Human Primary Osteocyte Differentiation in a 3D Culture System

Studies on primary osteocytes, which compose >90–95% of bone cells, embedded throughout the mineralized matrix, are a major challenge because of their difficult accessibility and the very rare models available in vitro. We engineered a 3D culture method of primary human osteoblast differentiation into osteocytes. These 3D‐differentiated osteocytes were compared with 2D‐cultured cells and with human microdissected cortical osteocytes obtained from bone cryosections. Human primary osteoblasts were seeded either within the interspace of calibrated biphasic calcium phosphate particles or on plastic culture dishes and cultured for 4 wk in the absence of differentiation factors. Osteocyte differentiation was assessed by histological and immunohistological analysis after paraffin embedding of culture after various times, as well as by quantitative RT‐PCR analysis of a panel of osteoblast and osteocyte markers after nucleic acid extraction. Histological analysis showed, after only 1 wk, the presence of an osteoid matrix including many lacunae in which the cells were individually embedded, exhibiting characteristics of osteocyte‐like cells. Real‐time PCR expression of a set of bone‐related genes confirmed their osteocyte phenotype. Comparison with plastic‐cultured cells and mature osteocytes microdissected from human cortical bone allowed to assess their maturation stage as osteoid‐osteocytes. This model of primary osteocyte differentiation is a new tool to gain insights into the biology of osteocytes. It should be a suitable method to study the osteoblast‐osteocyte differentiation pathway, the osteocyte interaction with the other bone cells, and orchestration of bone remodeling transmitted by mechanical loading and shear stress. It should be used in important cancer research areas such as the cross‐talk of osteocytes with tumor cells in bone metastasis, because it has been recently shown that gene expression in osteocytes is strongly affected by cancer cells of different origin. It could also be a very efficient tool for drug testing and bone tissue engineering applications.     

The Primary Function of gp130 Signaling in Osteoblasts Is To Maintain Bone Formation and Strength,Rather Than Promote Osteoclast Formation

Interleukin‐6 (IL‐6) family cytokines act via gp130 in the osteoblast lineage to stimulate the formation of osteoclasts (bone resorbing cells) and the activity of osteoblasts (bone forming cells), and to inhibit expression of the osteocyte protein, sclerostin. We report here that a profound reduction in trabecular bone mass occurs both when gp130 is deleted in the entire osteoblast lineage (Osx1Cre gp130 f/f) and when this deletion is restricted to osteocytes (DMP1Cre gp130 f/f). This was caused not by an alteration in osteoclastogenesis, but by a low level of bone formation specific to the trabecular compartment. In contrast, cortical diameter increased to maintain ultimate bone strength, despite a reduction in collagen type 1 production. We conclude that osteocytic gp130 signaling is required for normal trabecular bone mass and proper cortical bone composition. © 2014 American Society for Bone and Mineral Research.     

Bisphosphonates improve trabecular bone mass and normalize cortical thickness in ovariectomized, osteoblast connexin43 deficient mice

The gap junction protein, connexin43 (Cx43) controls both bone formation and osteoclastogenesis via osteoblasts and/or osteocytes. Cx43 has also been proposed to mediate an anti-apoptotic effect of bisphosphonates, potent inhibitors of bone resorption. We studied whether bisphosphonates are effective in protecting mice with a conditional Cx43 gene deletion in osteoblasts and osteocytes (cKO) from the consequences of ovariectomy on bone mass and strength. Ovariectomy resulted in rapid loss of trabecular bone followed by a slight recovery in wild type (WT) mice, and a similar degree of trabecular bone loss, albeit slightly delayed, occurred in cKO mice. Treatment with either risedronate (20μg/kg) or alendronate (40μg/kg) prevented ovariectomy-induced bone loss in both genotypes. In basal conditions, bones of cKO mice have larger marrow area, higher endocortical osteoclast number, and lower cortical thickness and strength relative to WT. Ovariectomy increased endocortical osteoclast number in WT but not in cKO mice. Both bisphosphonates prevented these increases in WT mice, and normalized endocortical osteoclast number, cortical thickness and bone strength in cKO mice. Thus, lack of osteoblast/osteocyte Cx43 does not alter bisphosphonate action on bone mass and strength in estrogen deficiency. These results support the notion that one of the main functions of Cx43 in cortical bone is to restrain osteoblast and/or osteocytes from inducing osteoclastogenesis at the endocortical surface.     

