Recent advances in optical techniques for dynamically probing cellular mechanobiology

Cellular mechanotransduction characterized by the transformation of mechanical stimuli into biochemical signals,represents a pivotal and complex process underpinning a multitude of cellular functionalities.This process is integral to diverse biological phenomena,including embryonic development,cell migration,tissue regeneration,and disease pathology,particularly in the context of cancer metastasis and cardiovascular diseases.Despite the profound biological and clinical significance of mechanotransduction,our understanding of this complex process remains incomplete.The recent development of advanced optical techniques enables in-situ force measurement and subcellular manipulation from the outer cell membrane to the organelles inside a cell.In this review,we delved into the current state-of-the-art techniques utilized to probe cellular mechanobiology,their principles,applications,and limitations.We mainly examined optical methodologies to quantitatively measure the mechanical properties of cells during intracellular transport,cell adhesion,and migration.We provided an introductory overview of various conventional and optical-based techniques for probing cellular mechanics.These techniques have provided into the dynamics of mechanobiology,their potential to unravel mechanistic intricacies and implications for therapeutic intervention.

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.

Preface:molecular,cellular,and tissue mechanobiology

Mechanics plays a crucial role in a diversity of biological processes at different length(from molecules,cells,tissues,organs to organisms)and time scales.As a rapidly growing field across the interfaces of mechanics,biology,and medical engineering,mechanobiology aims to identify the mechanical and biological responses of cells and tissues of

Designer substrates and devices for mechanobiology study

Both biological and engineering approaches have contributed significantly to the recent advance in the field of mechanobiology.Collaborating with biologists,bio-engineers and materials scientists have employed the techniques stemming from the conventional semiconductor industry to rebuild cellular milieus that mimic critical aspects of in vivo conditions and elicit cell/tissue responses in vitro.Such reductionist approaches have help to unveil important mechanosensing mechanism in both cellular and tissue level,including stem cell differentiation and proliferation,tissue expansion,wound healing,and cancer metastasis.In this mini-review,we discuss various microfabrication methods that have been applied to generate specific properties and functions of designer substrates/devices,which disclose cell-microenvironment interactions and the underlying biological mechanisms.In brief,we emphasize on the studies of cell/tissue mechanical responses to substrate adhesiveness,stiffness,topography,and shear flow.Moreover,we comment on the new concepts of measurement and paradigms for investigations of biological mechanotransductions that are yet to emerge due to on-going interdisciplinary efforts in the fields of mechanobiology and microengineering.

Combined OVX and Concurrent Mechanical Disuse Induced Osteocytes Morphological Alteration, and Mitigation by Mechanobiology and Sclerostin Antibody

Osteoporosis and osteopenia are major health issues that mainly affect elderly people,women after menopause and immobilized patients.Our previous studies have proved that sclerostin antibody(Scl-Ab)can dramatically enhance bone formation and reduce bone resorption in a severe osteoporosis rat model with the combination of ovariectomy(OVX)and hindlimb immobilization(HLS).However,the mechanism in the cellular level is unclear.The objective of this study is to assess the effect of Scl-Ab on osteocytic morphology change in a combined OVX and HLS rat model via quantification of long-and short-axis and the ratio and osteocyte volume in midshaft cortical bone.Four-month-old virgin female SD rats were divided into 7 groups(n=11 per group):Sham+Veh,Sham+HLS+Veh,Sham+HLS+Scl-Ab,OVX+Veh,OVX+Scl-Ab,OVX+HLS+Veh,OVX+HLS+Scl-Ab.HLS was performed 2 weeks after sham or OVX surgery;and treatment was initiated at the time of HLS.Scl-Ab(25 mg/kg)or vehicle was subcutaneously injected twice per week for 5 weeks.Femurs were harvested at the end of study and embedded in PMMA and polished for SEM imaging.Cortical bone mid shaft osteocyte number per bone area was quantified under 1K magnification;the ratios between long axis and short axis of osteocytes were quantified under 2K magnification;osteocyte dendrite number and surface area were quantified under 5K magnification.Osteocyte dendrites width was quantified using 10K magnification.All the quantification was done by ImageJ.We have reported that multiple morphological and structural changes in osteocytes,including a decreased osteocyte density and reduced osteocyte dendrite number in HLS,OVX or the combination group and Scl-Ab’s ability to abolish these unfavorable alterations.We continued our SEM analysis on osteocytes and discovered that the oval shape of osteocyte under HLS,OVX or HLS+OVX has been distorted toward a spindle-like shape,with relatively longer long axis and shorter short axis,assuming osteocyte has a perfect spheroid shape.The ratio between long-and short-axis showed an increased trend in OVX and HLS condition,but Scl-Ab inhibited these increases(P<0.001,P<0.01,respectively).The volume decreased in HLS,OVX group,but Scl-Ab maintained osteocytes’volume in HLS condition(P<0.001).It indicates that cortical bone responds to HLS and/or OVX and Scl-Ab treatment via multiple cellular mechanisms,including density of osteocyte,dendrite number and osteocyte shape.The change of osteocyte shape may imply an altered cytoskeleton system within osteocyte and a subsequent disruption of mechanosensing ability for osteocyte,which lead to bone loss macroscopically.These data suggest Scl-Ab’s therapeutic potential could be related with its ability to maintain osteocyte’s morphologic and structural changes induced by OVX,HLS or both.

