Osteocytic lacunar-canalicular system and pericellular matrix in mechanosensing
Date
2015
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
Bone is an important component of the skeletal system to ensure locomotion and provides mechanical support to the body. Bone tissue is able to adapt its mass and three-dimensional structure to the prevailing mechanical usage and thus obtain a higher efficiency of load bearing. My studies aimed to better understand the cellular mechanisms responsible for this adaptation phenomenon of bone.
As the most abundant cells in bone, osteocytes form an extensive cellular network through numerous cell processes emanating from individual cell bodies. These cellular protrusions and cell bodies are housed with an extensive pore system, the lacunar-canalicular system (LCS), and buried within the mineralized matrix of bones. This cellular network allows osteocytes to obtain nutrients from the blood supply, sense external mechanical signals, and communicate among themselves and with other cells on bone surfaces. The osteocytic pericellular matrix (PCM), a fibrous thin coating surrounding osteocytes and their long "dendritic" processes, is believed to play a critical role in osteocyte nutrition, cell-to-cell signaling, and mechanotransduction through regulation of fluid shear stress on the cell membrane and drag forces on the PCM fibers. The study of the osteocyte PCM in situ, however, is challenging because it is thin (~100 nm) and enclosed in mineralized processes. To fill the knowledge gap, our laboratory recently developed a novel tracer velocimetry approach that combined fluorescence recovery after photobleaching (FRAP) imaging with hydrodynamic modeling to quantify the structure of the osteocytic PCM in murine bone. In my first study, I applied the technique to mice with a deficiency in the expression of perlecan/HSPG2, a large heparan sulfate proteoglycan that is secreted in the osteocytic PCM of wild type mice. The aims of the study were to examine the effects of perlecan deficiency in the PCM on mechanosensing and test the sensitivity of the velocimetry approach. The results indicated increased diffusion and convection of tracer molecules and sparser osteocyte PCM in the perlecan-deficient bone. This result showed the usefulness of the new technique in studying osteocyte functions in vivo. Furthermore, the ultrastructure of the PCM and cellular stimulation forces were compared among young adult, aged and perlecan-deficient bones. Since the osteocyte LCS structure is an important parameter in the cellular stimulation, I adopted the TEM imaging in my second study to obtain the parameters such as canalicular density, canalicular annual gap and compared them between cortical and cancellous compartments, at different ages, and in two disease conditions. The LCS network showed both topological stability, in terms of the conservation of canalicular connectivity among osteocyte lacunae, and considerable variability in the pericellular annular fluid gap surrounding lacunae and canaliculi. This information may further help in estimating load-induced fluid flow in the LCS and the resultant cellular-level stimulation forces due to the interactions of the fluid flow and PCM within the LCS channels. My third study addressed the issue whether the PCM differs in different bone sites (long bone vs. flat bone) using Alcian Blue staining and FRAP diffusion experiments. Murine flat bones showed lower Alcian blue staining intensity, suggesting reduced expression of HSPG than that of long bones. Also tracer molecules diffused faster in the flat bones than in the long bones, indicating potential reduction of HSPG content in PCM of flat bone. The differences in PCM composition between long bone and calvaria may provide a potential explanation to calvaria's surprising resistance to disuse bone loss. Taken together, the studies suggest the PCM fibers act as osteocyte's sensing antennae, regulating the hydrodynamic forces experienced by the cell and thus bone's sensitivity and in vivo adaptation to its mechanical environments. Quantification of the PCM fiber density or related characteristics could be a powerful tool to identify an individual's sensitivity to loading and provide new targets to promote bone formation in osteoporotic patients.