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PubMed2005

Differences in osteocyte and lacunar density between Black and White American women.

We examined the differences in osteocyte and lacunar density between Black and White women, using previously obtained iliac bone biopsies from 34 healthy Black women, aged 21-70 years, and 94 White women, aged 20-73 years. For each subject, the density of osteocytes (Ot.N/B.Ar), empty lacunae (EL.N/B.Ar), and total lacunae (Tt.L.N/B.Ar) and the proportion of osteocyte-occupied lacunae (Ot.N/Tt.L.N) were separately measured in whole trabeculae, superficial bone (<25 microm from the bone surface), and deep bone (>45 microm from the bone surface). Compared with White women, Black women had higher values for osteocytes, empty lacunae, and total lacunae and lower values for percent occupied lacunae in superficial bone and whole trabeculae (P < 0.01 to <0.001). In deep bone there were more osteocytes and total lacunae in Black women, but the other measurements did not differ significantly between the two groups. As in White women, there were fewer osteocytes and total lacunae and more empty lacunae in deep than in superficial bone. The regressions of osteocyte and total lacunar density on age were not significant in Black women, but postmenopausal Black women had fewer osteocytes than premenopausal Black women, and percent occupied lacunae declined significantly with age in whole trabeculae and deep bone, which could only have resulted from osteocyte death. In contrast to White women, there was no inverse relationship between bone formation rate and osteocyte density in superficial bone and the observed bone formation rate was lower than predicted by osteocyte density. We conclude the following: (1) Cancellous bone is made with more osteocytes in Black than in White women, most likely because of diminished apoptosis of osteoblasts; this could contribute to increased bone strength in Black women. (2) In Black women, as in White women, there are fewer osteocytes and total lacunae and more empty lacunae in deep than in superficial bone. (3) There was moderate age-related loss of osteocytes in deep bone in Black women, indicating that osteocyte density depends more on the age of the bone than on the age of the subject. (4) The higher osteocyte density in Black women was not responsible for their lower bone formation rate.

Adult↗
PubMed2006

Quantitative regional associations between remodeling, modeling, and osteocyte apoptosis and density in rabbit tibial midshafts.

Evidence suggests that osteocyte apoptosis is involved in the adaptive response of bone, although the specific role of osteocytes in the signaling mechanism is unknown. Here, we examined and correlated regional variability in indices of remodeling, modeling, osteocyte apoptosis, and osteocyte density in rabbit tibia midshafts. Histomorphometric analysis indicated that remodeling parameters (BMU activation frequency, osteon density, forming osteon density, and resorption cavity density) were lower in the cranial region compared to other quadrants. In addition, pericortical subregions displayed less remodeling relative to intracortical and endocortical ones. Modeling indices also demonstrated regional variability in that periosteal surfaces exhibited a greater extent of bone forming surface than endosteal ones across all anatomic quadrants. In contrast, endosteal surfaces demonstrated significantly greater surface mineral apposition rates compared to periosteal surfaces in caudal, medial, and lateral but not cranial quadrants. Using TUNEL analysis to detect osteocytes undergoing apoptosis, the density of apoptotic osteocytes was found to be lower in cranial quadrants relative to medial ones. In addition, the densities of osteocyte lacunae, empty lacunae, and total osteocytes were higher in lateral fields relative to caudal quadrants. There was a strong, statistically significant linear correlation between the remodeling indices and apoptotic osteocyte density, supporting the theory that osteocytes undergoing apoptosis produce signals that attract or direct bone remodeling. In contrast, the modeling parameters did not exhibit a correlation with apoptotic osteocytes, although there was a strong correlation between the modeling indices and the density of empty osteocyte lacunae, corroborating previous studies that have found that osteocytes inhibit bone formation. It was found that osteocyte density and osteocyte lacunar density did not significantly correlate with modeling or remodeling parameters, suggesting that cell viability should be examined in studies correlating bone turnover parameters with the functional role of osteocytes in bone adaptation.

Animals↗
PubMed2004

Terminal differentiation of osteoblasts to osteocytes is accompanied by dramatic changes in the distribution of actin-binding proteins.

