Cell Communication (Osteoblasts)

Abstract

Mechanical loading regulates bone physiology; it inhibits bone resorption by osteoclasts and stimulates bone formation by osteoblasts. In this study we examine the mechanotransduction among bone cells, both osteoclasts and osteoblasts from mouse bone marrow, and different cell-lines, such as, RAW 264.7 (osteoclasts) and BMP-2-transfected C2C12 cells (osteoblasts).

In order to understand the mechanotransduction and the mechanisms through which cells convert mechanical stimulation into chemical signals, we stimulate them mechanically using AFM probe indentation.

Mechanical stimulation

Characterization of Mechanotransduction in Osteoblasts

Mechanical loading regulates bone physiology.
Cells of osteoblastic origin are considered to be the main mechanosensors in bone tissue
Atomic Force Microscopy enables precise application of a determined force at a set rate to a single cell.
One of the early responses to mechanical stimuli is an increase in intracellular free calcium concentration [Ca²⁺]_i

The objective of this work was to study the responses of osteoblasts to mechanical stimulations of different magnitude and duration.

Atomic Force Microscopy (AFM) was performed using a Molecular Force Probe-3D system from Asylum Research on top of an Inverted Optical Microscope (Olympus IX-71). This setup allows access to transparent samples optically as well as topographically. The samples were probed using blunt tips. Different injury levels were tested by varying the speed of indentation and the amount of force applied.

Mechanosensitivity of osteoblasts was assessed by AFM indentation. Force-distance curves provided mechanical stimulation. As AFM tip indents the cell (red), until the triggering force is reached. At this point, the tip retracts back (blue). The max-force and the speed of indentation were controlled.

When the maximum force is set, in response to mechanical stimulation, the cells exhibit an increase in [Ca²⁺]_i.Cells were treated with fluo-4-am for Ca labeling. On the right, the fluorescence sequence of a cell indented to penetration with the AFM tip. On the left the average fluorescence profile of the cell, indented at 0s, shows a global sharp increase in fluorescence and a transient decrease. The inset in in the fluorescence response demonstrates raw data before correction for photo-bleaching.

People

Researchers: Monserratt Lopez

Supervisors: Svetlana V Komarova* and Peter Grutter (*Dentistry Department, McGill University)

Collaborators:

- Osama Maria (Dentistry Department, McGill University)

- Jeffrey LeDue, Helene Bourque, Shu Xing, David Oliver (Physics)

Cell-cell Signaling

The Role of Mechanically-Induced ATP Release in Intercellular Communications among Bone Cells: Mathematical Model

Mechanical properties of vertebrate bone are determined by its micro-architecture and the material properties of the bone tissue. Both characteristics are highly responsive to change in mechanical usage. Extracellular ATP is known to mediate mechanotransduction among bone cells. ATP and other nucleotides act on bone cells through P2 receptors that belong to two families: P2X, the ligand-gated ion channels, and P2Y, the G-protein-coupled receptors. Multiple P2X and P2Y receptors are expressed on osteoblasts (P2X2,5,6,7 and P2Y1,2,4,6) and osteoclasts (P2X2,4,7 and P2Y1,2,6), however, it is not clear why so many receptors are needed and what are the specific roles for each of the receptors. The objective of this study was to construct a mathematical model to describe the intercellular signaling by ATP released due to a localized mechanical stimulation or injury.

(***Stella***)

People

Researchers: Shu Xing

Supervisors: Svetlana V Komarova* and Peter Grutter (*Dentistry Department, McGill University)

Collaborators:

- Osama Maria (Dentistry Department, McGill University)

- Monserratt Lopez, Helene Bourque (Physics)

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