Host response during unresolved urinary tract infection alters female mammary tissue homeostasis through collagen deposition and TIMP1.

Exposure to pathogens throughout a lifetime influences immunity and organ function. Here, we explore how the systemic host-response to bacterial urinary tract infection (UTI) induces tissue-specific alterations to the mammary gland. Utilizing a combination of histological tissue analysis, single cell transcriptomics, and flow cytometry, we identify that mammary tissue from UTI-bearing mice displays collagen deposition, enlarged ductal structures, ductal hyperplasia with atypical epithelial transcriptomes and altered immune composition. Bacterial cells are absent in the mammary tissue and blood of UTI-bearing mice, therefore, alterations to the distal mammary tissue are mediated by the systemic host response to local infection. Furthermore, broad spectrum antibiotic treatment resolves the infection and restores mammary cellular and tissue homeostasis. Systemically, unresolved UTI correlates with increased plasma levels of the metalloproteinase inhibitor, TIMP1, which controls extracellular matrix remodeling and neutrophil function. Treatment of nulliparous and post-lactation UTI-bearing female mice with a TIMP1 neutralizing antibody, restores mammary tissue normal homeostasis, thus providing evidence for a link between the systemic host response during UTI and mammary gland alterations.

Perineuronal net structure as a non-cellular mechanism contributing to affective state: A scoping review.

Affective state encompasses emotional responses to our physiology and influences how we perceive and respond within our environment. In affective disorders such as depression, cognitive adaptability is challenged, and structural and functional brain changes have been identified. However, an incomplete understanding persists of the molecular and cellular mechanisms at play in affective state. An exciting area of newly appreciated importance is perineuronal nets (PNNs); a specialised component of extracellular matrix playing a critical role in neuroprotection and synaptic plasticity. A scoping review found 24 studies demonstrating that PNNs are still a developing field of research with a promising general trend for stress in adulthood to increase the intensity of PNNs, whereas stress in adolescence reduced (potentially developmentally delayed) PNN numbers and intensity, while antidepressants correlated with reduced PNN numbers. Despite promising trends, limited research underscores the need for further exploration, emphasizing behavioral outcomes for validating affective states. Understanding PNNs' role may offer therapeutic insights for depression and inform biomarker development, advancing precision medicine and enhancing well-being.

Pericellular Matrix Formation and Atomic Force Microscopy of Single Primary Human Chondrocytes Cultured in Alginate Microgels.

One of the main components of articular cartilage is the chondrocyte's pericellular matrix (PCM), which is critical for regulating mechanotransduction, biochemical cues, and healthy cartilage development. Here, individual primary human chondrocytes (PHC) are encapsulated and cultured in 50 µm diameter alginate microgels using drop-based microfluidics. This unique culturing method enables PCM formation and manipulation of individual cells. Over ten days, matrix formation is observed using autofluorescence imaging, and the elastic moduli of isolated cells are measured using AFM. Matrix production and elastic modulus increase are observed for the chondrons cultured in microgels. Furthermore, the elastic modulus of cells grown in microgels increases ≈ten-fold over ten days, nearly reaching the elastic modulus of in vivo PCM. The AFM data is further analyzed using a Gaussian mixture model and shows that the population of PHCs grown in microgels exhibit two distinct populations with elastic moduli averaging 9.0 and 38.0 kPa. Overall, this work shows that microgels provide an excellent culture platform for the growth and isolation of PHCs, enabling PCM formation that is mechanically similar to native PCM. The microgel culture platform presented here has the potential to revolutionize cartilage regeneration procedures through the inclusion of in vitro developed PCM.

Macrophage migration is differentially regulated by fibronectin and laminin through altered adhesion and myosin II localization.

