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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

3D matrices induce Tenascin-C synthesis in lung fibroblasts. Levels of Tenascin-C (TNC) were measured from the supernatants of the 3D DMEM matrices by ELISA. The results were obtained from three independent experiments. Fibroblasts were cultured with 10 % of FBS. The synthesis of TNC was induced in a time-dependent manner, observing significant statistical differences between the 1st and the 14th and 21st days of culture (p < 0.05 for both comparisons). Although no differences were appreciated between the 14th and the 21st days of culture, a higher level of TNC was observed with 30 mM of ribose at the 21st day (p = 0.08). No statistical differences were observed between glycated and non-glycated matrices. These results suggest that the 3D microenvironment could be enough to produce changes in the secretion of TNC by normal fibroblasts in a time-dependent manner
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Fig7: 3D matrices induce Tenascin-C synthesis in lung fibroblasts. Levels of Tenascin-C (TNC) were measured from the supernatants of the 3D DMEM matrices by ELISA. The results were obtained from three independent experiments. Fibroblasts were cultured with 10 % of FBS. The synthesis of TNC was induced in a time-dependent manner, observing significant statistical differences between the 1st and the 14th and 21st days of culture (p < 0.05 for both comparisons). Although no differences were appreciated between the 14th and the 21st days of culture, a higher level of TNC was observed with 30 mM of ribose at the 21st day (p = 0.08). No statistical differences were observed between glycated and non-glycated matrices. These results suggest that the 3D microenvironment could be enough to produce changes in the secretion of TNC by normal fibroblasts in a time-dependent manner

Mentions: The supernatants of the DMEM matrices from three independent experiments were analyzed to evaluate the fibroblast tenascin-C (TNC) protein synthesis (Fig. 7). Results showed a progressive increase in the secretion of TNC in all the matrices from the 1st to the 21st day, with statistical differences between the 1st and the 14th day and the 1st and the 21st day (p < 0.05 for both comparisons). Although no differences were appreciated between the 14th and the 21st day of culture in most ribose conditions, a higher level of TNC at the 21st day was observed with 30 mM of ribose (p = 0.08). No statistical differences were found between ribosilated and non-ribosilated matrices. These results suggest that the 3D microenvironment could be enough to produce changes in the secretion of TNC by normal fibroblasts in a time-dependent manner.Fig. 7


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)

3D matrices induce Tenascin-C synthesis in lung fibroblasts. Levels of Tenascin-C (TNC) were measured from the supernatants of the 3D DMEM matrices by ELISA. The results were obtained from three independent experiments. Fibroblasts were cultured with 10 % of FBS. The synthesis of TNC was induced in a time-dependent manner, observing significant statistical differences between the 1st and the 14th and 21st days of culture (p < 0.05 for both comparisons). Although no differences were appreciated between the 14th and the 21st days of culture, a higher level of TNC was observed with 30 mM of ribose at the 21st day (p = 0.08). No statistical differences were observed between glycated and non-glycated matrices. These results suggest that the 3D microenvironment could be enough to produce changes in the secretion of TNC by normal fibroblasts in a time-dependent manner
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig7: 3D matrices induce Tenascin-C synthesis in lung fibroblasts. Levels of Tenascin-C (TNC) were measured from the supernatants of the 3D DMEM matrices by ELISA. The results were obtained from three independent experiments. Fibroblasts were cultured with 10 % of FBS. The synthesis of TNC was induced in a time-dependent manner, observing significant statistical differences between the 1st and the 14th and 21st days of culture (p < 0.05 for both comparisons). Although no differences were appreciated between the 14th and the 21st days of culture, a higher level of TNC was observed with 30 mM of ribose at the 21st day (p = 0.08). No statistical differences were observed between glycated and non-glycated matrices. These results suggest that the 3D microenvironment could be enough to produce changes in the secretion of TNC by normal fibroblasts in a time-dependent manner
Mentions: The supernatants of the DMEM matrices from three independent experiments were analyzed to evaluate the fibroblast tenascin-C (TNC) protein synthesis (Fig. 7). Results showed a progressive increase in the secretion of TNC in all the matrices from the 1st to the 21st day, with statistical differences between the 1st and the 14th day and the 1st and the 21st day (p < 0.05 for both comparisons). Although no differences were appreciated between the 14th and the 21st day of culture in most ribose conditions, a higher level of TNC at the 21st day was observed with 30 mM of ribose (p = 0.08). No statistical differences were found between ribosilated and non-ribosilated matrices. These results suggest that the 3D microenvironment could be enough to produce changes in the secretion of TNC by normal fibroblasts in a time-dependent manner.Fig. 7

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