Nuclear Mechanotransduction in Cellular Mechanobiology and Molecular Mechanomedicine

It is known that mechanical forces play critical roles in physiology and diseases but the underlying mechanisms remain largely unknown[1].Most studies on the role of forces focus on cell surface molecules and cytoplasmic proteins.However,increasing evidence suggests that nuclear mechanotransduction impacts nuclear activities and functions.Recently we have revealed that transgene dihydrofolate reductase(DHFR)gene expression is directly upregulated via cell surface forceinduced stretching of chromatin [2].Here we show that endogenous genes are also upregulated directly by force via integrins.We present evidence on an underlying mechanism of how gene transcription is regulated by force.We have developed a technique of elastic round microgels to quantify 3D tractions in vitro and in vivo[3].We report a synthetic small molecule(which has been stiffened structurally)that inhibits malignant tumor repopulating cell growth in a low-stiffness(force)microenvironment and cancer metastasis in mouse models without detectable toxicity[4].These findings suggest that direct nuclear mechanotransduction impacts mechanobiology and mechanomedicine at cellular and molecular levels.

Fung’s Theories on Vascular Mechanobiology

Mechanobiology is the study of how stress and strain are generated by cells and how these mechanical factors regulate cell morphology and function.The vascular system is subject to tensile and compressive stress and strain in the blood vessel wal that are generated by blood pressure and play a pivotal role in regulating vascular cell activities including proliferation,differentiation,apoptosis,and migration.These cellular processes are essential to vascular development,performance,and pathogenic alterations.Dr.Y.C.Fung has made significant contributions to vascular mechanobiology—establishing the uniform stress theory,addressing the generation and significance of uniform stress and strain across the blood vessel wall,and proposing the stress-growth theory,addressing the role of mechanical stress in regulating cell proliferative ac-tivities(Fung 1984,Fung 1990).These theories have exerted a profound impact on the development of Biomechanics and Mechanobiology in the vascular as well as other systems.