UNLABELLED: Immunofluorescence staining of actin-binding proteins in osteoblasts and osteocytes was performed. alpha-Actinin, myosin, and tropomyosin showed similar organization in both osteoblastic stress fibers and osteocyte processes. However, fimbrin, villin, filamin, and spectrin showed dramatic differences in distribution between osteoblasts and osteocytes. This study suggested that terminal differentiation of osteoblasts to osteocytes is accompanied by highly dramatic changes in the distribution of actin-binding proteins. INTRODUCTION: We previously reported that osteocyte shape is dependent on actin filaments. To analyze the terminal differentiation from osteoblasts to osteocytes, we investigated the actin-binding proteins, which are the control elements in the dynamic organization of the actin cytoskeleton. MATERIALS AND METHODS: We used primary chick osteocytes and osteoblasts, the phenotypes of which were confirmed by use of OB7.3, a chick osteocyte-specific monoclonal antibody and by detection of alkaline phosphatase activity, respectively. Immunofluorescence staining was performed for visualizing actin-binding proteins. Furthermore, we applied shear stress at 12 dyns/cm2 to the cells and compared the changes in fimbrin distribution. RESULTS: Immunofluorescence staining of fimbrin and alpha-actinin showed their presence in the processes of osteocytes, with especially strong signals of fimbrin at the sites of divarication of the processes. Anti-villin was reactive with the osteocyte cytoplasm but not with the processes. Interestingly, anti-villin immunoreactivity was much stronger in osteocytes than in osteoblasts. Filamin was localized along the stress fibers of osteoblasts but was seen only in those in the proximal base of osteocyte processes. Myosin and tropomyosin were found to have a similar pattern in both stress fibers of osteoblasts and osteocyte processes. The difference in the distribution of anti-spectrin staining was highly dramatic. Osteoblasts immunostained with anti-spectrin showed punctate signals on their cytoplasmic membranes, whereas anti-spectrin in osteocytes detected a filamentous organization; and the spectrin was totally colocalized with actin from the distal portion of the cytoplasmic processes to the cell center. In osteoblasts, shear stress induced recruitment of fimbrin to the end of stress fibers. However, fimbrin in the osteocyte processes did not change its localization. CONCLUSION: We found that terminal differentiation of osteoblasts to osteocytes was accompanied by highly dramatic changes in the distribution of actin-binding proteins, changes of which may affect cellular function.

Actinin↗
PubMed2004

Osteocyte viability and regulation of osteoblast function in a 3D trabecular bone explant under dynamic hydrostatic pressure.

UNLABELLED: A new trabecular bone explant model was used to examine osteocyte-osteoblast interactions under DHP loading. DHP loading enhanced osteocyte viability as well as osteoblast function measured by osteoid formation. However, live osteocytes were necessary for osteoblasts to form osteoids in response to DHP, which directly show osteoblast-osteocyte interactions in this in vitro culture. INTRODUCTION: A trabecular bone explant model was characterized and used to examine the effect of osteocyte and osteoblast interactions and dynamic hydrostatic pressure (DHP) loading on osteocyte viability and osteoblast function in long-term culture. MATERIALS AND METHODS: Trabecular bone cores obtained from metacarpals of calves were cleaned of bone marrow and trabecular surface cells and divided into six groups, (1) live cores + dynamic hydrostatic pressure (DHP), (2) live cores + sham, (3) live cores + osteoblast + DHP, (4) live cores + osteoblast + sham, (5) devitalized cores + osteoblast + DHP, and (6) devitalized cores + osteoblast + sham, with four culture durations (2, 8, 15, and 22 days; n = 4/group). Cores from groups 3-6 were seeded with osteoblasts, and cores from groups 5 and 6 were devitalized before seeding. Groups 1, 3, and 5 were subjected to daily DHP loading. Bone histomorphometry was performed to quantify osteocyte viability based on morphology and to assess osteoblast function based on osteoid surface per bone surface (Os/Bs). TUNEL staining was performed to evaluate the mode of osteocyte death under various conditions. RESULTS: A portion of osteocytes remained viable for the duration of culture. DHP loading significantly enhanced osteocyte viability up to day 8, whereas the presence of seeded osteoblasts significantly decreased osteocyte viability. Cores with live osteocytes showed higher Os/Bs compared with devitalized cores, which reached significant levels over a greater range of time-points when combined with DHP loading. DHP loading did not increase Os/Bs in the absence of live osteocytes. The percentage of apoptotic cells remained the same regardless of treatment or culture duration. CONCLUSION: Enhanced osteocyte viability with DHP suggests the necessity of mechanical stimulation for osteocyte survival in vitro. Furthermore, osteocytes play a critical role in the transmission of signals from DHP loading to modulate osteoblast function. This explant culture model may be used for mechanotransduction studies in long-term cultures.

Animals↗
PubMed1994

Characteristics and properties of osteocytes in culture.

