Physical Properties Of Extracellular Matrix Research Articles

Biomimetic Fibers Based on Equidistant Micropillar Arrays Determines Chondrocyte Fate via Mechanoadaptability.

It is recognized that the changes in the physical properties of extracellular matrix (ECM) result in fine-tuned cell responses including cell morphology, proliferation and differentiation. In this study, a novel patterned equidistant micropillar substrate based on polydimethylsiloxane (PDMS) is designed to mimic the collagen fiber-like network of the cartilage matrix. By changing the component of the curing agent to an oligomeric base, micropillar substrates with the same topology but different stiffnesses are obtained and it is found that chondrocytes seeded onto the soft micropillar substrate maintain their phenotype by gathering type II collagen and aggrecan more effectively than those seeded onto the stiff micropillar substrate. Moreover, chondrocytes sense and respond to micropillar substrates with different stiffnesses by altering the ECM-cytoskeleton-focal adhesion axis. Further, it is found that the soft substrate-preserved chondrocyte phenotype is dependent on the activation of Wnt/β-catenin signaling. Finally, it is indicated that the changes in osteoid-like region formation and cartilage phenotype loss in the stiffened sclerotic area of osteoarthritis cartilage to validate the changes triggered by micropillar substrates with different stiffnesses. This study provides the cell behavior changes that are more similar to those of real chondrocytes at tissue level during the transition from a normal state to a state of osteoarthritis.

Extracellular Matrix-Derived Biophysical Cues Mediate Interstitial Flow-Induced Sprouting Angiogenesis.

Sprouting angiogenesis is orchestrated by an intricate balance of biochemical and mechanical cues in the local tissue microenvironment. Interstitial flow has been established as a potent regulator of angiogenesis. Similarly, extracellular matrix (ECM) physical properties, such as stiffness and microarchitecture, have also emerged as important mediators of angiogenesis. However, the interplay between interstitial flow and ECM physical properties in the initiation and control of angiogenesis is poorly understood. Using a three-dimensional (3D) microfluidic tissue analogue of angiogenic sprouting with defined interstitial flow superimposed over ECM with well-characterized physical properties, we found that the addition of hyaluronan (HA) to collagen-based matrices significantly enhances sprouting induced by interstitial flow compared to responses in collagen-only hydrogels. We confirmed that both the stiffness and matrix pore size of collagen-only hydrogels were increased by the addition of HA. Interestingly, interstitial flow-potentiated sprouting responses in collagen/HA matrices were not affected when functionally blocking the HA receptor CD44. In contrast, enzymatic depletion of HA in collagen/HA matrices with hyaluronidase (HAdase) resulted in decreased stiffness, pore size, and interstitial flow-mediated sprouting to the levels observed in collagen-only matrices. Taken together, these results suggest that HA enhances interstitial flow-mediated angiogenic sprouting through its alterations to collagen ECM stiffness and pore size.

Extracellular matrix physical properties govern the diffusion of nanoparticles in tumor microenvironment

Nanoparticles (NPs) are confronted with limited and disappointing delivery efficiency in tumors clinically. The tumor extracellular matrix (ECM), whose physical traits have recently been recognized as new hallmarks of cancer, forms a main steric obstacle for NP diffusion, yet the role of tumor ECM physical traits in NP diffusion remains largely unexplored. Here, we characterized the physical properties of clinical gastric tumor samples and observed limited distribution of NPs in decellularized tumor tissues. We also performed molecular dynamics simulations and invitro hydrogel experiments through single-particle tracking to investigate the diffusion mechanism of NPs and understand the influence of tumor ECM physical properties on NP diffusion both individually and collectively. Furthermore, we developed an estimation matrix model with evaluation scores of NP diffusion efficiency through comprehensive analyses of the data. Thus, beyond finding that loose and soft ECM with aligned structure contribute to efficient diffusion, we now have a systemic model to predict NP diffusion efficiency based on ECM physical traits and provide critical guidance for personalized tumor diagnosis and treatment.

