Mechanical Loading Of Bone Research Articles

Early life vitamin D depletion and mechanical loading determine methylation changes in the RUNX2, RXRA, and osterix promoters in mice

BackgroundEarly life vitamin D exposure is linked to later skeletal health with maternal vitamin D status in pregnancy associated with neonatal bone mass. The MAVIDOS study has demonstrated that vitamin D supplementation leads to reduced RXRA DNA methylation. Mice exposed to early life vitamin D deficiency have reduced bone mass and bone accrual in response to mechanical loading. Using the tibiae of these mice, we have examined the effect of diet and mechanical loading on the DNA methylation of promoters of genetic loci important for bone growth and development and their association with bone strength.ResultsMechanical loading of mouse tibiae leads to a reduction of RXRA DNA methylation. Early life vitamin D deficiency is associated with altered methylation of osterix and Runx2 in these bones. Tibia strength was also demonstrated to be associated with a change in DNA methylation status in CpGs of the vitamin D receptor (VDR), ostrix, and RXRA genes.ConclusionsWe have shown for the first time that mechanical loading of bone and early life vitamin D deficiency leads to changes in the epigenome of this tissue in key genes in the vitamin D and osteoblast differentiation pathway.

Differential Effects of Neurectomy and Botox-induced Muscle Paralysis on Bone Phenotype and Titanium Implant Osseointegration.

Metabolic bone is highly innervated by both sensory and sympathetic nerves. In addition to skeletal development, neural regulation participates in local bone remodeling, which is important for successful osseointegration of titanium implants. Neurectomy is a model used to investigate the lack of neural function on bone homeostasis, but the relative impacts of direct denervation to bone or denervation-induced muscle paralysis are less well defined. To investigate this difference, we used two nerve intervention models, sciatic and femoral neurectomy (SFN) v. botox-induced muscle paralysis (BTX) and assessed the resulting femoral bone phenotype and Ti implant osseointegration. Male Sprague Dawley rats (19) were randomly divided into three groups: implant control (n=5), SFN (n=7), and BTX (n=7). Ti implants (microrough/hydrophilic [modSLA], Institut Straumann AG) were placed in the distal metaphysis of each femur on day 24 post-SFN or BTX. Bone and muscle were examined on day 28 after implant insertion. Both nerve intervention models impaired osseointegration. MicroCT and histology indicated that both models had reduced trabecular bone formation. Only BTX reduced cortical bone formation and increased cortical bone porosity. BTX resulted in more bone loss characterized by the least trabecular and cortical bone, as well as osseointegration. Osteoblasts isolated from the tibia exhibited a model-specific phenotype when they were grown on Ti substrates in vitro. Neurectomy caused more severe muscle atrophy than botox injection. These results indicate that neural regulation directly modulates bone formation and osseointegration. Muscle paralysis modulated the effects of loss of neural inputs into bone, supporting the hypothesis that mechanical loading of bone is a factor in achieving successful osseointegration. The different effects of botox and neurectomy on bone phenotype indicated that the sensory and sympathetic nerves had a role in the osseointegration process.

CaMKK2-Mediated Effects on Mechanically Induced Bone Formation in Male and Female Mice

Background and Hypothesis: Ca2+/calmo-dulin-dependent protein kinase kinase 2 (CaMKK2) is a serine-threonine protein kinase that plays a significant role in both anabolic and catabolic pathways of bone remodeling. Mechanical loading of bone translates an external force into both biochemical and structural changes. It has been shown that deletion or inhibition of CaMKK2 results in increased bone density in male and female mice. We hypothesize that the lack of CaMKK2 in bone cells will result in loading-induced bone mass accrual with no difference between male and female mice. 
 Experimental Design or Project Methods: The right tibia of anesthetized 16-week-old wild-type (WT) and CaMKK2 knockout (KO) mice were loaded at 2 Hz for 220 cycles and with peak forces specific to both sex and genotype. Loading was accomplished using an electro actuator (Bose ElectroForce 3200; EnduraTEC, Minnetonka, MN, USA). This was repeated 3, 5, 8 and 10 days after initial loading. The non-loaded left tibia served as an internal control. Calcein and alizarin red were administered intraperitoneally on days 9 and 16, respectively to metabolically label newly formed bone. Nineteen days after initial loading, mice were sacrificed. Blood and long bones of the lower limbs were collected for analysis. 
 Results: Using microcomputer tomography; dynamic histomorphometry; histology, immunohistochemistry, enzyme-linked immunosorbent assay and real-time reverse transcription-polymerase chain reaction, we will assess bone volume, bone formation rate, and underlying mechanisms at the cellular and molecular level. These data are forthcoming. 
 Conclusion and Potential Impact: With expanded knowledge on how bone growth is augmented, clinical outcomes related to osteoporosis and fracture healing, for example, may be improved. This may be accomplished through novel therapy related to these pathways that increases bone density or decreases fracture healing time.