Bone Mechanobiology On-a-Chip

Bone is able to adapt its composition and structure in order to suit its mechanical environment.Osteocytes,bone cells embedded in the calcified matrix,are believed to be the mechanosensors and responsible for orchestrating the bone remodeling process[1].However,detailed cellular and molecular mechanism underlying osteocyte mechanobiology is not well understood.Further,how osteocytes communicate with other cell populations under mechanical loading is unclear.Recently,we developed several microfluidic platforms to address these questions.In this talk,osteocyte intracellular response under mechanical loading in the microfluidic environment will first be presented.Next,inter cell-population communications under mechanical loading and its implication in bone disorder management such as bone metastasis prevention will be discussed.1.Study osteocyte response to mechanical loading in a microfluidic environment Current research has focused on observing bone cell mechanotransduction under different simulated physiological conditions(e.g.,shear stress,strain,pressure,etc.)using macro-scale devices.However,these devices often require large sample volumes,low through-put,extensive setup protocol,as well as very limited designs only suitable for general cell culture[2].On the other hand,in vitro microfluidic devices provide an optimal tool to better understand this biological process with its flexible design,physiologically-relevant dimensions,and high-throughput capabilities.Recent work on co-culture platform has demonstrated the feasibility of building more complex microfluidic devices for osteocyte mechanotransduction studies,while maintaining its biological relevance[3].However,there lacks a robust system where multi-physiological flow conditions are applied to bone cells to study their intercellular communication.We aim to fulfill this gap by designing and fabricating a multi-shear stress,co-culture platform to study interaction between osteocytes and other bone cells when exposed to an array of physical cues.The project will rely on standard microfluidic principles in designing devices that utilize changing geometric parameters to induce different flow rates that are directly proportional to the levels of shear stress.All channels within the same device will share a common inlet,while adjusting the resistance of each individual channel will result in a different flow rate.Devices are fabricated using PDMS,and bonded to glass slides of equal sizes.MLO-Y4 osteocyte like cells seeded in the device are stimulated with oscillatory fluid flow with a custom in-house pump.Significant differences in RANKL levels are observed between channels,demonstrating that proper cellular response to flow can be elicit from each distinct shear stress channels as designed.Furthermore,we aim to pair these multi-shear stress channels with corresponding culturing chambers connected through perfusion pores.Through perfusion between the multi-shear stress channels and culturing chambers,different cell population can communicate to each other as they are stimulated by varying levels of shear stress.Using this platform,we will be able to mimic the interaction between osteocytes and other bone cells in vitro.Due to the advantage of using microfluidic devices,various analytical methods can be used on-chip to determine cellular response,such as staining for biomarkers and differentiation factors.2.Microfluidic platform for investigation of mechanoregulation of breast cancer bone metastasis Approximately 70%of advanced breast cancer patients experience bone metastasis.Breast cancer cells(BCC)that extravasate across the endothelium to the bone reduce bone quality by disrupting the healthy bone remodeling balance.Exercise,a common cancer intervention strategy,can regulate bone remodeling,thus potentially affect BCC metastasis to bone through signals released by mechanical loaded osteocytes.Our recent in vitro studies showed that mechanically stimulatedosteocytes can regulate BCC migration via endothelial cells[5].However,a more physiologically relevant platform is needed to better investigate the mechanisms leading to interactions between BCC and bone microenvironment under mechanical loading.We present here a novel in vitro microfluidic tri-culture lumen system for studying mechanical regulation of breast cancer metastasis in bone.In this study,highly metastatic MDA-MB-231 human BCCs were cultured inside a cylindrical lumen lined with human umbilical vein endothelial cells(HUVECs),which is adjacent to a population of either static or mechanically-stimulated osteocyte-like MLO-Y4 cells.Physiologically relevant oscillatory fluid flow(OFF)(1 Pa,1 Hz)was produced by a custom pump to mechanically load the MLO-Y4 cells.Soluble factors were diffused through hydrogel-filled side channels to enable inter-cell population communication between MLO-Y4 cells and BCCs over 3 days.BCC extravasation distance and percentage were measured and normalized to the acellular control with MLO-Y4 media only.Paired t-tests(n=5)were used for statistical analysis and the Holm-Bonferroni method was applied for multiple comparison analysis.Statistical significance was taken at P<0.05.Photolithography and soft lithography were used to fabricate silicon SU-8 master and PDMS replicates,respectively.A HUVEC lumen was successfully cultured in the PDMS microfluidic device.Extravasation distance was significantly decreased in the flowed osteocytes(33.6%of control)compared to static osteocytes(108.0%of control),while the extravasation percentage showed a non-significant decreasing trend between the flow(58%of control)and static(106.3%of control)osteocytes.In summary,we developed the first microfluidic platform allowing the integration of physiologically relevant bone fluid stimulation and real-time intercellular signaling between different cell populations in vitro.Using this platform,the significantly reduced extravasation distance was found in the group where conditioned medium from osteocytes exposed to flow.We speculate this could be due to regulation of matrix metallopeptidase 9(MMP-9)used by cancer cells to degrade the surrounding matrix during extravasation.Future work with this platform will determine the key mechanisms involved in osteocyte regulation of BCC metastasis.

