Joseph D. Gardinier

In situ permeability measurement of the mammalian lacunar–canalicular system

Bone is capable of adapting its mass and structure under mechanical cues. Bone cells respond to various mechanical stimuli including substrate strain, fluid pressure, and fluid flow (shear stress) in vitro. Although tissue-level strains are well documented experimentally, microfluidic parameters around bone cells are quantified mainly through theoretical modeling. A key model parameter, the Darcy permeability of the bone lacunar-canalicular system (LCS), is difficult to measure using traditional methods due to the co-existence of the larger vascular and smaller LCS porosities. In this paper, we developed a novel method to measure the LCS permeability by rapid compaction of intact mammalian bones and recording the intramedullary pressure (IMP). Six canine metacarpals were subjected to three step compression tests with peak loads of 50, 100, or 200lbs, while the IMP was simultaneously recorded using a catheter pressure transducer. The loading ramp time was chosen to be ~2ms, which was long enough to allow pressure equilibrium to be established between the marrow cavity and the vascular pores, but short enough to observe the LCS fluid flowing into and out of the vascular pores. This loading scheme permitted us to differentiate the contribution of the two intermingled porosities to the IMP responses. The time constant of the IMP pressurization and relaxation due to the LCS was found to be 8.1+/-3.6s (n=18). The mid-shaft cortex of the metacarpals mainly consisted of osteons with an average radial thickness of 65+/-27microm, which served as the characteristic distance for the LCS fluid to relax. The LCS permeability was obtained via poroelastic analysis to be 2.8+/-1.8x10(-)(23)m(2), which was smaller than previous theoretical predictions (order of 10(-)(19) to 10(-)(22)m(2)), but within the range of previous experimentally based estimations (order of 10(-)(22) to 10(-)(25)m(2)). Our results also show that osteoblasts and osteocytes experience hydraulic pressures that differ by three orders of magnitude under physiological compressive strains. These estimates of the in vivo mechanical environments may be used to design in vitro models for elucidating the cellular and molecular mechanisms of bone adaptation and pathological bone loss.

PTH signaling during exercise contributes to bone adaptation

Improving the structural integrity of bone reduces fracture risk and development of osteoporosis later in life. Exercise can increase the mechanical properties of bone, and this increase is often attributed to the dynamic loading created during exercise. However, the increase in systemic parathyroid hormone (PTH) levels during exercise gives reason to hypothesize that PTH signaling also regulates bone adaptation in response to exercise. Therefore, the first aim of this study was to establish the impact PTH signaling has on bone adaptation during exercise by inhibiting PTH signaling with PTH(7‐34); the second aim was to determine whether increasing PTH levels during exercise with PTH(1‐34) can augment bone adaptation. Thirty minutes after a single bout of running on a treadmill, mice exhibited a twofold increase in systemic PTH levels. Under the same exercise regimen, the influence of PTH signaling on bone adaptation during exercise was then evaluated in mice after 21 consecutive days of exercise and treatment with PTH(7‐34), PTH(1‐34), or vehicle. Exercise alone caused a significant increase in trabecular bone volume with adaptation to a more platelike structure, which was inhibited with PTH(7‐34) during exercise. Changes in structural‐level and tissue‐level mechanical properties during exercise occurred in the absence of significant changes to cortical bone geometry. Inhibition of PTH signaling during exercise attenuated the changes in structural‐level mechanical properties, but not tissue‐level properties. Enhanced PTH signaling during exercise with PTH(1‐34) increased trabecular and cortical bone volume, but had little effect on the structural‐level and tissue‐level mechanical properties compared to exercise alone. Our study is the first to demonstrate that bone adaptation during exercise is not only a function of dynamic loading, but also PTH release, and that PTH signaling contributes differently at the structural and tissue levels.

P2Y2 receptors regulate osteoblast mechanosensitivity during fluid flow

Mechanical stimulation of osteoblasts activates many cellular mechanisms including the release of ATP. Binding of ATP to purinergic receptors is key to load-induced osteogenesis. Osteoblasts also respond to fluid shear stress (FSS) with increased actin stress fiber formation (ASFF) that we postulate is in response to activation of the P2Y2 receptor (P2Y2R). Furthermore, we predict that ASFF increases cell stiffness and reduces the sensitivity to further mechanical stimulation. We found that small interfering RNA (siRNA) suppression of P2Y2R attenuated ASFF in response to FSS and ATP treatment. In addition, RhoA GTPase was activated within 15 min after the onset of FSS or ATP treatment and mediated ASFF following P2Y2R activation via the Rho kinase (ROCK)1/LIM kinase 2/cofilin pathway. We also observed that ASFF in response to FSS or ATP treatment increased the cell stiffness and was prevented by knocking down P2Y2R. Finally, we confirmed that the enhanced cell stiffness and ASFF in response to RhoA GTPase activation during FSS drastically reduced the mechanosensitivity of the osteoblasts based on the intracellular Ca(2+) concentration ([Ca(2+)]i) response to consecutive bouts of FSS. These data suggest that osteoblasts can regulate their mechanosensitivity to continued load through P2Y2R activation of the RhoA GTPase signaling cascade, leading to ASFF and increased cell stiffness.

