Matrix–Matrix Interfaces Orchestrate Early Mechanosensitive Transition from Attractor To Track

Rebuilding the tooth-supporting tissues (periodontium) destroyed by periodontitis remains a clinical challenge. Periodontal ligament cells (PDLCs), multipotent cells within the periodontal ligament (PDL), differentiate and form new PDL and mineralized tissues (cementum and bone) during native tissue repair in response to specific extracellular matrix (ECM) cues. Thus, harnessing ECM cues to control PDLC activity ex vivo, and ultimately, to design a PDLC delivery vehicle for tissue regeneration is an important goal. In this study, poly(ethylene glycol) hydrogels were used as a synthetic PDL ECM to interrogate the roles of cell-matrix interactions and cell-mediated matrix remodeling in controlling PDLC activity. Results showed that PDLCs within matrix metalloproteinase (MMP)-degradable hydrogels expressed key PDL matrix genes and showed a six to eightfold increase in alkaline phosphatase (ALP) activity compared with PDLCs in nondegradable hydrogel controls. The increase in ALP activity, commonly considered an early marker of cementogenic/osteogenic differentiation, occurred independent of the presentation of the cell-binding ligand RGD or soluble media cues and remained elevated when inhibiting PDLC-matrix binding and intracellular tension. ALP activity was further increased in softer hydrogels regardless of degradability and was accompanied by an increase in PDLC volume. However, scaffolds that fostered PDLC ALP activity did not necessarily promote hydrogel ECM mineralization. Rather, matrix mineralization was greatest in stiffer, MMP-degradable hydrogels and required the presence of soluble media cues. These divergent outcomes illustrate the complexity of the PDLC response to ECM cues and the limitations of current scaffold materials. Nevertheless, key biomaterial design principles for controlling PDLC activity were identified for incorporation into scaffolds for periodontal tissue regeneration. Impact statement Engineered scaffolds are an attractive approach for delivering periodontal ligament cells (PDLCs) to rebuild the tooth-supporting tissues. Replicating key extracellular matrix (ECM) cues within tissue engineered scaffolds may maximize PDLC potential. However, the identity of important ECM cues and how they can be harnessed to control PDLC activity is still unknown. In this study, matrix degradability, cell-matrix binding, and stiffness were varied using synthetic poly(ethylene glycol) hydrogels for three-dimensional PDLC culture. PDLCs exhibited dramatic and divergent responses to these cues, supporting further investigation of ECM-replicating scaffolds for control of PDLC behavior and periodontal tissue regeneration.

Extracellular matrix cues as drivers of liver zonation: A framework for in vitro biomaterial design.

Synergistic effects of hypoxia and extracellular matrix cues in cardiomyogenesis

Piezo1 regulates remodeling of skin-derived extracellular matrix by embedded umbilical cord mesenchymal stem cells in a stiffness-dependent fashion

IntroductionG protein-coupled estrogen receptor (GPER) is a heptahelix estrogen-binding G protein-coupled receptor, and a potential therapeutic target for estrogen-related cancers and diseases. Recently, GPER has been recognized as a key mechano-regulator, but the effects on cell adhesion, spreading, morphology, migration, and differentiation are inconsistent or even contradicting in literature, due to the variations across cell lines and complex crosstalks with ER, and non-genomic actions of other hormones. Here, we focus on investigating the GPER effect on mesenchymal stem cell (MSC) mechanotransduction and differentiation.MethodsMSCs treated by synthetic agonist G1 and untreated cells were cultured on fibronectin-coated surfaces. Cell morphology and cytoskeletons were characterized by staining and imaging. RhoA signaling was evaluated by western blot assays. Cell migration was studied by both chemokinesis and chemotaxis experiments. MSC adipogenesis and osteogenesis were evaluated after two-week differentiation. In particular, since micropatterns have been widely used to mimic extracellular matrix (ECM) cues, modulate MSC mechanotransduction and differentiation, we investigate the GPER effect on both single-cell and sub-cellular fibronectin microline patterns prepared by microcontact printing.ResultsGPER activation regulates cytoskeleton organization, with reduced cell polarization and thinner ventral stress fibers; reduces MSC migration speed; significantly promotes osteogenesis and inhibits adipogenesis. Cell elongation by micropatterns and the reduction of cell polarization by GPER coexist in a sophisticated interplay.ConclusionsGPER directly mediates MSC mechanotransduction by RhoA inactivation, while its sustained effect on MSC differentiation promotes osteogenesis and inhibits adipogenesis despite reduced cell polarization and tension, suggesting potential mechanisms other than RhoA signaling. Our findings pave the way towards a deep understanding of GPER’s role and its interplay with ECM cues in mechanotransduction and differentiation, which will be important for developing GPER as a new therapeutic target, as well as considering its important effects in stem cell therapies and hormonal therapies.

