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Fibroblast viability and phenotypic changes within glycated stiffened three-dimensional collagen matrices.

Vicens-Zygmunt V, Estany S, Colom A, Montes-Worboys A, Machahua C, Sanabria AJ, Llatjos R, Escobar I, Manresa F, Dorca J, Navajas D, Alcaraz J, Molina-Molina M - Respir. Res. (2015)

Bottom Line: A promising approach is based on three-dimensional collagen type I matrices that are stiffened by cross-linking through non-enzymatic glycation with reducing sugars.Finally, a progressive contractile phenotype cell differentiation was associated with the contraction of these gels.The use of non-enzymatic glycation with a low ribose concentration may provide a suitable model with a mechanic and oxidative modified environment with cells embedded in it, which allowed cell proliferation and induced fibroblast phenotypic changes.

View Article: PubMed Central - PubMed

Affiliation: Department of Pneumology, Unit of Interstitial Lung Diseases, University Hospital of Bellvitge, Barcelona, Spain. vvicens@hotmail.com.

ABSTRACT

Background: There is growing interest in the development of cell culture assays that enable the rigidity of the extracellular matrix to be increased. A promising approach is based on three-dimensional collagen type I matrices that are stiffened by cross-linking through non-enzymatic glycation with reducing sugars.

Methods: The present study evaluated the biomechanical changes in the non-enzymatically glycated type I collagen matrices, including collagen organization, the advanced glycation end products formation and stiffness achievement. Gels were glycated with ribose at different concentrations (0, 5, 15, 30 and 240 mM). The viability and the phenotypic changes of primary human lung fibroblasts cultured within the non-enzymatically glycated gels were also evaluated along three consecutive weeks. Statistical tests used for data analyze were Mann-Whitney U, Kruskal Wallis, Student's t-test, two-way ANOVA, multivariate ANOVA, linear regression test and mixed linear model.

Results: Our findings indicated that the process of collagen glycation increases the stiffness of the matrices and generates advanced glycation end products in a ribose concentration-dependent manner. Furthermore, we identified optimal ribose concentrations and media conditions for cell viability and growth within the glycated matrices. The microenvironment of this collagen based three-dimensional culture induces α-smooth muscle actin and tenascin-C fibroblast protein expression. Finally, a progressive contractile phenotype cell differentiation was associated with the contraction of these gels.

Conclusions: The use of non-enzymatic glycation with a low ribose concentration may provide a suitable model with a mechanic and oxidative modified environment with cells embedded in it, which allowed cell proliferation and induced fibroblast phenotypic changes. Such culture model could be appropriate for investigations of the behavior and phenotypic changes in cells that occur during lung fibrosis as well as for testing different antifibrotic therapies in vitro.

No MeSH data available.


Related in: MedlinePlus

Increased collagen type I fiber reflection fluorescence with non-enzymatic glycation. Collagen type I fiber conformation observed with CRM (laser ex/em 568/568 nm) at different ribose (R) concentration over a period of twenty-one days. a. In post-glycated DMEM matrices, collagen type I fibers formed aggregates and reflected more fluorescence with elevated R concentrations. b. In post-glycated PBS matrices, collagen type I fibers were short, thin and homogeneous, with no increase of reflected fluorescence with higher R concentration (240 mM). a and b. Reflection fluorescence of non-glycated matrices (controls) was done to the reflective properties of the collagen. Time seems to not affect the reflection fluorescence of collagen. The increase in CRM signal in DMEM gels could also be contributed by the increased aggregation of collagen fibers observed in these gels, perhaps because the presence of glucose. The scale bar corresponds to 40 μm. CRM = Confocal reflection microscope; DMEM = Dulbecco’s modified Eagle medium; PBS = Phosphate-buffered saline
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Fig1: Increased collagen type I fiber reflection fluorescence with non-enzymatic glycation. Collagen type I fiber conformation observed with CRM (laser ex/em 568/568 nm) at different ribose (R) concentration over a period of twenty-one days. a. In post-glycated DMEM matrices, collagen type I fibers formed aggregates and reflected more fluorescence with elevated R concentrations. b. In post-glycated PBS matrices, collagen type I fibers were short, thin and homogeneous, with no increase of reflected fluorescence with higher R concentration (240 mM). a and b. Reflection fluorescence of non-glycated matrices (controls) was done to the reflective properties of the collagen. Time seems to not affect the reflection fluorescence of collagen. The increase in CRM signal in DMEM gels could also be contributed by the increased aggregation of collagen fibers observed in these gels, perhaps because the presence of glucose. The scale bar corresponds to 40 μm. CRM = Confocal reflection microscope; DMEM = Dulbecco’s modified Eagle medium; PBS = Phosphate-buffered saline

Mentions: Using CRM we examined the morphological and structural modifications of type I collagen post-glycated fibers (reflection fluorescence). Different changes occurred depending on the medium used in the preparation of the matrices (DMEM or PBS). DMEM matrices exhibited large, thick fibers that formed aggregates (Fig. 1a), while PBS matrices displayed short, thin collagen fibers with no evidence of aggregation (Fig. 1b). An increased intensity of collagen reflection fluorescence was observed in all the DMEM matrices with the highest ribose concentration (240 mM). However, this phenomenon did not occur in PBS matrices. This different reflection fluorescence intensity in DMEM and PBS matrices was attributed to different pattern of collagen fibers organization depending on the substrate used (DMEM or PBS).Fig. 1


Fibroblast viability and phenotypic changes within glycated stiffened three-dimensional collagen matrices.

