Function of osteocytes in bone

Although the structural design of cellular bone (i.e., bone containing osteocytes that are regularly spaced throughout the bone matrix) dates back to the first occurrence of bone as a tissue in evolution, and although osteocytes represent the most abundant cell type of bone, we know as yet little about the role of the osteocyte in bone metabolism. Osteocytes descend from osteoblasts. They are formed by the incorporation of osteoblasts into the bone matrix. Osteocytes remain in contact with each other and with cells on the bone surface via gap junction–coupled cell processes passing through the matrix via small channels, the canaliculi, that connect the cell body–containing lacunae with each other and with the outside world. During differentiation from osteoblast to mature osteocyte the cells lose a large part of their cell organelles. Their cell processes are packed with microfilaments. In this review we discuss the various theories on osteocyte function that have taken in consideration these special features of osteocytes. These are (1) osteocytes are actively involved in bone turnover; (2) the osteocyte network is through its large cell-matrix contact surface involved in ion exchange; and (3) osteocytes are the mechanosensory cells of bone and play a pivotal role in functional adaptation of bone. In our opinion, especially the last theory offers an exciting concept for which some biomechanical, biochemical, and cell biological evidence is already available and which fully warrants further investigations. © 1994 Wiley-Liss, Inc.     

Osteocyte-osteoclast morphological relationships and the putative role of osteocytes in bone remodeling

An osteocyte lacunae differential count under the light microscope (LM) (1-lacunae with live osteocytes, 2-empty lacunae and lacunae with degenerating osteocytes) was carried out outside the reversal lines of osteonic lamellar bone from various mammals and man to evaluate the possibility of osteocyte survival where osteoclast resorption had occurred. The polarized light microscope (PLM) was used to establish the curvature of bony lamellae outside the convexity of reversal lines: concave lamellae indicate osteocytes reabsorbed on their vascular side where they radiate long vascular dendrites; convex lamellae indicate bone resorption on the osteocyte mineral side, radiating short dendrites. In all samples it was found that: a) about 60% of osteocytes outside the reversal lines were live; b) the percentage of alive osteocytes close to reversal lines is higher when they are attacked on their mineral side. The present data support our view that surviving osteocytes, particularly those attacked from their mineral side, might intervene in the final phase of bone resorption (osteoclast inhibition?). The fact that under the transmission electron microscope (TEM) intercellular contacts were never observed between osteocytes and osteoclasts indicates that if a modulation should occur between these two cellular types it could take place by a paracrine route only. The putative role of the cells of the osteogenic system, particularly osteocytes, in the bone remodeling cycle is also discussed.     

Metabolic pathways of the fossil dinosaur bones. Part III. Intermediary and other osteocytes in the system of metabolic pathways of dinosaur bone

The fossil dinosaur bone material, 80 million years old was studied. Samples for analysis were prepared with specially elaborated methods. Images obtained in the light, transmission electron, and scanning electron microscopes revealed the spatial distribution of the osteocytes lying near and far from the vascular canal. Osteocytes of particular kind were found to be present in the immediate vicinity of the canal. The characteristic morphological structure and the localization of this osteocytes between the vascular canal and the osteocytes lying farther from the canal, as well as the mediation of this cell in the system of connections between those elements, served as a basis for the separation of this cells as the intermediary osteocyte. Among the osteocytes situated farther from the vascular canal, three kinds were distinguished: mono-, bi-, and multipolar, according to the kinds of processes and their distribution in relation to the mother cell body. By analogy with modern bone, in dinosaur bone specific functions may be ascribed to the distinguished types of osteocytes and to their differentiated processes in the conduction of nutrient and building elements and metabolites from the vascular canal to the intermediary osteocytes and, with their participation, to other osteocytes lying farther from the canal. This should naturally be analysed as the two-way tract.     

Morphological differentiation of the fossil dinosaur bone cells. Light, transmission electron-, and scanning electron-microscopic studies.

