LOCALIZING THE GENE EXPRESSION RESPONSE TO MECHANICAL LOADING IN TIBIAL BONE CELLS 633

Abstract

Gene expression for growth factors and bone matrix markers increases 5- to 12-fold in cortical bone periosteum after 4 d of in vivo mechanical loading [Raab-Cullen et al. JBMR 10:S255, 1995]. We used in situ hybridization to localize changes in mRNA expression for transforming growth factor β2 (TGFβ2) and Type I collagen (COL1) following loading (LOAD) or immobilization (IMM). Right hindlimbs of adult female Sprague-Dawley rats (5 mo, 265±5g) were IMM by taping (n=6) or loaded(n=6) with 4-point bending for 36 cycles at 33 N (1200 με), a protocol known to increase bone formation in the loaded region. Rats were sacrificed 18 hr after the 4th loading session or after 4 days of IMM; right and left(untreated control) tibiae were fixed, decalcified, and paraffin-embedded.In situ hybridization was performed with a 35S-UTP labelled RNA probes for TGFβ2 and COL1; slides were developed by emulsion autoradiography. Subjectively, signals for TGFβ2 and especially COL1 mRNA were increased in regions with thicker periosteal layers containing mature osteoblasts, and in regions with woven bone, which was observed in 3 of 6 LOAD tibiae. A significant increase in moderate-intensity staining (vs. background) for TGFβ2 (but not for COL1) was found for LOAD tibiae vs. within-animal control bones; no significant staining changes were noted in IMM tibiae vs controls. These preliminary results suggest that TGFβ2 plays a role in the response of periosteal bone cells to loading and is expressed in regions where bone matrix is actively forming.