The extracellular matrix (ECM) constitutes a three-dimensional network that surrounds all cells, organs and tissues in the body. It forms a biophysical filter for protection, nutrition and cell innervation, as well as the medium for facilitating immune response, angiogenesis, fibrosis and tissue regeneration. It is the mechanism by which mechanical forces are transmitted to the basement membrane which, through the integrins, supports the tensegrity system and activates the epigenetic mechanisms of the cell. A review and update on current knowledge on this topic reveals how disturbance of the ECM leads to a loss of efficient filtering, nutrition, elimination, and cell denervation functions, in addition to loss of regeneration capacity and disorders in mechanotransduction. Furthermore, such disturbance results in a loss of substrate, and with it the ability to provide a proper immune response against tumor, toxic and infectious agents. Reciprocal communication between ECM stromal and parenchymatous cells directs gene expression. The oncogenic capacity of the stroma derives from the associated cells as well as from the tumor cells, the angiogenic microenvironment and from an alteration in tensegrity; all of which are dependent on the ECM. It has been shown that the malignant phenotype is reversible by correction of the altered cues of the ECM.

Engineering the Extracellular Matrix to Model the Evolving Tumor Microenvironment.

Tumor microenvironment is a highly complex system consisting of non-cancerous cells, soluble factors, signaling molecules, extracellular matrix, and mechanical cues, which provides tumor cells with integrated biochemical and biophysical cues. It has been recognized as a significant regulator in cancer initiation, progression, metastasis, and drug resistance, which is becoming a crucial component of cancer biology. Modeling microenvironmental conditions of such complexity in vitro are particularly difficult and technically challenging. Significant advances in microfluidic technologies have offered an unprecedented opportunity to closely mimic the physiological microenvironment that is normally encountered by cancer cells in vivo. This review highlights the recent advances of microfluidic platform in recapitulating many aspects of tumor microenvironment from biochemical and biophysical regulations. The major events relevant in tumorigenesis, angiogenesis, and spread of cancer cells dependent on specific combinations of cell types and soluble factors present in microenvironmental niche are summarized. The questions and challenges that lie ahead if this field is expected to transform the future cancer research are addressed as well.

The physical properties of two-dimensional (2D) extracellular matrices (ECMs) modulate cell adhesion dynamics and motility, but little is known about the roles of local microenvironmental differences in three-dimensional (3D) ECMs. Here we generate 3D collagen gels of varying matrix microarchitectures to characterize their regulation of 3D adhesion dynamics and cell migration. ECMs containing bundled fibrils demonstrate enhanced local adhesion-scale stiffness and increased adhesion stability through balanced ECM/adhesion coupling, whereas highly pliable reticular matrices promote adhesion retraction. 3D adhesion dynamics are locally regulated by ECM rigidity together with integrin/ECM association and myosin II contractility. Unlike 2D migration, abrogating contractility stalls 3D migration regardless of ECM pore size. We find force is not required for clustering of activated integrins on 3D native collagen fibrils. We propose that efficient 3D migration requires local balancing of contractility with ECM stiffness to stabilize adhesions, which facilitates the detachment of activated integrins from ECM fibrils.

