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Background: Vascular smooth muscle cell (VSMC) contractility is critical for blood pressure regulation and vascular homeostasis. Identifying the key molecule that maintains VSMC contractility may provide a novel therapeutic target for vascular remodeling. ALK3 (activin receptor-like kinase 3) is a serine/threonine kinase receptor, and deletion of ALK3 causes embryonic lethality. However, little is known about the role of ALK3 in postnatal arterial function and homeostasis. Methods: We conducted in vivo studies in a tamoxifen-induced postnatal VSMC-specific ALK3 deletion mice suitable for analysis of blood pressure and vascular contractility. Additionally, the role of ALK3 on VSMC was determined using Western blot, collagen-based contraction assay and traction force microscopy. Furthermore, interactome analysis were performed to identify the ALK3-associated proteins and bioluminescence resonance energy transfer assay was used to characterize Gαq activation. Results: ALK3 deficiency in VSMC led to spontaneous hypotension and impaired response to angiotensin II in mice. In vivo and in vitro data revealed that ALK3 deficiency impaired contraction force generation by VSMCs, repressed the expression of contractile proteins, and inhibited the phosphorylation of myosin light chain. Mechanistically, Smad1/5/8 signaling mediated the ALK3-modulated contractile protein expressions but not myosin light chain phosphorylation. Furthermore, interactome analysis revealed that ALK3 directly interacted with and activated Gαq (guanine nucleotide-binding protein subunit αq)/Gα11 (guanine nucleotide-binding protein subunit α11), thereby stimulating myosin light chain phosphorylation and VSMC contraction. Conclusions: Our study revealed that in addition to canonical Smad1/5/8 signaling, ALK3 modulates VSMC contractility through direct interaction with Gαq/Gα11, and therefore, might serve as a potential target for modulating aortic wall homeostasis.

Cell traction mediates the biochemical and mechanical interactions between the cell and its extracellular matrix (ECM). Traction force microscopy (TFM) is a powerful technique for quantitative cellular scale traction analysis. However, it is challenging to characterize macromolecular scale traction events with current TFM due to the limited sampling density and algorithmic precision. In this article, we introduce a super-resolution TFM by utilizing a novel substrate surface modification method. Our TFM technique achieved a spatial resolution comparable to fluorescence microscopy and precision comparable to the rupture force of an integrin-ligand bond. Correlated imaging of TFM with fluorescence microscopy demonstrated that the residing paxillin highly correlated with traction while α5 integrin was located differently. Time-lapse TFM imaging captured a transient traction variation as the adhesion protein passed by. Thus, the novel super-resolution TFM benefits the studies on cellular biochemical and mechanical interactions.

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. 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. These pave the way for more precise exploration of the interaction between cells and extracellular matrix and the underlying mechanotransduction pathways in mechanobiology.

Interfacial interactions between cancer cells and surrounding microenvironment involve complex mechanotransduction mechanisms that are directly associated with tumor invasion and metastasis. Matrix remodeling triggers heterogeneity of stiffness in tumor microenvironment and thus generates anisotropic stiffness gradient (ASG). The migration of cancer cells mediated by ASG, however, still remains elusive. Based on a multi-layer polymerization method of microstructured hydrogels with surface topology, we develop an in vitro experimental platform for mechanical interactions of cancer cells with ASG matrix microenvironment. We show that mechanical guidance of mesenchymal cells is essentially modulated by ASG, leading to a spontaneous directional migration along the orientation parallel to the maximum stiffness although there is no stiffness gradient in the direction. The ASG-regulated mechanical guidance presents an alternative way of cancer cell directional migration. Further, our findings indicate that the mechanical guidance occurs only in mesenchymal cancer cells, but not in epithelial cancer cells, implying that cell contractility may contribute to ASG-regulated migration of cells. This work is not only helpful for elucidating the role of matrix remodeling in mediating tumor cell invasion and metastasis, but has potential implications for developing specific cancer treatments. STATEMENT OF SIGNIFICANCE: Local extracellular matrix (ECM) stiffening triggers mechanical heterogeneity in tumor microenvironment, which can exert a crucial impact on interfacial interactions between tumor cells and surrounding ECM. The underlying mechanobiological mechanism that tumor cells are modulated by mechanically heterogeneous ECM, however, still remains mysterious to a great extent. Through our established in vitro platform and analysis, we have demonstrated that anisotropic stiffness gradient (ASG) has the ability to elicit directional migration of cells, essentially depending on local stiffness gradients and the corresponding absolute stiffness values. This study is not only crucial for revealing the role of matrix remodeling in regulating tumor invasion and metastasis, but also offers a valuable guidance for developing anti-tumor therapies from the biomechanical perspective.