Macrophages are indispensable for proper immune surveillance and inflammatory regulation. They also exhibit dramatic phenotypic plasticity and are highly responsive to their local microenvironment, which includes the extracellular matrix (ECM). This work demonstrates that two fibrous ECM glycoproteins, fibronectin (FN) and laminin (LAM), elicit distinct morphological and migratory responses from macrophages in two-dimensional environments. LAM 111 inhibits macrophage cell spreading, but drives them to migrate rapidly and less persistently compared with cells on FN. Differential integrin engagement and ROCK/myosin II organization helps explain why macrophages alter their morphology and migration character on these two ECM components. This study also demonstrates that LAM 111 exerts a suppressive effect toward FN, as macrophages plated on a LAM/FN mixture adopt a morphology and migratory character almost identical to LAM alone. This suggests that distinct responses can be initiated downstream of receptor-ECM engagement, and that one component of the microenvironment may affect the cell's ability to sense another. Overall, macrophages appear intrinsically poised to rapidly switch between distinct migratory characters based on their ECM environments. The role of ECM composition in dictating motile and inflammatory responses in three-dimensional and in vivo contexts warrants further study.

High-strength and high-toughness ECM films with the potential for peripheral nerve repair.

Extracellular matrix (ECM) scaffolds are widely applied in the field of regeneration as the result of their irreplaceable biological advantages, and the preparation of ECM scaffolds into ECM hydrogels expands the applications to some extent. However, weak mechanical properties of current ECM materials limit the complete exploitation of ECM's biological advantages. To enable ECM materials to be utilized in applications requiring high strength, herein, we created a kind of new ECM material, ECM film, and evaluated its mechanical properties. ECM films exhibited outstanding toughness with no cracks after arbitrarily folding and crumpling, and dramatically high strength levels of 86 ± 17.25 MPa, the maximum of which was 115 MPa. Such spectacular high-strength and high-toughness films, containing only pure ECM without any crosslinking agents and other materials, far exceed current pure natural polymer gel films and even many composite gel films and synthetic polymer gel films. In addition, both PC12 cells and Schwann cells cultured on the surface of ECM films, especially Schwann cells, showed good proliferation, and the neurite outgrowth of the PC12 cells was promoted, indicating the application potential of ECM film in peripheral nerve repair.

Extracellular Matrix Deposition Defines the Duration of Cell Sheet Assembly from Human Adipose-Derived MSC.

Cell sheet (CS) engineering using mesenchymal stromal cells (MSC) draws significant interest for regenerative medicine and this approach translates to clinical use for numerous indications. However, little is known of factors that define the timing of CS assembly from primary cultures. This aspect is important for planning CS delivery in autologous and allogeneic modes of use. We used a comparative in vitro approach with primary donors' (n = 14) adipose-derived MSCs and evaluated the impact of healthy subject's sex, MSC culture features (population doubling time and lag-phase), and extracellular matrix (ECM) composition along with factors related to connective tissue formations (α-SMA and FAP-α) on CS assembly duration. Using qualitative and quantitative analysis methods, we found that, in seeded MSCs, high contents of collagen I and collagen IV had a direct correlation with longer CS assembly duration. We found that short lag-phase cultures faster turned to a ready-to-use CS, while age, sex, fibronectin, laminin, α-SMA, and FAP-α failed to provide a significant correlation with the timing of assembly. In detachable CSs, FAP-α was negatively correlated with the duration of assembly, suggesting that its concentration rose over time and contributed to MSC activation, transitioning to α-SMA-positive myofibroblasts and ECM turnover. Preliminary data on cell density and collagen I deposition suggested that the TGF-ß1 signaling axis is of pivotal importance for ECM composition and construct maturation.

Developmental biology: A hole in the matrix.

Neurons must access the environment to gather information, but this exposure must be carefully managed. New work finds that glial cells, the non-neuronal component of the nervous system, control environmental access by stage- and sex-specific patterning of the extracellular matrix.

Novel approaches to target fibroblast mechanotransduction in fibroproliferative diseases.

