Extracellular Matrix Alignment Research Articles

Generation of a tendon-like tissue from human iPS cells

Tendons and ligaments are essential connective tissues that connect the muscle and bone. Their recovery from injuries is known to be poor, highlighting the crucial need for an effective therapy. A few reports have described the development of artificial ligaments with sufficient strength from human cells. In this study, we successfully generated a tendon-like tissue (bio-tendon) using human induced pluripotent stem cells (iPSCs). We first differentiated human iPSCs into mesenchymal stem cells (iPSC-MSCs) and transfected them with Mohawk (Mkx) to obtain Mkx-iPSC-MSCs, which were applied to a newly designed chamber with a mechanical stretch incubation system. The embedded Mkx-iPSC-MSCs created bio-tendons and exhibited an aligned extracellular matrix structure. Transplantation of the bio-tendons into a mouse Achilles tendon rupture model showed host-derived cell infiltration with improved histological score and biomechanical properties. Taken together, the bio-tendon generated in this study has potential clinical applications for tendon/ligament-related injuries and diseases.

Fabrication of a Completely Biological and Anisotropic Human Mesenchymal Stem Cell-Based Vascular Graft.

Tissue-engineered small-diameter vascular grafts are required to match mechanical properties as well as cellular and extracellular architecture of native blood vessels. Although various engineering technologies have been developed, the most reliable strategy highlights the needs for incorporating completely biological components and anisotropic cellular and biomolecular organization into the tissue-engineered vascular graft (TEVG). Based on the antithrombogenic, immunoregulatory, and regenerative properties of human mesenchymal stem cells (hMSCs), this chapter provides a step-by-step protocol for generating a completely biological and anisotropic TEVG that comprises of hMSCs and highly aligned extracellular matrix (ECM) nanofibers. The hMSCs were grown on an aligned nanofibrous ECM scaffold derived from an oriented human dermal fibroblast (hDF) sheet and then wrapped around a temporary mandrel to form a tubular assembly, followed by a maturation process in a rotating wall vessel (RWV) bioreactor. The resulting TEVG demonstrates anisotropic structural and mechanical properties similar to that of native blood vessels. A completely biological, anisotropic, and mechanically strong TEVG that incorporates immunoregulatory hMSCs is promising to meet the urgent needs of a surgical intervention for bypass grafting.

A biofabrication method to align cells within bioprinted photocrosslinkable and cell-degradable hydrogel constructs via embedded fibers

The extracellular matrix (ECM) is composed of biochemical and biophysical cues that control cell behaviors and bulk mechanical properties. For example, anisotropy of the ECM and cell alignment are essential in the directional properties of tissues such as myocardium, tendon, and the knee meniscus. Technologies are needed to introduce anisotropic behavior into biomaterial constructs that can be used for the engineering of tissues as models and towards translational therapies. To address this, we developed an approach to align hydrogel fibers within cell-degradable bioink filaments with extrusion printing, where shear stresses during printing align fibers and photocrosslinking stabilizes the fiber orientation. Suspensions of hydrogel fibers were produced through the mechanical fragmentation of electrospun scaffolds of norbornene-modified hyaluronic acid, which were then encapsulated with meniscal fibrochondrocytes, mesenchymal stromal cells, or cardiac fibroblasts within gelatin-methacrylamide bioinks during extrusion printing into agarose suspension baths. Bioprinting parameters such as the needle diameter and the bioink flow rate influenced shear profiles, whereas the suspension bath properties and needle translation speed influenced filament diameters and uniformity. When optimized, filaments were formed with high levels of fiber alignment, which resulted in directional cell spreading during culture over one week. Controls that included bioprinted filaments without fibers or non-printed hydrogels of the same compositions either with or without fibers resulted in random cell spreading during culture. Further, constructs were printed with variable fiber and resulting cell alignment by varying print direction or using multi-material printing with and without fibers. This biofabrication technology advances our ability to fabricate constructs containing aligned cells towards tissue repair and the development of physiological tissue models.