Exosomes derived from mesenchymal stem cells (MSCs) have demonstrated regenerative potential for cell-free bone tissue engineering, nevertheless, certain challenges, including the confined therapeutic potency of exosomes and ineffective delivery method, are still persisted. Here, we confirmed that hypoxic precondition could induce enhanced secretion of exosomes from stem cells from human exfoliated deciduous teeth (SHEDs)

Cell phenotype heterogeneity within tumor tissue, especially which due to the emergence of epithelial-mesenchymal transition (EMT) in cancer cells, is associated with cancer invasion and metastasis. However, our understanding of the cellular mechanism(s) underlying the cooperation between EMT cell and epithelial cancer cell migration remains incomplete. Herein, heterotypic tumor spheroids containing both epithelial and EMT cancer cells were generated in vitro. We observed that EMT cells dominated the peripheral region of the self-organized heterotypic tumor spheroid. Furthermore, our results demonstrated that EMT cells could serve as leader cells to improve the collective migration efficiency of epithelial cancer cells and promote dispersion and invasion of the tumor spheroids, which was regulated by the force transition between EMT cells and epithelial cancer cells. Mechanistically, our data further suggest that force transmission is mediated by heterophilic N-cadherin/E-cadherin adhesion complexes between EMT and epithelial cancer cells. Impairment of N-cadherin/E-cadherin adhesion complex formation abrogated the ability of EMT cells to guide epithelial cancer cell migration and blocked the dispersion of tumor spheroids. Together, our data provide new insight into the mechanical interaction between epithelial and EMT cancer cells through heterophilic cadherin adhesion, which enables cooperative tumor cell migration, highlighting the role of EMT cells in tumor invasion.

Interactions between cells and their microenvironment trigger a series of complex mechanotransduction signals that regulate cell behavior. Mechanical characterization of cell interactions with extracellular matrix (ECM)/substrate is a prerequisite for quantitative investigations of the mechanisms of mechanotransduction. Here, we present recent progress in the quantification of traction forces generated during cell-ECM/substrate interaction, with an emphasis on force/stress reconstructions on functionalized ECM/substrates fabricated by microfluidics techniques such as elastomeric micropillar arrays, patterned and continuous substrates, and three-dimensional hydrogel substrates. We also present an overview of how to measure displacement fields induced by cell activity, and reliably and accurately capture cellular traction force/stress on different substrates. Lastly, we describe recent applications in mechano-biology.

Formation of the primitive streak (PS) marks one of the most important developmental milestones in embryonic development. However, our understanding of cellular mechanism(s) underlying cell fate diversification along the anterior-posterior axis of the PS remains incomplete. Furthermore, differences in biophysical phenotypes between anterior and posterior PS cells, which could affect their functions and regulate their fate decisions, remain uncharacterized. Herein, anterior and posterior PS cells were derived using human pluripotent stem cell (hPSC)-based in vitro culture systems. We observed that anterior and posterior PS cells displayed significantly different biophysical phenotypes, including cell morphology, migration, and traction force generation, which was further regulated by different levels of Activin A- and BMP4-mediated developmental signaling. Our data further suggested that intracellular cytoskeletal contraction could mediate anterior and posterior PS differentiation and phenotypic bifurcation through its effect on Activin A- and BMP4-mediated intracellular signaling events. Together, our data provide new information about biophysical phenotypes of anterior and posterior PS cells and reveal an important role of intracellular cytoskeletal contractility in regulating anterior and posterior PS differentiation of hPSCs. STATEMENT OF SIGNIFICANCE: Formation of the primitive streak (PS) marks one of the most important developmental milestones in embryonic development. However, molecular and cellular mechanism(s) underlying functional diversification of embryonic cells along the anterior-posterior axis of the PS remains incompletely understood. This work describes the first study to characterize the biophysical properties of anterior and posterior PS cells derived from human pluripotent stem cells (hPSCs). Importantly, our data showing the important role of cytoskeleton contraction in controlling anterior vs. posterior PS cell phenotypic switch (through its effect on intracellular Smad signaling activities downstream of Activin A and BMP4) should shed new light on biomechanical regulations of the development and anterior-posterior patterning of the PS. Our work will contribute significantly to uncovering new biophysical principles and cellular mechanisms driving cell lineage diversification and patterning during the PS formation.

Atomically thin transition-metal dichalcogenides (TMDCs) have received substantial interest due to their typical thickness-dependent optical and electronic properties and related applications in optoelectronics. However, the large-scale, thickness-tunable growth of such materials is still challenging. Herein, we report a fast growth of thickness-tunable wedding-cake-like MoS2 flakes on 6-in. soda-lime glass by using NaCl-coated Mo foils as metal precursors. The MoS2 thicknesses are tuned from one layer (1L) to >20L by controlling the concentrations of NaCl promoter. To attest to the ultrahigh crystal quality, related devices based on 1L–multilayer MoS2 lateral junctions have been constructed and display a relatively high rectification ratio (∼103) and extra high photoresponsitivity (∼104 A/W). Thanks to the scalable sizes, uniform distributions of the flakes and homogeneous optical properties, the applications in ultraviolet (UV) irradiation filtering eyewear are also demonstrated. Our work should hereby ...

