Regular ArticlePulsating Fluid Flow Increases Prostaglandin Production by Cultured Chicken Osteocytes—A Cytoskeleton-Dependent Process☆
Abstract
It has been postulated that the transduction of mechanical stress signals to bone cells occurs via loading-dependent flow of interstitial fluid through the lacuno-canalicular network of bone. We have shown earlier that chicken osteocytes release enhanced amounts of prostaglandin E2after 1 h treatment with pulsating fluid flow (PFF, 0.5 ± 0.02 Pa, 5 Hz). Here we study the acute response to PFF on three cell populations derived from fetal chick calvariae, namely periosteal fibroblasts (PF), an osteoblast and osteocyte containing population (OBmix), and osteocytes (OCY), and the involvement of the actin-cytoskeleton in this process. All three cell populations rapidly (OCY: within 5 min, OBmix, PF: within 10 min) increased their release of prostaglandins E2and I2in response to PFF, but the response by OCY was 2–4 times higher than that by OBmix or PF. Disruption of the actin-cytoskeleton by cytochalasin B completely abolished the response. We conclude that osteocytes are more sensitive to fluid shear stress than immature bone cells, and that the actin-cytoskeleton is involved in the response to fluid flow.
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Skeletal muscle and bone interact at the level of mechanical loading through the application of force by muscles to the skeleton. Recently considerable focus has been placed on signaling factors/molecules produced by these two tissues that may act to modulate the function of the other tissue. We sought to determine if muscle and muscle-derived factors were essential to the osteocyte response to loading. Botox® induced muscle paralysis was used to investigate the role of muscle contraction during in vivo tibia compression loading. 5–6 month-old female TOPGAL mice had their right hindlimb muscles surrounding the tibia injected with either BOTOX® or saline. At four days post injections when muscle paralysis peaked, the right tibia was subjected to a single session of in vivo compression loading at ∼2600 με. At 24 h post-load we observed a 2.5-fold increase in β-catenin signaling in osteocytes in the tibias of the saline injected mice, whereas loading of tibias from Botox® injected mice failed to active β-catenin signaling in osteocytes. This suggests that active muscle contraction produces a factor(s) that is necessary for or conditions the osteocyte's ability to respond to load. To further investigate the role of muscle derived factors, MLO-Y4 osteocyte-like cells and a luciferase based β-catenin reporter (TOPflash-MLO-Y4) cell line we developed were treated with conditioned media (CM) from C2C12 myoblasts (MB) and myotubes (MT) and ex vivo contracted Extensor Digitorum Longus (EDL) and Soleus (Sol) muscles under static or loading conditions using fluid flow shear stress (FFSS). 10 % C2C12 myotube CM, but not myoblast or NIH3T3 fibroblast cells CM, induced a rapid activation of the Akt signaling pathway, peaking at 15 min and returning to baseline by 1–2 h under static conditions. FFSS applied to MLO-Y4 cells for 2 h in the presence of 10 % MT-CM resulted in a 6–8 fold increase in pAkt compared to a 3–4 fold increase under control or when exposed to 10 % MB-CM. A similar response was observed in the presence of 10 % EDL-CM, but not in the presence of 10 % Sol-CM. TOPflash-MLO-Y4 cells were treated with 10 ng/ml Wnt3a in the presence or absence of MT-CM. While MT-CM resulted in a 2-fold activation and Wnt3a produced a 10-fold activation, the combination of MT-CM + Wnt3a resulted in a 25-fold activation of β-catenin signaling, implying a synergistic effect of factors in MT-CM with Wnt3a. These data provide clear evidence that specific muscles and myotubes produce factors that alter important signaling pathways involved in the response of osteocytes to mechanical load. These data strongly suggest that beyond mechanical loading there is a molecular coupling of muscle and bone.
Numerical simulations of fluid flow in trabecular–lacunar cavities under cyclic loading
2023, Computers in Biology and MedicineUnder external loading, the fluid shear stress (FSS) in the porous structures of bones, such as trabecular or lacunar–canalicular cavity, can influence the biological response of bone cells. However, few studies have considered both cavities. The present study investigated the characteristics of fluid flow at different scales in cancellous bone in rat femurs, as well as the effects of osteoporosis and loading frequency.
Sprague Dawley rats (3 months old) were divided into normal and osteoporotic groups. A multiscale 3D fluid–solid coupling finite element model considering trabecular system and lacunar–canalicular system was established. Cyclic displacement loadings with frequencies of 1, 2, and 4 Hz were applied.
Results showed that the wall FSS around the adhesion complexes of osteocyte on the canaliculi was higher than that on the osteocyte body. Under the same loading conditions, the wall FSS of the osteoporotic group was smaller than that of the normal group. The fluid velocity and FSS in trabecular pores exhibited a linear relationship with loading frequency. Similarly, the FSS around osteocytes also showed the loading frequency-dependent phenomenon.
The high cadence in movement can effectively increase the FSS level on osteocytes for osteoporotic bone, i.e., expand the space within the bone with physiological load. This study might help in understanding the process of bone remodeling under cyclic loading and provide the fundamental data for the development of strategies for osteoporosis treatment.