Morphodynamics facilitate cancer cells to navigate 3D extracellular matrix

Cell shape is linked to cell function. The significance of cell morphodynamics, namely the temporal fluctuation of cell shape, is much less understood. Here we study the morphodynamics of MDA-MB-231 cells in type I collagen extracellular matrix (ECM). We systematically vary ECM physical properties by tuning collagen concentrations, alignment, and gelation temperatures. We find that morphodynamics of 3D migrating cells are externally controlled by ECM mechanics and internally modulated by Rho/ROCK-signaling. We employ machine learning to classify cell shape into four different morphological phenotypes, each corresponding to a distinct migration mode. As a result, we map cell morphodynamics at mesoscale into the temporal evolution of morphological phenotypes. We characterize the mesoscale dynamics including occurrence probability, dwell time and transition matrix at varying ECM conditions, which demonstrate the complex phenotype landscape and optimal pathways for phenotype transitions. In light of the mesoscale dynamics, we show that 3D cancer cell motility is a hidden Markov process whereby the step size distributions of cell migration are coupled with simultaneous cell morphodynamics. Morphological phenotype transitions also facilitate cancer cells to navigate non-uniform ECM such as traversing the interface between matrices of two distinct microstructures. In conclusion, we demonstrate that 3D migrating cancer cells exhibit rich morphodynamics that is controlled by ECM mechanics, Rho/ROCK-signaling, and regulate cell motility. Our results pave the way to the functional understanding and mechanical programming of cell morphodynamics as a route to predict and control 3D cell motility.

Extracellular Matrix Mechanical Properties and Regulation of the Intestinal Stem Cells: When Mechanics Control Fate.

Intestinal stem cells (ISC) are crucial players in colon epithelium physiology. The accurate control of their auto-renewal, proliferation and differentiation capacities provides a constant flow of regeneration, maintaining the epithelial intestinal barrier integrity. Under stress conditions, colon epithelium homeostasis in disrupted, evolving towards pathologies such as inflammatory bowel diseases or colorectal cancer. A specific environment, namely the ISC niche constituted by the surrounding mesenchymal stem cells, the factors they secrete and the extracellular matrix (ECM), tightly controls ISC homeostasis. Colon ECM exerts physical constraint on the enclosed stem cells through peculiar topography, stiffness and deformability. However, little is known on the molecular and cellular events involved in ECM regulation of the ISC phenotype and fate. To address this question, combining accurately reproduced colon ECM mechanical parameters to primary ISC cultures such as organoids is an appropriated approach. Here, we review colon ECM physical properties at physiological and pathological states and their bioengineered in vitro reproduction applications to ISC studies.

The role of extracellular matrix in normal and pathological pregnancy: Future applications of microphysiological systems in reproductive medicine.

Extracellular matrix in the womb regulates the initiation, progression, and completion of a healthy pregnancy. The composition and physical properties of extracellular matrix in the uterus and at the maternal-fetal interface are remodeled at each gestational stage, while maladaptive matrix remodeling results in obstetric disease. As in vitro models of uterine and placental tissues, including micro-and milli-scale versions of these organs on chips, are developed to overcome the inherent limitations of studying human development in vivo, we can isolate the influence of cellular and extracellular components in healthy and pathological pregnancies. By understanding and recreating key aspects of the extracellular microenvironment at the maternal-fetal interface, we can engineer microphysiological systems to improve assisted reproduction, obstetric disease treatment, and prenatal drug safety.

Stiffness and topography of biomaterials dictate cell-matrix interaction in musculoskeletal cells at the bio-interface: A concise progress review.

Mutually interacted musculoskeletal tissues work together within the physiological environment full of varieties of external stimulus. Consistent with the locomotive function of the tissues, musculoskeletal cells are remarkably mechanosensitive to the physical cues. Signals like extracellular matrix (ECM) stiffness, topography, and geometry can be sensed and transduced into intracellular signaling cascades to trigger a series of cell responses, including cell adhesion, cell phenotype maintenance, cytoskeletal reconstruction, and stem cell differentiation (Du et al., 2011; Murphy et al., 2014; Lv et al., 2015; Kim et al., 2016; Kumar et al., 2017). With the development of tissue engineering and regenerative medicine, the potent effects of ECM physical properties on cell behaviors at the cell-matrix interface are drawing much attention. To mimic the interaction between cell and its ECM physical properties, developing advanced biomaterials with desired characteristics which could achieve the biointerface between cells and the surrounded matrix close to the physiological conditions becomes a great hotspot. In this review, based on the current publications in the field of biointerfaces, we systematically summarized the significant roles of stiffness and topography on musculoskeletal cell behaviors. We hope to shed light on the importance of physical cues in musculoskeletal tissue engineering and provide up to date strategies towards the natural or artificial replication of physiological microenvironment.

