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ABSTRACT Aim Materials and Methods Results Conclusion Periodontal disease is driven by oral pathogens, including Porphyromonas gingivalis, and the release of inflammatory cytokines. These cytokines (e.g., TNF) or their receptors (e.g., IL‐1R) are substrates of a disintegrin and metalloproteinases (ADAMs). In this study, we aimed to determine the effects of ADAMs on periodontal disease phenotypes.Western blot and FRET‐based activity measurements of the gingival crevicular fluid (GCF) of patients were compared with those of infected (P. gingivalis) or cytokine‐stimulated oral keratinocytes and primary human neutrophils, respectively. This was accompanied by an analysis of the released extracellular vesicles and MMP9 activity.In the GCF of patients, ADAM8 protein expression and activity were correlated with disease stage, whereas ADAM10 protein expression was inversely correlated with disease stage. Infection and the resulting cytokine release orchestrated the release of soluble ADAM8 by oral keratinocytes and primary neutrophils as soluble ectodomain and on exosomes, respectively. Furthermore, ADAM8 regulated the release of ADAM10 and MMP9.Dysregulation of cell‐associated and extracellular ADAM proteolytic activity may be an essential regulatory element in the progression of periodontal disease driven by ADAM8. The influence of ADAM8 on disease onset and the evaluation of targeting ADAM8 as a potential and novel local treatment option should be addressed in future translational in vivo studies. [ABSTRACT FROM AUTHOR]

Background & Aims: Chronic inflammatory illnesses are debilitating and recurrent conditions associated with significant comorbidities, including an increased risk of developing cancer. Extensive tissue remodeling is a hallmark of such illnesses, and is both a consequence and a mediator of disease progression. Despite previous characterization of epithelial and stromal remodeling during inflammatory bowel disease, a complete understanding of its impact on disease progression is lacking. Methods: A comprehensive proteomic pipeline using data-independent acquisition was applied to decellularized colon samples from the Muc2 knockout (Muc2KO) mouse model of colitis for an in-depth characterization of extracellular matrix remodeling. Unique proteomic profiles of the matrisomal landscape were extracted from prepathologic and overt colitis. Integration of proteomics and transcriptomics data sets extracted from the same murine model produced network maps describing the orchestrating role of matrisomal proteins in tissue remodeling during the progression of colitis. Results: The in-depth proteomic workflow used here allowed the addition of 34 proteins to the known colon matrisomal signature. Protein signatures of prepathologic and pathologic colitic states were extracted, differentiating the 2 states by expression of small leucine-rich proteoglycans. We outlined the role of this class and other matrisomal proteins in tissue remodeling during colitis, as well as the potential for coordinated regulation of cell types by matrisomal ligands. Conclusions: Our work highlights a central role for matrisomal proteins in tissue remodeling during colitis and defines orchestrating nodes that can be exploited in the selection of therapeutic targets.

Post-traumatic osteoarthritis in the temporomandibular joint (TMJ OA) is associated dysfunctional cellmatrix mediated signalling resulting from changes in the pericellular microenvironment after injury. Matrix metalloproteinase (MMP)-13 is a critical enzyme in biomineralisation and the progression of OA that can both degrade the extracellular matrix and modify extracellular receptors. This study focused on MMP-13 mediated changes in a transmembrane proteoglycan, Neuron Glial antigen 2 (NG2/CSPG4). NG2/CSPG4 is a receptor for type VI collagen and a known substrate for MMP-13. In healthy articular layer chondrocytes, NG2/CSPG4 is membrane bound but becomes internalised during TMJ OA. The objective of this study was to determine if MMP-13 contributed to the cleavage and internalisation of NG2/CSPG4 during mechanical loading and OA progression. Using preclinical and clinical samples, it was shown that MMP-13 was present in a spatiotemporally consistent pattern with NG2/CSPG4 internalisation during TMJ OA. In vitro, it was illustrated that inhibiting MMP-13 prevented retention of the NG2/CSPG4 ectodomain in the extracellular matrix. Inhibiting MMP-13 promoted the accumulation of membrane-associated NG2/CSPG4 but did not affect the formation of mechanical-loading dependent variant specific fragments of the ectodomain. MMP- 13 mediated cleavage of NG2/CSPG4 is necessary to initiate clathrin-mediated internalisation of the NG2/ CSPG4 intracellular domain following mechanical loading. This mechanically sensitive MMP-13-NG2/CSPG4 axis affected the expression of key mineralisation and OA genes including bone morphogenetic protein 2, and parathyroid hormone-related protein. Together, these findings implicated MMP-13 mediated cleavage of NG2/CSPG4 in the mechanical homeostasis of mandibular condylar cartilage during the progression of degenerative arthropathies such as OA.

The development of self‐healing conductive hydrogels is critical in electroactive nerve tissue engineering. Typical conductive materials such as polypyrrole (PPy) are commonly used to fabricate artificial nerve conduits. Moreover, the field of tissue engineering has advanced toward the use of products such as hyaluronic acid (HA) hydrogels. Although HA‐modified PPy films are prepared for various biological applications, the cell–matrix interaction mechanisms remain poorly understood; furthermore, there are no reports on HA‐modified PPy‐injectable self‐healing hydrogels for peripheral nerve repair. Therefore, in this study, a self‐healing electroconductive hydrogel (HASPy) from HA, cystamine (Cys), and pyrrole‐1‐propionic acid (Py‐COOH), with injectability, biodegradability, biocompatibility, and nerve‐regenerative capacity is constructed. The hydrogel directly targets interleukin 17 receptor A (IL‐17RA) and promotes the expression of genes and proteins relevant to Schwann cell myelination mainly by activating the interleukin 17 (IL‐17) signaling pathway. The hydrogel is injected directly into the rat sciatic nerve‐crush injury sites to investigate its capacity for nerve regeneration in vivo and is found to promote functional recovery and remyelination. This study may help in understanding the mechanism of cell–matrix interactions and provide new insights into the potential use of HASPy hydrogel as an advanced scaffold for neural regeneration. [ABSTRACT FROM AUTHOR]