Cancer cell migration is the hallmark of tumor metastasis; however, the mechanisms of cancer cell migration have not been fully understood. Considering the fact that biophysical and biochemical properties of the tumor microenvironment are altered during tumor progression, it is instinctive to think about whether the changed microenvironment can regulate cancer cell migration. Herein, we cultured human breast cancer cells (MDA-MB-231) on polyacrylamide gel substrates with different stiffnesses (1, 5, 10, and 20 kPa) with and without transforming growth factor-β1 (TGF-β1, 2 ng/mL) treatment to evaluate the effects of the altered tumor microenvironment on cancer cell migration in addition to the response of traction force generation and cytoskeleton remodeling. The results demonstrated that MDA-MB-231 migration increased with increasing substrate stiffness and was further enhanced with TGF-β1 addition. Traction forces and cytoskeleton remodeling were also found to be enhanced in response to TGF-β1 treatment. Furthermore, inhibiting myosin IIA-mediated contraction by blebbistatin decreased TGF-β1-enhanced traction force but increased TGF-β1-enhanced migration. These results imply that both biophysical (like stiffness) and biochemical (like TGF-β1) factors could orthogonally regulate cancer cell migration.

Transient receptor potential vanilloid 4 (TRPV4) is a calcium-permeable cation channel that has been associated with several types of cancer. However, its biological significance, as well as its related mechanism in endometrial cancer (EC) still remains elusive. In this study, we examined the function of calcium in EC, with a specific focus on TRPV4 and its downstream pathway. We reported here on the findings that a high level of serum ionized calcium was significantly correlated with advanced EC progression, and among all the calcium channels, TRPV4 played an essential role, with high levels of TRPV4 expression associated with cancer progression both in vitro and in vivo. Proteomic and bioinformatics analysis revealed that TRPV4 was involved in cytoskeleton regulation and Rho protein pathway, which regulated EC cell migration. Mechanistic investigation demonstrated that TRPV4 and calcium influx acted on the cytoskeleton via the RhoA/ROCK1 pathway, ending with LIMK/cofilin activation, which had an impact on F-actin and paxillin (PXN) levels. Overall, our findings indicated that ionized serum calcium level was significantly associated with poor outcomes and calcium channel TRPV4 should be targeted to improve therapeutic and preventive strategies in EC.

Yu Hei,1 Binhong Teng,2,3 Ziqian Zeng,2,3 Siqi Zhang,3 Qian Li,3 Jijia Pan,3 Zuyuan Luo,3 Chunyang Xiong,1 Shicheng Wei2,3 1Department of Mechanics and Engineering Science, College of Engineering, Peking University, Beijing, People’s Republic of China; 2Department of Oral and Maxillofacial Surgery, School and Hospital of Stomatology, Peking University, Beijing, People’s Republic of China; 3Laboratory of Biomaterials and Regenerative Medicine, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, People’s Republic of ChinaCorrespondence: Chunyang XiongDepartment of Mechanics and Engineering Science, College of Engineering, Peking University, Beijing, People’s Republic of ChinaTel +86 10 62757940Email cyxiong@pku.edu.cnShicheng WeiDepartment of Oral and Maxillofacial Surgery, School and Hospital of Stomatology, Peking University, Beijing, People’s Republic of ChinaTel +86 10 82195780Email sc-wei@pku.edu.cnBackground: Immune checkpoint blockades (ICBs) are a promising treatment for cancers such as melanoma by blocking important inhibitory pathways that enable tumor cells to evade immune attack. Programmed death ligand 1 monoclonal antibodies (aPDL1s) can be used as an ICB to significantly enhance the effectiveness of tumor immunotherapy by blocking the PD-1/PD-L1 inhibitory pathway. However, the effectiveness of aPDL1s may be limited by low selectivity in vivo and immunosuppressed tumor microenvironment including hypoxia.Purpose: To overcome the limitations, we develop a multifunctional immunoliposome, called CAT@aPDL1-SSL, with catalase (CAT) encapsulated inside to overcome tumor hypoxia and aPDL1s modified on the surface to enhance immunotherapeutic effects against melanoma.Methods: The multifunctional immunoliposomes (CAT@aPDL1-SSLs) are prepared using the film dispersion/post-insertion method. The efficacy of CAT@aPDL1-SSLs is verified by multiple experiments in vivo and in vitro.Results: The results of this study suggest that the multifunctional immunoliposomes preserve and protect the enzyme activity of CAT and ameliorate tumor hypoxia. Moreover, the enhanced cellular uptake of CAT@aPDL1-SSLs in vitro and their in vivo biodistribution suggest that CAT@aPDL1-SSLs have great targeting ability,resulting in improved delivery and accumulation of immunoliposomes in tumor tissue.Finally, by activating and increasing the infiltration of CD8+ T cells at the tumor site, CAT@aPDL1-SSLs inhibit the growth of tumor and prolong survival time of mice,with low systemic toxicity.Conclusion: In conclusion, the multifunctional immunoliposomes developed and proposed in this study are a promising candidate for melanoma immunotherapy, and could potentially be combined with other cancer therapies like radiotherapy and chemotherapy to produce positive outcomes.Keywords: immunotherapy, programmed death ligand 1 monoclonal antibodies, aPDL1s, tumor hypoxia, melanoma, liposomes