The ability of cells to sense and respond to changes in mechanical environment is vital in conditions of organ injury when the architecture of normal tissues is disturbed or lost. Among the various cellular players that respond to injury, fibroblasts take center stage in re-establishing tissue integrity by secreting and organizing extracellular matrix into stabilizing scar tissue. Activation, activity, survival, and death of scar-forming fibroblasts are tightly controlled by mechanical environment and proper mechanotransduction ensures that fibroblast activities cease after completion of the tissue repair process. Conversely, dysregulated mechanotransduction often results in fibroblast over-activation or persistence beyond the state of normal repair. The resulting pathological accumulation of extracellular matrix is called fibrosis, a condition that has been associated with over 40% of all deaths in the industrialized countries. Consequently, elements in fibroblast mechanotransduction are scrutinized for their suitability as anti-fibrotic therapeutic targets. We review the current knowledge on mechanically relevant factors in the fibroblast extracellular environment, cell-matrix and cell-cell adhesion structures, stretch-activated membrane channels, stress-regulated cytoskeletal structures, and co-transcription factors. We critically discuss the targetability of these elements in therapeutic approaches and their progress in pre-clinical and/or clinical trials to treat organ fibrosis.

Inference of long-range cell-cell force transmission from ECM remodeling fluctuations.

Cells sense, manipulate and respond to their mechanical microenvironment in a plethora of physiological processes, yet the understanding of how cells transmit, receive and interpret environmental cues to communicate with distant cells is severely limited due to lack of tools to quantitatively infer the complex tangle of dynamic cell-cell interactions in complicated environments. We present a computational method to systematically infer and quantify long-range cell-cell force transmission through the extracellular matrix (cell-ECM-cell communication) by correlating ECM remodeling fluctuations in between communicating cells and demonstrating that these fluctuations contain sufficient information to define unique signatures that robustly distinguish between different pairs of communicating cells. We demonstrate our method with finite element simulations and live 3D imaging of fibroblasts and cancer cells embedded in fibrin gels. While previous studies relied on the formation of a visible fibrous 'band' extending between cells to inform on mechanical communication, our method detected mechanical propagation even in cases where visible bands never formed. We revealed that while contractility is required, band formation is not necessary, for cell-ECM-cell communication, and that mechanical signals propagate from one cell to another even upon massive reduction in their contractility. Our method sets the stage to measure the fundamental aspects of intercellular long-range mechanical communication in physiological contexts and may provide a new functional readout for high content 3D image-based screening. The ability to infer cell-ECM-cell communication using standard confocal microscopy holds the promise for wide use and democratizing the method.

Development of Adaptive Immunity and Its Role in Lung Remodeling.

Asthma is characterized by airflow limitations resulting from bronchial closure, which can be either reversible or fixed due to changes in airway tissue composition and structure, also known as remodeling. Airway remodeling is defined as increased presence of mucins-producing epithelial cells, increased thickness of airway smooth muscle cells, angiogenesis, increased number and activation state of fibroblasts, and extracellular matrix (ECM) deposition. Airway inflammation is believed to be the main cause of the development of airway remodeling in asthma. In this chapter, we will review the development of the adaptive immune response and the impact of its mediators and cells on the elements defining airway remodeling in asthma.

Hydrogels to Recapture Extracellular Matrix Cues That Regulate Vascularization.

The ECM (extracellular matrix) is a 3-dimensional network that supports cellular responses and maintains structural tissue integrity in healthy and pathological conditions. The interactions between ECM and cells trigger signaling cascades that lead to phenotypic changes and structural and compositional turnover of the ECM, which in turn regulates vascular cell behavior. Hydrogel biomaterials are a powerful platform for basic and translational studies and clinical applications due to their high swelling capacity and exceptional versatility in compositions and properties. This review highlights recent developments and uses of engineered natural hydrogel platforms that mimic the ECM and present defined biochemical and mechanical cues for vascularization. Specifically, we focus on modulating vascular cell stimulation and cell-ECM/cell-cell interactions in the microvasculature that are the established biomimetic microenvironment.