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

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.     

Differences in Osteocyte Density and Bone Histomorphometry Between Men and Women and Between Healthy and Osteoporotic Subjects

Bone defects related to osteoporosis develop with increasing age and differ between males and females. It is currently thought that the bone remodeling process is supervised by osteocytes in a strain-dependent manner. We have shown an altered response of osteocytes from osteoporotic patients to mechanical loading, and osteocyte density is reduced in osteoporotic patients, which might relate to imperfect bone remodeling, leading to lack of bone mass and strength. Hence, information on osteocyte density will contribute to a better understanding of bone biology in males and females and to the assessment of osteoporosis. Osteocyte density as well as conventional histomorphometric parameters of trabecular bone were determined in cancellous iliac crest bone of healthy postmenopausal women and men and of osteoporotic women and men. Osteocyte density was higher in healthy females than in healthy males and lower in osteoporotic females than in healthy females. Bone mass was reduced in osteoporotic patients, both male and female. In females, trabecular number was reduced, whereas in males, trabecular thickness was reduced and eroded surface was increased. There were no correlations between the parameter groups bone architecture, bone formation, bone resorption, and osteocyte density. These results are consistent with impaired osteoblast function in osteoporotic patients and with a different mechanism of bone loss between men and women, in which osteocyte density might play a role. The reduced osteocyte numbers in female osteoporotic patients might relate to imperfect bone remodeling leading to lack of bone mass and strength. M. G. Mullender and S. D. Tan contributed equally to this work.     

Involvement of different ion channels in osteoblasts'' and osteocytes'' early responses to mechanical strain

The involvement of functional ion channels in previously documented early responses of osteocytes and osteoblasts to mechanical strain in bone tissue was investigated in explants of rat ulnae by the use of ion channel blockers. Gadolinium chloride (a blocker of stretch/shear-sensitive cation channels) elevated basal prostaglandin (PG) E₂ and prostacyclin (PGI₂) release and osteocyte glucose-6-phosphate dehydrogenase (G6PD) activity, but was associated with a reduction in basal nitric oxide (NO) production. Gadolinium abolished loading-related increases in the release of PGI₂ and NO and osteocyte G6PD activity. Gadolinium also reduced the loading-related release of PGE₂ assumed to originate from osteoblasts and the magnitude of loading-related increases in G6PD activity in these cells. Nifedipine (a blocker of L-type voltage-dependent calcium channels) had no effect on basal levels of prostanoid or NO release, or G6PD activity in osteocytes or osteoblasts, and did not affect loading-related release of PGI₂ or increase in osteocyte G6PD. However, nifedipine prevented loading-related increases in PGE₂ and NO release and osteoblast G6PD activity. These results are consistent with osteocytes' response to bone loading requiring activatable ion channels sensitive to gadolinium, but not those sensitive to nifedipine. In osteoblasts, the early responses to bone loading appear to be associated with ion channels sensitive to gadolinium and nifedipine; however, the nifedipine-sensitive channels seem to have the dominant effect.     

Novel explant model to study mechanotransduction and cell-cell communication.

To understand in situ behavior of osteocytes, we characterized a model of osteocytes in their native bone matrix and demonstrated real-time biologic activity of osteocytes while bending the bone matrix. Using 43 male Sprague-Dawley rats, dumbbell-shaped explants were harvested from stainless steel femoral implants after 6-12 weeks and incubated in culture medium or fixed. Sixteen specimens were used to determine bone volume density (BV/TV), volumetric bone mineral density (BMD) and histology for different implantation periods. Osteocyte viability was evaluated by L-lactate dehydrogenase (LDH) activity in 12 cultured explants. Confocal microscopy was used to assess tracer diffusion in three explants and changes in osteocyte pH of a mechanically loaded explant. From 6 to 12 weeks, explant BV/TV and volumetric BMD trended up 92.5% and 101%, respectively. They were significantly and highly correlated. Tissues were uniformly intramembranous and all bone cell types were present. Explants maintained LDH activity through culture day 8. Diffusion at 200 microM was limited to 1,209 Da. Explants appeared capable of reproducing complex bone biology. This model may be useful in understanding osteocyte mechanotransduction in the context of a physiologically relevant bone matrix.     

