying that the damage is irreversible.The simulation results also predict that strain induces a more frequent neuronal firing and a faster conduction.In a realistic situation,in addition to stretching,the loading condition is very complicated,which may induce complex axonal deformation(e.g., necking and swelling along the axons).We also simulated such necking deformation impairment and observed that the AP amplitude decreases at the necking region and recovers after that,indicating a blockage of the AP;and the conduction velocity decreases with the increase in deformation degree.Conclusions In this study,we developed a mechanoelectrical coupling model of neurons under stretching with consideration of axonal plastic deformation.With the model,we found that the effect of mechanical loading on electrophysiology mainly manifests as decreased membrane AP amplitude,a more frequent neuronal firing and a faster electrophysiological signal conduction.The model predicts not only stretch-induced injury but also a more gene ral necking deformation case,which may someday be revealed in future by experiments,providing a reference for the prediction and regulation of neuronal function under mechanical loadings.

Dynamic Change of Matrix Stiffness Switches Astrocyte Phenotype in Three Dimensions

Background Damage to the central nervous system(CNS)usually leads to the activation of astrocytes,followed by glial scar formation.For years,glial scar has been thought as a major obstacle for successful axon regeneration.However,increasing evidence suggests a beneficial role for this scar tissue as part of the endogenous local immune regulation and repair process.Surprisingly,in contrast to scars in other tissues,glial scars(mainly consist of reactive astrocytes)in both rat cortex and spinal cord were recently found to be significantly softer than healthy CNS tissues.Naive astrocytes have been found to change their phenotype to reactive astrocytes and gradually into scar-forming astrocytes,upregulating the astrocyte marker glial fibrillary acidic protein(GFAP),vimentin,and inflammatory proteins in almost all known brain disorders.Such phenotype transformation process has been widely thought unidirectional or irreversible.However,recent research revealed the environment-dependent plasticity of astrocyte phenotypes,with reactive astrocytes could revert in retrograde to naive astrocytes in proper microenvironment.In consideration of the important roles of mechanical cues in CNS and the unique softening behavior of glial scars,it is of great interesting to study the effects of dynamic changes of matrix stiffness on astrocyte phenotypic switch.Materials&methods Primary astrocyes were isolated from the cortex of SpragueDawley(SD)rats at PI.After cultured for 2 weeks,astrocytes were encapsulated into a set of three-dimensional(3D)hybrid hydrogel system composed of type I collagen and alginate.Immunofluorescence and Western blot expression analysis were applied for characterizting cell responses to different and dynamically changed matrix stiffness.A molecular dynamics model was developed for simulation.Results&discussion In this work,we established an in-vitro model to study the effects of dynamic changes of matrix stiffness on astrocyte phenotypic switchings in 3D.To simulate native cellular environment,we fabricated a set of hybrid hydrogel system composed of type I collagen and alginate.The stiffness of the hybrid hydrogels was demonstrated to be dynamically changed by adding calcium chloride or sodium citrate to crosslink or decrosslink alginate,respectively.Using 3D culture models,we showed that the decrease of matrix stiffness could promote astrocyte activation,with upregulated GFAP and IL-1β.In addition,3D cultured astrocytes spread greater with decreasing matrix stiffness.Moreover,we surprisingly found that astrocyte phenotype could be switched by dynamically changing matrix stiffness.Specifically,matrix stiffening reverted the activation of astrocytes,whereas matrix softening induced astrocyte activation.We further demonstrated that matrix stiffness-induced astrocyte activation was mediated through cytoskeletal tension and YAP protein.To some extent,YAP inhibition enhanced the responses of astrocytes to matrix stiffness.These may guide researchersto re-examine the role of matrix stiffness in reactive astrogliosis in vivo,and inspire the development of novel therapeutic approach for reducing glial scar following injury,enabling axonal regrowth and improving functional recovery by exploiting the benefits of mechanobiology studies.Conclusions Taken together,our results clarify the effects of matrix stiffness and its dynamic changes on phenotypic swtich of astrocytes in three dimensions and reveal environmental factors that regulate astrocytic phenotype transformation process,which may provide potential therapeutic approach for CNS injury.