The investigation was carried out on the fossil dinosaur bone from before 80 million years. Samples for examination were prepared with specially elaborated methods. Thus the isolated osteocytes of the dinosaur bone, which had previously undergone natural fossilizing processes, were obtained. This permitted their morphological assessment. On the basis of the images obtained in the light, transmission electron, and scanning electron microscopes, two types of osteocytes were distinguished. Type I was characterized by an elongated shape, its length exceeding the width several times; the mean dimensions of this osteocyte amounted to 28.8/7.03 micron. Type II was shorter, its mean dimensions being equal to 20.28/6.88 micron. Moreover, two types of osteocytes processes were differentiated: the first was represented by thick processes, so-called 'axial processes', whose diameter ranged from 0.5 to 1.5 micron, and which, as a rule, departed from the pole of the cell. They ran farther from the mother cell body to more distant osteocytes. The other type consisted of thin processes departing from various points of the cell body in no particular arrangement but always extrapolarly. They branched off in a close vicinity of the mother osteocyte. Their diameter ranged between 0.076 and 0.35 micron.     

Immunocytochemical demonstration of extracellular matrix proteins in isolated osteocytes

 Cultures of isolated osteocytes may offer an appropriate system to study osteocyte function, since isolated osteocytes in culture behave very much like osteocytes in vivo. In this paper we studied the capacity of osteocytes to change their surrounding extracellular matrix by production of matrix proteins. With an immunocytochemical method we determined the presence of collagen type I, fibronectin, osteocalcin, osteopontin and osteonectin in cultures of isolated chicken osteocytes, osteoblasts and periosteal fibroblasts. In osteoblast and periosteal fibroblast cultures, large extracellular networks of collagen type I and fibronectin were formed, but in osteocyte populations, extracellular threads of collagen or fibronectin were only rarely found. The percentage of cells positive for osteocalcin, osteonectin and osteopontin in the Golgi apparatus, on the other hand, was highest in the osteocyte population. These results show that osteocytes have the ability to alter the composition of their surrounding extracellular matrix by producing matrix proteins. We suggest this property is of importance for the regulation of the calcification of the bone matrix immediately surrounding the cells. More importantly, as osteocytes depend for their role as mechanosensor cells on their interaction with matrix proteins, the adaptation of the surrounding matrix offers a way to regulate their response to mechanical loading. Accepted: 9 July 1996     

Mechanically stimulated osteocytes regulate osteoblastic activity via gap junctions

The strong correlation between a bone's architectural properties and the mechanical forces that it experiences has long been attributed to the existence of a cell that not only detects mechanical load but also structurally adapts the bone matrix to counter it. One of the most likely cellular candidates for such a "mechanostat" is the osteocyte, which resides within the mineralized bone matrix and is perfectly situated to detect mechanically induced signals. However, as osteocytes can neither form nor resorb bone, it has been hypothesized that they orchestrate mechanically induced bone remodeling by coordinating the actions of cells residing on the bone surface, such as osteoblasts. To investigate this hypothesis, we developed a novel osteocyte-osteoblast coculture model that mimics in vivo systems by permitting us to expose osteocytes to physiological levels of fluid shear while shielding osteoblasts from it. Our results show that osteocytes exposed to a fluid shear rate of 4.4 dyn/cm² rapidly increase the alkaline phosphatase activity of the shielded osteoblasts and that osteocytic-osteoblastic physical contact is a prerequisite. Furthermore, both functional gap junctional intercellular communication and the mitogen-activated protein kinase, extracellular signal-regulated kinase 1/2 signaling pathway are essential components in the osteoblastic response to osteocyte communicated mechanical signals. By utilizing other nonosteocytic coculture models, we also show that the ability to mediate osteoblastic alkaline phosphatase levels in response to the application of fluid shear is a phenomena unique to osteocytes and is not reproduced by other mesenchymal cell types. osteocyte; osteoblast; fluid-flow; coculture; mechanical stimulation; gap junction; intercellular communication     

Estrogen protects primary osteocytes against glucocorticoid-induced apoptosis

Glucocorticoid-induced osteoporosis may be at least in part due to the increased apoptosis of osteocytes. To study the role of osteocyte apoptosis in glucocorticoid-induced osteoporosis, we isolated primary osteocytes from murine calvaria for the analysis of the effects of dexamethasone in in vitro culture. The cells were identified by morphology, cytochemical staining, immunocytochemical staining and mRNA expression of phosphate-regulating gene with homology to endopeptidases on the X chromosome (PHEX) and sclerosteosis/van Buchem disease gene (SOST). We found that dexamethasone induced osteocyte apoptosis in a dose-dependent manner. A glucocorticoid receptor antagonist, mifepristone (RU486), suppressed dexamethasone-induced osteocyte apoptosis, suggesting that it was mediated by glucocorticoid receptor. Immunocytochemical stainings showed that glucocorticoid receptors are present in primary osteocytes, and they were translocated to nuclei after the exposure to dexamethasone. Addition of estrogen prevented glucocorticoid receptor translocation into nuclei. Corresponding antiapoptotic effects in primary osteocytes were also seen after the pretreatment of primary osteocytes with a picomolar concentration of estrogen. The pure antiestrogen ICI 182,780 inhibited estrogen effect on apoptosis induced by dexamethasone. These data suggest that glucocorticoid receptors play an important role in glucocorticoid-induced osteocyte apoptosis. Most importantly, estrogen has a protective effect {against osteocyte}{ }{apoptosis}. To conclude, the mechanism of glucocorticoid-induced osteoporosis may be due to the apoptosis of osteocytes, which can be opposed by estrogen.     

