Collagen Stability in Long-Term 3-D Culture Systems for Corneal Stromal Tissue Engineering

Abstract

Purpose: : Our goal is to compare the stability of collagen in a primary human corneal fibroblast-based (PHCF) long-term stromal culture system grown scaffold-free or on reconstituted type I collagen substrate (RCS).

Methods: : PHCFs were cultured onto either bare or polycarbonate membranes coated with a 10-30 µm thick disorganized RCS. The cells were exposed to stabilized ascorbic acid to increase collagen synthesis. Optical brightfield and differential interference contrast (DIC) microscopy as well as transmission and quick-freeze/deep etch electron microscopy (TEM or QFDE) were used to assess matrix organization and collagen fibril morphology. Cuprolinic blue and TEM were used to localize PGs in the RCS constructs.

Results: : Total construct thickness was similar for the two culture systems, however, the amount of long-range organization in the constructs was significantly higher in the scaffold-free system over 11 weeks than in the RCS. Interestingly, collagen fibril morphology in the cell-derived matrix (CDM) degraded significantly with time in the scaffold-free system while CDM collagen fibril structure remained robust in the RCS culture. Cuprolinic blue staining revealed decoration of both the CDM collagen and the RCS collagen. Over time the RCS was remodeled into a loose network of fibrils and there was an increase in the number of GAG chains associated with RCS collagen.

Conclusions: : Though the RCS scaffold appears to interfere with the organization of CDM produced by PHCFs, the presence of the RCS appears to improve the stability of the cell-derived collagen. It is possible that the PHCFs are affected by the presence of the collagen scaffold such that secretion of matrix degrading enzymes (e.g MMPs) is reduced, secretion of MMP inhibitors (e.g TIMPs) is increased or that secretion of collagen is increased. It is also possible that the available RCS collagen acts as an enzyme "sink" sacrificially protecting the CDM matrix. Most intriguingly, it is possible that the large RCS collagen fibrils are being destabilized by the observed PG associations, freeing monomer to incorporate into the CDM collagen.

Keywords: cornea: basic science • cornea: stroma and keratocytes • proteoglycans/glycosaminoglycans