Decrease of Matrix Plasticity Promotes Fibroblast Activation in Fibrosis

This work identified the important role of matrix mechanical plasticity in mediating fibroblast activation.Many existing studies have highlighted the important effects of biochemical cues(e.g.,transforming growth factor-β1)and mechanicalstiffness on fibroblast activation.Our results indicated that self-assembled collagen hydrogels showed high plasticity and in which fibroblasts remain undifferentiated.However,when we decreased the plasticity of collagen hydrogels by increasing covalent crosslinking,fibroblasts showed a significant fibrotic response as reflected by the increasedα-SMA expression.Since the material systems we constructed have low and the same initial modulus,this process is stiffness independent.Although it has been reported that covalently crosslinked hydrogels are more difficult to degrade and matrix degradability has an important impact on cell behaviors,no significant changes of fibroblast activation were observed when proteases were broadly inhibited in our experiments.Importantly,the hydrogels we constructed showed similar plastic behaviors under creep and recovery tests compared to native normal and fibrotic tissues.These highlight the importance of matrix plasticity in mimicking the mechanical microenvironment of native fibrotic tissues.Mechanistically,we found that the enhanced fibroblast activation in low plastic matrix is mediated through integrin-actin pathway and nuclear localization of YAP.In high plastic collagen,matrix cannot provide effective resistance to actin contraction because of the rupture of weak crosslinks and the slippage of local fibers.On the contrary,in low plastic collagen,deformation energy can be stored in the network due to the existence of strong covalent crosslinks,thus enabling the build-up of cell traction and the formation of a robust cell-matrix interaction.Experiments of inhibiting or promoting cytoskeletal contractility and CGMD simulation both verified the above points.Our results clarify plasticity changes on the development of fibrotic diseases and highlight plasticity as an important mechanical cue in understanding cell-matrix interactions.

Matrix Stiffness Controls Cardiac Fibroblast Differentiation Through Regulating YAP via AT1R

Cardiac fibrosis is a common pathway to heart injury and failure,where continued activation of cardiac fibroblasts(CFs)during myocardium damage causes excessive deposition of the extracellular matrix and thus increases matrix stiffness.Increasing evidence has shown that stiff matrix plays an important positive role in promoting CF differentiation and cardiac fibrosis,with several signaling factors medicating CF mechanotransduction already identified.However,key moleculesthat perceive matrix stiffness to regulate CF differentiation remain to be fully defined.Recently,Hippo pathway transcriptional coactivators,i.e.,Yes-associated protein(YAP)and transcriptional coactivator with PDZ-binding motif(TAZ),have been found to work as mechanical signal transductors.Importantly,it has shown that YAP plays important roles in various types of fibrosis.Despite these findings,the role of YAP in CF mechanotransduction and cardiac fibrosis still remains elusive.Moreover,several several types of GPCRs have also been found to enable cells to sense mechanical cues,however,the relationship between these GPCRs and YAP in cell mechanotransduction is still not clear.Our recent work demonstrated that blocking of angiotensin II type 1 receptor(AT1R,the first GPCRs found to be mechanosensors)with losartan significantly inhibited the differentiation of CFs to myofibroblasts induced by stiff substrate.Taken these findings into account,we speculate that YAP may work as an important downstream signaling molecule of AT1R in mediating matrix stiffness-induced CF differentiation.In this work,we first characterized the expression of YAP in normal control(NC)and myocardial infarct(Ml)tissues of rats by using immunohistochemistry,immunofluorescence and Western blot analysis.We then investigated the role of YAP in matrix stiffness-induced CF differentiation in vitro by culturing CFs on mechanically tunable gelatin hydrogels.Finally,we explored the relationship between YAP and AT1R in CF mechanotransduction by selective transfection and inhibition experiments.The expression of YAP andα-SMA in cultured CFs were evaluated with immunofluorescence staining,Western blot and real-time quantitative PCR analysis.Immunohistochemical analysis revealed that both YAP andα-SMA significantly increased in Ml tissue compared with NC tissue.The expression and nuclear localization of YAP increased in CFs cultured on stiff matrix.YAP-deficient CFs cultured on soft and stiff matrix both showed decreased expression ofα-SMA.Meanwhile,YAP-overexpressing CFs cultured on soft and stiff matrix both showed increased expression ofα-SMA.Blocking of AT1R decreased the expression levels ofα-SMA and YAP and thus affected the responses of CFs to matrix stiffness.To sum up,our results identified an important role of YAP in mediating matrix stiffness-induced CF differentiation and also established the YAP pathway as an important signaling branch downstream of AT1R in CF mechanotransduction.This study may help to better understand the mechanism of fibrotic mechanotransduction and inspire the development of new approaches for treating cardiac fibrosis.

Development of a micro-indentation device for measuring the mechanical properties of soft materials

Indentation is a simple and nondestructive method to measure the mechanical properties of soft materials, such as hydrogels, elastomers and soft tissues. In this work, we have developed a micro-indentation system with high-precision to measure the mechanical properties of soft materials, where the shear modulus and Poisson＇s ratio of the materials can be obtained by analyzing the load relaxation curve. We have validated the accuracy and stability of the system by comparing the measured mechanical properties of a polyethylene glycol sample with that obtained from a commercial instrument. The mechanical properties of another typical polydimethylsiloxane sample submerged in heptane are measured by using conical and spherical indenters, respectively. The measured values of shear modulus and Poisson＇s ratio are within a reasonable range.