scholarly journals A novel collagen gel-based measurement technique for quantitation of cell contraction force

2015 ◽  
Vol 12 (106) ◽  
pp. 20141365 ◽  
Author(s):  
Tianrong Jin ◽  
Li Li ◽  
Richard C. M. Siow ◽  
Kuo-Kang Liu
Keyword(s):  
Kinase Inhibitor ◽  
Imaging System ◽  
Collagen Gel ◽  
Collagen Matrix ◽  
Collagen Gels ◽  
Contraction Force ◽  
Depth Sensing ◽  
Cell Contraction ◽  
Microscopy Imaging

Cell contraction force plays an important role in wound healing, inflammation, angiogenesis and metastasis. This study describes a novel method to quantify single cell contraction force in vitro using human aortic adventitial fibroblasts embedded in a collagen gel. The technique is based on a depth sensing nano-indentation tester to measure the thickness and elasticity of collagen gels containing stimulated fibroblasts and a microscopy imaging system to estimate the gel area. In parallel, a simple theoretical model has been developed to calculate cell contraction force based on the measured parameters. Histamine (100 µM) was used to stimulate fibroblast contraction while the myosin light chain kinase inhibitor ML-7 (25 µM) was used to inhibit cell contraction. The collagen matrix used in the model provides a physiological environment for fibroblast contraction studies. Measurement of changes in collagen gel elasticity and thickness arising from histamine treatments provides a novel convenient technique to measure cell contraction force within a collagen matrix. This study demonstrates that histamine can elicit a significant increase in contraction force of fibroblasts embedded in collagen, while the Young's modulus of the gel decreases due to the gel degradation.

scholarly journals The selective binding and transmigration of monocytes through the junctional complexes of human endothelium.

1988 ◽  
Vol 168 (5) ◽  
pp. 1865-1882 ◽  
Author(s):  
N A Pawlowski ◽  
G Kaplan ◽  
E Abraham ◽  
Z A Cohn
Keyword(s):  
Silver Staining ◽  
Umbilical Vein ◽  
Collagen Gel ◽  
Collagen Gels ◽  
Silver Stain ◽  
Term Survival ◽  
Topographical Distribution ◽  
Junctional Complexes

Human monocytes show a high affinity for vascular endothelium both in vitro and in vivo. To explore monocyte-endothelial interaction in greater detail, we have developed a new in vitro model for growth of human endothelial cells (EC). Human umbilical vein EC (HUVEC) cultured upon collagen gels form confluent monolayers of EC that bind silver at their intercellular border similar to cells in situ. Intercellular junctional structures, both adherens and tight junctions, were identified. In contrast, HUVEC grown on plastic surfaces did not stain with silver. The silver-staining characteristic of EC-collagen monolayers was reversible and related to their in vitro maturation and senescence. Silver staining of EC borders provided a grid by which the location of monocyte binding to the luminal surface of individual EC could be assessed. Using this technique, we found that monocytes preferentially bound to the margins of EC, in approximation to the silver-staining junctions. These results suggest that EC determinants recognized by monocytes occur in a unique topographical distribution on the apical face of EC. After binding, monocytes migrated through the EC monolayers at high basal rates. The lack of penetration of collagen gels in the absence of an EC monolayer suggested the generation of EC-specific chemotactic signal(s). Monocytes were observed to pass between EC without evidence of disruption of the monolayer. Silver stain remained present during all phases of migration, and under transmission electron microscopy, junctional complexes were found proximal to monocytes that had just completed their passage through the monolayer. After orientation to the basal surface of the EC monolayer, monocytes migrated randomly into the underlying collagen gel. Monocyte adherence, penetration, migration, and long term survival can be studied under these conditions.