The microenvironment and cytoskeletal remodeling in tumor cell invasion.

The physical cues of tumor microenvironment (TME) contribute greatly to the initiation and progression of cancer. Tumor tissues usually become stiffer than healthy tissues with more aligned fibers and changed porosity. In recent years, numerous studies attempted to investigate whether biophysical cues from the surrounding environment affect the biophysical, biochemical, and biological behavior of cells and consequently attribute to the development of cancer. Here, we review recent advances of our understanding of these physical cues in terms of extracellular matrix (ECM) stiffness and topography (alignment and porosity). We discuss the underlying mechanisms of changes in TME physical parameters. Then, we summarize how cancer cells sense the mechanical signals, transfer them to the downstream signaling pathways, and finally translate them to different cellular behaviors. Specifically, we discuss the role of mechanical changes of ECM in cancer cell stiffness, actin cytoskeleton organization, gene and protein expressions, the migration of cancer cells, and their response to specific treatments. We then review different methods which have been successfully utilized to model ECM physical properties. This review paper concluded with the limitations of current studies which followed by some insights into clarifying the therapeutic potential of ECM mechanical properties to target and control the development of cancer.

Extent of Cell Confinement in Microtracks Affects Speed and Results in Differential Matrix Strains

During metastasis, cancer cells navigate through a spatially heterogeneous extracellular matrix (ECM). Physical properties of ECM, including the degree of confinement, influence cell migration behavior. Here, utilizing in vitro three-dimensional collagen microtracks, we demonstrate that cell-ECM interactions, specifically the degree of spatial confinement, regulate migratory behavior. We found that cells migrate faster when they are fully confined, contacting all four walls (top, bottom, and two sides) of a collagen microtrack, compared with cells that are partially confined, contacting less than four walls. When fully confined, cells exhibit fewer but larger vinculin-containing adhesions and create greater strains in the surrounding matrix directed toward the cell body. In contrast, partially confined cells develop a more elongated morphology with smaller but significantly more vinculin-containing adhesions and displace the surrounding matrix less than fully confined cells. The resulting effect of increasing cell contractility via Rho activation is dependent on the number of walls with which the cell is in contact. Although matrix strains increase in both fully and partially confined cells, cells that are partially confined increase speed, whereas those in full confinement decrease speed. Together, these results suggest that the degree of cell-ECM contact during confined migration is a key determinant of speed, morphology, and cell-generated substrate strains during motility, and these factors may work in tandem to facilitate metastatic cell migration.

Preparation and Characterization of Human Adipose Tissue-Derived Extracellular Matrix, Growth Factors, and Stem Cells: A Concise Review.

Human adipose tissue is routinely discarded as medical waste. However, this tissue may have valuable clinical applications since methods have been devised to effectively isolate adipose-derived extracellular matrix (ECM), growth factors (GFs), and stem cells. In this review, we analyze the literature that devised these methods and then suggest an optimal method based on their characterization results. Methods that we analyze in this article include: extraction of adipose tissue, decellularization, confirmation of decellularization, identification of residual active ingredients (ECM, GFs, and cells), removal of immunogens, and comparing structural/physiological/biochemical characteristics of active ingredients. Human adipose ECMs are composed of collagen type I-VII, laminin, fibronectin, elastin, and glycosaminoglycan (GAG). GFs immobilized in GAG include basic fibroblast growth factor (bFGF), transforming growth factor beta 1(TGF-b1), insulin like growth factor 1 (IGF-1), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), BMP4 (bone morphogenetic protein 4), nerve growth factor (NGF), hepatocyte growth factor (HGF), and epithermal growth factor (EGF). Stem cells in the stromal-vascular fraction display mesenchymal markers, self-renewal gene expression, and multi-differentiation potential. Depending on the preparation method, the volume, biological activity, and physical properties of ECM, GFs, and adipose tissue-derived cells can vary. Thus, the optimal preparation method is dependent on the intended application of the adipose tissue-derived products.

