Enhanced Biomechanically Mediated “Phagocytosis” in Detached Tumor Cells

Uptake of particles by cells involves various natural mechanisms that are essential for their biological functions. The same mechanisms are used in the engulfment of synthetic colloidal drug carriers, while the extent of the uptake affects the biological performance and selectivity. Thus far, little is known regarding the effect of external biomechanical stimuli on the capacity of the cells to uptake nano and micro carriers. This is relevant for anchorage-dependent cells that have detached from surfaces or for cells that travel in the body such as tumor cells, immune cells and various circulating stem cells. In this study, we hypothesize that cellular deformability is a crucial physical effector for the successful execution of the phagocytosis-like uptake in cancer cells. To test this assumption, we develop a well-controlled tunable method to compare the uptake of inert particles by cancer cells in adherent and non-adherent conditions. We introduce a self-designed 3D-printed apparatus, which enables constant stirring while facilitating a floating environment for cell incubation. We reveal a mechanically mediated phagocytosis-like behavior in various cancer cells, that was dramatically enhance in the detached cell state. Our findings emphasize the importance of including proper biomechanical cues to reliably mimic certain physiological scenarios. Beyond that, we offer a cost-effective accessible research tool to study mixed cultures for both adherent and non-adherent cells.

Orthodontic Force–Induced BMAL1 in PDLCs Is a Vital Osteoclastic Activator

Orthodontic tooth movement (OTM) depends on periodontal ligament cells (PDLCs) sensing biomechanical stimuli and subsequently releasing signals to initiate alveolar bone remodeling. However, the mechanisms by which PDLCs sense biomechanical stimuli and affect osteoclastic activities are still unclear. This study demonstrates that the core circadian protein aryl hydrocarbon receptor nuclear translocator–like protein 1 (BMAL1) in PDLCs is highly involved in sensing and delivering biomechanical signals. Orthodontic force upregulates BMAL1 expression in periodontal tissues and cultured PDLCs in manners dependent on ERK (extracellular signal–regulated kinase) and AP1 (activator protein 1). Increased BMAL1 expression can enhance secretion of CCL2 (C-C motif chemokine 2) and RANKL (receptor activator of nuclear factor–κB ligand) in PDLCs, which subsequently promotes the recruitment of monocytes that differentiate into osteoclasts. The mechanistic delineation clarifies that AP1 induced by orthodontic force can directly interact with the BMAL1 promoter and activate gene transcription in PDLCs. Localized administration of the ERK phosphorylation inhibitor U0126 or the BMAL1 inhibitor GSK4112 suppressed ERK/AP1/BMAL1 signaling. These treatments dramatically reduced osteoclastic activity in the compression side of a rat orthodontic model, and the OTM rate was almost nonexistent. In summary, our results suggest that force-induced expression of BMAL1 in PDLCs is closely involved in controlling osteoclastic activities during OTM and plays a vital role in alveolar bone remodeling. It could be a useful therapeutic target for accelerating the OTM rate and controlling pathologic bone-remodeling activities.

Endothelial restoration of CAD GWAS gene PLPP3 by nanomedicine suppresses YAP/TAZ activity and reduces atherosclerosis in vivo

Genome-wide association studies (GWAS) have suggested new molecular mechanisms in vascular cells driving atherosclerotic diseases such as coronary artery disease (CAD) and ischemic stroke (IS). Nevertheless, a major challenge to develop new therapeutic approaches is to spatiotemporally manipulate these GWAS-identified genes in specific vascular tissues in vivo. YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif) have merged as critical transcriptional regulators in cells responding to biomechanical stimuli, such as in athero-susceptible endothelial cells activated by disturbed flow (DF). The molecular mechanisms by which DF activates while unidirectional flow (UF) inactivates YAP/TAZ remain incompletely understood. Recent studies demonstrated that DF and genetic predisposition (risk allele) of CAD/IS locus 1p32.2 converge to reduce phospholipid phosphatase 3 (PLPP3) expression in vascular endothelium. Restoration of endothelial PLPP3 in vivo, although remains challenging and unexplored, is hypothesized to reduce atherosclerosis. We devised a nanomedicine system integrating nanoparticles and Cdh5 promoter-driven plasmids to successfully restore PLPP3 expression in activated endothelium, resulting in suppressed YAP/TAZ activity and reduced DF-induced atherosclerosis in mice. Mechanistically, our studies discovered a molecular paradigm by which CAD/IS GWAS gene PLPP3 inactivates YAP/TAZ by reducing lysophosphatidic acid (LPA)-induced myosin II and ROCK in endothelium under UF. These results highlight a new mechanistic link between GWAS and YAP/TAZ mechano-regulation and moreover, establish a proof of concept of vascular wall-based therapies employing targeted nanomedicine to manipulate CAD/IS GWAS genes in vivo.

