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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

Collagen post-glycation increased the matrix stiffness. The stiffness of the non-cellular post-glycated matrices was measured using atomic force microscopy at days 7, 14 and 21. The values were normalized using that of the non-glycated matrices, i.e. control matrices (0 mM of ribose (R)) at the 7th day. The black arrows indicate the R concentration that rendered the collagen gels stiffer than the non-glycated gel at the 7th day. a. DMEM matrices. Collagen gels stiffened in a R dependent manner at the 14th day after glycation at 30 and 240 mM of ribose respect to the control non-glycated gels (p < 0.01). b. PBS matrices. The collagen gels stiffened with 240 mM of ribose at the 21st day after glycation (p < 0.01). a and b. This different stiffening dynamics suggests different cross-linking rates between these gels. The glucose present in DMEM could play a role in the stiffness developed, which could render a higher basal level of glycation before ribose treatment. Accordingly, the non-glycated DMEM gels (controls) showed greater stiffness than the glycated matrices at the 7th day. Phenomenon not observed in PBS matrices. The highest R concentration (240 mM) led to the stiffest conformation of both types of matrices (p < 0.01). p-values for 30 mM of ribose: + (0.01 < p < 0.05), ++ (p < 0.01); p-values for 240 mM of ribose: *(0.01 < p < 0.05), ** (p < 0.01)
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Fig3: Collagen post-glycation increased the matrix stiffness. The stiffness of the non-cellular post-glycated matrices was measured using atomic force microscopy at days 7, 14 and 21. The values were normalized using that of the non-glycated matrices, i.e. control matrices (0 mM of ribose (R)) at the 7th day. The black arrows indicate the R concentration that rendered the collagen gels stiffer than the non-glycated gel at the 7th day. a. DMEM matrices. Collagen gels stiffened in a R dependent manner at the 14th day after glycation at 30 and 240 mM of ribose respect to the control non-glycated gels (p < 0.01). b. PBS matrices. The collagen gels stiffened with 240 mM of ribose at the 21st day after glycation (p < 0.01). a and b. This different stiffening dynamics suggests different cross-linking rates between these gels. The glucose present in DMEM could play a role in the stiffness developed, which could render a higher basal level of glycation before ribose treatment. Accordingly, the non-glycated DMEM gels (controls) showed greater stiffness than the glycated matrices at the 7th day. Phenomenon not observed in PBS matrices. The highest R concentration (240 mM) led to the stiffest conformation of both types of matrices (p < 0.01). p-values for 30 mM of ribose: + (0.01 < p < 0.05), ++ (p < 0.01); p-values for 240 mM of ribose: *(0.01 < p < 0.05), ** (p < 0.01)

Mentions: To indirectly assess the extent of AGEs formation, we measured the autofluorescence intensity of DMEM and PBS matrices with different FBS concentrations at the wavelength of fluorescent AGEs (ex/em 360/440 nm) by a plate reader as described in the literature [20, 21, 27, 28, 31, 32]. As shown in Fig. 2 (a and b), the content of AGEs increased gradually in a ribose concentration-dependent manner in both conditions, DMEM and PBS (p < 0.05), independently of the serum concentration (p > 0.05). The highest value was found at the highest ribose concentration (240 mM, p < 0.01). An autofluorescence peak was found at 7th day (p < 0.01) in all conditions. Then, less autofluorescence was observed for all the matrices at day 14 and 21, probably due to a decreased amount of AGEs formation after changing the media every 2 days. Interestingly, autofluorescence measurements revealed that DMEM matrices (Fig. 2a) were more autofluorescent than PBS matrices (Fig. 2b) at days 1 and 7 (p < 0.05 in practically all conditions). Nevertheless, PBS matrices became more autofluorescent than DMEM ones after the 14th day (statistically significant in practically all conditions for FBS 1 % and 10 %). Because the accumulation of AGEs generated by non-enzymatic glycation induces modifications and cross-links that stiffen human tissue proteins [22, 25], we examined using the mechanical changes in glycated matrices at the micrometer scale by measuring the Young’s elastic modulus (E) through AFM (Fig. 3). The E value of the control gels (0 mM ribose) at day 7 was 1.6 ± 0.3 and 0.9 ± 0.5 kPa in DMEM and PBS post-glycated matrices (0 % FBS), respectively. All subsequent E values were normalized to the corresponding control values at day 7. Our results revealed that both DMEM and PBS matrices stiffened in a ribose-dependent manner with respect to the control non-glycated gels (p < 0.01). The highest value of stiffness was reached at day 21st for PBS matrices as shown in Table 1 (absolute E value: 2.5 ± 1.35 KPa). Nonetheless, the maximum point of stiffness was not always at the end of the experiment. Fold stiffness in DMEM gels remained rather unaltered after day 14th. In contrast we observed a moderate rise in fold gel stiffness in PBS gels up to 21st day (Fig. 3a and b). Consequently, stiffness of the matrix was greater after the 14th day of post-polymerization. However, the basal stiffness values in control gels (0 mM ribose) at day 7th were higher in DMEM than in PBS gels, perhaps because of glucose present in DMEM.Fig. 2


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)