MT1-MMP modulates the mechanosensitivity of osteocytes

Membrane-type matrix metalloproteinase-1 (MT1-MMP) is expressed by mechanosensitive osteocytes and affects bone mass. The extracellular domain of MT1-MMP is connected to extracellular matrix, while its intracellular domain is a strong modulator of cell signaling. In theory MT1-MMP could thus transduce mechanical stimuli into a chemical response. We hypothesized that MT1-MMP plays a role in the osteocyte response to mechanical stimuli. MT1-MMP-positive and knockdown (siRNA) MLO-Y4 osteocytes were mechanically stimulated with a pulsating fluid flow (PFF). Focal adhesions were visualized by paxillin immunostaining. Osteocyte number, number of empty lacunae, and osteocyte morphology were measured in long bones of MT1-MMP(+/+) and MT1-MMP(-/-) mice. PFF decreased MT1-MMP mRNA and protein expression in MLO-Y4 osteocytes, suggesting that mechanical loading may affect pericellular matrix remodeling by osteocytes. MT1-MMP knockdown enhanced NO production and c-jun and c-fos mRNA expression in response to PFF, concomitantly with an increased number and size of focal adhesions, indicating that MT1-MMP knockdown osteocytes have an increased sensitivity to mechanical loading. Osteocytes in MT1-MMP(-/-) bone were more elongated and followed the principle loading direction, suggesting that they might sense mechanical loading. This was supported by a lower number of empty lacunae in MT1-MMP(-/-) bone, as osteocytes lacking mechanical stimuli tend to undergo apoptosis. In conclusion, mechanical stimulation decreased MT1-MMP expression by MLO-Y4 osteocytes, and MT1-MMP knockdown increased the osteocyte response to mechanical stimulation, demonstrating a novel and unexpected role for MT1-MMP in mechanosensing.     

Phagocytosis of osteocytes by osteoclasts in femora of two week-old rabbits

Summary The osteoclast-osteocyte relationship at the endosteal surface of femora of two-week old rabbits was studied. Light microscopic observations suggest that during physiological resorption phagocytosis by osteoclasts of osteocytes takes place. Serial sections confirm that the cells are totally engulfed within the cytoplasm of the osteoclasts. Ultrastructural studies support these findings and indicate that the initial stage of phagocytosis of the osteocytes consists of the insinuation of an extension of the ruffled border into the osteocyte lacuna. These extensions are seen to make close contact with the osteocytes prior to their engulfment by the osteoclasts and their final digestion within phagosomes.     

E11/gp38 selective expression in osteocytes: regulation by mechanical strain and role in dendrite elongation

Within mineralized bone, osteocytes form dendritic processes that travel through canaliculi to make contact with other osteocytes and cells on the bone surface. This three-dimensional syncytium is thought to be necessary to maintain viability, cell-to-cell communication, and mechanosensation. E11/gp38 is the earliest osteocyte-selective protein to be expressed as the osteoblast differentiates into an osteoid cell or osteocyte, first appearing on the forming dendritic processes of these cells. Bone extracts contain large amounts of E11, but immunostaining only shows its presence in early osteocytes compared to more deeply embedded cells, suggesting epitope masking by mineral. Freshly isolated primary osteoblasts are negative for E11 expression but begin to express this protein in culture, and expression increases with time, suggesting differentiation into the osteocyte phenotype. Osteoblast-like cell lines 2T3 and Oct-1 also show increased expression of E11 with differentiation and mineralization. E11 is highly expressed in MLO-Y4 osteocyte-like cells compared to osteoblast cell lines and primary osteoblasts. Differentiated, mineralized 2T3 cells and MLO-Y4 cells subjected to fluid flow shear stress show an increase in mRNA for E11. MLO-Y4 cells show an increase in dendricity and elongation of dendrites in response to shear stress that is blocked by small interfering RNA specific to E11. In vivo, E11 expression is also increased by a mechanical load, not only in osteocytes near the bone surface but also in osteocytes more deeply embedded in bone. Maximal expression is observed not in regions of maximal strain but in a region of potential bone remodeling, suggesting that dendrite elongation may be occurring during this process. These data suggest that osteocytes may be able to extend their cellular processes after embedment in mineralized matrix and have implications for osteocytic modification of their microenvironment.     

