Andrew D. Baik

In situ intracellular calcium oscillations in osteocytes in intact mouse long bones under dynamic mechanical loading

Osteocytes have been hypothesized to be the major mechanosensors in bone. How in situ osteocytes respond to mechanical stimuli is still unclear because of technical difficulties. In vitro studies have shown that osteocytes exhibited unique calcium (Ca2+) oscillations to fluid shear. However, whether this mechanotransduction phenomenon holds for in situ osteocytes embedded within a mineralized bone matrix under dynamic loading remains unknown. Using a novel synchronized loading/imaging technique, we successfully visualized in real time and quantified Ca2+ responses in osteocytes and bone surface cells in situ under controlled dynamic loading on intact mouse tibia. The resultant fluid‐induced shear stress on the osteocyte in the lacunocanalicular system (LCS) was also quantified. Osteocytes, but not surface cells, displayed repetitive Ca2+ spikes in response to dynamic loading, with spike frequency and magnitude dependent on load magnitude, tissue strain, and shear stress in the LCS. The Ca2+ oscillations were significantly reduced by endoplasmic reticulum (ER) depletion and P2 purinergic receptor (P2R)/phospholipase C (PLC) inhibition. This study provides direct evidence that osteocytes respond to in situ mechanical loading by Ca2+ oscillations, which are dependent on the P2R/PLC/inositol trisphosphate/ER pathway. This study develops a novel approach in skeletal mechanobiology and also advances our fundamental knowledge of bone mechanotransduction.—Jing, D., Baik, A. D., Lu, X. L., Zhou, B., Lai, X., Wang, L., Luo, E., Guo, X. E. In situ intracellular calcium oscillations in osteocytes in intact mouse long bones under dynamic mechanical loading. FASEB J. 28, 28–1582 (1592). www.fasebj.org

Spreading area and shape regulate apoptosis and differentiation of osteoblasts

The in vivo observations have indicated that at the remodeling sites of bone, the spreading area or shape of preosteoblasts is confined by the mineralized matrix. But it remains unknown whether this spreading confinement regulates the differentiation or apoptosis of osteoblasts. In the present study, osteoblast-like cells (MC3T3-E1) were seeded on micropatterned islands with different area and shape. The expression of three osteogenic differentiation markers was measured by immunofluorescence staining and apoptotic cells were detected using a terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labelling assay kit. The membrane fluorescence staining results showed that the actual spreading area of micropatterned osteoblasts coincided with the designed value. When the area of a micropatterned cell was confined as 314 or 615 µm(2), which was lower than that of freely spreading osteoblasts, the circular shape promoted the expression of osteogenic differentiation markers and the percentage of apoptotic osteoblasts compared with the branched shape. This shape-regulated differentiation and apoptosis of osteoblasts with confined spreading area were abolished when actin polymerization was inhibited by cytochalasin D. The present study gives an insight into the roles of spreading morphology on osteoblastic differentiation and apoptosis.

Simultaneous tracking of 3D actin and microtubule strains in individual MLO-Y4 osteocytes under oscillatory flow

Osteocytes in vivo experience complex fluid shear flow patterns to activate mechanotransduction pathways. The actin and microtubule (MT) cytoskeletons have been shown to play an important role in the osteocytes biochemical response to fluid shear loading. The dynamic nature of physiologically relevant fluid flow profiles (i.e., 1Hz oscillatory flow) impedes the ability to image and study both actin and MT cytoskeletons simultaneously in the same cell with high spatiotemporal resolution. To overcome these limitations, a multi-channel quasi-3D microscopy technique was developed to track the actin and MT networks simultaneously under steady and oscillatory flow. Cells displayed high intercellular variability and intracellular cytoskeletal variability in strain profiles. Shear Exz was the predominant strain in both steady and oscillatory flows in the form of viscoelastic creep and elastic oscillations, respectively. Dramatic differences were seen in oscillatory flow, however. The actin strains displayed an oscillatory strain profile more often than the MT networks in all the strains tested and had a higher peak-to-trough strain magnitude. Taken together, the actin networks are the more responsive cytoskeletal networks in osteocytes under oscillatory flow and may play a bigger role in mechanotransduction pathway activation and regulation.

A noninvasive approach to determine viscoelastic properties of an individual adherent cell under fluid flow

Mechanical properties of cells play an important role in their interaction with the extracellular matrix as well as the mechanotransduction process. Several in vitro techniques have been developed to determine the mechanical properties of cells, but none of them can measure the viscoelastic properties of an individual adherent cell in fluid flow non-invasively. In this study, techniques of fluid-structure interaction (FSI) finite element method and quasi-3-dimensional (quasi-3D) cell microscopy were innovatively applied to the frequently used flow chamber experiment, where an adherent cell was subjected to fluid flow. A new non-invasive approach, with cells at close to physiological conditions, was established to determine the viscoelastic properties of individual cells. The results showed an instantaneous modulus of osteocytes of 0.49 ± 0.11 kPa, an equilibrium modulus of 0.31 ± 0.044 kPa, and an apparent viscosity coefficient of 4.07 ± 1.23 kPas. This new quantitative approach not only provides an excellent means to measure cell mechanical properties, but also may help to elucidate the mechanotransduction mechanisms for a variety of cells under fluid flow stimulation.