Focal adhesion protein Kindlin-2 regulates bone homeostasis in mice

Our recent studies demonstrate that the focal adhesion protein Kindlin-2 is critical for chondrogenesis and early skeletal development. Here, we show that deleting Kindlin-2 from osteoblasts using the 2.3-kb mouse Col1 a1-Cre transgene minimally impacts bone mass in mice, but deleting Kindlin-2 using the 10-kb mouse Dmp1-Cre transgene, which targets osteocytes and mature osteoblasts, results in striking osteopenia in mice. Kindlin-2 loss reduces the osteoblastic population but increases the osteoclastic and adipocytic populations in the bone microenvironment. Kindlin-2 loss upregulates sclerostin in osteocytes,downregulates β-catenin in osteoblasts, and inhibits osteoblast formation and differentiation in vitro and in vivo. Upregulation ofβ-catenin in the mutant cells reverses the osteopenia induced by Kindlin-2 deficiency. Kindlin-2 loss additionally increases the expression of RANKL in osteocytes and increases osteoclast formation and bone resorption. Kindlin-2 deletion in osteocytes promotes osteoclast formation in osteocyte/bone marrow monocyte cocultures, which is significantly blocked by an anti-RANKLneutralizing antibody. Finally, Kindlin-2 loss increases osteocyte apoptosis and impairs osteocyte spreading and dendrite formation.Thus, we demonstrate an important role of Kindlin-2 in the regulation of bone homeostasis and provide a potential target for the treatment of metabolic bone diseases.     

<Emphasis Type="Italic">In Vivo</Emphasis> Mechanical Loading Modulates Insulin-Like Growth Factor Binding Protein-2 Gene Expression in Rat Osteocytes

Mechanical stimulation is essential for maintaining skeletal integrity. Mechanosensitive osteocytes are important during the osteogenic response. The growth hormone-insulin-like growth factor (GH-IGF) axis plays a key role during regulation of bone formation and remodeling. Insulin-like growth factor binding proteins (IGFBPs) are able to modulate IGF activity. The aim of this study was to characterize the role of IGFBP-2 in the translation of mechanical stimuli into bone formation locally in rat tibiae. Female Wistar rats were assigned to three groups (n = 5): load, sham, and control. The four-point bending model was used to induce a single period of mechanical loading on the tibial shaft. The effect on IGFBP-2 mRNA expression 6 hours after stimulation was determined with nonradioactive in situ hybridization on decalcified tibial sections. Endogenous IGFBP-2 mRNA was expressed in trabecular and cortical osteoblasts, some trabecular and subendocortical osteocytes, intracortical endothelial cells of blood vessels, and periosteum. Megakaryocytes, macrophages, and myeloid cells also expressed IGFBP-2 mRNA. Loading and sham loading did not affect IGFBP-2 mRNA expression in osteoblasts, bone marrow cells, and chondrocytes. An increase of IGFBP-2 mRNA-positive osteocytes was shown in loaded (1.68-fold) and sham-loaded (1.35-fold) endocortical tibial shaft. In conclusion, 6 hours after a single loading session, the number of IGFBP-2 mRNA-expressing osteocytes at the endosteal side of the shaft and inner lamellae was increased in squeezed and bended tibiae. Mechanical stimulation modulates IGFBP-2 mRNA expression in endocortical osteocytes. We suggest that IGFBP-2 plays a role in the lamellar bone formation process.     

