Osteocyte Wnt/ß-catenin pathway activation upon mechanical loading is altered in ovariectomized mice.

Estrogen levels decline in both sexes with age, but more dramatically in females. Activation of the Wnt/ß-catenin signaling pathway is central to the regulation of bone mass accrual and maintenance and in response to mechanical loading. Using the ovariectomized mouse model we examined the effect of estrogen loss on the osteocyte's ability to activate the Wnt/ß-catenin pathway following mechanical loading. Female TOPGAL mice underwent ovariectomy (OVX) (n = 10) or sham surgery (n = 10) at 16 weeks of age. Four weeks post-surgery, a single loading session (global strain of 2200 µÎµ for 100 cycles at 2 Hz) was performed on the right forearm with the left as a non-loaded control. Mice (n = 5) were sacrificed at 1 or 24 hr post-load. Ulnae were stained for ß-catenin activation, femurs were used for µCT and 3-pt bending/biomechanical testing, and tibiae were used for histology analysis and to determine osteocyte lacunar size using SEM and high resolution micro-XCT. A 2.2-fold increase in ß-catenin signaling activation was observed 24 hr post-load in the Sham group but did not occur in the OVX group. The OVX group versus control had significant losses (p < 0.05) in trabecular BMD (-8%), BV/TV (-35%) and thickness (-23%), along with cortical thickness (-6%) and periosteal perimeter (-4%). The OVX group had significantly higher trabecular bone osteoclast numbers (63%), OCS/BS (77%) and N.OC/BPm (94%) and a significant decrease in osteoblast number (53%), OBS/BS (37%) and N.OB/BPm (40%) compared to the sham group (p < 0.05). Cortical bone lacunar number/lacunar volume and bone biomechanical properties did not change between groups. Given that the ulna is a cortical bone loading model and the lack of changes in osteocyte lacunar number/volume in cortical bone, which would alter strains experienced by osteocytes, these data suggest the absence of estrogen resulted in intrinsic changes in the ability of the osteocyte to respond to mechanical load, rather than changes in the biomechanical and architectural properties of bone.

Age and gender related differences in load-strain response in C57Bl/6 mice.

We examined the changes in mechanical strain response of male and female mouse tibia and ulna, using axial compression tests, to assess age-related changes in tibiae and ulnae by a non-contact strain measurement technique called the digital image correlation (DIC) and the standard strain gage. A unique aspect of the study was to compare bones from the same animal to study variations in behavior with aging. This study was conducted using male and female C57Bl/6 mice at 6, 12 and 22 months of age (N=6-7 per age and sex) using three load levels. The DIC technique was able to detect a greater number of statistically significant differences in comparison to the strain gaging method. Male ulna showed significantly higher DIC strains compared to strains captured from strain gage at all three levels of load at 6 months and in the lowest load at 12 months. DIC measurements revealed that the ulna becomes stiffer with aging for both males and females, which resulted in 0.4 to 0.8 times reduced strains in the 22-month group compared to the 6 month group. Male tibia showed three-fold increased strains in the 22 months group at 11.5 N load compared to 6 months group (p<.05).

Bone-Muscle Mutual Interactions.

PURPOSE OF REVIEW: The purpose of this review is to describe the current state of our thinking regarding bone-muscle interactions beyond the mechanical perspective. RECENT FINDINGS: Recent and prior evidence has begun to dissect many of the molecular mechanisms that bone and muscle use to communicate with each other and to modify each other's function. Several signaling factors produced by muscle and bone have emerged as potential mediators of these biochemical/molecular interactions. These include muscle factors such as myostatin, Irisin, BAIBA, IL-6, and the IGF family and the bone factors FGF-23, Wnt1 and Wnt3a, PGE2, FGF9, RANKL, osteocalcin, and sclerostin. The identification of these signaling molecules and their underlying mechanisms offers the very real and exciting possibility that new pharmaceutical approaches can be developed that will permit the simultaneous treatments of diseases that often occur in combination, such as osteoporosis and sarcopenia.

Age-related and sex-specific effects on architectural properties and biomechanical response of the C57BL/6N mouse femur, tibia and ulna.

