Stress-Dependent Effects of Platelet-Derived Growth Factor-BB on Fibroblast Migration and Traction Differ in Collagen and Fibrin

2000 ◽  
Author(s):  
David I. Shreiber ◽  
Paul A. J. Enever ◽  
Robert T. Tranquillo
Keyword(s):  
Type I Collagen ◽  
Cell Behavior ◽  
Environmental Cues ◽  
Type I ◽  
Collagen Gels ◽  
Fibrin Gels ◽  
Fibroblast Migration ◽  
Tissue Equivalents ◽  
Stress Dependent ◽  
Statistical Conclusion

Abstract We used our novel assays of cell behavior in tissue equivalents to study the dose-response effects of PDGF-BB on RDF migration and traction in mechanically stressed and stress-free type I collagen and fibrin gels. PDGF-BB increased fibroblast migration significantly in all assays, but the effects on traction depended on the presence of stress and the nature of the ECM. PDGF-BB decreased fibroblast traction in stressed collagen gels, but increased traction in stress-free gels. No statistical conclusion could be inferred for stressed fibrin gels, and increasing PDGF-BB decreased traction in stress-free fibrin gels. These results demonstrate the complex response of fibroblasts to environmental cues, and point to opportunities to orchestrate cell behavior to affect the outcome of wound healing.

scholarly journals Collagenolytic Potential of Rat Liver Myofibroblasts

2013 ◽  
pp. 15-25 ◽  
Author(s):  
A. JIROUTOVÁ ◽  
E. PETEROVÁ ◽  
L. BITTNEROVÁ ◽  
R. SLAVKOVSKÝ ◽  
P. ČEVELOVÁ ◽  
...  
Keyword(s):  
Rat Liver ◽  
Type I Collagen ◽  
Collagen Gel ◽  
Plastic Substrate ◽  
Type I ◽  
Collagen Gels ◽  
Rt Pcr ◽  
Fibrotic Liver ◽  
Fibrin Gels ◽  
Liver Myofibroblasts

Rat liver myofibroblasts (MFB) were isolated by repeated passaging of nonparenchymal liver cell fraction. They were cultured on polystyrene Petri dishes, on fibrin or on type I collagen gels for 5 days. Quantitative RT-PCR, Western blotting, zymography and immunocytochemistry were used to study differences in cell morphology and protein expression. MFB were large and spread on plastic substrate, with prominent α-smooth muscle (α-SMA) fibres. They turned much smaller and elongated on collagen which was accompanied by the rearrangement of the cytoskeleton and a decrease in α-SMA and β-actin content. Collagen gel induced the expression of a group of metalloproteinases (MMP-2, -3, -9, -13), on mRNA and protein level which resulted in the degradation of the gel. This response was accompanied by changes in the mRNA expression of cytokines of TGF-β family, CTGF and interleukin-6, as well as of osteopontin and thrombospondin-2 that are involved in metalloproteinases (MMPs) regulation. The expression of MMPs substrates, collagen types I, IV and XII did not change or decreased. The effects of fibrin gels on MFB were milder than those of collagen. MFB assumed to deposit collagen and other ECM components in fibrotic liver, besides hepatic stellate cells, also possess a great collagenolytic potential.

Human tracheobronchial epithelial cells direct migration of lung fibroblasts in three-dimensional collagen gels

1992 ◽  
Vol 262 (5) ◽  
pp. L535-L541 ◽  
Author(s):  
M. D. Infeld ◽  
J. A. Brennan ◽  
P. B. Davis
Keyword(s):  
Epithelial Cells ◽  
Type I Collagen ◽  
Three Dimensional ◽  
Cell Number ◽  
Tracheobronchial Tree ◽  
Lung Fibroblasts ◽  
Type I ◽  
Collagen Gels ◽  
Directed Migration ◽  
Fibroblast Migration

Normal airway morphogenesis and repair after injury depend in part on the interaction between the mesenchymal and epithelial cells in the tracheobronchial tree. We cultured human lung fibroblasts between layers of type I collagen gel and examined sections through these three-dimensional matrices to assess fibroblast migration. The migration assay used in these experiments allowed simultaneous assessment of directed and random fibroblast migration as well as cell number. We tested the hypothesis that human tracheobronchial epithelial (HTBE) cells direct the migration of fibroblasts. When fibroblasts were cultured alone, migration was nearly equivalent in the upper and lower collagen layers. When HTBE cells were plated on the upper collagen lattice, there was a net migration of fibroblasts toward the HTBE cells. The differential migration was evident early in culture but became maximal after 1 wk. Differences increased at higher HTBE cell inoculation densities. No epithelial chemokinetic or mitogenic influence was evident: total cell migration and total fibroblast number were not significantly different between the control and coculture sections. HTBE fibronectin production may contribute to directed migration because fibronectin, added to the upper lattice, reproduced a portion of the directed migration seen in coculture. Our data support the hypothesis that epithelial cells direct fibroblast migration.