Phenotypic characterization of osteoarthritic osteocytes from the sclerotic zones: a possible pathological role in subchondral bone sclerosis

Subchondral bone sclerosis is a well-recognised manifestation of osteoarthritis (OA). The osteocyte cell network is now considered to be central to the regulation of bone homeostasis; however, it is not known whether the integrity of the osteocyte cell network is altered in OA patients. The aim of this study was to investigate OA osteocyte phenotypic changes and its potential role in OA subchondral bone pathogenesis. The morphological and phenotypic changes of osteocytes in OA samples were investigated by micro-CT, SEM, histology, immunohistochemistry, TRAP staining, apoptosis assay and real-time PCR studies. We demonstrated that in OA subchondral bone, the osteocyte morphology was altered showing rough and rounded cell body with fewer and disorganized dendrites compared with the osteocytes in control samples. OA osteocyte also showed dysregulated expression of osteocyte markers, apoptosis, and degradative enzymes, indicating that the phenotypical changes in OA osteocytes were accompanied with OA subchondral bone remodelling (increased osteoblast and osteoclast activity) and increased bone volume with altered mineral content. Significant alteration of osteocytes identified in OA samples indicates a potential regulatory role of osteocytes in subchondral bone remodelling and mineral metabolism during OA pathogenesis.     

Matrix mineralization as a trigger for osteocyte maturation.

The morphology of the osteocyte changes during the cell's lifetime. Shortly after becoming buried in the matrix, an osteocyte is plump with a rich rough endoplasmic reticulum and a well-developed Golgi complex. This "immature" osteocyte reduces its number of organelles to become a "mature" osteocyte when it comes to reside deeper in the bone matrix. We hypothesized that mineralization of the surrounding matrix is the trigger for osteocyte maturation. To verify this, we prevented mineralization of newly formed matrix by administration of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) and then examined the morphological changes in the osteocytes in rats. In the HEBP group, matrix mineralization was disturbed, but matrix formation was not affected. The osteocytes found in the unmineralized matrix were immature. Mature osteocytes were seen in the corresponding mineralized matrix in the control group. The immature osteocytes in the unmineralized matrix failed to show immunoreactivity with anti-sclerostin antibody, whereas mature osteocytes in the mineralized matrix showed immunoreactivity in both control and HEBP groups. These findings suggest that mineralization of the matrix surrounding the osteocyte is the trigger for cytodifferentiation from a plump immature form to a mature osteocyte. The osteocyte appears to start secreting sclerostin only after it matures in the mineralized bone matrix.     