Combined finite element (FE) modeling and fluid shear experiment to determine the viscoelastic material properties of osteocytes

Osteocytes exhibit solid-like viscoelastic behavior in response to mechanical stresses. The goal of this study was to determine the viscoelastic properties of osteocytes using a combined finite element analysis (FE) modeling and experimental approach. The three-dimensional (3D) cell shape of the osteocyte under fluid flow was reconstructed using using a novel pseudo-3D microscopy technique. The cell shape was input into an ADINA fluid-structure FE software. The osteocyte was modeled using a linear and incompressible viscoelastic standard solid with a finite strain. The viscoelastic material parameters were determined by matching the predicted cell surface displacements with those measured experimentally. The instantaneous modulus of an osteocyte was 0.81 kPa and the equilibrium modulus was 0.11 kPa. The apparent viscosity were 0.85 kPa-s. The material properties measured in this study are comparable to the cell material properties reported in previous studies. This fluid-structure interaction cell model based on individual cell geometry may provide a novel technique to measure the viscoelastic properties of individual cells, as well as potential mechanisms of mechanical signal transduction.

Pseudo-3D Visualization of Cytoskeletal and Whole-Cell Deformation of MLO-Y4 Osteocytes Under Flow

Osteocytes respond to fluid shear loading by activating various biochemical pathways, mediating a dynamic process of bone formation and resorption. Whole-cell [1] and intracellular deformation [2] may be able to directly activate and modulate relevant biochemical pathways. Most studies on cell deformation have focused only on cell deformation in the plane parallel to the substrate surface. However, height-dependent cell deformation has not been well characterized even though it may contribute greatly to mechanotransduction mechanisms. Traditional techniques to obtain this additional height information of a cell-body include confocal and deconvolution microscopy, which require scanning a z-stack of the cell. However, this inherently limits the timescale under which the deformational information can be visualized. To further investigate this behavior at a high temporal resolution, we propose using a “pseudo-3D” microscopy method to better characterize osteocyte cell behavior. In this study, we present a novel technique that is able to image a single cell simultaneously in two orthogonal planes to obtain real-time images of cell at a millisecond timescale. The objectives of this study were to: (1) visualize actin or microtubule networks with the plasma membrane in two orthogonal planes simultaneously under fluid shear; (2) map out the deformations using digital image correlation; and (3) compare the depth-directional deformation of actin and microtubule networks of osteocytes.Copyright

Intercellular Calcium Wave Propagation in Linear and Circuit-Like Bone Cell Networks

Intracellular calcium ([Ca2+]i) transients in response to mechanical stimulation can be propagated to neighboring cells in bone cell networks, which provides an essential mechanism for cell-cell communication in bone. Transfer of intracellular second messengers (e.g., IP3 and Ca2+) through gap junction pores and the diffusion of extracellular ATP to activate membrane receptors have long been conjectured as the two major pathways for intercellular Ca2+ wave propagation [1]. In this study, by comparing the calcium wave in open-end linear and looped circuit-like cell chains, the roles of gap junction intercellular communication (GJIC) and extracellular ATP diffusion in calcium wave propagation in bone cell networks were examined. The results were further confirmed with pathway-inhibitor studies performed on linear cell chains.Copyright

Calcium Signaling in Bone Cell Networks Induced by Fluid Flow

Mechanical stimuli such as fluid flow can induce robust multiple intracellular calcium ([Ca2+]i) peaks in connected bone cell networks [1]. This fluid flow induced oscillation of [Ca2+]i can come from two sources: intracellular Ca2+ stores (e.g., endoplasmic reticulum, ER) and the extracellular environment. Moreover, [Ca2+]i signaling is mediated by various molecular pathways, such as IP3, ATP, PGE2, and NO. Osteocytes are believed to comprise a sensory network in bone tissue that monitors in vivo mechanical loading and triggers appropriate adaptive responses from osteoblasts and osteoclasts [2]. It is also well recognized that osteoblasts, the cells responsible for bone formation, can directly sense and respond to mechanical stimulation (e.g., fluid flow). In the present study, two types of cell networks were constructed in vitro with osteocyte-like and osteoblast-like cells, respectively, by using microcontact printing and self assembled monolayer (SAM) technologies. The calcium responses of the two types of cell networks to fluid flow were recorded, quantitatively analyzed, and compared. Then we examined how the [Ca2+]i response in the osteocyte cell network was influenced by gap junctions, intra/extracellular calcium sources, and other various molecular pathways.Copyright

A Semi-3D Real-Time Imaging Technique for Measuring Bone Cell Deformation Under Fluid Flow

Bone cells respond to fluid shear loading by activating various biochemical pathways, mediating a dynamic process of bone formation and resorption. The whole-cell volume dilatation [1] and regional deformation of intracellular structures [2] may be able to directly activate and modulate relevant biochemical pathways. Therefore, understanding how bone cells deform under fluid flow can help elucidate the fundamental mechanisms by which mechanical stimuli are able to initiate biochemical responses. Most studies on cell deformation have focused only on cell deformation in the plane parallel to the substrate surface. Height-dependent cell deformation has not been well characterized even though it may contribute greatly to mechanotransduction mechanisms. Traditional techniques to obtain this additional height information of a cell-body, such as confocal and deconvolution microscopy, are inherently limited by the timescale under which the deformational information can be visualized. Previous studies have investigated cell adhesion to substrate under flow using a single view side-view imaging technique [3, 4]. In this study, we present a novel technique that is able to image a single cell simultaneously in two orthogonal planes to obtain real-time images of a cell at a millisecond timescale. Thus, the objectives of this study were to: (1) develop an imaging technique to visualize the depth-directional information of a cell simultaneously with the traditional 2D view; (2) map out the strain fields of the cell by image analysis; and (3) investigate the viscoelastic behavior of osteoblasts under steady fluid flow.Copyright