Limits...
Measurement of biomolecular diffusion in extracellular matrix condensed by fibroblasts using fluorescence correlation spectroscopy.

Kihara T, Ito J, Miyake J - PLoS ONE (2013)

Bottom Line: Additionally, the diffusion coefficients of biomolecules were inversely correlated with molecular radius.Furthermore, molecular diffusion was greatly restricted, with a central focus on the populated cells.Thus, biomolecular diffusion is restricted in the vicinity of the cells where collagen fibers are highly condensed.

View Article: PubMed Central - PubMed

Affiliation: Department of Life and Environment Engineering, Faculty of Environmental Engineering, The University of Kitakyushu, 1-1 Hibikino, Wakamatsu, Kitakyushu, Fukuoka, Japan ; Department of Mechanical Science and Bioengineering, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama, Toyonaka, Osaka, Japan.

ABSTRACT
The extracellular matrix (ECM) comprises the heterogeneous environment outside of cells in a biological system. The ECM is dynamically organized and regulated, and many biomolecules secreted from cells diffuse throughout the ECM, regulating a variety of cellular processes. Therefore, investigation of the diffusive behaviors of biomolecules in the extracellular environment is critical. In this study, we investigated the diffusion coefficients of biomolecules of various sizes, measuring from 1 to 10 nm in radius, by fluorescence correlation spectroscopy in contracted collagen gel caused by fibroblasts, a traditional culture model of dynamic rearrangement of collagen fibers. The diffusion coefficients of the biomolecules in control collagen gel without cells decreased slightly as compared to those in solution, while the diffusion coefficients of biomolecules in the contracted gel at the cell vicinity decreased dramatically. Additionally, the diffusion coefficients of biomolecules were inversely correlated with molecular radius. In collagen gels populated with fibroblasts, the diffusion coefficient at the cell vicinity clearly decreased in the first 24 h of culture. Furthermore, molecular diffusion was greatly restricted, with a central focus on the populated cells. By using the obtained diffusion coefficients of biomolecules, we calculated the collagen fiber condensation ratio by fibroblasts in the cell vicinity at 3 days of culture to represent a 52-fold concentration. Thus, biomolecular diffusion is restricted in the vicinity of the cells where collagen fibers are highly condensed.

Show MeSH

Related in: MedlinePlus

Schematic diagram of probe diffusion in the contracted collagen gel.Collagen fibers are well condensed in the area surrounding the cell due to cellular activity. Biomolecules diffuse throughout the collagen fiber-containing space, in which the concentration of the collagen fibers varies from region to region. Diffusion behaviors of the biomolecules are probably affected by the local concentration of the collagen fibers. By measuring the diffusion behaviors of the biomolecules locally, we can develop a better understanding of the behaviors of these molecules in the heterogeneous ECM and of the physical environment surrounding the cells.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3842966&req=5

pone-0082382-g001: Schematic diagram of probe diffusion in the contracted collagen gel.Collagen fibers are well condensed in the area surrounding the cell due to cellular activity. Biomolecules diffuse throughout the collagen fiber-containing space, in which the concentration of the collagen fibers varies from region to region. Diffusion behaviors of the biomolecules are probably affected by the local concentration of the collagen fibers. By measuring the diffusion behaviors of the biomolecules locally, we can develop a better understanding of the behaviors of these molecules in the heterogeneous ECM and of the physical environment surrounding the cells.

Mentions: In this study, we measured the diffusion coefficients of size-varied biomolecules in fibroblast-contracted collagen gel by FCS. Since the collagen fibers are condensed into the area surrounding the fibroblasts due to the activities of these cells [13], the sampling space specificity and short measurement time of FCS are suitable for measurement of biomolecular diffusion in the condensed collagen fibers surrounding the cells (Fig. 1). By measuring the diffusion coefficients of size-varied biomolecules, we found features of the physical environment surrounding cells grown in the 3-dimensional collagen fiber environment.


