Osteocyte Processes Research Articles

Stereomorphologic observation of bone tissue response to hydroxyapatite using SEM with the EDTA-KOH method.

To obtain further information on the interaction of hydroxyapatite (HA) and the bony implantation bed, 20- to 40- mesh dense HA particles were implanted into the tibiae of dogs. Following healing periods of 2 weeks, 1 month, and 3 months, the specimens were retrieved and prepared by either conventional preparatory procedures for scanning electron microscopy (SEM) of the EDTA-KOH method. Under SEM observation, the interparticular osteogenesis among HA particles progressed in a programmed sequence. Ample blood supply and osteoblasts initially presented in the interparticular space. The secretion of bone matrix resulted in the formation of immature bone. This scaffold was then transformed into mature lamellar bone during the following bone remodeling process. The serial changes closely resembled the pattern viewed in controls that did not implant HA. A spatial relationship between bone cells and HA was clearly demonstrated. In particular, the osteoblasts displayed an extremely flat appearance with many microappendages. The microappendages anchored cells to the HA surface and fused with granular material covering the HA crystals. The more characteristic cellular morphology was revealed by the EDTA-KOH method. Microscopic pictures clearly identified the three-dimensional images of ruffled borders of osteoclasts and the slender cytoplasmic processes of osteocytes. This study provided further evidence for the favorable biological response of HA to bone cells as well as the value of the EDTA-KOH method in examining the stereomorphology of bone cells.

Confocal microscopy of cementocytes and their lacunae and canaliculi in rat molars.

The present study was designed to analyze the morphological characteristics of cementocytes and osteocytes. The maxillae of 10-week-old Wistar rats were used for observations. Non-decalcified ground sections stained vitally with fluorescence dyes and decalcified frozen sections stained with FITC-phalloidin were examined by confocal microscopy. Calcein and alizarin red stained the calcification front of bone, cementum, and dentin intensely. In addition, lacunae and canaliculi of cementocytes and osteocytes as well as dentinal canals were stained with the fluorescent dyes. The staining of lacunae and canaliculi was less intense than that of the calcification front of bone, cementum and dentin. The canaliculi of cementocytes and osteocytes were connected with the canaliculi extending from the calcification front of cementum and bone, respectively. The canalicular density was less in the cellular cementum than in the bone. Areas devoid of canaliculi were numerous in the cellular cementum, whereas areas devoid of canaliculi were scarce in the alveolar bone. Further, the lacunae of cementocytes showed various shapes, from oval to tubular, while the lacunae of osteocytes were invariably oval. The cell body and the cytoplasmic processes of cementocytes were positive for FITC-phalloidin within the extracellular matrix of cellular cementum, which was negative. The distribution of actin filaments in the osteocytes and the cementocytes was predominantly cortical and appeared to be closely associated with the cell membrane of the cell bodies and the cytoplasmic processes. Intense staining was seen at the proximal part of the cytoplasmic processes in both osteocytes and cementocytes, showing a punctuated structure of the cells that was more frequent in osteocytes than in cementocytes. The stress fiber known to be present in most of the cultured cells was not evident in the these cells in situ. The cells incorporated in the cementodentinal junction were strongly stained with FITC-phalloidin. The distribution pattern of the cytoplasmic processes stained with FITC-phalloidin was similar to that of the canaliculli stained vitally. The cytoplasmic processes of osteocytes and cementocytes were connected with those of cells lining the surface of bone and cementum. The present result-that lacunae and canaliculi of cementocytes were stained vitally with the fluorescence dyes-suggests that cementocytes may have a role in secondary calcification of cellular cementum. Further, the lower density of cytoplasmic processes in cementocytes than in osteocytes suggests a lack of complexity in the intercellular network within the cellular cementum.

Electrical signal transmission and gap junction regulation in a bone cell network: a cable model for an osteon.