Osteocytes subjected to fluid flow inhibit osteoclast formation and bone resorption

Bone has the capacity to alter its mass and structure to its mechanical environment. Osteocytes are the predominant bone cells and it is generally accepted that the osteocytes are the professional mechanosensors of bone. A strain-derived fluid flow through the lacuno-canalicular porosity seems to mechanically activate them, resulting in the production of signalling molecules such as nitric oxide (NO). We hypothesize that mechanically stimulated osteocytes modulate osteoclast formation and activity via soluble factors, thus affecting bone resorption. Osteocytes, osteoblasts, and periosteal fibroblasts were isolated from fetal chicken calvariae via enzymatic digestion. The periosteal fibroblasts were obtained from the periostea. Osteocytes were separated from osteoblasts by immunomagnetic separation. Cells were mechanically stimulated for 1 h with pulsating fluid flow (PFF, 0.70 +/- 0.30 Pa) at 5 Hz, or kept under static conditions. Conditioned medium was collected after 60 min. The effect of conditioned medium on osteoclastogenesis was tested on mouse bone marrow cells in the presence of macrophage colony stimulating factor and receptor activator of NF-kappaB ligand. After 6 days of culture, osteoclast formation and bone resorption was determined. Osteocytes subjected to 1 h pulsating fluid flow produced conditioned medium that inhibited the formation of osteoclasts. For osteoblast PFF-conditioned medium, such effect was, to a lesser extent, also observed, but not for periosteal fibroblast PFF-conditioned medium. Furthermore, PFF-treated osteocytes, but not osteoblast or periosteal fibroblast, produced conditioned medium that resulted in a decreased bone resorption. The NO synthase inhibitor N(G)-nitro-L-arginine methyl ester attenuated the inhibitory effects of osteocyte PFF-conditioned medium on osteoclast formation and resorption. We conclude that osteocytes subjected to PFF inhibit osteoclast formation and resorption via soluble factors, and the release of these factors was at least partially dependent on activation of an NO pathway in osteocytes in response to PFF. Thus, the osteocyte appears to be more responsive to PFF than the osteoblast or periosteal fibroblast regarding to the production of soluble factors affecting osteoclast formation and bone resorption.     

Actin and microtubule cytoskeletons of the processes of 3D-cultured MC3T3-E1 cells and osteocytes

Cell shape is the most critical determinant of cell function and is potentially influenced by the organization of a cell's cytoskeletal components. It has been reported that three-dimensionally cultured osteoblasts have a morphology that closely resembles that of osteocytes, most notably including formation of processes. We have previously shown the critical differences between cytoskeletal components in osteoblasts and osteocytes in two-dimensional culture. We have now extended that investigation to the cytoskeletal components of 3D-cultured osteoblasts and osteocytes using 3D cultures of the osteoblast cell line, MC3T3-E1, and primary osteocytes grown in collagen gel. Three-dimensional fluorescent image reconstructions for actin, fimbrin, alpha-actinin, myosin, tropomyosin, and microtubules were made using IMARIS software. Actin, fimbrin, alpha-actinin, myosin, and tropomyosin all appeared in the processes of both cell types, but fimbrin and myosin showed differences in their distribution patterns between cell types. Microtubules were limited in distribution to the proximal region of osteocyte processes but extended the entire length of MC3T3-E1 cell processes. Microtubules were essential for the integrity and formation of MC3T3-E1 cell processes, but osteocyte processes were dependent on actin. These results showed that there are significant differences between the actin and microtubule cytoskeletons in the processes of 3D-cultured MC3T3-E1 cells and in the processes of 3D-cultured primary osteocytes. These differences in the cytoskeleton of the processes of 3D-cultured osteoblasts and of osteocyte dendrites suggest that osteoblast processes may have a different functional role than the osteocyte dendritic network. An erratum to this article is available at .     

A quantitative evaluation of osteoblast-osteocyte relationships on growing endosteal surface of rabbit tibiae.

Scanning electron microscopy (SEM) was used to quantify the intercellular relationships between osteoblasts and osteocytes on the growing endosteal surfaces of the medullary canal of the tibia in four rabbits of different ages. The area of each osteoblast was measured on the SEM micrographs by means of an Image Analyzer. The number of osteocyte cytoplasmic processes was indirectly evaluated by counting the canalicular openings present on the same microscopic fields after the removal of the osteoblasts. The metabolic activity of the osteoblasts was indirectly evaluated from their shape, and the structure was analyzed by transmission electron microscope (TEM) in sections taken from the samples studied by SEM. In all four animals, the surface area of the osteoblasts (OA) was found to vary a great deal, whereas the density of canalicular openings was fairly uniform. Moreover, although the OA mean value increases significantly with the age of the animals, the density of canalicular openings does not; it would therefore appear that the older the animal and the more flattened the osteoblasts, the greater the number of canaliculi beneath them. Since osteoblast activity has previously been shown to be inversely proportional to the area of the protoplasm in contact with the bone surface, it appears that the less active osteoblasts should contact a greater number of osteocyte cytoplasmic processes. These findings suggest that osteocytes might play an important role in modulating osteoblast activity and in recruiting osteoblasts that differentiate into osteocytes, possibly by means of inhibitory signals transmitted via gap junctions.     

