Soluble heparin binding fraction from fibronectin tissue engineered scaffold inhibits neuronal growth

Abstract

Fibronectin (Fn) is an extracellular matrix glycoprotein involved in development and repair by promoting cell adhesion and consequent migration. It has been used experimentally as a scaffold for neural tissue engineered constructs to guide peripheral and central nervous system repair. Certain preparations of shear-aggregated fibronectin, however, inhibit neurite outgrowth and fibroblast adhesion. Washing this material abolished inhibition, suggesting that a soluble protein fragment from the material could be responsible. The aim of this study was to separate the fragment(s) responsible for this inhibition. The effects of medium conditioned with fibronectin and fibronectin derived fractions were tested in vitro on neurons from dorsal root ganglia. Fractions were separated according to heparin or gelatin binding affinity, and by ultrafiltration according to molecular weight. The extent of neurite outgrowth was measured, under these various conditions, using an immunofluorescent assay with quantitative image analysis. Unfractionated Fn-conditioned medium produced no significant reduction in neurite outgrowth compared to control medium. However, the individual fractions, obtained via affinity chromatography produced significant inhibition. In particular, the heparin binding fraction was the most inhibitory and blocked neurite outgrowth in a dose dependent manner. This reduction in neuronal growth did not increase cell death as determined by metabolic assay. The inhibitory fragment appeared to be less than 30 kDa molecular weight. These results indicate that the heparin binding fraction from certain preparations of shear-aggregated fibronectin can inhibit neuronal growth without cell death. This derivative of an endogenous protein therefore has therapeutic potential for reducing neuroma formation, control of tissue adhesion and in neural tissue engineering