Muscle-bone interactions: movement in the field of mechano-humoral coupling of muscle and bone.

Cyclical, mechanical loading of bone by skeletal muscle is widely recognized as a critical determinant of bone structure and mass. A growing body of evidence indicates that substances released from skeletal muscle into the bloodstream also regulate bone mass and metabolism. In this commentary, we discuss the status of research in the area of humoral regulation of bone mass by the skeletal muscle secretome, with an emphasis on the roles of myostatin, irisin, interleukin-6, and exosomes. The interplay between muscle, bone, and other modulators of bone mass, including circadian rhythm and sympathetic tone, is also discussed.

Comparison of strain measurement in the mouse forearm using subject-specific finite element models, strain gaging, and digital image correlation.

Mechanical loading in bone leads to the activation of bone-forming pathways that are most likely associated with a minimum strain threshold being experienced by the osteocyte. To investigate the correlation between cellular response and mechanical stimuli, researchers must develop accurate ways to measure/compute strain both externally on the bone surface and internally at the osteocyte level. This study investigates the use of finite element (FE) models to compute bone surface strains on the mouse forearm. Strains from three FE models were compared to data collected experimentally through strain gaging and digital image correlation (DIC). Each FE model was assigned subject-specific bone properties and consisted of one-dimensional springs representing the interosseous membrane. After three-point bending was performed on the ulnae and radii, moment of inertia was determined from microCT analysis of the bone region between the supports and then used along with standard beam analyses to calculate the Young's modulus. Non-contact strain measurements from DIC were determined to be more suitable for validating numerical results than experimental data obtained through conventional strain gaging. When comparing strain responses in the three ulnae, we observed a 3-14% difference between numerical and DIC strains while the strain gage values were 37-56% lower than numerical values. This study demonstrates a computational approach for capturing bone surface strains in the mouse forearm. Ultimately, strains from these macroscale models can be used as inputs for microscale and nanoscale FE models designed to analyze strains directly in the osteocyte lacunae.

Editorial: Mechanical Loading and Bone.

This Research Topic comprises 11 interesting and topical articles describing a mixture of clinical and laboratory approaches to how mechanical loading influences the skeleton. Of the five clinical papers, those by Tobias et al. and Janz et al. address important methodological issues concerning the use of accelerometers to record physical activity in a way that is relevant to mechanical loading of bone. Two separate papers by Toshihiro Sugiyama et al. discuss how understanding of skeletal responses to mechanical loading can help interpret the mechanism of action of bone anabolic drugs. The paper by Zengin et al. illustrates how understanding of mechanical loading responses can be used to interpret ethnic differences in skeletal structure. Of the six laboratory papers, those by Meakin et al. and Vazquez et al. focus on methodological approaches to study mechanical loading in vivo and in vitro, respectively. Four of the papers explore the mechanisms involved in mediating different aspects of mechanical loading responses of bone. Garcia-Lopez et al. report their findings concerning the mechanisms involved in the inhibition of bone resorption by cyclical mechanical strain in vitro; Kang and Robling review the role of the Wnt pathway in mechanotransduction; Alzahrani et al. review the mechanisms involved in distraction osteogenesis; and Betts and Muller review theories and models of mechanical regulation of bone regeneration after bone injury.

Paradoxical response to mechanical unloading in bone loss, microarchitecture, and bone turnover markers.

