JOURNAL OF BONE AND MINERAL RESEARCH Volume zyxwvutsrqpo I, Number I, 1993 Mary Ann Liebert, Inc., Publishers zyxwvutsrqp Electric Fields Modulate Bone Cell Function in a Density-Dependent Manner KENNETH J. McLEOD,’ HENRY J. DONAHUE,’ PAUL E. LEVIN,’ MARIE-ANNE FONTAINE,’ and CLINTON T. RUBIN’ ABSTRACT The influence of an extremely low frequency (ELF) electric field stimulus zyxw (30 Hz at 6 pV/cm rms), known to promote bone formation in vivo, was evaluated for its ability to affect bone cell function in vitro. To accom- plish this, we developed an apparatus for the exposure of monolayer cell systems to electric fields in a man- ner that provides relatively uniform electric field exposure of multiple cell samples as well as a rigorous sham exposure. We show that field exposure significantly limits the normal increase in osteoblastic cell number and enhances alkaline phosphatase activity compared to sham-exposed samples. Moreover, these alterations are shown to occur in a cell density-dependent manner. Samples plated at 6 zyxw x lo3 cells/cm’ show no effect of field exposure, In samples plated at 30 x lo’ cells/cm’, 72 h of field exposure resulted in 25% fewer cells in the exposed samples, and a doubling of alkaline phosphatase activity in those cells compared to sham ex- posure. Experiments using a 12 h exposure to preclude significant changes in cell number during the expo- sure show this density-dependent response to be biphasic. Sparse cultures (< 50 x lo3 cells/cm’) were not found to be affected by the field exposure, but increases in alkaline phosphatase activity occurred in cultures at densities of zyxwvuts 50-200 x lo3 and 200-350 x lo3 cells/cm’ and no effect on alkaline phosphatase activity was seen in confluent cell cultures of greater than 350 x lo3 cells/cm’. This work suggests that the demonstrated osteogenic effect of this specific electric field stimulus on bone tissue may be initiated by an alteration of the differentiated status of the osteoblasts in the tissue rather than by stimulating cell proliferation, as has been previously suggested. As importantly, a more thorough characterization of this density-dependent interac- tion with electric fields may help to identify the mechanism of transduction by which ELF electric fields in- teract with cells. INTRODUCTION HE EFFECTS OF EXTREMELY LOW FREQUENCY (ELF) elec- T tric fields on cell and tissue function has recently be- come a topic of considerable interest. Numerous investi- gations have begun to address the possible effect of field exposure on the neural and immune systems, and bone is perhaps the tissue most often studied in the context of elec- tric field effects. Bone tissue and cells are uniquely suited for studies on the interaction of electric fields with cells be- cause endogenous electric fields of substantial intensity normally occur within bone(’-” and exogenously applied fields are known to stimulate bone formation in viva.(') Using an in vivo model of disuse osteoporosis, we have pre- viously shown that induced electric fields zyx can both prevent the bone loss associated with disuse and also stimulate substantial new bone formation.(6)In addition, bone appears to be maxi- mally sensitive to electric fields induced within the endoge- nous frequency range, that is, at the frequencies of the electric currents that normally zyxw arise in bone as a result of functional activity.”) Specifically, we have shown that new bone forma- tion zyxwv can be maximized by using electric field stimuli in the fre- quency range of 10-30 Hz. Within this range, bone tissue will respond to fields as low as 1-10 microvolts per centi- meter (pV/cm),(’) field intensities on the order of those en- dogenously produced in bone tissue during normal activity. ‘Musculo-Skeletal Research Laboratory, Department of Orthopaedics. State University of New York, Stony Brook. ’University of Liege, Belgium. 977