Graphene has to be supported on a substrate in a variety of applications. The underlying substrate can exert a remarkable impact on the wettability of graphene, but the inherent mechanism remains elusive. By quantifying the static contact angles of water (polar liquid) and diiodomethane (nonpolar liquid) droplets on graphene supported on different kinds of substrate, we found that the apparent wetting transparency of the supported graphene with airborne hydrocarbon adsorption depends essentially on polarities of both the liquids and the underlying substrates, and that, in most cases, the supported graphene exhibits wetting translucency. With the Owens-Wendt theory, we then identified the specific components of the surface energy, which indicated that inserting graphene does not introduce a polar interaction and that the apparent transmitted polar energy of one layer of graphene is ∼20% for the investigated substrates. The apparent polarity of supported graphene can potentially be tuned by the underlying substrate. Practically, the wettability of graphene can be determined by investigating the competitive relationship between the reduced dispersive and polar interaction of the substrate and the increased dispersive one introduced via inserting the graphene layers. Our findings may have significant implications in designing functional nanostructures through tuning the surface properties of graphene by its surroundings.

Zinc oxide nanoparticles (ZnO-NPs) have been widely used in engineering and biomedicine. However, their adverse pathological effects and mechanisms, especially the biomechanical effects on respiratory system where airway smooth muscle cell (ASMC) contractility regulates the airway response and lung function, are not fully understood. Herein, we used traction force microscopy (TFM) method to investigate whether ZnO-NPs of different concentrations (0.1–10 μg/mL) can alter ASMC contractility (basal and agonist-stimulated) after a short-term exposure and the potential mechanisms. We found that ZnO-NPs exposure led to a decrease of ASMC viability in a dose-dependent manner. Notably, basal contractility was enhanced when the concentration of ZnO-NPs was less than 0.1 μg/mL and decreased afterwards, while KCl-stimulated contractility was reduced in all cases of ZnO-NPs treated groups. Cytoskeleton structure was also found to be significantly altered in ASMC with the stimulation of ZnO-NPs. More importantly, it seems that ZnO-NPs with low concentration (

Mechanical phenotypes of cells are found to hold vital clues to reveal cellular functions and behaviors, which not only has great physiological significance but also is crucial for disease diagnosis. To this end, we developed a set of electrodeformation-based biomechanical microchip assays to quantify mechanical phenotypes on the single-cell level. By investigating the spatiotemporal dynamics of cancer cells driven by dielectrophoresis forces, we captured the key global viscoelastic indexes including cellular elasticity, viscosity, and transition time that was defined as the ratio of the transient viscosity and elasticity, simultaneously, and thus explored their intrinsic correlation with cell cycle progression. Our results showed that both global elasticity and viscosity have a significant periodic variation with cell cycle progression, but the transition time remained unchanged in the process, indicating that it might be an intrinsic property of cancer cells that is independent of the cell cycle and the type of cell in the experiments. Further, we investigated the molecular mechanism regulating cellular viscoelastic phenotypes on the biomechanical chips through intracellular cytoskeletal perturbation assays. These findings, together with the electrodeformation-based microchip technique, not only reveal the relation between mechanical phenotypes of cancer cells and cell cycle progression but also provide a platform for implementing multi-index mechanical phenotype assays associated with cancer cell cycles in the clinic.

Graphene is the stiffest membrane and PDMS is one of the very compliant materials. The indentation response of a graphene monolayer mounted on a PDMS substrate (graphene/PDMS) is studied by both experimental and computational investigations. Due to the huge elastic modulus ratio between membrane and substrate (∼10 6 ) and the very large ratio of indentation depth to membrane thickness (∼10 3 ), the indentation deformation of graphene in the graphene/PDMS structure is analogous to that of free-standing (F-S) graphene but just with a relatively smaller indentation depth (i.e., the graphene deformation is insensitive to the appearance of PDMS substrate); the graphene can create a screening effect, which causes the PDMS substrate deformation to be insensitive to the tip geometry. In addition, with the aid of finite element method (FEM), the elastic modulus of graphene monolayer can be accurately determined from the overall indentation response of graphene/PDMS using an inverse analysis, which is determined as 0.982 TPa (very close to previously reported values). The present approach can be also extended to other 2D materials.

Background: Fibroblasts were the main seed cells in the studies of tissue engineering of the pelvic floor ligament. Basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) were widely studied but at various concentrations. This study aimed to optimize the concentrations of combined bFGF and EGF by evaluating their effects on proliferation and collagen secretion of fibroblasts. Methods: Fibroblasts were differentiated from rat adipose mesenchymal stem cells (ADSCs). Flow cytometry and immunohistochemistry were used for cell identification. The growth factors were applied at concentrations of 0, 1, 10, and 100 ng/ml as three groups: (1) bFGF alone, (2) EGF alone, and (3) bFGF mixed with EGF. Cell proliferation was evaluated by Cell Counting Kit-8 assays. Expression of Type I and III collagen (Col-I and Col-III) mRNAs was evaluated by real-time quantitative reverse transcription-polymerase chain reaction. Statistical analysis was performed with SPSS software and GraphPad Prism using one-way analysis of variance and multiple t-test. Results: ADSCs were successfully isolated from rat adipose tissue as identified by expression of typical surface markers CD29, CD44, CD90, and CD45 in flow cytometry. Fibroblasts induced from ADSC, compared with ADSCs, were with higher mRNA expression levels of Col I and Col III (F = 1.29, P = 0.0390). bFGF, EGF, and the mixture of bFGF with EGF can enhanced fibroblasts proliferation, and the concentration of 10 ng/ml of the mixture of bFGF with EGF displayed most effectively (all P < 0.05). The expression levels of Col-I and Col-III mRNAs in fibroblasts displayed significant increases in the 10 ng/ml bFGF combined with EGF group (all P < 0.05). Conclusions: The optimal concentration of both bFGF and EGF to promote cell proliferation and collagen expression in fibroblasts was 10 ng/ml at which fibroblasts grew faster and secreted more Type I and III collagens into the extracellular matrix, which might contribute to the stability of the pelvic floor microenvironment.

