Mechanobiology Research Research Articles

Nanoscale Cellular Traction Force Quantification: CRISPR-Cas12a Supercharged DNA Tension Sensors in Nanoclustered Ligand Patterns.

High-throughput measurement of cellular traction forces at the nanoscale remains a significant challenge in mechanobiology, limiting our understanding of how cells interact with their microenvironment. Here, we present a novel technique for fabricating protein nanopatterns in standard multiwell microplate formats (96/384-wells), enabling the high-throughput quantification of cellular forces using DNA tension gauge tethers (TGTs) amplified by CRISPR-Cas12a. Our method employs sparse colloidal lithography to create nanopatterned surfaces with feature sizes ranging from sub 100 to 800 nm on transparent, planar, and fully PEGylated substrates. These surfaces allow for the orthogonal immobilization of two different proteins or biomolecules using click-chemistry, providing precise spatial control over cellular signaling cues. We demonstrate the robustness and versatility of this platform through imaging techniques, including total internal reflection fluorescence microscopy, confocal laser scanning microscopy, and high-throughput imaging. Applying this technology, we measured the early stage mechanical forces exerted by 3T3 fibroblasts across different nanoscale features, detecting forces ranging from 12 to 56 pN. By integrating the Mechano-Cas12a Assisted Tension Sensor (MCATS) system, we achieved rapid and high-throughput quantification of cellular traction forces, analyzing over 2 million cells within minutes. Our findings reveal that nanoscale clustering of integrin ligands significantly influences the mechanical responses of cells. This platform offers a powerful tool for mechanobiology research, facilitating the study of cellular forces and mechanotransduction pathways in a high-throughput manner compatible with standard cell culture systems.

Leveraging computational modeling to explore epithelial and endothelial cell monolayer mechanobiology.

Endothelial cells (ENCs) and epithelial cells (EPCs) form monolayers whose barrier function is critical for the maintenance of physiological processes and extremely sensitive to mechanical cues. Computational models have emerged as powerful tools to elucidate how mechanical cues impact the behavior of these monolayers in health and disease. Herein, the importance of mechanics in regulating ENC and EPC monolayer behavior is established, highlighting similarities and differences in various biological contexts. Concurrently, computational approaches and their importance in accelerating mechanobiology studies are discussed, emphasizing their limitations and suggesting future directions. The aim is to inspire further synergies between cell biologists and modelers, which are crucial for accelerating cell mechanobiology research.

A Microfluidic-Based Cell-Stretching Culture Device That Allows for Easy Preparation of Slides for Observation with High-Magnification Objective Lenses.

Microfluidic-based cell-stretching devices are vital for studying the molecular pathways involved in cellular responses to mechanobiological processes. Accurate evaluation of these responses requires detailed observation of cells cultured in this cell-stretching device. This study aimed to develop a method for preparing microscope slides to enable high-magnification imaging of cells in these devices. The key innovation is creating a peelable bond between the cell culture membrane and the upper channel, allowing for easy removal of the upper layer and precise cutting of the membrane for high-magnification microscopy. Using the fabricated device, OP9 cells (15,000 cells/channel) were stretched, and the effects of focal adhesion proteins and the intracellular distribution of YAP1 were examined under a fluorescence microscope with 100× and 60× objectives. Stretch stimulation increased integrinβ1 expression and promoted integrin-vinculin complex formation by approximately 1.4-fold in OP9 cells. Furthermore, YAP1 nuclear localization was significantly enhanced (approximately 1.3-fold) during stretching. This method offers a valuable tool for researchers using microfluidic-based cell-stretching devices. The advancement of imaging techniques in microdevice research is expected to further drive progress in mechanobiology research.