Transforming growth factor-β (TGF-β) is a multifunctional cytokine that regulates the expression of ECM-associated genes during early injury. Tissue fibrosis development is driven by synergistic cues between the evolving biochemical and mechanical milieu. Few studies have addressed the role of substrate stiffness on TGF-β activity and extracellular matrix (ECM)-associated genes. We used a commercial formulation of polydimethylsiloxane (PDMS) to fabricate substrates of 40 kPa, 300 kPa, and 1.5 MPa stiffness, and cultured the HMF3S fibroblasts on substrates. We quantified TGF-β protein secreted by HMF3S cells on different substrates using a TGF-β responsive promoter reporter assay. We also tested for variations in gene expression levels on the substrates using RT-PCR and Western blotting and determined the MMP-2 and MMP-9 activities with gelatin zymography. The results showed that TGF-β protein activation was significantly compromised at lower stiffnesses. The expression of integrin α5 decreased on lower stiffness substrates and correlated with inefficient TGF-β protein activation. Collagen I, collagen III, and MMP-2 expression levels were lower on softer substrates; there was little MMP-9 activity on all substrates. Cell and nuclear morphologies were more rounded on compliant substrates, correlating with increased tubulin expression. Proliferations were higher on stiffer substrates, whereas cells on softer substrates showed cell cycle arrest. These results demonstrated critical feedback mechanisms between substrate stiffness and ECM regulation by fibroblasts, relevant in fibrosis. [ABSTRACT FROM AUTHOR]

Teeth consist of 3 mineralized tissues: enamel, dentin, and cementum. Tooth malformation, the most common craniofacial anomaly, arises from complex genetic and environmental factors affecting enamel structure, size, shape, and tooth eruption. Hyaluronic acid (HA), a primary extracellular matrix component, contributes to structural and physiological functions in periodontal tissue. Transmembrane protein 2 (TMEM2), a novel cell surface hyaluronidase, has been shown to play a critical role during embryogenesis. In this study, we demonstrate Tmem2 messenger RNA expression in inner enamel epithelium and presecretory, secretory, and mature ameloblasts. Tmem2 knock-in reporter mice reveal TMEM2 protein localization at the apical and basal ends of secretory ameloblasts. Micro–computed tomography analysis of epithelial-specific Tmem2 conditional knockout (Tmem2 - CKO) mice shows a significant reduction in enamel layer thickness and severe enamel deficiency. Enamel matrix protein expression was remarkably downregulated in Tmem2 - CKO mice. Scanning electron microscopy of enamel from Tmem2 - CKO mice revealed an irregular enamel prism structure, while the microhardness and density of enamel were significantly reduced, indicating impaired ameloblast differentiation and enamel matrix mineralization. Histological evaluation indicated weak adhesion between cells and the basement membrane in Tmem2 - CKO mice. The reduced and irregular expressions of vinculin and integrin β1 suggest that Tmem2 deficiency attenuated focal adhesion formation. In addition, abnormal HA accumulation in the ameloblast layer and weak claudin 1 immunoreactivity in Tmem2 - CKO mice indicate impaired tight junction gate function. Irregular actin filament assembly was also observed at the apical and basal ends of secretory ameloblasts. Last, we demonstrated that Tmem2 -deficient mHAT9d mouse ameloblasts exhibit defective adhesion to HA-containing substrates in vitro. Collectively, our data highlight the importance of TMEM2 in adhesion to HA-rich extracellular matrix, cell-to-cell adhesion, ameloblast differentiation, and enamel matrix mineralization. [ABSTRACT FROM AUTHOR]

Renal fibrosis is a hallmark of end-stage chronic kidney disease. It is characterized by increased accumulation of extracellular matrix (ECM), which disrupts cellular organization and function within the kidney. Here, we review the bi-directional interactions between cells and the ECM that drive renal fibrosis. We will discuss the cells involved in renal fibrosis, changes that occur in the ECM, the interactions between renal cells and the surrounding fibrotic microenvironment, and signal transduction pathways that are misregulated as fibrosis proceeds. Understanding the underlying mechanisms of cell–ECM crosstalk will identify novel targets to better identify and treat renal fibrosis and associated renal disease.

Nonlinear stiffening is a ubiquitous property of major types of biopolymers that make up the extracellular matrices (ECM) including collagen, fibrin, and basement membrane. Within the ECM, many types of cells such as fibroblasts and cancer cells have a spindle-like shape that acts like two equal and opposite force monopoles, which anisotropically stretch their surroundings and locally stiffen the matrix. Here, we first use optical tweezers to study the nonlinear force-displacement response to localized monopole forces. We then propose an effective-probe scaling argument that a local point force application can induce a stiffened region in the matrix, which can be characterized by a nonlinear length scale R* that increases with the increasing force magnitude; the local nonlinear force-displacement response is a result of the nonlinear growth of this effective probe that linearly deforms an increasing portion of the surrounding matrix. Furthermore, we show that this emerging nonlinear length scale R* can be observed around living cells and can be perturbed by varying matrix concentration or inhibiting cell contractility. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Engineered living microtissues such as cellular spheroids and organoids have enormous potential for the study and regeneration of tissues and organs. Microtissues are typically engineered via self‐assembly of adherent cells into cellular spheroids, which are characterized by little to no cell–material interactions. Consequently, 3D microtissue models currently lack structural biomechanical and biochemical control over their internal microenvironment resulting in suboptimal functional performance such as limited stem cell differentiation potential. Here, this work report on stimuli‐responsive cell‐adhesive micromaterials (SCMs) that can self‐assemble with cells into 3D living composite microtissues through integrin binding, even under serum‐free conditions. It is demonstrated that SCMs homogeneously distribute within engineered microtissues and act as biomechanically and biochemically tunable designer materials that can alter the composite tissue microenvironment on demand. Specifically, cell behavior is controlled based on the size, stiffness, number ratio, and biofunctionalization of SCMs in a temporal manner via orthogonal secondary crosslinking strategies. Photo‐based mechanical tuning of SCMs reveals early onset stiffness‐controlled lineage commitment of differentiating stem cell spheroids. In contrast to conventional encapsulation of stem cell spheroids within bulk hydrogel, incorporating cell‐sized SCMs within stem cell spheroids uniquely provides biomechanical cues throughout the composite microtissues' volume, which is demonstrated to be essential for osteogenic differentiation. [ABSTRACT FROM AUTHOR]

Objective: Cancer‐associated fibroblasts (CAFs) are abundantly infiltrated in oral squamous cell carcinoma (OSCC), but the contact‐dependent mechanisms that regulate CAFs phenotype in precursor cells, such as paracancerous fibroblasts (PFs), remain unclear. Here, a fibroblast‐attached organoid (FAO) model was initiated to determine phenotype transition of fibroblasts triggered by contact with OSCC. Material and methods: Organoids and fibroblasts were generated using OSCC and adjacent tissues. Cell‐clusters containing fibroblasts and tumour cells were aggregated to allow for FAOs expansion. Immunoblotting assay was performed to compare expression of Notch intracellular domain (NICD) in CAFs and PFs. Colony formation assay was employed to evaluate morphological activation of fibroblasts. Results: Compared to traditional 3D co‐culture, FAOs better modulated the spatial distribution of fibroblasts with tumour nests. The presence of CAFs with multiple branches was stably observed in FAOs during serial passage. Incorporation with organoids promoted the ability of PFs to form multiple branches. Immunoblotting assay confirmed higher NICD level in CAFs than PFs. Treatment with Notch inhibitor, N‐[N‐(3, 5‐difluorophenacetyl)‐l‐alanyl]‐S‐phenylglycine t‐butyl ester (i.e. DAPT) blocked morphological activation of fibroblasts incorporated into FAO. Conclusion: We developed a robust strategy to study contact‐dependent mechanisms underlying tumour‐stromal interaction, and suggested that Notch activity contributes to biogenesis of OSCC‐associated fibroblasts. [ABSTRACT FROM AUTHOR]