Measurement of biomolecular diffusion in extracellular matrix condensed by fibroblasts using fluorescence correlation spectroscopy.

Kihara T, Ito J, Miyake J - PLoS ONE (2013)

Schematic diagram of probe diffusion in the contracted collagen gel.Collagen fibers are well condensed in the area surrounding the cell due to cellular activity. Biomolecules diffuse throughout the collagen fiber-containing space, in which the concentration of the collagen fibers varies from region to region. Diffusion behaviors of the biomolecules are probably affected by the local concentration of the collagen fibers. By measuring the diffusion behaviors of the biomolecules locally, we can develop a better understanding of the behaviors of these molecules in the heterogeneous ECM and of the physical environment surrounding the cells.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3842966&req=5

pone-0082382-g001: Schematic diagram of probe diffusion in the contracted collagen gel.Collagen fibers are well condensed in the area surrounding the cell due to cellular activity. Biomolecules diffuse throughout the collagen fiber-containing space, in which the concentration of the collagen fibers varies from region to region. Diffusion behaviors of the biomolecules are probably affected by the local concentration of the collagen fibers. By measuring the diffusion behaviors of the biomolecules locally, we can develop a better understanding of the behaviors of these molecules in the heterogeneous ECM and of the physical environment surrounding the cells.
Mentions: In this study, we measured the diffusion coefficients of size-varied biomolecules in fibroblast-contracted collagen gel by FCS. Since the collagen fibers are condensed into the area surrounding the fibroblasts due to the activities of these cells [13], the sampling space specificity and short measurement time of FCS are suitable for measurement of biomolecular diffusion in the condensed collagen fibers surrounding the cells (Fig. 1). By measuring the diffusion coefficients of size-varied biomolecules, we found features of the physical environment surrounding cells grown in the 3-dimensional collagen fiber environment.

Bottom Line: Additionally, the diffusion coefficients of biomolecules were inversely correlated with molecular radius.Furthermore, molecular diffusion was greatly restricted, with a central focus on the populated cells.Thus, biomolecular diffusion is restricted in the vicinity of the cells where collagen fibers are highly condensed.

View Article: PubMed Central - PubMed

Affiliation: Department of Life and Environment Engineering, Faculty of Environmental Engineering, The University of Kitakyushu, 1-1 Hibikino, Wakamatsu, Kitakyushu, Fukuoka, Japan ; Department of Mechanical Science and Bioengineering, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama, Toyonaka, Osaka, Japan.

ABSTRACT
The extracellular matrix (ECM) comprises the heterogeneous environment outside of cells in a biological system. The ECM is dynamically organized and regulated, and many biomolecules secreted from cells diffuse throughout the ECM, regulating a variety of cellular processes. Therefore, investigation of the diffusive behaviors of biomolecules in the extracellular environment is critical. In this study, we investigated the diffusion coefficients of biomolecules of various sizes, measuring from 1 to 10 nm in radius, by fluorescence correlation spectroscopy in contracted collagen gel caused by fibroblasts, a traditional culture model of dynamic rearrangement of collagen fibers. The diffusion coefficients of the biomolecules in control collagen gel without cells decreased slightly as compared to those in solution, while the diffusion coefficients of biomolecules in the contracted gel at the cell vicinity decreased dramatically. Additionally, the diffusion coefficients of biomolecules were inversely correlated with molecular radius. In collagen gels populated with fibroblasts, the diffusion coefficient at the cell vicinity clearly decreased in the first 24 h of culture. Furthermore, molecular diffusion was greatly restricted, with a central focus on the populated cells. By using the obtained diffusion coefficients of biomolecules, we calculated the collagen fiber condensation ratio by fibroblasts in the cell vicinity at 3 days of culture to represent a 52-fold concentration. Thus, biomolecular diffusion is restricted in the vicinity of the cells where collagen fibers are highly condensed.

Show MeSH
Related in: MedlinePlus