Quantitative assessment of local collagen matrix remodeling in 3-D Culture: The role of Rho kinase

Kim, Areum; Lakshman, Neema; Petroll, Walter M

doi:10.1016/j.yexcr.2006.08.009

Cited by 106 publications

(138 citation statements)

References 52 publications

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“…However, little compaction and alignment of extracellular matrix is observed in the presence of Y-27632, consistent with previous findings (Fig. 6B) (Kim et al, 2006). Incubation with nocodazole reduced the amount of cell spreading and induced larger and more numerous stress fibers (Fig.…”

Section: Microtubules Regulate Cell Polarization and Morphology In 3-supporting

confidence: 92%

“…Collagen fibrils at the ends of cells generally appeared to be aligned parallel to the long axis of pseudopodia, consistent with previous studies (Fig. 2D) (Kim et al, 2006). Microtubules emanated from a microtubule organizing center, and were generally aligned with the long axis of the cells (Fig.…”

Section: Microtubule Disruption Induces Matrix Compaction and Alignmentsupporting

confidence: 90%

“…Following 24 hour exposure to nocodazole, there was an even greater increase in both the number of stress fibers and the amount of matrix compaction and alignment at the ends of cells. In contrast, when Rho kinase was inhibited, cells were elongated and had dendritic processes, as previously described (Kim et al, 2006). Subsequent addition of nocodazole induced retraction of some dendritic processes without associated pulling in of the ECM.…”

Section: Introductionsupporting

confidence: 66%

“…Cells treated with Y-27632 for 24 hours were elongated and had dendritic processes, as previously described (Kim et al, 2006). Nocodazole induced retraction of some dendritic processes without associated pulling in of the ECM.…”

Section: Discussionmentioning

confidence: 69%

“…Matrices were incubated for 24 hours with basal media (control), the Rho-kinase inhibitor Y-27632, nocodazole, or both Y-27632 and nocodazole. A standard concentration of 10μM of Y-27632 was used, which is optimal for specific inhibition of Rho-kinase (Anderson et al, 2004;Kim et al, 2006;Tamariz and Grinnel, 2002;Vishwanath et al, 2003). Cells were then fixed using 3% paraformaldehyde in phosphate buffer for 10 min, permeabalized with 0.5% Triton X-100 in phosphate buffer for 3 min, and blocked with 3% FBS (fetal bovine serum) containing buffer for 30 min.…”

Section: Assessment Of Cell Spreading and Morphologymentioning

confidence: 99%

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Microtubule regulation of corneal fibroblast morphology and mechanical activity in 3-D culture

Kim

Petroll

2007

Experimental Eye Research

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The purpose of this study was to investigate the role of microtubules in regulating corneal fibroblast structure and mechanical behavior using static (3-D) and dynamic (4-D) imaging of both cells and their surrounding matrix. Human corneal fibroblasts transfected to express GFP-zyxin (to label focal adhesions) or GFP-tubulin (to label microtubules) were plated at low density inside 100 μm thick type I collagen matrices. After 24 hours, the effects of nocodazole (to depolymerize microtubules), cytochalasin D (to disrupt f-actin), and/or Y-27632 (to block Rho-kinase) were evaluated using 3-D and 4-D imaging of both cells and ECM. After 24 hours of incubation, cells had well organized microtubules and prominent focal adhesions, and significant cell-induced matrix compaction was observed. Addition of nocodazole induced rapid microtubule disruption which resulted in Rho activation and additional cellular contraction. The matrix was pulled inward by retracting pseudopodial processes, and focal adhesions appeared to mediate this process, when present. Following 24 hour exposure to nocodazole, there was an even greater increase in both the number of stress fibers and the amount of matrix compaction and alignment at the ends of cells. When Rhokinase was inhibited, disruption of microtubules resulted in retraction of dendritic cell processes, and rapid formation and extension of lamellipodial processes at random locations along the cell body, eventually leading to a convoluted, disorganized cell shape. These data suggest that microtubules modulate both cellular contractility and local collagen matrix reorganization via regulation of Rho/ Rho kinase activity. In addition, microtubules appear to play a central role in dynamic regulation of cell spreading mechanics, morphology and polarity in 3-D culture.

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Section: Microtubules Regulate Cell Polarization and Morphology In 3-supporting

confidence: 92%

Section: Microtubule Disruption Induces Matrix Compaction and Alignmentsupporting

confidence: 90%

Section: Introductionsupporting

confidence: 66%

Section: Discussionmentioning

confidence: 69%

Section: Assessment Of Cell Spreading and Morphologymentioning

confidence: 99%

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Microtubule regulation of corneal fibroblast morphology and mechanical activity in 3-D culture

Kim

Petroll

2007

Experimental Eye Research

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Dynamic Hydrogels: Switching of 3D Microenvironments Using Two‐Component Naturally Derived Extracellular Matrices

et al. 2010

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Increasing Cell Seeding Density Improves Elastin Expression and Mechanical Properties in Collagen Gel-Based Scaffolds Cellularized with Smooth Muscle Cells

et al. 2018

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Vascular tissue engineering combines cells with scaffold materials in vitro aiming the development of physiologically relevant vascular models. For natural scaffolds such as collagen gels, where cells can be mixed with the material solution before gelation, cell seeding density is a key parameter that can affect extracellular matrix deposition and remodeling. Nonetheless, this parameter is often overlooked and densities sensitively lower than those of native tissues, are usually employed. Herein, the effect of seeding density on the maturation of tubular collagen gel-based scaffolds cellularized with smooth muscle cells is investigated. The compaction, the expression, and deposition of key vascular proteins and the resulting mechanical properties of the constructs are evaluated up to 1 week of maturation. Results show that increasing cell seeding density accelerates cell-mediated gel compaction, enhances elastin expression (more than sevenfold increase at the highest density, Day 7) and finally improves the overall mechanical properties of constructs. Of note, the tensile equilibrium elastic modulus, evaluated by stress-relaxation tests, reach values comparable to native arteries for the highest cell density, after a 7-day maturation. Altogether, these results show that higher cell seeding densities promote the rapid maturation of collagen gel-based vascular constructs toward structural and mechanical properties better mimicking native arteries.

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Quantitative assessment of local collagen matrix remodeling in 3-D Culture: The role of Rho kinase

Cited by 106 publications

References 52 publications

Microtubule regulation of corneal fibroblast morphology and mechanical activity in 3-D culture

Microtubule regulation of corneal fibroblast morphology and mechanical activity in 3-D culture

Dynamic Hydrogels: Switching of 3D Microenvironments Using Two‐Component Naturally Derived Extracellular Matrices

Increasing Cell Seeding Density Improves Elastin Expression and Mechanical Properties in Collagen Gel-Based Scaffolds Cellularized with Smooth Muscle Cells

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