PTH receptors and apoptosis in osteocytes

Osteocytes comprise a heterogenous population of terminally differentiated osteoblasts that direct bone remodeling in response to applied mechanical loading of bone. Increased osteocyte density accompanies the anabolic effect of PTH in vivo, whereas accelerated osteocyte death may be precipitated by estrogen deficiency or excess glucocorticoid exposure (conditions benefitted by intermittent PTH therapy) and by renal failure (where circulating intact PTH and, especially, PTH carboxylfragments are elevated). Osteocytes express type-1 PTH/ PTHrP receptors (PTH1Rs), which are fully activated by aminoterminal PTH fragments and couple to multiple signal transducers, including adenylyl cyclase and phospholipase C. Activation of PTH1Rs in osteocytes promotes gap junction-mediated intercellular coupling, increases expression of MMP-9, potentiates calcium influx via stretch-activated cation channels, amplifies the osteogenic response to mechanical loading in vivo, and regulates apoptosis. Control of osteocyte apoptosis by PTH1Rs is complex, in that intermittent PTH(1-34) administration reduces the fraction of vertebral apoptotic osteocytes at 1 month in adult mice but increases femoral metaphyseal osteocyte apoptosis at 1-2 weeks in young rats. In MLO-Y4 cells, PTH(1-34) prevents apoptosis otherwise induced within 6 hr by dexamethasone. In older studies, large doses of intact PTH(1-84) caused rapid "degenerative" morphologic changes in osteocytes, similar to those described in renal osteodystrophy. We isolated clonal conditionally immortalized osteocytic (OC) cell lines from mice homozygous for targeted ablation of the PTH1R gene. OC cells express abundant (2-3 x 10(6) per cell) receptors specific for the carboxyl(C)-terminus of intact PTH(1-84) ("CPTHRs") but, as expected, do not express PTH1Rs or respond to PTH(1-34). CPTHRs are expressed at much lower levels by other skeletally-derived cell lines. Several highly conserved ligand determinants of CPTHR binding have been identified, including PTH(24-27), PTH(53-54) and the sequence PTH(55-84), loss of which reduces binding affinity by over 100-fold. Human PTH(53-84), like PTH(1-84), PTH(24-84), and PTH(39-84), increases OC cell apoptosis. Ala-scanning mutagenesis to define sequences within PTH(55-84) important for binding and bioactivity is underway. We conclude that osteocytes may be important targets for CPTH fragments that are secreted by the parathyroid glands or generated by peripheral metabolism of intact PTH and that accumulate in blood, especially in renal failure. Studies of functional interplay between responses to CPTHRs and (transfected) PTH1Rs, using receptor-specific ligands in OC cells, should provide new insight into PTH regulation of osteocyte function and survival.     

Death of osteocytes turns off the inhibition of osteoclasts and triggers local bone resorption

Osteocytes have been suggested to play a role in the regulation of bone resorption, although their effect on bone turnover has remained controversial. In order to study this open question, we developed an organ culture system based on isolated rat calvaria, where the osteocyte viability and its effect on osteoclastic bone resorption can be monitored. Our results suggest that osteocytes are constitutively negative regulators of osteoclastic activity. Osteoclasts, which were cultured on calvarial slices with living osteocytes inside, failed to form actin rings which are the hallmarks of resorbing cells. A similar inhibitory effect was also achieved by the conditioned medium obtained from calvarial organ culture, suggesting that living osteocytes produce yet unrecognized osteoclast inhibitors. On the contrary, when osteocyte apoptosis was induced, this inhibitory effect disappeared and strong osteoclastic bone resorption activity was observed. Thus, local apoptosis of osteocytes may play a major role in triggering local bone remodeling.     

High mobility group box 1 protein regulates osteoclastogenesis through direct actions on osteocytes and osteoclasts in vitro

Old age and Cx43 deletion in osteocytes are associated with increased osteocyte apoptosis and osteoclastogenesis. We previously demonstrated that apoptotic osteocytes release elevated concentrations of the proinflammatory cytokine, high mobility group box 1 protein (HMGB1) and apoptotic osteocyte conditioned media (CM) promotes osteoclast differentiation. Further, prevention of osteocyte apoptosis blocks osteoclast differentiation and attenuates the extracellular release of HMGB1 and RANKL. Moreover, sequestration of HMGB1, in turn, reduces RANKL production/release by MLO-Y4 osteocytic cells silenced for Cx43 (Cx43^def), highlighting the possibility that HMGB1 promotes apoptotic osteocyte-induced osteoclastogenesis. However, the role of HMGB1 signaling in osteocytes has not been well studied. Further, the mechanisms underlying its release and the receptor(s) responsible for its actions is not clear. We now report that a neutralizing HMGB1 antibody reduces osteoclast formation in RANKL/M-CSF treated bone marrow cells. In bone marrow macrophages (BMMs), toll-like receptor 4 (TLR4) inhibition with LPS-RS, but not receptor for advanced glycation end products (RAGE) inhibition with Azeliragon attenuated osteoclast differentiation. Further, inhibition of RAGE but not of TLR4 in osteoclast precursors reduced osteoclast number, suggesting that HGMB1 produced by osteoclasts directly affects differentiation by activating TLR4 in BMMs and RAGE in preosteoclasts. Our findings also suggest that increased osteoclastogenesis induced by apoptotic osteocytes CM is not mediated through HMGB1/RAGE activation and that direct HMGB1 actions in osteocytes stimulate pro-osteoclastogenic signal release from Cx43^def osteocytes. Based on these findings, we propose that HMGB1 exerts dual effects on osteoclasts, directly by inducing differentiation through TLR4 and RAGE activation and indirectly by increasing pro-osteoclastogenic cytokine secretion from osteocytes.     

