A crucial role for matrix metalloproteinase 2 in osteocytic canalicular formation and bone metabolism

Extracellular matrix production and degradation by bone cells are critical steps in bone metabolism. Mutations of the gene encoding MMP-2, an extracellular matrix-degrading enzyme, are associated with a human genetic disorder characterized by subcutaneous nodules, arthropathy, and focal osteolysis. It is not known how the loss of MMP-2 function results in the pathology. Here, we show that Mmp2(-/-) mice exhibited opposing bone phenotypes caused by an impaired osteocytic canalicular network. Mmp2(-/-) mice showed decreased bone mineral density in the limb and trunk bones but increased bone volume in the calvariae. In the long bones, there was moderate disruption of the osteocytic networks and reduced bone density throughout life, whereas osteoblast and osteoclast function was normal. In contrast, aged but not young Mmp2(-/-) mice had calvarial sclerosis with osteocyte death. Severe disruption of the osteocytic networks preceded osteocyte loss in Mmp2(-/-) calvariae. Successful transplantation of wild-type periosteum restored the osteocytic canalicular networks in the Mmp2(-/-) calvariae, suggesting local roles of MMP-2 in determining bone phenotypes. Our results indicate that MMP-2 plays a crucial role in forming and maintaining the osteocytic canalicular network, and we propose that osteocytic network formation is a determinant of bone remodeling and mineralization.     

Quantitative associations between osteocyte density and biomechanics, microcrack and microstructure in OVX rats vertebral trabeculae

Osteocytes actively regulate bone modeling and remodeling, direct skeletal mineralization, and regulate calcium/phosphate homeostasis and extracellular matrix metabolism; yet the specific role of osteocytes in maintaining bone structural integrity and strength is unknown. Studies have shown that the density of osteocytes decreases with age and estrogen deficiency, as seen in postmenopausal women. Here, we examined the relationships between osteocyte density and the related variables, including biomechanics, bone mineral density, microcrack and microstructure of vertebral trabeculae, in ovariectomized rats. We found that osteocyte density correlated with some of the parameters that determine the biomechanical quality of bone. Our findings suggest that osteocytes could play a crucial role in maintaining the mechanical quality of bone, and osteocyte density could be considered as an alternative index in assessing bone quality.     

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.     

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.     

对骨细胞生物学功能的最新认识

骨细胞长久以来都被看作是一种终末分化的、代谢隋性的、深埋在骨基质之中的占位细胞．并不具有重要的生理作用和功能。然而近些年的研究发现,骨细胞是一种活跃的多功能细胞,参与机体诸多生物学过程的调控。骨细胞的生物学功能可以大致归纳为以下六类：调节骨重建平衡、感知和转导力学刺激、参与机体的神经-内分泌调节、与肌肉组织有密切的相互作用、降解与合成骨基质及调控骨组织内储存的钙和其他生物活性物质。     

Impact of extracellular matrix derived from osteoarthritis subchondral bone osteoblasts on osteocytes: role of integrinβ1 and focal adhesion kinase signaling cues

Introduction

Our recent study indicated that subchondral bone pathogenesis in osteoarthritis (OA) is associated with osteocyte morphology and phenotypic abnormalities. However, the mechanism underlying this abnormality needs to be identified. In this study we investigated the effect of extracellular matrix (ECM) produced from normal and OA bone on osteocytic cells function.

Methods

De-cellularized matrices, resembling the bone provisional ECM secreted from primary human subchondral bone osteoblasts (SBOs) of normal and OA patients were used as a model to study the effect on osteocytic cells. Osteocytic cells (MLOY4 osteocyte cell line) cultured on normal and OA derived ECMs were analyzed by confocal microscopy, scanning electron microscopy (SEM), cell attachment assays, zymography, apoptosis assays, qRT-PCR and western blotting. The role of integrinβ1 and focal adhesion kinase (FAK) signaling pathways during these interactions were monitored using appropriate blocking antibodies.