Cell autonomous requirement of connexin 43 for osteocyte survival: consequences for endocortical resorption and periosteal bone formation

Connexin 43 (Cx43) mediates osteocyte communication with other cells and with the extracellular milieu and regulates osteoblastic cell signaling and gene expression. We now report that mice lacking Cx43 in osteoblasts/osteocytes or only in osteocytes (Cx43(ΔOt) mice) exhibit increased osteocyte apoptosis, endocortical resorption, and periosteal bone formation, resulting in higher marrow cavity and total tissue areas measured at the femoral mid-diaphysis. Blockade of resorption reversed the increased marrow cavity but not total tissue area, demonstrating that endocortical resorption and periosteal apposition are independently regulated. Anatomical mapping of apoptotic osteocytes, osteocytic protein expression, and resorption and formation suggests that Cx43 controls osteoclast and osteoblast activity by regulating osteoprotegerin and sclerostin levels, respectively, in osteocytes located in specific areas of the cortex. Whereas empty lacunae and living osteocytes lacking osteoprotegerin were distributed throughout cortical bone in Cx43(ΔOt) mice, apoptotic osteocytes were preferentially located in areas containing osteoclasts, suggesting that osteoclast recruitment requires active signaling from dying osteocytes. Furthermore, Cx43 deletion in cultured osteocytic cells resulted in increased apoptosis and decreased osteoprotegerin expression. Thus, Cx43 is essential in a cell-autonomous fashion in vivo and in vitro for osteocyte survival and for controlling the expression of osteocytic genes that affect osteoclast and osteoblast function.     

Osteocytes and bone lining cells: Which are the best candidates for mechano-sensors in cancellous bone?

Previously, we have investigated the possible role of osteocytes as mechano-sensors, and mediators of bone turnover. It was found that the proposed regulatory mechanism produced morphologies of trabecular bone, under particular loading conditions, which were consistent with morphogenesis and adaptation as seen in reality. The main objective of this study was to descern whether lining cells or osteoblasts could possibly play a similar role as effectively with regard to their capacity for self-optimization of the trabecular architecture, in terms of a low apparent mass to stiffness ratio. For that purpose the earlier analyses with osteocytes as mechano-sensors, distributed throughout the bone, were repeated for mechano-sensors located at bone surfaces only. Compared to the osteocyte model, the surface cell remodelling algorithm was reluctant to change its architecture, which implies that it is less sensitive to changes in the loading pattern. This resulted in less efficient bone adaptation, which was reflected by a considerably higher relative mass for a similar apparent stiffness in the loading direction. In other words, more mass is needed to obtain an equally stiff structure, at the apparent level, with respect to the externally applied loads. Furthermore, stresses and strains at the tissue level vary across a much wider range, relative to the osteocyte model, where the higher incidence of elevated strains indicates an increased failure risk. Therefore, we conclude that mechanical information at the bone surface may not be sufficient to adequately regulate functional bone adaptation.     

Mechanical strain stimulates nitric oxide production by rapid activation of endothelial nitric oxide synthase in osteocytes.

