Sundar Srinivasan

Low-Magnitude Mechanical Loading Becomes Osteogenic When Rest Is Inserted Between Each Load Cycle

Strategies to counteract bone loss with exercise have had fairly limited success, particularly those regimens subjecting the skeleton to mild activity such as walking. In contrast, here we show that it is possible to induce substantial bone formation with low‐magnitude loading. In two distinct in vivo models of bone adaptation, we found that insertion of a 10‐s rest interval between each load cycle transformed a locomotion‐like loading regime that minimally influenced osteoblast activity into a potent anabolic stimulus. In the avian ulna model, the minimal mean (+SE) periosteal labeled surface (Ps.LS) observed in the intact contralateral bones (1.6 ± 1.5%) was doubled after 3 consecutive days of low‐magnitude loading (3.8 ± 1.5%; p = 0.03). However, modifying the regimen by inserting 10 s of rest between each load cycle significantly enhanced the periosteal response (21.9 + 4.5%; p = 0.03). In the murine tibia model, 5 consecutive days of 100 low‐magnitude loading cycles did not significantly alter mean periosteal bone formation rate (BFR) compared with contralateral bones (0.011 ± 0.005 μm3/μm2 per day vs. 0.021 ± 0.013 μm3/μm2 per day). In contrast, separating each of 10 of the same loading cycles with 10 s of rest significantly elevated periosteal BFR (0.167 ± 0.049 μm3/μm2 per day; p = 0.01). Endocortical bone formation parameters were not altered by any loading regimen in either model. We conclude that 10 s of rest between each load cycle of a low‐magnitude loading protocol greatly enhances the osteogenic potential of the regimen.

Enhanced osteoclastic resorption and responsiveness to mechanical load in gap junction deficient bone.

Emerging evidence suggests that connexin mediated gap junctional intercellular communication contributes to many aspects of bone biology including bone development, maintenance of bone homeostasis and responsiveness of bone cells to diverse extracellular signals. Deletion of connexin 43, the predominant gap junction protein in bone, is embryonic lethal making it challenging to examine the role of connexin 43 in bone in vivo. However, transgenic murine models in which only osteocytes and osteoblasts are deficient in connexin 43, and which are fully viable, have recently been developed. Unfortunately, the bone phenotype of different connexin 43 deficient models has been variable. To address this issue, we used an osteocalcin driven Cre-lox system to create osteoblast and osteocyte specific connexin 43 deficient mice. These mice displayed bone loss as a result of increased bone resorption and osteoclastogenesis. The mechanism underlying this increased osteoclastogenesis included increases in the osteocytic, but not osteoblastic, RANKL/OPG ratio. Previous in vitro studies suggest that connexin 43 deficient bone cells are less responsive to biomechanical signals. Interestingly, and in contrast to in vitro studies, we found that connexin 43 deficient mice displayed an enhanced anabolic response to mechanical load. Our results suggest that transient inhibition of connexin 43 expression and gap junctional intercellular communication may prove a potentially powerful means of enhancing the anabolic response of bone to mechanical loading.

Upregulation of osteopontin by osteocytes deprived of mechanical loading or oxygen.

The pathway(s) by which disuse is transduced into locally mediated osteoclastic resorption remain unknown. We found that both acute disuse (in vivo) and direct hypoxia (in vitro) induced rapid upregulation of OPN expression by osteocytes. Within the context of OPNs role in osteoclast migration and attachment, hypoxia‐induced osteocyte OPN expression may serve to mediate disuse‐induced bone resorption.

Why Rest Stimulates Bone Formation: A Hypothesis Based on Complex Adaptive Phenomenon

GROSS, T. S., S. L. POLIACHIK, B. J. AUSK, D. A. SANFORD, B. A. BECKER, and S. SRINIVASAN. Why rest stimulates bone formation: A hypothesis based on complex adaptive phenomenon. Exerc. Sport Sci. Rev., Vol. 32, No. 1, pp. 9–13, 2004. Moderate exercise is an ineffective strategy to build bone mass. The authors present data demonstrating that allowing bone to rest between each load cycle transforms low- and moderate-magnitude mechanical loading into a signal that potently induces bone accretion. They hypothesize that the osteogenic nature of rest-inserted loading arises by enabling osteocytes to communicate as a small world network.

