An ex vivo model to study transport processes and fluid flow in loaded bone

Load-induced fluid flow has been postulated to provide a mechanism for the transmission of mechanical signals (e.g. via shear stresses, enhancement of molecular transport, and/or electrical effects) and the subsequent elicitation of a functional adaptation response (e.g. modeling, remodeling, homeostasis) in bone. Although indirect evidence for such fluid flow phenomena can be found in the literature pertaining to strain generated potentials, actual measurement of fluid displacements in cortical bone is inherently difficult. This problem motivated us to develop and introduce an ex vivo perfusion model for the study of transport processes and fluid flow within bone under controlled mechanical loading conditions. To this end, a closed-loop system of perfusion was established in the explanted forelimb of the adult Swiss alpine sheep. Immediately prior to mechanical loading, a bolus of tracer was introduced intraarterially into the system. Thereafter, the forelimb of the left or right side (randomized) was loaded cyclically, via Schanz screws inserted through the metaphyses, producing a peak compressive strain of 0.2% at the middiaphysis of the anterior metacarpal cortex. In paired experiments with perfusion times totalling 2, 4, 8 and 16 min, the concentration of tracer measured at the middiaphysis of the cortex in cross section was significantly higher in the loaded bone than in the unloaded contralateral control. Fluorometric measurements of procion red concentration in the anterior aspect alone showed an enhancement in transport at early stages of loading (8 cycles, 2 min) but no effect in transport after higher number of cycles or increased perfusion times, respectively. This reflects both the small size of the molecular tracer, which would be expected to be transported rapidly by way of diffusive mechanisms alone, as well as the loading mode to which the anterior aspect was exposed. Thus, using our new model it could be shown that load-induced fluid flow represents a powerful mechanism to enhance molecular transport within the lacunocanalicular system of compact bone tissue. Based on these as well as previous studies, it appears that the degree of this effect is dependent on tracer size as well as the mechanical loading mode to which a given area of tissue is exposed.     

Effect of oscillating fluid shear on solute transport in cortical bone

The consequences of an oscillatory fluid shear mechanism on nutrient transport in bone during physical activity and ultrasonic therapy are discussed. During movement, periodic stress on bone creates transient pressure gradients that circulate interstitial fluid through calcified bone. A transport model derived from oscillatory Taylor-Aris dispersion phenomena was used to predict a ratio of effective-to-molecular diffusivity, K/D, for solutes of varying sizes up to 50 nm in diameter, in pores filled with interstitial fluid and pericellular matrix. The magnitude of the estimated transport enhancement depended on the molecular size, pore dimension, applied frequency and the displacement of the fluid during pressurization. For oscillation frequencies and amplitudes corresponding to those experienced during normal human activity, transport enhancements of up to 100 fold are expected for molecules larger than 5 nm in diameter. Enhancements of up to one order of magnitude, due to ultrasound stimulations in the MHz frequency range, are also expected for 7-nm-sized solutes. No effects are anticipated for ions, whose molecular diffusion time is too fast relative to the oscillation frequency. This model is expected to be useful for understanding differences in bone growth as a function of type of movement or to develop new physical therapies.     

Influence of cortical canal architecture on lacunocanalicular pore pressure and fluid flow

Bone is a dynamic tissue that undergoes structural modification in response to its mechanical environment, but how bone cells sense and respond to loading conditions remains incompletely understood. Current theories focus on strain-induced fluid flow for the primary means of mechanotransduction. To examine the influence of age-related cortical rarefaction on lacunocanalicular fluid characteristics, coupled fluid flow and mechanical computational models of bone specimens representing young, mid-age and aged samples were derived artificially from the same original micro-computed tomography image data. Simulated mechanical loading was applied to the bone models to induce pressure-driven interstitial fluid flow. Results demonstrated a decrease in pore pressure and fluid velocity magnitudes with age as a result of increased cortical porosity. Mean canal separation, as opposed to canal size, was implicated as a primary factor affecting age-related fluid dynamics. Future investigations through refinement of the model may implicate fluid stasis or inadequate nutrient transport experienced by osteocytes as a key factor in the initiation of cortical remodelling events.     

Influence of cortical canal architecture on lacunocanalicular pore pressure and fluid flow

Bone is a dynamic tissue that undergoes structural modification in response to its mechanical environment, but how bone cells sense and respond to loading conditions remains incompletely understood. Current theories focus on strain-induced fluid flow for the primary means of mechanotransduction. To examine the influence of age-related cortical rarefaction on lacunocanalicular fluid characteristics, coupled fluid flow and mechanical computational models of bone specimens representing young, mid-age and aged samples were derived artificially from the same original micro-computed tomography image data. Simulated mechanical loading was applied to the bone models to induce pressure-driven interstitial fluid flow. Results demonstrated a decrease in pore pressure and fluid velocity magnitudes with age as a result of increased cortical porosity. Mean canal separation, as opposed to canal size, was implicated as a primary factor affecting age-related fluid dynamics. Future investigations through refinement of the model may implicate fluid stasis or inadequate nutrient transport experienced by osteocytes as a key factor in the initiation of cortical remodelling events.     

