Original Full Length ArticleCalcium response in osteocytic networks under steady and oscillatory fluid flow
Highlights
► Intracellular calcium signaling of osteocytes is dependent on the profiles of mechanical stimulation. ► Osteocytic networks are more responsive to steady fluid flow than to oscillatory fluid flow. ► Responsive percentages of osteocytes have no significant difference under steady and oscillatory flows. ► Spatiotemporal characters of the calcium signaling transients in osteocytes vary significantly under different flow profiles. ► Endoplasmic reticulum calcium store, extracellular calcium source, and ATP pathways are critical for calcium signaling of osteocytes.
Introduction
Osteocyte networks in bone tissue can sense various mechanical stimuli generated by physical activities and act as an coordinator in the bone remodeling process by regulating both osteoclast and osteoblast activities [1], [2], [3]. The heterogeneous deformation across bone tissue induced by mechanical loading creates fluid pressure gradients in the lacunar–canalicular system (LCS), where the osteocytes reside in. This pressure gradient generates fluid flow across the LCS, which can induce shear stresses up to 5 Pa upon the osteocyte membrane and processes [4], [5], [6]. Fluid flow is regarded as an essential mechanical stimulation in the mechanobiology of osteocytes. Due to the versatile nature of human daily activities, the profiles of mechanical loading on bones are complex. Therefore the fluid flow upon osteocytes could be a combination of various patterns, including oscillating fluid flow and unidirectional flow. The oscillating flow is often correlated to cyclic movement of the skeletal system such as walking and running. The unidirectional steady flow can represent a physiological but unusual mechanical stimulus, e.g., a posture change of the human body from sitting to standing [7]. Previous studies showed that the mechanically adaptive responses of bone are dominated by “abnormal” strain changes under unusual loading rather than by the numerous cycles of “normal” loadings [8]. Therefore osteocytes respond more actively to new patterns or types of mechanical loadings than to ordinary mechanical stimulation. Osteocytes are known to be able to differentiate the temporal pattern of flow stimulation with distinct biochemical activities to regulate bone remodeling [1], [9], [10].
Little is known about the mechanobiology effects of different fluid flow profiles on osteocytic networks. To date there has only been one study that compared the responses of osteoblastic cells under the stimuli of steady and oscillatory flows [7]. Significant difference in calcium signaling was revealed in osteoblasts under two flow patterns. The oscillating flow, assumed to be the major stimulation on bone cells, was a much less potent stimulator than the steady flow on osteoblastic cells. We recently found that osteocytic networks, as the dedicated mechanical sensors in bone, are much more sensitive to fluid shear stress than the osteoblasts [11]. The intracellular calcium ([Ca2 +]i) signaling of osteocytes showed significantly different spatiotemporal characteristics with osteoblasts. Unlike the osteoblasts on bone surfaces, osteocyte networks are directly situated inside the LCS. Therefore it is important to understand and compare the responses of osteocytes under different flow patterns, which are associated with mechanical loading profiles on bone and further affect the bone remodeling process.
Intracellular calcium [Ca2 +]i signaling is one of the earliest responses in bone cells under mechanical stimulation that initiates a number of essential downstream signaling pathways, e.g., ATP and PGE2 release, and is typically observed to oscillate dramatically within seconds after mechanical stimulation [4], [12], [13], [14], [15]. This ubiquitous signaling molecule plays a critical role in a wide variety of physiological processes in bone cells including proliferation, differentiation, and cell responses to mechanical stimuli [16], [17], [18]. The calcium wave propagation across neighboring cells acts as an effective mechanism for the cell–cell communication in bone cell networks [14], [19], [20]. The characters of [Ca2 +]i signaling of bone cells are shown to be dependent on the mechanical loading profiles [21]. We have shown that osteocytes can release multiple spike-like [Ca2 +]i peaks under unidirectional fluid flow, up to 17 [Ca2 +]i peaks during a 9-minute flow stimulation. The spatiotemporal properties of the [Ca2 +]i transients are also found to be dependent on the magnitude of fluid flow. Therefore [Ca2 +]i oscillations of osteocytes can be employed as a sensitive signaling pathway to represent the cell responses with different external mechanical stimuli.
