Multiplex ultrasensitive detection of low abundance proteins remains a significant challenge in clinical applications, necessitating the development of innovative solutions. The integration of bead-based microfluidic chip platforms with their efficient target capture and separation capabilities, along with the advantages of miniaturization and low reagent consumption, holds great promise for building an integrated point-of-care testing (POCT) system that enables seamless sample input-result output. This review presents a comprehensive overview of recent advancements in bead-based microfluidic platforms for multiplex and ultrasensitive immunoassays, along with their potential applications in clinical diagnosis and treatment, which is organized into four sections: encoding techniques, the role of microfluidic platforms, applications, and future prospects.

Micropatterning is a sophisticated technique that precisely manipulates the spatial distribution of cell adhesion proteins on various substrates across multiple scales. This precise control over adhesive regions facilitates the manipulation of architectures and physical constraints for single or multiple cells. Furthermore, it allows for an in-depth analysis of how chemical and physical properties influence cellular functionality. In this comprehensive review, we explore the current understanding of the impact of geometrical confinement on cellular functions across various dimensions, emphasizing the benefits of micropatterning in addressing fundamental biological queries. We advocate that utilizing directed self-organization via physical confinement and morphogen gradients on micropatterned surfaces represents an innovative approach to generating functional tissue and controlling morphogenesis in vitro. Integrating this technique with cutting-edge technologies, micropatterning presents a significant potential to bridge a crucial knowledge gap in understanding core biological processes.

Among the various families of G protein-couple receptors (GPCR), the adhesion family of GPCRs is specialized by its expansive extracellular region, which facilitates the recruitment of various ligands. Previous hypothesis proposed that aGPCRs are activated by mechanical force, wherein a Stachel peptide is liberated from the GPCR autoproteolysis-inducing (GAIN) domain and subsequently binds to the transmembrane domain (7TM) upon activation. In this review, we summarize recent advancements in structural studies of aGPCRs, unveiling a conserved structural change of the Stachel peptide from the GAIN domain-embedded β-strand conformation to the 7TM-loaded α-helical conformation. Notably, using single-molecule studies, we directly observed the unfolding of GAIN domain and the release of Stachel peptide under physiological level of force, precisely supporting the mechanosensing mechanism for aGPCRs. We observed that the current complex structures of aGPCR adhesion domains with their respective ligands share a common pattern with the C-termini of each binding partner extending in opposite directions, suggesting a similar shearing stretch geometry for these aGPCRs to transmit the mechanical force generated in the circulating environment to the GAIN domain for its unfolding. Outstanding questions, including the relative orientations and interactions between 7TM and its preceding GAIN and adhesion domains of different aGPCRs, may require further structural and mechanical studies at the full-length receptor scale or cell-based level. Our analysis extends the current view of aGPCR structural organization and activation and offers valuable insights for the development of mechanosensor based on aGPCRs or discovery of mechanotherapy against aGPCRs.

Osteoarthritis (OA) is a progressive degenerative joint sickness related with mechanics, obesity, ageing, etc., mainly characterized by cartilage degeneration, subchondral bone damage and synovium inflammation. Coordinated mechanical absorption and conduction of the joint play significant roles in the prevalence and development of OA. Subchondral bone is generally considered a load-burdening tissue where mechanosensitive cells are resident, including osteocytes, osteoblast lineage cells, and osteoclast lineage cells (especially less concerned in mechanical studies). Mechano-signaling imbalances affect complicated cellular events and disorders of subchondral bone homeostasis. This paper will focus on the significance of mechanical force as the pathogenesis, involvement of various mechanical force patterns in mechanosensitive cells, and mechanobiology research of loading devices in vitro and in vivo, which are further discussed. Additionally, various mechanosensing structures (e.g., transient receptor potential channels, gap junctions, primary cilia, podosome-associated complexes, extracellular vesicles) and mechanotransduction signaling pathways (e.g., Ca²⁺ signaling, Wnt/β-catenin, RhoA GTPase, focal adhesion kinase, cotranscriptional activators YAP/TAZ) in mechanosensitive bone cells. Finally, we highlight potential targets for improving mechanoprotection in the treatment of OA. These advances furnish an integration of mechanical regulation of subchondral bone homeostasis, as well as OA therapeutic approaches by modulating mechanical homeostasis.

