Osteocytes are considered the primary mechanical sensor in bone tissue and orchestrate the coupled bone remodeling activity of adjacent osteoblast and osteoclast cells.In vivoinvestigation of mechanically induced signal propagation through networks of interconnected osteocytes is confounded by their confinement within the mineralized bone matrix, which cannot be modeled in conventional culture systems. In this study, we developed a new model that mimics thisin vivoconfinement using gelatin methacrylate (GelMA) hydrogel or GelMA mineralized using osteoblast-like model cells. This model also enables real-time optical examination of osteocyte calcium (Ca2+) signaling dynamics in response to fluid shear stimuli cultured under confined conditions. Using this system, we discovered several distinct and previously undescribed patterns of Ca2+responses that vary across networks of interconnected osteocytes as a function of space, time and connectivity. Heterogeneity in Ca2+signaling may provide new insights into bone remodeling in response to mechanical loading. Overall, such a model can be extended to study signaling dynamics within cell networks exposed to flow-induced mechanical stimuli under confined conditions.

Osteocytes are the main mechanosensory cells during orthodontic and physiologic bone remodeling. However, the question of how osteocytes transmit mechanical stimuli to biological responses remains largely unanswered. Intraflagellar transport (IFT) proteins are important for the formation and function of cilia, which are proposed to be mechanical sensors in osteocytes. In particular, IFT80 is highly expressed in mouse skulls and essential for ciliogenesis. This study aims to investigate the short- and long-term effects of IFT80 deletion in osteocytes on orthodontic bone remodeling and physiological bone remodeling in response to masticatory force. We examined 10-week-old experimental DMP1 CRE+.IFT80f/f and littermate control DMP1 CRE-.IFT80f/f mice. After 5 and 12 days of orthodontic force loading, the orthodontic tooth movement distance and bone parameters were evaluated using microCT. Osteoclast formation was assessed using TRAP-stained paraffin sections. The expression of sclerostin and RANKL was examined using immunofluorescence stain. We found that the deletion of IFT80 in osteocytes did not significantly impact either orthodontic or physiologic bone remodeling, as demonstrated by similar OTM distances, osteoclast numbers, bone volume fractions (bone volume/total volume), bone mineral densities, and the expressions of sclerostin and RANKL. Our findings suggest that there are other possible mechanosensory systems in osteocytes and anatomic limitations to cilia deflection in osteocytes in vivo.

BACKGROUND: Osteonecrosis of the femoral head (ONFH) is a common but intractable disease that appears to involve lipid metabolic disorders. Although numerous studies have demonstrated that high blood levels of low-density lipoprotein (LDL) are closely associated with ONFH, there is limited evidence to explain the pathological role of LDL. Pathological and in vitro studies were performed to investigate the role of disordered metabolism of LDL and oxidized LDL (ox-LDL) in the femoral head in the pathology of ONFH.
METHODS: Nineteen femoral head specimens from patients with ONFH were obtained for immunohistochemistry analysis. Murine long-bone osteocyte Y4 cells were used to study the effects of LDL/ox-LDL on cell viability, apoptosis, and metabolism process of LDL/ox-LDL in osteocytes in normoxic and hypoxic environments.
RESULTS: In the pathological specimens, marked accumulation of LDL/ox-LDL was observed in osteocytes/lacunae of necrotic regions compared with healthy regions. In vitro studies showed that ox-LDL, rather than LDL, reduced the viability and enhanced apoptosis of osteocytes. Pathological sections indicated that the accumulation of ox-LDL was significantly associated with impaired blood supply. Exposure to a hypoxic environment appeared to be a key factor leading to LDL/ox-LDL accumulation by enhancing internalisation and oxidation of LDL in osteocytes.
CONCLUSIONS: The accumulation of LDL/ox-LDL in the necrotic region may contribute to the pathology of ONFH. These findings could provide new insights into the prevention and treatment of ONFH.

This study aimed to evaluate the influence of estrogen deficiency and mechanical loading on bone around osseointegrated dental implants in a rat jaw model. The maxillary right first molars of 36 rats were extracted. One week later, the rats were divided into an unloaded group and a loaded group; short head implants and long head implants were inserted respectively. Nine weeks after implantation, the rats were further subjected to ovariectomy (OVX) or sham surgery. All animals were euthanized 21 weeks after OVX. Micro-computed tomography, histological and histomorphometrical evaluation were undertaken. Systemic bone mineral density and bone volume fraction decreased in OVX groups compared with the sham controls. Histomorphometrical observation indicated that unloaded OVX group showed significantly damaged osseointegration and bone loss versus the loaded OVX group. Both the bone density (BD) inside the peri-implant grooves and the percentage of bone-to-implant contact (BIC) were lower in the OVX groups than in the sham-surgery groups, although mechanical loading increased the BIC and BD in the loaded OVX group compared with the unloaded OVX group. An increased number of positive cells for tartrate-resistant acid phosphatase was observed in the OVX groups versus the sham controls. The percentage of sclerostin-positive osteocytes was lower under loaded compared with unloaded conditions in both the OVX groups and the sham controls. In conclusion, estrogen deficiency could be a risk factor for the long-term stability of osseointegrated implants, while mechanical loading could attenuate the negative influence of estrogen deficiency on bone formation and osseointegration.