The fluid flow induced elevation of cytosolic calcium comes mainly from two sources: intracellular stores (e.g., endoplasmic reticulum, ER) and the extracellular environment [13], [22]. The release of ER calcium store is mainly regulated by the inositol trisphosphate (IP3) pathway, which can be initiated by the activation of purinergic receptors on the cell membrane [16], [17]. After the cytosolic calcium concentration is elevated to a critical level by intra/extracellular sources, the depleted intracellular calcium stores tend to recover their calcium reservation to original levels and become ready for the next release of calcium [16], [23]. When a bone cell is under fluid flow induced shear stress, the activation of gap junction hemichannels (connexin 43) induces ATP efflux from the cytosol to the pericellular environment [24]. Extracellular ATP can elicit a significant [Ca2 +]i response by binding to the purinergic membrane receptors [25]. Fluid shear stress can also prompt the induction of COX-2 protein and further PGE2 release in osteoblast-like cells [41]. It was previously reported that fluid shear stress can elicit nitric oxide production in osteocytes and osteoblasts accompanied by an increased expression of nitric oxide synthase (NOS) [26], [27]. Nitric oxide modulates [Ca2 +]i signaling via a cyclic guanosine monophosphate (cGMP) dependent pathway [28] or nitrosylation of proteins [29]. Moreover, nitric oxide could directly contribute to the [Ca2 +]i release via triggering of an influx pathway that is, in part, responsible for the refilling of internal calcium stores [30].
Most studies of bone cell mechanotransduction under oscillatory flow used monolayer osteoblastic cells [7], [31], [32], [33], [34]. The osteocytes, unlike the osteoblasts residing on the bone surface, are embedded inside the mineralized bone tissue with a regular pattern. The intercellular spacing and the topology of connection dendrites of osteocytes are relatively regular across the tissue. The osteocytes are connected into an extensive network through the gap junctions on processes. Gap junctions are membrane-spanning channels, where each pair of connexons (i.e. hemichannels) forms a cylinder with a pore in the center through which small molecules (< 1 kDa) can pass from one cell to another [35]. It is widely accepted that this intercellular connection plays a significant role in coordinating bone cell network activities. Messenger molecules mentioned previously, such as IP3 and calcium, can directly transfer between the neighboring cells through gap junctions and thereby mediate propagation of [Ca2 +]i signaling in bone cells [20], [36]. The [Ca2 +]i signaling of osteocytes within a controlled cell network connected by gap junctions under different flow stimuli has yet to be obtained experimentally.
In this study, we hypothesize that the [Ca2 +]i signaling of osteocytic networks is dependent on the profiles of fluid flow. [Ca2 +]i response was employed as the primary outcome variable to compare the physiological responses of in vitro osteocytic networks under steady and oscillatory fluid flows. The roles of several essential [Ca2 +]i signaling pathways of osteocytic networks were also investigated and compared under two different flow profiles using pharmacological inhibitors.
Section snippets
Chemicals
Fetal bovine serum (FBS), calf serum (CS), and penicillin/streptomycin (P/S) were obtained from Hyclone Laboratories Inc. (Logan, UT). Trypsin/EDTA, octadecanethiol, dimethyl sulfoxide (DMSO), fibronectin, 18α-glycyrrhetinic acid (18α-GA), suramin, and thapsigargin (TG) were obtained from Sigma-Aldrich Co. (St. Louis, MO). Minimum essential alpha medium (α-MEM), calcium free Dulbecco's modified eagle medium (DMEM), and calcium-free Hank's balanced salt solution (HBSS) were obtained from
Results
A set of typical [Ca2 +]i transients of MLO-Y4 cells under oscillatory and steady fluid flow stimulation is shown in Fig. 3. Both flow patterns induced prominent [Ca2 +]i oscillations in MLO-Y4 cells. Under oscillating flow stimulation, the cell released a [Ca2 +]i peak at the onset of flow and a few weaker peaks afterwards with reduced magnitudes. Under steady flow stimulation, MLO-Y4 cells tended to release repetitive and spike-like [Ca2 +]i peaks with no attenuation in magnitude. A single cell
Discussion
In this study, the calcium signaling of osteocytic networks was investigated and compared under the stimulation of steady or oscillatory fluid flow. The results clearly demonstrated that the oscillatory flow is significantly less potent than the steady flow to osteocytes. Osteocytes tend to release more frequent [Ca2 +]i peaks with higher magnitudes under the stimulation of steady fluid flow than under the oscillatory flow. Previous studies on monolayer osteoblastic cells also showed that in
Conclusion
In summary, this study proved that fluid flow can induce robust intracellular calcium responses in osteocytic networks. The responsive percentage of osteocytes is not dependent on the flow patterns, but the spatiotemporal characteristics of calcium transients vary significantly under steady and oscillatory flows. Osteocytes can release more intracellular calcium peaks with higher magnitudes under steady flow than under oscillatory flow, i.e., steady flow is more stimulative to osteocytes than
Funding sources
NIH grants R21 AR052417, R01 AR052461, and RC1 AR058453 (XEG).
Disclosures/conflicts of interest
The authors have nothing to disclose, and have no conflicts of interest.
Acknowledgments
We thank Dr. L. Bonewald for her generous gift of MLO-Y4 cells.
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