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Cell adhesion molecules mediate cell-cell or cell-matrix interactions, some of which are mechanical sensors, such as integrins. Emerging evidence indicates that anthrax toxin receptor 1 (ANTXR1), a newly identified cell adhesion molecule, can also sense extracellular mechanical signals such as hydrostatic pressure and extracellular matrix (ECM) rigidity. ANTXR1 can interact with ECM through connecting intracellular cytoskeleton and ECM molecules (just like integrins) to regulate numerous biological processes, such as cell adhesion, cell migration or ECM homeostasis. Although with high structural similarity to integrins, its functions and downstream signal transduction are independent from those of integrins. In this perspective, based on existing evidence in literature, we analyzed the structural and functional evidence that ANTXR1 can act as a potential sensor for extracellular mechanical cues. To our knowledge, this is the first in-depth overview of ANTXR1 from the perspective of mechanobiology. An overview of ANTXR1 from the perspective of mechanobiology; An analysis of mechanical sensitivity of ANTXR1 in structure and function; A summary of existing evidence of ANTXR1 as a potential mechanosensor. [Display omitted] [ABSTRACT FROM AUTHOR]

Fibrillar collagen is a ubiquitous structural protein that plays a significant role in determining the mechanical properties of various tissues. The constituent collagen architecture can give direct insight into the respective functional role of the tissue due to the strong structure-function relationship that is exhibited. In such tissues, matrix structure can vary across local subregions contributing to mechanical heterogeneity which can be implicated in tissue function or failure. The post-myocardial infarction scar environment is an example of note where mechanically insufficient collagen can result in impaired cardiac function and possibly tissue rupture due to post-MI cellular response and matrix interactions. In order to further develop the understanding of cell-matrix interactions within heterogeneous environments, we developed a method of heterogeneous collagen gel fabrication which produces a region of randomly oriented fibers directly adjacent to an interconnected region of anisotropic alignment. To fully capture and evaluate the degree of alignment and spatial orientation heterogeneity, several image processing and automated analysis methods were employed. Our analysis revealed the successful fabrication of an interconnected spatially heterogeneous collagen gel possessing distinct regions of random or preferential alignment. Additionally, embedded cell populations were observed to recognize and reorient with their underlying and surrounding architectures through our cell-centric analysis techniques. Fibrillar collagen is a structural protein that contributes to the architecture-function relationship exhibited by various tissues where mechanically insufficient collagen architecture can lead to tissue failure. One environment where this can occur is the post-myocardial infarction scar environment where too much or too little collagen accumulation coupled with spatial fiber orientation heterogeneity can lead to environments incapable of normal mechanical functionality. While there are methodologies capable of generating aligned constructs, they do so with varying degrees of control and complexity with many producing uniform construct alignment. The presented platform is simple and produces continuous constructs possessing inherent spatial orientation heterogeneity. Coupling this with image processing and automated analysis methods enables the probing of fundamental cell-matrix interactions within heterogeneous environments. [Display omitted] [ABSTRACT FROM AUTHOR]

[Display omitted] Cells within living soft biological tissues seem to promote the maintenance of a mechanical state within a defined range near a so-called set-point. This mechanobiological process is often referred to as mechanical homeostasis. During this process, cells interact with the fibers of the surrounding extracellular matrix (ECM). It remains poorly understood, however, what individual cells actually regulate during these interactions, and how these micromechanical regulations are translated to the tissue-level to lead to what we observe as biomaterial properties. Herein, we examine this question by a combination of experiments, theoretical analysis, and computational modeling. We demonstrate that on short time scales (hours) - during which deposition and degradation of ECM fibers can largely be neglected - cells appear to not regulate the stress / strain in the ECM or their own shape, but rather only the contractile forces that they exert on the surrounding ECM. Cells in soft biological tissues sense and regulate the mechanical state of the extracellular matrix to ensure structural integrity and functionality. This so-called mechanical homeostasis plays an important role in the natural history of various diseases such as aneurysms in the cardiovascular system or cancer. Yet, it remains poorly understood to date which target quantity cells regulate on the mircroscale and how it translates to the macroscale. In this paper, we combine experiments, computer simulations, and theoretical analysis to compare different hypotheses about this target quantity. This allows us to identify a likely candidate for it at least on short time scales and in the simplified environment of tissue equivalents. [ABSTRACT FROM AUTHOR]