What are the key mechanical mechanisms governing integrin-mediated cell migration in three-dimensional fiber networks?

Cell migration plays a vital role in numerous processes such as development, wound healing, or cancer. It is well known that numerous complex mechanisms are involved in cell migration. However, so far it remains poorly understood what are the key mechanisms required to produce the main characteristics of this behavior. The reason is a methodological one. In experimental studies, specific factors and mechanisms can be promoted or inhibited. However, while doing so, there can always be others in the background which play key roles but which have simply remained unattended so far. This makes it very difficult to validate any hypothesis about a minimal set of factors and mechanisms required to produce cell migration. To overcome this natural limitation of experimental studies, we developed a computational model where cells and extracellular matrix fibers are represented by discrete mechanical objects on the micrometer scale. In this model, we had exact control of the mechanisms by which cells and matrix fibers interacted with each other. This enabled us to identify the key mechanisms required to produce physiologically realistic cell migration (including advanced phenomena such as durotaxis and a biphasic relation between migration efficiency and matrix stiffness). We found that two main mechanisms are required to this end: a catch-slip bond of individual integrins and cytoskeletal actin-myosin contraction. Notably, more advanced phenomena such as cell polarization or details of mechanosensing were not necessary to qualitatively reproduce the main characteristics of cell migration observed in experiments.

Extracellular matrix composition alters endothelial force transmission.

Extracellular matrix (ECM) composition is important in a host of pathophysiological processes such as angiogenesis, atherosclerosis, and diabetes, and during each of these processes ECM composition has been reported to change over time. However, the impact ECM composition has on the ability of endothelium to respond mechanically is currently unknown. Therefore, in this study, we seeded human umbilical vein endothelial cells (HUVECs) onto soft hydrogels coated with an ECM concentration of 0.1 mg/mL at the following collagen I (Col-I) and fibronectin (FN) ratios: 100% Col-I, 75% Col-I-25% FN, 50% Col-I-50% FN, 25% Col-I-75% FN, and 100% FN. We subsequently measured tractions, intercellular stresses, strain energy, cell morphology, and cell velocity. Our results revealed that tractions and strain energy are maximal at 50% Col-I-50% FN and minimal at 100% Col-I and 100% FN. Intercellular stress response was maximal on 50% Col-I-50% FN and minimal on 25% Col-I-75% FN. Cell area and cell circularity displayed a divergent relationship for different Col-I and FN ratios. We believe that these results will be of great importance to the cardiovascular field, biomedical field, and cell mechanics.NEW & NOTEWORTHY The endothelium constitutes the innermost layer of all blood vessels and plays an important role in vascular physiology and pathology. During certain vascular diseases, the extracellular matrix has been suggested to transition from a collagen-rich matrix to a fibronectin-rich matrix. In this study, we demonstrate the impact various collagen and fibronectin ratios have on endothelial biomechanical and morphological response.

Dynamic Biophysical Cues Near the Tip Cell Microenvironment Provide Distinct Guidance Signals to Angiogenic Neovessels.

The formation of new vascular networks via angiogenesis is a crucial biological mechanism to balance tissue metabolic needs, yet the coordination of factors that influence the guidance of growing neovessels remain unclear. This study investigated the influence of extracellular cues within the immediate environment of sprouting tips over multiple hours and obtained quantitative relationships describing their effects on the growth trajectories of angiogenic neovessels. Three distinct microenvironmental cues-fibril tracks, ECM density, and the presence of nearby cell bodies-were extracted from 3D time series image data. The prominence of each cue was quantified along potential sprout trajectories to predict the response to multiple microenvironmental factors simultaneously. Sprout trajectories significantly correlated with the identified microenvironmental cues. Specifically, ECM density and nearby cellular bodies were the strongest predictors of the trajectories taken by neovessels (p < 0.001 and p = 0.016). Notwithstanding, direction changing trajectories, deviating from the initial neovessel orientation, were significantly correlated with fibril tracks (p = 0.003). Direction changes also occurred more frequently with strong microenvironmental cues. This provides evidence for the first time that local matrix fibril alignment influences changes in sprout trajectories but does not materially contribute to persistent sprouting. Together, our results suggest the microenvironmental cues significantly contribute to guidance of sprouting trajectories. Further, the presented methods quantitatively distinguish the influence of individual microenvironmental stimuli during guidance.