Regulating Mechanotransduction in Three Dimensions using Sub‐Cellular Scale, Crosslinkable Fibers of Controlled Diameter, Stiffness, and Alignment

AbstractThe extracellular matrix (ECM) is a complex 3D framework of macromolecules, which regulate cell bioactivity via chemical and physical properties. The ECM's physical properties, including stiffness and physical constraints to cell shape, regulate actomyosin cytoskeleton contractions, which induce signaling cascades influencing gene expression and cell fate. Engineering such bioactivity, a.k.a., mechanotransduction, has been mainly achieved by 2D platforms such as micropatterns. These platforms cause cytoskeletal contractions with apico‐basal polarity and can induce mechanotransduction that is unnatural to most cells in native ECMs. An effective method to engineer mechanotransduction in 3D is needed. This work creates FiberGel, a 3D artificial ECM comprised of sub‐cellular scale fibers. These microfibers can crosslink into defined microstructures with the fibers' diameter, stiffness, and alignment independently tuned. Most importantly, cells are blended amongst the fibers prior to crosslinking, leading to homogeneously cellularized scaffolds. Studies using mesenchymal stem cells showed that the microfibers' diameter, stiffness, and alignment regulate 3D cell shape and the nuclei translocation of transcriptional coactivators YAP/TAZ (yes‐associated protein/transcriptional coactivator), which enables the control of cell differentiation and tissue formation. A novel technology based on repeated stretching and folding is created to synthesize FiberGel. This 3D platform can significantly contribute to mechanotransduction research and applications.

Impact of Hydrogel Elasticity and Adherence on Osteosarcoma Cells and Osteoblasts

Biochemical and physical properties of extracellular matrix (ECM) control cell behaviors, but how they affect osteosarcoma cells that do not require attachment and their normal counterparts (osteoblasts) that are anchorage-dependent has not been reported yet. In this study, the effects of matrix elasticity and adherence on osteosarcoma MG63 cells are investigated using four types of scaffolds (collagen type I, matrigel, alginate, and agarose) with varied adhesion ligands and rigidity, as compared with osteoblast hFOB1.19 cells. MG63 cells on 2D films are sensitive to ECM adherence, similar to the situation of hFOB1.19 cultured in both 2D and 3D. However, osteosarcoma cells in 3D hydrogels are sensitive to ECM elasticity rather than adherence, with tumor proliferation and malignancy varied with matrix rigidity. The results indicate that osteosarcomas cells might adopt unnatural characteristics on flat surfaces. But in 3D culture, they recover their normal state independent of adherence, as regulated mainly by ECM elasticity via the integrin-mediated focal adhesion pathway, which is further confirmed by in vivo studies. In contrast, osteoblasts and 2D cultured osteosarcoma cells are predominantly influenced by ECM bioactivity regulated by integrin-mediated adherens junction pathway. This study might provide new insights into rational design of scaffolds for tumor/tissue engineering.

The effects of liquid crystal-based composite substrates on cell functional responses of human umbilical cord-derived mesenchymal stem cells by mechano-regulatory process.

Physical properties of extracellular matrix, including elasticity and microstructure, have been considered as important factors inducing stem cell differentiation. This study developed a novel type of liquid crystal-based matrix by combining the elastic property of polyurethane with viscoelastic liquid crystal to generate a soft elastic response resembling physical microenvironment of stem cell niche, and explored the mechano-driving cell behaviors. Addition of varying liquid crystal concentration (10 wt%, 30 wt% and 50 wt%) had great effects on surface morphology and elastic modulus of liquid crystal/ polyurethane composite substrates. Changes in microstructure and elastic modulus of the substrates could cause intense cell responses that influenced cell properties, including proliferation, adhesion, and osteogenic differentiation. Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) cultured on both liquid crystal-10/polyurethane and liquid crystal-30/polyurethane substrates exhibited higher viability, more actin filament, and larger spreading area while liquid crystal-50/polyurethane substrates seemed not to favor cell attachment and spreading. Alkaline phosphatase activity and calcium deposition were significantly improved with hUC-MSCs on both liquid crystal-10/ polyurethane and liquid crystal-30/ polyurethane substrates, and the maximal alkaline phosphatase activity was observed in liquid crystal-10/ polyurethane while the lowest in liquid crystal-50/ polyurethane. Osteopontin was upregulated to a high level in both liquid crystal-10/ polyurethane and liquid crystal-30/ polyurethane groups after 14 days culturing; the maximal expression of osteocalcin and related transcription factor 2 were found in liquid crystal-10/ polyurethane group on day 21. Our findings revealed that hUC-MSCs could intensely sense the bioactive patterns and soft-matter feature of liquid crystal domains and subsequently modulated cell behaviors, which may prove useful in the development of new class of biomaterials for applications in tissue engineering and regenerative medicine.