Results

The ECM produced by OA SBOs contained less mineral content, showed altered organization of matrix proteins and matrix structure compared with the matrices produced by normal SBOs. Culture of osteocytic cells on these defective OA ECM resulted in a decrease of integrinβ1 expression and the de-activation of FAK cell signaling pathway, which subsequently affected the initial osteocytic cell’s attachment and functions including morphological abnormalities of cytoskeletal structures, focal adhesions, increased apoptosis, altered osteocyte specific gene expression and increased Matrix metalloproteinases (MMP-2) and -9 expression.

Conclusion

This study provides new insights in understanding how altered OA bone matrix can lead to the abnormal osteocyte phenotypic changes, which is typical in OA pathogenesis.     

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.     

The osteocyte as a wiring transmission system

The mechanism of transduction of mechanical strains into biological signals remains one of the more baffling problems of skeletal homeostasis. The updated literature ascribes to osteocytes the function of sensing the strains induced into the bone matrix by mechanical stresses. Whether the osteocytes perform such function by themselves or they are helped by other cells is also unknown. Indeed TEM investigations carried out in our laboratory pointed out the existence of a functional syncytium among all the cells of the osteogenic lineage (COL; stromal cells, osteoblasts or bone lining cells, osteocytes). On the basis of this finding, we suggested that COL may reciprocally modulate their function not only by volume transmission (paracrine and autocrine stimulation) but also by wiring transmission, namely in a neuronal like manner. Thanks to their location, osteocytes should theoretically be the first cells of COL functional syncytium to sense mechanical strains, whereas stromal cells should be the first to be activated by hormonal molecules diffusing across the endothelial lining. Since PTH and Estrogen receptors have also been localized on osteocytes, and considering that such hormones have been suggested to modulate the sensitivity to strain of the bone mechanosensor, we suggested that the osteocyte syncytium may constitute the microscopic bone structure that sense both mechanical strain and biochemical factors and, at any moment, after having combined the two types of stimuli, issues the appropriate signals to the other bone cells by volume and/or wiring-transmission. Stromal cells, on the other hand, besides transmitting signals from vascular endothelium to bone cells, may control the differentiation and then direct the course of the osteoblasts around the vascular framework.     

Inhibition of Osteocyte Apoptosis Prevents the Increase in Osteocytic Receptor Activator of Nuclear Factor κB Ligand (RANKL) but Does Not Stop Bone Resorption or the Loss of Bone Induced by Unloading

Apoptosis of osteocytes and osteoblasts precedes bone resorption and bone loss with reduced mechanical stimulation, and receptor activator of NF-κB ligand (RANKL) expression is increased with unloading in mice. Because osteocytes are major RANKL producers, we hypothesized that apoptotic osteocytes signal to neighboring osteocytes to increase RANKL expression, which, in turn, increases osteoclastogenesis and bone resorption. The traditional bisphosphonate (BP) alendronate (Aln) or IG9402, a BP analog that does not inhibit resorption, prevented the increase in osteocyte apoptosis and osteocytic RANKL expression. The BPs also inhibited osteoblast apoptosis but did not prevent the increase in osteoblastic RANKL. Unloaded mice exhibited high serum levels of the bone resorption marker C-telopeptide fragments of type I collagen (CTX), elevated osteoclastogenesis, and increased osteoclasts in bone. Aln, but not IG9402, prevented all of these effects. In addition, Aln prevented the reduction in spinal and femoral bone mineral density, spinal bone volume/tissue volume, trabecular thickness, mechanical strength, and material strength induced by unloading. Although IG9402 did not prevent the loss of bone mass, it partially prevented the loss of strength, suggesting a contribution of osteocyte viability to strength independent of bone mass. These results demonstrate that osteocyte apoptosis leads to increased osteocytic RANKL. However, blockade of these events is not sufficient to restrain osteoclast formation, inhibit resorption, or stop bone loss induced by skeletal unloading.     