Multiscale mechanobiology modeling for surgery assessment

This paper discusses some of the concept of modeling surgery outcome. It is also an attempt to offer a road map for progress. This paper may serve as a common ground of discussion for both communities i.e surgeons and computational scientist in its broadest sense. Predicting surgery outcome is a very difficult task. All patients are different, and multiple factors such as genetic, or environment conditions plays a role. The difficulty is to construct models that are complex enough to address some of these significant multiscale elements and simple enough to be used in clinical conditions and calibrated on patient data. We will pro- vide a multilevel progressive approach inspired by two applications in surgery that we have been working on. One is about vein graft adaptation after a transplantation, the other is the recovery of cosmesis outcome after a breast lumpectomy. This work, that is still very much in progress, may teach us some lessons. We are convinced that the digital revolution that is transforming the working environment of the surgeon makes closer collaboration between surgeons and computational scientist unavoidable. We believe that ＂computational surgery＂ will allow the community to develop predictive model of the surgery outcome and greatprogresses in surgery procedures that goes far beyond the operating room procedural aspect.

Molecular engineering and live-cell imaging in mechanobiology

Cells in the body are exposed to physiological and pathophysiological stimuli that encompass both chemical and mechanical factors,which coordinately modulate cellular functions. Compared to the large amount of information on cellular re-

Mechanical characterization of soft silicone gels via spherical nanoindentation for applications in mechanobiology

As a biocompatible material,soft silicone gels like CY52-276 have been applied to many engineering fields associated with interactions between mammalian cells and extracellular matrices/substrates,due to its nontoxicity,ease of preparation,optical transparency and tunable mechanical properties.Precise quantification of mechanical properties of silicone gels is crucial for quantitatively investigating mechanical responses of cells to microenvironments.Addressing the material with high surface energy,we design a new strategy for the nanoindentation technique to reduce or even eliminate the effect of interfacial adhesions with the aid of some specified buffers.Next,we dissect the dependence of its Young’s modulus on the ratios of monomers and crosslinkers and curing conditions,and therefore identify a dose-response relationship between its moduli and the corresponding prepolymer compositions.With the non-linear large deformation nanoindentation tests,we further showed that the two-parameter Mooney-Rivlin model may well characterize the hyperelastic deformations of the materials with different composition ratios.These pave the way for more precise exploration of the interaction between cells and extracellular matrix and the underlying mechanotransduction pathways in mechanobiology.

Vibration stimuli and the differentiation of musculoskeletal progenitor cells: Review of results in vitro and in vivo

Due to the increasing burden on healthcare budgets of musculoskeletal system disease and injury, there is a growing need for safe, effective and simple therapies. Conditions such as osteoporosis severely impact onquality of life and result in hundreds of hours of hospital time and resources. There is growing interest in the use of low magnitude, high frequency vibration(LMHFV) to improve bone structure and muscle performance in a variety of different patient groups. The technique has shown promise in a number of different diseases, but is poorly understood in terms of the mechanism of action. Scientific papers concerning both the in vivo and in vitro use of LMHFV are growing fast, but they cover a wide range of study types, outcomes measured and regimens tested. This paper aims to provide an overview of some effects of LMHFV found during in vivo studies. Furthermore we will review research concerning the effects of vibration on the cellular responses, in particular for cells within the musculoskeletal system. This includes both osteogenesis and adipogenesis, as well as the interaction between MSCs and other cell types within bone tissue.