Aging is known to reduce bone quality and bone strength. We sought to determine how aging affects the biomechanical and architectural properties of various long bones, and if sex influences age related differences/changes. While researchers have extensively studied these changes in individual bones of mice, there is no comprehensive study of the changes in the bones from the same mice to study the changes with aging. We performed three point bending tests and microcomputed tomography (microCT) analysis on femurs, tibiae and ulnae. Three point bending tests were utilized to calculate biomechanical parameters and imaging was also performed using high resolution microCT to reveal both cortical and trabecular microarchitecture C57BL/6N mice were divided into three age groups: 6, 12 and 22 months. Each age and sex group consisted of 6-7 mice. The ultimate load to failure (UL), elastic stiffness (ES), modulus of elasticity (E) and the moment of inertia about bending axis (MOI) for each bone was calculated using three point bending test. MicroCT scans of all the bones were analyzed to determine cortical bone volume per tissue volume (C.BV/TV), trabecular bone volume per tissue volume (Tb.BV/TV), cortical bone area (B.Ar) using CTAn's microCT analysis and tested for correlation with the biomechanical parameters. Mean (standard error) values of UL in femur decreased from 19.8(0.6) N to 12.8(1.1) N (p < .01) and 17.9(0.6) N to 14.6(1.0) N (p = .02) from 6 to 22 months groups in males and females respectively. Similarly, UL in tibia decreased from 19.8(0.5) N to 14.3(0.2) N (p < .01) and 14.4(0.6) N to 9.5(1.0) N (p < .01) from 6 to 22 months group in males and females respectively. ES in femur decreased from 113.2(7) N/mm to 69.6(6.7) N/mm (p < .01) from 6 to 22 months in males only. ES in tibia decreased from 78.6(3.2) N/mm to 65.0(2.3) N/mm (p = .01) and 53.1(2.9) N/mm to 44.0(1.7) N/mm (p = .02) from 6 to 22 months in males and females respectively. Interestingly, ES in ulna increased from 8.2(0.8) N/mm to 10.9(1.0) N/mm (p = .051) from 6 to 22 months of age in females only. E in femur decreased from 4.0(0.4) GPa to 2.8(0.2) GPa (p = .01) and 6.7(0.5) GPa to 4.5(0.4) GPa (p = .01) from 6 to 22 months of age in males and females respectively while tibia showed no change. However, E in ulna increased from 7.0(0.8) GPa to 11.0(1.1) GPa (p = .01) from 6 to 22 months of age in females only. Changes in age and sex-related bone properties were more pronounced in the femur and tibia, while the ulna showed fewer overall differences. Most of the changes were observed in biomechanical compared to architectural properties and female bones are more severely affected by aging. In conclusion, our data demonstrate that care must be taken to describe bone site and sex-specific, rather than making broad generalizations when describing age-related changes on the biomechanical and architectural properties of the skeleton.

Multiscale finite element modeling of mechanical strains and fluid flow in osteocyte lacunocanalicular system.

Osteocytes form over 90% of the bone cells and are postulated to be mechanosensors responsible for regulating the function of osteoclasts and osteoblasts in bone modeling and remodeling. Physical activity results in mechanical loading on the bones. Osteocytes are thought to be the main mechanosensory cells in bone. Upon load osteocytes secrete key factors initiating downstream signaling pathways that regulate skeletal metabolism including the Wnt/ß-catenin signaling pathway. Osteocytes have dendritic structures and are housed in the lacunae and canaliculi within the bone matrix. Mechanical loading is known to have two primary effects, namely a mechanical strain (membrane disruption by stretching) on the lacunae/cells, and fluid flow, in the form of fluid flow shear stress (FFSS), in the space between the cell membranes and the lacuna-canalicular walls. In response, osteocytes get activated via a process called mechanotransduction in which mechanical signals are transduced to biological responses. The study of mechanotransduction is a complex subject involving principles of engineering mechanics as well as biological signaling pathway studies. Several length scales are involved as the mechanical loading on macro sized bones are converted to strain and FFSS responses at the micro-cellular level. Experimental measurements of strain and FFSS at the cellular level are very difficult and correlating them to specific biological activity makes this a very challenging task. One of the methods commonly adopted is a multi-scale approach that combines biological and mechanical experimentation with in silico numerical modeling of the engineering aspects of the problem. Finite element analysis along with fluid-structure interaction methodologies are used to compute the mechanical strain and FFSS. These types of analyses often involve a multi-length scale approach where models of both the macro bone structure and micro structure at the cellular length scale are used. Imaging modalities play a crucial role in the development of the models and present their own challenges. This paper reviews the efforts of various research groups in addressing this problem and presents the work in our research group. A clear understanding of how mechanical stimuli affect the lacunae and perilacunar tissue strains and shear stresses on the cellular membranes may ultimately lead to a better understanding of the process of osteocyte activation.

Determination of Elastic Modulus in Mouse Bones Using a Nondestructive Micro-Indentation Technique Using Reference Point Indentation.

