The Journal of Bone and Joint Surgery 78:1337-47 (1996)
© 1996 The Journal of Bone and Joint Surgery, Inc.
The Biochemical Pathway Mediating the Proliferative Response of Bone Cells to a Mechanical Stimulus*
CARL T. BRIGHTON, M.D., PH.D. ,
J. RUSH S. FISHER, JR., M.D.,
STUART E. LEVINE, M.D.,
JOHN R. CORSETTI, M.D.,
THOMAS REILLY, M.D.,
ADAM S. LANDSMAN, PH.D. ,
JOHN L. WILLIAMS, PH.D. and
LAWRENCE E. THIBAULT, PH.D., PHILADELPHIA, PENNSYLVANIA
Investigation performed at the Department of Orthopaedic Surgery, University of Pennsylvania School of Medicine, and the Department of Bioengineering, University of Pennsylvania School of Engineering and Applied Science, Philadelphia
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 E2 had increased significantly (p < 0.01) as had cellular proliferation (p < 0.01), as indicated by the incorporation of [3H]-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 E2 synthesis but had no effect on the strain-induced increase in cellular proliferation, as indicated by the incorporation of [3H]-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 [3H]-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.
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