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]

Apical extracellular matrices (aECMs) form a physical barrier to the environment. In Caenorhabditis elegans, the epidermal aECM, the cuticle, is composed mainly of different types of collagen, associated in circumferential ridges separated by furrows. Here, we show that in mutants lacking furrows, the normal intimate connection between the epidermis and the cuticle is lost, specifically at the lateral epidermis, where, in contrast to the dorsal and ventral epidermis, there are no hemidesmosomes. At the ultrastructural level, there is a profound alteration of structures that we term ‘meisosomes,’ in reference to eisosomes in yeast. We show that meisosomes are composed of stacked parallel folds of the epidermal plasma membrane, alternately filled with cuticle. We propose that just as hemidesmosomes connect the dorsal and ventral epidermis, above the muscles, to the cuticle, meisosomes connect the lateral epidermis to it. Moreover, furrow mutants present marked modifications of the biomechanical properties of their skin and exhibit a constitutive damage response in the epidermis. As meisosomes co-localise to macrodomains enriched in phosphatidylinositol (4,5) bisphosphate, they could conceivably act, like eisosomes, as signalling platforms, to relay tensile information from the aECM to the underlying epidermis, as part of an integrated stress response to damage.

The aorta exhibits tremendous changes in geometry, composition, and mechanical properties during postnatal development. These changes are necessarily driven by transcriptional changes, both genetically programmed and mechano-responsive, but there has not been a careful comparison of time-course changes in the transcriptional profile and biomechanical phenotype. Here, we show that the greatest period of differential gene expression in the normal postnatal mouse aorta occurs prior to weaning at three weeks of age though with important evolution of many transcripts thereafter. We identify six general temporal patterns, including transcripts that monotonically decrease to lower or increase to higher steady state values as well as those that either peak or dip prior to or near weaning. We show that diverse transcripts within individual groupings correlate well over time, and that sub-sets of these groups correlate well with the developmental progression of different biomechanical metrics that are expected to be involved in mechano-sensing. In particular, expression of genes for elastin and elastin-associated glycoproteins tend to correlate well with the ratio of systolic-to-diastolic stress whereas genes for collagen fibers correlate well with the daily rate of change of systolic stress and genes for mechano-sensing proteins tend to correlate well with the systolic stress itself. We conclude that different groupings of genes having different temporal expression patterns correlate well with different measures of the wall mechanics, hence emphasizing a need for age-dependent, gene-specific computational modeling of postnatal development., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Damage and repair are recurring processes in tissues, with fibroblasts playing key roles by remodeling extracellular matrices (ECM) through protein synthesis, proteolysis, and cell contractility. Dysregulation of fibroblasts can lead to fibrosis and tissue damage, as seen in idiopathic pulmonary fibrosis (IPF). In advanced IPF, tissue damage manifests as honeycombing, or voids in the lungs. This study explores how transforming growth factor-beta (TGF-β), a crucial factor in IPF, induces lung fibroblast spheroids to create voids in reconstituted collagen through proteolysis and cell contractility, a process we termed as hole formation. These voids reduce when proteases are blocked. Spheroids mimic fibroblast foci observed in IPF. Results indicate that cell contractility mediates tissue opening by stretching fractures in the collagen meshwork. Matrix metalloproteinases (MMPs), including MMP1 and MT1-MMP, are essential for hole formation, with invadopodia playing a significant role. Blocking MMPs reduces hole size and promotes wound healing. This study shows how TGF-β induces excessive tissue destruction and how blocking proteolysis can reverse damage, offering insights into IPF pathology and potential therapeutic interventions., (© 2024 Wiley‐VCH GmbH.)

Cancer invasion through basement membranes represents the initial step of tumor dissemination and metastasis. However, little is known about how human cancer cells breach basement membranes. Here, we used a three-dimensional in vitro invasion model consisting of cancer spheroids encapsulated by a basement membrane and embedded in 3D collagen gels to visualize the early events of cancer invasion by confocal microscopy and live-cell imaging. Human breast cancer cells generated large numbers of basement membrane perforations, or holes, of varying sizes that expanded over time during cell invasion. We used a wide variety of small molecule inhibitors to probe the mechanisms of basement membrane perforation and hole expansion. Protease inhibitor treatment (BB94), led to a 63% decrease in perforation size. After myosin II inhibition (blebbistatin), the basement membrane perforation area decreased by only 15%. These treatments produced correspondingly decreased cellular breaching events. Interestingly, inhibition of actin polymerization dramatically decreased basement membrane perforation by 80% and blocked invasion. Our findings suggest that human cancer cells can primarily use proteolysis and actin polymerization to perforate the BM and to expand perforations for basement membrane breaching with a relatively small contribution from myosin II contractility. [ABSTRACT FROM AUTHOR]

The low-density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional endocytic receptor mediating the clearance of various molecules from the extracellular matrix. LRP1 also regulates cell surface expression of matrix receptors by modulating both extracellular and intracellular signals, though current knowledge of the underlying mechanisms remains partial in the frame of cancer cells interaction with matricellular substrates. In this study we identified that LRP1 downregulates calpain activity and calpain 2 transcriptional expression in an invasive thyroid carcinoma cell model. LRP1-dependent alleviation of calpain activity limits cellmatrix attachment strength and contributes to FTC133 cells invasive abilities in a modified Boyden chamber assays. In addition, using enzymatic assays and co-immunoprecipitation experiments, we demonstrated that LRP1 exerts post-translational inhibition of calpain activity through PKA-dependent phosphorylation of calpain-2. This LRP-1 dual mode of control of calpain activity fine-tunes carcinoma cell spreading. We showed that LRP1-mediated calpain inhibition participates in talin-positive focal adhesions dissolution and limits b1-integrin expression at carcinoma cell surface. In conclusion, we identified an additional and innovative intracellular mechanism which demonstrates LRP-1 pro-motile action in thyroid cancer cells. LRP-1 ability to specifically control calpain-2 expression and activity highlights a novel facet of its de-adhesion receptor status. [ABSTRACT FROM AUTHOR]

