scholarly journals PIEZO1 and TRPV4, which Are Distinct Mechano-Sensors in the Osteoblastic MC3T3-E1 Cells, Modify Cell-Proliferation

2019 ◽  
Vol 20 (19) ◽  
pp. 4960 ◽  
Author(s):  
Maki Yoneda ◽  
Hiroka Suzuki ◽  
Noriyuki Hatano ◽  
Sayumi Nakano ◽  
Yukiko Muraki ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Shear Stress ◽  
Fluid Flow ◽  
Mrna Expression ◽  
Mechanical Loading ◽  
Mechanical Stimulation ◽  
Mechanical Stimulus ◽  
Induced Response ◽  
Loading And Unloading ◽  
Mechanical Sensors

Mechanical-loading and unloading can modify osteoblast functioning. Ca2+ signaling is one of the earliest events in osteoblasts to induce a mechanical stimulus, thereby demonstrating the importance of the underlying mechanical sensors for the sensation. Here, we examined the mechano-sensitive channels PIEZO1 and TRPV4 were involved in the process of mechano-sensation in the osteoblastic MC3T3-E1 cells. The analysis of mRNA expression revealed a high expression of Piezo1 and Trpv4 in these cells. We also found that a PIEZO1 agonist, Yoda1, induced Ca2+ response and activated cationic currents in these cells. Ca2+ response was elicited when mechanical stimulation (MS), with shear stress, was induced by fluid flow in the MC3T3-E1 cells. Gene knockdown of Piezo1 in the MC3T3-E1 cells, by transfection with siPiezo1, inhibited the Yoda1-induced response, but failed to inhibit the MS-induced response. When MC3T3-E1 cells were transfected with siTrpv4, the MS-induced response was abolished and Yoda1 response was attenuated. Moreover, the MS-induced response was inhibited by a TRPV4 antagonist HC-067047 (HC). Yoda1 response was also inhibited by HC in MC3T3-E1 cells and HEK cells, expressing both PIEZO1 and TRPV4. Meanwhile, the activation of PIEZO1 and TRPV4 reduced the proliferation of MC3T3-E1, which was reversed by knockdown of PIEZO1, and TRPV4, respectively. In conclusion, TRPV4 and PIEZO1 are distinct mechano-sensors in the MC3T3-E1 cells. However, PIEZO1 and TRPV4 modify the proliferation of these cells, implying that PIEZO1 and TRPV4 may be functional in the osteoblastic mechano-transduction. Notably, it is also found that Yoda1 can induce TRPV4-dependent Ca2+ response, when both PIEZO1 and TRPV4 are highly expressed.

scholarly journals Effect of mechanical loading on insulin-like growth factor-I gene expression in rat tibia

2007 ◽  
Vol 192 (1) ◽  
pp. 131-140 ◽  
Author(s):  
Christianne M A Reijnders ◽  
Nathalie Bravenboer ◽  
Annechien M Tromp ◽  
Marinus A Blankenstein ◽  
Paul Lips
Keyword(s):  
Bone Formation ◽  
Mrna Expression ◽  
Mechanical Loading ◽  
Mechanical Stimulation ◽  
Molecular Mechanisms ◽  
Bone Marrow Cells ◽  
Mechanical Stimuli ◽  
Igf I ◽  
Female Wistar Rats ◽  
Tibia Shaft

