scholarly journals Crosstalk between invadopodia and the extracellular matrix

2020 ◽  
Author(s):  
Shinji Iizuka ◽  
Ronald P. Leon ◽  
Kyle P. Gribbin ◽  
Ying Zhang ◽  
Jose Navarro ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Type I Collagen ◽  
Complex Structure ◽  
Three Dimensional ◽  
3D Culture ◽  
Model Systems ◽  
Type I ◽  
Extracellular Matrix Components

ABSTRACTThe scaffold protein Tks5α is required for invadopodia-mediated cancer invasion both in vitro and in vivo. We have previously also revealed a role for Tks5 in tumor cell growth using three-dimensional (3D) culture model systems and mouse transplantation experiments. Here we use both 3D and high-density fibrillar collagen (HDFC) culture to demonstrate that native type I collagen, but not a form lacking the telopeptides, stimulated Tks5-dependent growth, which was dependent on the DDR collagen receptors. We used microenvironmental microarray (MEMA) technology to determine that laminin, collagen I, fibronectin and tropoelastin also stimulated invadopodia formation. A Tks5α-specific monoclonal antibody revealed its expression both on microtubules and at invadopodia. High- and super-resolution microscopy of cells in and on collagen was then used to place Tks5α at the base of invadopodia, separated from much of the actin and cortactin, but coincident with both matrix metalloprotease and cathepsin proteolytic activity. Inhibition of the Src family kinases, cathepsins or metalloproteases all reduced invadopodia length but each had distinct effects on Tks5α localization. These studies highlight the crosstalk between invadopodia and extracellular matrix components, and reveal the invadopodium to be a spatially complex structure.

scholarly journals Fibrin and Collagen Differentially but Synergistically Regulate Sprout Angiogenesis of Human Dermal Microvascular Endothelial Cells in 3-Dimensional Matrix

2013 ◽  
Vol 2013 ◽  
pp. 1-11 ◽  
Author(s):  
Xiaodong Feng ◽  
Marcia G. Tonnesen ◽  
Shaker A. Mousa ◽  
Richard A. F. Clark
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Type I Collagen ◽  
Three Dimensional ◽  
Tumor Stroma ◽  
Microvascular Endothelial Cells ◽  
Type I ◽  
Microvascular Endothelial

Angiogenesis is a highly regulated event involving complex, dynamic interactions between microvascular endothelial cells and extracellular matrix (ECM) proteins. Alteration of ECM composition and architecture is a hallmark feature of wound clot and tumor stroma. We previously reported that during angiogenesis, endothelial cell responses to growth factors are modulated by the compositional and mechanical properties of a surrounding three-dimensional (3D) extracellular matrix (ECM) that is dominated by either cross-linked fibrin or type I collagen. However, the role of 3D ECM in the regulation of angiogenesis associated with wound healing and tumor growth is not well defined. This study investigates the correlation of sprout angiogenesis and ECM microenvironment using in vivo and in vitro 3D angiogenesis models. It demonstrates that fibrin and type I collagen 3D matrices differentially but synergistically regulate sprout angiogenesis. Thus blocking both integrin alpha v beta 3 and integrin alpha 2 beta 1 might be a novel strategy to synergistically block sprout angiogenesis in solid tumors.

scholarly journals Extracellular matrix components of the mouse thymus microenvironment: ontogenetic studies and modulation by glucocorticoid hormones.

1991 ◽  
Vol 39 (11) ◽  
pp. 1539-1546 ◽  
Author(s):  
J Lannes-Vieira ◽  
M Dardenne ◽  
W Savino
Keyword(s):  
Extracellular Matrix ◽  
Basement Membrane ◽  
Type I Collagen ◽  
Basement Membranes ◽  
Thymic Epithelial Cell ◽  
Epithelial Cell Line ◽  
Type I ◽  
Extracellular Matrix Components

