Sost downregulation and local Wnt signaling are required for the osteogenic response to mechanical loading

Authors

Xiaolin Tu, Yumie Rhee, Keith W. Condon, Nicoletta Bivi, Matthew R. Allen, Denise Dwyer, Marina Stolina, Charles H. Turner, Alexander G. Robling, Lilian I. Plotkin, Teresita Bellido

Abstract

Sclerostin, the Wnt signaling antagonist encoded by the Sost gene, is secreted by osteocytes and inhibits bone formation by osteoblasts. Mechanical stimulation reduces sclerostin expression, suggesting that osteocytes might coordinate the osteogenic response to mechanical force by locally unleashing Wnt signaling. To investigate whether sclerostin downregulation is a pre-requisite for load-induced bone formation, we conducted experiments in transgenic mice (TG) engineered to maintain high levels of SOST expression during mechanical loading. This was accomplished by introducing a human SOST transgene driven by the 8 kb fragment of the DMP1 promoter that also provided osteocyte specificity of the transgene. Right ulnae were subjected to in vivo cyclic axial loading at equivalent strains for 1 min/day at 2 Hz; left ulnae served as internal controls. Endogenous murine Sost mRNA expression measured 24 h after 1 loading bout was decreased by about 50% in TG and wild type (WT) littermates. In contrast, human SOST, only expressed in TG mice, remained high after loading. Mice were loaded on 3 consecutive days and bone formation was quantified 16 days after initiation of loading. Periosteal bone formation in control ulnae was similar in WT and TG mice. Loading induced the expected strain-dependent increase in bone formation in WT mice, resulting from increases in both mineralizing surface (MS/BS) and mineral apposition rate (MAR). In contrast, load-induced bone formation was reduced by 70–85% in TG mice, due to lower MS/BS and complete inhibition of MAR. Moreover, Wnt target gene expression induced by loading in WT mice was absent in TG mice. Thus, downregulation of Sost/sclerostin in osteocytes is an obligatory step in the mechanotransduction cascade that activates Wnt signaling and directs osteogenesis to where bone is structurally needed.

Link to Article

http://dx.doi.org/10.1016/j.bone.2011.10.025

Low-magnitude high-frequency loading via whole body vibration enhances bone-implant osseointegration in ovariectomized rats

Authors

BaiLing Chen, YiQiang Li, DengHui Xie, XiaoXi Yang

Abstract

Osseointegration is vital to avoid long-time implants loosening after implantation surgery. This study investigated the effect of low-magnitude high-frequency (LMHF) loading via whole body vibration on bone-implant osseointegration in osteoporotic rats, and a comparison was made between LMHF vibration and alendronate on their effects. Thirty rats were ovariectomized to induce osteoporosis, and then treated with LMHF vibration (VIB) or alendronate (ALN) or a control treatment (OVX). Another 10 rats underwent sham operation to establish Sham control group. Prior to treatment, hydroxyapatite (HA)-coated titanium implants were inserted into proximal tibiae bilaterally. Both LMHF vibration and alendronate treatment lasted for 8 weeks. Histomorphometrical assess showed that both group VIB, ALN and Sham significantly increased bone-to-implant contact and peri-implant bone fraction (p < 0.05) when compared with group OVX. Nevertheless the bone-to-implant contact and peri-implant bone fraction of group VIB were inferior to group ALN and Sham (p < 0.05). Biomechanical tests also revealed similar results in maximum push out force and interfacial shear strength. Accordingly, it is concluded that LMHF loading via whole body vibration enhances bone-to-implant osseointegration in ovariectomized rats, but its effectiveness is weaker than alendronate.

Link to Article

http://dx.doi.org/10.1002/jor.22004

PTHrP drives breast tumor initiation, progression, and metastasis in mice and is a potential therapy target

Authors

Jiarong Li, Andrew C. Karaplis, Dao C. Huang, Peter M. Siegel, Anne Camirand, Xian Fang Yang, William J. Muller and Richard Kremer

Abstract

Parathyroid hormone–related protein (PTHrP) is a secreted factor expressed in almost all normal fetal and adult tissues. It is involved in a wide range of developmental and physiological processes, including serum calcium regulation. PTHrP is also associated with the progression of skeletal metastases, and its dysregulated expression in advanced cancers causes malignancy-associated hypercalcemia. Although PTHrP is frequently expressed by breast tumors and other solid cancers, its effects on tumor progression are unclear. Here, we demonstrate in mice pleiotropic involvement of PTHrP in key steps of breast cancer — it influences the initiation and progression of primary tumors and metastases. Pthrp ablation in the mammary epithelium of the PyMT-MMTV breast cancer mouse model caused a delay in primary tumor initiation, inhibited tumor progression, and reduced metastasis to distal sites. Mechanistically, it reduced expression of molecular markers of cell proliferation (Ki67) and angiogenesis (factor VIII), antiapoptotic factor Bcl-2, cell-cycle progression regulator cyclin D1, and survival factor AKT1. PTHrP also influenced expression of the adhesion factor CXCR4, and coexpression of PTHrP and CXCR4 was crucial for metastatic spread. Importantly, PTHrP-specific neutralizing antibodies slowed the progression and metastasis of human breast cancer xenografts. Our data identify what we believe to be new functions for PTHrP in several key steps of breast cancer and suggest that PTHrP may constitute a novel target for therapeutic intervention.