Background: Sclerostin, encoded by the SOST gene, has been implicated in the response to mechanical loading in bone. Some studies demonstrated that unloading leads to up-regulated SOST expression, which may induce bone loss.Purpose: Most reported studies regarding the changes caused by mechanical unloading were only based on a single site. Considering that the longitudinal bone growth leads to cells of different age with different sensitivity to unloading, we hypothesized that bone turnover in response to unloading is site specific.Methods: We established a disuse rat model by sciatic neurectomy in tibia. In various regions at two time-points, we evaluated the bone mass and microarchitecture in surgically-operated rats and control rats by micro-Computed Tomography (micro-CT) and histology, sclerostin/SOST by immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), and quantitative reverse transcription polymerase chain reaction (qPCR), tartrate resistant acid phosphatase 5b (TRAP 5b) by ELISA and TRAP staining, and other bone markers by ELISA. Results: Micro-CT and histological analysis confirmed bone volume in the disuse rats was significantly decreased compared with those in the time-matched control rats, and microarchitecture also changed 2 and 8 weeks after surgery. Compared with the control groups, SOST mRNA expression in the diaphysis was down-regulated at both week 2 and 8. On the contrary, the percentage of sclerostin-positive osteocytes showed an up-regulated response in the 5 - 6 mm region away from the growth plate, while in the 2.5 - 3.5 mm region, the percentage was no significant difference. Nevertheless, in 0.5 - 1.5 mm region, the percentage of sclerostin-positive osteocytes decreased after 8 weeks, consistent with serum SOST level. Besides, the results of TRAP also suggested that the expression in response to unloading may be opposite in different sites or system.Conclusion: Our data indicated that unloading-induced changes in bone turnover are probably site specific. This implies a more complex response pattern to unloading and unpredictable therapeutics which target SOST or TRAP 5b.

The Contribution of Experimental in vivo Models to Understanding the Mechanisms of Adaptation to Mechanical Loading in Bone.

Changing loading regimens by natural means such as exercise, with or without interference such as osteotomy, has provided useful information on the structure:function relationship in bone tissue. However, the greatest precision in defining those aspects of the overall strain environment that influence modeling and remodeling behavior has been achieved by relating quantified changes in bone architecture to quantified changes in bones’ strain environment produced by direct, controlled artificial bone loading. Jiri Hert introduced the technique of artificial loading of bones in vivo with external devices in the 1960s using an electromechanical device to load rabbit tibiae through transfixing stainless steel pins. Quantifying natural bone strains during locomotion by attaching electrical resistance strain gages to bone surfaces was introduced by Lanyon, also in the 1960s. These studies in a variety of bones in a number of species demonstrated remarkable uniformity in the peak strains and maximum strain rates experienced. Experiments combining strain gage instrumentation with artificial loading in sheep, pigs, roosters, turkeys, rats, and mice has yielded significant insight into the control of strain-related adaptive (re)modeling. This diversity of approach has been largely superseded by non-invasive transcutaneous loading in rats and mice, which is now the model of choice for many studies. Together such studies have demonstrated that over the physiological strain range, bone’s mechanically adaptive processes are responsive to dynamic but not static strains; the size and nature of the adaptive response controlling bone mass is linearly related to the peak loads encountered; the strain-related response is preferentially sensitive to high strain rates and unresponsive to static ones; is most responsive to unusual strain distributions; is maximized by remarkably few strain cycles, and that these are most effective when interrupted by short periods of rest between them.

Mechanoadaptation of developing limbs: shaking a leg

The proportion of total limb length taken up by the individual skeletal elements (limb proportionality), varies widely between species. These diverse skeletal forms have evolved to allow for a range of limb uses and they first emerge as the embryo develops, to achieve the characteristic skeletal architecture of each species. During this time, the developing skeleton experiences mechanical loading as a result of embryonic muscle contraction. The possibility that adaptation to such mechanical input may allow embryos to coordinate the appearance of skeletal design with their expanding range of movements has so far received little attention. This is surprising, given the critical role exerted by embryo movement in normal skeletal development; stage-specific in ovo immobilisation of embryonic chicks results in joint contractures and a reduction in longitudinal bone growth in the limbs. Epigenetic mechanisms allow for selective activation of genes in response to environmental signals, resulting in the production of phenotypic complexity in morphogenesis; mechanical loading of bone during movement appears to be one such signal. It may be that 'mechanosensitive' genes under regulation of mechanical input adjust proportionality along the bone's proximo-distal axis, introducing a level of phenotypic plasticity. If this hypothesis is upheld, species with more elongated distal limb elements will have a greater dependence on mechanical input for the differences in their growth, and mechanosensitive bone growth in the embryo may have evolved as an additional source of phenotypic diversity during skeletal development.

