Comparing histological, vascular and molecular responses associated with woven and lamellar bone formation induced by mechanical loading in the rat ulna.

Osteogenesis occurs by formation of woven or lamellar bone. Little is known about the molecular regulation of these two distinct processes. We stimulated periosteal bone formation at the ulnar mid-diaphysis of adult rats using a single bout of forelimb compression. We hypothesized that loading that stimulates woven bone formation induces higher over-expression of genes associated with cell proliferation, angiogenesis and osteogenesis compared to loading that stimulates lamellar bone formation. We first confirmed that a single bout of 100 cycles of loading using either a rest-inserted (0.1 Hz) or haversine (2 Hz) waveform (15 N peak force) was non-damaging and increased lamellar bone formation (LBF loading). Woven bone formation (WBF loading) was stimulated using a previously described, damaging fatigue loading protocol (2 Hz, 1.3 mm disp., 18 N peak force). There were dramatic differences in gene expression levels (based on qRT-PCR) between loading protocols that produced woven and lamellar bone. In contrast, gene expression levels were not different between LBF loading protocols using a rest-inserted or haversine waveform. Cell proliferation markers Hist4 and Ccnd1 were strongly upregulated (5- to 17-fold) 1 and 3 days after WBF loading, prior to woven bone formation, but not after LBF loading. The angiogenic genes Vegf and Hif1a were upregulated within 1 h after WBF loading and were strongly up on days 1-3 (3- to 15-fold). In sharp contrast, we observed only a modest increase (<2-fold) in Vegfa and Hif1a expression on day 3 following LBF loading. Consistent with these relative differences in gene expression, vascular perfusion 3 days after loading revealed significant increases in vessel number and volume following WBF loading, but not after LBF loading. Lastly, bone formation markers (Runx2, Osx, Bsp) were more strongly upregulated for woven (4- to 89-fold) than for lamellar bone (2-fold), consistent with the differences in new bone volume observed 10 days after loading. In summary, robust early increases both molecularly and histologically for cell proliferation and angiogenesis precede woven bone formation, whereas lamellar bone formation is associated with only a modest upregulation of molecular signals at later timepoints.