Neurite growth in 3D collagen gels with gradients of mechanical properties

2009 ◽  
Vol 102 (2) ◽  
pp. 632-643 ◽  
Author(s):  
Harini G. Sundararaghavan ◽  
Gary A. Monteiro ◽  
Bonnie L. Firestein ◽  
David I. Shreiber
Keyword(s):  
Mechanical Properties ◽  
Neurite Growth ◽  
Collagen Gels ◽  
3D Collagen

Gradients of Stiffness Guide Neurite Growth in 3D Collagen Gels

2007 ◽  
Author(s):  
Harini G. Sundararaghavan ◽  
David I. Shreiber
Keyword(s):  
Mechanical Properties ◽  
Crosslinking Agent ◽  
Collagen Gel ◽  
Neurite Growth ◽  
Mechanical Anisotropy ◽  
Spinal Cord Regeneration ◽  
Collagen Gels ◽  
Differential Growth ◽  
Biomaterial Scaffold ◽  
3D Collagen

One approach to enhance nerve and spinal cord regeneration following injury is to implant a biomaterial scaffold to ”bridge” the gap of the injury. Structural/mechanical anisotropy has been suggested as a means of orienting this growth axially. We have spatially varied the mechanical properties of a 3D collagen gel to direct growth axially and unidirectionally. Gradients of mechanical properties were generated in collagen gels by exposing the collagen to a 0–1mM gradient of genipin, a cell-tolerated crosslinking agent, for 12hrs via microfluidics. The gradient of stiffness was confirmed via a gradient of genipin-induced fluorescence intensity, which we have previously correlated to the storage modulus of collagen gels. The growth of neurites from isolated chick embryo dorsal root ganglia (DRG) in the presence of these gradients was evaluated after 5 days in culture. In control cases, neurites grew into the collagen gel and up either side of the cross-channel to approximately equal lengths. A 20% difference in differential growth was observed in control experiments. In contrast, when presented a gradient of shear modulus from ∼365Pa – 60Pa, neurites elected to grow down the gradient of stiffness to the compliant side, with an almost 300% difference. Interestingly, the length of neurites in gels with gradients was significantly greater than the length of those grown in gels with uniform, untreated gels with high compliance. Control of neurite growth, cell migration, and other aspects of cell behavior in 3D scaffolds via mechanical properties offers vast potential for tissue engineering and other regenerative therapies.

Guided Axon Growth by Gradients of Adhesion in Collagen Gels

2008 ◽  
Author(s):  
Harini G. Sundararaghavan ◽  
Gary A. Monteiro ◽  
David I. Shreiber
Keyword(s):  
Spinal Cord ◽  
Collagen Gel ◽  
Axon Growth ◽  
Neurite Growth ◽  
Gradient System ◽  
Spinal Cord Regeneration ◽  
Collagen Gels ◽  
Direct Growth ◽  
3D Collagen

During development, neurites are directed by gradients of attractive and repulsive soluble (chemotactic) cues and substrate-bound adhesive (haptotactic) cues. Many of these cues have been extensively researched in vitro, and incorporated into strategies for nerve and spinal cord regeneration, primarily to improve the regenerative environment. To enhance and direct growth, we have developed a system to create 1D gradients of adhesion through a 3D collagen gel using microfluidics. We test our system using collagen grafted with bioactive peptide sequences, IKVAV and YIGSR, from laminin — an extra-cellular matrix (ECM) protein known to strongly influence neurite outgrowth. Gradients are established from ∼0.37mg peptide/mg collagen – 0, and ∼0.18 mg peptide/mg collagen – 0 of each peptide and tested using chick dorsal root ganglia (DRG). Neurite growth is evaluated 5 days after gradient formation. Neurites show increased growth in the gradient system when compared to control and biased growth up the gradient of peptides. Growth in YIGSR-grafted collagen increased with steeper gradients, whereas growth in IKVAV-grafted collagen decreased with steeper gradients. These results demonstrate that neurite growth can be enhanced and directed by controlled, immobilized, haptotactic gradients through 3D scaffolds, and suggest that including these gradients in regenerative therapies may accelerate nerve and spinal cord regeneration.

Microfluidic Generation of Adhesion Gradients Through 3D Collagen Gels: Implications for Neural Tissue Engineering

2008 ◽  
Author(s):  
Harini G. Sundararaghavan ◽  
Gary A. Monteiro ◽  
David I. Shreiber
Keyword(s):  
Spinal Cord ◽  
Collagen Gel ◽  
Neurite Growth ◽  
Gradient System ◽  
Spinal Cord Regeneration ◽  
Collagen Gels ◽  
Direct Growth ◽  
3D Collagen ◽  
Regenerative Therapies

