Proliferation of anterior cruciate ligament cells in vitro by photo‐immobilized epidermal growth factor

Abstract

The purpose of this study was to explore the early treatment potential of anterior cruciate ligament (ACL) injuries using artificial juxtacrine stimulation by photo-immobilization of a growth factor. A photo-reactive epidermal growth factor complex (EGF-Az) was synthesized by conjugating EGF with N-(4-azidobenzoyloxy) succinimide followed by immobilization onto polystyrene culture plates using UV irradiation. ACL cells from human tissues (1 x 10(5)cells, 100 microl/well) were cultured as follows: control, no EGF; 50 microl native EGF; 50 microl EGF-Az immobilized; and 100 microl EGF-Az immobilized. The ACL cells were cultured long-term and evaluated for possible differences in their responses to EGF. An in vitro wound closure assay was developed to enable examination of cellular proliferation and migration. ACL cell proliferation was most evident in the photo-immobilized EGF culture group and was seen to increase in proportion to the amount of added EGF. In the in vitro wound closure assay, the lesioned area at 72 h after culture initiation was indistinguishable in the photo-immobilized cultures, but remained clearly visible in the controls. We conclude that photo-immobilized EGF induced rapid proliferation of ACL fibroblast cells by artificial juxtacrine stimulation and speculate that similar EGF immobilization onto bioabsorbable material (e.g., polyglycolic acid or polylactic acid) might contribute to a new therapy for the treatment of ACL injuries.