Strontium ranelate treatment of human primary osteoblasts promotes an osteocyte-like phenotype while eliciting an osteoprotegerin response

Summary  The effect of strontium ranelate (SR) on human osteoblast differentiation was tested. SR induced osteoblastic proliferation, in vitro mineralization, and increased the expression of osteocyte markers. SR also elicited an osteoprotegerin (OPG) secretory response. We conclude that SR promotes the osteoblast maturation and osteocyte differentiation while promoting an additional antiresorptive effect. Introduction  SR is a new treatment for osteoporosis that reduces the risk of hip and vertebral fractures in postmenopausal women. This study sought to investigate the extent, to which SR modulates human osteoblast differentiation. Methods  Adult human primary osteoblasts (NHBC) were exposed to SR under mineralizing conditions in long-term cultures. Osteoblast differentiation status was investigated by cell-surface phenotypic analysis. Expression of genes associated with osteoblast/osteocyte differentiation was examined using real-time RT-PCR. Secreted OPG was assayed by enzyme-linked immunosorbent assay. Results  SR significantly increased osteoblast replication. SR time- and dose-dependently induced an osteocyte-like phenotype, as determined by cell surface alkaline phosphatase and STRO-1 expression. SR at 5 mM or greater dramatically increased in vitro mineralization. In parallel, mRNA levels of dentin matrix protein (DMP)-1 and sclerostin were higher under SR treatment, strongly suggestive of the presence of osteocytes. SR also increased the OPG/RANKL ratio throughout the culture period, consistent with an effect to inhibit osteoblast-induced osteoclastogenesis. Conclusions  This study suggests that SR can promote osteoblast maturation and an osteocyte-like phenotype. Coupled with its effect on the OPG/RANKL system, these findings are consistent with in vivo effects in patients receiving SR for the treatment of osteoporosis.     

The dependency of solute diffusion on molecular weight and shape in intact bone

Solute transport through the bone lacunar–canalicular system (LCS) is essential for osteocyte survival and function, but quantitative data on the diffusivity of various biological molecules in the LCS are scarce. Using our recently developed approach based on fluorescence recovery after photobleaching (FRAP), diffusion coefficients of five exogenous fluorescent tracers (sodium fluorescein, dextran-3k, dextran-10k, parvalbumin, and ovalbumin) were measured in murine tibiae in situ. These tracers were chosen to test the dependency of solute diffusion on molecular weight (376–43,000 Da) and shape (linear vs. globular). Among the five tracers, no fluorescence recovery (and thus mobility) was detected for dextran-10k and the diffusion coefficients (D_LCS) of the other four tracers were 295 ± 46, 128 ± 32, 157 ± 88, 65 ± 21 μm² s^− 1 in the LCS, respectively. Overall, the rate of solute diffusion in the bone LCS showed strong dependency on molecular size and shape. Diffusivity decreased with increasing molecular weight for both linear and globular molecules, with the linear molecules decreasing at a faster rate. Compared with free diffusion (D_free) in aqueous solutions, the relative diffusivities (D_LCS / D_free) of the four tracers were not significantly different for sodium fluorescein, dextran-3k, parvalbumin, and ovalbumin (55.0 ± 8.6%, 68.1 ± 17.0%, 79.7 ± 44.7%, 61.0 ± 19.6%, respectively). This result did not agree with the homogenous molecular sieve model proposed for the osteocytic pericellular matrix structure. Instead, a heterogeneous porous model of the pericellular matrix may account for the observed solute transport in the LCS. In summary, the present study provides quantitative data on diffusion of various nutrients and signaling molecules in the LCS that are important for bone metabolism and mechanotransduction.     

Effect of Raloxifene Treatment on Osteocyte Apoptosis in Postmenopausal Women

Increased osteocyte apoptosis, as the result of estrogen deficiency, could play a role in the decrease of bone mass and bone strength seen in postmenopausal osteoporosis. We investigated whether treatment with raloxifene of postmenopausal women with osteoporosis affects osteocyte apoptosis. Transiliac bone biopsies were obtained from 26 osteoporotic women at baseline and after 2 years of treatment with placebo or raloxifene. Immunohistochemical detection of cleaved caspase-3 was performed on sections from nondecalcified bone biopsies to visualize apoptosis. In the trabecular bone total osteocytes, positively stained osteocytes and empty lacunae were counted and percent positive cells and percent empty lacunae determined. Statistical evaluation was performed by Wilcoxon’s paired t-test and Spearman’s rank correlations. There was no significant difference in percentage positive osteocytes between baseline and follow-up biopsies in both the placebo and the raloxifene groups. The percentage empty lacunae increased significantly in the placebo group (11.20 ± 1.43 vs. 9.00 ± 2.25, P = 0.014) but not in the raloxifene group. At baseline in both groups combined, there was a negative correlation between indices of bone remodeling and the percentage positive osteocytes (bone formation rate/bone volume r = −0.67, P = 0.001). We found no direct evidence for an effect of raloxifene treatment on osteocyte apoptosis, but small effects of raloxifene treatment cannot be excluded. The percent of apoptotic osteocytes was dependent on the level of bone remodeling in an individual.     