During development, neurites are directed by gradients of attractive and repulsive soluble (chemotactic) cues and substrate-bound adhesive (haptotactic) cues. Many of these cues have been extensively researched in vitro, and incorporated into strategies for nerve and spinal cord regeneration, primarily to improve the regenerative environment. To enhance and direct growth, we have developed a system to create 1D gradients of adhesion through a 3D collagen gel using microfluidics. We test our system using collagen grafted with bioactive peptide sequences, IKVAV and YIGSR, from laminin — an extra-cellular matrix (ECM) protein known to strongly influence neurite outgrowth [1, 2]. Gradients are established from 0.14 mg/ml–0, and 0.07 mg/ml–0 of each peptide and tested using chick dorsal root ganglia (DRG). Neurite growth is evaluated 5 days after gradient formation. Neurites show increased growth in the gradient system when compared to control and biased growth up the gradient of peptides. These results demonstrate that neurite growth can be enhanced and directed by controlled, immobilized, haptotactic gradients through 3D scaffolds, and suggest that including these gradients in regenerative therapies may accelerate nerve and spinal cord regeneration.

scholarly journals Microfluidic Generation of Haptotactic Gradients through 3D Collagen Gels for Enhanced Neurite Growth

2011 ◽  
Vol 28 (11) ◽  
pp. 2377-2387 ◽  
Author(s):  
Harini G. Sundararaghavan ◽  
Shirley N. Masand ◽  
David I. Shreiber
Keyword(s):  
Neurite Growth ◽  
Collagen Gels ◽  
3D Collagen

scholarly journals Image-based Characterization of 3D Collagen Networks and the Effect of Embedded Cells

2019 ◽  
Vol 25 (4) ◽  
pp. 971-981 ◽  
Author(s):  
Vanesa Olivares ◽  
Mar Cóndor ◽  
Cristina Del Amo ◽  
Jesús Asín ◽  
Carlos Borau ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Cell Activity ◽  
Three Dimensional ◽  
Collagen Fibers ◽  
Collagen Gels ◽  
Culture Dish ◽  
Collagen Concentration ◽  
A Cell ◽  
3D Collagen

AbstractCollagen microstructure is closely related to the mechanical properties of tissues and affects cell migration through the extracellular matrix. To study these structures, three-dimensional (3D) in vitro collagen-based gels are often used, attempting to mimic the natural environment of cells. Some key parameters of the microstructure of these gels are fiber orientation, fiber length, or pore size, which define the mechanical properties of the network and therefore condition cell behavior. In the present study, an automated tool to reconstruct 3D collagen networks is used to extract the aforementioned parameters of gels of different collagen concentration and determine how their microstructure is affected by the presence of cells. Two different experiments are presented to test the functionality of the method: first, collagen gels are embedded within a microfluidic device and collagen fibers are imaged by using confocal fluorescence microscopy; second, collagen gels are directly polymerized in a cell culture dish and collagen fibers are imaged by confocal reflection microscopy. Finally, we investigate and compare the collagen microstructure far from and in the vicinities of MDA-MB 23 cells, finding that cell activity during migration was able to strongly modify the orientation of the collagen fibers and the porosity-related values.

Mimicking osteocytes in vivo using 3D collagen gels: development of a novel tool to study osteocyte biology

2013 ◽  
pp. 1-1
Author(s):  
Nicole Scully ◽  
Sam L Evans ◽  
Deborah J Mason ◽  
Bronwen A J Evans
Keyword(s):  
Collagen Gels ◽  
3D Collagen

Effect of Freezing on Cell Viability and Mechanical Strength of Bioartificial Tissues

2001 ◽  
Author(s):  
Ramachandra V. Devireddy ◽  
Michael R. Neidert ◽  
John C. Bischof ◽  
Robert T. Tranquillo
Keyword(s):  
Mechanical Properties ◽  
Mechanical Strength ◽  
Cell Viability ◽  
Ultimate Tensile Stress ◽  
Collagen Gels ◽  
Cooling Rates ◽  
Human Foreskin Fibroblasts ◽  
Lower Strain ◽  
Maximum Stiffness ◽  
Eden Prairie

Abstract The effect of freezing on the viability and mechanical strength of bioartificial tissues was determined under a variety of cooling conditions, with the ultimate aim of optimizing the cryopreservation process. Bioartificial tissues (i.e. tissue-equivalents or TEs) were prepared by incubating entrapped human foreskin fibroblasts in collagen gels for a period of 2 weeks. The bioartificial tissues were frozen using a controlled rate freezer at various cooling rates (0.5, 2, 5, 20, 40 and > 1000°C/min or slam freezing). The viability (< 60 min after thawing) of the fibroblasts in the bioartificial tissue was assessed using the Ethidium Homodimer (dead cells stain red) and Hoechst Give cells stain blue) assay. Uniaxial tension experiments were performed on an MTS Microbionix System (Eden Prairie, MN) to assess the post-thaw mechanical properties (Maximum Stiffness; Ultimate Tensile Stress; and Strain to Failure) of the frozen-thawed bioartificial tissue (≤ 3 hours after thawing). The results suggest that cooling rates of either 2 or 5°C/min are optimal for preserving both the cell viability and mechanical properties of the bioartificial tissues, post-freeze. Bioartificial tissues were also frozen using a directional solidification stage at 5°C/min. The post-thaw viability results are comparable in both the directionally cooled and the controlled rate freezer samples. However, the mechanical properties of the directionally cooled samples are significantly different (with a higher maximum stiffness and a lower strain to failure) than those obtained for samples frozen using a controlled rate freezer. This suggests that the directionality of ice propagation into the sample affects the measured mechanical properties.

