Nerve fiber growth in culture on fibronectin, collagen, and glycosaminoglycan substrates

Abstract

In an initial report we described the use of 2-hydroxyethylmethacrylate (HEMA) in preparing defined culture substrates for studying nerve fiber growth. In those studies fibronectin and collagen were conspicuous, among a variety of HEMA-embedded proteins, in supporting fiber growth of embryonic neurons from chick dorsal root ganglia. Here we further document and extend our preliminary studies, especially with regard to the interaction of growing nerve fibers with fibronectin. HEMA substrates were prepared with proteolytic fragments of fibronectin, each of which had one or more of the functional sites of the intact molecule. Only those fragments of fibronectin that retained the region of the molecule known to mediate myoblast attachment were active in supporting nerve fiber growth. When added in excess to the culture medium, the samllest of the active fragments inhibited fiber growth on substrates that contained intact fibronectin. In culture medium depleted of serum fibronectin, HEMA gels containing collagens purified from connective tissues (types I and III) or from basement membranes (type IV) were about as effective as HEMA substrates containing fibronectin in supporting fiber growth. Nerve fiber growth on collagen substrates proceeded in the absence of fibronectin in the culture medium. Several glycosaminoglycans (heparin, chondroitin sulfate, hyaluronic acid) were ineffective as substrates for fiber growth. Treatment of HEMA/fibronectin gels with heparin or incorporation of heparin along with fibronectin in the gel profoundly diminished the efficacy of fibronectin in supporting nerve fiber growth. Our studies suggest that the growth of nerve fibers on fibronectin substrates results from direct interaction with a specific portion of the fibronectin molecule and that this interaction can be inhibited by heparin and possibly other glycosaminoglycans.