The immunosuppressive tumor microenvironment (TME) can significantly limit the immunotherapeutic effects of the PD-L1 antibody (aPDL1) by inhibiting the infiltration of CD8

Cells in living organisms live in multiphysics-coupled environments. There is growing evidence indicating that both exogenous electric field (EEF) and extracellular stiffness gradient (ESG) can regulate directional movement of cells, which are known as electrotaxis and durotaxis, respectively. How single cells respond to the ubiquitous electromechanical coupling cues, however, remains mysterious. Using microfluidic chip-based methodology and finite element-based electromechanical coupling design strategies, we develope an electromechanical coupling microchip system, enabling us to quantitatively investigate polarization and directional migration governed by EEF and ESG at the single cell level. It is revealed that both of electrotaxis and durotaxis nonlinearly depend on the physiological EEF and ESG, respectively. Specific combinations of EEF and ESG can subtly modify the polarization states of single cells and thus induce hyperpolarization and depolarization. Cells can integrate electrotaxis and durotaxis in response to multi-cue microenvironments via subtle mechanisms involving cooperation and competition during cellular electrosensing and mechanosensing. The work offers a platform for quantifying migration and polarization of cells driven by electromechanical cues, which is essential not only for elucidating physiological and pathological processes like embryo development, and invasion and metastasis of cancer cells, but for manipulating cell behaviors in a controllable and programmable fashion.

In recent years, the efficiency of digital image correlation (DIC) methods has attracted increasing attention because of its increasing importance for many engineering applications. Based on the classical affine optical flow (AOF) algorithm and the well-established inverse compositional Gauss–Newton algorithm, which is essentially a natural extension of the AOF algorithm under a nonlinear iterative framework, this paper develops a set of fast convolution-based DIC algorithms for high-efficiency subpixel image registration. Using a well-developed fast convolution technique, the set of algorithms establishes a series of global data tables (GDTs) over the digital images, which allows the reduction of the computational complexity of DIC significantly. Using the pre-calculated GDTs, the subpixel registration calculations can be implemented efficiently in a look-up-table fashion. Both numerical simulation and experimental verification indicate that the set of algorithms significantly enhances the computational efficiency of DIC, especially in the case of a dense data sampling for the digital images. Because the GDTs need to be computed only once, the algorithms are also suitable for efficiently coping with image sequences that record the time-varying dynamics of specimen deformations.

Cancer development and progression associated with epithelial-mesenchymal transition (EMT) not only results in biological and functional abnormalities but also leads to changes in mechanical and structural characteristics of cells. Mechanical characterization has been considered as a promising, label-free, alternative way to cancer diagnosis. In this paper, we fabricate a microchip to quantify mechanical deformability of cancer cells during EMT in an electrodeformation-based way. For three typical cancer cells, i.e., MCF-7, MDA-MB-231 and A549 cells, our experimental outcome manifests that there exists significant difference in the deformability before and after EMT induced by TGF-β1. Both MCF-7 and MDA-MB-231 cells after EMT show more than 40% reduction in elasticity and viscosity, whereas A549 cells appear to have a ∼25% decrease in elasticity and ∼35% in viscosity, respectively. These findings imply it is feasible to monitor the EMT process by means of mechanical clue of cells involved. It is expected that the present electrodeformation-based technique can be developed into a label-free, high-throughput method to evaluate metastatic potential of cancer cells, which is crucial to early detection and diagnosis of cancer.

Cell response to substrate rigidity, closely related to extracellular matrix protein composition, requires actomyosin-generated contractility. By introducing coefficients describing cell spreading and traction dynamics, and a revised high-resolution traction force microscopy, we analyzed the static and dynamic features of fibroblasts on fibronectin- or collagen- coated stiff or soft substrates. Large cell spreading area and branchlike morphology were more favorable on fibronectin than collagen. Cell spreading on fibronectin-coated substrates was more sensitive to rigidity compared with collagen. Low concentration fibronectin-coated substrate induced more dynamic lamellipodia movement than other conditions. Interestingly, the static average cell traction on high concentration fibronectin-coated stiff and soft substrates showed no difference. However, the lamellipodium traction dynamics was sensitive to rigidity on fibronectin. Particularly, lamellipodia on fibronectin-coated soft substrate performed much higher local traction dynamics compared with other groups. Together, dynamics of cell adhesion and traction are regulated by extracellular matrix protein composition, coupled with substrate rigidity.