Distal metastasis is the main cause of clinical treatment failure in patients with colon cancer. It is now known that the invasion and metastasis of cancer cells is precisely regulated by chemical and physical factors in vivo. However, the role of extracellular matrix (ECM) stiffness in colon cancer cell (CCCs) invasion and metastasis remains unclear. Here, bioinformatical analysis suggested that a high expression level of yes associated protein 1 (YAP1) was significantly associated with metastasis and poor prognosis in colon cancer patients. We further investigated the effects of polyacrylamide hydrogels with different stiffnesses (3, 20, and 38 ​kPa), which were simulated as ECM, on the mechanophenotype (F-actin cytoskeleton organization, electrophoretic rate, membrane fluidity, and Young's modulus) of CCCs. The results showed that a stiffer ECM could induce the maturation of focal adhesions and formation of stress fibers in CCCs, regulate their mechanophenotypes, and promote cell motility. We also demonstrated that the expression levels of YAP1 and paxillin were positively correlated in patients with colon cancer. YAP1 knockdown reduces paxillin clustering and cell motility and alters the cellular mechanophenotypes of CCCs. This is of great significance for an in-depth understanding of the invasion and metastatic mechanisms of colon cancer and for the optimization of clinical therapy from the perspective of mechanobiology.

Cellular behaviors such as migration, spreading, and differentiation arise from the interplay of cell-matrix interactions. The comprehension of this interplay has been advanced by the motor-clutch model, a theoretical framework that captures the binding-unbinding kinetics of mechanosensitive membrane-bound proteins involved in mechanochemical signaling, such as integrins. Since its introduction and subsequent development as a computational tool, the motor clutch model has been instrumental in elucidating the impact of biophysical factors on cellular mechanobiology. This review aims to provide a comprehensive overview of recent advances in the motor-clutch modeling framework, its role in elucidating the relationships between mechanical forces and cellular processes, and its potential applications in mechanomedicine.

Mechanical stimuli are known to play critical roles in mediating tissue repair and regeneration. Recently, this knowledge has led to a paradigm shift toward proactive programming of biological functionalities of biomaterials by leveraging mechanics-geometry-biofunction relationships, which are beginning to shape the newly emerging field of mechanobiomaterials. To profile this emerging field, this article aims to elucidate the fundamental principles in modulating biological responses with material-tissue mechanical interactions, illustrate recent findings on the relationships between material properties and biological responses, discuss the importance of mathematical/physical models and numerical simulations in optimizing material properties and geometry, and outline design strategies for mechanobiomaterials and their potential for tissue repair and regeneration. Given that the field of mechanobiomaterials is still in its infancy, this article also discusses open questions and challenges that need to be addressed.

Dendritic cells (DCs) play a pivotal role in bridging the innate and adaptive immune systems. From their immature state, scavenging tissue for foreign antigens to uptake, then maturation, to their trafficking to lymph nodes for antigen presentation, these cells are exposed to various forms of mechanical forces. Particularly, in the tumor microenvironment, it is widely known that microenvironmental biomechanical cues are heightened. The source of these forces arises from cell-to-extracellular matrix (ECM) and cell-to-cell interactions, as well as being exposed to increased microenvironmental pressures and fluid shear forces typical of tumors. DCs then integrate these forces, influencing their immune functions through mechanotransduction. This aspect of DC biology holds alternative, but important clues to understanding suppressed/altered DC responses in tumors, or allow the artificial enhancement of DCs for therapeutic purposes. This review discusses the current understanding of DC mechanobiology from the perspectives of DCs as sensors of mechanical forces and providers of mechanical forces.

Dysglycemia causes arterial endothelial damage, which is an early critical event in vascular complications for diabetes patients. Physiologically, moderate shear stress (SS) helps maintain endothelial cell health and normal function. Reactive oxygen species (ROS) and calcium ions (Ca²⁺) signals are involved in dysglycemia-induced endothelial dysfunction and are also implicated in SS-mediated regulation of endothelial cell function. Therefore, it is urgent to establish in vitro models for studying endothelial biomechanics and mechanobiology, aiming to seek interventions that utilize appropriate SS to delay or reverse endothelial dysfunction. Microfluidic technology, as a novel approach, makes it possible to replicate blood glucose environment and accurate pulsatile SS in vitro. Here, we reviewed the progress of microfluidic systems used for SS-mediated repair of dysglycemia-induced endothelial cell damage (ECD), revealing the crucial roles of ROS and Ca²⁺ during the processes. It holds significant implications for finding appropriate mechanical intervention methods, such as exercise training, to prevent and treat cardiovascular complications in diabetes.