Influence of interstitial bone microcracks on strain-induced fluid flow

It is well known that microcracks act as a stimulus for bone remodelling, initiating resorption by osteoclasts and new bone formation by osteoblasts. Moreover, microcracks are likely to alter the fluid flow and convective transport through the bone tissue. This paper proposes a quantitative evaluation of the strain-induced interstitial fluid velocities developing in osteons in presence of a microcrack in the interstitial bone tissue. Based on Biot theory in the low-frequency range, a poroelastic model is carried out to study the hydro-mechanical behaviour of cracked osteonal tissue. The finite element results show that the presence of a microcrack in the interstitial osteonal tissue may drastically reduce the fluid velocity inside the neighbouring osteons. This fluid inactive zone inside osteons can cover up to 10% of their surface. Consequently, the fluid environment of bone mechano-sensitive cells is locally modified.     

A fatigue microcrack alters fluid velocities in a computational model of interstitial fluid flow in cortical bone

Targeted remodeling is activated by fatigue microcracks and plays an important role in maintaining bone integrity. It is widely believed that fluid flow-induced shear stress plays a major role in modulating the mechanotransduction process. Therefore, it is likely that fluid flow-induced shear stress plays a major role in the initiation of the repair of fatigue damage. Since no in vivo measurements of fluid flow within bone exist, computational and mathematical models must be employed to investigate the fluid flow field and the shear stress occurring within cortical bone. We developed a computational fluid dynamic model of cortical bone to examine the effect of a fatigue microcrack on the fluid flow field. Our results indicate that there are alterations in the fluid flow field as far as 150 microm away from the crack, and that at distances farther than this, the fluid flow field is similar to the fluid flow field of intact bone. Through the crack and immediately above and below it, the fluid velocity is higher, while at the lateral edges it is lower than that calculated for the intact model, with a maximum change of 29%. Our results suggest that the presence of a fatigue microcrack can alter the shear stress in regions near the crack. These alterations in shear stress have the potential to significantly alter mechanotransduction and may play a role in the initiation of the repair of fatigue microcracks.     

Mechanosensation and fluid transport in living bone

The mechanosensory mechanisms in bone include (i) the cell system that is stimulated by external mechanical loading applied to the bone; (ii) the system that transduces that mechanical loading to a communicable signal; and (iii) the systems that transmit that signal to the effector cells for the maintenance of bone homeostasis and for strain adaptation of the bone structure. The effector cells are the osteoblasts and the osteoclasts. These systems and the mechanisms that they employ have not yet been unambiguously identified. The candidate systems will be reviewed. It will be argued that the current theoretical and experimental evidence suggests that osteocytes are the principal mechanosensory cells of bone, that they are activated by shear stress from fluid flowing through the osteocyte canaliculi, and that the electrically coupled three-dimensional network of osteocytes and lining cells is a communications system for the control of bone homeostasis and structural strain adaptation. The movement of bone fluid from the region of the bone vasculature through the canaliculi and the lacunae of the surrounding mineralized tissue accomplishes three important tasks. First, it transports nutrients to the osteocytes in the lacunae buried in the mineralized matrix. Second, it carries away the cell waste. Third, the bone fluid exerts a force on the cell process, a force that is large enough for the cell to sense. This is probably the basic mechanotransduction mechanism in bone, the way in which bone senses the mechanical load to which it is subjected. The mechanisms of bone fluid flow are described with particular emphasis on mechanotransduction. Also described is the cell to cell communication by which higher frequency signals might be transferred, a potential mechanism in bone by which the small whole tissue strain is amplified so the bone cells can respond to it. One of the conclusions is that higher frequency low amplitude strains can maintain bone as effectively as low frequency high amplitude strains. This conclusion leads to a paradigm shift in how to treat osteoporosis and how to cope with microgravity.     