Although the osteocyte is the most abundant among the highly differentiated cells of mature bone (osteocytes, lining cells, osteoblasts, and osteoclasts), its properties and functions are the least known and understood. Here we isolated osteocytes from mixed populations of bone cells liberated from fetal chick calvariae by alternate treatments with collagenase and EDTA. The osteocytes were removed from the bone cell populations by binding them via an osteocyte-specific antibody (MAb OB 7.3) to magnetic beads and removing the beads together with the coupled osteocytes from the population using a magnet. Isolated osteocytes were found to be highly differentiated, postmitotic cells that required their typical stellate morphology in culture. Osteocyte populations had alkaline phosphatase (ALP) activity somewhat lower than that of the osteoblast-like cell populations from which they were separated by the immunodissection procedure. On the single-cell level, the ALP activity was highly variable. Parathyroid hormone (PTH) receptors were found to be present on osteocytes as well as on osteoblast-like cells, but not on fibroblast-like cells of the outer periosteum. In response to PTH, osteocytes increased their intracellular levels of cAMP, as did the osteoblast-like cells. Osteocytes appeared to be somewhat more sensitive to PTH than osteoblasts. When seeded onto dentin slices, osteocytes did not corrode the dentin surface to any appraisable degree. We therefore found no evidence to support the notion that osteocytes play a role in the calcium homeostasis through osteocytic osteolysis. Whether osteocytes play an important role in perceiving and transducing hormonal and/or mechanical stimuli remains open for future research.

Alkaline Phosphatase↗
PubMed1996

Adhesive properties of isolated chick osteocytes in vitro.

Different functions have been proposed for osteocytes over time, but it is now generally accepted that their most important task lies in the sensing of strain caused by mechanical loading on bone. The fact that mechanical strain can be sensed as deformation of the extracellular matrix or as fluid shear stress along the cell, in the space between cell membrane and extracellular matrix, requires that osteocytes have close (specialized) contact with the bone matrix. We studied to which extracellular matrix proteins isolated chicken osteocytes adhere and whether this adhesion is mediated by specific cell adhesion receptors called integrins. The adhesive properties of the osteocytes were compared with that of osteoblasts. Osteocytes (and osteoblasts) adhere to the same substrates (i.e., collagen types I and II, collagen fibers, osteopontin, osteonectin, fibronectin, fibrinogen, thrombospondin, and laminin). Cell spreading varied between substrates, from all cells rounded on thrombospondin to all cells fully spread out on osteopontin, osteonectin, vitronectin, fibronectin, fibrinogen, and laminin. The percentage of osteocytes adhered was equivalent to that of osteoblasts adhered on all substrates except osteopontin and vitronectin, where osteocytes adhered less. The adhesion of osteocytes and osteoblasts to osteopontin, osteonectin, vitronectin, and fibrinogen was strongly inhibited, and to fibronectin and laminin moderately, by an RGD peptide. No RGD inhibition was found on collagen. An antibody against chicken integrin alpha v beta 3, the monoclonal antibody (MAb) 23C6, did not interfere with the adhesion of osteocytes and osteoblasts to matrix proteins, whereas an MAb against chicken integrin subunit beta 1 (CSAT) strongly inhibited adhesion to all substrates. Labeling with osteocyte-specific MAbs (OB7.3, OB37.4, and OB37.11) also did not hinder the adhesion of osteocytes to collagen type I, vitronectin, and osteopontin. Adhesion sites on osteocytes were small compared with the large adhesion plaques of osteoblasts, as demonstrated by interference reflection microscopy and immunocytochemically by staining for vinculin. Osteocyte adhesion is analogous to osteoblast adhesion with regard to the range of extracellular matrix proteins to which they adhere. The adhesion is mediated by the integrin subunit beta 1, but other integrins or nonintegrin adhesion receptors are also involved. Osteocytes make contact with the extracellular matrix via small attachment points which colocalize with vinculin. This connection between the bone matrix and the cytoskeleton may be important for osteocytic sensing of mechanical strain, as it supplies a transduction route of extracellular (mechanical) signals into intracellular messages.

Animals↗
PubMed2005

Three-dimensional reconstruction of chick calvarial osteocytes and their cell processes using confocal microscopy.

Osteocytes are surrounded by hard bone matrix. Therefore, it has not previously been possible to demonstrate the real architecture of the osteocyte network in bone. We previously reported that it is possible to observe osteocytes in bone by labeling the cells with fluorescence and using confocal laser scanning (CLS) microscopy. In this study, we for the first time conducted an extensive analysis of the morphology and morphometry of the three-dimensional (3D) osteocyte structure using three-dimensionally reconstructed fluorescent images. Sixteen-day-old embryonic chick calvariae were stained with fluorescently labeled phalloidin and observed using a confocal laser scanning microscope. Morphometry of osteocytes in the calvaria was analyzed using extensive three-dimensional reconstructing software IMARIS, process length measuring software NEURON TRACER and cell surface area-/cell volume-analyzing software SURPASS. From the IMARIS-derived images, we found that the average of 10 osteocytes is 52.7 +/- 5.7 processes, and the point-to-point distance between centers of the osteocytes was 24.1 +/- 2.8 microm. In addition, we could calculate that each osteocyte spans an average of 4180 +/- 673 microm3 of bone volume. NEURON TRACER showed that the length of osteocyte processes was 0.26 +/- 0.02 microm per 1 microm3 bone compartment. In addition, SURPASS indicated that the surface area of osteocytes was 0.36 +/- 0.03 microm2 per 1 microm3 bone compartment and that the volume ratio of osteocyte cell body to bone compartment was 9.42% +/- 1.18%. Together, the average total length of the processes, the average surface area, and the average volume of one osteocyte were 1070 +/- 145 microm, 1509 +/- 113 microm2, and 394 +/- 49 microm3, respectively. It is possible to reconstruct the real architecture of the osteocyte network and obtain morphometric data from fluorescently labeled osteocytes in chick calvaria.