Hepatocellular carcinoma (HCC) was usually coupled with increased stiffness of the extracellular matrix (ECM) and elevated level of transforming growth factor-β1 (TGF-β1). However, the mechanism by which substrate rigidity modulated TGF-β1 signaling transduction remained unknown. This paper investigated the molecular mechanism of how matrix stiffness regulating TGF-β1 signaling in HCC cells. By means of stiffness tunable collagen I-coated polyacrylamide (PA) gels, we found that the expressions of β1 integrin, p-FAK Y397 and p-Smad2 upregulated on stiffer gels as well as the content of TGF-β1 in culture media of HCC cells, which were inhibited by RGD blocking peptides, Y-27632 (ROCK inhibitor) or Blebbistatin (myosin II inhibitor). Cellular traction force was also significantly higher when plated on stiffer substrates but dramatically decreased after treatment with Y-27632 or Blebbistatin. Furthermore, the upregulation of p-Smad2 in the HCC cells on stiffer PA gels induced by exogenetic latent TGF-β1 was downregulated in the presence of RGD peptides. The nuclear translocation of Smad2 induced by latent TGF-β1 was inhibited by Y-27632 or Blebbistatin. Our results suggested that the extracellular matrix stiffness regulated latent TGF-β1 activation by cytoskeletal tension in HCC cells, showing that matrix stiffness was a key regulator involving the TGF-β1 activity in HCC cells. The current study presented a mechanism of how hepatocirrhosis developed into liver cancer.

Atomically thin transition-metal dichalcogenides (TMDCs) have received substantial interest due to their typical thickness-dependent optical and electronic properties and related applications in optoelectronics. However, the large-scale, thickness-tunable growth of such materials is still challenging. Herein, we report a fast growth of thickness-tunable wedding-cake-like MoS

Cell therapeutics hold tremendous regenerative potential and the therapeutic effect depends on the effective delivery of cells. However, current cell delivery carriers with unsuitable cytocompatibility and topological structure demonstrate poor cell viability during injection. Therefore, porous shape-memory cryogel microspheres (CMS) are prepared from methacrylated gelatin (GelMA) by combining an emulsion technique with gradient-cooling cryogelation. Pore sizes of the CMS are adjusted via the gradient-cooling procedure, with the optimized pore size (15.5 ± 6.0 µm) being achieved on the 30-min gradient-cooled variant (CMS-30). Unlike hydrogel microspheres (HMS), CMS promotes human bone marrow stromal cell (hBMSC) and human umbilical vein endothelial cell (HUVEC) adhesion, proliferated with high levels of stemness for 7 d, and protects cells during the injection process using a 26G syringe needle. Moreover, CMS-30 enhances the osteogenic differentiation of hBMSCs in osteoinductive media. CMS can serve as building blocks for delivering multiple cell types. Here, hBMSC-loaded and HUVEC-loaded CMS-30, mixed at a 1:1 ratio, are injected subcutaneously into nude mice for 2 months. Results show the development of vascularized bone-like tissue with high levels of OCN and CD31. These findings indicate that GelMA CMS of a certain pore size can effectively deliver multiple cells to achieve functional tissue regeneration.

The fracture behavior of graphene monolayer with the substrate binding effect can be investigated by the indentation response of graphene/poly-dimethylsiloxane (PDMS) nanocomposite (graphene monolayer mounted on super-soft substrate-PDMS) performed by atomic force microscopy (AFM). The nonlinear elastic constant of graphene is predicted by the reverse analysis of the experimental results using finite element method (FEM), based on which the intrinsic strength of graphene monolayer is evaluated as 37.6 N/m (corresponding to 112 GPa) and the corresponding strain is ∼0.23. It is found that the fracture stress of graphene is essentially not sensitive to the substrate binding effect (∼10% lower than their free-standing counterpart). On the basis of FEM, the deformation of graphene in graphene/PDMS under indentation is very similar to that of graphene in free-standing indentation, and the main strain of graphene is not transferred by the graphene/substrate interface but directly applied by the indentation load, which creates a very low deformation mismatch across the interface (or a very low interface stress). Therefore, there is no sliding/debonding occurrence for the graphene/PDMS interface in the indentation tests till the failure of graphene. The present work actually provides an alternative way to measure the fracture behavior of graphene with substrate sticking effect.

Cell volume growth occurs in all living tissues. The growth exerts mechanical stresses on surrounding tissues that may alter tissue microenvironment, and have significant implications in health and diseases. However, the level of growth stress generated by single cells in three-dimensional (3D) environment remains to be determined. To this end, we developed a growth force microscopy technique to determine 3D distribution of the stress. The technique was based on encapsulation of cells in elastic hydrogels, and involved 3D particle tracking and mechanical analysis of gel deformation. Data from the study demonstrated that the growth stress was dynamic, and the stress distribution at the gel-cell interface was correlated inversely to the mean surface curvature or the distance to the geometric center of the cell. The stress averaged over the cell surface increased with increasing gel stiffness, suggesting that cells could alter growth stress in response to stiffness change in microenvironment. These findings suggested that the elastic hydrogel-based microscopy technique had a potential to provide new insights into mechanisms of mechanical interactions between cell and its microenvironment.