Biomaterials combining biochemical and biophysical cues to establish close-to-extracellular matrix (ECM) models have been explored for cell expansion and differentiation purposes. Multivariate arrays are used as material-saving and rapid-to-analyze platforms, which enable selecting hit-spotted formulations targeting specific cellular responses. However, these systems often lack the ability to emulate dynamic mechanical aspects that occur in specific biological milieus and affect physiological phenomena including stem cells differentiation, tumor progression, or matrix modulation. We report a tailor-made strategy to address the combined effect of flow and biochemical composition of three-dimensional (3D) biomaterials on cellular response. We suggest a simple-to-implement device comprising (i) a perforated platform accommodating miniaturized 3D biomaterials and (ii) a bioreactor that enables the incorporation of the biomaterial-containing array into a disposable perfusion chamber. The system was upscaled to parallelizable setups, increasing the number of analyzed platforms per independent experiment. As a proof-of-concept, porous chitosan scaffolds with 1 mm diameter were functionalized with combinations of 5 ECM and cell-cell contact-mediating proteins, relevant for bone and dental regeneration, corresponding to 32 protein combinatorial formulations. Mesenchymal stem cells adhesion and production of an early osteogenic marker were assessed on-chip on static and under-flow dynamic perfusion conditions. Different hit-spotted biomaterial formulations were detected for the different flow regimes using direct image analysis. Cell-binding proteins still poorly explored as biomaterials components – amelogenin and E-cadherin – were here shown as relevant cell response modulators. Their combination with ECM cell-binding proteins – fibronectin, vitronectin, and type 1 collagen – rendered specific biomaterial combinations with high cell adhesion and ALP production under flow. The developed versatile system may be targeted at widespread tissue regeneration applications, and as a disease model/drug screening platform. A perfusion system that enables cell culture in arrays of three-dimensional biomaterials under dynamic flow is reported. The effect of 31 cell-binding protein combinations in the adhesion and alkaline phosphatase (ALP) production of mesenchymal stem cells was assessed using a single bioreactor chamber. Flow perfusion was not only assessed as a classical enhancer/accelerator of cell growth and early osteogenic differentiation. We hypothesized that flow may affect cell-protein interactions, and that key components driving cell response may differ under static or dynamic regimes. Indeed, hit-spotted formulations that elicited highest cell attachment and ALP production on static cell culture differed from the ones detected for dynamic flow assays. The impacting role of poorly studied proteins as E-cadherin and amelogenin as biomaterial components was highlighted. [ABSTRACT FROM AUTHOR]

Abstract Introduction In carious teeth, transforming growth factor beta 1 (TGF-β1) is released from the dentin matrix and possibly activated in an acidic environment. Conversely, EDTA solutions with a neutral to slightly alkaline pH are used in clinics to promote cell homing in regenerative endodontic procedures. We hypothesized that citric acid (CA) might be more beneficial. Methods TGF-β1 release from human dentin disks conditioned with either 10% CA (pH = 2) or 17% EDTA (pH = 8) and the behavior of human stem cells toward such pretreated dentin were studied. The protein concentration in conditioning solutions after 10 minutes of dentin exposure was determined using a pH-independent slot blot technique. Results There was a 5-fold higher concentration of the target protein in CA (382 ± 30 ng/disk) compared with EDTA (66 ± 3 ng/disk, P <.005). Using confocal laser scanning microscopy on immunofluorescent-labeled disks, we identified a high density of TGF-β1 in peritubular dentin after CA treatment. A migration assay showed that CA conditioning attracted significantly more stem cells toward the dentin after 24 hours compared with EDTA (P <.05) or phosphate-buffered saline (P <.005). To investigate whether the cell response to these dentin surfaces could be affected by different pretreatments, we cultured stem cells on conditioned dentin disks and found that CA had a significantly (P <.05) better effect than EDTA on cell attachment and cell survival. Conclusions CA conditioning could be useful and may have significant benefits over current treatments. Highlights • Under the conditions of the current study using human dentin disks, a 10% citric acid solution (pH = 2) was significantly (P <.05) better than 17% EDTA (pH = 8) regarding the following: -The release of transforming growth factor beta 1 (TGF-β1) into solution. -The exposure of TGF-β1 on the dentin surface. -The attraction of stem cells toward conditioned dentin. -Stem cell attachment on the conditioned surface. -Stem cell survival. [ABSTRACT FROM AUTHOR]