Animals↗
PubMed2002

Osteocytes: a proposed multifunctional bone cell.

Most cell types are ascribed a single function. The osteoclast holds the unique distinction of performing only one function in the body - that of resorbing bone. The osteoblast has been ascribed the major function of bone matrix production. Other less well-defined cell types include progenitor cells and the nebulous cell type that can support osteoclast formation upon stimulation with various bone resorbing cytokines. Obviously, these cells could have other functions. The definition of an osteocyte is descriptive of its location - cells surrounded by mineralized matrix - not its function. For this year's Sun Valley Workshop on osteocytes, several proposed functions will be presented. First, a general consensus exists that osteocytes are most likely sensitive to mechanotransduction and translate mechanical strain into biochemical signals. Consensus does not exist on the nature of the mechanical strain, the form of the biochemical signals, the target cell(s), or the viability status of the osteocyte. Second, it is also proposed that this cell is incredibly adaptable and expresses plasticity in response to mechanical stimuli. In other words, this cell can readjust its responses to strain in the presence of other bone agents such as hormones and bone factors. Third, it will also be presented that osteocytes maintain systemic mineral homeostasis by regulating mineral release and deposition over the enormous surface area over which these cells interface with the surrounding matrix. Although osteocytes are terminally differentiated osteoblasts, they appear to have separate and distinct properties from their predecessors. Bone cell biologists loaded with an arsenal of bone anabolic and catabolic factors are examining the expression and effects of these factors on osteocytes. Engineers trained in mathematical modeling have generated new models of strain and connectivity to be tested. The unique morphology of osteocytes suggests that the cytoskeleton in these cells may function differently from osteoblasts and other cell types. Osteocytes may consist of different subpopulations; some that possess receptors for parathyroid hormone (PTH) and others that only express receptors for carboxyl terminal PTH suggesting different functions and responses. Osteocytes may respond rapidly to strain through glutamate receptor-like mechanisms, through calcium influxes, through gap junctions, and less rapidly through the production of small molecules and factors. Strain may take the form of substrate stretching and/or fluid flow. Osteocytes may communicate with other osteocytes and/or bone surface cells such as lining cells, stromal cells, osteoblasts, and/or osteoclasts and their precursors. The viability status of the osteocyte may determine the type of signals sent from these cells. If the cells are deprived of oxygen or nutrients, the apoptotic cells may send signals for initiation of resorption. If the cells and/or their dendritic process are ripped or torn by microdamage, they may send signals of both resorption and formation. If the majority of these theories are correct, then the osteocyte is the 'smart' cell that can direct or orchestrate the bone resorbing and bone forming cells even in its death and dying.

Journal Article↗
PubMed2004

Upregulation of osteopontin by osteocytes deprived of mechanical loading or oxygen.

UNLABELLED: The pathway(s) by which disuse is transduced into locally mediated osteoclastic resorption remain unknown. We found that both acute disuse (in vivo) and direct hypoxia (in vitro) induced rapid upregulation of OPN expression by osteocytes. Within the context of OPN's role in osteoclast migration and attachment, hypoxia-induced osteocyte OPN expression may serve to mediate disuse-induced bone resorption. INTRODUCTION: We have recently reported that disuse induces osteocyte hypoxia. Because hypoxia upregulates osteopontin (OPN) in nonconnective tissue cells, we hypothesized that both disuse and hypoxia would rapidly elevate expression of OPN by osteocytes. MATERIALS AND METHODS: The response of osteocytes to 24 h of disuse was explored by isolating the left ulna diaphysis of adult male turkeys from loading (n = 5). Cortical osteocytes staining positive for OPN were determined using immunohistochemistry and confocal microscopy. In vitro experiments were performed to determine if OPN expression was altered in MLO-Y4 osteocytes by direct hypoxia (3, 6, 24, and 48 h) or hypoxia (3 and 24 h) followed by 24 h of reoxygenation. A final in vitro experiment explored the potential of protein kinase C (PKC) to regulate hypoxia-induced osteocyte OPN mRNA alterations. RESULTS: We found that 24 h of disuse significantly elevated osteocyte OPN expression in vivo (145% versus intact bones; p = 0.02). We confirmed this finding in vitro, by observing rapid and significant upregulation of OPN protein expression after 24 and 48 h of hypoxia. Whereas 24 h of reoxygenation after 3 h of hypoxia restored normal osteocyte OPN expression levels, 24 h of reoxygenation after 24 h of hypoxia did not mitigate elevated osteocyte OPN expression. Finally, preliminary inhibitor studies suggested that PKC serves as a potent upstream regulator of hypoxia-induced osteocyte OPN expression. CONCLUSIONS: Given the documented roles of OPN as a mediator of environmental stress (e.g., hypoxia), an osteoclast chemotaxant, and a modulator of osteoclastic attachment to bone, we speculate that hypoxia-induced osteocyte OPN expression may serve to mediate disuse-induced osteoclastic resorption. Furthermore, it seems that a brief window of time exists in which reoxygenation (as might be achieved by reloading bone) can serve to inhibit this pathway.