scholarly journals Matrix metalloproteinase-9 activates TGF-β and stimulates fibroblast contraction of collagen gels

2014 ◽  
Vol 306 (11) ◽  
pp. L1006-L1015 ◽  
Author(s):  
Tetsu Kobayashi ◽  
HuiJung Kim ◽  
Xiangde Liu ◽  
Hisatoshi Sugiura ◽  
Tadashi Kohyama ◽  
...  
Keyword(s):  
Matrix Metalloproteinase ◽  
Human Fibroblasts ◽  
Matrix Metalloproteinase 9 ◽  
Lung Fibroblasts ◽  
Type I ◽  
Image Analysis System ◽  
Collagen Gels ◽  
3D Collagen ◽  
Metalloproteinase 9 ◽  
Tgf Β1

Matrix metalloproteinase-9 (MMP-9) is a matrix-degrading enzyme implicated in many biological processes, including inflammation. It is produced by many cells, including fibroblasts. When cultured in three-dimensional (3D) collagen gels, fibroblasts contract the surrounding matrix, a function that is thought to model the contraction that characterizes both normal wound repair and fibrosis. The current study was designed to evaluate the role of endogenously produced MMP-9 in fibroblast contraction of 3D collagen gels. Fibroblasts from mice lacking expression of MMP-9 and human lung fibroblasts (HFL-1) transfected with MMP-9 small-interfering RNA (siRNA) were used. Fibroblasts were cast into type I collagen gels and floated in culture medium with or without transforming growth factor (TGF)-β1 for 5 days. Gel size was determined daily using an image analysis system. Gels made from MMP-9 siRNA-treated human fibroblasts contracted less than control fibroblasts, as did fibroblasts incubated with a nonspecific MMP inhibitor. Similarly, fibroblasts cultured from MMP-9-deficient mice contracted gels less than did fibroblasts from control mice. Transfection of the MMP-9-deficient murine fibroblasts with a vector expressing murine MMP-9 restored contractile activity to MMP-9-deficient fibroblasts. Inhibition of MMP-9 reduced active TGF-β1 and reduced several TGF-β1-driven responses, including activity of a Smad3 reporter gene and production of fibronectin. Because TGF-β1 also drives fibroblast gel contraction, this suggests the mechanism for MMP-9 regulation of contraction is through the generation of active TGF-β1. This study provides direct evidence that endogenously produced MMP-9 has a role in regulation of tissue contraction of 3D collagen gels mediated by fibroblasts.

The Effects of Geometry and Static Boundary Conditions on Microvessel Outgrowth in a 3D Model of Angiogenesis

2010 ◽  
Author(s):  
Clayton J. Underwood ◽  
Laxminarayanan Krishnan ◽  
Lowell T. Edgar ◽  
Steve Maas ◽  
James B. Hoying ◽  
...  
Keyword(s):  
Boundary Condition ◽  
Boundary Conditions ◽  
Direct Relationship ◽  
3D Model ◽  
Mechanical Strain ◽  
Matrix Stiffness ◽  
Collagen Gels ◽  
3D Collagen ◽  
In Vitro Angiogenesis

We reported previously that, in addition to mechanical strain, a constrained boundary condition alone can alter the organization of microvessel outgrowth during in vitro angiogenesis [1]. After 6 days of culture in vitro, microvessels aligned parallel to the long axis of rectangular 3D collagen gels that had constrained edges on the ends. However, unconstrained cultures did not show any alignment of microvessels. The ability to direct microvessel outgrowth during angiogenesis has significant implications for engineering prevascularized grafts and tissues in vitro, therefore an understanding of this process is important. Since there is direct relationship between the ability of endothelial cells to contract 3D gels and matrix stiffness [2], we hypothesize that some constrained boundary conditions will increase the apparent matrix stiffness and in turn will limit gel contraction, prevent microvessel alignment, and reduce microvessel outgrowth. The objective of this study was to compare microvessel growth and alignment under several different static boundary conditions.

scholarly journals Displacement fields using correlation methods as a tool to investigate cell migration in 3D collagen gels

2019 ◽  
Vol 275 (3) ◽  
pp. 172-182
Author(s):  
ARNOLD FERTIN ◽  
LAURE LAFORGUE ◽  
ALAIN DUPERRAY ◽  
VALÉRIE M. LAURENT ◽  
YVES USSON ◽  
...  
Keyword(s):  
Cell Migration ◽  
Collagen Gels ◽  
Displacement Fields ◽  
Correlation Methods ◽  
3D Collagen
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