Changes in the mechanical homeostasis of the temporomandibular joint (TMJ) can lead to the initiation and progression of degenerative arthropathies such as osteoarthritis (OA). Cells sense and engage with their mechanical microenvironment through interactions with the extracellular matrix. In the mandibular condylar cartilage, the pericellular microenvironment is composed of type VI collagen. NG2/CSPG4 is a transmembrane proteoglycan that binds with type VI collagen and has been implicated in the cell stress response through mechanical loading-sensitive signaling networks including ERK 1/2. The objective of this study is to define the role of NG2/CSPG4 in the initiation and progression of TMJ OA and to determine if NG2/CSPG4 engages ERK 1/2 in a mechanical loading-dependent manner. In vivo, we induced TMJ OA in control and NG2/CSPG4 knockout mice using a surgical destabilization approach. In control mice, NG2/CSPG4 is depleted during the early stages of TMJ OA and NG2/CSPG4 knockout mice have more severe cartilage degeneration, elevated expression of key OA proteases, and suppression of OA matrix synthesis genes. In vitro, we characterized the transcriptome and protein from control and NG2/CSPG4 knockout cells and found significant dysregulation of the ERK 1/2 signaling axis. To characterize the mechanobiological response of NG2/CSPG4, we applied mechanical loads on cell-agarose-collagen scaffolds using a compression bioreactor and illustrated that NG2/CSPG4 knockout cells fail to mechanically activate ERK 1/2 and are associated with changes in the expression of the same key OA biomarkers measured in vivo. Together, these findings implicate NG2/CSPG4 in the mechanical homeostasis of TMJ cartilage and in the progression of degenerative arthropathies including OA.

The low-density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional endocytic receptor mediating the clearance of various molecules from the extracellular matrix. LRP1 also regulates cell surface expression of matrix receptors by modulating both extracellular and intracellular signals, though current knowledge of the underlying mechanisms remains partial in the frame of cancer cells interaction with matricellular substrates. In this study we identified that LRP1 downregulates calpain activity and calpain 2 transcriptional expression in an invasive thyroid carcinoma cell model. LRP1-dependent alleviation of calpain activity limits cell-matrix attachment strength and contributes to FTC133 cells invasive abilities in a modified Boyden chamber assays. In addition, using enzymatic assays and co-immunoprecipitation experiments, we demonstrated that LRP1 exerts post-translational inhibition of calpain activity through PKA-dependent phosphorylation of calpain-2. This LRP-1 dual mode of control of calpain activity fine-tunes carcinoma cell spreading. We showed that LRP1-mediated calpain inhibition participates in talin-positive focal adhesions dissolution and limits β1-integrin expression at carcinoma cell surface. In conclusion, we identified an additional and innovative intracellular mechanism which demonstrates LRP-1 pro-motile action in thyroid cancer cells. LRP-1 ability to specifically control calpain-2 expression and activity highlights a novel facet of its de-adhesion receptor status.

The extracellular matrix (ECM) is a highly dynamic amalgamation of structural and signaling molecules whose quantitative and qualitative modifications drive the distinct programmed morphologic changes required for tissues to mature into their functional forms. The craniofacial complex houses a diverse array of tissues, including sensory organs, glands, and components of the musculoskeletal, neural, and vascular systems, alongside several other highly specialized tissues to form the most complex part of the vertebrate body. Through cell-ECM interactions, the ECM coordinates the cell movements, shape changes, differentiation, gene expression changes, and other behaviors that sculpt developing organs. In this review, we focus on several common key roles of the ECM to shape developing craniofacial organs and tissues. We summarize recent advances in our understanding of the ability of the ECM to biochemically and biomechanically orchestrate major events of craniofacial development, and we discuss how dysregulated ECM dynamics contributes to disease and disorders. As we expand our understanding of organ-specific matrix functionality and composition, we will improve our ability to rationally modify matrices to promote regeneration and/or prevent degenerative outcomes in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Opportunely arranged microscaled fibers offer an attractive 3D architecture for tissue regeneration as they may enhance and stimulate specific tissue regrowth. Among different scaffolding options, encapsulating cells in degradable hydrogel microfibers appears as particularly attractive strategy. Hydrogel patches, in fact, offer a highly hydrated environment, allow easy incorporation of biologically active molecules, and can easily adapt to implantation site. In addition, microfiber architecture is intrinsically porous and can improve mass transport, vascularization, and cell survival after grafting. Anionic polysaccharides, as pectin or the more popular alginate, represent a particularly promising choice for the fabrication of cell‐laden patches, due to their extremely mild gelation in the presence of divalent ions and widely accepted biocompatibility. In this study, to combine the favorable properties of hydrogel and fibrous architecture, a simple coaxial flow wet‐spinning system was used to prepare cell‐laden, 3D fibrous patches using RGD‐modified pectin. Rapid fabrication of coherent self‐standing patches, with diameter in the range of 100–200 μm and high cell density, was possible by accurate choice of pectin and calcium ions concentrations. Cells were homogeneously dispersed throughout the microfibers and remained highly viable for up to 2 weeks, when the initial stage of myotubes formation was observed. Modified‐pectin microfibers appear as promising scaffold to support muscle tissue regeneration, due to their inherent porosity, the favorable cell–material interaction, and the possibility to guide cell alignment toward a functional tissue. [ABSTRACT FROM AUTHOR]

In vitro models of human liver functions are used across a diverse range of applications in preclinical drug development and disease modeling, with particular increasing interest in models that capture facets of liver inflammatory status. This study investigates how the interplay between biophysical and biochemical microenvironment cues influences phenotypic responses, including inflammation signatures, of primary human hepatocytes (PHHs) cultured in a commercially available perfused bioreactor. A 3D printing‐based alginate microwell system is designed to form thousands of hepatic spheroids in a scalable manner as a comparator 3D culture modality to the bioreactor. Soft, synthetic extracellular matrix (ECM) hydrogel scaffolds with biophysical properties mimicking features of liver are engineered to replace polystyrene scaffolds, and the biochemical microenvironment is modulated with a defined set of growth factors and signaling modulators. The supplemented media significantly increases tissue density, albumin secretion, and CYP3A4 activity but also upregulates inflammatory markers. Basal inflammatory markers are lower for cells maintained in ECM hydrogel scaffolds or spheroid formats than polystyrene scaffolds, while hydrogel scaffolds exhibit the most sensitive response to inflammation as assessed by multiplexed cytokine and RNA‐Seq analyses. Together, these engineered 3D liver microenvironments provide insights for probing human liver functions and inflammatory response in vitro.