Lung cancer remains a leading cause of cancer-related deaths worldwide, largely due to late diagnosis and the complexity of the tumor microenvironment (TME).1 A key factor in lung cancer is the extracellular matrix (ECM), a 3D network composed of structural proteins, glycoproteins, proteoglycans, and growth factors that together regulate cell adhesion, proliferation, and signaling. ECM architecture and its changes are closely related to cancer mechanisms.2 Thus, physiological models that recapitulate ECM composition and mechanics are essential. 2D cultures fail to replicate the organization and biochemical and mechanical signals of the TME, whereas microfluidic platforms offer dynamic, 3D cell culture systems hydrogel-integrated.3 A broad range of biomaterials (synthetic, semi-synthetic, natural) is used to recapitulate the dynamics of ECM. Natural hydrogels such as collagen, gelatin methacrylate (GelMA), Matrigel, alginate, fibrin, and decellularized ECM (dECM) are widely used due to their inherent bioactivity and ability to support cell adhesion and proliferation.4 Synthetic hydrogels, such as polyethylene glycol (PEG) and polyacrylamide, provide tunable stiffness and control over matrix composition, while semi-synthetic hybrids (e.g., PEG-GelMA, GelMA-dECM) combine biological cues with structural stability (Figure 1).5. Cancer cell lines A549 (adenocarcinoma), H1299 (non-small cell lung cancer), and H460 (large cell carcinoma) are frequently used in cancer modelling. Co-culture systems integrate fibroblasts, endothelial cells, and immune cells (e.g., macrophages) to simulate the TME and study cell-matrix-cell interactions. Patient-derived organoids preserve tumor heterogeneity, genetic mutations, and drug response profiles, representing a personalized in vitro cancer model. Tumor spheroids embedded in hydrogels recapitulate diffusion gradients and are used to evaluate drug penetration and metastasis. Further, they can be integrated into microfluidic platforms or well plates. As a next step, it is important to investigate ECM rheology, determine mechanical structure and cytokine expression levels, and further validate cell-matrix interactions.6,7. These models have shown that tumor cells embedded in hydrogels exhibit enhanced invasive behavior and increased expression of matrix metalloproteinases (enzymes responsible for ECM degradation and remodeling). From a biomechanical perspective, rheological analyses revealed that cancer-associated hydrogels typically exhibit higher storage modulus than healthy matrices, reflecting a stiffer microenvironment and increased collagen levels.8. Collectively, recent findings underscore the central role of the ECM and hydrogel-based systems in modeling lung cancer progression. The development of tissue-specific, mechanically tunable, and microfluidic-integrated hydrogels has transformed in vitro modeling from static 2D monolayers to dynamic, physiologically relevant 3D systems. Increased stiffness is now recognized as a key regulator of cancer cell fate, governing proliferation, EMT, and metastasis through mechanotransduction pathways such as YAP/TAZ and integrin-FAK signaling.9 Despite significant progress, variability in dECM composition and crosslinking chemistry still challenges reproducibility and bioactivity. Moreover, current hydrogel-based models often lack immune cell components and vascular complexity, limiting their ability to fully emulate the native TME. Future efforts should integrate dECM-based hydrogels with organoid and microfluidic systems, supported by multi-omics profiling, to achieve patient-specific and physiologically relevant lung cancer models.

The generation of endothelial cells (ECs) from human pluripotent stem cells (PSCs) has been a promising approach for treating cardiovascular diseases for several years. Human PSCs, particularly induced pluripotent stem cells (iPSCs), are an attractive source of ECs for cell therapy. Although there is a diversity of methods for endothelial cell differentiation using biochemical factors, such as small molecules and cytokines, the efficiency of EC production varies depending on the type and dose of biochemical factors. Moreover, the protocols in which most EC differentiation studies have been performed were in very unphysiological conditions that do not reflect the microenvironment of native tissue. The microenvironment surrounding stem cells exerts variable biochemical and biomechanical stimuli that can affect stem cell differentiation and behavior. The stiffness and components of the extracellular microenvironment are critical inducers of stem cell behavior and fate specification by sensing the extracellular matrix (ECM) cues, adjusting the cytoskeleton tension, and delivering external signals to the nucleus. Differentiation of stem cells into ECs using a cocktail of biochemical factors has been performed for decades. However, the effects of mechanical stimuli on endothelial cell differentiation remain poorly understood. This review provides an overview of the methods used to differentiate ECs from stem cells by chemical and mechanical stimuli. We also propose the possibility of a novel EC differentiation strategy using a synthetic and natural extracellular matrix.

Introduction: Fibrin-matrices of different stiffness can be used for tissue engineering. The differentiation and extracellular matrix (ECM) remodeling properties of mesenchymal stem cells can be influenced by matrix stiffness. We hypothesized that stiffer fibrin matrices slow matrix degradation and favor the osteogenic differentiation of human adipose-derived stem cells (hASCs).Materials and Methods: hASCs were incorporated at different densities into soft and stiff fibrin matrices composed of 2 mg/ml fibrinogen and 0.1 or 1.0 IU/ml thrombin. The Young's moduli of the matrices were determined by nano-indentation. Fibrin degradation was determined during a 14 day culture period by ELISA. qPCR and histology were used to assess ECM remodeling and osteogenic differentiation.Results: Fibrin matrices polymerized with 1.0 IU/ml thrombin were 69% stiffer than those polymerized with 0.1 IU/ml. Stiffer matrices degraded more than soft matrices. Higher cell seeding densities increased matrix degradation. Cells in stiffer matrices produced more Alkaline Phosphatase and ECM than cells in softer matrices. RUNX-2 expression was almost ten times higher in stiff matrices than in soft matrices.Discussion: Only stiff fibrin matrices induced osteogenic differentiation of hASCs. Unexpectedly, this was accompanied by enhanced cell-mediated matrix remodeling. These results suggest that a mechanical threshold for differentiation and ECM-remodeling was reached for cells embedded in the stiff matrices.