In recent years, although several studies have demonstrated the potential of graphene-coated substrates in promoting attachment, proliferation and differentiation of osteoblasts and mesenchymal stem cells (MSCs), the effects of single-layer graphene on the osteogenic differentiation of human MSCs (hMSCs) remains unclear, especially in vivo. In this study, we transferred chemical vapor deposition (CVD) grown single-layer graphene to glass slides and observed its effects on adhesion, proliferation and osteogenic differentiation of human adipose-derived stem cells (hASCs) and human bone marrow mesenchymal stem cells (hBMMSCs) in vitro. Then, in vivo, we incubated hASCs and hBMMSCs on single-layer graphene-coated smooth titanium (Ti) disks before implanting them into the back subcutaneous area of nude mice. We found that single-layer graphene accelerated cell adhesion to the substrate without influencing cell proliferation of hMSCs. Moreover, we present the first study that explores the epigenetic role of single-layer graphene in determining stem cell fate. By utilizing epigenetic approaches, we reveal that single-layer graphene promotes osteogenic differentiation of hMSCs both in vitro and in vivo, potentially by upregulating methylation of H3K4 at the promoter regions of osteogenesis-associated genes. Overall, our results highlight the potential of this material in implants and injured tissues in clinical applications.

Digital image correlation (DIC) is essentially implicated in a class of inverse problem. Here, a regularization scheme is developed for the subset-based DIC technique to effectively inhibit potential ill-posedness that likely arises in actual deformation calculations and hence enhance numerical stability, accuracy and precision of correlation measurement. With the aid of a parameterized two-dimensional Butterworth window, a regularized subpixel registration strategy is established, in which the amount of speckle information introduced to correlation calculations may be weighted through equivalent subset size constraint. The optimal regularization parameter associated with each individual sampling point is determined in a self-adaptive way by numerically investigating the curve of 2-norm condition number of coefficient matrix versus the corresponding equivalent subset size, based on which the regularized solution can eventually be obtained. Numerical results deriving from both synthetic speckle images and actual experimental images demonstrate the feasibility and effectiveness of the set of newly-proposed regularized DIC algorithms.

B lymphocytes use B cell receptors (BCRs) to recognize membrane-bound antigens to further initiate cell spreading and contraction responses during B cell activation. We combined traction force microscopy and live-cell imaging to profile the origin, dynamics, and function of traction force generation in these responses. We showed that B cell activation required the generation of 10 to 20 nN of traction force when encountering antigens presented by substrates with stiffness values from 0.5 to 1 kPa, which mimic the rigidity of antigen-presenting cells in vivo. Perturbation experiments revealed that F-actin remodeling and myosin- and dynein-mediated contractility contributed to traction force generation and B cell activation. Moreover, membrane-proximal BCR signaling molecules (including Lyn, Syk, Btk, PLC-γ2, BLNK, and Vav3) and adaptor molecules (Grb2, Cbl, and Dok-3) linking BCR microclusters and motor proteins were also required for the sustained generation of these traction forces. We found a positive correlation between the strength of the traction force and the mean fluorescence intensity of the BCR microclusters. Furthermore, we demonstrated that isotype-switched memory B cells expressing immunoglobulin G (IgG)-BCRs generated greater traction forces than did mature naive B cells expressing IgM-BCRs during B cell activation. Last, we observed that primary B cells from patients with rheumatoid arthritis generated greater traction forces than did B cells from healthy donors in response to antigen stimulation. Together, these data delineate the origin, dynamics, and function of traction force during B cell activation.

In recent years, dental implants have become the preferred approach for the restoration of missing teeth. At present, most dental implants are made of pure titanium, and are affected by peri-implantitis and bone resorption, which usually start from the implant neck, due to the complex environment in this region. To address these issues, in this study we modified the surface of titanium (Ti) implants to exploit the antibacterial and osteoinductive effects of single-layer graphene sheets. Chemical vapor deposition (CVD)-grown single-layer graphene sheets were transferred to titanium discs, and a method for improving the adhesion strength of graphene on Ti was developed due to compromised adhesion strength between graphene and titanium surface. A thermal treatment of 2 h at 160 °C was found to enhance the adhesion strength of graphene on Ti to facilitate clinical transformation. Graphene coatings of Ti enhanced cell adhesion and osteogenic differentiation, and imparted antibacterial activity to Ti substrate; these favorable effects were not affected by the thermal treatment. In summary, the present study elucidated the effects of a thermal treatment on the adhesion strength and osteoinductive activity of single-layer graphene sheets on titanium substrates.

B cells use B-cell receptors (BCRs) to sense antigens that are usually presented on substrates with different stiffness. However, it is not known how substrate stiffness affects B-cell proliferation, class switch, and in vivo antibody responses. We addressed these questions using polydimethylsiloxane (PDMS) substrates with different stiffness (20 or 1100 kPa). Live cell imaging experiments suggested that antigens on stiffer substrates more efficiently trigger the synaptic accumulation of BCR and phospho-Syk molecules compared with antigens on softer substrates. In vitro expansion of mouse primary B cells shows different preferences for substrate stiffness when stimulated by different expansion stimuli. LPS equally drives B-cell proliferation on stiffer or softer substrates. Anti-CD40 antibodies enhance B-cell proliferation on stiffer substrates, while antigens enhance B-cell proliferation on softer substrates through a mechanism involving the enhanced phosphorylation of PI3K, Akt, and FoxO1. In vitro class switch differentiation of B cells prefers softer substrates. Lastly, NP67-Ficoll on softer substrates accounted for an enhanced antibody response in vivo. Thus, substrate stiffness regulates B-cell activation, proliferation, class switch, and T cell independent antibody responses in vivo, suggesting its broad application in manipulating the fate of B cells in vitro and in vivo.