The dependence of transversely isotropic elasticity of human femoral cortical bone on porosity

The objective of this study was to examine the dependence of the elastic properties of cortical bone as a transversely isotropic material on its porosity. The longitudinal Young's modulus, transverse Young's modulus, longitudinal shear modulus, transverse shear modulus, and longitudinal Poisson's ratio of cortical bone were determined from eighteen groups of longitudinal and transverse specimens using tensile and torsional tests on a servo-hydraulic material testing system. These cylindrical waisted specimens of cortical bone were harvested from the middle diaphysis of three pairs of human femora. The porosity of these specimens was assessed by means of histology. Our study demonstrated that the longitudinal Young's and shear moduli of human femoral cortical bone were significantly (p<0.01) negatively correlated with the porosity of cortical bone. Conversely, the elastic properties in the transverse direction did not have statistically significant correlations with the porosity of cortical bone. As a result, the transverse elastic properties of cortical bone were less sensitive to changes in porosity than those in the longitudinal direction. Additionally, the anisotropic ratios of cortical bone elasticity were found to be significantly (p<0.01) negatively correlated with its porosity, indicating that cortical bone tended to become more isotropic when its porosity increased. These results may help a number of researchers develop more accurate micromechanics models of cortical bone.     

Microstructural changes associated with osteoporosis negatively affect loading-induced fluid flow around osteocytes in cortical bone

Loading-induced interstitial fluid flow in the microporosities of bone is critical for osteocyte mechanotransduction and for the maintenance of tissue health, enhancing convective transport in the lacunar-canalicular system. In recent studies, our group has reported alterations of bone’s vascular porosity and lacunar-canalicular system microarchitecture in a rat model of postmenopausal osteoporosis. In this work, poroelastic finite element analysis was used to investigate whether these microstructural changes can affect interstitial fluid flow around osteocytes. Animal-specific finite element models were developed combining micro-CT reconstructions of bone microstructure and measures of the poroelastic material properties. These models were used to quantify and compare loading-induced fluid flow in the lacunar-canalicular system of ovariectomized and sham-operated rats. A parametric analysis was also used to quantify the influence of the lacunar-canalicular permeability and vascular porosity on the fluid velocity magnitude. Results show that mechanically-induced interstitial fluid velocity can be significantly reduced in the lacunar-canalicular system of ovariectomized rats. Interestingly, the vascular porosity is shown to have a major influence on interstitial fluid flow, while the lacunar-canalicular permeability influence is limited when larger than ${10}^{-20}{\text{m}}^2$. Altogether our results suggest that microstructural changes associated with the osteoporotic condition can negatively affect interstitial fluid flow around osteocytes in the lacunar-canalicular system of cortical bone. This fluid flow reduction could impair mechanosensation of the osteocytic network, possibly playing a role in the initiation and progression of age-related bone loss and postmenopausal osteoporosis.     

Root cortical senescence decreases root respiration,nutrient content and radial water and nutrient transport in barley

The functional implications of root cortical senescence (RCS) are poorly understood. We tested the hypotheses that RCS in barley (1) reduces the respiration and nutrient content of root tissue; (2) decreases radial water and nutrient transport; and (3) is accompanied by increased suberization to protect the stele. Genetic variation for RCS exists between modern germplasm and landraces. Nitrogen and phosphorus deficiency increased the rate of RCS. Maximal RCS, defined as the disappearance of the entire root cortex, reduced root nitrogen content by 66%, phosphorus content by 63% and respiration by 87% compared with root segments with no RCS. Roots with maximal RCS had 90, 92 and 84% less radial water, nitrate and phosphorus transport, respectively, compared with segments with no RCS. The onset of RCS coincided with 30% greater aliphatic suberin in the endodermis. These results support the hypothesis that RCS reduces root carbon and nutrient costs and may therefore have adaptive significance for soil resource acquisition. By reducing root respiration and nutrient content, RCS could permit greater root growth, soil resource acquisition and resource allocation to other plant processes. RCS merits investigation as a trait for improving the performance of barley, wheat, triticale and rye under edaphic stress.     

The relative contributions of non-enzymatic glycation and cortical porosity on the fracture toughness of aging bone

The risk of fracture increases with age due to the decline of bone mass and bone quality. One of the age-related changes in bone quality occurs through the formation and accumulation of advanced glycation end-products (AGEs) due to non-enzymatic glycation (NEG). However as a number of other changes including increased porosity occur with age and affect bone fragility, the relative contribution of AGEs on the fracture resistance of aging bone is unknown. Using a high-resolution nonlinear finite element model that incorporate cohesive elements and micro-computed tomography-based 3d meshes, we investigated the contribution of AGEs and cortical porosity on the fracture toughness of human bone. The results show that NEG caused a 52% reduction in propagation fracture toughness (R-curve slope). The combined effects of porosity and AGEs resulted in an 88% reduction in propagation toughness. These findings are consistent with previous experimental results. The model captured the age-related changes in the R-curve toughening by incorporating bone quantity and bone quality changes, and these simulations demonstrate the ability of the cohesive models to account for the irreversible dynamic crack growth processes affected by the changes in post-yield material behavior. By decoupling the matrix-level effects due to NEG and intracortical porosity, we are able to directly determine the effects of NEG on fracture toughness. The outcome of this study suggests that it may be important to include the age-related changes in the material level properties by using finite element analysis towards the prediction of fracture risk.     

