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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

Post-glycated matrices induce a contractile phenotype. Primary human normal lung fibroblasts cultured within three-dimensional glycated DMEM matrices with 10 % of serum were analyzed to determine the level of alpha-smooth muscle actin (α-SMA) gene expression at the 7th, 14th and 21st day of culture. α-SMA gene expression was observed in the glycated matrices prepared with 5 mM, 15 mM ribose (R) and in the non-glycated matrices (controls) on the 21st day (only statistically significant (p < 0.05) for 5 mM). This phenomenon corresponded to the occurrence of gel contraction. In contrast, α-SMA gene expression and gels contraction were not observed in the 30 mM R glycated matrices on the 21st day. Interestingly, a progressive increase of α-SMA gene expression was observed with a gradual increase of R on day 7th, suggesting that the differentiation of fibroblasts to a contractile phenotype depends not only on time but also on the ribose concentration, which is associated with the extent of collagen cross-linking. The data are presented as percentages respect to the values on the 1st day. The experiments were repeated three times, with similar results obtained. * p-value < 0.05
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Fig5: Post-glycated matrices induce a contractile phenotype. Primary human normal lung fibroblasts cultured within three-dimensional glycated DMEM matrices with 10 % of serum were analyzed to determine the level of alpha-smooth muscle actin (α-SMA) gene expression at the 7th, 14th and 21st day of culture. α-SMA gene expression was observed in the glycated matrices prepared with 5 mM, 15 mM ribose (R) and in the non-glycated matrices (controls) on the 21st day (only statistically significant (p < 0.05) for 5 mM). This phenomenon corresponded to the occurrence of gel contraction. In contrast, α-SMA gene expression and gels contraction were not observed in the 30 mM R glycated matrices on the 21st day. Interestingly, a progressive increase of α-SMA gene expression was observed with a gradual increase of R on day 7th, suggesting that the differentiation of fibroblasts to a contractile phenotype depends not only on time but also on the ribose concentration, which is associated with the extent of collagen cross-linking. The data are presented as percentages respect to the values on the 1st day. The experiments were repeated three times, with similar results obtained. * p-value < 0.05

Mentions: A higher increase of α-SMA gene expression was observed in 0–15 mM of ribose at the 21st day (Fig. 5). α-SMA protein detection appeared from day 7th in all conditions, with and without ribose, and was significantly increased at days 14 and 21 compared to the 7th day (p < 0.01), which suggests that the induction of α-SMA is modified by the 3D microambient ‘per se’ (Fig. 6).Fig. 5


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)

Post-glycated matrices induce a contractile phenotype. Primary human normal lung fibroblasts cultured within three-dimensional glycated DMEM matrices with 10 % of serum were analyzed to determine the level of alpha-smooth muscle actin (α-SMA) gene expression at the 7th, 14th and 21st day of culture. α-SMA gene expression was observed in the glycated matrices prepared with 5 mM, 15 mM ribose (R) and in the non-glycated matrices (controls) on the 21st day (only statistically significant (p < 0.05) for 5 mM). This phenomenon corresponded to the occurrence of gel contraction. In contrast, α-SMA gene expression and gels contraction were not observed in the 30 mM R glycated matrices on the 21st day. Interestingly, a progressive increase of α-SMA gene expression was observed with a gradual increase of R on day 7th, suggesting that the differentiation of fibroblasts to a contractile phenotype depends not only on time but also on the ribose concentration, which is associated with the extent of collagen cross-linking. The data are presented as percentages respect to the values on the 1st day. The experiments were repeated three times, with similar results obtained. * p-value < 0.05
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: Post-glycated matrices induce a contractile phenotype. Primary human normal lung fibroblasts cultured within three-dimensional glycated DMEM matrices with 10 % of serum were analyzed to determine the level of alpha-smooth muscle actin (α-SMA) gene expression at the 7th, 14th and 21st day of culture. α-SMA gene expression was observed in the glycated matrices prepared with 5 mM, 15 mM ribose (R) and in the non-glycated matrices (controls) on the 21st day (only statistically significant (p < 0.05) for 5 mM). This phenomenon corresponded to the occurrence of gel contraction. In contrast, α-SMA gene expression and gels contraction were not observed in the 30 mM R glycated matrices on the 21st day. Interestingly, a progressive increase of α-SMA gene expression was observed with a gradual increase of R on day 7th, suggesting that the differentiation of fibroblasts to a contractile phenotype depends not only on time but also on the ribose concentration, which is associated with the extent of collagen cross-linking. The data are presented as percentages respect to the values on the 1st day. The experiments were repeated three times, with similar results obtained. * p-value < 0.05
Mentions: A higher increase of α-SMA gene expression was observed in 0–15 mM of ribose at the 21st day (Fig. 5). α-SMA protein detection appeared from day 7th in all conditions, with and without ribose, and was significantly increased at days 14 and 21 compared to the 7th day (p < 0.01), which suggests that the induction of α-SMA is modified by the 3D microambient ‘per se’ (Fig. 6).Fig. 5

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