Conditioned medium from osteocytes stimulates the proliferation of bone marrow mesenchymal stem cells and their differentiation into osteoblasts

Osteocytes are the most abundant cells in bone and there is increasing evidence that they control bone remodeling via direct cell-to-cell contacts and by soluble factors. In the present study, we have used the MLO-Y4 cell line to study the effect of osteocytes on the proliferation, differentiation and bone-forming capacity of bone marrow mesenchymal stem cells (MSC). Conditioned media (CM) from osteocytic MLO-Y4 and osteoblastic MC3T3-E1 cell lines were collected and added on mouse bone marrow cultures, in which MSC were induced to osteoblasts. There was a significant increase in alkaline phosphatase activity and osteocalcin expression in the presence of MLO-Y4 CM. No such stimulus could be observed with MC3T3-E1 CM. There was almost 4-fold increase in bone formation and up to 2-fold increase in the proliferation of MSC with MLO-Y4 CM. The highly proliferating bone marrow cells were negative for ALP and OCN, suggesting that they could represent early osteoblast precursors. MLO-Y4 CM did not enhance the viability of mature osteoblasts nor protected them of apoptosis. This is the first study to describe soluble signals between osteocytes and osteoblasts and there most likely are several still unidentified or unknown factors in osteocyte CM. We conclude that osteocytes have an active stimulatory role in controlling bone formation.     

Resorption Controls Bone Anabolism Driven by Parathyroid Hormone (PTH) Receptor Signaling in Osteocytes

The contribution of remodeling-based bone formation coupled to osteoclast activity versus modeling-based bone formation that occurs independently of resorption, to the anabolic effect of PTH remains unclear. We addressed this question using transgenic mice with activated PTH receptor signaling in osteocytes that exhibit increased bone mass and remodeling, recognized skeletal effects of PTH elevation. Direct inhibition of bone formation was accomplished genetically by overexpressing the Wnt antagonist Sost/sclerostin; and resorption-dependent bone formation was inhibited pharmacologically with the bisphosphonate alendronate. We found that bone formation induced by osteocytic PTH receptor signaling on the periosteal surface depends on Wnt signaling but not on resorption. In contrast, bone formation on the endocortical surface results from a combination of Wnt-driven increased osteoblast number and resorption-dependent osteoblast activity. Moreover, elevated osteoclasts and intracortical/calvarial porosity is exacerbated by overexpressing Sost and reversed by blocking resorption. Furthermore, increased cancellous bone is abolished by Wnt inhibition but further increased by blocking resorption. Thus, resorption induced by PTH receptor signaling in osteocytes is critical for full anabolism in cortical bone, but tempers bone gain in cancellous bone. Dissecting underlying mechanisms of PTH receptor signaling would allow targeting actions in different bone compartments, enhancing the therapeutic potential of the pathway.     

Osteocyte calcium signaling response to bone matrix deformation

Osteocytes embedded in calcified bone matrix have been widely believed to play important roles in mechanosensing to achieve adaptive bone remodeling in a changing mechanical environment. In vitro studies have clarified several types of mechanical stimuli such as hydrostatic pressure, fluid shear stress, and direct deformation influence osteocyte functions. However, osteocyte response to mechanical stimuli in the bone matrix has not been clearly understood. In this study, we observed the osteocyte calcium signaling response to the quantitatively applied deformation in the bone matrix. A novel experimental system was developed to apply deformation to cultured bone tissue with osteocytes on a microscope stage. As a mechanical stimulus to the osteocytes in bone matrix, in-plane shear deformation was applied using a pair of glass microneedles to bone fragments, obtained from 13-day-old embryonic chick calvariae. Deformation of bone matrix and cells was quantitatively evaluated using an image correlation method by applying for differential interference contrast images of the matrix and fluorescent images of immunolabeled osteocytes, together with imaging of the cellular calcium transient using a ratiometric method. As a result, it was confirmed that the newly developed system enables us to apply deformation to bone matrix and osteocytes successfully under the microscope without significant focal plane shift or deviation from the observation view field. The system could be a basis for further development to investigate the mechanosensing mechanism of osteocytes in bone matrix through examination of various types of rapid biochemical signaling responses and intercellular communication induced by matrix deformation.     

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.     