Bone adapts to mechanical loading by changing its shape and mass. Osteocytes, as major mechanosensors, are critical for bone modeling/remodeling in response to mechanical stimuli. Intracellular calcium oscillation is one of the early responses in osteocytes, and this further facilitates bone cell communication through released biochemical signals. Our previous study has found that mechanically induced calcium oscillations in osteocytes enhance the release of extracellular vesicles (EVs), and those released EVs can elevate bone formation activity. However, the mechanism of mechanically stimulated EVs’ regulation of bone formation and resorption is still unclear. Here, using in vitro studies, we exposed OCY454 cells, with relatively high sclerostin expression, to steady fluid flow (SFF) and characterized the functions of rapidly released EVs in osteoblast and osteoclast regulation. Our study demonstrates that SFF stimulates intracellular calcium response in OCY454 cells and further induces sclerostin, osteoprotegerin (OPG), receptor activator of NF-κB ligand (RANKL) inside or outside EVs to regulate osteoblast and osteoclast activities. This load-induced protein and EVs release is load-duration dependent. Moreover, stimulated osteocytes rapidly regulate osteoclast maturation through EVs capsulated RANKL. In contrast, other regulating proteins, OPG, and sclerostin, are mainly released directly into the medium without EV capsulation.

A definitive understanding of the interplay between protein binding/migration and membrane curvature evolution is emerging but needs further study. The mechanisms defining such phenomena are critical to intracellular transport and trafficking of proteins. Among trafficking modalities, exosomes have drawn attention in cancer research as these nano-sized naturally occurring vehicles are implicated in intercellular communication in the tumor microenvironment, suppressing anti-tumor immunity and preparing the metastatic niche for progression. A significant question in the field is how the release and composition of tumor exosomes are regulated. In this perspective article, we explore how physical factors such as geometry and tissue mechanics regulate cell cortical tension to influence exosome production by co-opting the biophysics as well as the signaling dynamics of intracellular trafficking pathways and how these exosomes contribute to the suppression of anti-tumor immunity and promote metastasis. We describe a multiscale modeling approach whose impact goes beyond the fundamental investigation of specific cellular processes toward actual clinical translation. Exosomal mechanisms are critical to developing and approving liquid biopsy technologies, poised to transform future non-invasive, longitudinal profiling of evolving tumors and resistance to cancer therapies to bring us one step closer to the promise of personalized medicine.

A recent study published in Nature Communications demonstrated that restoring SERCA2 pump deficiency can enhance bone mechano-responsiveness in type 2 diabetes (T2D) by modulating osteocyte calcium dynamics. The findings revealed that in T2D mice, the ability of the bone to respond to mechanical stress is compromised, primarily due to attenuated calcium oscillatory dynamics within osteocytes rather than in osteoblasts or osteoclasts. The underlying mechanism of this reduction in bone mechano-responsiveness in T2D was identified as a specific decrease in osteocytic SERCA2 expression mediated by PPARα. Additionally, mice overexpressing SERCA2 in osteocytes exhibited reduced deterioration of bone mechano-responsiveness induced by T2D. Collectively, this study provides mechanistic insights into T2D-induced deterioration in bone mechano-responsiveness and identifies a promising therapeutic approach to counteract T2D-associated fragility fractures.

A recent study published in Nature Communications showed that essential modulatory roles of interfacial adhesion and mechanical microenvironments such as geometric constraints and extracellular matrix stiffness, in microbe-host cell interactions. This study utilized single-cell force spectroscopy and RNA sequencing to gain insight into the intrinsic mechanisms by which the mechanical microenvironment regulates bacterial-host interactions and therefore reveal potential interventions against bacterial invasion. Meanwhile, the adhesion forces involved in the bacterial-host interactions were recognized as a new indicator for assessing the extent of bacterial infection. Taken together, these findings demonstrate that interfacial adhesion forces and mechanical microenvironments play a dominant role in modulating functions and behaviors of microorganisms and host cells, which also provide a mechanobiology-inspired idea for the development of subsequent drug-resistant antimicrobials and broad-spectrum antiviral drugs.

The significance of early detection and isolation of infected individuals, along with the quantitative assessment of antibodies against the virus, has gained widespread recognition during the ongoing covid-19 pandemic. This necessitates the development of cost-effective, user-friendly, decentralized testing methods characterized by both high sensitivity and specificity. In this article, we present a comprehensive review of an innovative, low-cost rapid decentralized immunoassay technology, applicable across various diagnostic and quantitative testing scenarios. Distinguishing itself from conventional immunoassay technologies, this method is featured with mechanically enhanced specificity without compromising sensitivity. We delve into the basic principle of the technology and a comparative analysis of this technology in relation to other immunodiagnostic methods, highlighting its potential applications in a wide spectrum of diagnostic tests.