Animals↗
PubMed2004

Osteocytic expression of constitutive NO synthase isoforms in the femoral neck cortex: a case-control study of intracapsular hip fracture.

UNLABELLED: NO is an osteocytic signaling molecule that can inhibit osteoclasts. The NO synthases eNOS and nNOS were expressed by >50% of osteonal osteocytes in controls. Hip fracture cases showed +NOS osteocytes only in deep osteonal bone, and 25-35% reduced expression overall. These data are consistent with increased osteonal vulnerability to deep osteoclastic attack. INTRODUCTION: Osteocytes may regulate the response to mechanical stimuli in bone through the production of local signaling molecules such as NO derived from the NO synthase eNOS. Because NO is inhibitory to osteoclastic resorption, it has been suggested that osteocytes expressing eNOS act as sentinels, confining resorption within single osteons. Recently, nNOS has been shown to be present in osteocytes of adult human bone. MATERIALS AND METHODS: Cross-sections of the femoral neck (eight female cases of intracapsular hip fracture and seven postmortem controls; age, 68-91 years) were analyzed by immunohistochemistry. The percentages of osteocytes expressing each of these two isoforms were calculated, and their distances to the nearest canal surface were measured. RESULTS: The percentage of +nNOS osteocytes was lower in the fracture cases than in the controls (cases: 43.12 +/- 1.49, controls: 56.68 +/- 1.45; p < 0.0001). Compared with nNOS, eNOS expression was further reduced (p = 0.009) in the cases but was not different in the controls (cases: 36.41 +/- 1.53, controls: 56.47 +/- 2.41; p < 0.0001). The minimum distance of +eNOS or +nNOS osteocytes to a canal surface was higher in the cases compared with controls (eNOS: controls; 44.4 +/- 2.2 microm, cases: 61.7 +/- 2.0 microm; p < 0.0001; nNOS: controls: 52.4 +/- 1.7 microm, cases: 60.2 +/- 2.1 microm; p = 0.0039). +eNOS osteocytes were closer to the canal surfaces than +nNOS osteocytes in the controls by 8.00 +/- 4.0 microm (p = 0.0012). CONCLUSION: The proportions of osteocytes expressing nNOS and eNOS were both reduced in the fracture cases, suggesting that the capacity to generate NO might be reduced. Furthermore, the reduction in NOS expression occurs in those osteocytes closest to the canal surface, suggesting that the ability of NO to minimize resorption depth might be impaired. Further studies are needed on the regulation of the expression and activity of these distinct NOS isoforms.

Aged↗
PubMed2002

Spatial distribution of Bax and Bcl-2 in osteocytes after bone fatigue: complementary roles in bone remodeling regulation?

Osteocyte apoptosis appears to play a key role in the mechanism by which osteoclastic resorption activity targets bone for removal, because osteocyte apoptosis occurs in highly specific association with microdamage and subsequent remodeling after fatigue. However, beyond terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-biotin nick end labeling (TUNEL) assay, little is known about the mechanisms controlling osteocyte apoptosis in vivo. In the current studies, expression of Bax, a proapoptotic gene product, and Bcl-2, an antiapoptotic gene product, was determined in osteocytes of fatigued rat bone using immunocytochemical staining and compared with TUNEL staining patterns. Bax and Bcl-2 were evident in osteocytes by 6 h after loading. Moreover, Bax and Bcl-2 in osteocytes were expressed differently as a function of distance from microdamage sites. The peak of Bax expression and TUNEL+ staining in osteocytes was observed immediately at the microcrack locus, which is where bone resorption occurs in this system; in contrast, Bcl-2 expression, the antiapoptotic signal, reached its greatest level at some distance (1-2 mm) from microcracks. These data suggest that near sites of microinjury in bone, those osteocytes that do not undergo apoptosis are prevented from doing so by active protection mechanisms. Moreover, the zone of apoptotic osteocytes around microcracks was effectively "walled in" by a surrounding halo of surviving osteocytes actively expressing Bc1-2. Thus, the expression pattern of apoptosis-inhibiting gene products by osteocytes surrounding the apoptotic osteocyte at microdamage sites also may provide important signals in the guidance of resorption processes that occur in association with osteocyte apoptosis after fatigue.