Cilia-assisted flow of viscoelastic fluid in a divergent channel under porosity effects

Biomechanics and Modeling in Mechanobiology - Cilia-driven laminar flow of an incompressible viscoelastic fluid in a divergent channel has been conducted numerically using the BVP4C technique. The...     

Interstitial fluid velocity is decreased around cortical bone vascular pores and depends on osteocyte position in a rat model of disuse osteoporosis

Biomechanics and Modeling in Mechanobiology - Muscle paralysis induced with botulinum toxin (Botox) injection increases vascular porosity and reduces osteocyte lacunar density in the tibial...     

Ultrasound speed in equine cortical bone: effects of orientation, density, porosity and temperature

Ultrasound speed, as measured by a transmission technique in equine cortical bone, was found to vary markedly with the direction of the ultrasound path through the bone. Using bone samples from the mid-site of the third metacarpus of 20 horses, the ultrasound speed was measured as 4125 m s-1 in the longitudinal direction, 3442 m s-1 in the circumferential or transverse direction, and 3428 m s-1 in the radial direction. These results confirm the anisotropic properties of compact bone. Ultrasound speed had a positive linear relationship when compared with bone specific gravity of cortical bone (r = 0.773, n = 35, p less than 0.0001), and an inverse linear relationship with porosity. Specific gravity has an inverse correlation with porosity (r = 0.857, n = 35, p less than 0.0001). Over the temperature range of 4-42 degrees C, ultrasound speed varied inversely according to temperature with a logarithmic function giving the best fit. These results have important implications for the clinical applications of ultrasound speed in assessing bone quality in racehorses and provide important basic information for the understanding of the passage of ultrasound through cortical bone, which has possible clinical applications in humans.     

Root cortical aerenchyma inhibits radial nutrient transport in maize (Zea mays)

Background and Aims

Formation of root cortical aerenchyma (RCA) can be induced by nutrient deficiency. In species adapted to aerobic soil conditions, this response is adaptive by reducing root maintenance requirements, thereby permitting greater soil exploration. One trade-off of RCA formation may be reduced radial transport of nutrients due to reduction in living cortical tissue. To test this hypothesis, radial nutrient transport in intact roots of maize (Zea mays) was investigated in two radiolabelling experiments employing genotypes with contrasting RCA.

Methods

In the first experiment, time-course dynamics of phosphate loading into the xylem were measured from excised nodal roots that varied in RCA formation. In the second experiment, uptake of phosphate, calcium and sulphate was measured in seminal roots of intact young plants in which variation in RCA was induced by treatments altering ethylene action or genetic differences.

Key Results

In each of three paired genotype comparisons, the rate of phosphate exudation of high-RCA genotypes was significantly less than that of low-RCA genotypes. In the second experiment, radial nutrient transport of phosphate and calcium was negatively correlated with the extent of RCA for some genotypes.

Conclusions

The results support the hypothesis that RCA can reduce radial transport of some nutrients in some genotypes, which could be an important trade-off of this trait.     

Scaling of tree vascular transport systems along gradients of nutrient supply and altitude

A recent metabolic scaling theory predicts that plants minimize resistance to hydraulic conduction in the bulk transport network by narrowing the diameter of xylem conduits distally. We hypothesized that trees growing at high altitude or on nutrient-depleted soils would prioritize survival over minimizing hydraulic resistance, and that their vascular systems would be structured differently from those of trees growing under more benign conditions. In fact, conduits were observed to narrow towards the periphery of vascular system within all 45 trees of three species we investigated, and scaling relationships were indistinguishable across a range of environments. Thus, conduit tapering relationships appear to be invariant with respect to environmental conditions.     

Viscoelastic dissipation in compact bone: implications for stress-induced fluid flow in bone

Viscoelastic properties of wet and dry human compact bone were studied in torsion and in bending for both the longitudinal and transverse directions at frequencies from 5 mHz to 5 kHz in bending to more than 50 kHz in torsion. Two series of tests were done for different longitudinal and transverse specimens from a human tibia. Wet bone exhibited a larger viscoelastic damping tan delta (phase between stress and strain sinusoids) than dry bone over a broad range of frequency. All the results had in common a relative minimum in tan delta over a frequency range, 1 to 100 Hz, which is predominantly contained in normal activities. This behavior is inconsistent with an optimal "design" for bone as a shock absorber. There was no definitive damping peak in the range of frequencies explored, which could be attributed to fluid flow in the porosity of bone.