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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

α-SMA protein detection in the 3D matrices. alpha-smooth muscle actin (α-SMA) protein expression in the primary human normal lung fibroblasts was evaluated using western blot. Fibroblasts were cultured within 3D glycated DMEM matrices containing 10 % serum. a. Western Blot. α-SMA band, 42 kDa; α-tubulin band, 50 kDa. Albumin band (66 kDa, due to FBS and visible in cellular and acellular matrices blots’, probably because of the difficulty in rinsing all the media from the matrices prior to protein extraction). The experiments were repeated three times. a1. An insignificant amount of α-SMA was detected in all the cellular matrices. a2 and a3. Higher amounts of α-SMA were observed in all the cellular matrices on the 14th and 21st days of culture. a1, a2 and a3. No tubulin or α-SMA bands were detected in the non-cells matrices. b. Densitometric analysis of α-SMA levels (Ratio of α-SMA to α-tubulin). The results were obtained from three independent experiments. *p < 0.05, **p < 0.01. A gradual decrease of α-SMA was observed at higher ribose concentrations on the 7th day of culture (p > 0.05). Contractile phenotype was most strongly detected on the 14th and 21st days of culture. It was significantly higher at all ribose concentrations, also for controls without ribose, compared with the level on the 7th day. It suggests that the microambient ‘per se’ could induce a phenotype change and that cells contribute to the matrix contraction
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Fig6: α-SMA protein detection in the 3D matrices. alpha-smooth muscle actin (α-SMA) protein expression in the primary human normal lung fibroblasts was evaluated using western blot. Fibroblasts were cultured within 3D glycated DMEM matrices containing 10 % serum. a. Western Blot. α-SMA band, 42 kDa; α-tubulin band, 50 kDa. Albumin band (66 kDa, due to FBS and visible in cellular and acellular matrices blots’, probably because of the difficulty in rinsing all the media from the matrices prior to protein extraction). The experiments were repeated three times. a1. An insignificant amount of α-SMA was detected in all the cellular matrices. a2 and a3. Higher amounts of α-SMA were observed in all the cellular matrices on the 14th and 21st days of culture. a1, a2 and a3. No tubulin or α-SMA bands were detected in the non-cells matrices. b. Densitometric analysis of α-SMA levels (Ratio of α-SMA to α-tubulin). The results were obtained from three independent experiments. *p < 0.05, **p < 0.01. A gradual decrease of α-SMA was observed at higher ribose concentrations on the 7th day of culture (p > 0.05). Contractile phenotype was most strongly detected on the 14th and 21st days of culture. It was significantly higher at all ribose concentrations, also for controls without ribose, compared with the level on the 7th day. It suggests that the microambient ‘per se’ could induce a phenotype change and that cells contribute to the matrix contraction

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)

α-SMA protein detection in the 3D matrices. alpha-smooth muscle actin (α-SMA) protein expression in the primary human normal lung fibroblasts was evaluated using western blot. Fibroblasts were cultured within 3D glycated DMEM matrices containing 10 % serum. a. Western Blot. α-SMA band, 42 kDa; α-tubulin band, 50 kDa. Albumin band (66 kDa, due to FBS and visible in cellular and acellular matrices blots’, probably because of the difficulty in rinsing all the media from the matrices prior to protein extraction). The experiments were repeated three times. a1. An insignificant amount of α-SMA was detected in all the cellular matrices. a2 and a3. Higher amounts of α-SMA were observed in all the cellular matrices on the 14th and 21st days of culture. a1, a2 and a3. No tubulin or α-SMA bands were detected in the non-cells matrices. b. Densitometric analysis of α-SMA levels (Ratio of α-SMA to α-tubulin). The results were obtained from three independent experiments. *p < 0.05, **p < 0.01. A gradual decrease of α-SMA was observed at higher ribose concentrations on the 7th day of culture (p > 0.05). Contractile phenotype was most strongly detected on the 14th and 21st days of culture. It was significantly higher at all ribose concentrations, also for controls without ribose, compared with the level on the 7th day. It suggests that the microambient ‘per se’ could induce a phenotype change and that cells contribute to the matrix contraction
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4494165&req=5

Fig6: α-SMA protein detection in the 3D matrices. alpha-smooth muscle actin (α-SMA) protein expression in the primary human normal lung fibroblasts was evaluated using western blot. Fibroblasts were cultured within 3D glycated DMEM matrices containing 10 % serum. a. Western Blot. α-SMA band, 42 kDa; α-tubulin band, 50 kDa. Albumin band (66 kDa, due to FBS and visible in cellular and acellular matrices blots’, probably because of the difficulty in rinsing all the media from the matrices prior to protein extraction). The experiments were repeated three times. a1. An insignificant amount of α-SMA was detected in all the cellular matrices. a2 and a3. Higher amounts of α-SMA were observed in all the cellular matrices on the 14th and 21st days of culture. a1, a2 and a3. No tubulin or α-SMA bands were detected in the non-cells matrices. b. Densitometric analysis of α-SMA levels (Ratio of α-SMA to α-tubulin). The results were obtained from three independent experiments. *p < 0.05, **p < 0.01. A gradual decrease of α-SMA was observed at higher ribose concentrations on the 7th day of culture (p > 0.05). Contractile phenotype was most strongly detected on the 14th and 21st days of culture. It was significantly higher at all ribose concentrations, also for controls without ribose, compared with the level on the 7th day. It suggests that the microambient ‘per se’ could induce a phenotype change and that cells contribute to the matrix contraction
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