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

Abstract

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 restinserted (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 hr 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.

Effects of low intensity pulsed ultrasound stimulation on bone regeneration in rat parietal bone defect model

“…Although the precise mechanism of the present enhancement of bone regeneration is not clear, it is likely that LIPUS stimulated cellular migration, proliferation and differentiation which were controlled by these signal molecules [25,26]. It has been reported that LIPUS stimulated angiogenesis and the most effective time point of LIPUS application was at the second week when extensive angiogenesis generally occurs after the surgery [27,28]. Thus, promotion of angiogenesis by LIPUS seemed to contribute to the present enhancement of bone regeneration.…”

Hypoxia-inducible Factor-1α Protein Negatively Regulates Load-induced Bone Formation

“…This situation differs from the exogenous mechanical stimuli implemented in the current study, which are not associated with significant hypoxia or increased angiogenesis (45), but rather, are considered anabolic. On the other hand, application of much more intensive loads that induce bone fatigue with significant tissue damage is associated with increased skeletal vascularity likely due to tissue hypoxia (46,47).…”

Age-Related Impairment of Bones' Adaptive Response to Loading in Mice Is Associated With Sex-Related Deficiencies in Osteoblasts but No Change in Osteocytes

“…Left limbs were used as internal controls as previously validated 21,24. The protocol for noninvasively loading the mouse tibia has been reported previously 21,25.…”