This study will examine the hypothesis that exposure to 60 Hz magnetic fields may produce alterations in endochonral ossification, the physiological process central to skeletal embryogenesis and longitudinal growth. In particular, we will examine 60 Hz field effects on proliferation and differentiation and the genomic regulation of these key events in development.

This study will utilize an experimental model of endochondral ossification which has been well characterized in morphological, biochemical, and molecular terms and has been used to examine proliferation and differentiation in the context of development. This model will be used to explore dosimetric considerations of 60 Hz exposure and to determine if there are developmental stages of particular sensitivity to these fields. Based on the results of previous studies, our studies are expected to reveal changes in cell differentiation and extracellular matrix synthesis in response to field stimulation. The specific amplitude and exposure duration producing these effects will be determined and the cell population responding to the stimulation will be identified. Finally, the molecular mechanisms by which 60 Hz exposure has its effects will be identified and related to amplitude and exposure parameters. The questions posed by the hypothesis will be answered by the correlation of the biological response and dose parameters.

This approach will extend the observations of 60 Hz field effects on morphology and gene expression in detail previously unavailable in a model relevant to limb development. These data will yield insight into the effects of 60 Hz exposure on fetal skeletal development.

The second aim will describe the sequence of gene expression involved in the differentiation of chondrocytes from a mesenchymal progenitor cell pool in order to gain insight into the mechanism of activity of ELF field stimulation. Specific proliferation - and differentiation - associated genes will be identified which are expressed during chondrogenic differentiation. The genes studied will be those associated with growth (histone H4, c-fos, c-jun, c-myc cyclins) and phenotypic expression (proteoglycan core protein, type II and X collagen, alkaline phosphatase, osteocalcin, and osteopontin, and for bone remodeling: TRAP and carbonic anhydrase).

Finally, since the developmental model of endochondral ossification contains a heterogeneous cell population, the third aim will be to identify the specific cell types expressing growth- and differentiation-associated genes. This will be done with in situ hybridization. This analysis will enable a further assessment of the timing of differentiation and the quantity and localization of differentiating cells. For example, since TGF is an important regulator of cartilage and bone formation and bone remodeling, it will be necessary to localize differentiation TGF expression to specific cell populations.

The exposure apparatus for the 60 Hz sinusoidal magnetic fields will be obtained from Columbia Magnetics, Inc. Kennewick, WA. The in vivo exposure apparatus (Quad-4x) consists of two exposure units. This allows for one unit to be the sham or control unit and the other to be the exposed unit. A set-up of this nature will allow for the performance of double blind experimentation. Animals will be placed in the cages mounted in the frame of the Quad-4. Exposure to the chosen dose and amplitude will be randomized and controlled by an IBM PC utilizing Texas Instruments Lab View software for Windows. The Quad-4x unit consists of: 1) one magnetic exposure apparatus (4,4 coil Merritt cells) with free standing base; 2) control switching center allowing operation in either the sham or exposed modes and 3) animal cage support racks to accommodate 4 animal cages (approximately 6"H x 7.5'W x 12"L). The unit is pre-wired at the site of manufacture to be connected to a AC veriac type power supply upon delivery.

The dimensions of the uniform field exposure is sufficient to accommodate 4 animal cages in each cell for a total of 16 cages per unit. Each unit requires a floor space area of about 2 x 4 ft. and is about 5.5 ft. tall. Two units can be positioned as close as 8 ft. apart without the stray fields of the exposed mode unit interfering with the sham mode unit.

In specific aim two, the absence of proliferation in our early studies, together with earlier expression of differentiation - associated genes for proteoglycan, core protein, and type II collagen, suggests that we will observe a down-regulation of growth-associated genes and an earlier expression of differentiation-associated genes. This would suggest an abbreviation of proliferation and an acceleration of differentiation.