Engineering and Modeling the Lung Mesenchyme.

The structure of the mammalian lung controls the flow of air through the airways and into the distal alveolar region where gas exchange occurs. Specialized cells in the lung mesenchyme produce the extracellular matrix (ECM) and growth factors required for lung structure. Historically, characterizing the mesenchymal cell subtypes was challenging due to their ambiguous morphology, overlapping expression of protein markers, and limited cell-surface molecules needed for isolation. The recent development of single-cell RNA sequencing (scRNA-seq) complemented with genetic mouse models demonstrated that the lung mesenchyme comprises transcriptionally and functionally heterogeneous cell-types. Bioengineering approaches that model tissue structure clarify the function and regulation of mesenchymal cell types. These experimental approaches demonstrate the unique abilities of fibroblasts in mechanosignaling, mechanical force generation, ECM production, and tissue regeneration. This chapter will review the cell biology of the lung mesenchyme and experimental approaches to study their function.

Structure-Function relationships in the skeletal muscle extracellular matrix.

The vast majority of skeletal muscle biomechanical studies have rightly focused on its active contractile properties. However, skeletal muscle passive biomechanical properties have significant clinical impact in aging and disease and are yet incompletely understood. This review focuses on the passive biomechanical properties of the skeletal muscle extracellular matrix (ECM) and suggests aspects of its structural basis. Structural features of the muscle ECM such as perimysial cables, collagen cross-links and endomysial structures have been described, but the way in which these structures combine to create passive biomechanical properties is not completely known. We highlight the presence and organization of perimysial cables. We also demonstrate that the analytical approaches that define passive biomechanical properties are not necessarily straight forward. For example, multiple equations, such as linear, exponential, and polynomial are commonly used to fit raw stress-strain data. Similarly, multiple definitions of zero strain exist that affect muscle biomechanical property calculations. Finally, the appropriate length range over which to measure the mechanical properties is not clear. Overall, this review summarizes our current state of knowledge in these areas and suggests experimental approaches to measuring the structural and functional properties of skeletal muscle.

Synthetic fibrous hydrogels as a platform to decipher cell-matrix mechanical interactions.

Cells continuously sense external forces from their microenvironment, the extracellular matrix (ECM). In turn, they generate contractile forces, which stiffen and remodel this matrix. Although this bidirectional mechanical exchange is crucial for many cell functions, it remains poorly understood. Key challenges are that the majority of available matrices for such studies, either natural or synthetic, are difficult to control or lack biological relevance. Here, we use a synthetic, yet highly biomimetic hydrogel based on polyisocyanide (PIC) polymers to investigate the effects of the fibrous architecture and the nonlinear mechanics on cell-matrix interactions. Live-cell rheology was combined with advanced microscopy-based approaches to understand the mechanisms behind cell-induced matrix stiffening and plastic remodeling. We demonstrate how cell-mediated fiber remodeling and the propagation of fiber displacements are modulated by adjusting the biological and mechanical properties of this material. Moreover, we validate the biological relevance of our results by demonstrating that cellular tractions in PIC gels develop analogously to those in the natural ECM. This study highlights the potential of PIC gels to disentangle complex bidirectional cell-matrix interactions and to improve the design of materials for mechanobiology studies.

Extracellular matrix assembly stress initiates Drosophila central nervous system morphogenesis.