2: Genetic- and Mobilization-Based Alterations in Matrix Alignment to Mitigate Aberrant Cell Fate Determination

Purpose:Cells recognize mechanical cues from the extracellular environment through interactions with matrix proteins, such as collagen. Following injury, fibrotic deposition of collagen can lead to aberrant cell differentiation and failed healing. Aligned collagen matrices can drive PDGFRα+ mesenchymal progenitor cells (MPCs) towards osteochondral lineages, leading to heterotopic ossification (HO). Previous work has shown that reducing mechanical strain by immobilizing a limb prevents the formation of an aligned extracellular matrix (ECM), leading to altered MPC differentiation, however, the cellular mechanisms of matrix reorganization following injury remain unclear. Using novel bioinformatics approach, we identified discoidin domain receptor 2 (DDR2) as a key MPC specific tyrosine kinase receptor that interacts with the fibrillar collagen matrix. We hypothesized that DDR2 activation leads to regulation of ECM alignment and therefore serves as a novel upstream regulator of mechanotransductive signaling following musculoskeletal repair.Methods:Heterotopic ossification was induced using a proven mouse model of 30% total body surface area burn with concurrent Achilles’ transection in Ddr2 deletion (Ddr2slie/slie) and littermate control mice on the C57/BL6J background. In separate experiments, ankle joint immobilizers were placed on injured mice for 1, 2 or 3 weeks after burn/tenotomy. Tissue from Ddr2 deleted, immobilized and mobile control mice were harvested for immunofluorescence histology, second harmonic generation of collagen fiber histology at 1, 3 and 9 weeks after injury (SHG) (n=4/group). HO site tissue was also harvested prior to injury and at 1 and 6 weeks after burn/tenotomy and processed for Single-Cell RNA sequencing (scRNA seq) and single nucleus assay for transposase-accessible chromatin (snATAC) using 10X Sequencing and downstream analysis using Seurat 4.0.1 and Signac 3.1.5.Results:Single-cell RNA sequencing showed that genes for collagen type 1 and 3 and Ddr2 are highly upregulated in MPCs following injury. SnATAC showed increased open chromatin reads in the promoter of MPCs following injury (not shown). Utilizing SHG, we found that the collagen matrix is aligned at three-weeks following injury, but one week of limb immobilization was enough to prevent aligned collagen. The active phosphorylated form of DDR2 (pDDR2) was decreased in PDGFRα+ MPCs within immobilized samples, suggesting that DDR2 may affect matrix alignment. Ddr2slie/slie had more disorganized collagen matrix nine weeks following injury and significantly reduced HO.Conclusions:This is the first work to alter collagen matrix organization by genetic modification and mobilization protocols and identifies DDR2 as a critical upstream receptor in matrix organization and aberrant progenitor differentiation following injury. DDR2 antagonism could have therapeutic potential in preventing heterotopic ossification following traumatic injury.

Reticulocalbin 3 is involved in postnatal tendon development by regulating collagen fibrillogenesis and cellular maturation

Tendon plays a critical role in the joint movement by transmitting force from muscle to bone. This transmission of force is facilitated by its specialized structure, which consists of highly aligned extracellular matrix consisting predominantly of type I collagen. Tenocytes, fibroblast-like tendon cells residing between the parallel collagen fibers, regulate this specialized tendon matrix. Despite the importance of collagen structure and tenocyte function, the biological mechanisms regulating fibrillogenesis and tenocyte maturation are not well understood. Here we examine the function of Reticulocalbin 3 (Rcn3) in collagen fibrillogenesis and tenocyte maturation during postnatal tendon development using a genetic mouse model. Loss of Rcn3 in tendon caused decreased tendon thickness, abnormal tendon cell maturation, and decreased mechanical properties. Interestingly, Rcn3 deficient mice exhibited a smaller collagen fibril distribution and over-hydroxylation in C-telopeptide cross-linking lysine from α1(1) chain. Additionally, the proline 3-hydroxylation sites in type I collagen were also over-hydroxylated in Rcn3 deficient mice. Our data collectively suggest that Rcn3 is a pivotal regulator of collagen fibrillogenesis and tenocyte maturation during postnatal tendon development.