A cable model is formulated to estimate the spatial distribution of intracellular electric potential and current, from the cement line to the lumen of an osteon, as the frequency of the loading and the conductance of the gap junction are altered. The model predicts that the characteristic diffusion time for the spread of current along the membrane of the osteocytic processes, 0.03 sec, is nearly the same as the predicted pore pressure relaxation time in Zeng et al. (Annals of Biomedical Engineering. 1994) for the draining of the bone fluid into the osteonal canal. This approximate equality of characteristic times causes the cable to behave as a high-pass, low-pass filter cascade with a maximum in the spectral response for the intracellular potential at approximately 30 Hz. This behavior could be related to the experiments of Rubin and McLeod (Osteoporosis, Academic Press, 1996) which show that live bone appears to be selectively responsive to mechanical loading in a specific frequency range (15-30 Hz) for several species.

Ultrastructural localization of alpha-parvalbumin in the epiphyseal plate cartilage and bone of growing rats.

The distribution of the calcium-binding protein, alpha-parvalbumin, in the epiphyseal plate cartilage and bone of growing rats was examined by electron microscope immunocytochemistry of undecalcified samples. Parvalbumin immunoreactivity, as revealed by gold particles, increased with maturation of chondrocytes and was maximal in the zone of calcification. It was found in the cytoplasm of chondrocytes, osteoblasts and osteocytes, corroborating light microscope observations. The immunolabeling was associated with amorphous electron-dense material in the cytoplasm and not bound to membranes. There was moderate parvalbumin immunolabeling over the dense chromatin in the nuclei of chondrocytes and bone cells, but none in the cell processes of mature and hypertrophic chondrocytes, in the matrix vesicles themselves, or in the cell processes of osteoblasts. However, there was parvalbumin immunoreactivity in the cell processes of the osteocytes of compact cortical bone. The uncalcified and calcified matrix of the epiphyseal cartilage, the osteoid, and the fully mineralized cortical bone were devoid of parvalbumin immunoreactivity. Thus, immunoreactive parvalbumin is confined to the cell bodies of chondrocytes and osteoblasts, and is unlikely to be directly involved in mineral deposition. The maximal parvalbumin immunoreactivity in the last terminal chondrocytes of the zone of calcification suggests that the protein is involved in buffering intracellular Ca2+, preventing the stimulation of degenerative processes by high intracellular calcium. The parvalbumin immunoreactivity in the cell processes of osteocytes of compact cortical bone seems to indicate that this calcium-binding protein may be involved in the regulation of Ca2+ fluxes and hence in calcium homeostasis in bone.

Localization of the Ca 2+-binding α-Parvalbumin and its mRNA in epiphyseal plate cartilage and bone of growing rats

This study describes the localization of α-parvalbumin, in undecalcified tibial epiphyseal cartilage and bone of growing rats by immunocytochemistry in the light microscope, and of parvalbumin mRNA by in situ hybridization. They were compared to the distribution of the calbindin-D9K and its mRNA in rat epiphyseal cartilage. All the chondrocytes of the epiphyseal cartilage were parvalbumin-immunopositive, but there was no parvalbumin immunoreactivity in the uncalcified or calcified extracellular cartilage matrix. The intensity of the immunostaining increased from the resting and proliferative to the mature and hypertrophic chondrocytes, with the greatest intensity in the terminal hypertrophic chondrocytes in the calcifying zone. The parvalbumin immunostaining was located in the cytoplasm, but no immunoreactivity was detected in any chondrocyte processes. The parvalbumin mRNA distribution and levels, as revealed by in situ hybridization, exactly mirrored those of the parvalbumin protein. In contrast to parvalbumin, calbindin-D9K and its mRNA appeared in mature chondrocytes and decreased in hypertrophic up to calcifying chondrocytes. Calbindin-D9K was located in the cytoplasm and all along the cell processes. In bone, the osteoblasts and the osteocytes of trabecular and compact cortical bones were immunoreactive for parvalbumin and contained parvalbumin mRNA. Parvalbumin lay in their cytoplasm, but there was no parvalbumin immunostaining in the extracellular uncalcified or mineralized bone matrix. The long processes of osteocytes, in compact bone only, were parvalbumin immunoreactive. Osteoclasts contained cytoplasmic parvalbumin immunoreactivity. Thus, the pattern of immunoreactive parvalbumin distribution indicates that the protein is not involved in the extracellular mineralization of cartilage and bone matrix. It appears to be associated with specific calcium-related intracellular functions in chondrocytes and in osteoblasts, osteocytes, and osteoclasts. As the highest cytoplasmic concentration of parvalbumin is in the terminal hypertrophic chondrocytes, parvalbumin could act as a calcium buffer to delay the death of chondrocytes. In compact bone, parvalbumin could also have a role throughout the osteocyte processes in regulating the fluxes of calcium ions for mineral homeostatis. ( Bone 17:121–130; 1995)