Abstract 418: Co-Culture of Endothelial Cells, Smooth Muscle Cells and Monocytes in Collagen Scaffold: A Novel i n vitro Model of Atherosclerosis

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Martin Liu ◽  
Angelos Karagiannis ◽  
Matthew Sis ◽  
Srivatsan Kidambi ◽  
Yiannis Chatzizisis
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Type I Collagen ◽  
Smooth Muscle Actin ◽  
Muscle Cells ◽  
Type I ◽  
Collagen Gels ◽  
Human Coronary Artery

Objectives: To develop and validate a 3D in-vitro model of atherosclerosis that enables direct interaction between various cell types and/or extracellular matrix. Methods and Results: Type I collagen (0.75 mg/mL) was mixed with human artery smooth muscle cells (SMCs; 6x10 5 cells/mL), medium, and water. Human coronary artery endothelial cells (HCAECs; 10 5 /cm 2 ) were plated on top of the collagen gels and activated with oxidized low density lipoprotein cholesterol (LDL-C). Monocytes (THP-1 cells; 10 5 /cm 2 ) were then added on top of the HCAECs. Immunofluorescence showed the expression of VE-cadherin by HCAECs (A, B) and α-smooth muscle actin by SMCs (A). Green-labelled LDL-C particles were accumulated in the subendothelial space, as well as in the cytoplasm of HCAECs and SMCs (C). Activated monocytes were attached to HCAECs and found in the subendothelial area (G-I). Both HCAECs and SMCs released IL-1β, IL-6, IL-8, PDGF-BB, TGF-ß1, and VEGF. Scanning and transmission electron microscopy showed the HCAECs monolayer forming gap junctions and the SMCs (D-F) and transmigrating monocytes within the collagen matrix (G-I). Conclusions: In this work, we presented a novel, easily reproducible and functional in-vitro experimental model of atherosclerosis that has the potential to enable in-vitro sophisticated molecular and drug development studies.

scholarly journals Migration of breast cancer cells into reconstituted type I collagen gels assessed via a combination of frozen sectioning and azan staining

2014 ◽  
Vol 8 (4) ◽  
pp. 212-216 ◽  
Author(s):  
Kyohei Fukuda ◽  
Yo Kamoshida ◽  
Taisuke Kurokawa ◽  
Mioto Yoshida ◽  
Yoko Fujita-Yamaguchi ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Type I Collagen ◽  
Type I ◽  
Collagen Gels

The synthesis of type X collagen by bovine and human growth-plate chondrocytes

1991 ◽  
Vol 99 (3) ◽  
pp. 641-649 ◽  
Author(s):  
A. Marriott ◽  
S. Ayad ◽  
M.E. Grant
Keyword(s):  
Growth Plate ◽  
Type I Collagen ◽  
Polyacrylamide Gel Electrophoresis ◽  
Human Growth ◽  
Type I ◽  
Collagen Gels ◽  
Type X Collagen ◽  
Growth Plate Chondrocytes ◽  
Growth Plate Cartilage ◽  
Disulphide Bonds

Chondrocytes were isolated from bovine growth-plate cartilage and cultured within type I collagen gels. A major collagen with chains of Mr 59,000, decreasing to 47,000 on pepsinization, was synthesized and identified as type X collagen. This collagen was cleaved at two sites by mammalian collagenase, resulting in a major triple-helical fragment with chains of Mr 32,000. The species of Mr 59,000, 47,000 and 32,000 were not detected by SDS-polyacrylamide gel electrophoresis before reduction, indicating the presence of disulphide bonds within the triple helix. In contrast, similar biosynthetic studies with human growth-plate cartilage in organ culture, indicated that human type X collagen does not contain disulphide bonds. A polyclonal antiserum was raised to bovine type X collagen and used in immunolocalization studies to provide direct evidence for the association of type X collagen with the hypertrophic chondrocytes in both bovine and human growth plates during development.