The determination of the elastic modulus of bone is important in studying the response of bone to loading and is determined using a destructive three-point bending method. Reference point indentation (RPI), with one cycle of indentation, offers a nondestructive alternative to determine the elastic modulus. While the elastic modulus could be determined using a nondestructive procedure for ex vivo experiments, for in vivo testing, the three-point bending technique may not be practical and hence RPI is viewed as a potential alternative and explored in this study. Using the RPI measurements, total indentation distance (TID), creep indentation distance, indentation force, and the unloading slope, we have developed a numerical analysis procedure using the Oliver-Pharr (O/P) method to estimate the indentation elastic modulus. Two methods were used to determine the area function: (1) Oliver-Pharr (O/P-based on a numerical procedure) and (2) geometric (based on the calculation of the projected area of indentation). The indentation moduli of polymethyl methacrylate (PMMA) calculated by the O/P (3.49-3.68 GPa) and geometric (3.33-3.49 GPa) methods were similar to values in literature (3.5-4 GPa). In a study using femurs from C57Bl/6 mice of different ages and genders, the three-point bending modulus was lower than the indentation modulus. In femurs from 4 to 5 months old TOPGAL mice, we found that the indentation modulus from the geometric (5.61 ± 1.25 GPa) and O/P (5.53 ± 1.27 GPa) methods was higher than the three-point bending modulus (5.28 ± 0.34 GPa). In females, the indentation modulus from the geometric (7.45 ± 0.86 GPa) and O/P (7.46 ± 0.92 GPa) methods was also higher than the three-point bending modulus (7.33 ± 1.13 GPa). We can conclude from this study that the RPI determined values are relatively close to three-point bending values.

Muscle-Bone Crosstalk: Emerging Opportunities for Novel Therapeutic Approaches to Treat Musculoskeletal Pathologies.

Osteoporosis and sarcopenia are age-related musculoskeletal pathologies that often develop in parallel. Osteoporosis is characterized by a reduced bone mass and an increased fracture risk. Sarcopenia describes muscle wasting with an increasing risk of injuries due to falls. The medical treatment of both diseases costs billions in health care per year. With the impact on public health and economy, and considering the increasing life expectancy of populations, more efficient treatment regimens are sought. The biomechanical interaction between both tissues with muscle acting on bone is well established. Recently, both tissues were also determined as secretory endocrine organs affecting the function of one another. New exciting discoveries on this front are made each year, with novel signaling molecules being discovered and potential controversies being described. While this review does not claim completeness, it will summarize the current knowledge on both the biomechanical and the biochemical link between muscle and bone. The review will highlight the known secreted molecules by both tissues affecting the other and finish with an outlook on novel therapeutics that could emerge from these discoveries.

Larger therapeutic window for steroid versus VEGF-A inhibitor in inflammatory angiogenesis: surprisingly similar impact on leukocyte infiltration.

PURPOSE: The current treatments against inflammatory angiogenesis are steroids and anti-VEGF-A, such as dexamethasone and bevacizumab, respectively. However, the therapeutic windows for dexamethasone and bevacizumab against inflammatory angiogenesis are unknown. METHODS: To investigate the therapeutic windows for dexamethasone and bevacizumab, we used the corneal pocket assay. IL-1ß pellets were implanted in corneas of BALB/c mice that were then treated with dexamethasone or bevacizumab at different time points. Angiogenesis (area, number of vessels, and sprouting) was quantitated at various time points after implantation. Nuclear Factor-κB (NF-κB) signaling and leukocyte accumulation in inflammatory angiogenesis were examined by Western blotting, by immunohistochemistry, and in the authors' novel leukocyte transmigration assay. RESULTS: Dexamethasone inhibited IL-1ß-induced angiogenesis when treatment started 4 days after IL-1ß implantation, while bevacizumab only inhibited angiogenesis by day 2 after implantation. Both bevacizumab and dexamethasone inhibited angiogenic sprouting. Interestingly, bevacizumab did not affect NF-κB activation, which is one of the main signaling targets for steroid action. The authors' new imaging approach revealed that bevacizumab and steroid treatment blocked leukocyte infiltration into implanted corneas. CONCLUSIONS: VEGF-A inhibition affected angiogenic sprouting, while it was not effective against matured vessels. Both dexamethasone and bevacizumab inhibited leukocyte transmigration from angiogenic vessels; however, dexamethasone had a larger therapeutic window. These insights improve the treatment strategy in angiogenic disorders.

Blood vessel endothelial VEGFR-2 delays lymphangiogenesis: an endogenous trapping mechanism links lymph- and angiogenesis.