Novel mechanism for OSM-promoted extracellular matrix remodeling in breast cancer: LOXL2 upregulation and subsequent ECM alignment

BackgroundInvasive ductal carcinoma (IDC) is a serious problem for patients as it metastasizes, decreasing 5-year patient survival from > 95 to ~ 27%. The breast tumor microenvironment (TME) is often saturated with proinflammatory cytokines, such as oncostatin M (OSM), which promote epithelial-to-mesenchymal transitions (EMT) in IDC and increased metastasis. The extracellular matrix (ECM) also plays an important role in promoting invasive and metastatic potential of IDC. Specifically, the reorganization and alignment of collagen fibers in stromal ECM leads to directed tumor cell motility, which promotes metastasis. Lysyl oxidase like-2 (LOXL2) catalyzes ECM remodeling by crosslinking of collagen I in the ECM. We propose a novel mechanism whereby OSM induces LOXL2 expression, mediating stromal ECM remodeling of the breast TME.MethodsBioinformatics was utilized to determine survival and gene correlation in patients. IDC cell lines were treated with OSM (also IL-6, LIF, and IL-1β) and analyzed for LOXL2 expression by qRT-PCR and immunolabelling techniques. Collagen I contraction assays, 3D invasion assays, and confocal microscopy were performed with and without LOXL2 inhibition to determine the impact of OSM-induced LOXL2 on the ECM.ResultsOur studies demonstrate that IDC patients with high LOXL2 and OSM co-expression had worse rates of metastasis-free survival than those with high levels of either, individually, and LOXL2 expression is positively correlated to OSM/OSM receptor (OSMR) expression in IDC patients. Furthermore, human IDC cells treated with OSM resulted in a significant increase in LOXL2 mRNA, which led to upregulated protein expression of secreted, glycosylated, and enzymatically active LOXL2. The expression of LOXL2 in IDC cells did not affect OSM-promoted EMT, and LOXL2 was localized to the cytoplasm and/or secreted. OSM-induced LOXL2 promoted an increase in ECM collagen I fiber crosslinking, which led to significant fiber alignment between cells and increased IDC cell invasion.ConclusionsAligned collagen fibers in the ECM provide pathways for tumor cells to migrate more easily through the stroma to nearby vasculature and tissue. These results provide a new paradigm through which proinflammatory cytokine OSM promotes tumor progression. Understanding the nuances in IDC metastasis will lead to better potential therapeutics to combat against the possibility.

Extracellular Matrix Alignment Directs Provisional Matrix Assembly and Three Dimensional Fibrous Tissue Closure.

Gap closure is a dynamic process in wound healing, in which a wound contracts and a provisional matrix is laid down, to restore structural integrity to injured tissues. The efficiency of wound closure has been found to depend on the shape of a wound, and this shape dependence has been echoed in various in vitro studies. While wound shape itself appears to contribute to this effect, it remains unclear whether the alignment of the surrounding extracellular matrix (ECM) may also contribute. In this study, we investigate the role both wound curvature and ECM alignment have on gap closure in a 3D culture model of fibrous tissue. Using microfabricated flexible micropillars positioned in rectangular and octagonal arrangements, seeded 3T3 fibroblasts embedded in a collagen matrix formed microtissues with different ECM alignments. Wounding these microtissues with a microsurgical knife resulted in wounds with different shapes and curvatures that closed at different rates. Observing different regions around the wounds, we noted local wound curvature did not impact the rate of production of provisional fibronectin matrix assembled by the fibroblasts. Instead, the rate of provisional matrix assembly was lowest emerging from regions of high fibronectin alignment and highest in the areas of low matrix alignment. Our data suggest that the underlying ECM structure affects the shape of the wound as well as the ability of fibroblasts to build provisional matrix, an important step in the process of tissue closure and restoration of tissue architecture. The study highlights an important interplay between ECM alignment, wound shape, and tissue healing that has not been previously recognized and may inform approaches to engineer tissues. Impact statement Current models of tissue growth have identified a role for curvature in driving provisional matrix assembly. However, most tissue repair occurs in fibrous tissues with different levels of extracellular matrix (ECM) alignment. Here, we show how this underlying ECM alignment may affect the ability of fibroblasts to build new provisional matrix, with implications for in vivo wound healing and providing insight for engineering of new tissues.

