The surface of biodegradable chitosan nanofibers was functionalized with pristine amine groups and used for immobilization of substance-P, which mediates pain perception and regulates wound healing, inflammation, and angiogenesis. The objectives of this study were to develop substance-P immobilized chitosan nanofibers and to evaluate the biological effects of the immobilized substance-P. Under the same conditions, the amount of substance-P covalently immobilized on chitosan nanofibers was 72.5 +/- 16.9ng compared to 7.3 +/- 2.2ng in passively absorbed samples. Substance-P on chitosan nanofibers released slowly and promoted greater proliferation (similar to 1.4-fold) of human mesenchymal stem cells than pristine samples after 7days in culture.