Histomorphological study on pattern of fluid movement in cortical bone in goats

Abstract

Streaming potential is considered one of the most important mechanisms to moderate the function of osteoblasts and osteocytes in bone growth, remodeling and fracture repair. The present study was designed to demonstrate the fluid flow pattern in the cortical bone matrix in an animal model using undecalcified histological techniques. Immediately after injection of ferritin into the tibia nutrient artery of four adult goats, the animals were euthanized. Undecalcified transverse and longitudinal blocks of cortical bone obtained from the tibial diaphysis were immersed in Perl's reagent and embedded in methyl methacrylate. Sections were cut and ground to 30-50 µm thickness for histomorphological evaluation at different magnifications and focusing levels. A serial grinding technique was used to validate the observations made at different focusing levels. As expected, ferritin was observed in the interstitial compartment in both transverse and longitudinal sections. In osteons sectioned transversely, the pattern of centrifugal movement of ferritin marker was demonstrated as single or multiple halos around the Haversian canal. The most apparent halo in osteons with multiple halos was the one found closest to the Haversian canal. The total number of identifiable single or multiple halos increased or was altered when counting was made with higher magnification or at different focusing levels, respectively. Irregular and incomplete ferritin halos indicated structural complexity of the osteons. Overall, the pattern of ferritin movement was consistent with bulk interstitial fluid flow influenced by both hydrostatic pressure and transudation. This study demonstrated for the first time multiple concentric halos of the fluid flow marker ferritin around Haversian canals in the cortical interstitial compartment. The results suggest that the undecalcified technique might be a useful method for qualitative and quantitative studies on cortical fluid flow. Anat Rec 255: 380-387, 1999. 1999 Key words: fluid flow; cortical bone; ferritin transudation; undecalcification; histology; microscopy Mature cortical bone is structurally organized into osteons. Each osteon is roughly cylindrical in shape with concentric lamellae around the vasculature-containing Haversian canal. Between the lamellae lie the ellipsoidal lacunae where the osteocytes reside. Cortical bone has a hierarchical network of flow channels, i.e. the longitudinal Haversian and the transverse Volkmann's canals to the tiny lacunae-canaliculi channels around the bone cells, and the microporosity of the collagen-hydroxyapatite bone matrix (Cooper et al

Delineating bone's interstitial fluid pathway in vivo

“…For example, ferritin (approximately 10 nm in diameter) injected into mature dogs [18] and goats [17,20] has demonstrated halo-like labeling surrounding the vascular canals, shown as discrete lines with a spacing similar to that observed for canaliculi [18] or multiple rings within the matrix with a clear margin from the osteonal canal surface [17,20]. These results suggest that ferritin may be small enough to pass through the canalicular pores and possibly through the collagen-apatite pores in the mineralized matrix.…”

Mapping bone interstitial fluid movement: Displacement of ferritin tracer during histological processing

“…By only considering three subareas of the cross section, we likely overlooked the ~4% halo labeling found for the FROZ1 sections in the present study. Another difference between the present study and our previous study that could possibly affect the results was that Karnovsky's fixative was used instead of alcohol to better mimic processing by previous investigators [5,15,16,19].…”

“…In addition, appositional fronts along the bone surfaces have been said to demonstrate centrifugal interstitial fluid movement from the medullary canal to the periosteal surface [5,6,[9][10][11]15,16,19,26]. The purpose of this study was to analyze the effect of different histological processes that could lead to the presence of ferritin in the mineralized matrix and the characteristic ferritin halo labeling previously demonstrated by other investigators.…”

“…Bone interstitial fluid flow is also believed to aid in delivering nutrients and transporting metabolic waste products from the bone cells [17]. To better understand interstitial fluid movement in bone, techniques using markers or tracers such as procion red (300-400 Da, diameter <1 nm), reactive red (1470 Da, diameter ~1 nm), microperoxidase (1860 Da, diameter ~2 nm), horseradish peroxidase (40 kDa, diameter ~6 nm), ferritin (440 kDa, diameter ~12 nm [20]), and different-sized dextrans (range 300 Da-2000 kDa, diameter ~1 nm-60 nm) have been used to map how different sized molecules travel through the various porosities in bone [1,5,8,[13][14][15][16]19,[22][23][24].…”

“…However, other investigators have shown that ferritin forms halo-shaped labeling that appears to enter the mineralized matrix around blood vessels, along with appositional fronts along the bone surfaces [5,15,16,19]. This ferritin labeling is widely used to explain normal interstitial fluid movement in bone: the halos are said to demonstrate bulk centrifugal interstitial fluid movement away from a highly pressurized vascular porosity, and the appositional fronts are said to demonstrate centrifugal interstitial fluid movement from the medullary canal to the periosteal surface [5,6,[9][10][11]15,16,19,26]. These previous studies suggest that ferritin may be small enough to pass through the canalicular pores and possibly through the collagenapatite pores in the mineralized matrix.…”