Viscoelastic characterization of the tissue‐engineered corneal stromal model is important for our understanding of the cell behaviors in the pathophysiologic altered corneal extracellular matrix (ECM). The effects of the interactions between stromal cells and different ECM characteristics on the viscoelastic properties during an 11‐day culture period were explored. Collagen‐based hydrogels seeded with keratocytes were used to replicate human corneal stroma. Keratocytes were seeded at 8 × 103 cells per hydrogel and with collagen concentrations of 3, 5 and 7 mg/ml. Air‐pulse‐based surface acoustic wave optical coherence elastography (SAW‐OCE) was employed to monitor the changes in the hydrogels' dimensions and viscoelasticity over the culture period. The results showed the elastic modulus increased by 111%, 56% and 6%, and viscosity increased by 357%, 210% and 25% in the 3, 5 and 7 mg/ml hydrogels, respectively. To explain the SAW‐OCE results, scanning electron microscope was also performed. The results confirmed the increase in elastic modulus and viscosity of the hydrogels, respectively, arose from increased fiber density and force‐dependent unbinding of bonds between collagen fibers. This study reveals the influence of cell‐matrix interactions on the viscoelastic properties of corneal stromal models and can provide quantitative guidance for mechanobiological investigations which require collagen ECM with tuneable viscoelastic properties. [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]

Natural polymer material-based hydrogels normally show inferior mechanical stability and strength to bear large deformation and cyclic loading, therefore their applications in food, biomedical and tissue engineering fields are greatly limited. In this study, gelatin-based hydrogels with remarkable stability, as well as tunable mechanical properties, were prepared via a facile method known as the Hofmeister effect. The higher concentration of potassium sulfatesolution resulted in more dehydration and molecular chain folding, thus the treated hydrogels showed significantly improved tensile and compressive modulus, and decreased equilibrium swelling ratio, as revealed by scanning electron microscopy (SEM), Fourier transform infraredspectroscopy (FTIR), and mechanical tests, etc. Additionally, the reinforced hydrogels were recoverable and biocompatible to modulate the proliferation behavior of human umbilical vein endothelial cells. In conclusion, this paper provides a facile reference for tuning mechanical properties of gelatin-based hydrogels and cell-hydrogel interactions, which show potential capacity in tissue engineering and biomedical fields. [ABSTRACT FROM AUTHOR]

Bone loss caused by trauma, neoplasia, congenital defects, or periodontal disease is a major cause of disability and human suffering. Skeletal progenitor cell–extracellular matrix interactions are critical for bone regeneration. Discoidin domain receptor 2 (DDR2), an understudied collagen receptor, plays an important role in skeletal development. Ddr2 loss-of-function mutations in humans and mice cause severe craniofacial and skeletal defects, including altered cranial shape, dwarfing, reduced trabecular and cortical bone, alveolar bone/periodontal defects, and altered dentition. However, the role of this collagen receptor in craniofacial regeneration has not been examined. To address this, calvarial subcritical-size defects were generated in wild-type (WT) and Ddr2- deficient mice. The complete bridging seen in WT controls at 4 wk postsurgery was not observed in Ddr2- deficient mice even after 12 wk. Quantitation of defect bone area by micro–computed tomography also revealed a 50% reduction in new bone volume in Ddr2- deficient mice. Ddr2 expression during calvarial bone regeneration was measured using Ddr2-LacZ knock-in mice. Expression was restricted to periosteal surfaces of uninjured calvarial bone and, after injury, was detected in select regions of the defect site by 3 d postsurgery and expanded during the healing process. The impaired bone healing associated with Ddr2 deficiency may be related to reduced osteoprogenitor or osteoblast cell proliferation and differentiation since knockdown/knockout of Ddr2 in a mesenchymal cell line and primary calvarial osteoblast cultures reduced osteoblast differentiation while Ddr2 overexpression was stimulatory. In conclusion, Ddr2 is required for cranial bone regeneration and may be a novel target for therapy. [ABSTRACT FROM AUTHOR]

Syndecans are membrane proteoglycans regulating extracellular matrix assembly, cell adhesion and signaling. Their ectodomains can be shed from the cell surface, and act as paracrine and autocrine effectors or as competitors of full-length syndecans. We report the first biophysical characterization of the recombinant ectodomains of the four human syndecans using biophysical techniques, and show that they behave like flexible random-coil intrinsically disordered proteins, and adopt several conformation ensembles in solution. We have characterized their conformational landscapes using native mass spectrometry (MS) and ion-mobility MS, and demonstrated that the syndecan ectodomains explore the majority of their conformational landscape, from minor compact, globular-like, conformations to extended ones. We also report that the ectodomain of syndecan-4, corresponding to a natural isoform, is able to dimerize via a disulfide bond. We have generated a three-dimensional model of the C-terminus of this dimer, which supports the dimerization via a disulfide bond. Furthermore, we have mapped the NXIP adhesion motif of syndecans and their sequences involved in the formation of ternary complexes with integrins and growth factor receptors on the major conformations of their ectodomains, and shown that these sequences are not accessible in all the conformations, suggesting that only some of them are biologically active. Lastly, although the syndecan ectodomains have a far lower number of amino acid residues than their membrane partners, their intrinsic disorder and flexibility allow them to adopt extended conformations, which have roughly the same size as the cell surface receptors (e.g., integrins and growth factor receptors) they bind to.

In the adult organism, hematopoietic stem and progenitor cells (HSPC) reside in the bone marrow (BM) in specialized hematopoietic stem cell niches of which the extracellular matrix (ECM) is an integral component. Laminins (LM) are a family of heterotrimeric ECM molecules of which mainly family members containing an α4 or α5 chain are expressed in cells from BM niches and involved in HSPC homing and proliferation. Various integrin and non-integrin laminin receptors have been identified and characterized. Among these, the integrins α6β1 and α3β1 were reported to be strongly expressed on human and mouse HSPC. In the present study, we focus on two further specific laminin receptors, namely integrin α7β1 and basal cell adhesion molecule/Lutheran (BCAM/Lu). Using RT-PCR analyses, immunofluorescence staining, immunoblotting and flow cytometry, we show that both are strongly expressed by human lineage-negative CD34 + HSPC. Treatment with function-blocking antibodies against BCAM/Lu neither inhibits the strong adhesive interaction of CD34 + HSPC with LM-511/LM-521 nor the LM-511/LM-521 mediated changes in CD34 + HSPC proliferation, but however, influences the cytokine-induced differentiation of HSPC in colony formation assays. In addition, stromal-derived factor (SDF) 1α-mediated transmigration of CD34 + HSPC through an endothelial cell layer was effectively diminished by BCAM/Lu antibodies, suggesting a direct involvement of BCAM/Lu in the migration process. This study indicates that both laminin receptors newly identified on human CD34 + HSPC should be taken into consideration in future studies.