Link to Article

http://www.jci.org/articles/view/46134

Three-Dimensionally Printed Polycaprolactone and β-Tricalcium Phosphate Scaffolds for Bone Tissue Engineering: An In Vitro Study

Authors

Basel Sharaf, Caroline B. Faris, Harutsugi Abukawa, Srinivas M. Susarla, Joseph P. Vacanti, Leonard B. Kaban, Maria J. Troulis

Abstract

The purpose of this study was to evaluate porcine bone marrow–derived progenitor cell (pBMPC) proliferation and penetration into a novel 3-dimensionally printed scaffold. Four different tissue engineering scaffolds to evaluate pBMPC proliferation and penetration were examined. Scaffolds were fabricated from polycaprolactone (PCL) or the combination of β-tricalcium phosphate (β-TCP) and PCL (50:50), with 2 separate channel sizes (1 mm [small (S)] vs 2 mm [large (L)]). Scaffolds were fabricated into 20 × 20 × 7–mm blocks by use of a TheriForm machine (Integra Life Sciences, Akron, OH). Four groups of scaffolds were examined for pBMPC proliferation and penetration: group 1, β-TCP/PCL S; group 2, β-TCP/PCL L; group 3, PCL S; and group 4, PCL L. Nonparametric mean (Kruskal-Wallis) and multiple comparisons tests were used to compare the 4 groups. No shrinkage or deformation was noted in any of the scaffold groups after 2 weeks of culture. Mean surface cell counts ranged from 13.4 to 87.8 cells/0.57 mm2, with group 1 (β-TCP/PCL S) having statistically significantly higher counts than the other groups (P < .001). Mean interior cell counts ranged from 10.9 to 75.6 cells/0.57 mm2, with group 1 having the greatest interior cell count (P < .001). Total collagen formation ranged from 0.2% to 86%, with group 1 having the highest collagen formation (P < .001). The 3-dimensionally printed scaffold (β-TCP/PCL) with 1-mm channels showed greater cellular proliferation, penetration, and collagen formation after a 2-week in vitro culture than the other scaffolds evaluated. β-TCP/PCL S scaffolds warrant further evaluation for bone tissue engineering in vivo.

Link to Article

http://dx.doi.org/10.1016/j.joms.2011.07.029

Transgenic overexpression of bone morphogenetic protein 11 propeptide in skeleton enhances bone formation

Authors

Zicong Li, Fang Zeng, Alva Mitchell, Yong Soo Kim, Zhenfang Wu, Jinzeng Yang

Abstract

Bone morphogenetic protein 11 (BMP11) is a key regulatory protein in skeletal development. BMP11 propeptide has been shown to antagonize GDF11 activity in vitro. To explore the role of BMP11 propeptide in skeletal formation in vivo, we generated transgenic mice with skeleton-specific overexpression of BMP11 propeptide cDNA. The mice showed a transformation of the seventh cervical vertebra into a thoracic vertebra in our previous report. Presently, further characterizations of the transgenic mice indicated that ossification in calvatia was dramatically enhanced in transgenic fetuses at 16.5 dpc in comparison with their wild-type littermates. At 10 weeks of age, bone mineral content and bone mineral density were significantly (P < 0.05) higher in transgenic mice than that in their wild-type littermates based on dual energy X-ray absorptiometry analysis. The relative trabecular bone volume measured by histological analysis was dramatically increased in transgenic mice compared with their wild-type littermates. The enhanced bone formations in the transgenic mice appear to result from increase osteoblast activities as the expressions of four osteoblast markers – α1 type 1 collagen, osteocalcin, alkaline phosphatase and phex were significantly higher in transgenic fetuses than that in their wild-type littermates. These results suggest that over-expression of BMP11 propeptide stimulates bone formation by increasing osteoblast cell functions.

Link to Article

http://dx.doi.org/10.1016/j.bbrc.2011.11.019

Femoral metaphysis bending test of rat: introduction and validation of a novel biomechanical testing protocol for osteoporosis

Authors

BaiLing Chen, YiQiang Li, XiaoXi Yang and DengHui Xie

Abstract

The diaphysis bending test is generally accepted to assess the biomechanical properties of bone in osteoporotic animals. However, bone strength loss was more pronounced at the metaphysis than diaphysis. Therefore, the biomechanical test should be focused on the metaphysis. This study aimed to validate a novel biomechanical test for femoral metaphysis in ovariectomized rats. Twenty 5-month-old female Sprague-Dawley rats were randomly divided into the ovariectomized (OVX) and sham-operated (Sham) groups. Examination of femur bone mineral density (BMD) and histomorphometry of the distal femur were performed. Femur biomechanical parameters (maximal load, yield load, and stiffness) were determined by the diaphysis bending test and a novel designed metaphysis bending test. Pearson's correlations were used to analyze the relationships between the biomechanical parameters and BMD or bone histomorphometry indexes (%Tb.Ar, Tb.N, Tb.Th), respectively. The femur BMD, bone histomorphometry indexes, and biomechanical parameters of OVX were inferior to those of the Sham group (P < 0.05). In the diaphysis bending test, the mean difference of the maximum load and yield load between the OVX and Sham groups were 13.83 ± 5.27 and 15.69 ± 4.15 N, which were significantly lower than in the metaphysis bending test (43.34 ± 4.27, 48.90 ± 4.35 N; all P < 0.05). Positive correlations between biomechanical parameters and femur BMD or bone histomorphometry indexes were observed in both the diaphysis bending and metaphysis bending test. The biomechanical parameters in the metaphysis bending test showed stronger correlations with BMD and bone histomorphometry indexes. The femoral metaphysis bending test was validated to assess osteoporosis in our study, and it was more sensitive than the diaphysis bending test in evaluating the change of biomechanical properties of the femur in osteoporotic rats.

Link to Article

http://dx.doi.org/10.1007/s00776-011-0167-7