Cytoskeleton and thyroglobulin expression change during transformation of thyroid epithelium to mesenchyme-like cells

Development ◽  
1988 ◽  
Vol 102 (3) ◽  
pp. 605-622 ◽  
Author(s):  
G. Greenburg ◽  
E.D. Hay
Keyword(s):  
Type I Collagen ◽  
Collagen Gel ◽  
Type I ◽  
Basal Cells ◽  
Collagen Gels ◽  
Expression Change ◽  
Cell Functions ◽  
The Matrix ◽  
3D Matrix ◽  
Mesenchymal Transformation

In considering the mechanism of transformation of epithelium to mesenchyme in the embryo, it is generally assumed that the ability to give rise to fibroblast-like cells is lost as epithelia mature. We reported previously that a definitive embryonic epithelium, that of the anterior lens, gives rise to freely migrating mesenchyme-like cells when suspended in type I collagen matrices. Here, we show that a highly differentiated epithelium that expresses cytokeratin changes to a vimentin cytoskeleton and loses thyroglobulin during epithelial-mesenchymal transformation induced by suspension in collagen gel. Using dispase and collagenase, we isolated adult thyroid follicles devoid of basal lamina and mesenchyme, and we suspended the follicles in 3D collagen gels. Cells bordering the follicle lumen retain epithelial polarity and thyroid phenotype, but basal cell surface organization is soon modified as a result of tissue multilayering and elongation of basal cells into the collagenous matrix. Cytodifferentiation, determined by thyroglobulin immunoreactivity, is lost as the basal epithelial cells move into the matrix after 3–4 days in collagen. By TEM, it can be seen that the elongating cells acquire pseudopodia, filopodia and mesenchyme-like nuclei and RER. Immunofluorescence examination of intermediate filaments showed that freshly isolated follicles and follicles cultured on planar substrata react only with anticytokeratin. However, all of the mesenchyme-like cells express vimentin and they gradually lose cytokeratin. These results suggest that vimentin may be necessary for cell functions associated with migration within a 3D matrix. The mesenchymal cells do not revert to epithelium when grown on planar substrata and the transformation of epithelium to mesenchyme-like cells does not occur within basement membrane gels. The results are relevant to our understanding of the initiation of epithelial-mesenchymal transformation in the embryo and the genetic mechanisms controlling cell shape, polarity and cytoskeletal phenotype.

scholarly journals The Influence of a Nanopatterned Scaffold that Mimics Abnormal Renal Mesangial Matrix on Mesangial Cell Behavior

2019 ◽  
Vol 20 (21) ◽  
pp. 5349
Author(s):  
Chia-Jung Chang ◽  
Rin Minei ◽  
Takeshi Sato ◽  
Akiyoshi Taniguchi
Keyword(s):  
Molecular Mechanisms ◽  
Type I Collagen ◽  
Transforming Growth Factor ◽  
Cell Function ◽  
Glomerular Disease ◽  
Cell Behavior ◽  
Type I ◽  
Mesangial Matrix ◽  
Cell Functions ◽  
In Cells

The alteration of mesangial matrix (MM) components in mesangium, such as type IV collagen (COL4) and type I collagen (COL1), is commonly found in progressive glomerular disease. Mesangial cells (MCs) responding to altered MM, show critical changes in cell function. This suggests that the diseased MM structure could play an important role in MC behavior. To investigate how MC behavior is influenced by the diseased MM 3D nanostructure, we fabricated the titanium dioxide (TiO2)-based nanopatterns that mimic diseased MM nanostructures. Immortalized mouse MCs were used to assess the influence of disease-mimic nanopatterns on cell functions, and were compared with a normal-mimic nanopattern. The results showed that the disease-mimic nanopattern induced disease-like behavior, including increased proliferation, excessive production of abnormal MM components (COL1 and fibronectin) and decreased normal MM components (COL4 and laminin α1). In contrast, the normal-mimic nanopattern actually resulted in cells displaying normal proliferation and the production of normal MM components. In addition, increased expressions of α-smooth muscle actin (α-SMA), transforming growth factor β1 (TGF-β1) and integrin α5β1 were detected in cells grown on the disease-mimic nanopattern. These results indicated that the disease-mimic nanopattern induced disease-like cell behavior. These findings will help further establish a disease model that mimics abnormal MM nanostructures and also to elucidate the molecular mechanisms underlying glomerular disease.