Digital image correlation (DIC) is nowadays widely applied to many engineering areas as an effective optical displacement measurement technique. To minimize the potential effect of spatial sampling locations on full-field displacement measurement, this paper develops a self-adaptive sampling DIC algorithm for accurate and reliable displacement computation over entire specimen surfaces. Depending on local deformation states, the algorithm can automatically optimize spatial distribution of sampling points over specimen surfaces in a self-adaptive manner in combination with the well-developed DIC algorithm with Gaussian windows. Both a series of well-designed computer-simulated speckle images and actual cell-substrate deformation ones are employed to verify the feasibility and effectiveness of the proposed algorithm, which demonstrates that the set of self-adaptive sampling algorithm is capable of recovering more accurate and precise full-field displacements compared to the conventional DIC algorithm with equidistant sampling.

The mechanosensing ability of lymphocytes regulates their activation in response to antigen stimulation, but the underlying mechanism remains unexplored. Here, we report that B cell mechanosensing-governed activation requires BCR signaling molecules. PMA-induced activation of PKCβ can bypass the Btk and PLC-γ2 signaling molecules that are usually required for B cells to discriminate substrate stiffness. Instead, PKCβ-dependent activation of FAK is required, leading to FAK-mediated potentiation of B cell spreading and adhesion responses. FAK inactivation or deficiency impaired B cell discrimination of substrate stiffness. Conversely, adhesion molecules greatly enhanced this capability of B cells. Lastly, B cells derived from rheumatoid arthritis (RA) patients exhibited an altered BCR response to substrate stiffness in comparison with healthy controls. These results provide a molecular explanation of how initiation of B cell activation discriminates substrate stiffness through a PKCβ-mediated FAK activation dependent manner. DOI: http://dx.doi.org/10.7554/eLife.23060.001, eLife digest The human immune system protects the body from harmful bacteria, viruses and other microbes. Immune cells called B cells use proteins called B cell receptors on their surface to identify these invaders. When the B cell receptors detect molecules called antigens on the surface of the microbes, they produce signals that activate the B cell and enable it to combat the infection. Previous research has found that B cells react differently to antigens depending on the stiffness of the surface to which an antigen is attached. Now, Shaheen, Wan, Li et al. have attached antigens to artificial surfaces that were either stiff or soft and examined how B cells responded to them. Some of the B cells were modified to lack particular molecules that are important for B cell receptor signaling. The results of the experiments suggest that two signaling molecules – called protein kinase C beta (PKCβ) and focal adhesion kinase (FAK) – enable B cells to distinguish between the stiffness of different surfaces. PKCβ activates FAK, which causes the B cell to spread onto the surface and stick to it. However, B cells that had an inactive version of FAK – or lacked the protein entirely – did not efficiently spread onto the surfaces and were less able to discriminate between stiff and soft surfaces. In autoimmune diseases such as rheumatoid arthritis, B cells are overactive and attack the body’s own cells. Shaheen et al. found that the B cells of people with rheumatoid arthritis are less able to distinguish between stiff and soft surfaces than normal cells. Further research that investigates how to change the ability of a B cell to detect stiffness could therefore help researchers to develop treatments or vaccines for rheumatoid arthritis and other autoimmune conditions. DOI: http://dx.doi.org/10.7554/eLife.23060.002

Item does not contain fulltext Experimental findings indicate that cell function and behavior such as cell growth, division, migration and differentiation, are subtly regulated via integrin-dependent cell adhesion. Cell adhesion is influenced by nanoscale ligand spacing and rigidity of extracellular substrates, as cell adhesion drops greatly when the ligand spacing is larger than ~60nm, and cell adhesion is stronger on stiff than soft substrates. However, how nanoscale ligand spacing and substrate stiffness jointly affect integrin clustering and hence nascent cell adhesion remains to be elucidated. To quantitatively investigate the phenomena and the underlying mechanochemical mechanism of integrin clustering modulated by ligand spacing and substrate stiffness, we introduced Monte Carlo simulations varying the values of ligand spacing and substrate stiffness. Moreover, the effects of integrin number, integrin binding free energy, integrin association free energy, and local ligand spacing were investigated. The simulation results showed that integrin clustering decreased sharply, when ligand spacing was relatively large such as dL>60nm in the current simulations, regardless of substrate rigidities, though with close spacing, the clustering increased with the substrate stiffness. The investigation contributes to the goals of understanding and predicting experimental phenomena, directing and optimizing biomaterial design, and manipulating integrin-dependent cell-substrate adhesion in tissue engineering. 01 augustus 2017