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The aim of this study is to demonstrate the application of focused ion beam-scanning electron microscopy, FIB-SEM for revealing the three-dimensional features of osteocytic cytoplasmic processes in metaphyseal (immature) and diaphyseal (mature) trabeculae. Tibiae of eight-week-old male mice were fixed with aldehyde solution, and treated with block staining prior to FIB-SEM observation. While two-dimensional backscattered SEM images showed osteocytes' cytoplasmic processes in a fragmented fashion, three-dimensional reconstructions of FIB-SEM images demonstrated that osteocytes in primary metaphyseal trabeculae extended their cytoplasmic processes randomly, thus maintaining contact with neighboring osteocytes and osteoblasts. In contrast, diaphyseal osteocytes extended thin cytoplasmic processes from their cell bodies, which ran perpendicular to the bone surface. In addition, these osteocytes featured thick processes that branched into thinner, transverse cytoplasmic processes; at some point, however, these transverse processes bend at a right angle to run perpendicular to the bone surface. Osteoblasts also possessed thicker cytoplasmic processes that branched off as thinner processes, which then connected with cytoplasmic processes of neighboring osteocytes. Thus, FIB-SEM is a useful technology for visualizing the three-dimensional structures of osteocytes and their cytoplasmic processes.

Canalicular fluid flow is acknowledged to play a major role in bone functioning, allowing bone cells' metabolism and activity and providing an efficient way for cell-to-cell communication. Bone canaliculi are small canals running through the bone solid matrix, hosting osteocyte's dendrites, and saturated by an interstitial fluid rich in ions. Because of the small size of these canals (few hundred nanometers in diameter), fluid flow is coupled with electrochemical phenomena. In our previous works, we developed a multi-scale model accounting for coupled hydraulic and chemical transport in the canalicular network. Unfortunately, most of the physical and geometrical information required by the model is hardly accessible by nowadays experimental techniques. The goal of this study was to numerically assess the influence of the physical and material parameters involved in the canalicular fluid flow. The focus was set on the electro-chemo-mechanical features of the canalicular milieu, hopefully covering any in vivo scenario. Two main results were obtained. First, the most relevant parameters affecting the canalicular fluid flow were identified and their effects quantified. Second, these findings were given a larger scope to cover also scenarios not considered in this study. Therefore, this study gives insight into the potential interactions between electrochemistry and mechanics in bone and provides the rational for further theoretical and experimental investigations.

Bone cells are connected to one another in a network, via their dendritic cellular processes. Previously, we hypothesized that these processes could be ruptured by microcracks. We proposed this as a mechanism by which osteoctyes could detect the presence of microcracks. In order for this mechanism to be effective, the number of ruptured processes would have to increase with microcrack length and also with the applied cyclic stress. This paper presents for the first time experimental data, which shows that this is indeed the case. We examined samples of bovine, ovine and murine bone ex vivo and observed processes passing across crack faces: some were still intact whilst others had ruptured. The number of intact processes per unit crack length decreased significantly with increasing crack length and also decreased in samples, which had been tested in vitro at higher stress levels. A theoretical model that we had developed previously was able to predict the overall magnitude and general trends in the experimental data. This work has provided further support for our "scissors" model, which proposes that microcracks can be detected because they disturb the osteocyte network, specifically by rupturing cellular processes where they pass across the crack faces.

Murine calvariae pre-osteoblasts (MC3T3-E1), grown in a novel bioreactor, proliferate into a mineralizing 3D osteoblastic tissue that undergoes progressive phenotypic maturation into osteocyte-like cells. Initially, the cells are closely packed (high cell/matrix ratio), but transform into a more mature phenotype (low cell/matrix ratio) after about 5 mo, a process that recapitulates stages of bone development observed in vivo. The cell morphology concomitantly evolves from spindle-shaped pre-osteoblasts through cobblestone-shaped osteoblasts to stellate-shaped osteocyte-like cells interconnected by many intercellular processes. Gene-expression profiles parallel cell morphological changes, up-to-and-including increased expression of osteocyte-associated genes such as E11, DMP1, and sclerostin. X-ray scattering and infrared spectroscopy of contiguous, square centimeter-scale macroscopic mineral deposits are consistent with bone hydroxyapatite, showing that bioreactor conditions can lead to ossification reminiscent of bone formation. Thus, extended-term osteoblast culture (< or =10 mo) in a bioreactor based on the concept of simultaneous growth and dialysis captures the full continuum of bone development otherwise inaccessible with conventional cell culture, resulting in an in vitro model of osteogenesis and a source of terminally differentiated osteocytes that does not require demineralization of fully formed bone.