The mechanics and dynamics of cancer cells sensing noisy 3D contact guidance

Contact guidance is a major physical cue that modulates cancer cell morphology and motility, and is directly linked to the prognosis of cancer patients. Under physiological conditions, particularly in the three-dimensional (3D) extracellular matrix (ECM), the disordered assembly of fibers presents a complex directional bias to the cells. It is unclear how cancer cells respond to these noncoherent contact guidance cues. Here we combine quantitative experiments, theoretical analysis, and computational modeling to study the morphological and migrational responses of breast cancer cells to 3D collagen ECM with varying degrees of fiber alignment. We quantify the strength of contact guidance using directional coherence of ECM fibers, and find that stronger contact guidance causes cells to polarize more strongly along the principal direction of the fibers. Interestingly, sensitivity to contact guidance is positively correlated with cell aspect ratio, with elongated cells responding more strongly to ECM alignment than rounded cells. Both experiments and simulations show that cell-ECM adhesions and actomyosin contractility modulate cell responses to contact guidance by inducing a population shift between rounded and elongated cells. We also find that cells rapidly change their morphology when navigating the ECM, and that ECM fiber coherence modulates cell transition rates between different morphological phenotypes. Taken together, we find that subcellular processes that integrate conflicting mechanical cues determine cell morphology, which predicts the polarization and migration dynamics of cancer cells in 3D ECM.

Abstract PO010: Engineering T cells to enhance 3D migration through mechanically and structurally complex tumor microenvironments

Abstract While chemical signals play a strong role in drawing T cells into solid tumors, the physical features of the stroma, such as architecture and mechanics, also strongly influence T cell infiltration as well as their ability to effectively distribute throughout, and sample, the entire tumor volume. Indeed, the mechanically and chemically complex stromal reaction in solid tumors can limit access and effective distribution of T cells creating antitumor immunity-free sanctuaries. Furthermore, many solid tumors are rich with aligned extracellular matrix (ECM) networks, which provide contact guidance for carcinoma cells, but can also direct migration of infiltrated T cells within solid tumors. Yet, our understanding of how native and engineered T cells migrate through mechanically complex tumor microenvironments (TMEs) is quite incomplete. As such, defining the principles of T cell migration in structurally and mechanically complex tumor microenvironments is critical to understanding sanctuaries from antitumor immunity and for optimizing T cell-related therapeutic strategies. In order to decipher and enhance migration of T cells through complex microenvironments, we initially engineered nanotextured elastic platforms that mimic ECM architectures observed in breast and pancreatic carcinomas. These platforms allowed us to define how the balance between contractility localization-dependent T cell phenotypes (i.e. high cortical contractility distributed along the curved cell membrane versus forces imparted onto substrates) influences migration in response to tumor-mimetic mechanical and structural and cues. From this information we characterized a mechanical optimum for migration that can be perturbed by manipulating an axis between microtubule stability and contractile force generation. In three-dimensional (3D) environments and live pancreatic tumors, we demonstrate that microtubule instability, leading to increased Rho pathway-dependent cortical contractility, promotes migration while clinically used microtubule-stabilizing chemotherapies profoundly decrease effective migration. Indeed, we show that rational manipulation of the microtubule-contractility axis, either pharmacologically or through genome engineering with CRISPR results in engineered T cells that more effectively move through and interrogate 3D matrix and tumor volumes. This suggests that T cell engineering to generate “mechanically optimized” therapeutic cells that better navigate through 3D microenvironments could be part of an effective strategy to enhance efficacy of immune therapeutics. Citation Format: Erdem Tabdanov, Nelson Rodriguez-Merced, Alexander Cartagena-Rivera, Vikram Puram, Mackenzie Callaway, Ethan Ensminger, Emily Pomeroy, Kenta Yamamoto, Walker Lahr, Beau Webber, Branden Moriarity, Alexander Zhovmer, Paolo Provenzano. Engineering T cells to enhance 3D migration through mechanically and structurally complex tumor microenvironments [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO010.