Vicens-Zygmunt V, Estany S, Colom A, Montes-Worboys A, Machahua C, Sanabria AJ, Llatjos R, Escobar I, Manresa F, Dorca J, Navajas D, Alcaraz J, Molina-Molina M - Respir. Res. (2015)

Increased collagen type I fiber reflection fluorescence with non-enzymatic glycation. Collagen type I fiber conformation observed with CRM (laser ex/em 568/568 nm) at different ribose (R) concentration over a period of twenty-one days. a. In post-glycated DMEM matrices, collagen type I fibers formed aggregates and reflected more fluorescence with elevated R concentrations. b. In post-glycated PBS matrices, collagen type I fibers were short, thin and homogeneous, with no increase of reflected fluorescence with higher R concentration (240 mM). a and b. Reflection fluorescence of non-glycated matrices (controls) was done to the reflective properties of the collagen. Time seems to not affect the reflection fluorescence of collagen. The increase in CRM signal in DMEM gels could also be contributed by the increased aggregation of collagen fibers observed in these gels, perhaps because the presence of glucose. The scale bar corresponds to 40 μm. CRM = Confocal reflection microscope; DMEM = Dulbecco’s modified Eagle medium; PBS = Phosphate-buffered saline
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4494165&req=5

Fig1: Increased collagen type I fiber reflection fluorescence with non-enzymatic glycation. Collagen type I fiber conformation observed with CRM (laser ex/em 568/568 nm) at different ribose (R) concentration over a period of twenty-one days. a. In post-glycated DMEM matrices, collagen type I fibers formed aggregates and reflected more fluorescence with elevated R concentrations. b. In post-glycated PBS matrices, collagen type I fibers were short, thin and homogeneous, with no increase of reflected fluorescence with higher R concentration (240 mM). a and b. Reflection fluorescence of non-glycated matrices (controls) was done to the reflective properties of the collagen. Time seems to not affect the reflection fluorescence of collagen. The increase in CRM signal in DMEM gels could also be contributed by the increased aggregation of collagen fibers observed in these gels, perhaps because the presence of glucose. The scale bar corresponds to 40 μm. CRM = Confocal reflection microscope; DMEM = Dulbecco’s modified Eagle medium; PBS = Phosphate-buffered saline
Mentions: Using CRM we examined the morphological and structural modifications of type I collagen post-glycated fibers (reflection fluorescence). Different changes occurred depending on the medium used in the preparation of the matrices (DMEM or PBS). DMEM matrices exhibited large, thick fibers that formed aggregates (Fig. 1a), while PBS matrices displayed short, thin collagen fibers with no evidence of aggregation (Fig. 1b). An increased intensity of collagen reflection fluorescence was observed in all the DMEM matrices with the highest ribose concentration (240 mM). However, this phenomenon did not occur in PBS matrices. This different reflection fluorescence intensity in DMEM and PBS matrices was attributed to different pattern of collagen fibers organization depending on the substrate used (DMEM or PBS).Fig. 1

Bottom Line: A promising approach is based on three-dimensional collagen type I matrices that are stiffened by cross-linking through non-enzymatic glycation with reducing sugars.Finally, a progressive contractile phenotype cell differentiation was associated with the contraction of these gels.The use of non-enzymatic glycation with a low ribose concentration may provide a suitable model with a mechanic and oxidative modified environment with cells embedded in it, which allowed cell proliferation and induced fibroblast phenotypic changes.

View Article: PubMed Central - PubMed

Affiliation: Department of Pneumology, Unit of Interstitial Lung Diseases, University Hospital of Bellvitge, Barcelona, Spain. vvicens@hotmail.com.

ABSTRACT

Background: There is growing interest in the development of cell culture assays that enable the rigidity of the extracellular matrix to be increased. A promising approach is based on three-dimensional collagen type I matrices that are stiffened by cross-linking through non-enzymatic glycation with reducing sugars.

Methods: The present study evaluated the biomechanical changes in the non-enzymatically glycated type I collagen matrices, including collagen organization, the advanced glycation end products formation and stiffness achievement. Gels were glycated with ribose at different concentrations (0, 5, 15, 30 and 240 mM). The viability and the phenotypic changes of primary human lung fibroblasts cultured within the non-enzymatically glycated gels were also evaluated along three consecutive weeks. Statistical tests used for data analyze were Mann-Whitney U, Kruskal Wallis, Student's t-test, two-way ANOVA, multivariate ANOVA, linear regression test and mixed linear model.

Results: Our findings indicated that the process of collagen glycation increases the stiffness of the matrices and generates advanced glycation end products in a ribose concentration-dependent manner. Furthermore, we identified optimal ribose concentrations and media conditions for cell viability and growth within the glycated matrices. The microenvironment of this collagen based three-dimensional culture induces α-smooth muscle actin and tenascin-C fibroblast protein expression. Finally, a progressive contractile phenotype cell differentiation was associated with the contraction of these gels.

Conclusions: The use of non-enzymatic glycation with a low ribose concentration may provide a suitable model with a mechanic and oxidative modified environment with cells embedded in it, which allowed cell proliferation and induced fibroblast phenotypic changes. Such culture model could be appropriate for investigations of the behavior and phenotypic changes in cells that occur during lung fibrosis as well as for testing different antifibrotic therapies in vitro.

No MeSH data available.


Related in: MedlinePlus