Prolactin effects on ion transport across cultured mouse mammary epithelium

1981 ◽  
Vol 240 (3) ◽  
pp. C110-C115 ◽  
Author(s):  
C. A. Bisbee
Keyword(s):  
Cell Cultures ◽  
Short Circuit ◽  
Collagen Gel ◽  
Mammary Epithelium ◽  
Short Circuit Current ◽  
Sodium Absorption ◽  
Collagen Gels ◽  
Mouse Mammary Epithelium

Prolactin is a known osmoregulatory hormone in lower vertebrates, and recent evidence indicates that this hormone modulates ionic concentrations in milk. In an ultrastructurally and biochemically differentiated primary cell culture system in which mouse mammary epithelium is maintained on floating collagen gels, prolactin causes an increase in short-circuit current (Isc) of monolayers of cells derived from midpregnant (24.6 to 48.0 microA . cm-2) and lactating (10.4 to 16.1 microA . cm-2) glands. Transepithelial potential differences (basal side ground) average about -12 mV and are similar to those seen in vivo. Prelactating mammary epithelial cell cultures have transepithelial resistances ranging from 374 omega . cm2 (prolactin present) to 507 omega . cm2 (prolactin absent), and lactating cell cultures have resistances averaging almost 1,000 omega . cm2. Prolactin effects require at most one day of culture maintenance in prolactin-containing medium, and the effects are not due to known contamination of prolactin preparations with arginine vasopressin or growth hormone. Medium concentrations of prolactin as low as 1 ng/ml can elicit these effects. In prelactating cell cultures not treated with prolactin, the Isc is equal to the rate of sodium absorption. Prolactin increases sodium absorption fourfold but increases Isc only twofold. Clearly, prolactin induces other active transport; neither potassium nor chloride movements can account for this additional transport. Resistance values, current-voltage plots, and permeability coefficients indicate that in vitro mammary epithelium is a moderately “tight” tissue. Comparisons with intact glands indicate that in vitro mammary epithelium closely resembles its in vivo counterpart. Floating collagen gel cultures appear suitable for elucidating transport properties in cellularly heterogeneous and structurally complex mammalian tissues.

Effect of shear stress on microvessel network formation of endothelial cells with in vitro three-dimensional model

2004 ◽  
Vol 287 (3) ◽  
pp. H994-H1002 ◽  
Author(s):  
Akinori Ueda ◽  
Masaki Koga ◽  
Mariko Ikeda ◽  
Susumu Kudo ◽  
Kazuo Tanishita
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Network Formation ◽  
Three Dimensional ◽  
Collagen Gel ◽  
Flow Chamber ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Collagen Gels

Shear stress stimulus is expected to enhance angiogenesis, the formation of microvessels. We determined the effect of shear stress stimulus on three-dimensional microvessel formation in vitro. Bovine pulmonary microvascular endothelial cells were seeded onto collagen gels with basic fibroblast growth factor to make a microvessel formation model. We observed this model in detail using phase-contrast microscopy, confocal laser scanning microscopy, and electron microscopy. The results show that cells invaded the collagen gel and reconstructed the tubular structures, containing a clearly defined lumen consisting of multiple cells. The model was placed in a parallel-plate flow chamber. A laminar shear stress of 0.3 Pa was applied to the surfaces of the cells for 48 h. Promotion of microvessel network formation was detectable after ∼10 h in the flow chamber. After 48 h, the length of networks exposed to shear stress was 6.17 (±0.59) times longer than at the initial state, whereas the length of networks not exposed to shear stress was only 3.30 (±0.41) times longer. The number of bifurcations and endpoints increased for networks exposed to shear stress, whereas the number of bifurcations alone increased for networks not exposed to shear stress. These results demonstrate that shear stress applied to the surfaces of endothelial cells on collagen gel promotes the growth of microvessel network formation in the gel and expands the network because of repeated bifurcation and elongation.