Probing the mechanosensitivity in cell adhesion and migration: Experiments and modeling

Cell adhesion and migration are basic physiolog- ical processes in living organisms. Cells can actively probe their mechanical micro-environment and respond to the ex- ternal stimuli through cell adhesion. Cells need to move to the targeting place to perform function via cell migration. For adherent cells, cell migration is mediated by cell-matrix adhesion and cell-cell adhesion. Experimental approaches, especially at early stage of investigation, are indispensable to studies of cell mechanics when even qualitative behaviors of cell as well as fundamental factors in cell behaviors are unclear. Currently, there is increasingly accumulation of ex- perimental data of measurement, thus a quantitative formula- tion of cell behaviors and the relationship among these fun- damental factors are highly needed. This quantitative under- standing should be crucial to tissue engineering and biomed- ical engineering when people want to accurately regulate or control cell behaviors from single cell level to tissue level. In this review, we will elaborate recent advances in the ex- perimental and theoretical studies on cell adhesion and mi- gration, with particular focuses laid on recent advances in experimental techniques and theoretical modeling, through which challenging problems in the cell mechanics are sug- gested.

Cell Nanomechanics Based on Dielectric Elastomer Actuator Device

As a frontier of biology,mechanobiology plays an important role in tissue and biomedical engineering.It is a common sense that mechanical cues under extracellular microenvironment affect a lot in regulating the behaviors of cells such as proliferation and gene expression,etc.In such an interdisciplinary field,engineering methods like the pneumatic and motor-driven devices have been employed for years.Nevertheless,such techniques usually rely on complex structures,which cost much but not so easy to control.Dielectric elastomer actuators(DEAs)are well known as a kind of soft actuation technology,and their research prospect in biomechanical field is gradually concerned due to their properties just like large deformation(>100%)and fast response(<1 ms).In addition,DEAs are usually optically transparent and can be fabricated into small volume,which make them easy to cooperate with regular microscope to realize real-time dynamic imaging of cells.This paper first reviews the basic components,principle,and evaluation of DEAs and then overview some corresponding applications of DEAs for cellular mechanobiology research.We also provide a comparison between DEA-based bioreactors and current custom-built devices and share some opinions about their potential applications in the future according to widely reported results via other methods.

In situ measurement of ECM rheology and microheterogeneity in embedded and overlaid 3D pancreatic tumor stroma co-cultures via passive particle tracking

Tumor growth is regulated by a diverse set of extraellular infuences,including paracrine crosstalk with stromal partners,and biophysical interactions with surrounding cells and tissues.Studies elucidating the role of physical force and the mechanical properties of the extracellular matrix(ECM)itself as regulators of tumor growth and invasion have been greatly catalyzed by the use of in ritro three-dimensional(3D)tumor models.These systems provide the ability to systematically isolate,manipulate,and evaluate impact of stromal components and extracellular mechanics in a platform that is both conducive to imaging and biologically relevant.However,recognizing that mechanoregulatory crosstalk is bi-directional and fully utilizing these models requires complementary methods for in situ measurements of the local mechanical environment.Here,in 3D tumor/fbroblast co-culture models of pancreatic canocer,a discase characterized by its prominent stromal involvement,we evaluate the use of particle-tracking microrhoology to probe dynamic mechanical changes.Using videos of fuorescently labeled polystyrene micro-spheres embedded in ollagen I ECM,we measure spatiotemporal changes in the Brownian motion of probes to report local ECM shear modulus and microheterogeneity.This approach reveals st ffening of collagen in fibroblast co-cultures relative to cultures with cancer cells only,which exhibit degraded ECM with heterogeneous microstructure.We further show that these effects are dependent on culture geometry with contrast ing behavior for embedded and overlay cultures.In addition to potential application to screening stroma targeted therapeutics,this work also provides insight into how the compoition and plating geometry impact the mechanical propertios of 3D cell cultures that are increasingly widey used in cancer biology.

Role of Mechanical Stimuli in Oral Implantation

Prosthetic implantation has been a prevalent surgical procedure in dentistry. Insertion of dental implant significantly changes local oral conditions and leads to the surrounding bone to remodel to a new morphology. To predict how the bone responds such a biomechanical change, finite element analysis (FEA) based remodeling simulation has proven effective. For a range of mechanical stimuli, which should be used remains controversial arguable? This paper aims to compare how the different mechanical stimuli, including mechostat model (effective strain), daily stress and strain energy density (SED) affect the predictions of bone remodeling.

