Local, Three-Dimensional Strain Measurements Within Largely Deformed Extracellular Matrix Constructs

The ability to create extracellular matrix (ECM) constructs that are mechanically and biochemically similar to those found in vivo and to understand how their properties affect cellular responses will drive the next generation of tissue engineering strategies. To date, many mechanisms by which cells biochemically communicate with the ECM are known. However, the mechanisms by which mechanical information is transmitted between cells and their ECM remain to be elucidated. “Self-assembled” collagen matrices provide an in vitro-model system to study the mechanical behavior of ECM. To begin to understand how the ECM and the cells interact mechanically, the three-dimensional (3D) mechanical properties of the ECM must be quantified at the micro-(local) level in addition to information measured at the macro-(global) level. Here we describe an incremental digital volume correlation (IDVC) algorithm to quantify large (>0.05) 3D mechanical strains in the microstructure of 3D collagen matrices in response to applied mechanical loads. Strain measurements from the IDVC algorithm rely on 3D confocal images acquired from collagen matrices under applied mechanical loads. The accuracy and the precision of the IDVC algorithm was verified by comparing both image volumes collected in succession when no deformation was applied to the ECM (zero strain) and image volumes to which simulated deformations were applied in both 1D and 3D (simulated strains). Results indicate that the IDVC algorithm can accurately and precisely determine the 3D strain state inside largely deformed collagen ECMs. Finally, the usefulness of the algorithm was demonstrated by measuring the microlevel 3D strain response of a collagen ECM loaded in tension.

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In vitro rapid organization of endothelial cells into capillary-like networks is promoted by collagen matrices.

The Journal of Cell Biology ◽

10.1083/jcb.97.5.1648 ◽

1983 ◽

Vol 97 (5) ◽

pp. 1648-1652 ◽

Cited By ~ 428

Author(s):

R Montesano ◽

L Orci ◽

P Vassalli

Keyword(s):

Endothelial Cells ◽

Three Dimensional ◽

Collagen Matrix ◽

Collagen Gels ◽

Collagen Matrices ◽

Narrow Lumen ◽

Capillary Endothelial Cells ◽

Vitro Model

We have studied the behavior of cloned capillary endothelial cells grown inside a three dimensional collagen matrix. Cell monolayers established on the surface of collagen gels were covered with a second layer of collagen. This induced the monolayers of endothelial cells to reorganize into a network of branching and anastomosing capillary-like tubes. As seen by electron microscopy, the tubes were formed by at least two cells (in transverse sections) delimiting a narrow lumen. In addition, distinct basal lamina material was present between the abluminal face of the endothelial cells and the collagen matrix. These results showed that capillary endothelial cells have the capacity to form vessel-like structures with well-oriented cell polarity in vitro. They also suggest that an appropriate topological relationship of endothelial cells with collagen matrices, similar to that occurring in vivo, has an inducive role on the expression of this potential. This culture system provides a simple in vitro model for studying the factors involved in the formation of new blood vessels (angiogenesis).

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Interaction between vascular endothelial cells and vascular intimal spindle-shaped cells in vitro

Journal of Cell Science ◽

10.1242/jcs.60.1.89 ◽

1983 ◽

Vol 60 (1) ◽

pp. 89-102

Author(s):

D de Bono ◽

C. Green

Keyword(s):

Extracellular Matrix ◽

Endothelial Cells ◽

Vascular Endothelial Cells ◽

Three Dimensional ◽

Vascular Endothelial ◽

Pure Cultures ◽

Species Specific ◽

Endothelial Growth Factor

The interactions between human or bovine vascular endothelial cells and fibroblast-like vascular intimal spindle-shaped cells have been studied in vitro, using species-specific antibodies to identify the different components in mixed cultures. Pure cultures of endothelial cells grow as uniform, nonoverlapping monolayers, but this growth pattern is lost after the addition of spindle cells, probably because the extracellular matrix secreted by the latter causes the endothelial cells to modify the way they are attached to the substrate. The result is a network of tubular aggregates of endothelial cells in a three-dimensional ‘polylayer’ of spindle-shaped cells. On the other hand, endothelial cells added to growth-inhibited cultures of spindle-shaped cells will grow in sheets over the surface of the culture. Human endothelial cells grown in contact with spindle-shaped cells have a reduced requirement for a brain-derived endothelial growth factor. The interactions of endothelial cells and other connective tissue cells in vitro may be relevant to the mechanisms of endothelial growth and blood vessel formation in vivo, and emphasize the potential importance of extracellular matrix in controlling endothelial cell behaviour.

