ASGSB 1999 Annual Meeting Abstracts

GENE EXPRESSION OF CULTURED OSTEOBLASTS SUBJECTED TO HYPERGRAVITY. W.J. Landis, V. Towe¹, K.J. Hodgens¹, J. DiCanzio¹ and L.C. Gerstenfeld². The Northeastern Ohio Universities College of Medicine, Rootstown, OH, ¹Children’s Hospital, Boston, MA, and ²Boston University School of Medicine, Boston, MA.

To understand more completely the adaptive nature of bone in response to loading, cultures of osteoblasts isolated from normal 17-day old embryonic chick calvaria were introduced into hollow fiber bioreactors (Spectrum Laboratories, Laguna Hills, CA) and placed in an incubator (37C, 5% CO₂) at the end of a 9-ft centrifuge arm at the Hypergravity Facility for Cell Culture (HyFaCC), NASA/Ames Research Center, Moffett Field, CA. Cells were subjected to 3.3G or 4.0G for a period of 2 wks on the centrifuge, cell media (DME, 10% FBS, 12.5 µg/ml ascorbate, 10 mM -glycerophosphate) being changed (~1 hr duration) every 2 days.  Osteoblasts under identical conditions were maintained at 1G as controls over the same 2 wks.  Cells were harvested and total RNA extracted using Tri-Reagent (Molecular Research Center, Cincinnati, OH). RNA was resolved on agarose gels and hybridized to cDNA probes of chicken fibronectin (FN), osteopontin (OPN), osteocalcin (OC), type I collagen (C), and bone sialoprotein (BSP) to examine respective levels of message expression. Autoradiogram values were normalized to 18S rRNA. Three independent measurements were made for each blot. After 2 wks of centrifugation, all mRNA levels had decreased in magnitude at both 3.3G and 4.0G compared to 1G and at 4.0G compared to 3.3G. A multiple comparisons procedure (Tukey’s Studentized Range test) indicated statistically significant differences (p < 0.05) between both 3.3G and 4.0G compared to 1G for C and BSP expression; between 3.3G and 4.0G for C expression; and between 4.0G and 1G for FN expression. No significant differences with respect to G forces were observed for either OPN or OC. These results indicate that loading osteoblasts under hypergravity conditions produces demonstrable down-regulation of expression levels of certain genes important in bone matrix production and mineralization. The data yield insight into a possible mechanism underlying the adaptation of bone to mechanical forces, including gravitational loading.

This work was supported by NASA grant NAG5-7789.

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