Atelo-Collagen Monomers Encode the Information Necessary to Produce Aligned Collagen "Lamellae" De Novo

Abstract

Purpose: : In the cornea (and in other load-bearing extracellular matrices-ECMs) collagen fibrils are arranged into highly-organized arrays. The specific structure of these arrays depends on the function of each tissue. The goal of this investigation is to demonstrate that such structures may be reproduced simply de novo by controlling atelo-collagen monomer concentration and providing geometric confinement during fibrillogenesis.

Methods: : 3 mg/mL of acid soluble solution of atelo-collagen type I (PureCol, Inamed Biomaterials, Fremont, CA) was concentrated to ~175 mg/mL using 20K MWCO at 4°C. The solution was then neutralized using physiological buffer, confined between two coverslips, and left in the 37°C oven for 6 hours. Differential Interference Contrast microscopy (DIC), Quick-Freeze, Deep-Etch (QFDE), and Transmission Electron Microscopy (TEM) were used to assess the resulting collagen fibril's organization and morphology. The results were compared to the ECM derived from the fibroblast cells in culture.

Results: : DIC imaging demonstrated that the constructs possess long-range organization in the form of layers of aligned fibrillar structures which can alternate in direction (30-90 degrees between layers). QFDE and TEM images revealed three levels of collagen fibrillar structures: 1) Large diameter (17.5±1 nm) fibrils which appeared aligned (TEM and QFDE), 2) A dense and isotropic network of small diameter (5.5±1 nm) fibrils (QFDE) and 3) Medium diameter (9.3±1 nm) fibrils which are also organized and appear to be transitional structures from the small to the larger diameter fibrils. QFDE was also able to resolve areas of aligned monomers (1.5 nm diameter) which were not incorporated into fibrils. TEM and QFDE comparisons of the de novo matrix with the cell synthesized ECM revealed numerous structural similarities.

Conclusions: : These results demonstrate that in addition to the ability to self-assemble into fibrils, collagen also has longer range organizational information coded into the triple helix (in this case with no telopeptides or pro-collagen present). It seems unlikely that during development, mesenchymal cells would not take advantage of this remarkable liquid-crystalline property of collagen by confining and concentrating monomers to produce both local and global alignment.

Keywords: development • cornea: stroma and keratocytes • extracellular matrix