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Human induced pluripotent stem cell-derived three-dimensional cardiomyocyte tissues ameliorate the rat ischemic myocardium by remodeling the extracellular matrix and cardiac protein phenotype

PLoS ONE ◽

10.1371/journal.pone.0245571 ◽

2021 ◽

Vol 16 (3) ◽

pp. e0245571

Author(s):

Junya Yokoyama ◽

Shigeru Miyagawa ◽

Takami Akagi ◽

Mitsuru Akashi ◽

Yoshiki Sawa

Keyword(s):

Myocardial Infarction ◽

Extracellular Matrix ◽

Pluripotent Stem Cell ◽

Heart Function ◽

Three Dimensional ◽

Induced Pluripotent Stem Cell ◽

Left Ventricular ◽

Induced Pluripotent

The extracellular matrix (ECM) plays a key role in the viability and survival of implanted human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). We hypothesized that coating of three-dimensional (3D) cardiac tissue-derived hiPSC-CMs with the ECM protein fibronectin (FN) would improve the survival of transplanted cells in the heart and improve heart function in a rat model of ischemic heart failure. To test this hypothesis, we first explored the tolerance of FN-coated hiPSC-CMs to hypoxia in an in vitro study. For in vivo assessments, we constructed 3D-hiPSC cardiac tissues (3D-hiPSC-CTs) using a layer-by-layer technique, and then the cells were implanted in the hearts of a myocardial infarction rat model (3D-hiPSC-CTs, n = 10; sham surgery control group (without implant), n = 10). Heart function and histology were analyzed 4 weeks after transplantation. In the in vitro assessment, cell viability and lactate dehydrogenase assays showed that FN-coated hiPSC-CMs had improved tolerance to hypoxia compared with the control cells. In vivo, the left ventricular ejection fraction of hearts implanted with 3D-hiPSC-CT was significantly better than that of the sham control hearts. Histological analysis showed clear expression of collagen type IV and plasma membrane markers such as desmin and dystrophin in vivo after implantation of 3D-hiPSC-CT, which were not detected in 3D-hiPSC-CMs in vitro. Overall, these results indicated that FN-coated 3D-hiPSC-CT could improve distressed heart function in a rat myocardial infarction model with a well-expressed cytoskeletal or basement membrane matrix. Therefore, FN-coated 3D-hiPSC-CT may serve as a promising replacement for heart transplantation and left ventricular assist devices and has the potential to improve survivability and therapeutic efficacy in cases of ischemic heart disease.

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Porcine hepatocytes culture on biofunctionalized 3D inverted colloidal crystal scaffolds as an in vitro model for predicting drug hepatotoxicity

RSC Advances ◽

10.1039/c9ra03225h ◽

2019 ◽

Vol 9 (31) ◽

pp. 17995-18007 ◽

Cited By ~ 2

Author(s):

Lingyan Wu ◽

Gaia Ferracci ◽

Yan Wang ◽

Teng Fei Fan ◽

Nam-Joon Cho ◽

...

Keyword(s):

In Vitro Model ◽

Colloidal Crystal ◽

Three Dimensional ◽

Drug Induced ◽

Attrition Rates ◽

Common Causes ◽

Vitro Model ◽

Colloidal Crystal Scaffolds

As drug-induced hepatotoxicity represents one of the most common causes of drug failure, three-dimensional in vitro liver platforms represent a fantastic toolbox to predict drug toxicity and reduce in vivo studies and drug attrition rates.

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RGD-modified injectable hydrogel maintains islet beta-cell survival and function

Journal of Applied Biomaterials & Functional Materials ◽

10.1177/2280800020963473 ◽

2020 ◽

Vol 18 ◽

pp. 228080002096347

Author(s):

Tianshu Lan ◽

Jingyi Guo ◽

Xiaoming Bai ◽

Zengjiong Huang ◽

Zhimin Wei ◽

...

Keyword(s):

Extracellular Matrix ◽

Islet Transplantation ◽

Pancreatic Beta Cells ◽

Three Dimensional ◽

High Concentration ◽

Β Cells ◽

Glucose Levels ◽

And Function

Objective: A potential solution for islet transplantation and drug discovery vis-à-vis treating diabetes is the production of functional islets in a three-dimensional extracellular matrix. Although several scaffold materials have been reported as viable candidates, a clinically applicable one that is injectable and can maintain long-term functionality and survival of islet pancreatic beta-cells (β-cells) is far from being established. Results: In the current study, we evaluated a ready-to-use and injectable hydrogel’s impact on β-cells’ function and viability, both in vitro and in vivo. We found that β-cells in high concentration with hydrogels functionalized via Arg-Gly-Asp (RGD) demonstrated better viability and insulin secretory capacity in vitro. Moreover, it is a biocompatible hydrogel that can maintain β-cell proliferation and vascularization without stimulating inflammation after subcutaneous injection. Meanwhile, modifying the hydrogel with RGD can maintain β-cells’ secretion of insulin, regulating the blood glucose levels of mice with streptozotocin-induced diabetes. Conclusions: Thus, these preliminary results indicate that this RGD-modified hydrogel is a potential extracellular matrix for islet transplantation at extrahepatic sites, and they also provide a reference for future tissue engineering study.