Animals↗
PubMed1994

Histological identification of osteocytes in the allegedly acellular bone of the sea breams Acanthopagrus australis, Pagrus auratus and Rhabdosargus sarba (Sparidae, Perciformes, Teleostei).

The bone of advanced teleost fishes such as those of the family Sparidae is said to lack osteocytes or to be acellular. Acellularity has been determined by apparent lack of osteocyte lacunae. This study questions the validity of this criterion. Scanning electron and light microscopy of paraffin and resin sections were used to show that the sides of sea bream mandibles consist of laminar parallel-fibred bone that we call tubular bone, because it contains tubules, and localised regions of Sharpey fibre bone. Osteocytes lie along the walls of tubules that also contain collagen fibril bundles (T-fibres), or in the lumens of tubules that do not contain T-fibres. We show that the osteocytes are derived from osteoblasts. The T-fibre system is different from other fibre systems that have been described. The tubules enclose wide T-fibres (lenticular in cross-section, maximum width about 8 microns) that taper at their ends and continue as thin T-fibres (round in cross-section, about 2 microns wide). The T-fibres originate in the periosteum. In mature tubular bone, spaces of increasing size develop around the osteocytes. Osteocytes are released from the bone matrix and become postosteocytes or bone-lining cells. Secondary bone lines the largest spaces. In Sharpey fibre bone, small osteocytes in small lacunae (about 2 microns wide) are found in columns parallel to the Sharpey fibres. Large osteocytes are found in large round spaces and are much larger than comparable osteocytes in lacunae in the bone of the salmon Salmo salar. We conclude that an absence of visible or conventional osteocyte lacunae does not mean that the cells themselves are absent. There are cells and two types of collagen fibre bundle in the tubules. The cells are osteocytes derived from osteoblasts, and these osteocytes apparently resorb bone with the result that large amounts of bone are destroyed. "Acellular" tubular and Sharpey fibre bone are types of cellular bone that differ from each other and from conventional cellular bone.

Animals↗
PubMed2006

Osteocytes subjected to pulsating fluid flow regulate osteoblast proliferation and differentiation.

Osteocytes are thought to orchestrate bone remodeling, but it is unclear exactly how osteocytes influence neighboring bone cells. Here, we tested whether osteocytes, osteoblasts, and periosteal fibroblasts subjected to pulsating fluid flow (PFF) produce soluble factors that modulate the proliferation and differentiation of cultured osteoblasts and periosteal fibroblasts. We found that osteocyte PFF conditioned medium (CM) inhibited bone cell proliferation, and osteocytes produced the strongest inhibition of proliferation compared to osteoblasts and periosteal fibroblasts. The nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) attenuated the inhibitory effects of osteocyte PFF CM, suggesting that a change in NO release is at least partially responsible for the inhibitory effects of osteocyte PFF CM. Furthermore, osteocyte PFF CM stimulated osteoblast differentiation measured as increased alkaline phosphatase activity, and l-NAME decreased the stimulatory effects of osteocyte PFF CM on osteoblast differentiation. We conclude that osteocytes subjected to PFF inhibit proliferation but stimulate differentiation of osteoblasts in vitro via soluble factors and that the release of these soluble factors was at least partially dependent on the activation of a NO pathway in osteocytes in response to PFF. Thus, the osteocyte appears to be more responsive to PFF than the osteoblast or periosteal fibroblast with respect to the production of soluble signaling molecules affecting osteoblast proliferation and differentiation.

Animals↗
PubMed2000

Decline in osteocyte lacunar density in human cortical bone is associated with accumulation of microcracks with age.

