In situ measurement of solute transport in the bone lacunar‐canalicular system

Solute transport through the bone lacunar-canalicular system is believed to be essential for osteocyte survival and function but has proved difficult to measure. We report an approach that permits direct measurement of real-time solute movement in intact bones. By using fluorescence recovery after photobleaching, the movement of a vitally injected fluorescent dye (sodium fluorescein) among individual osteocytic lacunae was visualized in situ beneath the periosteal surface of mouse cortical bone at depths up to 50 microm with laser scanning confocal microscopy. Transport was analyzed by using a two-compartment mathematical model of solute diffusion that accounted for the characteristic anatomical features of the lacunar-canalicular system. The diffusion coefficient of fluorescein (376 Da) was determined to be 3.3 +/- 0.6 x 10(-6) cm2/sec, which is 62% of its diffusion coefficient in water and is similar to diffusion coefficients measured for comparably sized molecules in cartilage. The diffusion of fluorescein in bone is also consistent with the presence of an osteocyte pericellular matrix whose structure resembles that proposed for the endothelial glycocalyx [Squire, J. M., Chew, M., Nneji, G., Neal, C., Barry, J. & Michel, C. (2001) J. Struct. Biol. 136, 239-255]. To our knowledge, this is the first instance where the dynamics of molecular movement has been measured directly in the bone lacunar-canalicular system. This in situ imaging approach should also facilitate the analysis of convection-based transport mechanisms in bones of living animals.

[1]  R K Jain,et al.  Direct in vivo measurement of targeted binding in a human tumor xenograft. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[2]  S. Cowin,et al.  Ultrastructure of the osteocyte process and its pericellular matrix. , 2004, The anatomical record. Part A, Discoveries in molecular, cellular, and evolutionary biology.

[3]  S. Cowin,et al.  Candidates for the mechanosensory system in bone. , 1991, Journal of biomechanical engineering.

[4]  Felix W Wehrli,et al.  Diffusion of exchangeable water in cortical bone studied by nuclear magnetic resonance. , 2002, Biophysical journal.

[5]  R. Peters,et al.  A microfluorimetric study of translational diffusion in erythrocyte membranes. , 1974, Biochimica et biophysica acta.

[6]  Subrata Saha,et al.  A theoretical model for stress-generated fluid flow in the canaliculi-lacunae network in bone tissue. , 1990, Journal of biomechanics.

[7]  Peter Niederer,et al.  Investigation of the Morphology of the Lacunocanalicular System of Cortical Bone Using Atomic Force Microscopy , 2001, Annals of Biomedical Engineering.

[8]  R. Robinson,et al.  Morphology of the osteon. An electron microscopic study. , 1966, The Journal of bone and joint surgery. American volume.

[9]  A F Mak,et al.  A histomorphometric observation of flows in cortical bone under dynamic loading. , 2000, Microvascular research.

[10]  D P Fyhrie,et al.  Intracortical remodeling in adult rat long bones after fatigue loading. , 1998, Bone.

[11]  Alice Maroudas,et al.  Diffusion and partition of solutes in cartilage under static load. , 2003, Biophysical chemistry.

[12]  P. Niederer,et al.  In vivo tracer transport through the lacunocanalicular system of rat bone in an environment devoid of mechanical loading. , 1998, Bone.

[13]  Stephen C. Cowin,et al.  Modeling Tracer Transport in an Osteon under Cyclic Loading , 2004, Annals of Biomedical Engineering.

[14]  Scott J Hollister,et al.  Osteocyte lacuna size and shape in women with and without osteoporotic fracture. , 2004, Journal of biomechanics.

[15]  Sheldon Weinbaum,et al.  Mechanotransduction and strain amplification in osteocyte cell processes. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[16]  B. Gabryel,et al.  Piracetam and vinpocetine exert cytoprotective activity and prevent apoptosis of astrocytes in vitro in hypoxia and reoxygenation. , 2002, Neurotoxicology.

[17]  J. James,et al.  Death of osteocytes. Electron microscopy after in vitro ischaemia. , 1986, The Journal of bone and joint surgery. British volume.

[18]  P. Bullough,et al.  Permeability of articular cartilage. , 1968, Nature.

[19]  J. Squire,et al.  Quasi-periodic substructure in the microvessel endothelial glycocalyx: a possible explanation for molecular filtering? , 2001, Journal of structural biology.

[20]  Olivier Verborgt,et al.  Spatial Distribution of Bax and Bcl‐2 in Osteocytes After Bone Fatigue: Complementary Roles in Bone Remodeling Regulation? , 2002, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[21]  G. Cochran,et al.  Transcortical streaming potentials are generated by circulatory pressure gradients in living canine tibia , 1990, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[22]  Stephen B Doty,et al.  Delineating bone's interstitial fluid pathway in vivo. , 2004, Bone.

[23]  R K Jain,et al.  Direct measurement of interstitial convection and diffusion of albumin in normal and neoplastic tissues by fluorescence photobleaching. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[24]  D B Burr,et al.  Alterations to the en bloc basic fuchsin staining protocol for the demonstration of microdamage produced in vivo. , 1995, Bone.

[25]  Sheldon Weinbaum,et al.  On bone adaptation due to venous stasis. , 2003, Journal of biomechanics.

[26]  Farshid Guilak,et al.  Site-Specific Molecular Diffusion in Articular Cartilage Measured using Fluorescence Recovery after Photobleaching , 2003, Annals of Biomedical Engineering.

[27]  A. van der Plas,et al.  Sensitivity of osteocytes to biomechanical stress in vitro , 1995, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[28]  S. Lang,et al.  DIFFUSION OF GLUCOSE IN STRESSED AND UNSTRESSED CANINE FEMUR IN VITRO * , 1974, Annals of the New York Academy of Sciences.

[29]  Stephen C. Cowin,et al.  Mechanotransduction and flow across the endothelial glycocalyx , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[30]  K. Piekarski,et al.  Transport mechanism operating between blood supply and osteocytes in long bones , 1977, Nature.

[31]  T. Tanaka,et al.  Differences in the transport systems between cementocytes and osteocytes in rats using microperoxidase as a tracer. , 1992, Archives of oral biology.

[32]  S. Cowin,et al.  A model for the excitation of osteocytes by mechanical loading-induced bone fluid shear stresses. , 1994, Journal of biomechanics.

[33]  Gustavo Carrero,et al.  Using FRAP and mathematical modeling to determine the in vivo kinetics of nuclear proteins. , 2003, Methods.

[34]  P. B. Allen.  Conduction of Heat. , 1983 .

[35]  M. K. Knothe Tate,et al.  An ex vivo model to study transport processes and fluid flow in loaded bone. , 2000, Journal of biomechanics.

[36]  J. C. Jaeger,et al.  Conduction of Heat in Solids , 1952 .