Loading-induced Fluid and Disuse Bone Loss

Principal Investigator: Christopher R. Jacobs, PhD

Investigators: Henry J. Donahue, PhD and R. Lane Smith, PhD

Project Category: Bone & Joint, Spinal Cord Injury, and Stroke - 2005

Objective: Microgravity conditions result in a dramatic reduction of normal skeletal loading due to decreased weight bearing leading to rapid bone loss. Also, disuse bone loss occurs in veteran patients with spinal cord injury and stroke. A variety of countermeasures flown to date have not been effective in combating this bone loss. Interestingly, exposure to microgravity has been shown to result in decreased numbers of osteoblasts, but has little effect on osteoclasts. Cellular biophysical signals due to loading have been shown to regulate osteoblastic bone formation in vitro, however it is not known if such signals can regulate the formation of new osteoblasts. The population of osteoblasts is continually replenished by marrow stromal cells (MSCs), a population of multipotential cells that give rise to several mesenchymal phenotypes. Our central hypothesis is that loading induced fluid flow may regulate the proliferation and differentiation of MSCs down the osteogenic pathway. In this three-year project we will examine the effect of loading-induced oscillatory fluid flow on MSCs in vitro.

Research Plan: This project is divided into three specific aims:

1. Identify the intracellular calcium signalling pathway activated by loading-induced oscillatory fluid flow. hMSC cells will be cultured in vitro and placed in our oscillatory flow system. Cells exposed to fluid flow will be monitored for increased intracellular calcium concentration with a real-time microspectrofluorometer. The flow profile will be sinusoidal superposed with a low level of constant wash-through perfusion to provide fresh media. A pharmacological blocking strategy will be employed to characterize the calcium increases in terms of its source (intracellular or extracellular), second messengers involved (eg IP3), and the involvement of stretch activated cation channels.

2. Determine the effects of exposure to loading-induced fluid flow on the proliferation rate and the expression of markers of differentiation of hMSCs. hMSCs will be exposed to two hours of oscillatory fluid flow with continuous wash-out perfusion and the number of viable cells and proliferation rate determined relative to non-flow exposed controls as a function of time.

3. Determine the role of intracellular calcium in changes in proliferation rate and the expression of osteoblastic differentiation markers. hMSCs will be exposed to one hour of oscillatory fluid flow as in Aim 2 in the presence of blockers shown in Aim 1 to inhibit the flow-induced intracellular calcium increase or their vehicle control.

Work Accomplished: To date, Aims 1 and 2 have been completed and published in the Journal of Orthopaedic Research. Aim 3 is in progress.

Expected Outcome: The long-term goal of these studies is to better understand the how mechanical loading influences the behavior of bone. Increased understanding of this relationship will lead to the identification of novel targets of therapeutic interventions in bone diseases with a mechanical component such as osteoporosis.

Funding Source: NASA

Funding Status: Active