Human Corneal Fibroblasts Secrete Locally-Organized Collagen Arrays on Disorganized Collagen Substrates

Abstract

Purpose: : To determine if the structure of extracellular matrix secreted by stimulated primary human corneal fibroblasts (PHCFs) is influenced by the structure of a disorganized collagenous substrate.

Methods: : Human corneal stromal cells were isolated from donor rims and stimulated with stabilized Vitamin C (to facilitate collagen synthesis) in the presence of 10% FBS (to induce a fibroblastic phenotype transition). The resulting PHCFs were plated at high density onto transwell membranes coated with disorganized type I collagen and allowed to produce matrix for up to 12 weeks. Structural assessments included standard and quick-freeze deep etch electron microscopy and differential interference contrast optical microscopy.

Results: : The fibroblasts formed a stratified construct on top of the reconstituted collagen substrate. The construct comprised significant amounts of ECM interspersed with flattened fibroblastic cells. The synthesized ECM contained alternating arrays of striated fibrils in stroma-like lamellae. The local organization of the synthesized collagen did not , reflect the structure of the reconstituted, disorganized collagen fibrils. There was typically a discernible organizational demarcation line between the synthesized collagen and the substrate collagen (even around cells which penetrated the substrate). Interestingly, there were numerous vesicles and a secondary matrix of thin fibrils observed in the culture. The role of these structures is not known. In addition, the standard TEM suggested the presence of significant empty space. While QFDE revealed a rich, dense ECM throughout the synthesized matrix.

Conclusions: : The investigation demonstrated that human corneal fibroblasts are capable of synthesizing organized ECM resembling developing mammalian stroma in culture on reconstituted collagen gels. The local organization of the synthesized collagen does not appear to be influenced by the disorganized collagen substrate even at the interface. We conclude that the mechanisms which control synthesized collagen short-range organization in our culture system are exerted locally by the fibroblasts. These results are consistent with observed ability of mammalian neural-crest derived mesenchymal cells to synthesize organized stroma during development without a primary stromal template.

Keywords: cornea: stroma and keratocytes • cornea: basic science