Angio- and lymphangiogenesis are inherently related processes. However, how blood and lymphatic vessels regulate each other is unknown. This work introduces a novel mechanism explaining the temporal and spatial relation of blood and lymphatic vessels. Vascular endothelial growth factor-A (VEGF-A) surprisingly reduced VEGF-C in the supernatant of blood vessel endothelial cells, suggesting growth factor (GF) clearance by the growing endothelium. The orientation of lymphatic sprouting toward angiogenic vessels and away from exogenous GFs was VEGF-C dependent. In vivo molecular imaging revealed higher VEGF receptor (R)-2 in angiogenic tips compared with normal vessels. Consistently, lymphatic growth was impeded in the angiogenic front. VEGF-C/R-2 complex in the cytoplasm of VEGF-A-treated endothelium indicated that receptor-mediated internalization causes GF clearance from the extracellular matrix. GF clearance by receptor-mediated internalization is a new paradigm explaining various characteristics of lymphatics.

Activation of ß-catenin signaling in MLO-Y4 osteocytic cells versus 2T3 osteoblastic cells by fluid flow shear stress and PGE2: Implications for the study of mechanosensation in bone.

The osteocyte is hypothesized to be the mechanosensory cell in bone. However, osteoblastic cell models have been most commonly used to investigate mechanisms of mechanosensation in bone. Therefore, we sought to determine if differences might exist between osteocytic and osteoblastic cell models relative to the activation of ß-catenin signaling in MLO-Y4 osteocytic, 2T3 osteoblastic and primary neonatal calvarial cells (NCCs) in response to pulsatile fluid flow shear stress (PFFSS). ß-catenin nuclear translocation was observed in the MLO-Y4 cells at 2 and 16 dynes/cm(2) PFFSS, but only at 16 dynes/cm(2) in the 2T3 or NCC cultures. The MLO-Y4 cells released high amounts of PGE(2) into the media at all levels of PFFSS (2-24 dynes/cm(2)) and we observed a biphasic pattern relative to the level of PFFSS. In contrast PGE(2) release by 2T3 cells was only detected during 16 and 24 dynes/cm(2) PFFSS starting at >1h and never reached the levels produced by the MLO-Y4 cells. Exogenously added PGE(2) was able to induce ß-catenin nuclear translocation in all cells suggesting that the differences between the cell lines observed for ß-catenin nuclear translocation were associated with the differences in PGE(2) production. To investigate a possible mechanism for the differences in PGE(2) release by the MLO-Y4 and 2T3 cells we examined the regulation of Ptgs2 (Cox-2) gene expression by PFFSS. 2T3 cell Ptgs2 mRNA levels at both 0 and 24h after 2h of PFFSS showed biphasic increases with peaks at 4 and 24 dynes/cm(2) and 24-hour levels were higher than zero-hour levels. MLO-Y4 cell Ptgs2 expression was similarly biphasic; however at 24-hour post-flow Ptgs2 mRNA levels were lower. Our data suggest significant differences in the sensitivity and kinetics of the response mechanisms of the 2T3 and neonatal calvarial osteoblastic versus MLO-Y4 osteocytic cells to PFFSS. Furthermore our data support a role for PGE(2) in mediating the activation of ß-catenin signaling in response to the fluid flow shear stress.

Atrial natriuretic peptide reduces vascular leakage and choroidal neovascularization.

Atrial natriuretic peptide (ANP) is a hormone with diuretic, natriuretic, and vasodilatory properties. ANP blocks vascular endothelial growth factor (VEGF) production and signaling in vitro; however, its role in vascular leakage and angiogenesis is unknown. In vitro, retinal barrier permeability (transepithelial electrical resistance (TEER)) was measured in cultured retinal endothelial (HuREC) and retinal epithelial (ARPE-19) cells with VEGF (10 ng/ml), ANP (1 pM to 1 micromol/L), and/or isatin, an ANP receptor antagonist. In vivo, blood-retinal barrier (BRB) leakage was studied using the Evans Blue dye technique in rats treated with intravitreal injections of ANP, VEGF, or vehicle. Choroidal neovascularization was generated by laser injury, and 7 days later, lesion size and leakage was quantitated. ANP significantly reversed VEGF-induced BRB TEER reduction in both HuREC and ARPE-19 cells, modeling the inner and the outer BRB, respectively. Isatin, a specific ANP receptor antagonist, reversed ANP's effect. ANP reduced the response of ARPE-19 cells to VEGF apically but not basolaterally, suggesting polarized expression of the ANP receptors in these cells. ANP's TEER response was concentration but not time dependent. In vivo, ANP significantly reduced VEGF-induced BRB leakage and the size of laser-induced choroidal neovascularization lesions. In sum, ANP is an effective inhibitor of VEGF-induced vascular leakage and angiogenesis in vivo. These results may lead to new treatments for ocular diseases where VEGF plays a central role, such as age-related macular degeneration or diabetic retinopathy.