Collagen post-glycation increased the matrix stiffness. The stiffness of the non-cellular post-glycated matrices was measured using atomic force microscopy at days 7, 14 and 21. The values were normalized using that of the non-glycated matrices, i.e. control matrices (0 mM of ribose (R)) at the 7th day. The black arrows indicate the R concentration that rendered the collagen gels stiffer than the non-glycated gel at the 7th day. a. DMEM matrices. Collagen gels stiffened in a R dependent manner at the 14th day after glycation at 30 and 240 mM of ribose respect to the control non-glycated gels (p < 0.01). b. PBS matrices. The collagen gels stiffened with 240 mM of ribose at the 21st day after glycation (p < 0.01). a and b. This different stiffening dynamics suggests different cross-linking rates between these gels. The glucose present in DMEM could play a role in the stiffness developed, which could render a higher basal level of glycation before ribose treatment. Accordingly, the non-glycated DMEM gels (controls) showed greater stiffness than the glycated matrices at the 7th day. Phenomenon not observed in PBS matrices. The highest R concentration (240 mM) led to the stiffest conformation of both types of matrices (p < 0.01). p-values for 30 mM of ribose: + (0.01 < p < 0.05), ++ (p < 0.01); p-values for 240 mM of ribose: *(0.01 < p < 0.05), ** (p < 0.01)
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4494165&req=5

Fig3: Collagen post-glycation increased the matrix stiffness. The stiffness of the non-cellular post-glycated matrices was measured using atomic force microscopy at days 7, 14 and 21. The values were normalized using that of the non-glycated matrices, i.e. control matrices (0 mM of ribose (R)) at the 7th day. The black arrows indicate the R concentration that rendered the collagen gels stiffer than the non-glycated gel at the 7th day. a. DMEM matrices. Collagen gels stiffened in a R dependent manner at the 14th day after glycation at 30 and 240 mM of ribose respect to the control non-glycated gels (p < 0.01). b. PBS matrices. The collagen gels stiffened with 240 mM of ribose at the 21st day after glycation (p < 0.01). a and b. This different stiffening dynamics suggests different cross-linking rates between these gels. The glucose present in DMEM could play a role in the stiffness developed, which could render a higher basal level of glycation before ribose treatment. Accordingly, the non-glycated DMEM gels (controls) showed greater stiffness than the glycated matrices at the 7th day. Phenomenon not observed in PBS matrices. The highest R concentration (240 mM) led to the stiffest conformation of both types of matrices (p < 0.01). p-values for 30 mM of ribose: + (0.01 < p < 0.05), ++ (p < 0.01); p-values for 240 mM of ribose: *(0.01 < p < 0.05), ** (p < 0.01)
Mentions: To indirectly assess the extent of AGEs formation, we measured the autofluorescence intensity of DMEM and PBS matrices with different FBS concentrations at the wavelength of fluorescent AGEs (ex/em 360/440 nm) by a plate reader as described in the literature [20, 21, 27, 28, 31, 32]. As shown in Fig. 2 (a and b), the content of AGEs increased gradually in a ribose concentration-dependent manner in both conditions, DMEM and PBS (p < 0.05), independently of the serum concentration (p > 0.05). The highest value was found at the highest ribose concentration (240 mM, p < 0.01). An autofluorescence peak was found at 7th day (p < 0.01) in all conditions. Then, less autofluorescence was observed for all the matrices at day 14 and 21, probably due to a decreased amount of AGEs formation after changing the media every 2 days. Interestingly, autofluorescence measurements revealed that DMEM matrices (Fig. 2a) were more autofluorescent than PBS matrices (Fig. 2b) at days 1 and 7 (p < 0.05 in practically all conditions). Nevertheless, PBS matrices became more autofluorescent than DMEM ones after the 14th day (statistically significant in practically all conditions for FBS 1 % and 10 %). Because the accumulation of AGEs generated by non-enzymatic glycation induces modifications and cross-links that stiffen human tissue proteins [22, 25], we examined using the mechanical changes in glycated matrices at the micrometer scale by measuring the Young’s elastic modulus (E) through AFM (Fig. 3). The E value of the control gels (0 mM ribose) at day 7 was 1.6 ± 0.3 and 0.9 ± 0.5 kPa in DMEM and PBS post-glycated matrices (0 % FBS), respectively. All subsequent E values were normalized to the corresponding control values at day 7. Our results revealed that both DMEM and PBS matrices stiffened in a ribose-dependent manner with respect to the control non-glycated gels (p < 0.01). The highest value of stiffness was reached at day 21st for PBS matrices as shown in Table 1 (absolute E value: 2.5 ± 1.35 KPa). Nonetheless, the maximum point of stiffness was not always at the end of the experiment. Fold stiffness in DMEM gels remained rather unaltered after day 14th. In contrast we observed a moderate rise in fold gel stiffness in PBS gels up to 21st day (Fig. 3a and b). Consequently, stiffness of the matrix was greater after the 14th day of post-polymerization. However, the basal stiffness values in control gels (0 mM ribose) at day 7th were higher in DMEM than in PBS gels, perhaps because of glucose present in DMEM.Fig. 2

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