Passive stiffness of fibrotic skeletal muscle in mdx mice relates to collagen architecture.

The amount of fibrotic material in dystrophic mouse muscles relates to contractile function, but not passive function. Collagen fibres in skeletal muscle are associated with increased passive muscle stiffness in fibrotic muscles. The alignment of collagen is independently associated with passive stiffness in dystrophic skeletal muscles. These outcomes demonstrate that collagen architecture rather than collagen content should be a target of anti-fibrotic therapies to treat muscle stiffness. Fibrosis is prominent in many skeletal muscle pathologies including dystrophies, neurological disorders, cachexia, chronic kidney disease, sarcopenia and metabolic disorders. Fibrosis in muscle is associated with decreased contractile forces and increased passive stiffness that limits joint mobility leading to contractures. However, the assumption that more fibrotic material is directly related to decreased function has not held true. Here we utilize novel measurement of extracellular matrix (ECM) and collagen architecture to relate ECM form to muscle function. We used mdx mice, a model for Duchenne muscular dystrophy that becomes fibrotic, and wildtype mice. In this model, extensor digitorum longus (EDL) muscle was significantly stiffer, but with similar total collagen, while the soleus muscle did not change stiffness, but increased collagen. The stiffness of the EDL was associated with increased collagen crosslinking as determined by collagen solubility. Measurement of ECM alignment using polarized light microscopy showed a robust relationship between stiffness and alignment for wildtype muscle that broke down in mdx muscles. Direct visualization of large collagen fibres with second harmonic generation imaging revealed their relative abundance in stiff muscles. Collagen fibre alignment was linked to stiffness across all muscles investigated and the most significant factor in a multiple linear regression-based model of muscle stiffness from ECM parameters. This work establishes novel characteristics of skeletal muscle ECM architecture and provides evidence for a mechanical function of collagen fibres in muscle. This finding suggests that anti-fibrotic strategies to enhance muscle function and excessive stiffness should target large collagen fibres and their alignment rather than total collagen.

Radiofrequency ablation alters the microstructural organization of healthy and enzymatically digested porcine mitral valves.

Myxomatous mitral valve degeneration is a common cause of mitral regurgitation and is often associated with mitral valve prolapse. With no known targets to pharmacologically treat mitral valve prolapse, surgery is often the only treatment option. Recently, radiofrequency ablation has been proposed as a percutaneous alternative to surgical resection for the reduction of mitral valve leaflet area. Using an in vitro model of porcine mitral valve anterior leaflet enlargement following enzymatic digestion, we sought to investigate mechanisms by which radiofrequency ablation alters the geometry, microstructural organization, and mechanical properties of healthy and digested leaflets. Paired measurements before and after ablation revealed the impact of radiofrequency ablation on leaflet properties. Multiphoton imaging was used to characterize changes in the structure and organization of the valvular extracellular matrix; planar biaxial mechanical testing and constitutive modeling were used to estimate mechanical properties of healthy and digested leaflets. Enzymatic digestion increased leaflet area and thickness to a similar extent as clinical mitral valve disease. Radiofrequency ablation altered extracellular matrix alignment and reduced the area of digested leaflets to that of control. Additionally, enzymatic digestion resulted in fiber alignment and reorientation toward the radial direction, causing increased forces during ablation and a structural stiffening which was improved by radiofrequency ablation. Radiofrequency ablation induces radial extracellular matrix alignment and effectively reduces the area of enlarged mitral valve leaflets. Hence, this technique may be a therapeutic approach for myxomatous mitral valve disease and is thus an avenue for future study.

