Chemically-bound nerve growth factor for neural tissue engineering applications.
In order to promote regeneration after spinal cord injury, growth factors have been applied in vivo to rescue ailing neurons and provide a path finding signal for regenerating neurites. We previously demonstrated that soluble growth factor concentration gradients can guide axons over long distances, but this model is inherently limited to in vitro applications. To translate the use of growth factor gradients to an implantible device for in vivo studies, we developed a photochemical method to bind nerve growth factor (NGF) to microporous poly(2-hydroxyethylmethacrylate) (PHEMA) gels and tested bioactivity in vitro. A cell adhesive photoreactive poly(allylamine) (PAA) was synthesized and characterized. This photoreactive PAA was applied to the surface of the PHEMA gels to provide both a cell adhesive layer and a photoreactive handle for further NGF immobilization. Using a direct ELISA technique, the amount of NGF immobilized on the surface of PHEMA after UV exposure was determined to be 5.65 +/- 0.82 ng/cm2 or 3.4% of the originally applied NGF. A cell-based assay was performed to determine the bioactivity of the immobilized NGF. Using pheochromocytoma (PC-12) cells, 30 +/- 7% of the cell population responded to bound NGF, a response statistically similar to that of cells cultured on collagen in the presence of 40 ng/ml soluble NGF of 39 +/- 12%. These results demonstrate that PHEMA with photochemically bound NGF is bioactive. This photochemical technique may be useful to spatially control the amount of NGF bound to PHEMA using light and thus build a stable concentration gradient.
Department of Chemical Engineering and Applied Chemistry University of Toronto, 200 College Street, Toronto, ON M5S 3E5, Canada.
Terri Adams
TA
Kapur
Molly S
MS
Shoichet
D000818:Animals; D005782:Gels; D020932:Nerve Growth Factor / Q000737:chemistry*; D009417:Nerve Tissue / Q000166:cytology*; D016716:PC12 Cells; D010777:Photochemistry / Q000379:methods; D011102:Polyhydroxyethyl Methacrylate / Q000737:chemistry; D011485:Protein Binding / Q000528:radiation effects; D051381:Rats; D023822:Tissue Engineering / Q000379:methods* / Q000592:standards; D014466:Ultraviolet Rays
D005782:Gels; D011102:Polyhydroxyethyl Methacrylate; D020932:Nerve Growth Factor