Vascularisation of pluripotent stem cell–derived myocardium: biomechanical insights for physiological relevance in cardiac tissue engineering

The myocardium is a diverse environment, requiring coordination between a variety of specialised cell types. Biochemical crosstalk between cardiomyocytes (CM) and microvascular endothelial cells (MVEC) is essential to maintain contractility and healthy tissue homeostasis. Yet, as myocytes beat, heterocellular communication occurs also through constantly fluctuating biomechanical stimuli, namely (1) compressive and tensile forces generated directly by the beating myocardium, and (2) pulsatile shear stress caused by intra-microvascular flow. Despite endothelial cells (EC) being highly mechanosensitive, the role of biomechanical stimuli from beating CM as a regulatory mode of myocardial-microvascular crosstalk is relatively unexplored. Given that cardiac biomechanics are dramatically altered during disease, and disruption of myocardial-microvascular communication is a known driver of pathological remodelling, understanding the biomechanical context necessary for healthy myocardial-microvascular interaction is of high importance. The current gap in understanding can largely be attributed to technical limitations associated with reproducing dynamic physiological biomechanics in multicellular in vitro platforms, coupled with limited in vitro viability of primary cardiac tissue. However, differentiation of CM from human pluripotent stem cells (hPSC) has provided an unlimited source of human myocytes suitable for designing in vitro models. This technology is now converging with the diverse field of tissue engineering, which utilises in vitro techniques designed to enhance physiological relevance, such as biomimetic extracellular matrix (ECM) as 3D scaffolds, microfluidic perfusion of vascularised networks, and complex multicellular architectures generated via 3D bioprinting. These strategies are now allowing researchers to design in vitro platforms which emulate the cell composition, architectures, and biomechanics specific to the myocardial-microvascular microenvironment. Inclusion of physiological multicellularity and biomechanics may also induce a more mature phenotype in stem cell–derived CM, further enhancing their value. This review aims to highlight the importance of biomechanical stimuli as determinants of CM-EC crosstalk in cardiac health and disease, and to explore emerging tissue engineering and hPSC technologies which can recapitulate physiological dynamics to enhance the value of in vitro cardiac experimentation.

Modeling of Endothelial Calcium Responses within a Microfluidic Generator of Spatio-Temporal ATP and Shear Stress Signals

Intracellular calcium dynamics play essential roles in the proper functioning of cellular activities. It is a well known important chemosensing and mechanosensing process regulated by the spatio-temporal microenvironment. Nevertheless, how spatio-temporal biochemical and biomechanical stimuli affect calcium dynamics is not fully understood and the underlying regulation mechanism remains missing. Herein, based on a developed microfluidic generator of biochemical and biomechanical signals, we theoretically analyzed the generation of spatio-temporal ATP and shear stress signals within the microfluidic platform and investigated the effect of spatial combination of ATP and shear stress stimuli on the intracellular calcium dynamics. The simulation results demonstrate the capacity and flexibility of the microfluidic system in generating spatio-temporal ATP and shear stress. Along the transverse direction of the microchannel, dynamic ATP signals of distinct amplitudes coupled with identical shear stress are created, which induce the spatio-temporal diversity in calcium responses. Interestingly, to the multiple combinations of stimuli, the intracellular calcium dynamics reveal two main modes: unimodal and oscillatory modes, showing significant dependence on the features of the spatio-temporal ATP and shear stress stimuli. The present study provides essential information for controlling calcium dynamics by regulating spatio-temporal biochemical and biomechanical stimuli, which shows the potential in directing cellular activities and understanding the occurrence and development of disease.

Tools, techniques, and future opportunities for characterizing the mechanobiology of uterine myometrium

The myometrium is the smooth muscle layer of the uterus that generates the contractions that drive processes such as menstruation and childbirth. Aberrant contractions of the myometrium can result in preterm birth, insufficient progression of labor, or other difficulties that can lead to maternal or fetal complications or even death. To investigate the underlying mechanisms of these conditions, the most common model systems have conventionally been animal models and human tissue strips, which have limitations mostly related to relevance and scalability, respectively. Myometrial smooth muscle cells have also been isolated from patient biopsies and cultured in vitro as a more controlled experimental system. However, in vitro approaches have focused primarily on measuring the effects of biochemical stimuli and neglected biomechanical stimuli, despite the extensive evidence indicating that remodeling of tissue rigidity or excessive strain is associated with uterine disorders. In this review, we first describe the existing approaches for modeling human myometrium with animal models and human tissue strips and compare their advantages and disadvantages. Next, we introduce existing in vitro techniques and assays for assessing contractility and summarize their applications in elucidating the role of biochemical or biomechanical stimuli on human myometrium. Finally, we conclude by proposing the translation of “organ on chip” approaches to myometrial smooth muscle cells as new paradigms for establishing their fundamental mechanobiology and to serve as next-generation platforms for drug development.

