In pursuit of accurate structural and mechanical osteocyte mechanotransduction models.

Abstract

To enhance preventative and therapeutic strategies for age-related bone fragility increases, several investigators have begun to explore the relationship between bone matrix mechanics and osteocyte physiology. Progress remains restricted by the absence of models to study osteocytes while interacting with their native bone matrix and by superficial knowledge of their local mechanical environment. To address these issues, a novel model has been created that allows one to: (1) study osteocytes in the context of their native three dimensional extracellular matrix; and (2) monitor biologic activity of these osteocytes while controlled forces (or displacements) are applied to the matrix. An implant-explant system has been produced in a Sprague-Dawley rat that allows one to generate regular bone samples and maintain them in tissue culture for several days. Implant-explant system tissue has been studied with microcomputed tomography and histology to determine that it is a solid model of mostly intramembranous bone and likely capable of reproducing complex biologic processes in an appropriate environment. Explant viability in culture has been detailed along with practical diffusion information for introducing biologic probes. Depth dependent changes in relative fluorescence have been quantified and must be taken into account when interpreting changes secondary to mechanical perturbations. Finally, preliminary studies confirm that intracellular pH can be monitored during mechanical loading by measuring changes in relative fluorescence. Additionally, a technique to quantify perilacunar strains has been described along with a reproducible protocol for application, characterization and calibration on any digital imaging modality. Using a laser scanning confocal microscope, displacement resolutions of 45 nm could be achieved and strain calculations were within 10% of analytic values. Microscopic strains around osteocytes were compared with tissue-level values that approximate routine functional activity. Perilacunar strains were nearly an order of magnitude greater than strains measured at the more global scale. Strains also appeared to escalate around the lacunar border, which is consistent with elementary mechanics. Local strain values are in excess of tissue-level yield strains, which has implications for osteocyte physiology and perilacunar material properties.