The present investigation was an ontogenetic study on the distribution of extracellular matrix (ECM) components in the thymic microenvironment of C57BL/6 mice (comprising young and old adults and developing embryos) and NZB mice. In addition, we evaluated the in vivo and in vitro influence of hydrocortisone treatment on basement membrane protein production by a thymic epithelial cell line. In young normal animals, Type I collagen was restricted to the interstitial spaces of the capsule and septa, where Type IV collagen, fibronectin, and laminin could be detected in the basement membranes. In addition, fibronectin-containing fibers were seen within the medulla of the thymic lobules. The ECM distribution pattern in the developing embryos was distinct from that observed in adults, since a fine meshwork of basement membrane-containing proteins was clearly seen throughout the parenchyma. Moreover, aging normal and NZB mice exhibited a denser ECM pattern than young adult normal animals. Treatment with hydrocortisone, both in vivo and in vitro, resulted in enhancement of ECM expression, detected in mice as early as 2 hr post injection and lasting for several days. Considering that the fluctuations of ECM expression parallel important events in thymocyte differentiation, we discuss the possibility that the two phenomena may be associated.

Morphological and Functional Characteristics of Three-Dimensional Engineered Bone-Ligament-Bone Constructs Following Implantation

2009 ◽  
Vol 131 (10) ◽  
Author(s):  
Jinjin Ma ◽  
Kristen Goble ◽  
Michael Smietana ◽  
Tatiana Kostrominova ◽  
Lisa Larkin ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Type I Collagen ◽  
Three Dimensional ◽  
Functionally Graded ◽  
Neonatal Rat ◽  
Ligament Injury ◽  
Type I ◽  
Tangent Moduli

The incidence of ligament injury has recently been estimated at 400,000/year. The preferred treatment is reconstruction using an allograft, but outcomes are limited by donor availability, biomechanical incompatibility, and immune rejection. The creation of an engineered ligament in vitro solely from patient bone marrow stromal cells (has the potential to greatly enhance outcomes in knee reconstructions. Our laboratory has developed a scaffoldless method to engineer three-dimensional (3D) ligament and bone constructs from rat bone marrow stem cells in vitro. Coculture of these two engineered constructs results in a 3D bone-ligament-bone (BLB) construct with viable entheses, which was successfully used for medial collateral ligament (MCL) replacement in a rat model. 1 month and 2 month implantations were applied to the engineered BLBs. Implantation of 3D BLBs in a MCL replacement application demonstrated that our in vitro engineered tissues grew and remodeled quickly in vivo to an advanced phenotype and partially restored function of the knee. The explanted 3D BLB ligament region stained positively for type I collagen and elastin and was well vascularized after 1 and 2 months in vivo. Tangent moduli of the ligament portion of the 3D BLB 1 month explants increased by a factor of 2.4 over in vitro controls, to a value equivalent to those observed in 14-day-old neonatal rat MCLs. The 3D BLB 1 month explants also exhibited a functionally graded response that closely matched native MCL inhomogeneity, indicating the constructs functionally adapted in vivo.

scholarly journals MT1-MMP–dependent neovessel formation within the confines of the three-dimensional extracellular matrix

2004 ◽  
Vol 167 (4) ◽  
pp. 757-767 ◽  
Author(s):  
Tae-Hwa Chun ◽  
Farideh Sabeh ◽  
Ichiro Ota ◽  
Hedwig Murphy ◽  
Kevin T. McDonagh ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Cell Surface ◽  
Type I Collagen ◽  
Ex Vivo ◽  
Three Dimensional ◽  
Collagenolytic Activity ◽  
Type I ◽  
Ex Vivo Model

During angiogenesis, endothelial cells initiate a tissue-invasive program within an interstitial matrix comprised largely of type I collagen. Extracellular matrix–degradative enzymes, including the matrix metalloproteinases (MMPs) MMP-2 and MMP-9, are thought to play key roles in angiogenesis by binding to docking sites on the cell surface after activation by plasmin- and/or membrane-type (MT) 1-MMP–dependent processes. To identify proteinases critical to neovessel formation, an ex vivo model of angiogenesis has been established wherein tissue explants from gene-targeted mice are embedded within a three-dimensional, type I collagen matrix. Unexpectedly, neither MMP-2, MMP-9, their cognate cell-surface receptors (i.e., β3 integrin and CD44), nor plasminogen are essential for collagenolytic activity, endothelial cell invasion, or neovessel formation. Instead, the membrane-anchored MMP, MT1-MMP, confers endothelial cells with the ability to express invasive and tubulogenic activity in a collagen-rich milieu, in vitro or in vivo, where it plays an indispensable role in driving neovessel formation.