The phylogenetic origin and evolution of acellular bone in teleost fishes: insights into osteocyte function in bone metabolism

Vertebrate bone is composed of three main cell types: osteoblasts, osteoclasts and osteocytes, the latter being by far the most numerous. Osteocytes are thought to play a fundamental role in bone physiology and homeostasis, however they are entirely absent in most extant species of teleosts, a group that comprises the vast majority of bony ‘fishes’, and approximately half of vertebrates. Understanding how this acellular (anosteocytic) bone appeared and was maintained in such an important vertebrate group has important implications for our understanding of the function and evolution of osteocytes. Nevertheless, although it is clear that cellular bone is ancestral for teleosts, it has not been clear in which specific subgroup the osteocytes were lost. This review aims to clarify the phylogenetic distribution of cellular and acellular bone in teleosts, to identify its precise origin, reversals to cellularity, and their implications. We surveyed the bone type for more than 600 fossil and extant ray‐finned fish species and optimised the results on recent large‐scale molecular phylogenetic trees, estimating ancestral states. We find that acellular bone is a probable synapomorphy of Euteleostei, a group uniting approximately two‐thirds of teleost species. We also confirm homoplasy in these traits: acellular bone occurs in some non‐euteleosts (although rarely), and cellular bone was reacquired several times independently within euteleosts, in salmons and relatives, tunas and the opah (Lampris sp.). The occurrence of peculiar ecological (e.g. anadromous migration) and physiological (e.g. red‐muscle endothermy) strategies in these lineages might explain the reacquisition of osteocytes. Our review supports that the main contribution of osteocytes in teleost bone is to mineral homeostasis (via osteocytic osteolysis) and not to strain detection or bone remodelling, helping to clarify their role in bone physiology.     

The reaction of osteoclasts when releasing osteocytes from osteocytic lacunae in the bone during bone modeling

Osteocytes are released from the osteocytic lacunae when osteoclasts resorb the bone matrix during bone modeling and remodeling. It remains unknown how osteoclasts react when releasing osteocytes during bone modeling, and the fate of these released osteocytes is also unclear. Femoral mid-shafts of 2-day-old kittens were sectioned into serial 0.5 microm-thick semithin or 0.1 microm-thick ultrathin sections, and examined by light microscopy (LM) and transmission electron microscopy (TEM). The sections showed many osteoclasts at the endosteum but there were no osteoblasts. There were many half-released, fully released, half-exposed, and fully exposed osteocytes on the bone surfaces. Many cell-like structures were seen in the cell bodies of osteoclasts by LM, and some semithin sections were re-sectioned into ultrathin sections for re-observation by TEM. By TEM, these were determinated to be mononuclear cells. The serial ultrathin sections showed that the mononuclear cells appeared to be engulfed in osteoclasts on one section but that the cell was connected with the bone surface of the osteocytic lacuna on another section. These results show that the mononuclear cells in the osteoclasts were osteocytes. The present study suggests that osteoclasts engulf some osteocytes but do not engulf others when releasing osteocytes during bone modeling.     

The turnover of mineralized growth plate cartilage into bone may be regulated by osteocytes

During endochondral ossification, growth plate cartilage is replaced with bone. Mineralized cartilage matrix is resorbed by osteoclasts, and new bone tissue is formed by osteoblasts. As mineralized cartilage does not contain any cells, it is unclear how this process is regulated. We hypothesize that, in analogy with bone remodeling, osteoclast and osteoblast activity are regulated by osteocytes, in response to mechanical loading. Since the cartilage does not contain osteocytes, this means that cartilage turnover during endochondral ossification would be regulated by the adjacent bone tissue. We investigated this hypothesis with an established computational bone adaptation model. In this model, osteocytes stimulate osteoblastic bone formation in response to the mechanical bone tissue loading. Osteoclasts resorb bone near randomly occurring microcracks that are assumed to block osteocyte signals. We used finite element modeling to evaluate our hypothesis in a 2D-domain representing part of the growth plate and adjacent bone. Cartilage was added at a constant physiological rate to simulate growth. Simulations showed that osteocyte signals from neighboring bone were sufficient for successful cartilage turnover, since equilibrium between cartilage remodeling and growth was obtained. Furthermore, there was good agreement between simulated bone structures and rat tibia histology, and the development of the trabecular architecture resembled that of infant long bones. Additionally, prohibiting osteoclast invasion resulted in thickened mineralized cartilage, similar to observations in a knock-out mouse model. We therefore conclude that it is well possible that osteocytes regulate the turnover of mineralized growth plate cartilage.     

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.