Skeletal Aging
2022, Mayo Clinic ProceedingsAging represents the single greatest risk factor for chronic diseases, including osteoporosis, a skeletal fragility syndrome that increases fracture risk. Optimizing bone strength throughout life reduces fracture risk. Factors critical for bone strength include nutrition, physical activity, and vitamin D status, whereas unhealthy lifestyles, illnesses, and certain medications (eg, glucocorticoids) are detrimental. Hormonal status is another important determinant of skeletal health, with sex steroid concentrations, particularly estrogen, having major effects on bone remodeling. Aging exacerbates bone loss in both sexes and results in imbalanced bone resorption relative to formation; it is associated with increased marrow adiposity, osteoblast/osteocyte apoptosis, and accumulation of senescent cells. The mechanisms underlying skeletal aging are as diverse as the factors that determine the strength (and thus fragility) of bone. This review updates our current understanding of the epidemiology, pathophysiology, and treatment of osteoporosis and provides an overview of the underlying hallmark mechanisms that drive skeletal aging.
Osteocytes are embedded dendritic bone cells; by virtue of their position in bone tissue, ability to coordinate bone building osteoblasts and resorbing osteoclasts, and sensitivity to tissue level mechanical loading, they serve as the resident bone mechanosensor. The mechanisms osteocytes use to change mechanical loading into biological signals that drive tissue level changes has been well studied over the last 30 years, however the ways loading parameters are encoded at the cellular level are still not fully understood. Calcium signaling is a first messenger signal exhibited by osteocytes in response to mechanical forces. A body of work interrogating the mechanisms of osteocyte calcium signaling exists and is presently expanding, presenting the opportunity to better understand the relationship between calcium signaling characteristics and tuned osteocyte responses to tissue level strain features (e.g. magnitude, duration, frequency). This review covers the history of osteocyte load induced calcium signaling and highlights potential cellular mechanisms used by osteocytes to turn details about loading parameters into biological events.
Alterations in osteocyte lacunar morphology affect local bone tissue strains
2021, Journal of the Mechanical Behavior of Biomedical MaterialsOsteocytes are capable of remodeling their perilacunar bone matrix, which causes considerable variations in the shape and size of their lacunae. If these variations in lacunar morphology cause changes in the mechanical environment of the osteocytes, in particular local strains, they would subsequently affect bone mechanotransduction, since osteocytes are likely able to directly sense these strains. The purpose of this study is to quantify the effect of alterations in osteocyte lacunar morphology on peri-lacunar bone tissue strains. To this end, we related the actual lacunar shape in fibulae of six young-adult (5-month) and six old (23-month) mice, quantified by high-resolution micro-computed tomography, to microscopic strains, analyzed by micro-finite element modeling. We showed that peak effective strain increased by 12.6% in osteocyte cell bodies (OCYs), 9.6% in pericellular matrix (PCM), and 5.3% in extra cellular matrix (ECM) as the lacunae volume increased from 100-200 μm3 to 500–600 μm3. Lacunae with a larger deviation (>8°) in orientation from the longitudinal axis of the bone are exposed to 8% higher strains in OCYs, 6.5% in PCM, 4.2% in ECM than lacunae with a deviation in orientation below 8°. Moreover, increased lacuna sphericity from 0 to 0.5 to 0.7–1 led to 25%, 23%, and 13% decrease in maximum effective strains in OCYs, PCM, and ECM, respectively. We further showed that due to the presence of smaller and more round lacunae in old mice, local bone tissue strains are on average 5% lower in the vicinity of lacunae and their osteocytes of old mice compared to young. Understanding how changes in lacunar morphology affect the micromechanical environment of osteocytes presents a first step in unraveling their potential role in impaired bone mechanoresponsiveness with e.g. aging.
Adenylyl cyclase 3 regulates osteocyte mechanotransduction and primary cilium
2021, Biochemical and Biophysical Research CommunicationsOsteocytes are accepted as the primary mechanosensing cell in bone, but how they translate mechanical signals into biochemical signals remains unclear. Adenylyl cyclases (AC) are enzymes that catalyze the production of second messenger cyclic adenosine monophosphate (cAMP). Osteocytes display a biphasic, cAMP response to fluid shear with an initial decrease in cAMP concentrations and then an increased concentration after sustained mechanical stimulation. To date, AC6, a calcium-inhibited AC, is the primary isoform studied in bone. Since osteocytes are calcium-responsive mechanosensors, we asked if a calcium-stimulated isoform contributes to mechanotransduction. Using a transcriptomic dataset of MLO-Y4 osteocyte-like cells from the NIH Gene Expression Omnibus, we identified AC3 as the only calcium-stimulated isoform expressed. We show that inhibiting AC3 in MLO-Y4 cells results in decreased cAMP-signaling with fluid shear and increased osteogenic response to fluid flow (measured as Ptgs2 expression) of longer durations, but not shorter. AC3 likely contributes to osteocyte mechanotransduction through a signaling axis involving the primary cilium and GSK3β. We demonstrate that AC3 localizes to the primary cilium, as well as throughout the cytosol and that fluid-flow regulation of primary cilia length is altered with an AC3 knockdown. Regulation of GSK3β is downstream of the primary cilium and cAMP signaling, and with western blots we found that GSK3β inhibition by phosphorylation is increased after fluid shear in AC3 knockdown groups. Our data show that AC3 contributes to osteocyte mechanotransduction and warrants further investigation to pave the way to identifying new therapeutic targets to treat bone disease like osteoporosis.
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Abbreviations used: AA, arachidonic acid; PFF, pulsating fluid flow; PGE2, prostaglandin E2; PGI2, prostaglandin I2; OCY, osteocytes; PF, periosteal fibroblasts; OBmix, osteoblast- and osteocyte-containing population.