Calvarial bone cells of rats were subjected to either a cyclic biaxial strain of 0.17 per cent (1700 microstrain) or a hydrostatic pressure of 2.5, five, or ten pounds per square inch (17.2, 34.5, or sixty-nine kilopascals). The frequency was held constant at one hertz for both types of mechanical stimulation, When cultured bone cells that had been subjected to a cyclic biaxial strain for two hours were harvested twenty-two hours later, it was found that the level of prostaglandin E(2) had increased significantly (p < 0.01) as had cellular proliferation (p < 0.01), as indicated by the incorporation of [H-3]-thymidine. The addition to the medium of indomethacin, an inhibitor of prostaglandin synthesis, at a ten-micromolar concentration significantly inhibited (p < 0.01) the increase in prostaglandin E(2) synthesis but had no effect on the strain-induced increase in cellular proliferation, as indicated by the incorporation of [H-3]-thymidine, Twenty-four hours after exposure to the same cyclic biaxial strain for thirty seconds, other cultured bone cells showed a significant increase in the level of cytoskeletal calmodulin (p < 0.05) and in the DNA content (p < 0.05), N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7), a calmodulin antagonist, was added to the medium at a one-micromolar concentration, which had been shown to have no effect on the increase in the DNA content of control cells; W-7 completely blocked the increase in the level of cytoskeletal calmodulin and in the DNA content in the cells that were subjected to a cyclic biaxial strain, The bone cells subjected to a hydrostatic pressure showed a dose-dependent increase in the concentration of cytosolic Ca2+, as measured with Fura 2-AM, a fluorescent indicator of intracellular calcium, With a pressure of ten pounds per square inch (sixty-nine kilopascals), the increase in the concentration of cytosolic Ca2+ was nearly eight times greater than that at 2.5 pounds per square inch (17.2 kilopascals) (126 +/- 15.2 compared with 16 +/- 8.0 nanomolar, mean and standard deviation), The addition to the medium of neomycin, an inhibitor of the inositol phosphate cascade, at a ten-millimolar concentration completely blocked the increase in the concentration of cytosolic Ca2+ in these cells; this concentration of neomycin had been shown to have no effect on proliferation in control bone cells, There was also a dose-dependent relationship between the duration of the stimulus and the cellular proliferation, Remarkably, one cycle of pressure at ten pounds per square inch (sixty-nine kilopascals) and a frequency of approximately one hertz produced a 57 per cent increase in the incorporation of [H-3]-thymidine at twenty-four hours (p < 0.001), From these findings, we hypothesized that the inositol phosphate cascade-cytosolic Ca2+-cytoskeletal calmodulin system plays a dominant role in the signal transduction of a mechanical stimulus into increased proliferation of bone cells, at least under the conditions reported here, CLINICAL RELEVANCE: Understanding the mechanisms by which bone cells convert a mechanical signal into a biological response is the beginning of an understanding of Wolff's law which states that form follows function, A complete understanding of Wolff's law eventually should lead to an understanding of the cellular and molecular processes governing bone-remodeling and may allow therapeutic manipulation of bone-remodeling in clinical practice.