The Application of Bioreactors for Cartilage Tissue Engineering: Advances, Limitations, and Future Perspectives

Tissue engineering (TE) has brought new hope for articular cartilage regeneration, as TE can provide structural and functional substitutes for native tissues. The basic elements of TE involve scaffolds, seeded cells, and biochemical and biomechanical stimuli. However, there are some limitations of TE; what most important is that static cell culture on scaffolds cannot simulate the physiological environment required for the development of natural cartilage. Recently, bioreactors have been used to simulate the physical and mechanical environment during the development of articular cartilage. This review aims to provide an overview of the concepts, categories, and applications of bioreactors for cartilage TE with emphasis on the design of various bioreactor systems.

The Effect of Intense Exercise on Equine Serum Proteoglycan-4/Lubricin

Objective: Local biological and biomechanical-stimuli modulate proteoglycan-4 secretion within synovial joints. For the horse, changes to proteoglycan-4 concentration and function are notable in acute joint injury and osteoarthritis. Proteoglycan-4 (also known as Lubricin) is present in the blood, however the effect of exercise on equine serum levels is unknown. The overall objective of this study was, therefore, to investigate the effect of intense exercise on serum proteoglycan-4 in thoroughbred horses.Methods: Samples of blood were taken from thoroughbreds (n = 12) during a chuckwagon racing event (Alberta, Canada). The chuckwagon race is a sprint racing event where teams of horses pull a combined 1,325 lbs (601 kg) of wagon and driver around a 5/8th mile (1 km) of dirt track, racing at full gallop to the finish. Blood samples were collected 30-min before the race start, and several timepoints post-race: 5-min, 90-min, 3-h, 12-h, and 23-h. Proteoglycan-4 concentrations in serum were quantified by enzyme-linked-immunosorbent-assay using recombinant-human proteoglycan-4 standards and anti-proteoglycan-4 mAb 9G3. The molecular weight of immunoreactive proteoglycan-4 in serum was assessed by western blot.Results: Proteoglyan-4 in serum demonstrated the expected high MW immunoreactivity to mAb 9G3, consistent with that of full length PRG4. Serum proteoglycan-4 decreased five-minutes post-race from baseline concentration (0.815 ± 0.175 to 0.466 ± 0.090 μg/mL, μ ± SEM, p < 0.01).Conclusions: The concentration of serum proteoglycan-4 in horses decreased significantly five min post-exercise. A potential explanation for this finding could be increased proteoglycan-4 clearance from the circulation. Further investigations could extend to complete the detailed characterization of proteoglycan-4 structure and its potential function within the blood as it relates to joint health and exercise.

Stem Cell Mechanobiology and the Role of Biomaterials in Governing Mechanotransduction and Matrix Production for Tissue Regeneration

Mechanobiology has underpinned many scientific advances in understanding how biophysical and biomechanical cues regulate cell behavior by identifying mechanosensitive proteins and specific signaling pathways within the cell that govern the production of proteins necessary for cell-based tissue regeneration. It is now evident that biophysical and biomechanical stimuli are as crucial for regulating stem cell behavior as biochemical stimuli. Despite this, the influence of the biophysical and biomechanical environment presented by biomaterials is less widely accounted for in stem cell-based tissue regeneration studies. This Review focuses on key studies in the field of stem cell mechanobiology, which have uncovered how matrix properties of biomaterial substrates and 3D scaffolds regulate stem cell migration, self-renewal, proliferation and differentiation, and activation of specific biological responses. First, we provide a primer of stem cell biology and mechanobiology in isolation. This is followed by a critical review of key experimental and computational studies, which have unveiled critical information regarding the importance of the biophysical and biomechanical cues for stem cell biology. This review aims to provide an informed understanding of the intrinsic role that physical and mechanical stimulation play in regulating stem cell behavior so that researchers may design strategies that recapitulate the critical cues and develop effective regenerative medicine approaches.

Mechanical Stimulation: A Crucial Element of Organ-on-Chip Models

Organ-on-chip (OOC) systems recapitulate key biological processes and responses in vitro exhibited by cells, tissues, and organs in vivo. Accordingly, these models of both health and disease hold great promise for improving fundamental research, drug development, personalized medicine, and testing of pharmaceuticals, food substances, pollutants etc. Cells within the body are exposed to biomechanical stimuli, the nature of which is tissue specific and may change with disease or injury. These biomechanical stimuli regulate cell behavior and can amplify, annul, or even reverse the response to a given biochemical cue or drug candidate. As such, the application of an appropriate physiological or pathological biomechanical environment is essential for the successful recapitulation of in vivo behavior in OOC models. Here we review the current range of commercially available OOC platforms which incorporate active biomechanical stimulation. We highlight recent findings demonstrating the importance of including mechanical stimuli in models used for drug development and outline emerging factors which regulate the cellular response to the biomechanical environment. We explore the incorporation of mechanical stimuli in different organ models and identify areas where further research and development is required. Challenges associated with the integration of mechanics alongside other OOC requirements including scaling to increase throughput and diagnostic imaging are discussed. In summary, compelling evidence demonstrates that the incorporation of biomechanical stimuli in these OOC or microphysiological systems is key to fully replicating in vivo physiology in health and disease.