[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]

Mechanical interactions between cells and the extracellular matrix (ECM) lead to the formation of biophysical cues, notably in the form of cell-generated tension, stiffness, and concentration profiles in the ECM. Fibrillar ECMs have nonlinear stiffnesses, linked to the reorientation of fibers under stress and strain, and nonelastic properties, resulting from the force-induced unbinding of transient bonds (crosslinks) that interconnect fibers. Mechanical forces generated by cells can lead to local ECM stiffening and densification. Cell tension is also propagated through the ECM network. The underlying factors that regulate the relative emergence of these signals are not well understood. Here, through computational simulations of 3D ECM fiber networks, we show that the composition of ECM crosslinks is a key determinant of the degree of densification and stiffening that can be achieved by cell-generated forces. This also regulates the sustainability of tensions propagated through the ECM. In particular, highly transient force-sensitive crosslinks promote nonelastic densification and rapid tension relaxation, whereas permanent crosslinks promote nonlinear stiffening and stable tension profiles. A heterogeneous population of crosslinks with different unbinding kinetics enables ECMs to exhibit accumulation, tension propagation, and stiffening simultaneously in response to mechanical interactions with cells.

Abstract Background Multi-walled carbon nanotubes (MWCNT) have been shown to elicit the release of inflammatory and pro-fibrotic mediators, as well as histopathological changes in lungs of exposed animals. Current standards for testing MWCNTs and other nanoparticles (NPs) rely on low-throughput in vivo studies to assess acute and chronic toxicity and potential hazard to humans. Several alternative testing approaches utilizing two-dimensional (2D) in vitro assays to screen engineered NPs have reported conflicting results between in vitro and in vivo assays. Compared to conventional 2D in vitro or in vivo animal model systems, three-dimensional (3D) in vitro platforms have been shown to more closely recapitulate human physiology, providing a relevant, more efficient strategy for evaluating acute toxicity and chronic outcomes in a tiered nanomaterial toxicity testing paradigm. Results As inhalation is an important route of nanomaterial exposure, human lung fibroblasts and epithelial cells were co-cultured with macrophages to form scaffold-free 3D lung microtissues. Microtissues were exposed to multi-walled carbon nanotubes, M120 carbon black nanoparticles or crocidolite asbestos fibers for 4 or 7 days, then collected for characterization of microtissue viability, tissue morphology, and expression of genes and selected proteins associated with inflammation and extracellular matrix remodeling. Our data demonstrate the utility of 3D microtissues in predicting chronic pulmonary endpoints following exposure to MWCNTs or asbestos fibers. These test nanomaterials were incorporated into 3D human lung microtissues as visualized using light microscopy. Differential expression of genes involved in acute inflammation and extracellular matrix remodeling was detected using PCR arrays and confirmed using qRT-PCR analysis and Luminex assays of selected genes and proteins. Conclusion 3D lung microtissues provide an alternative testing platform for assessing nanomaterial-induced cell-matrix alterations and delineation of toxicity pathways, moving towards a more predictive and physiologically relevant approach for in vitro NP toxicity testing.

Cancer invasion through basement membranes represents the initial step of tumor dissemination and metastasis. However, little is known about how human cancer cells breach basement membranes. Here, we used a three-dimensional in vitro invasion model consisting of cancer spheroids encapsulated by a basement membrane and embedded in 3D collagen gels to visualize the early events of cancer invasion by confocal microscopy and live-cell imaging. Human breast cancer cells generated large numbers of basement membrane perforations, or holes, of varying sizes that expanded over time during cell invasion. We used a wide variety of small molecule inhibitors to probe the mechanisms of basement membrane perforation and hole expansion. Protease inhibitor treatment (BB94), led to a 63% decrease in perforation size. After myosin II inhibition (blebbistatin), the basement membrane perforation area decreased by only 15%. These treatments produced correspondingly decreased cellular breaching events. Interestingly, inhibition of actin polymerization dramatically decreased basement membrane perforation by 80% and blocked invasion. Our findings suggest that human cancer cells can primarily use proteolysis and actin polymerization to perforate the BM and to expand perforations for basement membrane breaching with a relatively small contribution from myosin II contractility.

New strategies for tissue engineering have great potential for restoring and revitalizing impaired tissues and organs, including the use of smart hydrogels that can be modified to enhance organization and functionality of the salivary glands. For instance, monomers of laminin-111 peptides chemically conjugated to fibrin hydrogel (L1pM-FH) promote cell cluster formation in vitro and salivary gland regeneration in vivo when compared with fibrin hydrogel (FH) alone; however, L1pM-FH produce only weak expression of acinar differentiation markers in vivo (e.g., aquaporin-5 and transmembrane protein 16). Since previous studies demonstrated that a greater impact can be achieved when trimeric forms were used as compared with monomeric or dimeric forms, we investigated the extent to which trimers of laminin-111 chemically conjugated to FH (L1pT-FH) can increase the expression of acinar differentiation markers and elevate saliva secretion. In vitro studies using Par-C10 acinar cells demonstrated that when compared with L1pM-FH, L1pT-FH induced similar levels of acinar-like cell clustering, polarization, lumen formation, and calcium signaling. To assess the performance of the trimeric complex in vivo, we compared the ability of L1pM-FH and L1pT-FH to increase acinar differentiation markers and restore saliva flow rate in a salivary gland wound model of C57BL/6 mice. Our results show that L1pT-FH applied to wounded mice significantly improved the expression of the acinar differentiation markers and saliva secretion when compared with the monomeric form. Together, these positive effects of L1pT-FH warrant its future testing in additional models of hyposalivation with the ultimate goal of applying this technology in humans. [ABSTRACT FROM AUTHOR]

Ameloblastin (Ambn) has the potential to regulate cell-matrix adhesion through familiar cell-binding domains, but the proposed sequence motifs are not highly conserved across species. Here, we report that Ambn binds to ameloblast-like cell membranes through a highly evolutionary conserved amphipathic helix-forming (AH) motif encoded by exon 5. We applied high-resolution confocal microscopy to show colocalization of Ambn with ameloblast membrane surfaces in developing mouse incisors. Using a series of Ambn-derived peptides and Ambn variants, we showed that Ambn binds to cell membranes through a motif within the sequence encoded by exon 5. Using peptides derived from the N- or C-termini of this sequence, as well as Ambn variants that lacked or had a disrupted AH motif, we demonstrated that the AH motif located at the N-terminus of the sequence is involved in cell-Ambn adhesion. Sequence analysis revealed that this highly conserved AH motif is absent from other enamel matrix proteins, including amelogenin, enamelin, and amelotin. Collectively, these data suggest that Ambn binds to the cell surface membrane via a helix-forming motif and provide insight into the molecular mechanism and function of Ambn in enamel cell-matrix interaction. [ABSTRACT FROM AUTHOR]

