Additive manufacturing, or 3D printing, has become significantly more commonplace in tissue engineering over the past decade, as a variety of new printing materials have been developed. In ...extrusion‐based printing, materials are used for applications that range from cell free printing to cell‐laden bioinks that mimic natural tissues. Beyond single tissue applications, multi‐material extrusion based printing has recently been developed to manufacture scaffolds that mimic tissue interfaces. Despite these advances, some material limitations prevent wider adoption of the extrusion‐based 3D printers currently available. This progress report provides an overview of this commonly used printing strategy, as well as insight into how this technique can be improved. As such, it is hoped that the prospective report guides the inclusion of more rigorous material characterization prior to printing, thereby facilitating cross‐platform utilization and reproducibility.
Extrusion‐based 3D printing has demonstrated significant promise for the fabrication of cell‐free and cell‐laden engineered tissues. This progress report discusses extrusion‐based 3D printing and recent advances in this field with examples of how they are approaching biomedical engineering problems. These highlights illustrate the advancements that are leading the way for development, characterization, and design of materials for 3D printing.
Since its conceptualization in the 1980s, the provisional matrix has often been characterized as a simple fibrin-containing scaffold for wound healing that supports the nascent blood clot and is ...functionally distinct from the basement membrane. However subsequent advances have shown that this matrix is far from passive, with distinct compositional differences as the wound matures, and providing an active role for wound remodeling. Here we review the stages of this matrix, provide an update on the state of our understanding of provisional matrix, and present some of the outstanding issues related to the provisional matrix, its components, and their assembly and use in vivo.
Abstract One of the recent paradigm shifts in stem cell biology has been the discovery that stem cells can begin to differentiate into mature tissue cells when exposed to intrinsic properties of the ...extracellular matrix (ECM), such as matrix structure, elasticity, and composition. These parameters are known to modulate the forces a cell can exert upon its matrix. Mechano-sensitive pathways subsequently convert these biophysical cues into biochemical signals that commit the cell to a specific lineage. Just as with well-studied growth factors, ECM parameters are extremely dynamic and are spatially- and temporally-controlled during development, suggesting that they play a morphogenetic role in guiding differentiation and arrangement of cells. Our ability to dynamically regulate the stem cell niche as the body does is likely a critical requirement for developing differentiated cells from stem cells for therapeutic applications. Here, we present the emergence of stem cell mechanobiology and its future challenges with new biomimetic, three-dimensional scaffolds that are being used therapeutically to treat disease.
Mesenchymal stem cell (MSC) differentiation is regulated in part by tissue stiffness, yet MSCs can often encounter stiffness gradients within tissues caused by pathological, e.g., myocardial ...infarction ∼8.7±1.5 kPa/mm, or normal tissue variation, e.g., myocardium ∼0.6±0.9 kPa/mm; since migration predominantly occurs through physiological rather than pathological gradients, it is not clear whether MSC differentiate or migrate first. MSCs cultured up to 21 days on a hydrogel containing a physiological gradient of 1.0±0.1 kPa/mm undergo directed migration, or durotaxis, up stiffness gradients rather than remain stationary. Temporal assessment of morphology and differentiation markers indicates that MSCs migrate to stiffer matrix and then differentiate into a more contractile myogenic phenotype. In those cells migrating from soft to stiff regions however, phenotype is not completely determined by the stiff hydrogel as some cells retain expression of a neural marker. These data may indicate that stiffness variation, not just stiffness alone, can be an important regulator of MSC behavior.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Aim: Studies of environmental niche shift/niche conservatism that are based on species distribution modelling require a quantification of niche purity and potential overlap. Although various metrics ...have been proposed for this task, no comparisons of their performance are available yet that express the linearity of range shifts and error-proneness. Herein, we assess the performance of six niche overlap metrics using three sister pairs of plethodontid salamanders as well as artificial species to test for linearity of overlap curves, impacts of varying potential distribution sizes and study area sizes. Location: North America, artificial environments. Methods: Species distribution models for the salamanders were performed with Maxent, and artificial species were created in the R environment. Potential distributions of species with varying range sizes and extents of the study area were compared using the Bray-Curtis distance BC, Schoener's D, two different modifications of the Hellinger distance I mod, I cor, Pianka's O and Horn's R. Niche overlaps in ecological space were compared using linear discriminant analyses based on principal components. Results: Simulations of niche overlaps revealed strong variations in the performance of the niche overlap metrics. In artificial species, BC and D performed best, followed by O, R and I cor , but the modified Hellinger distance I mod showed a nonlinear slope and a truncated range. Furthermore, the simulations suggest that, in proportionally small potential distributions on large grids, an inclusion of a high proportion of grid cells with low occurrence probabilities representing background noise may bias assessments of niche overlaps. Main conclusions: Both the salamander examples and simulations suggest that Schoener's D and the Bray-Curtis distance BC are best suited to compute niche overlaps from potential distributions derived from species distribution models. However, like all analysed metrics, both D and BC are seriously affected by the inclusion of high numbers of grid cells where the species are probably absent, i. e. with low occurrence probabilities. Therefore, pre-processing to eliminate background noise in the potential distribution grids is highly recommended.