An open-source membrane stretcher for simultaneous mechanical and structural characterizations of soft materials and biological tissues

The ability to simultaneously measure material mechanics and structure is central for understanding their nonlinear relationship that underlies the mechanical properties of materials, such as hysteresis, strain-stiffening and -softening, and plasticity. This experimental capability is also critical in biomechanics and mechanobiology research, as it enables direct characterizations of the intricate interplay between cellular responses and tissue mechanics. Stretching devices developed over the past few decades, however, do not often allow simultaneous measurements of the structural and mechanical responses of the sample. In this work, we introduce an open-source stretching system that can apply uniaxial strain at a submicron resolution, report the tensile force response of the sample, and be mounted on an inverted microscope for real-time imaging. Our system consists of a pair of stepper-based linear motors that stretch the sample symmetrically, a force transducer that records the sample tensile force, and an optically clear sample holder that allows for high-magnification microscopy. Using polymer samples and cellular specimens, we characterized the motion control accuracy, force measurement robustness, and microscopy compatibility of our stretching system. We envision that this uniaxial stretching system will be a valuable tool for characterizing soft and living materials.

Meta Data Analysis of Sex Distribution of Study Samples Reported in Summer Biomechanics, Bioengineering, and Biotransport Annual Conference Abstracts.

The biased use of male subjects in biomedical research has created limitations, underscoring the importance of including women to enhance the outcomes of evidence-based medicine and to promote human health. While federal policies (e.g., the 1993 Revitalization Act and the 2016 Sex as a Biological Variable Act) have aimed to improve sex balance in studies funded by the National Institutes of Health (NIH), data on sex inclusivity in non-NIH funded research remain limited. The objective of this study was to analyze the trend of sex inclusion in abstracts submitted to the Summer Biomechanics, Bioengineering, & Biotransport Conference (SB3C) over 7 years. We scored every abstract accepted to SB3C, and the findings revealed that approximately 20% of total abstracts included sex-related information, and this trend remained stable. Surprisingly, there was no significant increase in abstracts, including both sexes and those with balanced female and male samples. The proportion of abstracts with balanced sexes was notably lower than those including both sexes. Additionally, we examined whether the exclusion of one sex from the corresponding studies was justified by the research questions. Female-only studies had a 50% justification rate, while male-only studies had only 2% justification. Disparity in sex inclusion in SB3C abstracts was apparent, prompting us to encourage scientists to be more mindful of the sex of the research samples. Addressing sex inclusivity in biomechanics and mechanobiology research is essential for advancing medical knowledge and for promoting better healthcare outcomes for everyone.

Hot Topics and Emerging Trends in Mechanobiology Research

Mechanobiology focuses on a series of important physiopathological processes, such as how cells perceive different mechanomechanical stimuli, the process of intracellular mechanotransduction, and how mechanical signals determine the behavior and fate of cells. From the initial stage of embryogenesis, to developmental biology and regenerative medicine, or even through the whole life process, mechanical signaling cascades and cellular mechanical responses in mechanobiology are of great significance in biomedical research. In recent years, research in the field of mechanobiology has undergone remarkable development. Several scientific consortia around the world have been analyzing mechanobiological processes from different perspectives, aiming to gain insights into the regulatory mechanisms by which mechanical factors affect cell fate determination. In this article, we summarized and reviewed the topics that have attracted more research interests in recent years in the field of mechanobiology, for example, arterial blood vessels, stem cell, and ion channel. We also discussed the potential trends that may emerge, such as nuclear deformation, fibrous extracellular matrix, tumor mechanobiology, cellular mechanotransduction, and piezo ion channels. In addition, we put forward new ideas concerning the limitations of mechanism research and the importance of big data analysis and mining in this field, thereby providing objective support and a systematic framework for grasping the hot research topics and exploring new research directions in the field of mechanobiology.

Light-induced modulation of viscoelastic properties in azobenzene polymers.

Photo-induced isomerization of azobenzene molecules drives mass migrations in azopolymer samples. The resulting macroscopic directional photo-deformation of the material morphology has found many applications in literature, although the fundamental mechanisms behind this mass transfer are still under debate. Hence, it is of paramount importance to find quantitative observables that could drive the community toward a better understanding of this phenomenon. In this regard, azopolymer mechanical properties have been intensively studied, but the lack of a nanoscale technique capable of quantitative viscoelastic measurements has delayed the progress in the field. Here, we use bimodal atomic force microscopy (AFM) as a powerful technique for nanomechanical characterizations of azopolymers. With this multifrequency AFM approach, we map the azopolymer local elasticity and viscosity, with high resolution, after irradiation. We find that, while in the (previously) illuminated region, a general photo-softening is measured; locally, the Young modulus and the viscosity depend upon the inner structuring of the illuminating light spot. We then propose a possible interpretation based on a light-induced expansion plus a local alignment of the polymer chains (directional hole-burning effect), which explains the experimental observations. The possibility to access, in a reliable and quantitative way, both Young modulus and viscosity could trigger new theoretical-numerical investigations on the azopolymer mass migration dynamics since, as we show, both parameters can be considered measurable. Furthermore, our results provide a route for engineering the nanomechanical properties of azopolymers, which could find interesting applications in cell mechanobiology research.