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Mechanobiology of Epithelia From the Perspective of Extracellular Matrix Heterogeneity

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2020.596599 ◽

2020 ◽

Vol 8 ◽

Author(s):

Aleksandra N. Kozyrina ◽

Teodora Piskova ◽

Jacopo Di Russo

Keyword(s):

Extracellular Matrix ◽

Three Dimensional ◽

Cell Sheets ◽

Ecm Remodeling ◽

Cellular Behavior ◽

New Concepts ◽

Matrix Heterogeneity ◽

The Impact

Understanding the complexity of the extracellular matrix (ECM) and its variability is a necessary step on the way to engineering functional (bio)materials that serve their respective purposes while relying on cell adhesion. Upon adhesion, cells receive messages which contain both biochemical and mechanical information. The main focus of mechanobiology lies in investigating the role of this mechanical coordination in regulating cellular behavior. In recent years, this focus has been additionally shifted toward cell collectives and the understanding of their behavior as a whole mechanical continuum. Collective cell phenomena very much apply to epithelia which are either simple cell-sheets or more complex three-dimensional structures. Researchers have been mostly using the organization of monolayers to observe their collective behavior in well-defined experimental setups in vitro. Nevertheless, recent studies have also reported the impact of ECM remodeling on epithelial morphogenesis in vivo. These new concepts, combined with the knowledge of ECM biochemical complexity are of key importance for engineering new interactive materials to support both epithelial remodeling and homeostasis. In this review, we summarize the structure and heterogeneity of the ECM before discussing its impact on the epithelial mechanobiology.

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Crosstalk between invadopodia and the extracellular matrix

10.1101/2020.02.26.966762 ◽

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.

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

Bioengineering ◽

10.3390/bioengineering9010035 ◽

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.

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TGF-β Plays a Key Role in Morphogenesis of the Pancreatic Islets of Langerhans by Controlling the Activity of the Matrix Metalloproteinase MMP-2

The Journal of Cell Biology ◽

10.1083/jcb.143.3.827 ◽

1998 ◽

Vol 143 (3) ◽

pp. 827-836 ◽

Cited By ~ 126

Author(s):

Francisco Miralles ◽

Tadej Battelino ◽

Paul Czernichow ◽

Raphael Scharfmann

Keyword(s):

Extracellular Matrix ◽

Matrix Metalloproteinase ◽

Islets Of Langerhans ◽

Neutralizing Antibody ◽

Matrix Metalloproteinase 2 ◽

Pancreatic Islets Of Langerhans ◽

The Matrix ◽

Vitro Model

Islets of Langerhans are microorgans scattered throughout the pancreas, and are responsible for synthesizing and secreting pancreatic hormones. While progress has recently been made concerning cell differentiation of the islets of Langerhans, the mechanism controlling islet morphogenesis is not known. It is thought that these islets are formed by mature cell association, first differentiating in the primitive pancreatic epithelium, then migrating in the extracellular matrix, and finally associating into islets of Langerhans. This mechanism suggests that the extracellular matrix has to be degraded for proper islet morphogenesis. We demonstrated in the present study that during rat pancreatic development, matrix metalloproteinase 2 (MMP-2) is activated in vivo between E17 and E19 when islet morphogenesis occurs. We next demonstrated that when E12.5 pancreatic epithelia develop in vitro, MMP-2 is activated in an in vitro model that recapitulates endocrine pancreas development (Miralles, F., P. Czernichow, and R. Scharfmann. 1998. Development. 125: 1017–1024). On the other hand, islet morphogenesis was impaired when MMP-2 activity was inhibited. We next demonstrated that exogenous TGF-β1 positively controls both islet morphogenesis and MMP-2 activity. Finally, we demonstrated that both islet morphogenesis and MMP-2 activation were abolished in the presence of a pan-specific TGF-β neutralizing antibody. Taken together, these observations demonstrate that in vitro, TGF-β is a key activator of pancreatic MMP-2, and that MMP-2 activity is necessary for islet morphogenesis.

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