Mechanical Stress to Cell Nucleus Inhibits Proliferation and Differentiation of Vascular Smooth Muscle Cells

Cells sense the external environment such as a surface topography and change many cellular functions. Cell nucleus has been proposed to act as a cellular mechanosensor, and the changes in nuclear shape possibly affect the functional regulation of cells. This study demonstrated a large-scale mechanical deformation of the intracellular nucleus using polydimethylsiloxane (PDMS)-based micropillar substrates and investigated the effects of nuclear deformation on migration, proliferation, and differentiation of vascular smooth muscle cells (VSMCs). VSMCs spread completely between the fibronectin-coated pillars, leading to strong deformations of their nuclei resulted in a significant inhibition of the cell migration. The proliferation and smooth muscle differentiation of VSMCs with deformed nuclei were dramatically inhibited on the micropillars. These results indicate that the inhibition of proliferation and VSMC differentiation resulted from deformation of the nucleus with high internal stress, and this type of large-scale nuclear mechanical stress might lead the cells to a “quiescent state”.

Propagation of Acoustic Waves Caused by the Accelerations of Vibrating Hand-Held Tools in Viscoelastic Soft Tissues of Human Hands and a Mechanobiological Picture for the Related Injuries

As is well known, hand-arm vibration syndrome (HAVS), or vibration-induced white finger (VWF), which is a secondary form of Raynaud’s syndrome, is an industrial injury triggered by regular use of vibrating hand-held tools. According to the related biopsy tests, the main vibration-caused lesion is an increase in the thickness of the artery walls of the small arteries and arterioles resulted from enlarged vascular smooth muscle cells (VSMCs) in the wall layer known as tunica media. The present work develops a mechanobiological picture for the cell enlargement. The work deals with acoustic variables in solid materials, i.e., the non-equilibrium components of mechanical variables in the materials in the case where these components are weakly non-equilibrium. The work derives an explicit expression for the infinite-time cell-volume relative enlargement. This enlargement is directly affected by the acoustic pressure in the soft living tissue (SLT). In order to reduce the enlargement, one can reduce either the ratio of the acoustic pressure in the SLT to the cell bulk modulus or the relaxation time induced by the cell osmosis, or both the characteristics. Also, a mechanoprotective role of the above relaxation time in the cell-volume maintenance is noted. The above mechanobiological picture focuses attention on the pressure in an SLT and, thus, modeling of propagation of acoustic waves caused by the acceleration of a vibrating hand-held tool. The present work analyzes the propagation along the thickness of an infinite planar layer of an SLT. The work considers acoustic modeling. As a general viscoelastic acoustic model, the work suggests linear non-stationary partial integro-differential equation (PIDE) for the weakly non-equilibrium component of the average normal stress (ANS) or, briefly, the acoustic ANS. The PIDE is, in the exponential approximation for the normalized stress-relaxation function (NSRF) reduced to the third-order linear non-stationary partial differential equation (PDE), which is of the Zener type. The unique advantage of the PIDE is that it presents a compact model for the acoustic ANS in an SLT, which explicitly includes the NSRF, thereby enabling a consistent description of the lossy-propagation effects inherent in SLTs. The one-spatial-coordinate version of this PDE in the planar SLT layer with the corresponding boundary conditions is considered. The relevance of these settings is motivated by a conclusion of other authors, which is based on the results of the frequency-domain simulation in three spatial coordinates. The boundary-value problem at arbitrary value of the stress-relaxation time (SRT) and arbitrary but sufficiently regular shape of the external acceleration is analytically solved by means of the Fourier method. The obtained solution is the steady-state acoustic ANS and allows calculation of the corresponding steady-state acoustic pressure as well. The derived analytical representations are computationally implemented. Propagation of the pressure waves in the SLT layer at zero and different nonzero values of the SRT, and the single-pulse external acceleration is presented. They complement the zero-SRT and zero-SRT-asymptote results with the results for various values of the SRT. The obtained pressure values are, at all of the space-time points under consideration, meeting the condition for the adequateness of the linear model. In the case where the SRT is zero, the results well agree with the ones obtained by using the simulation software package LS-DYNA. The dependence of the damping of acoustic variables in an SLT on the SRT in the present third-order case significantly generalizes the one in the second-order linear systems. The related resonance effect in the waves of the acoustic pressure propagating in an SLT is also discussed. The effects of the NSRF-originated memory function provided by the present third-order PDE model are necessary for proper simulation of the pressure, which is of special importance in the aforementioned mechanoboiological picture. The results obtained in the work present a viscoelastic acoustic framework for SLTs. These results open a way to quantitatively specific evaluation of technological strategies for reduction of the vibration-caused injuries or, loosely speaking, achieving “zero’’ injury.