Remodeling of aligned fibrous extracellular matrix by encapsulated cells under mechanical stretching

Extracellular matrix (ECM) remodeling is essential for the development and functions of connective tissues (e.g., heart, muscle and the periodontal ligament), and entails the highly anisotropic response of cells and their organized ECM molecules to mechanical stimulation. However, the nature of how cells remodel their surrounding ECM under mechanical stimulation remains elusive. Here, we encapsulated human periodontal ligament stem cells (hPDLSCs) within an aligned rat collagen scaffold labeled with fluorescein isothiocyanate (FITC) and applied mechanical stimulation on the scaffold using magnetic stretching. Through tracking the FITC-labeled rat collagen scaffold and the newly secreted human type I collagen, we studied the effect of magnetic stretching on the mechanism of aligned ECM remodeling by the encapsulated cells. We found that the aligned topography combined with magnetic stretching could significantly promote initial ECM degradation and new ECM secretion: expression of matrix metalloproteinases 1 and 9 is increased markedly, and the elastic modulus of the stretched scaffold (75 kPa) is significantly higher than that of the random scaffold (50 kPa). The data support a model whereby the cells remodel their surrounding ECM under continuous stretching through degradation and then secretion of new ECM to integrate with the aligned ECM and maintain tissue function. Our study offers a valuable basis for future optimized design of biomaterial scaffolds for clinical translation. Statement of significanceExtracellular matrix (ECM) remodeling is essential for the development and functions of connective tissues. However, the nature of how cells remodel their surrounding aligned ECM under mechanical stimulation remains elusive. Herein, we developed a method to reveal the remodeling of aligned rat collagen scaffold by the encapsulated human periodontal ligament stem cells (hPDLSCs) using fluorescence imaging. We found that the aligned topography combined with magnetic stretching could significantly promote initial ECM degradation and new ECM secretion: the expression of matrix metalloproteinase 1 and 9 are significantly higher, and the elastic modulus increases from 50 kPa to 75 kPa as compared to the random collagen scaffold encapsulating hPDLSCs. Our study holds great potential in optimization of bio-scaffold design for clinical translation.

Spatial-omics: Novel approaches to probe cell heterogeneity and extracellular matrix biology

Complex intercellular interactions as well as biomolecular and biomechanical cues from the extracellular matrix (ECM) profoundly affect cellular functions. Traditional transcriptomic and proteomic approaches have provided insight into disease progression by identifying discrete cellular subpopulations or microenvironmental signatures characteristic of normal or pathological tissues, however these techniques do not examine how a given cellular state relates to its interactions with neighboring cells or its surrounding ECM with multiparametric characterization (i.e. ECM alignment, mechanical forces, crosslinking, etc.). Emerging spatial-omic techniques can provide high-resolution mapping of expression profiles similar to scRNA-seq and mass spectroscopy directly within tissues. The ability to preserve the spatial context of cells within samples, their cellular geometry, as well as their surrounding ECM gives spatial-omics the opportunity to interrogate previously unexplored signaling modalities, which has the potential to revolutionize ECM research and our understanding of fibrotic diseases. In this review, we present current spatial transcriptomic and proteomic techniques and discuss how they may be applied to investigate cell-ECM interactions.

Delineating the role of membrane blebs in a hybrid mode of cancer cell invasion in three-dimensional environments.

The study of cancer cell invasion in 3D environments in vitro has revealed a variety of invasive modes, including amoeboid migration, characterized by primarily round cells that invade in a protease- and adhesion-independent manner. Here, we delineate a contractility-dependent migratory mode of primarily round breast cancer cells that is associated with extensive integrin-mediated extracellular matrix (ECM) reorganization that occurs at membrane blebs, with bleb necks sites of integrin clustering and integrin-dependent ECM alignment. We show that the spatiotemporal distribution of blebs and their utilization for ECM reorganization is mediated by functional β1 integrin receptors and other components of focal adhesions. Taken together, the work presented here characterizes a migratory mode of primarily round cancer cells in complex 3D environments and reveals a fundamentally new function for membrane blebs in cancer cell invasion.