Bone mechanotransduction may require augmentation in order to strengthen the senescent skeleton

Physical exercise is thought to hold promise as a non-invasive countermeasure against skeletal fragility arising from post-menopausal and age-related osteoporosis. Importantly, mechanical loading and exercise are capable of increasing bone size via periosteal expansion, which by far, is the most effective means of strengthening the structure of a given bone. The focus of this review was to therefore explore whether exercise has the potential to increase periosteal modeling and bone size in the senescent skeleton. A survey of exercise trials in humans suggests that exercise interventions that enhance periosteal modeling in the young skeleton fail to do the same in the elderly skeleton. Underlying this ineffectiveness, in vitro studies indicate that aging lowers basal levels of cell function and degrades bone mechanotransduction at a variety of levels from altered second messenger signaling to gene expression driving proliferation and/or differentiation. Given these age-related alterations, the ultimate efficacy of an exercise intervention may depend upon concurrent supplementation that directly address deficits in signaling and/or cell function. In this context, in vivo animal models of mechanical loading that simulate the muted periosteal adaptation in the elderly hold potential to examine the efficacy of countermeasures. Preliminary in vivo experiments suggest that pharmacologically counteracting age-related deficits in cellular function can restore exercise induced periosteal modeling in the senescent skeleton to levels observed in young animals. If the safety and efficacy of this strategy were to be confirmed for human use, it would enable the utilization of exercise as a viable countermeasure against skeletal fragility at senescence.

Rescuing Loading Induced Bone Formation at Senescence

The increasing incidence of osteoporosis worldwide requires anabolic treatments that are safe, effective, and, critically, inexpensive given the prevailing overburdened health care systems. While vigorous skeletal loading is anabolic and holds promise, deficits in mechanotransduction accrued with age markedly diminish the efficacy of readily complied, exercise-based strategies to combat osteoporosis in the elderly. Our approach to explore and counteract these age-related deficits was guided by cellular signaling patterns across hierarchical scales and by the insight that cell responses initiated during transient, rare events hold potential to exert high-fidelity control over temporally and spatially distant tissue adaptation. Here, we present an agent-based model of real-time Ca2+/NFAT signaling amongst bone cells that fully described periosteal bone formation induced by a wide variety of loading stimuli in young and aged animals. The model predicted age-related pathway alterations underlying the diminished bone formation at senescence, and hence identified critical deficits that were promising targets for therapy. Based upon model predictions, we implemented an in vivo intervention and show for the first time that supplementing mechanical stimuli with low-dose Cyclosporin A can completely rescue loading induced bone formation in the senescent skeleton. These pre-clinical data provide the rationale to consider this approved pharmaceutical alongside mild physical exercise as an inexpensive, yet potent therapy to augment bone mass in the elderly. Our analyses suggested that real-time cellular signaling strongly influences downstream bone adaptation to mechanical stimuli, and quantification of these otherwise inaccessible, transient events in silico yielded a novel intervention with clinical potential.

Metaphyseal and diaphyseal bone loss in the tibia following transient muscle paralysis are spatiotemporally distinct resorption events

When the skeleton is catabolically challenged, there is great variability in the timing and extent of bone resorption observed at cancellous and cortical bone sites. It remains unclear whether this resorptive heterogeneity, which is often evident within a single bone, arises from increased permissiveness of specific sites to bone resorption or localized resorptive events of varied robustness. To explore this question, we used the mouse model of calf paralysis induced bone loss, which results in metaphyseal and diaphyseal bone resorption of different timing and magnitude. Given this phenotypic pattern of resorption, we hypothesized that bone loss in the proximal tibia metaphysis and diaphysis occurs through resorption events that are spatially and temporally distinct. To test this hypothesis, we undertook three complimentary in vivo/μCT imaging studies. Specifically, we defined spatiotemporal variations in endocortical bone resorption during the 3weeks following calf paralysis, applied a novel image registration approach to determine the location where bone resorption initiates within the proximal tibia metaphysis, and explored the role of varied basal osteoclast activity on the magnitude of bone loss initiation in the metaphysis using μCT based bone resorption parameters. A differential response of metaphyseal and diaphyseal bone resorption was observed throughout each study. Acute endocortical bone loss following muscle paralysis occurred almost exclusively within the metaphyseal compartment (96.5% of total endocortical bone loss within 6days). Using our trabecular image registration approach, we further resolved the initiation of metaphyseal bone loss to a focused region of significant basal osteoclast function (0.03mm(3)) adjacent to the growth plate. This correlative observation of paralysis induced bone loss mediated by basal growth plate cell dynamics was supported by the acute metaphyseal osteoclastic response of 5-week vs. 13-month-old mice. Specifically, μCT based bone resorption rates normalized to initial trabecular surface (BRRBS) were 3.7-fold greater in young vs. aged mice (2.27±0.27μm(3)/μm(2)/day vs. 0.60±0.44μm(3)/μm(2)/day). In contrast to the focused bone loss initiation in the metaphysis, diaphyseal bone loss initiated homogeneously throughout the long axis of the tibia predominantly in the second week following paralysis (81.3% of diaphyseal endocortical expansion between days 6 and 13). The timing and homogenous nature are consistent with de novo osteoclastogenesis mediating the diaphyseal resorption. Taken together, our data suggests that tibial metaphyseal and diaphyseal bone loss induced by transient calf paralysis are spatially and temporally discrete events. In a broader context, these findings are an essential first step toward clarifying the timing and origins of multiple resorptive events that would require targeting to fully inhibit bone loss following neuromuscular trauma.