Polarized Light Microscopy for Analyzing Tissue Mechanics During In Vitro Acupuncture

2008 ◽  
Author(s):  
Lowell Taylor Edgar ◽  
Margaret Julias ◽  
David I. Shreiber ◽  
Helen M. Buettner
Keyword(s):  
Connective Tissue ◽  
Light Microscopy ◽  
Type I Collagen ◽  
Polarized Light ◽  
Tissue Mechanics ◽  
Polarized Light Microscopy ◽  
Tissue Response ◽  
Type I ◽  
Collagen Gels

Acupuncture is a traditional therapy originating in China almost 2000 years ago. Acupuncture has slowly been growing in popularity in the West, and clinical evidence has shown the potential for acupuncture as a low-cost ‘alternative’ therapy for an assortment of ailments [1]. The practice of acupuncture involves inserting fine needles into the skin followed by needle manipulation, usually by rotation. Recent studies by Langevin et al demonstrate that this rotation causes the subcutaneous connective tissue to couple to and wind around the needle [2–4], which suggests that mechanotransduction in the connective tissue might play a role in the therapeutic mechanisms that underlay acupuncture [2, 3]. To begin to decompose and quantify this complex mechanism at the tissue level in a controlled setting, we have simulated acupuncture in type I collagen gels in vitro, and have developed algorithms to quantify the tissue response following imaging with polarized light microscopy (PLM).

scholarly journals Dynamic Stretching of Fibrillar Collagen Enhances Cross-linking by Transglutaminas

2019 ◽  
Vol 4 (4) ◽  
pp. 2473011419S0038
Author(s):  
Nicolas Shealy ◽  
James Rex ◽  
Amy Bradshaw ◽  
Christopher Gross
Keyword(s):  
Molecular Weight ◽  
Type I Collagen ◽  
Cross Linking ◽  
Type I ◽  
Fibrillar Collagen ◽  
Collagen Gels ◽  
Cross Link ◽  
Dynamic Stretching ◽  
Insoluble Collagen ◽  
Static Conditions

Category: Basic Sciences/Biologics Introduction/Purpose: New approaches to improve tendon repair after injury are an active area of research. Critical properties of tendons are governed by the production and assembly of fibrillar collagens. Cross-linking of fibrillar collagen is a primary factor in determining the function and mechanical properties of the collagen fibers comprising Enzymatic cross-linking by lysyl oxidase in the telopeptide domain of collagen I and III is one determinant of collagen fibril assembly and is the best characterized biochemical cross-link. Transglutaminase catalyzes the modification of lysine residues that in turn form an n-e-glutamyl lysine bond between proteins in the extracellular space. We hypothesize that transglutaminase-dependent modification of collagen in tendons is also a principal determinant of tendon strength and function and is dependent upon tension. Methods: 3-D collagen gels were generated from acid solubilized type I collagen with telopeptides (Advanced BioMatrix). Collagen gels were plated and loaded into a MechanoCulture FX apparatus (CellScale). Gels were subjected to a 10% stretch for 24 hrs at 37°C at 2hz (dynamic) or no stretch, static controls. Gels exposed to enzymatic cross-linking were incubated with either 2.4 ng of recombinant Transglutaminase 2 (Axxora) in a 10 mM Ca2+ solution. Inhibition and labeling of transglutaminase substrates was performed by incubation of collagen gels with 0.2 mM aminopentyl biotinamide in DMSO. Soluble collagen was separated from insoluble collagen by centrifugation at 10,000G. Insoluble fractions were boiled in SDS-Laemmli buffer prior to separation by SDS-PAGE. Collagen in soluble and insoluble fractions was evaluated by Coomassie stain whereas transglutaminase modification was detected via western blot using streptavidin conjugated horse radish peroxidase to detect biotinylated proteins. Results: Evaluation of collagen gels subjected to dynamic versus static stretch revealed minor differences in insoluble collagen incorporation in the two conditions. Notably, higher molecular weight cross-linked forms of collagen appeared to be higher in dynamic versus static gels. In the presence of transglutaminase, differences in higher molecular weight cross-linked forms of collagen, beta-bands, were also detected. Finally, incorporation of biotinylated transglutaminase substrate into collagen alpha bands was enriched in dynamic versus static cultures. Hence, preliminary results support a differential role for transglutaminase modification in collagen under cyclic tension versus static conditions. Conclusion: A better understanding of the role of dynamic stretching and differential tension in the regulation of collagen cross- link formation is predicted to contribute to improved strategies to treat injured tendons.

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