A Low‐Cost, User‐Friendly Rheo‐Optical Compression Assay to Measure Mechanical Properties of Cell Spheroids in Standard Cell Culture Plates

AbstractThe mechanical characterization of cell spheroids, one of the most widely used 3D biology models in vitro, is a hotspot of current research on the role played by the mechanical response of cells and tissues. The techniques proposed so far in the literature, while providing important scientific insights, require specialized equipment and technical skills that are not usually available in cell biology facilities. Here, an innovative rheo‐optical compression assay is presented: it is based on microscopy glass coverslips as the load is applied to cell spheroids in standard cell culture plates and on image acquisition with an optical microscope or even a smartphone equipped with adequate magnification lenses. Mechanical properties can be simply obtained by correlating the applied load to the deformation of cell spheroids measured by image analysis. The low‐cost, user‐friendly features of the proposed technique can boost mechanobiology research making it easily affordable to any biomedical lab equipped with cell culture facilities.

Parametric optimization of culture chamber for cell mechanobiology research.

Mechanical signals influence the morphology, function, differentiation, proliferation, and growth of cells. Due to the small size of cells, it is essential to analyze their mechanobiological responses with an in vitro mechanical loading device. Cells are cultured on an elastic silicone membrane substrate, and mechanical signals are transmitted to the cells by the substrate applying mechanical loads. However, large areas of non-uniform strain fields are generated on the elastic membrane, affecting the experiment's accuracy. In the study, finite-element analysis served as the basis of optimization, with uniform strain as the objective. The thickness of the basement membrane and loading constraints were parametrically adjusted. Through finite-element cycle iteration, the "M" profile basement membrane structure of the culture chamber was obtained to enhance the uniform strain field of the membrane. The optimized strain field of culture chamber was confirmed by three-dimensional digital image correlation (3D-DIC) technology. The results showed that the optimized chamber improved the strain uniformity factor. The uniform strain area proportion of the new chamber reached 90%, compared to approximately 70% of the current chambers. The new chamber further improved the uniformity and accuracy of the strain, demonstrating promising application prospects.

SFAlab: image-based quantification of mechano-active ventral actin stress fibers in adherent cells.

Ventral actin stress fibers (SFs) are a subset of actin SFs that begin and terminate at focal adhesion (FA) complexes. Ventral SFs can transmit forces from and to the extracellular matrix and serve as a prominent mechanosensing and mechanotransduction machinery for cells. Therefore, quantitative analysis of ventral SFs can lead to deeper understanding of the dynamic mechanical interplay between cells and their extracellular matrix (mechanoreciprocity). However, the dynamic nature and organization of ventral SFs challenge their quantification, and current quantification tools mainly focus on all SFs present in cells and cannot discriminate between subsets. Here we present an image analysis-based computational toolbox, called SFAlab, to quantify the number of ventral SFs and the number of ventral SFs per FA, and provide spatial information about the locations of the identified ventral SFs. SFAlab is built as an all-in-one toolbox that besides analyzing ventral SFs also enables the identification and quantification of (the shape descriptors of) nuclei, cells, and FAs. We validated SFAlab for the quantification of ventral SFs in human fetal cardiac fibroblasts and demonstrated that SFAlab analysis i) yields accurate ventral SF detection in the presence of image imperfections often found in typical fluorescence microscopy images, and ii) is robust against user subjectivity and potential experimental artifacts. To demonstrate the usefulness of SFAlab in mechanobiology research, we modulated actin polymerization and showed that inhibition of Rho kinase led to a significant decrease in ventral SF formation and the number of ventral SFs per FA, shedding light on the importance of the RhoA pathway specifically in ventral SF formation. We present SFAlab as a powerful open source, easy to use image-based analytical tool to increase our understanding of mechanoreciprocity in adherent cells.