In situ collagen gelation is a method that combines a static three-dimensional culture technique with rotating bioreactors. This method was designed for large dense tissue engineering ex vivo. To challenge the current limitations on size, we combined the static collagen gel embedding method with high-aspect ratio rotating bioreactors. Rat calvarial cells in gelated collagens were cultured in rotating vessels with 5 mM beta-glycerophosphate-containing medium for 1, 2, or 3 wk and then analyzed for cell morphology, cell distribution, and viability, as well as for contraction of the collagen gel. The size of collagen gels with rat calvarial cells averaged 2.8 cm in diameter x 0.25 cm in thickness at the end of 3 wk. Scanning electron microscopy and laser scanning confocal microscopy of collagen gels revealed a homogeneous distribution of living cells. Despite the barrier effects from induced calcification, in collagen gels, cell metabolic activity (alkaline phosphatase assay and 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide assay) increased over the 3 wk, and cell viability (trypan blue exclusion and flow cytometry analysis) remained at about 90% at the end of 3 wk. Based on our results, we determined that in situ collagen gelation provides a feasible method for engineering large dense tissue ex vivo.

The effect of mechanical stresses on osteogenesis, the viability of osteocytes and their metabolic activity in organ culture of bones intermittently loaded "in vitro" are reported. Metatarsal bones, isolated from 12-day-old rats, were cultured in BGJb medium (with 10% foetal calf serum, 75 micrograms/ml of ascorbic acid, 100 U/ml of penicillin and 100 micrograms/ml of streptomycin), in humidified air enriched by 5% CO2 and 30% O2, and loaded in our original device for 1/2 an hour at 1 Hz. homotypic isolated and unloaded bones, cultured in the same medium, were taken as controls. The ALP (alkaline phophatase activity) increases in the media of loaded bones in comparison with the control bones. The percentage of viable osteocytes is significantly greater in loaded than in control bones. TEM observations demonstrate that in both loaded and control unloaded bones, osteocytes show well developed organelle machinery and several gap junctions with adjacent cellular processes. In the cells of loaded bones, however, a higher number of cytoplasmic organelles and gap junctions were found. In particular, RER increases twice, gap junctions three times. The induced osteogenesis and the TEM observations demonstrate the suitability of this experimental model and support the recent advanced hypothesis according to which the mechanical loading may exert a trophic function on osteocytes, stimulating both the proteic synthesis in the above-mentioned cells and the cell-to-cell communication. Furthermore, the loading is likely to exert a biological stimulus on osteoblasts via signalling molecules produced by osteocytes.

Metallic chambers were implanted into the proximal tibiae of rabbits to permit microscopic examination of living bone in situ. The bone repair process secondary to the injury produced during installation of the chamber, was visualized. Six to 8 weeks after implantation, osteoid and/or bone could be seen. The effects of various doses of disodium ethane-1-hydroxy-1, 1-diphosphonate (EHDP) on the repair and regeneration processes following chamber implantation were studied. Data from various techniques indicated that: (1) following low dose EHDP (0.25 mg/kg/day) chambers contained bone tissue morphologically and ultrastructurally indistinguishable from controls; and (2) with higher doses of EHDP (2.5 or 10 mg/kg/day) chamber contained spicules of normal osteoid, osteoblasts and osteocytes, but were devoid of osteoclasts. The effects of the various regimes of EHDP also were assessed on regenerated, trabecular bone contained within the tibia chambers three months after implantation of the chambers. Data from various methods of analysis supported the following conclusions: (1) the low dose of EHDP (0.25 mg/kg/day) had no toxic effects on the trabecular bone within the chambers but there appeared to be an increase in bone formation as compared to saline control; (2) higher doses of EHDP (2.5 or 10mg/kg/day) were not toxic to bone cells but thick osteoid seams formed on the trabecular bone within the chambers. No osteoclasts were found associated with the bone apparently due to the coverage of bone surfaces by osteoid seams; and (3) osteoid which accumulated after EHDP treatment of 2.5 mg/kg/day for 2 months remained uncalcified for as long as 2 months following withdrawal of EHDP administration. The results showed the value of tibial chamber for examining microscopically living bone in situ and demonstrated the inhibitory effect of EHDP on mineralization of newly formed osteoid and a lack of effect on bone cells.

Electron microscopy of bone from the distal ends of femurs of six 3-month-old male C3H/Fg mice revealed varying modes and quantities of C-type virus production by cells in the preosteoblast to osteocyte developmental gradient. Production occurred by budding from the cell surface, either from very short pedicles or from longer microvillous-like processes. Although apparently absent in preosteoblasts, limited numbers of budding viruses (90-110 nM) were seen in 6 of 53 osteoblasts examined. Examination of 200 osteocytes revealed evidence of virus production in all but eight. In these cells, budding occurred from the cell body and its processes with release of viruses into the pericellular lacunar space. Budding from the longer microvillous-like processes sometimes resulted in binding together of viruses in bead-like fashion by thin strands of limiting membrane. In a few cases budding was also seen on the cell processes within the canaliculi. The quantities of viruses present within the lacunae usually increased with increasing age of the osteocytes, indicating continual virus production. Most of the viruses observed, however, were pre C-types along with lesser numbers of mature C particles. Thus, factors contributing to the production of mature C particles may be diminished in osteocytes of mice at this age.