Antibacterial Wound Healing Gels from Oligochitosan Salts

2012 ◽  
Vol 506 ◽  
pp. 31-34
Author(s):  
W. Janvikul ◽  
P. Ngamviriyavong ◽  
P. Uppanun ◽  
P. Tanjak ◽  
N. Sangjun
Keyword(s):  
Staphylococcus Epidermidis ◽  
Wound Closure ◽  
Collagen Gel ◽  
Antibacterial Activities ◽  
Dermal Fibroblasts ◽  
Collagen Gels ◽  
Collagen Gel Matrix ◽  
Growth Of Bacteria

Oligochitosan salt-based antibacterial wound gels were developed and evaluated in both in vitro and in vivo models. The antibacterial activities of the oligochitosan salts and the wound gels were investigated against Staphylococcus epidermidis RP625 and Escherichia coli ATCC 11775. The minimum inhibitory concentrations (MIC) of the oligochitosan salts were found in the range of 16-256 μg/mL. The wound gels demonstrated their in vitro activities on inhibiting the growth of bacteria. The 3-D collagen gel matrix containing human dermal fibroblasts cultured with each test gel was used as an in vitro model for the examination of cell proliferation and secretion of interleukin-8 (IL-8). The gels appeared to promote the proliferation and formation of cellular process of the fibroblasts in the 3-D collagen gels and stimulate the fibroblasts to produce more IL-8. In the in vivo model, it was noted that the gels could accelerate the wound closure process. The wounds were completely closed within 14 days.

Bacterial Endotoxin Inhibits Migration, Attachment, and Orientation of Human Gingival Fibroblasts in vitro and Delays Collagen Gel Contraction

1987 ◽  
Vol 66 (9) ◽  
pp. 1449-1455 ◽  
Author(s):  
S. Pitaru ◽  
M. Soldinger ◽  
D. Madgar ◽  
Z. Metzger
Keyword(s):  
Collagen Type ◽  
Cell Attachment ◽  
Collagen Gel ◽  
Collagen Type I ◽  
Human Gingival Fibroblasts ◽  
Bacterial Endotoxin ◽  
Type I ◽  
Gingival Fibroblasts ◽  
Collagen Gels

The purpose of this study was to assess the effect of endotoxin adsorbed to dental surfaces and to collagen type I on the migration, attachment, and orientation of human gingival fibroblasts (HGF). Transversely cut porcine tooth root slices (RS), 200 μm thick, were prepared. Half of the RS obtained were partially demineralized in EDTA. Half of the demineralized and non-demineralized RS were incubated with 400 μg/mL of endotoxin for 24 hr, whereas the other half were maintained in PBS and served as controls. Experimental and control RS were placed on confluent layers of HFG and cultured for six days. Cell migration toward and cell attachment to the periphery of the RS and the formation of oriented cell sheets were assessed by means of photographic techniques. Additionally, six-day-old cultures were fixed and processed for SEM observation. In separate experiments, the effect of endotoxin on cell attachment to collagen type I and on contraction of three-dimensional collagen gels was assessed. It was found that: (i) bacterial endotoxin inhibited migration and attachment of HGF to both demineralized and non-demineralized cementum and interfered with the development of oriented cellular structure ; (ii) the inhibitory effect was significantly more pronounced for non-demineralized than for demineralized cementum; (iii) the morphology of HGF attached to endotoxin-treated dental surfaces was altered compared with that of their controls; and (iv) bacterial endotoxin inhibited cell attachment to collagen type I and delayed the contraction of collagen gel.

scholarly journals Design and construction of a novel measurement device for mechanical characterization of hydrogels: A case study

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0247727
Author(s):  
Shayan Shahab ◽  
Mehran Kasra ◽  
Alireza Dolatshahi-Pirouz
Keyword(s):  
Mechanical Properties ◽  
Shear Modulus ◽  
Magnetic Beads ◽  
Mechanical Characterization ◽  
Collagen Gel ◽  
Biological Tissues ◽  
Elastic Behavior ◽  
Collagen Gels ◽  
Collagen Concentration