Large Polyacrylamide Hydrogels for Large-Batch Cell Culture and Mechanobiological Studies.

The rigidity of a cell's substrate or extracellular matrix plays a vital role in regulating cell and tissue functions. Polyacrylamide (PAAm) hydrogels are one of the most widely used cell culture substrates that provide a physiologically relevant range of stiffness. However, it is still arduous and time-consuming to prepare PAAm substrates in large batches for high-yield or multiscale cell cultures. In this communication, a simple method to prepare PAAm hydrogels with less time cost and easily accessible materials is presented. The hydrogel is mechanically uniform and supports cell culture in a large batch. It is further shown that the stiffness of the hydrogel covers a large range of Young's modulus and is sensed by cells, regulating various cell features including changes in cell morphology, proliferation, and contractility. This method improves the reproducibility of mechanobiology studies and can be easily applied for mechanobiology research requiring large numbers of cells or experimental groups.

CellVisioner: A Generalizable Cell Virtual Staining Toolbox based on Few-Shot Transfer Learning for Mechanobiological Analysis.

Visualizing cellular structures especially the cytoskeleton and the nucleus is crucial for understanding mechanobiology, but traditional fluorescence staining has inherent limitations such as phototoxicity and photobleaching. Virtual staining techniques provide an alternative approach to addressing these issues but often require substantial amount of user training data. In this study, we develop a generalizable cell virtual staining toolbox (termed CellVisioner) based on few-shot transfer learning that requires substantially reduced user training data. CellVisioner can virtually stain F-actin and nuclei for various types of cells and extract single-cell parameters relevant to mechanobiology research. Taking the label-free single-cell images as input, CellVisioner can predict cell mechanobiological status (e.g., Yes-associated protein nuclear/cytoplasmic ratio) and perform long-term monitoring for living cells. We envision that CellVisioner would be a powerful tool to facilitate on-site mechanobiological research.

Monitoring Mechano-Regulation of Gene Expression by RNA Sequencing.

The advent of high-throughput sequencing techniques has revolutionized biological research. One such method is RNA sequencing, which has become a relatively affordable and routine method for quantifying and comparing gene expression changes over desired experimental conditions. Along with the popularity of the method, a myriad of user-friendly, open-source computational tools have also emerged for differential gene expression analyses. Correspondingly, decades of mechanobiology research have established that mechanical cues, both alone and/or in combination with biochemical signals, can be powerful regulators of transcriptional programs and consequently cell state/fate transitions. Thus, it has become possible to investigate both universal and specific temporally resolved transcriptional responses upon mechanical stimulation genome-wide. This chapter will describe methods to analyze transcriptional changes in response to extrinsic mechanical stretch.

Testing 3D printed biological platform for advancing simulated microgravity and space mechanobiology research

The advancement of microgravity simulators is helping many researchers better understanding the impact of the mechanically unloaded space environment on cellular function and disfunction. However, performing microgravity experiments on Earth, using simulators such as the Random Positioning Machine, introduces some unique practical challenges, including air bubble formation and leakage of growth medium from tissue culture flask and plates, all of which limit research progress. Here, we developed an easy-to-use hybrid biological platform designed with the precision of 3D printing technologies combined with PDMS microfluidic fabrication processes to facilitate reliable and reproducible microgravity cellular experiments. The system has been characterized for applications in the contest of brain cancer research by exposing glioblastoma and endothelial cells to 24 h of simulated microgravity condition to investigate the triggered mechanosensing pathways involved in cellular adaptation to the new environment. The platform demonstrated compatibility with different biological assays, i.e., proliferation, viability, morphology, protein expression and imaging of molecular structures, showing advantages over the conventional usage of culture flask. Our results indicated that both cell types are susceptible when the gravitational vector is disrupted, confirming the impact that microgravity has on both cancer and healthy cells functionality. In particular, we observed deactivation of Yap-1 molecule in glioblastoma cells and the remodeling of VE-Cadherin junctional protein in endothelial cells. The study provides support for the application of the proposed biological platform for advancing space mechanobiology research, also highlighting perspectives and strategies for developing next generation of brain cancer molecular therapies, including targeted drug delivery strategies.