32 wk old C3H/HeJ mice actively respond to mechanical loading

Numerous studies indicate that C3H/HeJ (C3H) mice are mildly responsive to mechanical loading compared to C57BL/6J (C57) mice. Guided by data indicating high baseline periosteal osteoblast activity in 16 wk C3H mice, we speculated that simply allowing the C3H mice to age until basal periosteal bone formation was equivalent to that of 16 wk C57 mice would restore mechanoresponsiveness in C3H mice. We tested this hypothesis by subjecting the right tibiae of 32 wk old C3H mice and 16 wk old C57 mice to low magnitude rest-inserted loading (peak strain: 1235 mu epsilon) and then exposing the right tibiae of 32 wk C3H mice to low (1085 mu epsilon) or moderate (1875 mu epsilon) magnitude cyclic loading. The osteoblastic response to loading on the endocortical and periosteal surfaces was evaluated via dynamic histomorphometry. At 32 wk of age, C3H mice responded to low magnitude rest-inserted loading with significantly elevated periosteal mineralizing surface, mineral apposition rate and bone formation compared to unloaded contralateral bones. Surprisingly, the periosteal bone formation induced by low magnitude rest-inserted loading in C3H mice exceeded that induced in 16 wk C57 mice. At 32 wk of age, C3H mice also demonstrated an elevated response to increased magnitudes of cyclic loading. We conclude that a high level of basal osteoblast function in 16 wk C3H mice appears to overwhelm the ability of the tissue to respond to an otherwise anabolic mechanical loading stimulus. However, when basal surface osteoblast activity is equivalent to that of 16 wk C57 mice, C3H mice demonstrate a clear ability to respond to either rest-inserted or cyclic loading.

Cortical bone resorption following muscle paralysis is spatially heterogeneous.

Mechanical loading of the skeleton, as induced by muscle function during activity, plays a critical role in maintaining bone homeostasis. It is not understood, however, whether diminished loading (and thus diminished mechanical stimuli) directly mediates the bone resorption that is associated with disuse. Our group has recently developed a murine model in which we have observed rapid and profound bone loss in the tibia following transient paralysis of the calf muscles. As cortical bone loss is achieved via rapid endocortical expansion without alterations in periosteal morphology, we believe this model holds unique potential to explore the spatial relation between altered mechanical stimuli and subsequent bone resorption. Given the available literature, we hypothesized that endocortical resorption following transient muscle paralysis would be spatially homogeneous. To test this hypothesis, we first validated an image registration algorithm that quantified site-specific cortical bone alterations with high precision and accuracy. We then quantified endocortical expansion in the tibial diaphysis within 21 days following transient muscle paralysis and found that, within the analyzed mid-diaphyseal region (3.15 mm), site-specific bone loss was focused on the anterior surface in the proximal region but shifted to the posterior surface at the distal end of the analyzed volume. This site-specific, and highly repeatable biologic response suggests active osteoclast chemotaxis or focal activation of osteoclastic resorption underlies the spatially consistent endocortical resorption induced by transient muscle paralysis. Clarifying this relation holds potential to yield unique insight into how the removal of factors critical for bone homeostasis acutely precipitates local modulation of cellular responses within bone.

Botulinum Toxin Induces Muscle Paralysis and Inhibits Bone Regeneration in Zebrafish

Intramuscular administration of Botulinum toxin (BTx) has been associated with impaired osteogenesis in diverse conditions of bone formation (eg, development, growth, and healing), yet the mechanisms of neuromuscular‐bone crosstalk underlying these deficits have yet to be identified. Motivated by the emerging utility of zebrafish (Danio rerio) as a rapid, genetically tractable, and optically transparent model for human pathologies (as well as the potential to interrogate neuromuscular‐mediated bone disorders in a simple model that bridges in vitro and more complex in vivo model systems), in this study, we developed a model of BTx‐induced muscle paralysis in adult zebrafish, and we examined its effects on intramembranous ossification during tail fin regeneration. BTx administration induced rapid muscle paralysis in adult zebrafish in a manner that was dose‐dependent, transient, and focal, mirroring the paralytic phenotype observed in animal and human studies. During fin regeneration, BTx impaired continued bone ray outgrowth, morphology, and patterning, indicating defects in early osteogenesis. Further, BTx significantly decreased mineralizing activity and crystalline mineral accumulation, suggesting delayed late‐stage osteoblast differentiation and/or altered secondary bone apposition. Bone ray transection proximal to the amputation site focally inhibited bone outgrowth in the affected ray, implicating intra‐ and/or inter‐ray nerves in this process. Taken together, these studies demonstrate the potential to interrogate pathological features of BTx‐induced osteoanabolic dysfunction in the regenerating zebrafish fin, define the technological toolbox for detecting bone growth and mineralization deficits in this process, and suggest that pathways mediating neuromuscular regulation of osteogenesis may be conserved beyond established mammalian models of bone anabolic disorders.