Three-Dimensional-Printed External Scaffolds Mitigate Loss of Volume and Topography in Engineered Elastic Cartilage Constructs

Cartilage ◽  
2021 ◽  
pp. 194760352110495
Author(s):  
Xue Dong ◽  
Ishani D. Premaratne ◽  
Jaime L. Bernstein ◽  
Arash Samadi ◽  
Alexandra J. Lin ◽  
...  
Keyword(s):  
Injection Molding ◽  
Type I Collagen ◽  
Three Dimensional ◽  
Type I ◽  
Base Area ◽  
Elastic Cartilage ◽  
3D Printed ◽  
Injection Molded

Objective: A major obstacle in the clinical translation of engineered auricular scaffolds is the significant contraction and loss of topography that occur during maturation of the soft collagen-chondrocyte matrix into elastic cartilage. We hypothesized that 3-dimensional-printed, biocompatible scaffolds would “protect” maturing hydrogel constructs from contraction and loss of topography. Design: External disc-shaped and “ridged” scaffolds were designed and 3D-printed using polylactic acid (PLA). Acellular type I collagen constructs were cultured in vitro for up to 3 months. Collagen constructs seeded with bovine auricular chondrocytes (BAuCs) were prepared in 3 groups and implanted subcutaneously in vivo for 3 months: preformed discs with (“Scaffolded/S”) or without (“Naked/N”) an external scaffold and discs that were formed within an external scaffold via injection molding (“Injection Molded/SInj”). Results: The presence of an external scaffold or use of injection molding methodology did not affect the acellular construct volume or base area loss. In vivo, the presence of an external scaffold significantly improved preservation of volume and base area at 3 months compared to the naked group ( P < 0.05). Construct contraction was mitigated even further in the injection molded group, and topography of the ridged constructs was maintained with greater fidelity ( P < 0.05). Histology verified the development of mature auricular cartilage in the constructs within external scaffolds after 3 months. Conclusion: Custom-designed, 3D-printed, biocompatible external scaffolds significantly mitigate BAuC-seeded construct contraction and maintain complex topography. Further refinement and scaling of this approach in conjunction with construct fabrication utilizing injection molding may aid in the development of full-scale auricular scaffolds.

A hydrogel derived from acellular blood vessel extracellular matrix to promote angiogenesis

2019 ◽  
Vol 33 (10) ◽  
pp. 1301-1313
Author(s):  
Wei Fu ◽  
Peng Xu ◽  
Bei Feng ◽  
Yang Lu ◽  
Jie Bai ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Blood Vessel ◽  
Type I Collagen ◽  
Umbilical Vein ◽  
Skin Flap ◽  
Therapeutic Effects ◽  
Type I ◽  
Hematoxylin And Eosin

The biocompatibility and bioactivity of injectable acellular extracellular matrix nominates its use as an optimal candidate for cell delivery, serving as a reconstructive scaffold. In this study, we investigated the feasibility of preparing a blood vessel matrix (BVM) hydrogel, which revealed its pro-angiogenic effects in vitro and its therapeutic effects in an in vivo skin flap model. Aortic and abdominal aortic arteries from pigs were acellularized by Triton-X 100 and confirmed by hematoxylin and eosin and 4,6-diamidino-2-phenylindole staining. Different concentrations of blood vessel matrix hydrogel were generated successfully through enzymatic digestion, neutralization, and gelation. Hematoxylin and eosin staining, Masson’s trichrome staining, collagen type I immunohistochemistry staining, and enzyme-linked immunosorbent assays showed that type I collagen and some growth factors were retained in the hydrogel. Scanning electron microscopy demonstrated the different diametric fibrils in blood vessel matrix hydrogels. A blood vessel matrix hydrogel-coated plate promoted the tube formation of human umbilical vein endothelial cells in vitro. After injection into skin flaps, the hydrogel improved the flap survival rate and increased blood perfusion and capillary density. These results indicated that we successfully prepared a blood vessel matrix hydrogel and demonstrated its general characteristics and angiogenic effects in vitro and in vivo.

scholarly journals Matrix metalloproteinase-2 governs lymphatic vessel formation as an interstitial collagenase