Introduction: Mammographic density (MD) is associated with greater risk to malignancy. MD is also correlated to high collagen content in the extra cellular matrix (ECM). Data from our group and others have highlighted the importance of mechanical cues from the ECM in breast tissue homeostasis and tumor progression to invasion [1; 2; reviewed in 3]. Whether the stiffness of the ECM could also initiate breast cancer and if so how remains unknown. Because elevated collagen levels increases ECM stiffness, we hypothesize that MD increases breast cancer risk because the ECM is stiffer. Materials and Methods: We studied breast tissues obtained through prophylactic mastectomy from women with low (BIRADS 1) versus high MD (BIRADS 4). From each surgically excised breast, samples of 0.5cm x 0.5cm x 1cm dimension were removed from the retroareolar region and from 4 peripheral quadrants. Sample sections were subjected to biophysical, morphological and biochemical analysis. Biophysical analysis included the application of Atomic Force Microscopy to obtain an extensive force map of distinct anatomical regions of the ECM associated with the intra-lobular and inter-lobular ECM. Topological analysis of ECM architecture was performed using two photons and SIM-POL imaging coupled with picrosirius staining, polarized light imaging and image quantification. Biochemical and morphological analysis consisted of immunohistochemistry for markers that detect mechano-signaling in the epithelium and stromal fibroblasts, and H&E to visualize cellular and ECM organization. Results and Discussion: We found that the intra-lobular ECM associated with the terminal end-buds in the breast contained anisotropic relaxed collagen fibrils and was very compliant. By contrast, the inter-lobular ECM of the breast contained oriented collagen fibrils and was relatively stiffer. Notably, the ECM associated with the retroareolar region, which is typically detected as very dense using mammographic imaging, contained oriented collagen fibrils, and was significantly stiffer than the ECM associated with the peripheral quadrants. Intriguingly, preliminary data suggested that the ECM associated with the terminal end-buds in the upper outer quadrant showed a trend towards greater stiffness in women with high MD (BIRADS 4) than low MD (BIRADS 1). Although, it is tempting to speculate that ECM stiffness could enhance risk to malignancy, further sample analysis is now necessary. Conclusions: • In the human breast there is anatomical heterogeneity with respect to ECM organization and mechanical properties. High MD appears to reflect elevated ECM stiffness. The intra-lobular ECM is considerably stiffer in the upper outer quadrant than in the other peripheral regions of the breast. Atomic Force Microscopy is a tractable method to monitor ECM stiffness and mechanical heterogeneity in the human breast. Acknowledgements: supported by W81XWH-05-1-0330 and R01 CA138818-01A1 to VMW, 1U01 ES019458-01 to VMW and ZW, and P50 CA 58207 to JG, VW, SH and LC, U54CA143836-01 to JL and VW.

By shaping biochemical gradients and extracellular matrix cues within the local microenvironment, cellular spatial organization plays a critical role in regulating tissue development, homeostasis, and disease progression. Microfluidic platforms are highly suitable for the study of these cell-cell and cell-matrix interactions as they precisely control cell arrangement and gradients compared to conventional experimental systems. Cells are often embedded within hydrogels to improve physiological relevance by enabling matrix-mediated signaling. However, many designs restrict the number and arrangement of hydrogels or generate gradients in only one dimension, limiting their ability to recapitulate complex tissue architectures. To address this need, we introduce a 3D printed microfluidic insert compatible with microplates that allows patterning of up to ten unique hydrogel arrays in two dimensions and generation of parallel or orthogonal concentration gradients. We first develop a physics-based computational model of hydrogel filling to define design parameters that ensure robust hydrogel patterning. We then establish perpendicular concentration gradients on timescales relevant to biological experiments. Furthermore, we demonstrate high cell viability in our 3D-printed devices and control of fibroblast migration across multiple patterned hydrogels. Finally, we monitor the recruitment of primary human monocyte towards cell-free and fibroblast-seeded 3D collagen matrices. Our microfluidic insert platform is compatible with high-throughput automation workflows and allows for interrogation of spatially variant signals that regulate cell migration and cell-cell signaling in physiologically-relevant 3D microenvironments.

Chemical, mechanical, and topographic extracellular matrix (ECM) cues have been extensively studied for their influence on cell behavior. These ECM cues alter cell adhesion, cell shape, and cell migration and activate signal transduction pathways to influence gene expression, proliferation, and differentiation. ECM elasticity and topography, in particular, have emerged as material properties of intense focus based on strong evidence these physical cues can partially dictate stem cell differentiation. Cells generate forces to pull on their adhesive contacts, and these tractional forces appear to be a common element of cells' responses to both elasticity and topography. This review focuses on recently published work that links ECM topography and mechanics and their influence on differentiation and other cell behaviors. We also highlight signaling pathways typically implicated in mechanotransduction that are (or may be) shared by cells subjected to topographic cues. Finally, we conclude with a brief discussion of the potential implications of these commonalities for cell based therapies and biomaterial design.