Mechanical loading plays an essential role in maintaining skeletal integrity. Mechanical stimulation leads to increased bone formation. However, the cellular and molecular mechanisms that are involved in the translation of mechanical stimuli into bone formation, are not completely understood. Growth factors and osteocytes, which act as mechanosensors, play a key role during the bone formation after mechanical stimulation. The aim of this study was to characterize the role of IGF-I in the translation of mechanical stimuli into bone formation locally in rat tibiae. Fifteen female Wistar rats were randomly assigned to three groups (n = 5): load, sham-loaded, and control. The four-point bending model of Forwood and Turner was used to induce a single period of mechanical loading on the tibia shaft. The effects of mechanical loading on IGF-I mRNA expression were determined with non-radioactive in situ hybridization on decalcified tibiae sections, 6 h after the loading session. Endogenous IGF-I mRNA was expressed in trabecular and cortical osteoblasts, some trabecular and sub-endocortical osteocytes, intracortical endothelial cells of blood vessels, and periosteum. Megakaryocytes, macrophages, and myeloid cells also expressed IGF-I mRNA. In the growth plate, IGF-I mRNA was located in proliferative and hypertrophic chondrocytes. Mechanical loading did not affect the IGF-I mRNA expression in osteoblasts, bone marrow cells, and chondrocytes, but the osteocytes at the endosteal side of the shaft showed a twofold increase of IGF-I mRNA expression. The proportion of IGF-I mRNA positive osteocytes in loaded tibiae was 29.3 ± 12.9% (mean ± s.d.; n = 5), whereas sham-loaded and contra-lateral control tibiae exhibited 16.7 ± 4.4% (n = 5) and 14.7 ± 4.2% (n = 10) respectively (P < 0.05). Lamellar bone formation after a single mechanical loading session was observed at the endosteal side of the shaft. In conclusion, a single loading session results in a twofold up-regulation of IGF-I mRNA synthesis in osteocytes which are present in multiple layers extending into the cortical bone of mechanically stimulated tibia shaft 6 h after loading. This supports the hypothesis that IGF-I, which is located in osteocytes, is involved in the translation of mechanical stimuli into bone formation.

scholarly journals Purinergic Signaling Mediates PTH and Fluid Flow-Induced Osteoblast Proliferation

2021 ◽  
Vol 2021 ◽  
pp. 1-8
Author(s):  
Yanghui Xing ◽  
Liang Song ◽  
Yingying Zhang ◽  
Tengyu Zhang ◽  
Jian Li ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Fluid Flow ◽  
Mechanical Stimulation ◽  
Purinergic Receptors ◽  
Bone Growth ◽  
Purinergic Signaling ◽  
Atp Release ◽  
Osteoblast Proliferation ◽  
Creb Phosphorylation ◽  
Mechanical Signals

Both parathyroid hormone (PTH) and mechanical signals are able to regulate bone growth and regeneration. They also can work synergistically to regulate osteoblast proliferation, but little is known about the mechanisms how PTH and mechanical signals interact with each other during this process. In this study, we investigated responses of MC3T3-E1 osteoblasts to PTH and oscillatory fluid flow. We found that osteoblasts are more sensitive to mechanical signals in the presence of PTH according to ERK1/2 phosphorylation, ATP release, CREB phosphorylation, and cell proliferation. PTH may also reduce the osteoblast refractory period after desensitization due to mechanical signals. We further found that the synergistic responses of osteoblasts to fluid flow or ATP with PTH had similar patterns, suggesting that synergy between fluid flow and PTH may be through the ATP pathway. After we inhibited ATP effects using apyrase in osteoblasts, their synergistic responses to mechanical stimulation and PTH were also inhibited. Additionally, knocking down P2Y2 purinergic receptors can significantly attenuate osteoblast synergistic responses to mechanical stimulation and PTH in terms of ERK1/2 phosphorylation, CREB phosphorylation, and cell proliferation. Thus, our results suggest that PTH enhances mechanosensitivity of osteoblasts via a mechanism involving ATP and P2Y2 purinergic receptors.