Transmission electron microscopic demonstration of vimentin in rat osteoblast and osteocyte cell bodies and processes using the immunogold technique.

The immunogold labeling technique and transmission electron microscopy were used to demonstrate the expression and position of the intermediate filament vimentin in rat osteoblast and osteocyte cell bodies and cell processes. Conventional light and transmission electron microscopic studies of bone cells demonstrated adjacent cell linkage to be mediated by osteoblast and osteocyte processes present within the canalicular system traversing the bone matrix. The cell processes were filled with densely packed filaments, many of which have been shown previously to be actin microfilaments. The appearance, however, of 10 nm diameter filaments in some cell processes and the fact the intermediate filament vimentin has been defined in many cells of mesenchymal origin raised the possibility that some of these filaments might be vimentin. The ultrastructural colloidal gold immunochemical technique allowed for demonstration in situ of the expression of vimentin filaments plus accurate definition of their position. The studies were performed in newborn rat femoral and tibial diaphyseal cortical bone and in 1-week-old repair bone from 2.4 mm diameter defects made through the lateral cortex in 6-week-old rat femurs and tibias. The bone tissues for the immunochemical study were fixed in 1% glutaraldehyde, 4% paraformaldehyde, and 0.1 M phosphate buffer (pH 7.4) for 2 days. Decalcification was performed in 6% EDTA for 2-3 days. Infiltration involved use of Lowicryl resin K4M, and the embedding and curing processes were performed in a cryostat with temperatures -30 degrees C. An antivimentin monoclonal antibody was used for labeling using the postembedding technique. Effective antibody dilutions ranged from 1:10 to 1:200, with the dilutions of 1:25 and 1:100 showing the best combination of filament labeling with the least matrix background. The grids were exposed to 10 nanometer gold colloid conjugated goat anti-mouse IgM for demonstration of binding. Vimentin immunolabeling was defined clearly in relation to filaments within the osteoblast and osteocyte cell body cytoplasm, throughout the entire length of the osteoblast and osteocyte cell processes, and in close relationship to the intracellular gap junctions which were present within the cell processes both close to the cell bodies and within the canaliculi well away from them. Immunogold labeling demonstrates the presence of the intermediate filament vimentin in osteoblast and osteocyte cell bodies and processes of rat bone. Vimentin distribution is not concentrated to specific areas, is present throughout the extent of the bodies and processes, and is seen immediately adjacent to gap junctions.

Transmission electron and high-voltage electron microscopy of osteocyte cellular processes extending to the dental implant surface.

Examination of the morphology of osteocytes within the bone supporting endosteal dental implants was performed using conventional transmission and high-voltage transmission electron microscopy (HVEM). The in vivo dog model used 72 implants inserted into the premolar region of 18 experimental animals. Forty-eight implants in 12 dogs were used as anterior abutments for fixed bridges for periods up to 12 months. The mineralized matrix of the supporting bone was either directly apposed to the implant surface or was separated from the implant by a narrow region of unmineralized matrix. Osteocytes were routinely observed to be closely associated with the bone-implant interface, as well as at a distance from the implant. Osteocytes were found to extend cellular processes directly to the implant surface through canaliculi. The osteocyte processes contained microfilaments. The three-dimensional capabilities of HVEM elucidated the nature of these cell processes at the point of exit from the osteocyte, as the processes extended through the mineralized matrix, and as the processes terminated at the implant interface. This report suggests that avenues of communication may exist between the implant and the osseous cells, providing intriguing hypotheses regarding biomechanical forces and osteogenesis at the implant interface. Furthermore, an electron-dense deposit was observed upon the inner confines of the canalicular wall, upon the outer aspects of the osteocyte lacuna, and upon the outer aspect of the bone interfacing the implant.