Natural biopolymer-based hydrogels especially agarose and collagen gels, considering their biocompatibility with cells and their capacity to mimic biological tissues, have widely been used for in-vitro experiments and tissue engineering applications in recent years; nevertheless their mechanical properties are not always optimal for these purposes. Regarding the importance of the mechanical properties of hydrogels, many mechanical characterization studies have been carried out for such biopolymers. In this work, we have focused on understanding the mechanical role of agarose and collagen concentration on the hydrogel strength and elastic behavior. In this direction, Amirkabir Magnetic Bead Rheometry (AMBR) characterization device equipped with an optimized electromagnet, was designed and constructed for the measurement of hydrogel mechanical properties. The operation of AMBR set-up is based on applying a magnetic field to actuate magnetic beads in contact with the gel surface in order to actuate the gel itself. In simple terms the magnetic beads leads give rise to mechanical shear stress on the gel surface when under magnetic influence and together with the associated bead-gel displacement it is possible to calculate the hydrogel shear modulus. Agarose and Collagen gels with respectively 0.2–0.6 wt % and 0.2–0.5 wt % percent concentrations were prepared for mechanical characterization in terms of their shear modulus. The shear modulus values for the different percent concentrations of the agarose gel were obtained in the range 250–650 Pa, indicating the shear modulus increases by increasing in the agar gel concentration. In addition to this, the values of shear modulus for the collagen gel increase as function of concentration in the range 240–520 Pa in accordance with an approximately linear relationship between collagen concentration and gel strength.

Isotonic Biaxial Loading: An Economic, Versatile Method for the Study of Fibroblast-Populated Collagen Gels

1999 ◽  
Author(s):  
Jeffrey W. Holmes ◽  
Thomas K. Borg
Keyword(s):  
Biaxial Loading ◽  
Cardiac Fibroblasts ◽  
Collagen Gel ◽  
Simple System ◽  
Neonatal Rat ◽  
Collagen Gels ◽  
Maximal Contraction ◽  
Contraction Force ◽  
A Value ◽  
Collagen Gel Contraction

Abstract Studies of collagen gel contraction by embedded fibroblasts have characterized the response of free (unloaded) or isometric (maximally loaded) gels but not the response to intermediate loads. An inexpensive, simple system was devised to isotonically load fibroblast-populated collagen gels using freely hanging weights. This system provided excellent repeatability. Maximal contraction force was estimated at 1 mN per million cells in neonatal rat cardiac fibroblasts, a value that agreed well with reported isometric experiments in fibroblasts from other tissues. The ability to load uniaxially or biaxially and with variable loads will facilitate exploration of the regulation of fibroblast mechanics and biology by stress.

A role for netrin-1 in the guidance of cortical efferents

Development ◽  
1997 ◽  
Vol 124 (24) ◽  
pp. 5063-5074 ◽  
Author(s):  
C. Metin ◽  
D. Deleglise ◽  
T. Serafini ◽  
T.E. Kennedy ◽  
M. Tessier-Lavigne
Keyword(s):  
Short Range ◽  
Collagen Gel ◽  
Collagen Gels ◽  
Ganglionic Eminence ◽  
Promoting Effect ◽  
Initial Portion ◽  
Diffusible Factor ◽  
The Central Nervous System

An intermediate target for axons leaving the cerebral cortex in embryonic mammals is the ganglionic eminence (GE), the embryonic precursor of the basal ganglia. The cues that direct these axons over the initial portion of their trajectory are not well understood, but could include both short-range and long-range attractants and repellents. In the present study, we provide evidence that corticofugal axons might be guided at least partly by a diffusible factor or factors originating in the lateral GE and the sulcus between the lateral and medial ridges of the GE (ISS), as well as evidence implicating the axonal chemoattractant netrin-1 in mediating these effects. Explants of lateral GE and ISS obtained from E12.5 and E13.5 mouse forebrain have a strong effect on both the outgrowth and orientation of corticofugal axons when cultured at a distance with explants of embryonic cortex in collagen gels. Netrin-1 mRNA is detected in these target tissues by in situ hybridization, and both netrin-1 protein and heterologous cells secreting netrin-1 can mimic the outgrowth-promoting effect of these target tissues in vitro. Furthermore, the growth of corticofugal axons is oriented toward an ectopic source of netrin-1 in vitro, and a function blocking anti-netrin-1 antiserum specifically abolishes the cortical axon outgrowth elicited by explants of lateral GE and the ISS in collagen gel cocultures. Taken together, these results suggest a role for netrin-1 in the attraction at a distance of early cortical axons by the GE. Thus in mammals -- as is also observed in nematodes -- the development of non-commissural projections in anterior regions of the embryo might be directed by mechanisms similar to those involved in directing the development of commissural projections in more posterior regions of the central nervous system.