Temporal gradients in shear stimulate osteoblastic proliferation via ERK1/2 and retinoblastoma protein

2002 ◽  
Vol 283 (2) ◽  
pp. E383-E389 ◽  
Author(s):  
Guang-Liang Jiang ◽  
Charles R. White ◽  
Hazel Y. Stevens ◽  
John A. Frangos
Keyword(s):  
Cell Proliferation ◽  
Fluid Flow ◽  
Steady Flow ◽  
Mechanical Loading ◽  
Pulsatile Flow ◽  
Venous Pressure ◽  
Retinoblastoma Protein ◽  
S Phase ◽  
Osteoblastic Cells ◽  
Fluid Shear

Bone cells are subject to interstitial fluid flow (IFF) driven by venous pressure and mechanical loading. Rapid dynamic changes in mechanical loading cause transient gradients in IFF. The effects of pulsatile flow (temporal gradients in fluid shear) on rat UMR106 cells and rat primary osteoblastic cells were studied. Pulsatile flow induced a 95% increase in S-phase UMR106 cells compared with static controls. In contrast, ramped steady flow stimulated only a 3% increase. Similar patterns of S-phase induction were also observed in rat primary osteoblastic cells. Pulsatile flow significantly increased relative UMR106 cell number by 37 and 62% at 1.5 and 24 h, respectively. Pulsatile flow also significantly increased extracellular signal-regulated kinase (ERK1/2) phosphorylation by 418%, whereas ramped steady flow reduced ERK1/2 activation to 17% of control. Correspondingly, retinoblastoma protein was significantly phosphorylated by pulsatile fluid flow. Inhibition of mitogen-activated protein (MAP)/ERK kinase (MEK)1/2 by U0126 (a specific MEK1/2 inhibitor) reduced shear-induced ERK1/2 phosphorylation and cell proliferation. These findings suggest that temporal gradients in fluid shear stress are potent stimuli of bone cell proliferation.

Flow-induced calcium oscillations in rat osteoblasts are age, loading frequency, and shear stress dependent

2001 ◽  
Vol 281 (5) ◽  
pp. C1635-C1641 ◽  
Author(s):  
Seth W. Donahue ◽  
Christopher R. Jacobs ◽  
Henry J. Donahue
Keyword(s):  
Shear Stress ◽  
Fluid Flow ◽  
Mechanical Loading ◽  
Cell Metabolism ◽  
Bone Cell ◽  
Bone Adaptation ◽  
Calcium Oscillations ◽  
Loading Frequency ◽  
Spontaneous Oscillations ◽  
Stress Dependent

Bone adaptation to mechanical loading is dependent on age and the frequency and magnitude of loading. It is believed that load-induced fluid flow in the porous spaces of bone is an important signal that influences bone cell metabolism and bone adaptation. We used fluid flow-induced shear stress as a mechanical stimulus to study intracellular calcium (Ca[Formula: see text]) signaling in rat osteoblastic cells (ROB) isolated from young, mature, and old animals. Fluid flow produced higher magnitude and more abundant [Ca2+]ioscillations than spontaneous oscillations, suggesting that flow-induced Ca[Formula: see text] signaling encodes a different cellular message than spontaneous oscillations. ROB from old rats showed less basal [Ca2+]i activity and were less responsive to fluid flow. Cells were more responsive to 0.2 Hz than to 1 or 2 Hz and to 2 Pa than to 1 Pa. These data suggest that the frequency and magnitude of mechanical loading may be encoded by the percentage of cells displaying [Ca2+]ioscillations but that the ability to transduce this information may be altered with age.

scholarly journals A fluid–structure interaction model to characterize bone cell stimulation in parallel-plate flow chamber systems

2013 ◽  
Vol 10 (81) ◽  
pp. 20120900 ◽  
Author(s):  
T. J. Vaughan ◽  
M. G. Haugh ◽  
L. M. McNamara
Keyword(s):  
Shear Stress ◽  
Fluid Flow ◽  
Parallel Plate ◽  
Bone Cells ◽  
Mechanical Stimulus ◽  
Flow Chamber ◽  
Parallel Plate Flow Chamber ◽  
Structure Interaction