A fiber matrix model for fluid flow and streaming potentials in the canaliculi of an osteon.

A theoretical model is developed to predict the fluid shear stress and streaming potential at the surface of osteocytic processes in the lacunar-canalicular porosity of an osteon when the osteon is subject to mechanical loads that are parallel or perpendicular to its axis. The theory developed in Weinbaum et al. (31) for the flow through a proteoglycan matrix in a canaliculus is employed in a poroelastic model for the osteon. Our formulation is a generalization of that of Petrov et al. (17). Our model predicts that, in order to satisfy the measured frequency dependence of the phase and magnitude of the SGP in macroscopic bone samples, the fiber spacing in the fluid annulus must lie in the narrow range 6-7 nm typical of the spacing of GAG sidechains along a protein monomer. The model predictions for the local SGP profiles in the osteon agree with the experimental observations of Starkebaum et al. (24). The theory predicts that the pore pressure relaxation time, tau d, for a 150-300 microns diameter osteon with the foregoing matrix structure is approximately 0.03-0.13 sec, and that the amplitude of the mean fluid shear stress on the membrane of the osteocytic process at the mean areal radius of the osteon has a maximum at 28 Hz if tau d = 0.06 sec. This maximum, which is independent of the magnitude of the loading, could be important in vivo since the recent experiments of Turner et al. (28) and McLeod et al. (15) have a peak in the strain frequency spectrum between 20 and 30 Hz that also appears to be independent of the type (magnitude) of loading. Numerical predictions for the amplitude of the average fluid shear stress on the osteocytic membrane at the mean areal radius of the osteon show that the fluid shear stress associated with the low amplitude 20-30 Hz spectral strain component is at least as large as the average fluid shear stress associated with the high amplitude 1 Hz stride component, although the latter loading is an order of magnitude larger, and has a magnitude that lies within the middle of the range, 6-30 dynes/cm2, where fluid shear stresses in tissue culture studies with osteoblast monolayers have elicited an intracellular Ca++ response (31). The implications of these results for intracellular electrical communication are discussed.

A model for the excitation of osteocytes by mechanical loading-induced bone fluid shear stresses

A new experimentally testable hypothesis is advanced for the mechanosensory transduction mechanism by which communicating osteocytes sense the very small in vivo strains in the calcified matrix components of bone. We propose that the osteocytes, although not responsive to substantial fluid pressures, can be stimulated by relatively small fluid shear stresses acting on the membranes of their osteocytic processes. Biot's porous media theory is used to relate the combined axial and bending loads applied to a whole bone to the flow past the osteocytic processes in their canaliculi. In this theory, the bone pores of interest are the proteoglycan filled fluid annuli that surround the osteocytic processes in the canaliculi. We show that previously predicted fluid pore pressure relaxation times were a hundred-fold too short for the lacunar-canalicular porosity because they neglected the fluid drag associated with proteoglycan matrix on the surface membrane of the osteocyte and its cell processes. The recent theory developed in Tsay and Weinbaum [ J. Fluid Mech. 226, 125–148 (1991)] for flow through cross-linked fiber filled channels is used to model the flow through this proteoglycan matrix. The predicted pore relaxation time, 1–2 s, closely corresponds to the times measured by Salzstein and Pollack [ J. Biomechanics 20, 271–280 (1987)]. Furthermore, using this model, the magnitude of the predicted fluid induced shear stresses, 8–30 dyn cm −2, is shown to be similar to the fluid shear stresses measured in osteoblasts and other cells in which an intracellular Ca 2+ shear stress response had been observed. This model is also used, in conjunction with anatomical data and the pore fluid pressure relaxation time data, to show that the spacing between the fibers is approximately 7 nm. The result is consistent with the notion that the canalicular pore space is filled with glycosaminoglycans that are ordered by albumin according to the model of Michel [ J. Physiol. 404, 1–29 (1988)]. The new hypothesis is also shown to be consistent with the experiments of McLeod et al. [ J. Biomechanics (submitted)] which suggest that high-frequency low-amplitude postural strains can maintain and even increase bone mass.