Osteocyte network; a negative regulatory system for bone mass augmented by the induction of Rankl in osteoblasts and Sost in osteocytes at unloading

Reduced mechanical stress is a major cause of osteoporosis in the elderly, and the osteocyte network, which comprises a communication system through processes and canaliculi throughout bone, is thought to be a mechanosensor and mechanotransduction system; however, the functions of osteocytes are still controversial and remain to be clarified. Unexpectedly, we found that overexpression of BCL2 in osteoblasts eventually caused osteocyte apoptosis. Osteoblast and osteoclast differentiation were unaffected by BCL2 transgene in vitro. However, the cortical bone mass increased due to enhanced osteoblast function and suppressed osteoclastogenesis at 4 months of age, when the frequency of TUNEL-positive lacunae reached 75%. In the unloaded condition, the trabecular bone mass decreased in both wild-type and BCL2 transgenic mice at 6 weeks of age, while it decreased due to impaired osteoblast function and enhanced osteoclastogenesis in wild-type mice but not in BCL2 transgenic mice at 4 months of age. Rankl and Opg were highly expressed in osteocytes, but Rankl expression in osteoblasts but not in osteocytes was increased at unloading in wild-type mice but not in BCL2 transgenic mice at 4 months of age. Sost was locally induced at unloading in wild-type mice but not in BCL2 transgenic mice, and the dissemination of Sost was severely interrupted in BCL2 transgenic mice, showing the severely impaired osteocyte network. These findings indicate that the osteocyte network is required for the upregulation of Rankl in osteoblasts and Sost in osteocytes in the unloaded condition. These findings suggest that the osteocyte network negatively regulate bone mass by inhibiting osteoblast function and activating osteoclastogenesis, and these functions are augmented in the unloaded condition at least partly through the upregulation of Rankl expression in osteoblasts and that of Sost in osteocytes, although it cannot be excluded that low BCL2 transgene expression in osteoblasts contributed to the enhanced osteoblast function.     

Strain uses gap junctions to reverse stimulation of osteoblast proliferation by osteocytes

Identifying mechanisms by which cells of the osteoblastic lineage communicate in vivo is complicated by the mineralised matrix that encases osteocytes, and thus, vital mechanoadaptive processes used to achieve load‐bearing integrity remain unresolved. We have used the coculture of immunomagnetically purified osteocytes and primary osteoblasts from both embryonic chick long bone and calvariae to examine these mechanisms. We exploited the fact that purified osteocytes are postmitotic to examine both their effect on proliferation of primary osteoblasts and the role of gap junctions in such communication. We found that chick long bone osteocytes significantly increased basal proliferation of primary osteoblasts derived from an identical source (tibiotarsi). Using a gap junction inhibitor, 18β‐glycyrrhetinic acid, we also demonstrated that this osteocyte‐related increase in osteoblast proliferation was not reliant on functional gap junctions. In contrast, osteocytes purified from calvarial bone failed to modify basal proliferation of primary osteoblast, but long bone osteocytes preserved their proproliferative action upon calvarial‐derived primary osteoblasts. We also showed that coincubated purified osteocytes exerted a marked inhibitory action on mechanical strain–related increases in proliferation of primary osteoblasts and that this action was abrogated in the presence of a gap junction inhibitor. These data reveal regulatory differences between purified osteocytes derived from functionally distinct bones and provide evidence for 2 mechanisms by which purified osteocytes communicate with primary osteoblasts to coordinate their activity.     

Apoptotic osteocytes regulate osteoclast precursor recruitment and differentiation in vitro