Substrate Stiffness Determines the Establishment of Apical-Basal Polarization in Renal Epithelial Cells but Not in Tubuloid-Derived Cells.

Mechanical guidance of tissue morphogenesis is an emerging method of regenerative medicine that can be employed to steer functional kidney architecture for the purpose of bioartificial kidney design or renal tissue engineering strategies. In kidney morphogenesis, apical-basal polarization of renal epithelial cells is paramount for tubule formation and subsequent tissue functions like excretion and resorption. In kidney epithelium, polarization is initiated by integrin-mediated cell-matrix adhesion at the cell membrane. Cellular mechanobiology research has indicated that this integrin-mediated adhesion is responsive to matrix stiffness, raising the possibility to use matrix stiffness as a handle to steer cell polarization. Herein, we evaluate apical-basal polarization in response to 2D substates of different stiffness (1, 10, 50 kPa and glass) in Madin Darby Canine Kidney cells (MDCKs), a classic canine-derived cell model of epithelial polarization, and in tubuloid-derived cells, established from human primary cells derived from adult kidney tissue. Our results show that sub-physiological (1 kPa) substrate stiffness with low integrin-based adhesion induces polarization in MDCKs, while MDCKs on supraphysiological (>10 kPa) stiffness remain unpolarized. Inhibition of integrin, indeed, allows for polarization on the supraphysiological substrates, suggesting that increased cellular adhesion on stiff substrates opposes polarization. In contrast, tubuloid-derived cells do not establish apical-basal polarization on 2D substrates, irrespective of substrate stiffness, despite their ability to polarize in 3D environments. Further analysis implies that the 2D cultured tubuloid-derived cells have a diminished mechanosensitive capacity when presented with different substrate stiffnesses due to immature focal adhesions and the absence of a connection between focal adhesions and the cytoskeleton. Overall, this study demonstrates that apical-basal polarization is a complex process, where cell type, the extracellular environment, and both the mechanical and chemical aspects in cell-matrix interactions performed by integrins play a role.

Intervertebral Disc-on-a-Chip as Advanced In Vitro Model for Mechanobiology Research and Drug Testing: A Review and Perspective.

Discogenic back pain is one of the most diffused musculoskeletal pathologies and a hurdle to a good quality of life for millions of people. Existing therapeutic options are exclusively directed at reducing symptoms, not at targeting the underlying, still poorly understood, degenerative processes. Common intervertebral disc (IVD) disease models still do not fully replicate the course of degenerative IVD disease. Advanced disease models that incorporate mechanical loading are needed to investigate pathological causes and processes, as well as to identify therapeutic targets. Organs-on-chip (OoC) are microfluidic-based devices that aim at recapitulating tissue functions in vitro by introducing key features of the tissue microenvironment (e.g., 3D architecture, soluble signals and mechanical conditioning). In this review we analyze and depict existing OoC platforms used to investigate pathological alterations of IVD cells/tissues and discuss their benefits and limitations. Starting from the consideration that mechanobiology plays a pivotal role in both IVD homeostasis and degeneration, we then focus on OoC settings enabling to recapitulate physiological or aberrant mechanical loading, in conjunction with other relevant features (such as inflammation). Finally, we propose our view on design criteria for IVD-on-a-chip systems, offering a future perspective to model IVD mechanobiology.

Nano-Precision Tweezers for Mechanosensitive Proteins and Beyond.

Mechanical forces play pivotal roles in regulating cell shape, function, and fate. Key players that govern the mechanobiological interplay are the mechanosensitive proteins found on cell membranes and in cytoskeleton. Their unique nanomechanics can be interrogated using single-molecule tweezers, which can apply controlled forces to the proteins and simultaneously measure the ensuing structural changes. Breakthroughs in high-resolution tweezers have enabled the routine monitoring of nanometer-scale, millisecond dynamics as a function of force. Undoubtedly, the advancement of structural biology will be further fueled by integrating static atomic-resolution structures and their dynamic changes and interactions observed with the force application techniques. In this minireview, we will introduce the general principles of single-molecule tweezers and their recent applications to the studies of force-bearing proteins, including the synaptic proteins that need to be categorized as mechanosensitive in a broad sense. We anticipate that the impact of nano-precision approaches in mechanobiology research will continue to grow in the future.