Immunoreactive calbindin-D9K in bone matrix vesicle

This electron microscope study describes the subcellular occurrence and distribution of immunoreactive calbindin-D9K in the trabecular metaphyseal and compact cortical bone of normal rats, rachitic vitamin-D-deficient rats, and rachitic rats given 1,25-(OH)2D3. Undecalcified bones were embedded in Lowicryl K4M and calbindin-D9K antigenicity was detected by the protein A-gold method. Immunoreactive calbindin-D9K was localized in the cytoplasm and cell processes of osteoblasts and osteocytes. Immunoreactive calbindin-D9K was also found within matrix vesicles and calcifying matrix vesicles, where it lay over the needle-shaped crystallites, at the apparent site of initial crystal formation, but not along the whole crystallites. In fully mineralized bone it occurred at the same site, over the crystallites. Calibindin-D9K was vitamin-D-dependent in the osteoblasts and matrix vesicles, where its presence was correlated with the reappearance of crystallites in 1,25-(OH)2D3-treated vitamin-D-deficient rats. This suggests that immunoreactive calbindin-D9K is involved in mineral deposition in bone matrix vesicles. Abnormal intracellular calcification associated with calbindin-D9K antigenicity in the osteoblasts of 1,25-(OH)2D3-treated vitamin-D-deficient rats indicates that immunoreactive calbindin-D9K may also play a part in abnormal intracellular mineral deposition.

Morphology of Osteosarcoma: New Qualitative and Quantitative Investigations 1

Results on seven cases of osteosarcoma are reported, based on new morphologic methods and quantitative procedures. Tumor tissue was embedded without prior decalcification in plastic and sectioned. Imprint cytology preparations were produced from fresh tumor tissue, and cell nuclei were measured with an electronic image analysing computer system. The loss of differentiation seen in osteosarcomas differs among osteoblasts, osteocytes, and osteoclasts. The differentiation of osteoclasts, namely their resorptive characteristics, disappears relatively early. Tumor osteocytes show loss of differentiation in their osteocyte processes. The new formation of tumor bone tissue remains in the near normal range of volume density when nuclear polymorphy is limited. The formation of ground substance and mineralization are apparently closely couplet to one another, since in our cases mostly ordered osteoid seams were observed. The capacity for mineralization of bone tissue is lost with marked polymorphy. The significance of these results for diagnostic statements and therapeutic consequences will be further discussed in long term studies.

Mechanical similarities between alternate osteons and cross-ply laminates

Fully calcified osteon samples with alternating lamellae were prepared from longitudinal sections of human femoral shafts according to the technique described by Ascenzi and Bonucci. Osteons of this type reveal an alternation of dark and bright lamellae under the polarizing microscope. The samples were loaded by tension in a direction parallel with their longitudinal axis using a specially designed microwave extensimeter based on cavity and pulse techniques. All the samples were tested wet at a temperature of ca. 20°C. The stress-strain curves recorded from each osteon show a change in slope or knee at low stresses. A useful model for understanding this phenomenon is furnished by the mechanical properties of that type of fiber-reinforced plastic material called cross-ply laminate. The comparison of the data obtained from this material with those furnished by an electron microscope investigation carried out on the loaded osteons suggests that the change in slope at low stresses is due to failure of the interfibrillar cementing substance in those lamellae having fiber bundles oriented perpendicularly to the loading direction, and to yielding of the canaliculi previously filled with osteocyte processes. The physiological implications of these results are discussed.

Tight junctions between osteocyte processes.

abstract— Rat alveolar bone was studied by electron microscopy. In some of the specimens tight junctions were observed between overlapping osteocyte processes. The possible function of the tight junctions is discussed.