Previous studies have indicated that physiological levels of dynamic mechanical strain produce rapid increases in nitric oxide (NO) release from rat ulna explants and primary cultures of osteoblast-like cells and embryonic chick osteocytes derived from long bones. To establish the mechanism by which loading-induced NO production may be regulated, we have examined: nitric oxide synthase (NOS) isoform mRNA and protein expression, the effect of mechanical loading in vivo on NOS mRNA expression, and the effect of mechanical strain on NO production by bone cells in culture. Using Northern blot analyses, in situ hybridization, and immunocytochemistry we have established that the predominant NOS isoform expressed in rat long bone periosteal osteoblasts and in a distinct population of cortical bone osteocytes is the endothelial form of NOS (eNOS), with little or no expression of the inducible NOS or neuronal NOS isoforms. In contrast, in non-load-bearing calvariae there are no detectable levels of eNOS in osteocytes and little in osteoblasts. Consistent with these observations, ulnar explants release NO rapidly in response to loading in vitro, presumably through the activation of eNOS, whereas calvarial explants do not. The relative contribution of different bone cells to these rapid increases in strain-induced NO release was established by assessment of medium nitrite (stable NO metabolite) concentration, which showed that purified populations of osteocytes produce significantly greater quantities of NO per cell in response to mechanical strain than osteoblast-like cells derived from the same bones. Using Northern blot hybridization, we have also shown that neither a single nor five consecutive daily periods of in vivo mechanical loading produced any significant effect on different NOS isoform mRNA expression in rat ulnae. In conclusion, our results indicate that eNOS is the prevailing isoform expressed by cells of the osteoblast/osteocyte lineage and that strain produces increases in the activity of eNOS without apparently altering the levels of eNOS mRNA.     

Osteocyte recruitment declines as the osteon fills in: interacting effects of osteocytic sclerostin and previous hip fracture on the size of cortical canals in the femoral neck

There is little information on the distribution of osteocytes within the individual cortical osteon, but using direct 3-D imaging in a single subject, Hannah et al. found a gradient with a two-fold higher density of cells adjacent to the cement line compared to near the canal. Since a limiting factor for bone formation might be the availability of osteoblasts due to their recruitment as osteocytes, we studied distributions of osteonal osteocytes in frozen sections of the femoral neck cortex. Osteocytes were stained with an anti-sclerostin antibody and counter-stained with toluidine blue. Adjacent sections were stained for alkaline phosphatase (ALP). Each osteonal osteocyte was categorised as being sclerostin-positive (scl+) or negative (scl-). ImageJ was used to measure the perimeter and area of each osteon and canal, while special purpose routines were used to measure the minimum distances of each osteocyte from the cement line and the canal. Canal area was strongly correlated with osteon area. Osteocytes were most dense close to the cement line; and their areal density within the matrix declined up to three-fold between the cement line and the canal, depending on osteon diameter. Large and small osteons had similar densities of osteocytes close to the cement line, but fractured neck of femur cases had significantly lower densities of osteocytes close to the canal. Higher osteocyte density close to the canal was associated with ALP expression. It is concluded that entombment of osteocytes newly drawn from the osteoblast pool into the mineralising matrix is independent of preceding bone resorption depth. As osteonal infilling proceeds, osteocyte formation declines more rapidly than matrix formation, leading to a progressive reduction in osteocyte density. A shrinking supply of precursor osteoblasts due to previous osteocyte recruitment, apoptosis, or both could produce this effect. In a statistically significant contrast, sclerostin negative osteocytes adjacent to the canal had the expected effect of reducing canal size in controls but this was not seen in hip fracture. This demonstrated the failure of osteonal osteoblasts to sustain bone formation through a complete remodelling cycle in osteoporosis, perhaps due to insufficient osteoblasts remaining capable of mineralized matrix formation. The failure of osteocytic sclerostin suppression to associate with bone formation in these osteons might alternatively be explained by downstream interference with sclerostin's effect on wnt signalling.     

Androgen receptor (AR) in osteocytes is important for the maintenance of male skeletal integrity: Evidence from targeted AR disruption in mouse osteocytes