Forces controlling tissue morphogenesis are attributed to cellular-driven activities, and any role for extracellular matrix (ECM) is assumed to be passive. However, all polymer networks, including ECM, can develop autonomous stresses during their assembly. Here, we examine the morphogenetic function of an ECM before reaching homeostatic equilibrium by analyzing de novo ECM assembly during Drosophila ventral nerve cord (VNC) condensation. Asymmetric VNC shortening and a rapid decrease in surface area correlate with the exponential assembly of collagen IV (Col4) surrounding the tissue. Concomitantly, a transient developmentally induced Col4 gradient leads to coherent long-range flow of ECM, which equilibrates the Col4 network. Finite element analysis and perturbation of Col4 network formation through the generation of dominant Col4 mutations that affect assembly reveal that VNC morphodynamics is partially driven by a sudden increase in ECM-driven surface tension. These data suggest that ECM assembly stress and associated network instabilities can actively participate in tissue morphogenesis.

Role of glia and extracellular matrix in controlling neuroplasticity in the central nervous system.

Neuronal plasticity is critical for the maintenance and modulation of brain activity. Emerging evidence indicates that glial cells actively shape neuroplasticity, allowing for highly flexible regulation of synaptic transmission, neuronal excitability, and network synchronization. Astrocytes regulate synaptogenesis, stabilize synaptic connectivity, and preserve the balance between excitation and inhibition in neuronal networks. Microglia, the brain-resident immune cells, continuously monitor and sculpt synapses, allowing for the remodeling of brain circuits. Glia-mediated neuroplasticity is driven by neuronal activity, controlled by a plethora of feedback signaling mechanisms and crucially involves extracellular matrix remodeling in the central nervous system. This review summarizes the key findings considering neurotransmission regulation and metabolic support by astrocyte-neuronal networks, and synaptic remodeling mediated by microglia. Novel data indicate that astrocytes and microglia are pivotal for controlling brain function, indicating the necessity to rethink neurocentric neuroplasticity views.

Introducing aesthetic regenerative scaffolds: An immunological perspective.

BACKGROUND: Skin aging arises from immunological responses to tissue deterioration and damage. Tissue repair processes encompass the regeneration of original tissue and 'scarless' wound healing seen in foetuses, and the extreme fibrotic responses and scarring seen in adults. Anti-aging aesthetic medicine uses interventions like biomaterial-based fillers to influence these immunological responses and renew aged tissue structure and function. At filler injection sites, an inflammatory response occurs that causes a spectrum of outcomes, ranging from tissue regeneration to fibrosis and filler encapsulation. Importantly, the resulting inflammatory pathway can be predetermined by the biomaterial injected. AIMS: By understanding this immunological process, we can develop Aesthetic Regenerative Scaffolds (ARS) - aesthetic injectable biomaterials - to direct inflammatory wound healing away from chronic, fibrotic responses, and towards physiological tissue regeneration. MATERIALS AND METHODS: We identified and reviewed literature on the immunological and cellular responses to injected dermal fillers, whereby the wound healing response to the injection was moderated under the influence of an injected biomaterial. RESULTS: We described the mechanisms of dermal wound healing and the use of ARS to direct healing towards tissue regeneration instead of scarring. We also summarised studies on extracellular matrix remodeling by calcium hydroxylapatite. We found that Calcium hydroxylapatite fillers produce collagen as they gradually degrade and their spherical structures serve as a scaffold for tissue regeneration. Furthermore, CaHA improved fibroblast contractility, collagen type III and elastin production, proliferation and angiogenesis with less inflammation than hyaluronic acid fillers. DISCUSSION: Regneration pathways can be influenced at specific points between a facial filler biomaterial and the wound healingmechanisms at its site of implantaion. CONCLUSION: Physicians can select scaffolds that direct the immune response away from a fibrotic chronic inflammatory pathway and towards regeneration to enable true repair of the aging skin.