Bone loss during partial weight bearing (1/6th gravity) is mitigated by resistance and aerobic exercise in mice

Astronauts on long duration missions continue to experience bone loss, as much as 1–2% each month, for up to 4.5 years after a mission. Mechanical loading of bone with exercise has been shown to increase bone formation, mass, and geometry. The aim of this study was to compare the efficacy of two exercise protocols during a period of reduced gravitational loading (1/6th body weight) in mice. Since muscle contractions via resistance exercise impart the largest physiological loads on the skeleton, we hypothesized that resistance training (via vertical tower climbing) would better protect against the deleterious musculoskeletal effects of reduced gravitational weight bearing when compared to endurance exercise (treadmill running). Young adult female BALB/cBYJ mice were randomly assigned to three groups: 1/6g (G/6; n=6), 1/6g with treadmill running (G/6+RUN; n=8), or 1/6g with vertical tower climbing (G/6+CLB; n=9). Exercise was performed five times per week. Reduced weight bearing for 21 days was achieved through a novel harness suspension system. Treadmill velocity (12–20m/min) and daily run time duration (32–51min) increased incrementally throughout the study. Bone geometry and volumetric bone mineral density (vBMD) at proximal metaphysis and mid-diaphysis tibia were assessed by in vivo peripheral quantitative computed tomography (pQCT) on days 0 and 21 and standard dynamic histomorphometry was performed on undemineralized sections of the mid-diaphysis after tissue harvest. G/6 caused a significant decrease (P<0.001) in proximal tibia metaphysis total vBMD (−9.6%). These reductions of tibia metaphyseal vBMD in G/6 mice were mitigated in both G/6+RUN and G/6+CLB groups (P<0.05). After 21 days of G/6, we saw an absolute increase in tibia mid-diaphysis vBMD and in distal metaphysis femur vBMD in both G/6+RUN and G/6+CLB mice (P<0.05). Substantial increases in endocortical and periosteal mineralizing surface (MS/BS) at mid-diaphysis tibia in G/6+CLB demonstrate that bone formation can be increased even in the presence of reduced weight bearing. These data suggest that moderately vigorous endurance exercise and resistance training, through treadmill running or climb training mitigates decrements in vBMD during 21 days of reduced weight bearing. Consistent with our hypothesis, tower climb training, most pronounced in the tibia mid-diaphysis, provides a more potent osteogenic response compared to treadmill running.

Paradoxical Sost gene expression response to mechanical unloading in metaphyseal bone

The Sost gene encodes Sclerostin, an inhibitor of Wnt-signaling, generally considered a main response gene to mechanical loading in bone. Several papers describe that unloading leads to upregulation of Sost, which in turn may lead to loss of bone. These studies were based on whole bone homogenates or cortical bone. By serendipity, we noted an opposite response to unloading in the proximal rat tibia. Therefore, we hypothesized that Sost-expression in response to changes in mechanical load is bone site specific.One hind limb of male, 3 month old rats was unloaded by paralyzing the extensors with Botulinium toxin A (Botox) injections. A series of experiments compared the expression of Sost mRNA in the unloaded and contralateral, loaded limbs, after 3 or 10 days, in metaphyseal cancellous bone, metaphyseal cortical bone, and diaphyseal cortical bone. We also conducted μCT to confirm changes in bone volume density related to unloading.Sost mRNA expression in the cancellous metaphyseal bone was downregulated almost 2-fold, both 3 days and 10 days after unloading (P<0.05). A similar tendency was seen in the metaphyseal cortical bone, in which Sost was 1.5-fold downregulated (P<0.05) after 10days, but not significantly changed after 3days. In contrast, diaphyseal cortical Sost expression was instead upregulated 1.4-fold (P<0.05) following 3-day unloading, while there was no significant change after 10days. Cancellous bone volume density was 58% lower (P<0.001, compared to cage controls) in the unloaded limb but not significantly affected in the loaded limb.The results suggest that Sost mRNA expression in metaphyseal bone responds to mechanical unloading in an opposite direction to that observed in diaphyseal cortical bone. This proposes a more complex expression pattern for Sost in response to unloading. Therapeutics that target Sclerostin during altered loading conditions may result in local bone mass changes that are difficult to predict.

