Biochemical study of human periodontal ligament: preparation of cell attachment materials induced by pulsed electromagnetic fields.

The periodontium, especially the periodontal ligament and alveolar bone, are tissues constantly subjected to physical stress such as occlusion and mastication. This study was designed to explore the effect of the pulsed electromagnetic fields (PEMF) on the cell attachment and the spread of human periodontal ligament fibroblasts (HPLF) and rat osteoblasts (ROB). PEMF are categorized as one type of mechanical stress. HPLF were obtained by the explantation method described by Saito et al. They were then subcultured in Dulbecco's modified Eagle's medium (D-MEM) and supplemented with 2 mg/ml dialyzed fetal calf serum protein (FCSP), 50 micrograms/ml ascorbic acid and penicillin/streptomycin after trypsinization. ROB were isolated from a two-day-old rat calvaria by the sequential bacterial collagenase digestion method described by Dziak and Brand and were subcultured in D-MEM supplemented with FCSP, ascorbic acid and penicillin/streptomycin. After the confluent HPLF were cultured with serum-free MCDB 107 medium, the quiescent HPLF were exposed with or without PEMF for 24 hr. This was followed by the collection of the control conditioned medium (C-CM) and PEMF exposed conditioned medium (PEMF-CM). The cell attachment assay was performed so that the hydrophobic 24 multiwells were coated with the whole conditioned medium or fractionated conditioned medium by a PO-60K column. After coating, heat inactivated BSA blocked nonspecific sites for cell adhesion, and 3H-TdR labeled HPLF or ROB were cultured on the precoated wells. The activity of cell attachment and spreading was determined by the radioactivity of 3H-TdR using a scintillation counter. The characters of cell attachment factors derived from HPLF were hydrophobic, heat labile and proteolytic enzyme digestible. In addition, the fractionated PEMF-CM enhanced the spreading activity of ROB. PEMF induced the 10 KDa which can enhance the HPLF and ROB spreading. Therefore, the cell attachment and spreading factors secreted by HPLF exposed with PEMF may regulate HPLF and also ROB.