DEVELOPMENT OF A MECHANICALLY ACTIVE PERFUSION CHAMBER FOR INVESTIGATIONS OF MECHANICAL SIGNAL TRANSDUCTION IN TRABECULAR BONE IN VIVO

Participants: M.R. Moalli, B. Roessler, N. Caldwell, C. Maynard, S.A. Goldstein

Keywords: mechanotransduction, trabecular bone, in vivo, mechanical loading

Introduction

The mechanisms for the coupling of cell-level mechanical signals into intracellular biochemical signals are currently under intense investigation, and several candidate pathways have been proposed. Due to the inherent difficulty of manipulating specific components of the signaling cascades triggered by mechanical signals at the cellular level, these investigations have largely been conducted in vitro. We have developed a perfusion chamber in which bone cells can potentially be genetically or biochemically manipulated prior to the application of a controlled mechanical load in vivo.

Methods

A hollowed, large volume titanium bone chamber implant was surgically implanted into the proximal tibial metaphyses of two adult, male canines as previously described¹ using protocols approved by the Institutional Animal Care and Use Committee. Four weeks post-operatively the chambers were cleared of tissue and connected with a novel perfusion system. This system consisted of a hemi-spherical titanium cap with a swedged-on 22 gauge needle and a polysulfone subcutaneous access port (AccessÔ Technologies). Once the cap is engaged, the needle is centrally positioned and extends to the bottom of the chamber. The hub of the needle is occluded and radial fenestrations were drilled along its length for uniform delivery of the perfusate. The access port is buried within the subcutaneous tissue of the medial thigh and is connected to the chamber cap by 5-6 cm of polyurethane tubing. Thus the direction of fluid flow is from the access port reservoir, through the tubing, into the cap and out the fenestrated needle to the woven trabecular bone developing within the chamber.

Results

The infusion cap was placed in two dogs and after 8 weeks of bone formation, genistein or DMSO vehicle was infused into each chamber via the access port. Two hours after infusion, each tibial chamber was simultaneously loaded (17.8 N, 1800 cycles, 1 Hz). Specimens were harvested immediately and analyzed by Western blot for FAK phosphorylation. Infusion of genistein inhibited load-induced FAK activation, while FAK was phosphorylated in the DMSO-infused control chamber.

Discussion

The results demonstrate that biochemical mediators which are commonly used to treat cells in in vitro studies can also be used to inhibit/manipulate cellular processes occurring in the microenvironment of the bone chamber. Given the previous results which demonstrated moderate transfection efficiency with the adenoviral constructs, we are confident that the cells in the bone chamber will uptake the DNA encoded in the CMV-promoter-based expression plasmids. Current studies are aimed at utilizing this novel model to manipulate specific components of the proposed signaling cascades in a reproducible, controlled manner.