Cyclic hydrostatic pressure stimulates enhanced bone development in the foetal chick femur in vitro

Mechanical loading of bone and cartilage in vivo results in the generation of cyclic hydrostatic forces as bone compression is transduced to fluid pressure in the canalicular network and the joint synovium. It has therefore been suggested that hydrostatic pressure is an important stimulus by which osteochondral cells and their progenitors sense and respond to mechanical loading in vivo. In this study, hydrostatic pressure regimes of 0–279kPa at 0.005–2Hz were applied to organotypically cultured ex vivo chick foetal femurs (e11) for 1hour per day in a custom designed bioreactor for 14days and bone formation assessed by X-ray microtomography and qualified by histology. We found that the mineralised portion of the developing femur cultured under any cyclic hydrostatic pressure regime was significantly larger and/or denser than unstimulated controls but that constant (non-cycling) hydrostatic pressure had no effect on bone growth. Further experiments showed that the increase in bone formation was directly proportional to stimulation frequency (R2=0.917), but independent of the magnitude of the pressure applied, whilst even very low frequencies of stimulation (0.005Hz) had significant effects on bone growth. Expression of Type-II collagen in both epiphyses and diaphysis was significantly upregulated (1.48-fold and 1.95-fold respectively), together with osteogenic genes (osteonectin and osteopontin) and the osteocyte maturation marker CD44. This work demonstrates that cyclic hydrostatic pressure promotes bone growth and mineralisation in a developmental model and supports the hypothesis that hydrostatic forces play an important role in regulating bone growth and remodelling in vivo.

Bone remodelling around a cementless glenoid component

Post-operative change in the mechanical loading of bone may trigger its (mechanically induced) adaptation and hamper the mechanical stability of prostheses. This is especially important in cementless components, where the final fixation is achieved by the bone itself. The aim of this study is, first, to gain insight into the bone remodelling process around a cementless glenoid component, and second, to compare the possible bone adaptation when the implant is assumed to be fully bonded (best case scenario) or completely loose (worst case scenario). 3D finite element models of a scapula with and without a cementless glenoid component were created. 3D geometry of the scapula, material properties, and several physiological loading conditions were acquired from or estimated for a specific cadaver. Update of the bone density after implantation was done according to a node-based bone remodelling scheme. Strain energy density for different loading conditions was evaluated, weighted according to their frequencies in activities of daily life and used as a mechanical stimulus for bone adaptation. The average bone density in the glenoid increased after implantation. However, local bone resorption was significant in some regions next to the bone-implant interface, regardless of the interface condition (bonded or loose). The amount of bone resorption was determined by the condition imposed to the interface, being slightly larger when the interface was loose. An ideal screw, e.g. in which material fatigue was not considered, was enough to keep the interface micromotions small and constant during the entire bone adaptation simulation.

The Bone-Muscle Relationship in Men and Women

Muscle forces are a strong determinant of bone structure, particularly during the process of growth and development. The gender divergence in the bone-muscle relationship becomes strongly evident during adolescence. In females, growth is characterized by increased estrogen levels and increased mass and strength of bone relative to that of muscle, whereas in men, increases in testosterone fuel large increases in muscle, resulting in muscle forces that coincide with a large growth in bone dimensions and strength. In adulthood, significant age-related losses are observed for both bone and muscle tissues. Large decrease in estrogen levels in women appears to diminish the skeleton's responsiveness to exercise more than in men. In contrast, the aging of the muscle-bone axis in men is a function of age related declines in both hormones. In addition to the well-known age related changes in the mechanical loading of bone by muscle, newer studies appear to provide evidence of age- and gender-related variations in molecular signaling between bone and muscle that are independent of purely mechanical interactions. In summary, gender differences in the acquisition and age-related loss in bone and muscle tissues may be important for developing gender-specific strategies for using exercise to reduce bone loss with aging.