Fibroblastic Reticular Cells Control Conduit Matrix Deposition during Lymph Node Expansion

SummaryLymph nodes (LNs) act as filters, constantly sampling peripheral cues. This is facilitated by the conduit network, a tubular structure of aligned extracellular matrix (ECM) fibrils ensheathed by fibroblastic reticular cells (FRCs). LNs undergo rapid 3- to 5-fold expansion during adaptive immune responses, but these ECM-rich structures are not permanently damaged. Whether conduit flow or filtering function is affected during LN expansion is unknown. Here, we show that conduits are partially disrupted during acute LN expansion, but FRC-FRC contacts remain connected. We reveal that polarized FRCs deposit ECM basolaterally using LL5-β and that ECM production is regulated at transcriptional and secretory levels by the C-type lectin CLEC-2, expressed by dendritic cells. Inflamed LNs maintain conduit size exclusion, and flow is disrupted but persists, indicating the robustness of this structure despite rapid tissue expansion. We show how dynamic communication between peripheral tissues and LNs provides a mechanism to prevent inflammation-induced fibrosis in lymphoid tissue.

Cancer Cells Sense Fibers by Coiling on them in a Curvature-Dependent Manner.

SummaryMetastatic cancer cells sense the complex and heterogeneous fibrous extracellular matrix (ECM) by formation of protrusions, and our knowledge of how cells physically recognize these fibers remains in its infancy. Here, using suspended ECM-mimicking isodiameter fibers ranging from 135 to 1,000 nm, we show that metastatic breast cancer cells sense fiber diameters differentially by coiling (wrapping-around) on them in a curvature-dependent manner, whereas non-tumorigenic cells exhibit diminished coiling. We report that coiling occurs at the tip of growing protrusions and the coil width and coiling rate increase in a curvature-dependent manner, but time to maximum coil width occurs biphasically. Interestingly, bundles of 135-nm diameter fibers recover coiling width and rate on 1,000-nm-diameter fibers. Coiling also coincides with curvature-dependent persistent and ballistic transport of endogenous granules inside the protrusions. Altogether, our results lay the groundwork to link biophysical sensing with biological signaling to quantitate pro- and anti-invasive fibrous environments.Video

A 3-Dimensional In Vitro Model of Zonally Organized Extracellular Matrix.

Functional cartilage repair requires the new formation of organized hyaline cartilaginous matrix to avoid the generation of fibrous repair tissue. The potential of mesenchymal progenitors was used to assemble a 3-dimensional structure in vitro, reflecting the zonation of collagen matrix in hyaline articular cartilage. The 3-dimensional architecture of collagen alignment in pellet cultures of chondroprogenitors (CPs) was assessed with Picrosirius red staining analyzed under polarized light. In parallel assays, the trilineage capability was confirmed by calcium deposition during osteogenesis by alizarin S staining and alkaline phosphatase staining. Using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), mRNA levels of ALP, RUNX2, and BGLAP were assessed after 21 days of osteoinduction. Lipid droplets were stained with oil red O and adipogenic differentiation was confirmed by RT-qPCR analysis of PPARG and LPL gene expression. Under conditions promoting the chondrogenic signature in self-assembling constructs, CPs formed an aligned extracellular matrix, positive for glycosaminoglycans and collagen type II, showing developing zonation of birefringent collagen fibers along the cross section of pellets, which reflect the distribution of collagen fibers in hyaline cartilage. Induced osteogenic and adipogenic differentiation confirmed the trilineage potential of CPs. This model promotes the differentiation and self-organization of postnatal chondroprogenitors, resulting in the formation of zonally organized engineered hyaline cartilage comparable to the 3 zones of native cartilage.

