A method for preparation of etched collagen fibers that support neurite outgrowth

Abstract

Use of collagenous substrates for growth of attachment-dependent and attachmentindependent cells have been reported in the literature. Growth of fibroblasts and epithelial cells on collagen sponges and fibers has been previously studied in our laboratory. This article reports the result of studies of neurite outgrowth on etched collagen fibers in cell culture. Collagen fibers with a mean diameter of about 134 p m were etched on glass coverslips using a collagenase solution until individual fibrils of 1 p m in diameter were observed. The kinetics of etching were optimized at a calcium chloride concentration of 5 mM and a collagenase activity of 300 unit/mL. At room temperature an etching time of 10 h maximized the number of fibrils exposed per fiber. Cell culture studies on etched collagen fibers indicate that neurites from rat fetal cortical neurons elongate along the longitudinal axes of collagen fibrils. In some instances the cell body observed was not on top of the collagen fibrils but situated between two parallel fibrils. Results of these experiments indicate that growth of neurites along collagen fibrils in cell culture may be a useful model to evaluate neurite extension in the presence of neurotrophic factors as well as to study optimization of substrates for nerve regeneration.