Graphical abstract Abstract While tissue form and function is highly dependent upon tissue-specific collagen composition and organization, little is known of the mechanisms controlling the bundling of collagen fibrils into fibers and larger structural designs that lead to the formation of bones, tendons and other tissues. Using the cornea as a model system, our previous 3 dimensional mapping of collagen fiber organization has demonstrated that macrostructural organization of collagen fibers involving interweaving, branching and anastomosing plays a critical role in controlling mechanical stiffness, corneal shape and refractive power. In this work, the cellular and mechanical mechanisms regulating critical events in the assembly of collagen macrostructure are analysed in the developing chicken cornea. We elucidated the temporal events leading to adult corneal structure and determined the effects of intraocular pressure (IOP) on the organization of the collagen macrostructure. Our findings indicate that the complex adult collagen organization begins to appear on embryonic day 10 (E10) after deposition of the primary stroma and full invasion of keratocytes. Importantly, organizational changes in keratocytes appearing at E9 preceded and predicted later changes in collagen organization. Corneal collagen organization remained unaffected when the development of IOP was blocked at E4. These findings support a primary role for keratocytes in controlling stromal organization, mechanical stiffness and corneal shape that are not regulated by the IOP. Our findings also suggest that the avian cornea represents an excellent experimental model for elucidating key regulatory steps and mechanisms controlling the collagen fiber organization that is critical to determining tissue form and function. Statement of significance This work by using an ex ovo model system, begins to investigate the potential mechanisms controlling collagen fibril macrostructure. In particular, this work highlights a convergent role for the corneal keratocytes in organizing the complex collagen macrostructure, necessary to support high visual acuity. Our data supports that the intraocular pressure does not influence collagen fibril macrostructure and suggest that the avian cornea represents an excellent experimental model for elucidating key regulatory steps and mechanisms controlling the collagen fiber organization that is critical to determining tissue form and function. Clearly understanding the cellular and molecular mechanisms that underlie collagen fibril macrostructure will be highly beneficial for future tissue engineering and regenerative medicine applications. [ABSTRACT FROM AUTHOR]

Nucleus pulposus (NP) cells are derived from the notochord and differ from neighboring cells of the intervertebral disc in phenotypic marker expression and morphology. Adult human NP cells lose this phenotype and morphology with age in a pattern that contributes to progressive disc degeneration and pathology. Select laminin-mimetic peptide ligands and substrate stiffnesses were examined for their ability to regulate human NP cell phenotype and biosynthesis through the expression of NP-specific markers aggrecan, N-cadherin, collagen types I and II, and GLUT1. Peptide-conjugated substrates demonstrated an ability to promote expression of healthy NP-specific markers, as well as increased biosynthetic activity. We show an ability to re-express markers of the juvenile NP cell and morphology through control of peptide presentation and stiffness on well-characterized polyacrylamide substrates. NP cells cultured on surfaces conjugated with α3 integrin receptor peptides P4 and P678, and on α2, α5, α6, β1 integrin-recognizing peptide AG10, show increased expression of aggrecan, N-cadherin, and types I and II collagen, suggesting a healthier, more juvenile-like phenotype. Multi-cell cluster formation was also observed to be more prominent on peptide-conjugated substrates. These findings indicate a critical role for cell-matrix interactions with specific ECM-mimetic peptides in supporting and maintaining a healthy NP cell phenotype and bioactivity. Statement of Significance NP cells reside in a laminin-rich environment that deteriorates with age, including a loss of water content and changes in the extracellular matrix (ECM) structure that may lead to the development of a degenerated IVD. There is great interest in methods to re-express healthy, biosynthetically active NP cells using laminin-derived biomimetic peptides toward the goal of using autologous cell sources for tissue regeneration. Here, we describe a novel study utilizing several laminin mimetic peptides conjugated to polyacrylamide gels that are able to support an immature, healthy NP phenotype after culture on “soft” peptide gels. These findings can support future studies in tissue regeneration where cells may be directed to a desired regenerative phenotype using niche-specific ECM peptides. [ABSTRACT FROM AUTHOR]