Stem cells regulate their fate by binding to, and contracting against, the extracellular matrix. Recently, it has been proposed that in addition to matrix stiffness and ligand type, the degree of ...coupling of fibrous protein to the surface of the underlying substrate, that is, tethering and matrix porosity, also regulates stem cell differentiation. By modulating substrate porosity without altering stiffness in polyacrylamide gels, we show that varying substrate porosity did not significantly change protein tethering, substrate deformations, or the osteogenic and adipogenic differentiation of human adipose-derived stromal cells and marrow-derived mesenchymal stromal cells. Varying protein-substrate linker density up to 50-fold changed tethering, but did not affect osteogenesis, adipogenesis, surface-protein unfolding or underlying substrate deformations. Differentiation was also unaffected by the absence of protein tethering. Our findings imply that the stiffness of planar matrices regulates stem cell differentiation independently of protein tethering and porosity.
Cardiac fibrosis is a serious condition currently lacking effective treatments. It occurs as a result of cardiac fibroblast (CFB) activation and differentiation into myofibroblasts, characterized by ...proliferation, extracellular matrix (ECM) production and stiffening, and contraction due to the expression of smooth muscle α-actin. The mechanical properties of myocardium change regionally and over time after myocardial infarction (MI). Although mechanical cues are known to activate CFBs, it is unclear which specific mechanical stimuli regulate which specific phenotypic trait; thus we investigated these relationships using three in vitro models of CFB mechanical activation and found that 1) paracrine signaling from stretched cardiomyocytes induces CFB proliferation under mechanical conditions similar to those of the infarct border region; 2) direct stretch of CFBs mimicking the mechanical environment of the infarct region induces a synthetic phenotype with elevated ECM production; and 3) progressive matrix stiffening, modeling the mechanical effects of infarct scar maturation, causes smooth muscle α-actin fiber formation, up-regulation of collagen I, and down-regulation of collagen III. These findings suggest that myocyte stretch, fibroblast stretch, and matrix stiffening following MI may separately regulate different profibrotic traits of activated CFBs.
The spatial presentation of mechanical information is a key parameter for cell behavior. We have developed a method of polymerization control in which the differential diffusion distance of unreacted ...cross-linker and monomer into a prepolymerized hydrogel sink results in a tunable stiffness gradient at the cell–matrix interface. This simple, low-cost, robust method was used to produce polyacrylamide hydrogels with stiffness gradients of 0.5, 1.7, 2.9, 4.5, 6.8, and 8.2 kPa/mm, spanning the in vivo physiological and pathological mechanical landscape. Importantly, three of these gradients were found to be nondurotactic for human adipose-derived stem cells (hASCs), allowing the presentation of a continuous range of stiffnesses in a single well without the confounding effect of differential cell migration. Using these nondurotactic gradient gels, stiffness-dependent hASC morphology, migration, and differentiation were studied. Finally, the mechanosensitive proteins YAP, Lamin A/C, Lamin B, MRTF-A, and MRTF-B were analyzed on these gradients, providing higher-resolution data on stiffness-dependent expression and localization.
Breast cancer development is associated with increasing tissue stiffness over years. To more accurately mimic the onset of gradual matrix stiffening, which is not feasible with conventional static ...hydrogels, mammary epithelial cells (MECs) were cultured on methacrylated hyaluronic acid hydrogels whose stiffness can be dynamically modulated from “normal” (<150 Pascals) to “malignant” (>3,000 Pascals) via two-stage polymerization. MECs form and remain as spheroids, but begin to lose epithelial characteristics and gain mesenchymal morphology upon matrix stiffening. However, both the degree of matrix stiffening and culture time before stiffening play important roles in regulating this conversion as, in both cases, a subset of mammary spheroids remained insensitive to local matrix stiffness. This conversion depended neither on colony size nor cell density, and MECs did not exhibit “memory” of prior niche when serially cultured through cycles of compliant and stiff matrices. Instead, the transcription factor Twist1, transforming growth factor β (TGFβ), and YAP activation appeared to modulate stiffness-mediated signaling; when stiffness-mediated signals were blocked, collective MEC phenotypes were reduced in favor of single MECs migrating away from spheroids. These data indicate a more complex interplay of time-dependent stiffness signaling, spheroid structure, and soluble cues that regulates MEC plasticity than suggested by previous models.
Mechanical cues from the extracellular matrix (ECM) regulate various cellular processes via distinct mechanotransduction pathways. In breast cancer, increased ECM stiffness promotes ...epithelial-to-mesenchymal transition (EMT), cell invasion, and metastasis. Here, we identify a mechanosensitive EPHA2/LYN protein complex regulating EMT and metastasis in response to increasing ECM stiffness during tumor progression. High ECM stiffness leads to ligand-independent phosphorylation of ephrin receptor EPHA2, which recruits and activates the LYN kinase. LYN phosphorylates the EMT transcription factor TWIST1 to release TWIST1 from its cytoplasmic anchor G3BP2 to enter the nucleus, thus triggering EMT and invasion. Genetic and pharmacological inhibition of this pathway prevents breast tumor invasion and metastasis in vivo. In human breast cancer samples, activation of this pathway correlates with collagen fiber alignment, a marker of increasing ECM stiffness. Our findings reveal an EPHA2/LYN/TWIST1 mechanotransduction pathway that responds to mechanical signals from the tumor microenvironment to drive EMT, invasion, and metastasis.
•High ECM stiffness activates LYN kinase to promote EMT and invasion•LYN directly phosphorylates TWIST1 to promote nuclear localization of TWIST1•High ECM stiffness promotes ligand-independent EPHA2 signaling to activate LYN•EPHA2/LYN axis promotes breast tumor invasion and metastasis
Fattet et al. identified a EPHA2/LYN/TWIST1 signaling cascade activated by mechanical forces from the extracellular matrix to promote epithelial-mesenchymal transition and cell invasion. They demonstrate that activation of this mechanotransduction pathway promotes breast tumor invasion and metastasis and is involved in human breast cancer metastasis.
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