Inhibition of Osteoclastogenesis by Mechanically Loaded Osteocytes: Involvement of MEPE

In regions of high bone loading, the mechanoresponsive osteocytes inhibit osteoclastic bone resorption by producing signaling molecules. One possible candidate is matrix extracellular phosphoglycoprotein (MEPE) because acidic serine- and aspartate-rich MEPE-associated motif peptides upregulate osteoprotegerin (OPG) gene expression, a negative regulator of osteoclastogenesis. These peptides are cleaved from MEPE when relatively more MEPE than PHEX (phosphate-regulating gene with homology to endopeptidases on the X chromosome) is present. We investigated whether mechanical loading of osteocytes affects osteocyte-stimulated osteoclastogenesis by involvement of MEPE. MLO-Y4 osteocytes were mechanically loaded by 1-h pulsating fluid flow (PFF; 0.7 ± 0.3 Pa, 5 Hz) or kept under static control conditions. Recombinant MEPE (0.05, 0.5, or 5 μg/ml) was added to some static cultures. Mouse bone marrow cells were seeded on top of the osteocytes to determine osteoclastogenesis. Gene expression of MEPE, PHEX, receptor activator of nuclear factor kappa-B ligand (RANKL), and OPG by osteocytes was determined after PFF. Osteocytes supported osteoclast formation under static control conditions. Both PFF and recombinant MEPE inhibited osteocyte-stimulated osteoclastogenesis. PFF upregulated MEPE gene expression by 2.5-fold, but not PHEX expression. PFF decreased the RANKL/OPG ratio at 1-h PFF treatment. Our data suggest that mechanical loading induces changes in gene expression by osteocytes, which likely contributes to the inhibition of osteoclastogenesis after mechanical loading of bone. Because mechanical loading upregulated gene expression of MEPE but not PHEX, possibly resulting in the upregulation of OPG gene expression, we speculate that MEPE is a soluble factor involved in the inhibition of osteoclastogenesis by osteocytes.

Inhibition of bone resorption reduces osteoarthritis in mice with high bone turn over

Mechanical adaptation of bone is a cellular process that allows bone to adjust its mass and structure to its mechanical environment. Such remodeling occurs by recruitment and activation of osteoclasts and osteoblasts in relation to strain distribution over bone tissue. These mechanical changes are detected by osteocytes, which results in production of signalling molecules such as nitric oxide (NO). We have shown earlier that mechanically stimulated osteocytes inhibit osteoclast formation via soluble factors, thus affecting bone resorption. We hypothesize that mechanically stimulated osteocytes modulate osteoclast formation by changing expression of osteoclastogenesis-related genes. Therefore, we investigated whether mechanical stimulation affects osteoclastogenesis-related gene expression in osteocytes. MLO-Y4 osteocytes were seeded at 10 cells/cm on glass slides in α-MEM with 5% FBS and 5% FCS. Osteocytes were mechanically stimulated by 1 h pulsating fluid flow (PFF;0.7±0.3 Pa,5 Hz), and post-incubated for 1 h and 6 h without PFF. Controls were kept under static culture conditions. NO production was determined as parameter of bone cell responsiveness. After 1 h PFF and 1 h and 6 h postincubation, cells were lysed for total RNA isolation. Gene expression of MEPE, PHEX, sclerostin, RANKL, and OPG was studied using real time-PCR. Within 5 min PFF increased NO production by 2.0-fold. PFF upregulated MEPE gene expression by 23.0-fold after 1 h, but not at 1 h and 6 h post-incubation. PFF did not affect PHEX expression. Sclerostin expression was not detectable. PFF upregulated RANKL expression by 2.1 fold, and OPG expression by 2.7 fold. PFF decreased the RANKL/OPG ratio by 20% after 1 h, but not at 1 h and 6 h post-incubation. Our data suggest that mechanical loading induces changes in osteoclastogenesis-related gene expression in osteocytes, which likely contributes to the inhibition of osteoclastogenesis after mechanical loading of bone. Since mechanical loading upregulated gene expression of MEPE but not of PHEX, possibly resulting in the upregulation of OPG gene expression, we speculate that MEPE might be a key soluble factor produced after mechanical loading of osteocytes leading to the inhibition of osteoclastogenesis.