Matrix and Soluble Factor Pathways to Lineage Specification

Integration of soluble factors and physical properties of extracellular matrix is likely key to stem cell differentiation but the extent to which those pathways overlap remains unclear. Here motivated by the micromechanics of osteogenic niche we looked at the synergy between matrix stimuli and pharmacological perturbation of Retinoic Acid (RA) pathway on primary and iPSC-derived mesenchymal stem cells (MSCs) including iPSC- derived cells from progeria patients towards osteogenesis. Retinoic acid receptor RARG transcription factor is known to regulate nucleoskeletal protein Lamin-A. A cell-by-cell analysis showed that rigid matrix favor higher LMNA and correlates with increased nuclear-to-cytoplasmic ratio of RARG. We found that the Progerin allele of lamin-A is similarly regulated by specific RARG agonist/antagonists. A mechanochemical gene circuit in which tension on lamin-A ultimately favors RARG activity describes well the experimentally observed trend. Scatter-plots of single-cell analyses also show that some cells on stiff substrates fall within the response envelopes of cells cultured on soft substrates and that shared sub- population of non-responding cells we also found that don’t respond to RA agonist or antagonist regulation of Lamin-A. RA antagonist drove lamin-A dependent upregulation of osteogenic markers on rigid substrates and pretreated xenografts showed bone-level calcification suggesting a synergistic effect of soluble and insoluble factors on subpopulation of stem cells that are highly mechanoresponsive.

Preparation of Nanofibers with Renewable Polymers and Their Application in Wound Dressing

Renewable polymers have attracted considerable attentions in the last two decades, predominantly due to their environmentally friendly properties, renewability, good biocompatibility, biodegradability, bioactivity, and modifiability. The nanofibers prepared from the renewable polymers can combine the excellent properties of the renewable polymer and nanofiber, such as high specific surface area, high porosity, excellent performances in cell adhesion, migration, proliferation, differentiation, and the analogous physical properties of extracellular matrix. They have been widely used in the fields of wound dressing to promote the wound healing, hemostasis, skin regeneration, and treatment of diabetic ulcers. In the present review, the different methods to prepare the nanofibers from the renewable polymers were introduced. Then the recent progress on preparation and properties of the nanofibers from different renewable polymers or their composites were reviewed; the application of them in the fields of wound dressing was emphasized.

Microfluidic Assay To Study the Combinatorial Impact of Substrate Properties on Mesenchymal Stem Cell Migration.

As an alternative to complex and costly in vivo models, microfluidic in vitro models are being widely used to study various physiological phenomena. It is of particular interest to study cell migration in a controlled microenvironment because of its vital role in a large number of physiological processes, such as wound healing, disease progression, and tissue regeneration. Cell migration has been shown to be affected by variations in the biochemical and physical properties of the extracellular matrix (ECM). To study the combinatorial impact of the ECM physical properties on cell migration, we have developed a microfluidic assay to induce migration of human bone marrow derived mesenchymal stem cells (hBMSCs) on polydimethylsiloxane (PDMS) substrates with varying combinatorial properties (hydrophobicity, stiffness, and roughness). The results show that although the initial cell adhesion and viability appear similar on all PDMS samples, the cell spreading and migration are enhanced on PDMS samples exhibiting intermediate levels of hydrophobicity, stiffness, and roughness. This study suggests that there is a particular range of substrate properties for optimal cell spreading and migration. The influence of substrate properties on hBMSC migration can help understand the physical cues that affect cell migration, which may facilitate the development of optimized engineered scaffolds with desired properties for tissue regeneration applications.

A Biomimetic Core-Shell Platform for Miniaturized 3D Cell and Tissue Engineering.