Blood ◽  
2012 ◽  
Vol 119 (21) ◽  
pp. 5048-5056 ◽  
Author(s):  
Benoit Detry ◽  
Charlotte Erpicum ◽  
Jenny Paupert ◽  
Silvia Blacher ◽  
Catherine Maillard ◽  
...  
Keyword(s):  
Matrix Metalloproteinase ◽  
Lymphatic Vessel ◽  
Type I Collagen ◽  
3D Culture ◽  
Matrix Composition ◽  
Matrix Metalloproteinase 2 ◽  
Type I ◽  
Vessel Formation

Abstract Lymphatic dysfunctions are associated with several human diseases, including lymphedema and metastatic spread of cancer. Although it is well recognized that lymphatic capillaries attach directly to interstitial matrix mainly composed of fibrillar type I collagen, the interactions occurring between lymphatics and their surrounding matrix have been overlooked. In this study, we demonstrate how matrix metalloproteinase (MMP)–2 drives lymphatic morphogenesis through Mmp2-gene ablation in mice, mmp2 knockdown in zebrafish and in 3D-culture systems, and through MMP2 inhibition. In all models used in vivo (3 murine models and thoracic duct development in zebrafish) and in vitro (lymphatic ring and spheroid assays), MMP2 blockage or down-regulation leads to reduced lymphangiogenesis or altered vessel branching. Our data show that lymphatic endothelial cell (LEC) migration through collagen fibers is affected by physical matrix constraints (matrix composition, density, and cross-linking). Transmission electron microscopy and confocal reflection microscopy using DQ-collagen highlight the contribution of MMP2 to mesenchymal-like migration of LECs associated with collagen fiber remodeling. Our findings provide new mechanistic insight into how LECs negotiate an interstitial type I collagen barrier and reveal an unexpected MMP2-driven collagenolytic pathway for lymphatic vessel formation and morphogenesis.

scholarly journals Cell-extracellular matrix interactions in the fluidic phase direct the topology and polarity of self-organized epithelial structures

2020 ◽  
Author(s):  
Mingxing Ouyang ◽  
Jiun-Yann Yu ◽  
Yenyu Chen ◽  
Linhong Deng ◽  
Chin-Lin Guo
Keyword(s):  
Extracellular Matrix ◽  
Large Scale ◽  
Type I Collagen ◽  
Mdck Cells ◽  
Early Stage ◽  
Single Cells ◽  
Confocal Imaging ◽  
Type I

AbstractIn vivo, cells are surrounded by extracellular matrix (ECM). To build organs from single cells, it is generally believed that ECM serves as a large-scale scaffold to coordinate cell positioning and differentiation. Nevertheless, how cells utilize cell-ECM interactions to spatiotemporally coordinate their positioning and differentiation to different ECM at the whole-tissue scale is not fully understood. Here, using in vitro assay with engineered MDCK cells co-expressing H2B-mCherry (nucleus) and gp135 (Podocalyxin)-GFP (apical marker), we show that such spatiotemporal coordination for epithelial morphogenesis and polarization can be initiated and determined by cell-soluble ECM interaction in the fluidic phase. The coordination depends on the native topology of ECM components such as sheet-like basement membrane (BM, mimicked by Matrigel in experiments) and linear fiber-like type I collagen (COL). Two types of coordination are found: scaffold formed by BM (COL) facilitates a close-ended (open-ended) coordination that leads to the formation of lobular (tubular) epithelium, where polarity is preserved throughout the entire lobule/tubule. During lobular formation with BM, polarization of individual cells within the same cluster occurs almost simultaneously, whereas the apicobasal polarization in the presence of COL can start at local regions and proceed in a collective way along the axis of tubule, which might suggest existence of intercellular communications at the cell-population level. Further, in the fluidic phase, we found that cells can form apicobasal polarity throughout the entire lobule/tubule without a complete coverage of ECM at the basal side. Based on reconstructions from time-lapse confocal imaging, this is likely derived from polarization occurring at early stage and being maintained through growth of the epithelial structures. Under suspension culture with COL, the polarization was impaired with formation of multi-lumens on the tubes, implying the importance of ECM microenvironment for tubulogenesis. Our results suggest a mechanism for cells to form polarity and coordinate positioning in vivo, and a strategy for engineering epithelial structures through cell-soluble ECM interaction and self-assembly in vitro.