Epithelial tissues mechanically deform the surrounding extracellular matrix during embryonic development, wound repair, and tumor invasion. Ex vivo measurements of such multicellular tractions within three-dimensional (3D) biomaterials could elucidate collective dissemination during disease progression and enable preclinical testing of targeted antimigration therapies. However, past 3D traction measurements have been low throughput due to the challenges of imaging and analyzing information-rich 3D material deformations. Here, we demonstrate a method to profile multicellular clusters in a 96-well-plate format based on spatially heterogeneous contractile, protrusive, and circumferential tractions. As a case study, we profile multicellular clusters across varying states of the epithelial-mesenchymal transition, revealing a successive loss of protrusive and circumferential tractions, as well as the formation of localized contractile tractions with elongated cluster morphologies. These cluster phenotypes were biochemically perturbed by using drugs, biasing toward traction signatures of different epithelial or mesenchymal states. This higher-throughput analysis is promising to systematically interrogate and perturb aberrant mechanobiology, which could be utilized with human-patient samples to guide personalized therapies. [ABSTRACT FROM AUTHOR]

The cornea, as a dynamic and responsive tissue, constantly interacts with mechanical forces in order to maintain its structural integrity, barrier function, transparency and refractive power. Cells within the cornea sense and respond to various mechanical forces that fundamentally regulate their morphology and fate in development, homeostasis and pathophysiology. Corneal cells also dynamically regulate their extracellular matrix (ECM) with ensuing cell-ECM crosstalk as the matrix serves as a dynamic signaling reservoir providing biophysical and biochemical cues to corneal cells. Here we provide an overview of mechanotransduction signaling pathways then delve into the recent advances in corneal mechanobiology, focusing on the interplay between mechanical forces and responses of the corneal epithelial, stromal, and endothelial cells. We also identify species-specific differences in corneal biomechanics and mechanotransduction to facilitate identification of optimal animal models to study corneal wound healing, disease, and novel therapeutic interventions. Finally, we identify key knowledge gaps and therapeutic opportunities in corneal mechanobiology that are pressing for the research community to address especially pertinent within the domains of limbal stem cell deficiency, keratoconus and Fuchs' endothelial corneal dystrophy. By furthering our understanding corneal mechanobiology, we can contextualize discoveries regarding corneal diseases as well as innovative treatments for them. [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]

Zirconia (ZrO2) dental implants provide good biocompatibility, have good corrosion resistance, and have a color that is similar to that of natural teeth. Unfortunately, ZrO2 is a bioinert material and therefore achieves osseointegration difficultly. In this study, we sought to enhance osseointegration by producing rough ZrO2 surfaces that contain hydroxyl groups (designated ZSA) through the use of sandblasting in conjunction with alkaline treatment. We immobilized type I collagen on ZSA surfaces using the natural cross-linker, procyanidin. Our results further showed that surfaces produced in ZSA-P/C featured more and steadier type I collagen than surfaces produced in ZSA-C. The ZSA-P/C also presented superior cell responses in terms of adhesion, proliferation, and mineralization of human bone marrow mesenchymal stem cells. The enhanced cell responses in the ZSA-P/C were induced through the prolonged activation of focal adhesion kinase, AKT (the phosphoinositide 3-kinase pathway), and p38 (the mitogen-activated protein kinase pathway). The simple and novel approach to immobilize type I collagen on roughened ZrO2 surfaces presented in this article can likely benefit dental implant applications. [ABSTRACT FROM AUTHOR]

The fibrous network of an extracellular matrix (ECM) possesses mechanical properties that convey critical biological functions in cell mechanotransduction. Engineered fibrous hydrogels show promise in emulating key aspects of ECM structure and functions. However, varying hydrogel mechanics without changing its architecture remains a challenge. A composite fibrous hydrogel is developed to vary gel stiffness without affecting its structure by controlling intrafibrillar crosslinking. The hydrogel is formed from aldehyde-modified cellulose nanocrystals and gelatin methacryloyl that provide the capability of intrafibrillar photocrosslinking. By varying the degree of gelatin functionalization with methacryloyl groups and/or photoirradiation time, the hydrogel's elastic modulus is changed by more than an order of magnitude, while preserving the same fiber diameter and pore size. The hydrogel is used to seed primary mouse lung fibroblasts and test the role of ECM stiffness on fibroblast contraction and activation. Increasing hydrogel stiffness by stronger intrafibrillar crosslinking results in enhanced fibroblast activation and increased fibroblast contraction force, yet at a reduced contraction speed. The developed approach enables the fabrication of biomimetic hydrogels with decoupled structural and mechanical properties, facilitating studies of ECM mechanics on tissue development and disease progression., (© 2023 Wiley-VCH GmbH.)

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]

Temporomandibular joint (TMJ) osteoarthritis (TMJOA) disrupts extracellular matrix (ECM) homeostasis, leading to cartilage degradation. Upregulated a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-5 leads to cleavage of its substrate aggrecan (Acan) and is considered a hallmark of TMJOA. However, most research on ADAMTS5-Acan turnover has focused on hyaline cartilage, not fibrocartilage, which comprises the TMJ. The mandibular condylar cartilage (MCC) of the TMJ is organized in zones, and chondrocytes are arranged in axial rows, yet the molecular mechanisms required to generate the MCC zonal architecture have not been elucidated. Here, we test the hypothesis that ADAMTS5 is required for development of the TMJ MCC. Adamts5+/+ and Adamts5-/- murine TMJs were harvested at postnatal day 7 (P7), P21, 2 mo, and 6 mo of age; histomorphometrics indicated increased ECM. Immunohistochemistry and Western blots demonstrated the expanded ECM correlated with increased Acan localization in Adamts5-/- compared to Adamts5+/+. Cell volume was also decreased in the MCC of Adamts5-/- due to both a reduction in cell size and less mature hypertrophic chondrocytes. Analysis of chondrogenic maturation markers by quantitative real-time polymerase chain reaction indicated Col2a1, Col10a1, and Sox9 were significantly reduced in Adamts5-/- MCC compared to that of Adamts5+/+. The older (6 mo) Adamts5-/- MCC exhibited changes in chondrogenic cell arrangements, including clustering and chondrogenic atrophy, that correlated with early stages of TMJOA using modified Mankin scoring. These data indicate a potentially novel and critical role of ADAMTS5 for maturation of hypertrophic chondrocytes and establishment of the zonal architecture that, when disrupted, may lead to early onset of TMJOA. [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]