Three-month effects of corneal cross-linking on corneal fibroblasts

Corneal collagen crosslinking(CXL)has revolutionized the treatment of keratoconus in the past decade.In order to evaluate the 3-month effects of CXL on corneal fibroblasts,a longitudinal study at the tissue and cellular level was carried out with a total of 16 rabbits that underwent CXL,deepithelialization(DEP),or non-treatment(control)and kept for 1 to 3 months.The duration of corneal stromal remodeling after CXL was determined by examining the differentiation,apoptosis,and number changes of keratocytes in tissue sections from animals 1,2,or 3 months post-treatment.Upon the finish of tissue remodeling,separate rabbits were used to extract keratocytes and set up cell culture for vimentin immunofluorescence staining.The same cell culture was used for(1)migration measurement through the wound-healing assay;(2)elastic modulus measurement by atomic force microscope(AFM);(3)the proliferation,apoptosis,cytoskeleton andα-SMA expression tests through EdU(5-ethynyl-2’-deoxyuridine)assay,TUNEL(TdT-mediated dUTP Nick-End Labeling)assay,phalloidin andα-SMA(alpha-smooth muscle actin)immunofluorescence analysis,respectively.Results showed that the migratory activity,elastic modulus,andα-SMA expression of the corneal fibroblasts increased after CXL treatment,while apoptosis,proliferation,and morphology of F-actin cytoskeleton of the fibroblasts had no significant change after 3 months.In contrast,measured cellular parameters(migratory,elastic moduli,α-SMA expression,apoptosis,proliferation,and morphology of F-actin cytoskeleton of fibroblasts)did not change significantly after DEP.In conclusion,the dynamic changes of keratocytes were nearly stable 3 months after CXL treatment.CXL has an impact on corneal fibroblasts,including migration,elastic modulus andα-SMA expression,while epithelialization may not alter the biological behavior of cells significantly.

Portal Hypertension in Nonalcoholic Fatty Liver Disease: Challenges and Paradigms

Portal hypertension in cirrhosis is defined as an increase in the portal pressure gradient(PPG)between the portal and hepatic veins and is traditionally estimated by the hepatic venous pressure gradient(HVPG),which is the difference in pressure between the free-floating and wedged positions of a balloon catheter in the hepatic vein.By convention,􀀫HVPG≥10mmHg indicates clinically significant portal hypertension,which is associated with adverse clinical outcomes.Nonalcoholic fatty liver disease(NAFLD)is a common disorder with a heterogeneous clinical course,which includes the development of portal hypertension.There is increasing evidence that portal hypertension in NAFLD deserves special considerations.First,elevated PPG often precedes fibrosis in NAFLD,suggesting a bidirectional relationship between these pathological processes.Second,HVPG underestimates PPG in NAFLD,suggesting that portal hypertension is more prevalent in this condition than currently believed.Third,cellular mechanoresponses generated early in the pathogenesis of NAFLD provide a mechanistic explanation for the pressurefibrosis paradigm.Finally,a better understanding of liver mechanobiology in NAFLD may aid in the development of novel pharmaceutical targets for prevention and management of this disease.