Abstract
BackgroundThere 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.MethodsThe 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.ResultsOur 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12931-015-0237-z) contains supplementary material, which is available to authorized users.
Highlights
There is growing interest in the development of cell culture assays that enable the rigidity of the extracellular matrix to be increased
An increased intensity of collagen reflection fluorescence was observed in all the Dulbecco’s modified Eagle medium (DMEM) matrices with the highest ribose concentration (240 mM)
Most in vitro studies in lung fibrosis have been performed with fibroblasts growing in a plate (2D) or in 3D based on polyacrylamide hydrogels [8, 38, 39] or collagen type I gels [17, 18, 27, 40] with fibroblasts growing on top
Summary
There is growing interest in the development of cell culture assays that enable the rigidity of the extracellular matrix to be increased. Myofibroblasts and other cell types, are normally surrounded by this fibrous three-dimensional (3D) extracellular matrix and are mainly responsible for the synthesis, secretion and degradation of the ECM components These cells are responsible for the correct turnover of ECM proteins, preserving the lung architecture and function [1]. Most in vitro studies of fibroblasts behavior have been performed using conventional two-dimensional (2D) cultures, which lack the structural three-dimensionality provided by the ECM in the original tissue This limitation is important because it can cause the loss or change of important tissue-specific cell functions due to the lack of essential ECM signals [2, 3]. These studies have clearly shown that cell functions and phenotypes depend on the structure of the microenvironment, such as the ECM [2,3,4] and have revealed the profound impact of extracellular forces on the behavior of fibroblasts and other cell types [3, 7, 8]
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