Dimensionality can have a profound impact on stiffness-mediated differentiation of mesenchymal stem cells (MSCs). However, while we have begun to understand cellular response when encapsulated within 3D substrates, the behavior of cells within macro-porous substrates is relatively underexplored. The goal of this study was to determine the influence of macro-porous topographies on stiffness-mediated differentiation of MSCs. We developed macro-porous recombinant elastin-like protein (ELP) substrates that allow independent control of mechanical properties and ligand chemistry. We then used computational modeling to probe the impact of pore topography on the mechanical stimulus that cells are exposed to within these substrates, and finally we investigated stiffness induced biases towards adipogenic and osteogenic differentiation of MSCs within macro-porous substrates. Computational modeling revealed that there is significant heterogeneity in the mechanical stimuli that cells are exposed to within porous substrates and that this heterogeneity is predominantly due to the wide range of possible cellular orientations within the pores. Surprisingly, MSCs grown within 3D porous substrates respond to increasing substrate stiffness by up-regulating both osteogenesis and adipogenesis. These results demonstrate that within porous substrates the behavior of MSCs diverges from previously observed responses to substrate stiffness, emphasizing the importance of topography as a determinant of cellular behavior. [ABSTRACT FROM AUTHOR]

Animal cells in tissues are supported by biopolymer matrices, which typically exhibit highly nonlinear mechanical properties. While the linear elasticity of the matrix can significantly impact cell mechanics and functionality, it remains largely unknown how cells, in turn, affect the nonlinear mechanics of their surrounding matrix. Here, we show that living contractile cells are able to generate a massive stiffness gradient in three distinct 3D extracellular matrix model systems: collagen, fibrin, and Matrigel. We decipher this remarkable behavior by introducing nonlinear stress inference microscopy (NSIM), a technique to infer stress fields in a 3D matrix from nonlinear microrheology measurements with optical tweezers. Using NSIM and simulations, we reveal large long-ranged cell-generated stresses capable of buckling filaments in the matrix. These stresses give rise to the large spatial extent of the observed cell-induced matrix stiffness gradient, which can provide a mechanism for mechanical communication between cells. [ABSTRACT FROM AUTHOR]

Temporomandibular joint (TMJ) disorders are often associated with development of osteoarthritis-like changes in the mandibular condyle. Discoidin domain receptor 2 (DDR2), a collagen receptor preferentially activated by type I and III collagen found in the TMJ and other fibrocartilages, has been associated with TMJ degeneration, but its role in normal joint development has not been previously examined. Using Ddr2 LacZ-tagged mice and immunohistochemistry, we found that DDR2 is preferentially expressed and activated in the articular zone of TMJs but not knee joints. To assess the requirement for Ddr2 in TMJ development, studies were undertaken to compare wild-type and smallie ( slie) mice, which contain a spontaneous deletion in Ddr2 to produce an effective null allele. Analysis of TMJs from newborn Ddr2slie/slie mice revealed a developmental delay in condyle mineralization, as measured by micro-computed tomography and histologic analysis. In marked contrast, knee joints of Ddr2slie/slie mice were normal. Analysis of older Ddr2slie/slie mice (3 and 10 mo) revealed that the early developmental delay led to a dramatic and progressive loss of TMJ articular integrity and osteoarthritis-like changes. Mutant condyles had a rough and flattened bone surface, accompanied by a dramatic loss of bone mineral density. Mankin scores showed significantly greater degenerative changes in the TMJs of 3- and 10-mo-old Ddr2slie/slie mice as compared with wild-type controls. No DDR2-dependent degenerative changes were seen in knees. Analysis of primary cultures of TMJ articular chondrocytes from wild-type and Ddr2slie/slie mice showed defects in chondrocyte maturation and mineralization in the absence of Ddr2. These studies demonstrate that DDR2 is necessary for normal TMJ condyle development and homeostasis and that these DDR2 functions are restricted to TMJ fibrocartilage and not seen in the hyaline cartilage of the knee. [ABSTRACT FROM AUTHOR]

Intestinal epithelial cell differentiation is a highly controlled and orderly process occurring in the crypt so that cells migrating out to cover the villi are already fully functional. Absorptive cell precursors, which originate from the stem cell population located in the lower third of the crypt, are subject to several cycles of amplification in the transit amplifying (TA) zone, before reaching the terminal differentiation compartment located in the upper third. There is a large body of evidence that absorptive cell differentiation is halted in the TA zone through various epigenetic, transcriptional and intracellular signalling events or mechanisms allowing the transient expansion of this cell population but how these mechanisms are themself regulated remains obscure. One clue can be found in the epithelial cell-matrix microenvironment located all along the crypt-villus axis. Indeed, a previous study from our group revealed that α5-subunit containing laminins such as lamimin-511 and 512 inhibit early stages of differentiation in Caco-2/15 cells. Among potential receptors for laminin 511/512 is the integrin α7β1, which has previously been reported to be expressed in the human intestinal crypts and in early stages of Caco-2/15 cell differentiation. In this study, the effects of knocking down ITGA7 in Caco-2/15 cells were studied using shRNA and CRISPR/Cas9 strategies. Abolition of the α7 integrin subunit resulted in a significant increase in the level of differentiation and polarization markers as well as the morphological features of intestinal cells. Activities of focal adhesion kinase and Src kinase were both reduced in α7-knockdown cells while the three major intestinal pro-differentiation factors CDX2, HNFα1 and HNF4α were overexpressed. Two epigenetic events associated with intestinal differentiation, the reduction of tri-methylated lysine 27 on histone H3 and the increase of acetylation of histone H4 were also observed in α7-knockdown cells. On the other hand, the ablation of α7 had no effect on cell proliferation. In conclusion, these data indicate that integrin α7β1 acts as a major repressor of absorptive cell terminal differentiation in the Caco-2/15 cell model and suggest that the laminin-α7β1 integrin interaction occurring in the transit amplifying zone of the adult intestine is involved in the transient halting of absorptive cell terminal differentiation. [Display omitted] • Integrin α7Bβ1 is detected in the transit amplification zone of the intestinal crypt. • α7Bβ1 acts as a major repressor of differentiation in the Caco-2/15 cell model. • ITGA7 abolition reduces focal adhesion kinase and Src phosphorylation. • ITGA7 abolition increases expression of CDX2, HNFα1 and HNF4α. • ITGA7 knockout has no effect on cell proliferation. [ABSTRACT FROM AUTHOR]