MATRIX STRUCTURE IN THE LACUNAR/CANALICULAR POROSITY

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DESCRIPTION (Adapted from the Applicant's Abstract): The applicants have proposed a new hypothesis for the cellular level mechanosensory mechanism via which the cellular components of bone sense mechanical strain, and developed a theoretical model to test its quantitative feasibility. According to this hypothesis, the fluid annuli surrounding the osteocytic processes in the canaliculi are filled with a fiber matrix that is similar in pore structure to the surface glycocalyx on vascular endothelium. The interstitial fluid flow through this matrix caused by mechanical loading generates a drag force on the matrix which is manifested as a shearing force on the membranes of the osteocytic processes. It is this shear force that the investigators propose is the mechanosensory signal via which osteocytes sense mechanical strain in bone. Several experimental studies have already shown that there are intracellular biochemical responses at the theoretically predicted shear stress levels. The applicants have also proposed that the stress-generated potentials measured in bone are due to streaming currents which are directed along the axes of the fibers, rather than, as previously believed, flow through pores in the mineralized bone. The presently proposed research involves three Specific Aims. Although ultrastructural studies have suggested the presence of gel-like structural components in the canalicular fluid space, there has been no definitive study which has characterized the pore structure of this region or the thickness of the matrix layer. In the applicants' model the fibers are assumed to be ordered by plasma proteins and form a molecular sieve for albumin (7nm dia.). In Specific Aim 1, the investigators propose to make the first direct measurements of the thickness of the surface glycocalyx on bone cells and its pore size, using a cationic tracer which is a little larger than albumin. An important debate has arisen over the site of SGP. Supporters of the original hypothesis that the SGP resides in the pores of the mineralized matrix have argued that while the walls of the canaliculi may not be permeable to macromolecules, they could permit the passage of water and small ions, providing for a connectivity between the two porosities. In Specific Aim 2, the applicants propose to explore this possibility, using small fluorescent molecular probes, and develop a model to quantitatively examine the consequences of this connectivity if it exists. Whereas the high molecular weight tracer used in Aim 1 above should reveal the pore structure of the glycocalyx and its thickness, it is unlikely to tell much about the charge distribution in the glycocalyx and the nature of its anionic groups. In Specific Aim 3, the investigators will seek to apply a "critical electrolyte staining" technique in which one uses a small penetrating osmiophilic dye, Alcian blue, in combination with MgCl2. The latter molecule selectively blocks sulphate and carboxyl groups, depending on its concentration, and thus will identify the anionic groups that constitute the matrix and their spatial distribution.