Conduit Integrity is Compromised During Acute Lymph Node Expansion

Lymph nodes (LNs) act as filters, constantly sampling peripheral cues. This is facilitated by the conduit network, a tubular structure of aligned extracellular matrix (ECM) fibrils ensheathed by fibroblastic reticular cells (FRCs). LNs undergo 3-5-fold expansion during each adaptive immune response but these ECM-rich structures are not permanently damaged. Whether conduit integrity and filtering function is affected during LN expansion is unknown. Here we show that conduits are disrupted during acute LN expansion but FRC-FRC contacts remain connected. We reveal that polarised FRCs deposit ECM basolaterally utilising LL5-β, and that ECM production is regulated at transcriptional and secretory levels by the C-type lectin CLEC-2, expressed by dendritic cells. Inflamed LNs maintain conduit size-exclusion, but flow becomes leaky, allowing soluble molecules increased access to antigen-presenting cells. We show how dynamic communication between peripheral tissues and LNs changes during immune responses, and describe a mechanism enabling LNs to prevent inflammation-induced fibrosis.

Spatial Regulation of Valve Interstitial Cell Phenotypes within Three-Dimensional Micropatterned Hydrogels.

Calcific aortic valve disease (CAVD) is the third leading cause of cardiovascular disease. CAVD exhibits progressive disruption of the normally highly organized and aligned extracellular matrix (ECM) structure within the valve leaflets simultaneously with myofibroblastic and/or osteogenic differentiation of indigenous endogenous valve interstitial cells (VIC). It is unclear how the alignment of VIC within their 3D microenvironment drives VIC phenotype or how alignment affects cellular responses to biochemical cues in physiological or pathological conditions. In this study, we implement a photolithographic technique to control the alignment and elongation of both normal and diseased human aortic VIC (HAVIC) within microengineered 3D hydrogels consisting of methacrylated hyaluronic acid and methacrylated gelatin. Stripe micropatterning created distinct alignment of HAVIC within a 3D culture system, which promoted spreading and enhanced their activation and osteogenic differentiation in pro-osteogenic conditions. HAVIC from a patient with CAVD exhibited greater susceptibility to myofibroblastic and osteogenic differentiation in culture. The roles of conjugated basic fibroblastic growth factor (bFGF) and RhoA/ROCK pathway in regulating HAVIC phenotypes were also investigated in the presence of aligned microtopography. The addition of bFGF was preventative to osteogenic differentiation for healthy HAVIC; however, it promoted osteogenic differentiation in diseased HAVIC. Inhibition of the ROCK pathway only decreased osteogenic differentiation for diseased HAVIC in the aligned formation. Collectively, these results improve our knowledge of the effects that VIC alignment has on VIC phenotypes and valve disease progression. The cell culture platform also enables a better understanding of the interplay between topography, biochemical cues, and VIC differentiation and provides information useful for directing differentiation as well as valve tissue regeneration.

Remodeling of the Collagen Matrix in Aging Skin Promotes Melanoma Metastasis and Affects Immune Cell Motility.

Physical changes in skin are among the most visible signs of aging. We found that young dermal fibroblasts secrete high levels of extracellular matrix (ECM) constituents, including proteoglycans, glycoproteins, and cartilage-linking proteins. The most abundantly secreted was HAPLN1, a hyaluronic and proteoglycan link protein. HAPLN1 was lost in aged fibroblasts, resulting in a more aligned ECM that promoted metastasis of melanoma cells. Reconstituting HAPLN1 inhibited metastasis in an aged microenvironment, in 3-D skin reconstruction models, and in vivo. Intriguingly, aged fibroblast-derived matrices had the opposite effect on the migration of T cells, inhibiting their motility. HAPLN1 treatment of aged fibroblasts restored motility of mononuclear immune cells, while impeding that of polymorphonuclear immune cells, which in turn affected regulatory T-cell recruitment. These data suggest that although age-related physical changes in the ECM can promote tumor cell motility, they may adversely affect the motility of some immune cells, resulting in an overall change in the immune microenvironment. Understanding the physical changes in aging skin may provide avenues for more effective therapy for older patients with melanoma. SIGNIFICANCE: These data shed light on the mechanochemical interactions that occur between aged skin, tumor, and immune cell populations, which may affect tumor metastasis and immune cell infiltration, with implications for the efficacy of current therapies for melanoma.See related commentary by Marie and Merlino, p. 19.This article is highlighted in the In This Issue feature, p. 1.