Computational modeling based on confocal imaging predicts changes in osteocyte and dendrite shear stress due to canalicular loss with aging.

Exercise and physical activity exert mechanical loading on the bones which induces bone formation. However, the relationship between the osteocyte lacunar-canalicular morphology and mechanical stress experienced locally by osteocytes transducing signals for bone formation is not fully understood. In this study, we used computational modeling to predict the effect of canalicular density, the number of fluid inlets, and load direction on fluid flow shear stress (FFSS) and bone strains and how these might change following the microstructural deterioration of the lacunar-canalicular network that occurs with aging. Four distinct computational models were initially generated of osteocytes with either ten or eighteen dendrites using a fluid-structure interaction method with idealized geometries. Next, a young and a simulated aged osteocyte were developed from confocal images after FITC staining of the femur of a 4-month-old C57BL/6 mouse to estimate FFSS using a computational fluid dynamics approach. The models predicted higher fluid velocities in the canaliculi versus the lacunae. Comparison of idealized models with five versus one fluid inlet indicated that with four more inlets, one-half of the dendrites experienced FFSS greater than 0.8 Pa, which has been associated with osteogenic responses. Confocal image-based models of real osteocytes indicated a six times higher ratio of canalicular to lacunar surface area in the young osteocyte model than the simulated aged model and the average FFSS in the young model (FFSS = 0.46 Pa) was three times greater than the aged model (FFSS = 0.15 Pa). Interestingly, the surface area with FFSS values above 0.8 Pa was 23 times greater in the young versus the simulated aged model. These findings may explain the impaired mechano-responsiveness of osteocytes with aging.

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).

Osteocyte lacunar strain determination using multiscale finite element analysis.

Osteocytes are thought to be the primary mechanosensory cells within bone, regulating both osteoclasts and osteoblasts to control load induced changes in bone resorption and formation. Osteocytes initiate intracellular responses including activating the Wnt/ß-catenin signaling pathway after experiencing mechanical forces. In response to changing mechanical loads (strain) the osteocytes signal to cells on the bone surface. However, this process of osteocyte activation appears heterogeneous since it occurs in sub-populations of osteocytes, even within regions predicted to be experiencing similar global strain magnitudes determined based on traditional finite element modeling approaches. Several studies have investigated the strain responses of osteocyte lacunae using finite element (FE) models, but many were limited by the use of idealized geometries (e.g., ellipsoids) and analysis of a single osteocyte. Finite element models by other groups included more details, such as canaliculi, but all were done on models consisting of a single osteocyte. We hypothesized that variation in size and orientation of the osteocyte lacunae within bone would give rise to micro heterogeneity in the strain fields that could better explain the observed patterns of osteocyte activation following load. The osteocytes in our microscale and nanoscale models have an idealized oval shape and some are based on confocal scans. However, all the FE models in this preliminary study consist of multiple osteocytes. The number of osteocytes in the 3D confocal scan models ranged from five to seventeen. In this study, a multi-scale computational approach was used to first create an osteocyte FE model at the microscale level to examine both the theoretical lacunar and perilacunar strain responses based on two parameters: 1) lacunar orientation and 2) lacunar size. A parametric analysis was performed by steadily increasing the perilacunar modulus (5, 10, 15, and 20 GPa). Secondly, a nanoscale FE model was built using known osteocyte dimensions to determine the predicted strains in the perilacunar matrix, fluid space, and cell body regions. Finally, 3-D lacunar models were created using confocal image stacks from mouse femurs to determine the theoretical strain in the lacunae represented by realistic geometries. Overall, lacunar strains decreased by 14% in the cell body, 15% in the fluid space region and 25% in the perilacunar space as the perilacunar modulus increased, indicating a stress shielding effect. Lacunar strains were lower for the osteocytes aligned along the loading axis compared to those aligned perpendicular to axis. Increases in lacuna size also led to increased lacunar strains. These finite element model findings suggest that orientation and lacunar size may contribute to the heterogeneous initial pattern of osteocyte strain response observed in bone following in vivo applied mechanical loads. A better understanding of how mechanical stimuli directly affect the lacunae and perilacunar tissue strains may ultimately lead to a better understanding of the process of osteocyte activation in response to mechanical loading.

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.