Fatigue loading causes a spatial distribution of osteocyte apoptosis co-localized with bone resorption spaces peaking around microdamage sites. Since osteocytes have been shown to regulate osteoclast formation and activity, we hypothesize that osteocyte apoptosis regulates osteoclastogenesis. In this study, we used serum-starvation to mimic reduced nutrient transport in microdamaged bone and induce apoptosis in MLO-Y4 osteocyte-like cells; conditioned medium was used to apply soluble factors released by apoptotic osteocytes (aOCY) to healthy non-apoptotic MLO-Y4 cells. Osteoclast precursor (RAW264.7 monocyte) migration and differentiation were assessed in the presence of conditioned media (CM) from: (A) aOCY, (B) osteocytes treated with apoptosis conditioned medium (i.e., healthy osteocytes in the presence of apoptosis cues; apoptosis CM-treated osteocytes (atOCY)), and (C) osteocytes treated with non-apoptosis conditioned medium (i.e., healthy osteocytes in the absence of apoptosis cues; non-apoptosis CM-treated osteocytes (natOCY)). Receptor activator for nuclear factor-κB ligand (RANKL), macrophage colony stimulating factor (M-CSF), vascular endothelial growth factor (VEGF) and osteoprotegerin (OPG) mRNA, and protein expression were measured. Our findings indicate that soluble factors released by aOCY and atOCY promoted osteoclast precursor migration (up to 64% and 24% increase, respectively) and osteoclast formation (up to 450% and 265% increase, respectively). Osteoclast size increased up to 233% in the presence of aOCY and atOCY CM. Recruitment, formation and size were unaltered by natOCY. RANKL mRNA and protein expression were upregulated only in aOCY, while M-CSF and VEGF increased in atOCY. Addition of RANKL-blocking antibody abolished aOCY-induced osteoclast precursor migration and osteoclast formation. VEGF and M-CSF blocking antibodies abolished atOCY-induced osteoclastogenesis. These findings suggest that aOCY directly and indirectly (through atOCY) initiate targeted bone resorption by regulating osteoclast precursor recruitment and differentiation.     

Isolation and culture of primary osteocytes from the long bones of skeletally mature and aged mice

The purpose of this work was to establish a methodology to enable the isolation and study of osteocytes from skeletally mature young (4-month-old) and old (22-month-old) mice. The location of osteocytes deep within bone is ideal for their function as mechanosensors. However, this location makes the observation and study of osteocytes in vivo technically difficult. Osteocytes were isolated from murine long bones through a process of extended collagenase digestions combined with EDTA-based decalcification. A tissue homogenizer was used to reduce the remaining bone fragments to a suspension of bone particles, which were placed in culture to yield an outgrowth of osteocyte-like cells. All of the cells obtained from this outgrowth that displayed an osteocyte-like morphology stained positive for the osteocyte marker E11/GP38. The osteocyte phenotype was further confirmed by a lack of staining for alkaline phosphatase and the absence of collagen1a1 expression. The outgrowth of osteocytes also expressed additional osteocyte-specific genes such as Sost and Mepe. This technique facilitates the isolation of osteocytes from skeletally mature bone. This novel enabling methodology should prove useful in advancing our understanding of the roles mature osteocytes play in bone health and disease.     

Matrix metalloproteinases (MMPs) safeguard osteoblasts from apoptosis during transdifferentiation into osteocytes: MT1-MMP maintains osteocyte viability

Osteoblasts undergo apoptosis or differentiate into either osteocytes or bone-lining cells after termination of bone matrix synthesis. In this study, we investigated the role of matrix metalloproteinases (MMPs) in differentiation of osteoblasts, bone formation, transdifferentiation into osteocytes, and osteocyte apoptosis. This was accomplished by using calvarial sections from the MT1-MMP-deficient mouse and by culture of the mouse osteoblast cell line MC3T3-E1 and primary mouse calvarial osteoblasts. We found that a synthetic matrix metalloprotease inhibitor, GM6001, strongly inhibited bone formation in vitro of both primary osteoblasts and MC3T3 cells by approximately 75%. To further investigate at which level of osteoblast differentiation MMP inhibition was attenuating osteoblast function, we found that neither preosteoblast nor mature osteoblast activity was affected. In contrast, cell survival of osteoblasts forced to transdifferentiate into osteocytes in 3D type I collagen gels were inhibited by more than 50% when exposed to 10 microM GM6001 and to Tissue Inhibitor of Metalloproteinase-2 (TIMP-2), a natural MT1-MMP inhibitor. This shows the importance of MMPs in safeguarding osteoblasts from apoptosis when transdifferentiating into osteocytes. By examination of osteoblasts and osteocytes embedded in calvarial bone in the MT1-MMP deficient mice, we found that MT1-MMP deficient mice had 10-fold higher levels of apoptotic osteocytes than wild-type controls. We have previously shown that MT1-MMP activates latent Transforming Growth Factorbeta (TGF-beta). These findings strongly suggest that MT1-MMP-activated TGF-beta maintains osteoblast survival during transdifferentiation into osteocytes, and maintains mature osteocyte viability. Thus, the interrelationship of MMPs and TGF-beta may play an important role in bone formation and maintenance.