Despite osteocytes' ideal position to sense the local environment and thereby influence bone remodeling, the function of osteocytes in bone remains controversial. In this study, histomorphometric examination of male and female femoral middiaphyseal cortical bone was conducted to determine if bone's remodeling response, indicated by tissue porosity and accumulation of damage, is associated with osteocyte lacunar density (number of osteocyte lacunae per bone area). The results support the sensory role of the osteocyte network as the decline in osteocyte lacunar density in human cortical bone is associated with the accumulation of microcracks and increase in porosity with age. Porosity and microcrack density increased exponentially with a decline in osteocyte lacunar density indicating that a certain minimum number of osteocytes is essential for an "operational" network. No gender-related differences were found in the relationship of osteocyte lacunar density to age, porosity, or microcrack density. The coefficient of variation of osteocyte lacunar density increased linearly with age, indicating that aging bone tissue is characterized by increased heterogeneity in the spatial organization of osteocytes. Osteocyte lacunar density, porosity, and microcrack density exhibited the same exponential probability density distribution in the donor population, indicating their regulation by similar biological phenomena.

Adolescent↗
PubMed2004

The metalloproteinase MT1-MMP is required for normal development and maintenance of osteocyte processes in bone.

The osteocyte is the terminally differentiated state of the osteogenic mesenchymal progenitor immobilized in the bone matrix. Despite their numerical prominence, little is known about osteocytes and their formation. Osteocytes are physically separated in the bone matrix but seemingly compensate for their seclusion from other cells by maintaining an elaborate network of cell processes through which they interact with other osteocytes and bone-lining cells at the periosteal and endosteal surfaces of the bone. This highly organized architecture suggests that osteocytes make an active contribution to the structure and maintenance of their environment rather than passively submitting to random embedding during bone growth or repair. The most abundant matrix protein in the osteocyte environment is type-I collagen and we demonstrate here that, in the mouse, osteocyte phenotype and the formation of osteocyte processes is highly dependent on continuous cleavage of type-I collagen. This collagenolytic activity and formation of osteocyte processes is dependent on matrix metalloproteinase activity. Specifically, a deficiency of membrane type-1 matrix metalloproteinase leads to disruption of collagen cleavage in osteocytes and ultimately to the loss of formation of osteocyte processes. Osteocytogenesis is thus an active invasive process requiring cleavage of collagen for maintenance of the osteocyte phenotype.

Animals↗
PubMed2006

Osteocyte apoptosis is induced by weightlessness in mice and precedes osteoclast recruitment and bone loss.

UNLABELLED: Mechanical stimulation of cultured osteocytic cells attenuates their apoptosis. We report here that, conversely, reduced mechanical forces in the murine model of unloading by tail suspension increases the prevalence of osteocyte apoptosis, followed by bone resorption and loss of mineral and strength. INTRODUCTION: Mechanical loading is critical for the maintenance of bone mass; weightlessness, as with reduced physical activity in old age, bed rest, or space flight, invariably leads to bone loss. However, the cellular and molecular mechanisms responsible for these phenomena are poorly understood. Based on our earlier findings that physiologic levels of mechanical strain prevent apoptosis of osteocytic cells in vitro, we examined here whether, conversely, reduced mechanical forces increase the prevalence of osteocyte apoptosis in vivo and whether this event is linked to bone loss. MATERIALS AND METHODS: Swiss Webster mice or OG2-11beta-hydroxysteroid dehydrogenase type 2 (OG2-11beta-HSD2) transgenic mice and wildtype littermates were tail-suspended or kept under ambulatory conditions. Static and dynamic histomorphometry and osteocyte and osteoblast apoptosis by in situ end-labeling (ISEL) were assessed in lumbar vertebra; spinal BMD was measured by DXA; and bone strength was measured by vertebral compression. RESULTS: We show that within 3 days of tail suspension, mice exhibited an increased incidence of osteocyte apoptosis in both trabecular and cortical bone. This change was followed 2 weeks later by increased osteoclast number and cortical porosity, reduced trabecular and cortical width, and decreased spinal BMD and vertebral strength. Importantly, whereas in ambulatory animals, apoptotic osteocytes were randomly distributed, in unloaded mice, apoptotic osteocytes were preferentially sequestered in endosteal cortical bone--the site that was subsequently resorbed. The effect of unloading on osteocyte apoptosis and bone resorption was reproduced in transgenic mice in which osteocytes are refractory to glucocorticoid action, indicating that stress-induced hypercortisolemia cannot account for these effects. CONCLUSIONS: We conclude that diminished mechanical forces eliminate signals that maintain osteocyte viability, thereby leading to apoptosis. Dying osteocytes in turn become the beacons for osteoclast recruitment to the vicinity and the resulting increase in bone resorption and bone loss.

Animals↗
PubMed2006

Fluid shear stress induces less calcium response in a single primary osteocyte than in a single osteoblast: implication of different focal adhesion formation.