Bone continuously adapts its internal structure to accommodate the functional demands of its mechanical environment and strain-induced flow of interstitial fluid is believed to be the primary mediator of mechanical stimuli to bone cells in vivo. In vitro investigations have shown that bone cells produce important biochemical signals in response to fluid flow applied using parallel-plate flow chamber (PPFC) systems. However, the exact mechanical stimulus experienced by the cells within these systems remains unclear. To fully understand this behaviour represents a most challenging multi-physics problem involving the interaction between deformable cellular structures and adjacent fluid flows. In this study, we use a fluid–structure interaction computational approach to investigate the nature of the mechanical stimulus being applied to a single osteoblast cell under fluid flow within a PPFC system. The analysis decouples the contribution of pressure and shear stress on cellular deformation and for the first time highlights that cell strain under flow is dominated by the pressure in the PPFC system rather than the applied shear stress. Furthermore, it was found that strains imparted on the cell membrane were relatively low whereas significant strain amplification occurred at the cell–substrate interface. These results suggest that strain transfer through focal attachments at the base of the cell are the primary mediators of mechanical signals to the cell under flow in a PPFC system. Such information is vital in order to correctly interpret biological responses of bone cells under in vitro stimulation and elucidate the mechanisms associated with mechanotransduction in vivo .

Evaluation of different culture techniques of osteoblasts on 3D scaffolds

2010 ◽  
Vol 5 (4) ◽  
pp. 456-465 ◽  
Author(s):  
Ying-ying Wu ◽  
Yu Ban ◽  
Ning Geng ◽  
Yong-yue Wang ◽  
Xiao-guang Liu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Shear Stress ◽  
Fluid Flow ◽  
Fluid Shear Stress ◽  
Static Condition ◽  
Fluid Shear ◽  
Perfusion Fluid ◽  
Culture Techniques ◽  
Cells Cultured ◽  
Continuous Perfusion

AbstractBones adjust their structure to withstand the mechanical demands they experience. It is suggested that flow-derived shear stress may be the most significant and primary mediator of mechanical stimulation. In this study, we designed and fabricated a fluid flow cell culture system that can load shear stress onto cells cultured on 3D scaffolds. We evaluated the effect of different culture techniques, namely, (1) continuous perfusion fluid flow, (2) intermittent perfusion fluid flow, and (3) static condition, on the proliferation of osteoblasts seeded on partially deproteinized bones. The flow rate was set at 1 ml/min for all the cells cultured using flow perfusion and the experiment was conducted for 12 days. Scanning electron microscopy analysis indicated an increase in cell proliferation for scaffolds subjected to fluid shear stress. In addition, the long axes of these cells lengthened along the flowing fluid direction. Continuous perfusion significantly enhanced cell proliferation compared to either intermittent perfusion or static condition. All the results demonstrated that fluid shear stress is able to enhance the proliferation of cells and change the form of cells.

Fluid-Structure Interaction Modelling Predicts That Strain Transfer Through Focal Attachments of Osteoblast Cells are the Primary Mediators of Mechanical Stimulation Under Fluid Flow

2013 ◽  
Author(s):  
T. J. Vaughan ◽  
M. G. Haugh ◽  
L. M. McNamara
Keyword(s):  
Fluid Flow ◽  
Mechanical Stimulation ◽  
Interstitial Fluid ◽  
Bone Cells ◽  
Mechanical Stimulus ◽  
Mechanical Stimuli ◽  
Interstitial Fluid Flow ◽  
Interaction Modelling

Bone continuously adapts its internal structure to accommodate the functional demands of its mechanical environment. It has been proposed that indirect strain-induced flow of interstitial fluid surrounding bone cells may be the primary mediator of mechanical stimuli in-vivo [1]. Due to the practical difficulties in ascertaining whether interstitial fluid flow is indeed the primary mediator of mechanical stimuli in the in vivo environment, much of the evidence supporting this theory has been established through in vitro investigations that have observed cellular activity in response to fluid flow imposed by perfusion chambers [2]. While such in vitro experiments have identified key mechanisms involved in the mechanotransduction process, the exact mechanical stimulus being imparted to cells within a monolayer is unknown [3]. Furthermoreit is not clear whether the mechanical stimulation is comparable between different experimental systems or, more importantly, is representative of physiological loading conditions experienced by bone cells in vivo.

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