Does blood pressure enhance solute transport in the bone lacunar-canalicular system?

Solute transport through bone plays an important role in tissue metabolism and cellular mechanotransduction. Due to limited diffusion within the mineralized bone matrix, both mechanical loading and vascular pressure have been proposed to drive interstitial fluid flow within the lacunar-canalicular system (LCS); thereby augmenting solute diffusion in bone. Although blood supply is critical for bone nutrition, growth, and fracture healing, whether physiological blood pressures can drive significant fluid and solute convection remains controversial within the literature. The goal of this study was to directly test the hypothesis that in vivo blood pressures enhance solute transport in the bone LCS. Using a newly developed imaging approach based on fluorescence recovery after photobleaching (FRAP), we first measured the transport rate of sodium fluorescein (M.W. 376 Da) through the tibial LCS in four anesthetized mice (in the presence of vascular pressure). These data were then compared with the tracer transport rates at the same locations/lacunae after sacrifice (in the absence of vascular pressure). Using paired FRAP experiments we did not detect differences in tracer transport rates between bones from live anesthetized animals versus those in postmortem bodies (p>0.05, N=18). In a separate cohort of four anesthetized mice a mean jugular pulse pressure of approximately 10 mmHg at approximately 10 Hz was measured. Further theoretical analysis showed that for bones from both small and large animal species the blood pressure-driven convection of either small (376 Da) or large (43,000 Da) molecules was at least one order of magnitude smaller than diffusion under either normal or elevated pressure conditions. We conclude that despite the extreme importance of vasculature in bone physiology, vascular pressure itself does not enhance acute solute transport within the bone LCS. Therefore, mechanisms other than the vascular pressure-induced fluid flow such as altered biochemical factors may account for the bone adaptation associated with altered circulation. The present study helped clarify a long-standing controversy regarding vascular pressure-induced bone fluid flow and provided a better understanding of bone adaptation in both physiological and pathological conditions.

Exercise increases pyridinoline cross-linking and counters the mechanical effects of concurrent lathyrogenic treatment

The collagen cross-link profile of bone, associated with bone strength and fracture toughness, is tightly regulated (affecting cross-link quantity, type, lysine hydroxylation and maturity) and may contribute to the improvements in bone quality during exercise. We hypothesized that 1) exercise promotes mature cross-link formation, 2) increased mature cross-linking is accompanied by shifts in lysine hydroxylation, and 3) these changes in collagen cross-link profile have positive effects on mechanical properties. Growing male C57Bl6 mice were treated with 30 min/day of running exercise, 350 mg/kg/day β-aminopropionitrile (BAPN) injected subcutaneously to inhibit enzymatic collagen cross-linking, or both exercise and BAPN, from 5 to 8 weeks of age. Bone collagen cross-linking profile, mechanical properties, morphology, and mineralization were measured from the tibiae. Cross-link measures, including immature, pyridinoline, pyrrole and pentosidine cross-links, ratios reflecting cross-link maturity and hydroxylation, and mineralization were tested for their importance to mechanical properties across 8 week groups through correlation analyses and step-wise linear regressions. BAPN treatment significantly reduced lysylpyridinoline, pyrrole, hydroxylysinorleucine, and total mature collagen cross-linking, resulting in decreased bone elastic modulus and increased yield strain despite a marginal increase in TMD. Exercise caused a shift toward pyridinoline cross-linking, with increased hydroxylysylpyridinoline and decreased pyrrole cross-linking resulting in total mature cross-linking and estimated tissue level mechanical properties matching sedentary control levels. Exercise superimposed on BAPN treatment increased total mature cross-linking from BAPN to control levels, but did so by increasing pyridinoline, not pyrrole, cross-links. Exercise also counteracted the BAPN effects on modulus and strain, without a change in TMD. Pyrrole cross-linking was the strongest correlate of modulus (r=0.470, p<0.01) and yield strain (r=-0.467, p<0.01). Cross-links with similar levels of telopeptide lysine hydroxylation to pyrrole (lysylpyridinoline and hydroxylysinorleucine) also correlated with modulus and strain to a lesser extent. In conclusion, exercise in growing mice promotes pyridinoline collagen cross-linking in bone, the resulting increase in total mature cross-linking is sufficient to counteract the mechanical effects of concurrent cross-link inhibition, and this responsiveness to loading is a potential means by which exercise might improve bone quality in diseased or otherwise compromised bone.

Systemic PTH Release During Exercise Enhances Trabecular Bone Architecture

Bone mineral density gained during adolescent years is a significant determinant of bone quality maintained during adult-hood [1]. The ability to enhance the structural integrity of bone at a young age reduces the risk of bone fracture as well as the development of musculoskeletal diseases, such as osteoporosis [1].Copyright