scholarly journals The Impact of the Extracellular Matrix Environment on Sost Expression by the MLO-Y4 Osteocyte Cell Line

2022 ◽  
Vol 9 (1) ◽  
pp. 35
Author(s):  
Robert T. Brady ◽  
Fergal J. O’Brien ◽  
David A. Hoey
Keyword(s):  
Extracellular Matrix ◽  
Cell Line ◽  
Three Dimensional ◽  
3D Culture ◽  
Specific Gene ◽  
Collagen Scaffolds ◽  
Sost Gene ◽  
The Impact

Bone is a dynamic organ that can adapt its structure to meet the demands of its biochemical and biophysical environment. Osteocytes form a sensory network throughout the tissue and orchestrate tissue adaptation via the release of soluble factors such as a sclerostin. Osteocyte physiology has traditionally been challenging to investigate due to the uniquely mineralized extracellular matrix (ECM) of bone leading to the development of osteocyte cell lines. Importantly, the most widely researched and utilized osteocyte cell line: the MLO-Y4, is limited by its inability to express sclerostin (Sost gene) in typical in-vitro culture. We theorised that culture in an environment closer to the in vivo osteocyte environment could impact on Sost expression. Therefore, this study investigated the role of composition and dimensionality in directing Sost expression in MLO-Y4 cells using collagen-based ECM analogues. A significant outcome of this study is that MLO-Y4 cells, when cultured on a hydroxyapatite (HA)-containing two-dimensional (2D) film analogue, expressed Sost. Moreover, three-dimensional (3D) culture within HA-containing collagen scaffolds significantly enhanced Sost expression, demonstrating the impact of ECM composition and dimensionality on MLO-Y4 behaviour. Importantly, in this bone mimetic ECM environment, Sost expression was found to be comparable to physiological levels. Lastly, MLO-Y4 cells cultured in these novel conditions responded accordingly to fluid flow stimulation with a decrease in expression. This study therefore presents a novel culture system for the MLO-Y4 osteocyte cell line, ensuring the expression of an important osteocyte specific gene, Sost, overcoming a major limitation of this model.

A Collagen/DBP Sponge System Designed for in Vitro Analysis of Chondroinduction

1991 ◽  
Vol 252 ◽  
Author(s):  
Shuichi Mizuno ◽  
Chris Lycette ◽  
Charlene Quinto ◽  
Julie Glowacki
Keyword(s):  
Type I Collagen ◽  
Three Dimensional ◽  
Dermal Fibroblasts ◽  
Type I ◽  
Cellular Mechanisms ◽  
Fresh Tissue ◽  
Freeze Dried ◽  
Bone Powder

ABSTRACTIn response to subcutaneous implants of demineralized bone powder (DBP), cells are attracted to the DBP, are converted to chondroblasts, and produce a cartilage matrix that is resorbed and replaced by bone. In order to define the cellular mechanisms of this induction, we developed a collagen sponge model for simulating the in vivo environment and for promoting the ingrowth and viability of cells cultured in them in vitro. Reconstituted pepsin–digested type I collagen from bovine hide was neutralized. Rat DBP (75–250 εm) was added into the collagen mixture (20 mg/ml). In order to simulate the connective tissue environment, modified chondroitin sulfate, heparan sulfate, or hyaluronic acid was added into the mixture. Aliquots (0.2 ml) were placed in 3/8 inch diameter molds and freeze-dried. Human dermal fibroblasts were cultured from minced fresh tissue and inoculated at 1.5 × 105 cells/sponge. Fifteen hours later, some sponges were transferred to medium which contained growth factors (PDGF or TGF-β). At intervals, samples were examined histologically. The inoculated cells attached to the collagen fibers and migrated into the sponge. Eventually the sponges contracted and acquired an oval shape. Cells on or near DBP were ovoid or stellate in shape. Cell morphology was modulated by glycosaminoglycan composition of the sponge. Increasing doses of PDGF or TGF-β promoted cellularity within the sponges. In conclusion, this system simulates the in vivo environment but allows accessibility for analysis. This three-dimensional matrix culture system will enable investigation of mechanisms of chondroinduction by morphogenic material.

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