Development of a three-dimensional extracellular matrix equivalent for neural cells and nerve regeneration.

摘要

The ability to organize cellular responses in three dimensions (3-D) constitutes an important step in tissue engineering and reconstruction, especially for nervous tissue. This dissertation deals with the development of an hydrogel based extracellular matrix (ECM) equivalent whose 3-D structure and biological activity are optimized for neurite extension from neural cells. Rat embryonic day 14 striatal cells and chick embryonic day 9 dorsal root ganglia extended neurites in agarose hydrogels in a concentration dependent manner. Primary neural cells did not extend neurites above an agarose gel concentration of 1.25% wt/vol which yielded an average pore radius of 150 nm. Gel pore size studies revealed that the pore size of agarose gels fell exponentially as the gel concentration increased. We surmise that the gel porosity plays an important role in determining the ability of agarose gels to support neurite extension. Lamination of permissive and non-permissive gel concentrations facilitates the creation of 3-D neural 'tracts' in vitro. The phenotypic expression of various cells is influenced by ECM molecules. Therefore the agarose hydrogel with the optimal physical structure for neurite extension was subsequently derivatized with various ECM oligopeptides. The oligopeptide domains GRGDSP, CDPGYIGSR, and IKVAV, which are partly responsible for the cell attachment and neurite promoting properties of laminin (LN), were covalently immobilized to agarose gels. Embryonic day 9 (E9) chick dorsal root ganglia (DRG) and PC12 cells were suspended in derivatized agarose gels and their neurite extension was evaluated in three dimensions. Agarose gels derivatized with CDPGYIGSR and a cocktail of all three peptides, enhanced neurite extension from E9 DRGs while IKVAV derivatized gels inhibited neurite extension. However, IKVAV derivatized gels enhanced neurite extension from PC12 cells compared to underivatized agarose gels and gels derivatized with other LN oligopeptides. The effect of derivatized agarose gels on the regeneration of transected rat spinal dorsal roots was evaluated by using 6 mm long polymer guidance channels filled with CDPGYIGSR-agarose to bridge a 4 mm gap in a transected dorsal root model. After 4 weeks, significantly greater numbers of myelinated axons were observed in the channels filled with CDPGYIGSR-agarose gels compared to channels filled with underivatized agarose gels. Thus 3-D matrices carrying appropriate neuroactive peptides can be tailored to evoke specific responses from cells and tissues exposed to them.; The ability of agarose hydrogels to organize, support and direct neurite extension coupled with the possibility of specifically 'tailoring' them to suit particular cellular environments, makes them attractive candidates for nervous tissue engineering applications such as 3-D neural cell culture, directed 3-D neurite extension in vitro. In the in vivo scenario, they may also serve to enhance regeneration by optimally presenting neurite promoting molecules to the regeneration environment.