Androgens play a key role in the maintenance of male skeletal integrity. The regulation of this integrity by androgen receptor (AR) signaling has been mainly attributed to osteoblasts. Although osteocytes have emerged as key regulators of bone remodeling, the influence of sex steroids on these cells has been poorly studied. We aimed to investigate the role of AR signaling, specifically in osteocytes using the Cre/LoxP system in male mice (driven by dentin matrix protein 1 [ocy‐ARKOs]). Osteocyte fractions of control (AR(ex2)/Y) and ocy‐ARKO (ARflox(ex2)/Y; DMP1‐cre) mice isolated through sequential collagenase digestion showed increasing AR expression toward the mature osteocyte fraction of control males compared with the more immature fractions, whereas this was reduced by >80% in ocy‐ARKO osteocytes. The skeletal phenotype of mutant mice was further assessed by histomorphometry and quantitative micro‐computed tomography at 12 and 32 weeks of age. Ocy‐ARKOs had significantly lower trabecular bone volume and number in femora and tibias at 32 weeks as well as decreased trabecular number in the L₅ vertebra at 12 weeks. Biomechanical testing showed that ocy‐ARKO femora were also stiffer and required a lower ultimate force to induce failure at 32 weeks. However, femoral cortical structure was not significantly different at any time point. The absence of AR in osteocyte also did not appear to affect trabecular bone formation nor its response to mechanical loading. In conclusion, selective inactivation of the AR in osteocytes of male mice accelerates age‐related deterioration of skeletal integrity. These findings provide evidence for a direct role of androgens in the maintenance of trabecular bone through actions of the AR in osteocytes. © 2012 American Society for Bone and Mineral Research.     

Talking among Ourselves: Paracrine Control of Bone Formation within the Osteoblast Lineage

While much research focuses on the range of signals detected by the osteoblast lineage that originate from endocrine influences, or from other cells within the body, there are also multiple interactions that occur within this family of cells. Osteoblasts exist as teams and form extensive communication networks both on, and within, the bone matrix. We provide four snapshots of communication pathways that exist within the osteoblast lineage between different stages of their differentiation, as follows: (1) PTHrP, a factor produced by early osteoblasts that stimulates the activity of more mature bone-forming cells and the most mature osteoblast embedded within the bone matrix, the osteocyte; (2) sclerostin, a secreted factor, released by osteocytes into their extensive communication network to restrict the activity of younger osteoblasts on the bone surface; (3) oncostatin M, a member of the IL-6/gp130 family of cytokines, expressed throughout osteoblast differentiation and acting to stimulate osteoblast activity that works on a different receptor in the mature osteocyte compared to the preosteoblast; and (4) Eph/ephrins, cell-contact-dependent kinases, and the osteoblast-lineage-specific interaction of EphB4 and ephrinB2, which provides a checkpoint for entry to the late stages of osteoblast differentiation and restricts RANKL expression.     

Osteocytes Acidify Their Microenvironment in Response to PTHrP In Vitro and in Lactating Mice In Vivo

Osteocytes appear to mobilize calcium within minutes in response to PTH injections; we have previously shown that osteocytes remove their perilacunar matrix during lactation through activation of the PTH type 1 receptor. Mechanisms utilized by osteocytes to mobilize calcium are unknown but we hypothesized that the molecular components may be similar to those used by osteoclasts. Here we show, using IDG‐SW3 cells that ATP6V0D2, an essential component of vacuolar ATPase in osteoclasts, and other genes associated with osteoclastic bone resorption, increase with osteoblast to osteocyte differentiation. Furthermore, PTHrP increases ATP6V0D2 expression and induces proton generation by primary osteocytes, which is blocked by bafilomycin, a vacuolar ATPase inhibitor. These in vitro proton measurements raised the question of osteocyte viability in an acidic environment. Interestingly, osteocytes, showed enhanced viability at pH as low as 5 compared to osteoblasts and fibroblasts in vitro. To study in vivo acidification by osteocytes, virgin and lactating CD1 mice on a low calcium diet were injected with the pH indicator dye, acridine orange, and their osteocyte lacuno‐canalicular system imaged by confocal microscopy. Lower pH was observed in lactating compared to virgin animals. In addition, a novel transgenic mouse line with a topaz variant of green fluorescent protein (GFPtpz)‐tagged collagen α2(I) chain was used. Instead of the expected reduction in GFP‐fluorescence only in the perilacunar matrix, reduced fluorescence was observed in the entire bone matrix of lactating mice. Based on our experiments showing quenching of GFP in vitro, we propose that the observed reduction in GFP fluorescence in lactating mice is due to quenching of GFP by the acidic pH generated by osteocytes. Together these findings provide novel mechanistic insight into how osteocytes remove calcium from their perilacunar/pericanalicular matrices through active acidification of their microenvironment and show that osteocytes, like osteoclasts, are resistant to the negative effects of acid on viability. © 2017 American Society for Bone and Mineral Research.