This article describes a biomimetic core-shell platform with a collagen-based core and an alginate hydrogel shell for cell and tissue culture. With this system, chemical and physical properties of extracellular matrix (ECM) in the core microenvironment can be controlled to regulate proliferation and development of cells/tissues under miniaturized three-dimensional (3D) culture.

Union is strength: matrix elasticity and microenvironmental factors codetermine stem cell differentiation fate

Stem cells are an attractive cellular source for regenerative medicine and tissue engineering applications due to their multipotency. Although the elasticity of the extracellular matrix (ECM) has been shown to have crucial impacts in directing stem cell differentiation, it is not the only contributing factor. Many researchers have recently attempted to design microenvironments that mimic the stem cell niche with combinations of ECM elasticity and other cues, such as ECM physical properties, soluble biochemical factors and cell-cell interactions, thereby driving cells towards their preferred lineages. Here, we briefly discuss the effect of matrix elasticity on stem cell lineage specification and then summarize recent advances in the study of the combined effects of ECM elasticity and other cues on the differentiation of stem cells, focusing on two aspects: biophysical and biochemical factors. In the future, biomedical scientists will continue investigating the union strength of matrix elasticity and microenvironmental cues for manipulating stem cell fates.

Cross-Linked Matrix Rigidity and Soluble Factors Induce Differentiation via Distinct but Overlapping Pathways

Stem cell differentiation is regulated by both soluble factors and the physical properties of extracellular matrix, but the extent to which differentiation pathways are distinct or overlap is often unclear. Here, the micromechanical stiffness of the collagenous bone surface together with broad compositional correlations with collagen-I across many soft tissues suggests enzymatic cross-linking of matrix correlates with nucleoskeletal protein lamin-A, with a retinoid receptor RARG, and with induction toward osteogenesis. Collagen films just 2 nm thick on mica were stiffened or not by transglutaminase cross-linking and used as minimal culture substrates for Mesenchymal stem cells (MSCs). Cells pulling on pristine nano-films visibly deformed and aligned with the collagen fibrils, but on cross-linked films, cells spread isotropically as if adhering to a substrate of greater effective stiffness. Cell nuclei also spread and stiffened, with an increase of lamin-A, nuclear localization of RARG, and upregulation of key early and late osteogenic factors. RARG antagonists also increased lamin-A, and enhanced osteogenesis on rigid substrates in vitro as well as in xenografts of MSCs in mice. A model of the underlying Mechanochemical Gene Circuit couples the sensitivity of stem cells to both insoluble and soluble factors, while a proteomic comparison underscores both differences and overlaps in differentiation pathways.

Angiogenic responses are enhanced in mechanically and microscopically characterized, microbial transglutaminase crosslinked collagen matrices with increased stiffness

During angiogenesis, endothelial cells (ECs) use both soluble and insoluble cues to expand the existing vascular network to meet the changing trophic needs of the tissue. Fundamental to this expansion are physical interactions between ECs and extracellular matrix (ECM) that influence sprout migration, lumen formation and stabilization. These physical interactions suggest that ECM mechanical properties may influence sprouting ECs and, therefore, angiogenic responses. In a three-dimensional angiogenic model in which a monolayer of ECs is induced to invade an underlying collagen matrix, angiogenic responses were measured as a function of collagen matrix stiffness by inducing collagen crosslinking with microbial transglutaminase (mTG). By biaxial mechanical testing, stiffer collagen matrices were measured with both mTG treatment and incubation time. Using two-photon excited fluorescence (TPF) and second harmonic generation (SHG), it was shown that collagen TPF intensity increased with mTG treatment, and the TPF/SHG ratio correlated with biaxially tested mechanical stiffness. SHG and OCM were further used to show that other ECM physical properties such as porosity and pore size did not change with mTG treatment, thus verifying that matrix stiffness was tuned independently of matrix density. The results showed that stiffer matrices promote more angiogenic sprouts that invade deeper. No differences in lumen size were observed between control and mTG stiffened matrices, but greater remodeling was revealed in stiffer gels using SHG and OCM. The results of this study show that angiogenic responses are influenced by stiffness and suggest that ECM properties may be useful in regenerative medicine applications to engineer angiogenesis.