Abstract: Mesenchymal stem cells (MSCs) can be made to rearrange into microtissues in response to specific matrix cues, a process that depends on a balance between cell–matrix and cell–cell interactions. The effect of such cues, and especially their interplay, is still not fully understood, particularly in three-dimensional (3-D) systems. Here, the behaviour of human MSCs cultured within hydrogel matrices with tailored stiffness and composition was evaluated. MSC aggregation occurred only in more compliant matrices (G′⩽120Pa), when compared to stiffer ones, both in the presence and in the absence of matrix-bound arginine–glycine–aspartic acid cell–adhesion ligands (RGD; 0, 100 and 200μM). Fibronectin assembly stabilized cell–cell contacts within aggregates, even in non-adhesive matrices. However, MSCs were able to substantially contract the artificial matrix only when RGD was present. Moreover, compliant matrices facilitated cell proliferation and provided an environment conducive for MSC osteogenic differentiation, even without RGD. Cell interactions with the original matrix became less important as time progressed, while the de novo-produced extracellular matrix became a more critical determinant of cell fate. These data provide further insights into the mechanisms by which MSCs sense their microenvironment to organize into tissues, and provide new clues to the design of cell-instructive 3-D matrices. [Copyright &y& Elsevier]

Abstract: The pericellular matrix (PCM) surrounding chondrocytes is thought to play an important role in transmitting biochemical and biomechanical signals to the cells, which regulates many cellular functions including tissue homeostasis. To better understand chondrocytes interactions with their PCM, three-dimensional poly(ethylene glycol) (PEG) hydrogels containing Arg–Gly–Asp (RGD), the cell-adhesion sequence found in fibronectin and which is present in the PCM of cartilage, were employed. RGD was incorporated into PEG hydrogels via tethers at 0.1, 0.4 and 0.8mM concentrations. Bovine chondrocytes were encapsulated in the hydrogels and subjected to dynamic compressive strains (0.3Hz, 18% amplitude strain) for 48h, and their response assessed by cell morphology, ECM gene expression, cell proliferation and matrix synthesis. Incorporation of RGD did not influence cell morphology under free swelling conditions. However, the level of cell deformation upon an applied strain was greater in the presence of RGD. In the absence of dynamic loading, RGD appears to have a negative effect on chondrocyte phenotype, as seen by a 4.7-fold decrease in collagen II/collagen I expressions in 0.8mM RGD constructs. However, RGD had little effect on early responses of chondrocytes (i.e. cell proliferation and matrix synthesis/deposition). When isolating RGD as a biomechanical cue, cellular response was very different. Chondrocyte phenotype (collagen II/collagen I ratio) and proteoglycan synthesis were enhanced with higher concentrations of RGD. Overall, our findings demonstrate that RGD ligands enhance cartilage-specific gene expression and matrix synthesis, but only when mechanically stimulated, suggesting that cell–matrix interactions mediate chondrocyte response to mechanical stimulation. [Copyright &y& Elsevier]

Abstract: The extracellular matrix is an insoluble aggregate of large proteins and glycosoaminoglycans that comprises the microenvironment of cells in tissue. The matrix displays a host of ligands that interact with cell-surface receptors to mediate the attachment and spreading of cells and regulate signaling processes. Studies of cell–matrix interactions and downstream signaling processes commonly employ substrates having an adsorbed layer of protein and are challenged by the difficulty in controlling the structure and activity of the immobilized protein. Significant effort has been directed towards the development of model substrates that present adhesion ligands in defined densities, orientations and environments. Among these approaches, self-assembled monolayers of alkanethiolates on gold offer a high level of control over the molecular structure of the surface and are well-suited to studies of cell adhesion. This review describes the design and use of monolayers for applications in cell biology, including the use of monolayers to evaluate the roles of peptide and protein ligands in cell–matrix interactions, the development of methods to pattern ligands on monolayers and applications to cell biology, the development of dynamic monolayers that can switch the activities of ligands presented to an adherent cell, and the rewiring of interactions between a cell and its substrate. These examples illustrate the flexibility inherent to monolayers for applications in cell biology. [Copyright &y& Elsevier]