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Human primary corneal fibroblasts synthesize and deposit proteoglycans in long-term 3-D cultures.

ABSTRACT: Our goal was to develop a 3-D multi-cellular construct using primary human corneal fibroblasts cultured on a disorganized collagen substrate in a scaffold-free environment and to use it to determine the regulation of proteoglycans over an extended period of time (11 weeks). Electron micrographs revealed multi-layered constructs with cells present in between alternating parallel and perpendicular arrays of fibrils. Type I collagen increased 2-4-fold. Stromal proteoglycans including lumican, syndecan4, decorin, biglycan, mimecan, and perlecan were expressed. The presence of glycosaminoglycan chains was demonstrated for a subset of the core proteins (lumican, biglycan, and decorin) using lyase digestion. Cuprolinic blue-stained cultures showed that sulfated proteoglycans were present throughout the construct and most prominent in its mid-region. The size of the Cuprolinic-positive filaments resembled those previously reported in a human corneal stroma. Under the current culture conditions, the cells mimic a development or nonfibrotic repair phenotype.

SUBMITTER: Ren R

PROVIDER: S-EPMC3760227 | biostudies-literature | 2008 Oct

REPOSITORIES: biostudies-literature

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Human primary corneal fibroblasts synthesize and deposit proteoglycans in long-term 3-D cultures.

Ren R R Hutcheon A E K AE Guo X Q XQ Saeidi N N Melotti S A SA Ruberti J W JW Zieske J D JD Trinkaus-Randall V V

Developmental dynamics : an official publication of the American Association of Anatomists 20081001 10

Our goal was to develop a 3-D multi-cellular construct using primary human corneal fibroblasts cultured on a disorganized collagen substrate in a scaffold-free environment and to use it to determine the regulation of proteoglycans over an extended period of time (11 weeks). Electron micrographs revealed multi-layered constructs with cells present in between alternating parallel and perpendicular arrays of fibrils. Type I collagen increased 2-4-fold. Stromal proteoglycans including lumican, synde ...[more]

PMID: 18624285

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Project description:BackgroundCoinfections with fungi and bacteria in ocular pathologies are increasing at an alarming rate. Two of the main etiologic agents of infections on the corneal surface, such as Aspergillus fumigatus and Staphylococcus aureus, can form a biofilm. However, mixed fungal-bacterial biofilms are rarely reported in ocular infections. The implementation of cell cultures as a study model related to biofilm microbial keratitis will allow understanding the pathogenesis in the cornea. The cornea maintains a pathogen-free ocular surface in which human limbo-corneal fibroblast cells are part of its cell regeneration process. There are no reports of biofilm formation assays on limbo-corneal fibroblasts, as well as their behavior with a polymicrobial infection.ObjectiveTo determine the capacity of biofilm formation during this fungal-bacterial interaction on primary limbo-corneal fibroblast monolayers.ResultsThe biofilm on the limbo-corneal fibroblast culture was analyzed by assessing biomass production and determining metabolic activity. Furthermore, the mixed biofilm effect on this cell culture was observed with several microscopy techniques. The single and mixed biofilm was higher on the limbo-corneal fibroblast monolayer than on abiotic surfaces. The A. fumigatus biofilm on the human limbo-corneal fibroblast culture showed a considerable decrease compared to the S. aureus biofilm on the limbo-corneal fibroblast monolayer. Moreover, the mixed biofilm had a lower density than that of the single biofilm. Antibiosis between A. fumigatus and S. aureus persisted during the challenge to limbo-corneal fibroblasts, but it seems that the fungus was more effectively inhibited.ConclusionThis is the first report of mixed fungal-bacterial biofilm production and morphological characterization on the limbo-corneal fibroblast monolayer. Three antibiosis behaviors were observed between fungi, bacteria, and limbo-corneal fibroblasts. The mycophagy effect over A. fumigatus by S. aureus was exacerbated on the limbo-corneal fibroblast monolayer. During fungal-bacterial interactions, it appears that limbo-corneal fibroblasts showed some phagocytic activity, demonstrating tripartite relationships during coinfection.

Dataset Information

Human primary corneal fibroblasts synthesize and deposit proteoglycans in long-term 3-D cultures.

Publications

Human primary corneal fibroblasts synthesize and deposit proteoglycans in long-term 3-D cultures.

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