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Does matrix-damage influence solute transport in osteocyte lacunar-canalicular system?

INTRODUCTION: Microcracks develop in vivo as a result of cyclic loading during normal daily activity and tissue repair is critical to prevent fracture [1]. Microdamage removal is believed to be accomplished through bone remodeling [2]. Osteocytes, the most abundant cell-type embedded in bone tissue, have been shown to initiate bone remodeling by signaling to other effecter cells via soluble autocrine and paracrine signals and/or direct cell-cell contact [3]. Despite its critical role in damage repair [4], how solute transport in the osteocyte lacunar-canalicular system (LCS) is altered after tissue damage is not fully understood. In the present study, we investigated the effects of matrix-damage on diffusive transport through the LCS in static or reloaded conditions. Our hypothesis was that solute diffusivity within the LCS increases at the damaged region, which becomes more pronounced when bone is mechanically loaded. METHODS: Sample preparation: Block beams of bovine femur were prepared using a diamond saw and a V-notch (half depth of the cortical thickness) introduced at the beam center [5]. Block beams were sectioned into thin wafers (50×4×0.2 mm). Three notched wafers were loaded and unloaded for five cycles at a rate of 6 μm/s using a tensile test device (Ernest F. Fullam Inc. Latham, NY), which was fixed on the stage of a light microscope (Olympus BX51). Opaque process zones size of ~0.5x0.5mm were observed in association with post-yield matrix damage using transmission images at the roots of the machined notches (Fig. 1A) as demonstrated earlier [5]. The regions 5-10 mm from the notch with no apparent transparency changes were used as undamaged controls. To investigate the transport characteristics of the damaged and undamaged tissues, the samples were immersed in sodium fluorescein (0.1 mg/ml) dissolved in phosphate buffered saline (PBS) at 4C overnight before being subjected to fluorescence after photobleaching (FRAP) tests. FRAP experiments: Damage induced cortical wafers were held on a custom loading apparatus [6] consisting of an eletromagnetically actuated loading device (LM1 Testbench, Bose Corp., MN, USA) integrated with a confocal laser scanning microscope (Zeiss LSM 510, Carl Zeiss, Inc, NY, USA). The bone was then subjected to 10-15 FRAP tests at the dark zone and one control region, respectively, using a 40× (0.6 NA) air objective (Korr). The procedure for FRAP experiments was described in our previous study [7]. Briefly a cluster of fluorescently labeled lacunae approximately 15-20 μm below the wafer’s top surface was identified. The central lacuna was selected as the target of the FRAP experiment and tightly enclosed with an elliptical region of interest (ROI) tool. The selected lacuna was photobleached under high intensity laser illumination and its intensity recovery was recorded to extract the transport characteristics (transport rate or diffusivity) [6]. Wafers were first studied when they held at a small tare load (0.2 N), followed by sessions when they were loaded at 10 N, 20 N, and 30 N in tension. Transport characteristics: Using a custom FRAP image analysis program [6, 7], we obtained the transport rate for each FRAP test, which described the reciprocal time constant for the tracer’s refilling the photobeached lacuna. Briefly, The time course of fluorescence intensity I(t) within the photobleached lacuna, including that prior to photobleaching I0, immediately after photobleaching Ib, and the postbleach steady-state intensity I∞, were calculated from the time series. The intensity data were subjected to linear curve fitting with Eq. 1 to obtain the transport rate k [7], which was used to back calculate solute diffusivity based on the anatomy of the LCS.