The role of osteocytes during experimental orthodontic tooth movement: A review

ObjectiveTo explore the types of orthodontic force-induced mechanical stimuli that regulate osteocyte function.DesignIn orthodontics, a tooth can be moved through the alveolar bone when an appropriate orthodontic force is applied. These mechanical loads stimulate cells within the bone tissue around the tooth. These cellular responses lead to bone resorption on the side of the tooth where the pressure has been applied and bone deposition on the side of the tooth experiencing tension. Recently, osteocytes were identified to function as mechano-sensory cells in bone tissue that direct bone resorption and bone formation. Based on recent literature, the proposed function of osteocytes during orthodontic tooth movement is explored with better understanding.ResultsSeveral stimuli regulating osteocyte function have been highlighted, and their potential roles in events initiating osteocyte sensing of orthodontic force have been explored in detail. The most popular hypotheses for osteocyte response include stress-induced bone matrix deformation/microcrack formation and fluid-flow shear stress.ConclusionsUnderstanding osteocyte function under mechanical stress may have profound implications in future orthodontic treatments.     

Osteocytes: Mechanosensors of Bone and Orchestrators of Mechanical Adaptation

Significant progress has been made in the field of mechanotransduction in bone cells. The knowledge about the role of osteocytes as the professional mechanosensor cells of bone as well as the lacuno-canalicular porosity as the structure that mediates mechanosensing is increasing. New insights might result in a paradigm for understanding the bone formation response to mechanical loading, and the bone resorption response to disuse. Under physiological loading conditions the strain-derived flow of interstitial fluid through the lacuno-canalicular porosity seems to mechanically activate the osteocytes, which subsequently alter the bone remodeling activity of osteoblasts and/or osteoclasts. Fatigue loading results in local microdamage, disruption of normal flow patterns, and osteocyte apoptosis. Apoptotic osteocytes likely attract osteoclasts to resorb the damaged bone. This concept allows explanation of local bone gain and loss, as well as remodeling in response to fatigue damage, as processes supervised by mechanosensitive osteocytes. Uncovering the cellular and mechanical basis of the osteocyte’s response to loading would greatly contribute to our understanding of the cellular basis for bone remodeling, and could contribute to the discovery of new treatment modalities for bone mass disorders, such as osteoporosis.     

Osteocyte density in aging subjects is enhanced in bone adjacent to remodeling haversian systems

The osteocyte is a candidate regulatory cell for bone remodeling. Previously, we demonstrated that there is a substantial (approximately 50%) loss of osteocytes from their lacunae in the cortex of the elderly femoral neck. Higher occupancy was evident in tissue exhibiting high remodeling and high porosity. The present study examines the distribution of osteocytes within individual osteonal systems at differing stages of the remodeling cycle. In 22 subjects, lacunar density, osteocyte density, and their quotient, the percent lacunar occupancy, was assessed up to a distance of 65 μm from the canal surface in six quiescent, resorbing, and forming osteons. In both forming (p = 0.024) and resorbing (p = 0.034) osteons, osteocyte densities were significantly higher in cases of hip fracture than controls. However, there were no significant between-group differences in lacunar occupancy. In both cases and controls, osteocyte density (p < 0.0001; mean difference ±SEM: 157 ± 34/mm²) and lacunar occupancy (p = 0.025; mean difference: 8.1 ± 3.4%) were shown to be significantly higher in forming compared with quiescent osteons. Interestingly, resorbing systems also exhibited significantly elevated osteocyte density in both the fracture and the control group combined (mean difference 76 ± 23/mm²; p = 0.003). Lacunar occupancy was also greater in resorbing compared with quiescent osteons (both groups combined: p = 0.022; mean difference: 5.7 ± 2.3%). Elevated osteocyte density and lacunar occupancy in forming compared with quiescent systems was expected because of the likely effects of aging on quiescent osteons. However, the higher levels of these parameters in resorbing compared with quiescent systems was the opposite of what we expected and suggests that, in addition to their postulated mechanosensory role in the suppression of remodeling and bone loss, osteocytes might also contribute to processes initiating or maintaining bone resorption.     

The fate of donor osteocytes in fine particulate bone powders during repair of bone defects in experimental rats

The aim of the study was to investigate the fate of donor osteocytes in fine particulate bone powders during repair of bone defects in experimental rats. The iliac bone of male inbred DA rats was harvested and used as the larger bone grafts and also prepared as fine particulate (granulated) bone powders (300-500 μm size particles) for transplantation into radial defects in female rats. The presence and relative amounts of genes specific to the sex-determining region of the Y-chromosome (Sry) originating from the bone grafts were evaluated by polymerase chain reaction and by in situ hybridization, respectively. Additional samples were evaluated histologically. In the larger bone grafts, the expression of Sry decreased relatively early, disappeared by 1 week, reappeared at 4 weeks and continued to increase with time. In the fine particulate bone powders, Sry was detected all the time and its expression was statistically greater than in the larger bone grafts at each time point. Both bone grafts provided donor cells to repair the defects. The donor cells seemed to function differently between the two groups. The fine particulate bone powders contained more living osteocytes in comparison with the larger bone grafts and may accelerate the healing of bone defects compared with conventional autografts.     

Influence of a diphosphonate on the cellular aspect of young bone tissue

Summary Stimulated by the rather sparse information in the literature on cellular changes induced by EHDP, we carried out electron microscopic investigations on young bone tissue and on de novo bone formation. Cellular changes could be observed during continuous administration of EHDP. The osteoblasts demonstrated temporary storing of crystalloid structures in the mitochondria, and atypical osteocytes showed persistent changes indicative of hyperactivity. The osteoclasts exhibited varying ultrastructural features with respect to the number and appearance of nuclei, Golgi, RER, and lysosomes. These changes under the influence of EHDP could be an indication of altered activity of the osteoclast. The possible interference of EHDP with bone cell metabolism is discussed.