Technological perspectives on laser speckle micro-rheology for cancer mechanobiology research.

.Significance: The ability to measure the micro-mechanical properties of biological tissues and biomaterials is crucial for numerous fields of cancer research, including tumor mechanobiology, tumor-targeting drug delivery, and therapeutic development.Aim: Our goal is to provide a renewed perspective on the mainstream techniques used for micro-mechanical evaluation of biological tissues and biomimetic scaffoldings. We specifically focus on portraying the outlook of laser speckle micro-rheology (LSM), a technology that quantifies the mechanical properties of biomaterials and tissues in a rapid, non-contact manner.Approach: First, we briefly explain the motivation and significance of evaluating the tissue micro-mechanics in various fields of basic and translational cancer research and introduce the key concepts and quantitative metrics used to explain the mechanical properties of tissue. This is followed by reviewing the general active and passive themes of measuring micro-mechanics. Next, we focus on LSM and elaborate on the theoretical grounds and working principles of this technique. Then, the perspective for measuring the micro-mechanical properties via LSM is outlined. Finally, we draw an overview picture of LSM in cancer mechanobiology research.Results: With the continued emergence of new approaches for measuring the mechanical attributes of biological tissues, the field of micro-mechanical imaging is at its boom. As one of these competent innovations, LSM presents a tremendous potential for both technical maturation and prospective applications in cancer biomechanics and mechanobiology research.Conclusion: By elaborating the current viewpoint of LSM, we expect to accelerate the expansion of this approach to new territories in both technological domains and applied fields. This renewed perspective on LSM may also serve as a road map for other micro-mechanical measurement concepts to be applied for answering mechanobiological questions.

Porous Geometry Guided Micro-mechanical Environment Within Scaffolds for Cell Mechanobiology Study in Bone Tissue Engineering.

Mechanobiology research is for understanding the role of mechanics in cell physiology and pathology. It will have implications for studying bone physiology and pathology and to guide the strategy for regenerating both the structural and functional features of bone. Mechanobiological studies in vitro apply a dynamic micro-mechanical environment to cells via bioreactors. Porous scaffolds are commonly used for housing the cells in a three-dimensional (3D) culturing environment. Such scaffolds usually have different pore geometries (e.g. with different pore shapes, pore dimensions and porosities). These pore geometries can affect the internal micro-mechanical environment that the cells experience when loaded in the bioreactor. Therefore, to adjust the applied micro-mechanical environment on cells, researchers can tune either the applied load and/or the design of the scaffold pore geometries. This review will provide information on how the micro-mechanical environment (e.g. fluid-induced wall shear stress and mechanical strain) is affected by various scaffold pore geometries within different bioreactors. It shall allow researchers to estimate/quantify the micro-mechanical environment according to the already known pore geometry information, or to find a suitable pore geometry according to the desirable micro-mechanical environment to be applied. Finally, as future work, artificial intelligent – assisted techniques, which can achieve an automatic design of solid porous scaffold geometry for tuning/optimising the micro-mechanical environment are suggested.

Mechanobiology of the female reproductive system.

BackgroundMechanobiology in the field of human female reproduction has been extremely challenging technically and ethically.MethodsThe present review provides the current knowledge on mechanobiology of the female reproductive system. This review focuses on the early phases of reproduction from oocyte development to early embryonic development, with an emphasis on current progress.Main findings (Results)Optimal, well‐controlled mechanical cues are required for female reproductive system physiology. Many important questions remain unanswered; whether and how mechanical imbalances among the embryo, decidua, and uterine muscle contractions affect early human embryonic development, whether the biomechanical properties of oocytes/embryos are potential biomarkers for selecting high‐quality oocytes/embryos, whether mechanical properties differ between the two major compartments of the ovary (cortex and medulla) in normally ovulating human ovaries, whether durotaxis is involved in several processes in addition to embryonic development. Progress in mechanobiology is dependent on development of technologies that enable precise physical measurements.ConclusionMore studies are needed to understand the roles of forces and changes in the mechanical properties of female reproductive system physiology. Recent and future technological advancements in mechanobiology research will help us understand the role of mechanical forces in female reproductive system disorders/diseases.