Focal Adhesion Kinase Is Important for Fluid Shear Stress-Induced Mechanotransduction in Osteoblasts

Mechanical loading of bone is important for maintenance of bone mass and structural stability of the skeleton. When bone is mechanically loaded, movement of fluid within the spaces surrounding bone cells generates fluid shear stress (FSS) that stimulates osteoblasts, resulting in enhanced anabolic activity. The mechanisms by which osteoblasts convert the external stimulation of FSS into biochemical changes, a process known as mechanotransduction, remain poorly understood. Focal adhesions are prime candidates for transducing external stimuli. Focal adhesion kinase (FAK), a nonreceptor tyrosine kinase found in focal adhesions, may play a key role in mechanotransduction, although its function has not been directly examined in osteoblasts. We examined the role of FAK in osteoblast mechanotransduction using short interfering RNA (siRNA), overexpression of a dominant negative FAK, and FAK(-/-) osteoblasts to disrupt FAK function in calvarial osteoblasts. Osteoblasts were subjected to varying periods oscillatory fluid flow (OFF) from 5 min to 4 h, and several physiologically important readouts of mechanotransduction were analyzed including: extracellular signal-related kinase 1/2 phosphorylation, upregulation of c-fos, cyclooxygenase-2, and osteopontin, and release of prostaglandin E(2). Osteoblasts with disrupted FAK signaling exhibited severely impaired mechanical responses in all endpoints examined. These data indicate the importance of FAK for both short and long periods of FSS-induced mechanotransduction in osteoblasts.

Mechanical loading by fluid shear stress enhances IGF-1 receptor signaling in osteoblasts in a PKCzeta-dependent manner.

Maintenance of optimal bone physiology requires the coordinated activity of osteoclasts that resorb old bone and osteoblasts that deposit new bone. Mechanical loading of bone and the resulting movement of interstitial fluid within the spaces surrounding bone cells is thought to play a key role is maintaining optimal bone mass. One way in which fluid movement may promote bone formation is by enhancing osteoblast survival. We have shown previously that application of fluid flow to osteoblasts in vitro confers a protective effect by inhibiting osteoblast apoptosis (Pavalko et al., 2003, J. Cell Physiol., 194: 194-205). To investigate the cellular mechanisms that regulate the response of osteoblasts to fluid shear stress, we have examined the possible interaction between fluid flow and growth factors in MC3T3-E1 osteoblast-like cells. We found that insulin-like growth factor-I (IGF-I) was significantly more effective at preventing TNF-alpha-induced apoptosis when cells were first subjected to mechanical loading by exposure to either unidirectional or oscillatory fluid flow compared to cells that were maintained in static culture. Additionally, downstream signaling in response to treatment with IGF-I, including ERK and Akt activation, was enhanced in cells that were subjected to fluid flow, compared to cells maintained in static culture. Furthermore, we found that PKC activity is essential for fluid shear stress sensitization of IGF-IR, since a specific inhibitor of PCKzeta function blocked the flow-enhanced IGF-I-activated Akt and ERK phosphorylation. Together, our results suggest that fluid shear stress may regulate IGF-I signaling in osteoblasts in a PKC-zeta-dependent manner.

Osteocyte: a Cellular Basis for Mechanotransduction in Bone

Osteocyte is the most abundant cell type in bone, and the only cell type located inside the mineralized matrix. The striking structural design of bone predicts an important role for osteocytes in determining bone structure and function. Osteocytes are connected with each other via gap junctions and form a three dimensional cellular network in mineralized bone matrix. Recently, it has been shown that osteocytes are not only passive bystanders, but also have an active regulatory role in whole body phosphate and calcium metabolism. Osteocytes are cells which sense mechanical loading in bone. They respond to mechanical stimuli by producing and secreting several bioactive substances including nitric oxide and prostaglandins and thus transmit messages of loading to effector cells, like osteoblasts and osteoclasts. Present data suggest that osteocytes actively inhibit osteoclastic bone resorption. Whenever osteocytes die, this inhibitory effect is turned off and osteoclasts are activated. This mechanism of action could explain targeted remodeling in the region of stress induced microcracks happened. Osteocytes can also modulate osteoblasts function. In conclusion, osteocytes sense mechanical stimuli, transmit signals through cellular network and regulate osteoblast and osteoclast function during bone remodeling.

Wnt/β-Catenin Signaling Is a Normal Physiological Response to Mechanical Loading in Bone

Wnt/β-Catenin Signaling Is a Normal Physiological Response to Mechanical Loading in Bone