Bone and immune cells typically inhabit the same microenvironment and engage in mutual interactions to collectively execute the functions of the “osteoimmune system.” Establishing a harmonized and enduring osteoimmune system significantly enhances bone regeneration, necessitating the maintenance of bone and immune homeostasis. Recently, mechanobiology has garnered increasing interest in bone tissue engineering, with matrix stiffness emerging as a crucial parameter that has been extensively investigated. The effect of matrix stiffness on bone homeostasis remains relatively clear. Soft substrates tend to significantly affect the chondrogenic differentiation of bone marrow mesenchymal stem cells, whereas increasing matrix stiffness is advantageous for osteogenic differentiation. Increased stiffness increases osteoclast differentiation and activity. Additionally, there is increasing emphasis on immune homeostasis, which necessitates dynamic communication between immune cells. Immune cells are crucial in initiating bone regeneration and driving early inflammatory responses. Functional changes induced by matrix stiffness are pivotal for determining the outcomes of engineered tissue mimics. However, inconsistent and incomparable findings regarding the responses of different immune cells to matrix stiffness can be perplexing owing to variations in the stiffness range, measurement methods, and other factors. Therefore, this study aimed to provide a comprehensive review of the specific effects of matrix stiffness on diverse immune cells, with a particular focus on its implications for bone regeneration, which would offer theoretical insights into the treatment of large segmental bony defects and assist in the clinical development of new engineering strategies.

The dynamic protrusions of lamellipodia and filopodia have emerged as crucial players in tumor progression and metastasis. These membrane structures, governed by intricate actin cytoskeletal rearrangements, facilitate cancer cell migration, invasion, and interaction with the tumor microenvironment. This review provides a comprehensive examination of the structural and functional attributes of lamellipodia and filopodia, shedding light on their pivotal roles in mediating cancer invasion. Navigating through the intricate landscape of cancer biology, the review illuminates the intricate signaling pathways and regulatory mechanisms orchestrating the formation and activity of these protrusions. The discussion extends to the clinical implications of lamellipodia and filopodia, exploring their potential as diagnostic and prognostic markers, and delving into therapeutic strategies that target these structures to impede cancer progression. As we delve into the future, the review outlines emerging technologies and unexplored facets that beckon further research, emphasizing the need for collaborative efforts to unravel the complexities of lamellipodia and filopodia in cancer, ultimately paving the way for innovative therapeutic interventions.

As local regions in the tumor outstrip their oxygen supply, hypoxia can develop, affecting not only the cancer cells, but also other cells in the microenvironment, including cancer associated fibroblasts (CAFs). Hypoxia is also not necessarily stable over time, and can fluctuate or oscillate. Hypoxia Inducible Factor-1 is the master regulator of cellular response to hypoxia, and can also exhibit oscillations in its activity. To understand how stable, and fluctuating hypoxia influence breast CAFs, we measured changes in gene expression in CAFs in normoxia, hypoxia, and oscillatory hypoxia, as well as measured change in their capacity to resist, or assist breast cancer invasion. We show that hypoxia has a profound effect on breast CAFs causing activation of key pathways associated with fibroblast activation, but reduce myofibroblast activation and traction force generation. We also found that oscillatory hypoxia, while expectedly resulted in a “sub-hypoxic” response in gene expression, it resulted in specific activation of pathways associated with actin polymerization and actomyosin maturation. Using traction force microscopy, and a nanopatterned stromal invasion assay, we show that oscillatory hypoxia increases contractile force generation vs stable hypoxia, and increases heterogeneity in force generation response, while also additively enhancing invasibility of CAFs to MDA-MB-231 invasion. Our data show that stable and unstable hypoxia can regulate many mechnobiological characteristics of CAFs, and can contribute to transformation of CAFs to assist cancer dissemination and onset of metastasis.

The gastrointestinal (GI) tract's primary role is food digestion, relying on coordinated fluid secretion and bowel movements triggered by mechanosensation. Enteroendocrine cells (EECs) are specialized mechanosensitive cells that convert mechanical forces into electrochemical signals, culminating in serotonin release to regulate GI motility. Despite their pivotal role, knowledge of EEC mechanical properties has been lacking due to their rarity and limited accessibility. In this brief report, we present the first single-cell mechanical characterization of human ECCs isolated from healthy intestinal organoids. Using single-cell optical tweezers, we measured EEC stiffness profiles at the physiological temperature and investigated changes following tryptophan metabolism inhibition. Our findings not only shed light on EEC mechanics but also highlight the potential of adult stem cell-derived organoids for studying these elusive cells.