UNLABELLED: The immediate calcium response to fluid shear stress was compared between osteocytes and osteoblasts on glass using real-time calcium imaging. The osteoblasts were responsive to fluid shear stress of up to 2.4 Pa, whereas the osteocytes were not. The difference in flow-induced calcium may be related to differences in focal adhesion formation. INTRODUCTION: To explore the immediate response to mechanical stress in a bone cell population, we examined flow-induced calcium transients. In addition, the involvement of focal adhesion-related calcium transients in response to fluid flow in the cells was studied. MATERIALS AND METHODS: Bone cells were isolated from 16-day-old embryonic chicken calvaria by serial treatment with EDTA and collagenase. Single cells on glass without intercellular connections were subjected to fluid flow, and intracellular calcium concentration was measured using imaging with fluo-3. The identification of cell populations in the same field was performed with a chick osteocyte-specific antibody, OB7.3, and an alkaline phosphatase substrate, ELF-97, for osteoblast identification afterward. Immunofluorescence staining of vinculin was performed to visualize focal adhesions. RESULTS: The percentage of cells responding to fluid shear stress at 1.2 Pa was 5.5% in osteocytes, 32.4% in osteoblasts, and 45.6% in OB7.3/ELF-97-negative cells. Furthermore, osteoblasts and OB7.3/ELF-97-negative cells were more responsive to 2.4 Pa than 1.2 Pa, whereas osteocytes were less responsive. The elevation of calcium transients over baseline did not show any significant differences in the populations. To elucidate the mechanism accounting for the fact that single osteocytes are less sensitive to fluid shear stress of up to 2.4 Pa than osteoblasts, we studied focal adhesion-related calcium transients. First, we compared focal adhesion formation between osteocytes and osteoblasts and found a larger number of focal adhesions in osteoblasts than in osteocytes. Next, when the cells were pretreated with GRGDS (0.5 mM) before flow treatment, a significant reduction of calcium transients in osteoblasts (18%) was observed, whereas calcium transients in osteocytes were not changed by GRGDS. Control peptide GRGES did not reduce the calcium transients in either cell type. Furthermore, we confirmed that osteoblasts in calvaria showed a marked formation of vinculin plaques in the periphery of the cells. However, osteocytes in calvaria showed faint vinculin plaques only at the base of the processes. CONCLUSIONS: On glass, single osteocytes are less sensitive to fluid shear stress up to 2.4 Pa than osteoblasts. The difference in calcium transients might be related to differences in focal adhesion formation. Shear stress of a higher magnitude or direct deformation may be responsible for the mechanical response of osteocytes in bone.

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PubMed2003

Reduced iliac cancellous osteocyte density in patients with osteoporotic vertebral fracture.

UNLABELLED: Iliac cancellous osteocyte density declines with age, but its relationship to vertebral fracture pathogenesis is unknown. We performed iliac bone biopsy in 44 women with clinical vertebral fracture and 56 healthy women. The fracture patients had 34% fewer osteocytes but no reduction in percent occupied lacunae. Some patients destined to sustain vertebral fracture make cancellous bone with fewer osteocytes. INTRODUCTION: Patient's with vertebral fracture have less bone than appropriate healthy controls, but other factors may contribute to bone fragility. Iliac cancellous osteocyte density declines with age in healthy women; we asked whether this variable differed between fracture patients and healthy controls. METHODS: Two groups of women were assembled. Forty-four (mean age, 66.2 years) had unequivocal evidence of bone fragility manifested as painful nontraumatic vertebral fracture, and 56 (mean age, 62.2 years) were skeletally healthy. All subjects underwent iliac bone biopsy. From archival embedded biopsy cores, new sections were stained with Goldner's trichrome, in which we enumerated osteocyte-occupied lacunae (stained), empty lacunae (unstained), and total lacunae per bone area. RESULTS: Cancellous osteocyte density was 34% lower in the fracture group than in the controls (p < 0.001); this difference was not a consequence of higher turnover, having less bone, or the small difference in age. The area under the receiver operating characteristic (ROC) curve for discrimination between the groups was >90% for osteocyte density and <75% for bone volume/tissue volume (BV/TV). The disease-related osteocyte deficit was accompanied by a proportionate reduction in empty lacunae and no change in percent occupied lacunae; therefore, it was not the result of premature death. Both superficial bone (<25 microm from the surface) and deep bone (>45 microm from the surface) were affected. In contrast, the age-related deficit is accompanied by an increase in empty lacunae and fall in percent osteocyte-occupied lacunae and occurs only in deep bone, but not in superficial bone. CONCLUSIONS: In some patients destined to sustain spontaneous vertebral compression fracture, iliac cancellous bone is made with fewer osteocytes than normal; the mechanism of osteocyte incorporation into bone needs more detailed study. Osteocyte deficiency could contribute to bone fragility, either by impairing the detection of fatigue microdamage or by reducing canalicular fluid flow. Current practices of defining vertebral fracture based on morphometry alone regardless of symptoms, and diagnosing osteoporosis based on bone densitometry alone regardless of fracture history, should be reexamined.

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