scholarly journals A Screening System for Evaluating Cell Extension Formation, Collagen Compaction, and Degradation in Drug Discovery

2017 ◽  
Vol 23 (2) ◽  
pp. 132-143 ◽  
Author(s):  
Asuka Yuda ◽  
Christopher A. McCulloch
Keyword(s):  
Cancer Cell ◽  
Kinase Inhibitor ◽  
Collagen Gel ◽  
Fibroblast Cell ◽  
Collagen Degradation ◽  
Matrix Remodeling ◽  
Collagen Gels ◽  
Latrunculin B ◽  
Cell Extension ◽  
The Mean

The generation of cell extensions is critical for matrix remodeling in tissue invasion by cancer cells, but current methods for identifying molecules that regulate cell extension formation and matrix remodeling are not well adapted for screening purposes. We applied a grid-supported, floating collagen gel system (~100 Pa stiffness) to examine cell extension formation, collagen compaction, and collagen degradation in a single assay. With the use of cultured diploid fibroblasts, a fibroblast cell line, and two cancer cell lines, we found that compared with attached collagen gels (~2800 Pa), the mean number and length of cell extensions were respectively greater in the floating gels. In assessing specific processes in cell extension formation, compared with controls, the number of cell extensions was reduced by latrunculin B, β1 integrin blockade, and a formin FH2 domain inhibitor. Screening of a kinase inhibitor library (480 compounds) with the floating gel assay showed that compared with vehicle-treated cells, there were large reductions of collagen compaction, pericellular collagen degradation, and number of cell extensions after treatment with SB431542, SIS3, Fasudil, GSK650394, and PKC-412. These data indicate that the grid-supported floating collagen gel model can be used to screen for inhibitors of cell extension formation and critical matrix remodeling events associated with cancer cell invasion.

The Development of Structural and Mechanical Anisotropy in Fibroblast Populated Collagen Gels

2005 ◽  
Vol 127 (5) ◽  
pp. 742-750 ◽  
Author(s):  
Stavros Thomopoulos ◽  
Gregory M. Fomovsky ◽  
Jeffrey W. Holmes
Keyword(s):  
Structure Function ◽  
Collagen Gel ◽  
Cell Types ◽  
Model System ◽  
Mechanical Anisotropy ◽  
Collagen Gels ◽  
Structural Anisotropy ◽  
Mechanical Constraints ◽  
Collagenous Tissues

An in vitro model system was developed to study structure-function relationships and the development of structural and mechanical anisotropy in collagenous tissues. Fibroblast-populated collagen gels were constrained either biaxially or uniaxially. Gel remodeling, biaxial mechanical properties, and collagen orientation were determined after 72h of culture. Collagen gels contracted spontaneously in the unconstrained direction, uniaxial mechanical constraints produced structural anisotropy, and this structural anisotropy was associated with mechanical anisotropy. Cardiac and tendon fibroblasts were compared to test the hypothesis that tendon fibroblasts should generate greater anisotropy in vitro. However, no differences were seen in either structure or mechanics of collagen gels populated with these two cell types, or between fibroblast populated gels and acellular gels. This study demonstrates our ability to control and measure the development of structural and mechanical anisotropy due to imposed mechanical constraints in a fibroblast-populated collagen gel model system. While imposed constraints were required for the development of anisotropy in this system, active remodeling of the gel by fibroblasts was not. This model system will provide a basis for investigating structure-function relationships in engineered constructs and for studying mechanisms underlying the development of anisotropy in collagenous tissues.

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