Wnt3a involved in the mechanical loading on improvement of bone remodeling and angiogenesis in a postmenopausal osteoporosis mouse model

Abstract

Osteoporosis is a major health problem, making bones fragile and susceptible to fracture. Previous works showed that mechanical loading stimulated bone formation and accelerated fracture healing. Focusing on the role of Wnt3a (wingless/integrated 3a), this study was aimed to assess effects of mechanical loading to the spine, using ovariectomized (OVX) mice as a model of osteoporosis. Two-week daily application of this novel loading (4 N, 10 Hz, 5 min/d) altered bone remodeling with an increase in Wnt3a. Spinal loading promoted osteoblast differentiation, endothelial progenitor cell migration, and tube formation and inhibited osteoclast formation, migration, and adhesion. A transient silencing of Wnt3a altered the observed loading effects. Spinal loading significantly increased bone mineral density, bone mineral content, and bone area per tissue area. The loaded OVX group showed a significant increase in the number of osteoblasts and reduction in osteoclast surface/bone surface. Though expression of osteoblastic genes was increased, the levels of osteoclastic genes were decreased by loading. Spinal loading elevated a microvascular volume as well as VEGF expression. Collectively, this study supports the notion that Wnt3a-mediated signaling involves in the effect of spinal loading on stimulating bone formation, inhibiting bone resorption, and promoting angiogenesis in OVX mice. It also suggests that Wnt3a might be a potential therapeutic target for osteoporosis treatment.-Li, X., Liu, D., Li, J., Yang, S., Xu, J., Yokota, H., Zhang, P. Wnt3a involved in the mechanical loading on improvement of bone remodeling and angiogenesis in a postmenopausal osteoporosis mouse model.

Keywords: OVX; bone formation; bone resorption; neovascularization; spinal load.

Figure 1

Role of Wnt3a in the effect of spinal loading on bone remodeling and angiogenesis in the OVX mice. A) Experimental setup. Timeline and loading site for spinal loading. B) Immunohistochemistry staining of Wnt3a in the femur was conducted in vivo. Scale bars, 50 µm; n = 10. C) Western blot analysis of Wnt3a and β‐catenin in the femur of OVX treated with mechanical loading in vivo (n = 6). D) Western blot analysis of Wnt3a, ALP, NFATc1, and VEGF in bone marrow‐derived cells, which were transfected with siRNA Wnt3a in vitro (n = 6). NS, not significant. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

Effect of silencing Wnt3a on osteoblast differentiation and mineralization with spinal loading in OVX mice in vitro. A) Representative images of osteoblast differentiation by ALP staining. Scale bar, 50 µm. B) Representative images of osteoblast mineralization by Alizarin Red staining. Scale bar, 500 µm. C) Quantitative analysis of ALP⁺ cells (percent). D) Optical density of calcium deposition after Alizarin Red staining. NS, not significant, n = 6 per treatment group. *P< 0.05, **P< 0.01, ***P< 0.001.

Figure 3

Effect of silencing Wnt3a on osteoclast formation, migration, and adhesion with spinal loading in OVX mice in vitro. A) Representative images of osteoclast formation by TRAP staining. B) Representative images of preosteoclasts migration by crystal violet staining. C) Representative images of preosteoclasts adhesion by crystal violet staining. D–F) Quantitative analysis of formation (D), migration (E), and adhesion (F) cells. LFP, low power field; NS, not significant, n = 6 per treatment group. *P< 0.05, **P< 0.01, ***P< 0.001. Scale bars, 200 µm.

Figure 4

Effect of silencing Wnt3a on EPCs migration and tube formation with spinal loading in OVX mice in vitro. A) Representative images of EPCs tube formation. B) Representative images of EPCs migration by crystal violet staining. C) Quantitative analysis of cumulative tube lengths. D) Quantitative analysis of EPCs migratory. FV, field of vision; NS, not significant, n = 6 per treatment group. Scale bars, 100 µm. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

Effects of ovariectomy and spinal loading on BMD and BMC. A) BMD of whole body. B) BMC of whole body. C) BMD of lumbar spine. D) BMC of lumbar spine. E) BMD of femur and tibia. F) BMC of femur and tibia, n = 10 per treatment group; n = 10 for lumbar spine; n = 20 for femur and tibia. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6

Effects of ovariectomy and spinal loading on bone remodeling and angiogenesis in vivo. A) The histologic parameters of trabecular bone on the proximal side of the growth plate in distal femur were determined by H&E staining. Scale bar, 200 µm; n = 10. The arrows indicate trabecular bone. B) MacNeal's staining was used to determine the number of osteoblasts in trabecular bone surface in distal metaphysis of femur. Scale bar, 50 µm; n = 10. Osteoblasts, located on trabecular bone surface, are indicated by the arrows. Loading‐induced increase in osteoblasts numbers in OVX mice. C) TRAP staining was used to evaluate bone resorption in the distal metaphysis of femur. Scale bar, 50 µm; n = 10. TRAP‐positive cells, in red, are indicated by the arrows. D) Ink blood perfusion angiography was used to analyze the density of microvessels. Scale bar, 100 µm; n = 10. E) Western blot showed the protein level of OCN, RUNX2, ALP, NFATc1, RANKL, CTSK, and VEGF in OVX treated with spinal loading (n = 6). F) Proposed mechanism of Wnt3a in the effect of mechanical loading on postmenopausal osteoporosis. *P < 0.05, **P < 0.01, ***P< 0.001.