The female skeleton undergoes significant material and ultrastructural changes to meet high calcium demands during reproduction and lactation. Through the peri-lacunar/canalicular remodeling (PLR), osteocytes actively resorb surrounding matrix and enlarge their lacunae and canaliculi during lactation, which are quickly reversed after weaning. How these changes alter the physicochemical environment of osteocytes, the most abundant and primary mechanosensing cells in bone, are not well understood. In this study, we developed a multiscale poroelastic modeling technique to investigate lactation-induced changes in stress, fluid pressurization, fluid flow, and solute transport across multiple length scales (whole bone, porous midshaft cortex, lacunar-canalicular pore system (LCS), and pericellular matrix (PCM) around osteocytes) in murine tibiae subjected to axial compression at 3 N peak load (~320 με) at 0.5, 2, or 4 Hz. Based on previously reported skeletal anatomical measurements from lactating and nulliparous mice, our models demonstrated that loading frequency, LCS porosity, and PCM density were major determinants of fluid and solute flows responsible for osteocyte mechanosensing, cell-cell signaling, and metabolism. When loaded at 0.5 Hz, lactation-induced LCS expansion and potential PCM reduction promoted solute transport and osteocyte mechanosensing via primary cilia, but suppressed mechanosensing via fluid shear and/or drag force on the cell membrane. Interestingly, loading at 2 or 4 Hz was found to overcome the mechanosensing deficits observed at 0.5 Hz and these counter effects became more pronounced at 4 Hz and with sparser PCM in the lactating bone. Synergistically, higher loading frequency (2, 4 Hz) and sparser PCM enhanced flow-mediated mechanosensing and diffusion/convection of nutrients and signaling molecules for osteocytes. In summary, lactation-induced structural changes alter the local environment of osteocytes in ways that favor metabolism, mechanosensing, and post-weaning recovery of maternal bone. Thus, osteocytes play a role in balancing the metabolic and mechanical functions of female skeleton during reproduction and lactation.

OBJECTIVE: The purpose of this pilot porcine cadaver study was to evaluate the feasible temperature thresholds, which affect osteocyte viability and bone matrix in a preclinical setup, assessing the potential of thermal necrosis for implant removal for further in vivo investigations.
METHODS: After implant bed preparation in the upper and lower jaw, temperature effects on the bone were determined, using two tempering pistons with integrated thermocouples. To evaluate threshold temperature and time intervals leading to bone necrosis, one piston generated warm temperatures at 49 to 56 °C for 10 s and the other generated cold temperatures at 5 to 1 °C for 30 s. Effects were assessed by a semi-quantitative, histomorphometrical scoring system, scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), and transmission electron microscopy (TEM).
RESULTS: The bone matrix was significantly degenerated starting at 51 °C for 10 s and 5 °C for 30 s. The osteocyte condition indicated significant bone damage beginning at cold temperatures of 2 °C. Temperature inputs starting at 53 °C led to decalcification and swollen mitochondria, which lost the structure of their inner cristae.
CONCLUSIONS: This study identified temperatures and durations, in both heat and cold, so that the number of samples may be kept low in further studies regarding temperature-induced bone necrosis. Levels of 51 °C for 10 s and 5 °C for 30 s have presented significant matrix degeneration.
CONCLUSIONS: Temperature thresholds, potentially leading to thermo-explantation of dental implants and other osseointegrated devices, were identified.

This study sought to derive an enhanced understanding of the complex intracellular interactions that drive bone loss in postmenopausal osteoporosis. We applied an in-vitro multicellular niche to recapitulate cell-cell signalling between osteocytes, osteoblasts and osteoclasts to investigate (1) how estrogen-deficient and mechanically loaded osteocytes regulate osteoclastogenesis and (2) whether ROCK-II inhibition affects these mechanobiological responses. We report that mechanically stimulated and estrogen-deficient osteocytes upregulated RANKL/OPG and M-CSF gene expression, when compared to those treated with 10 nM estradiol. Osteoclast precursors (RAW 264.7) cultured within this niche underwent significant reduction in osteoclastogenic gene expression (CTSK), and there was an increasing trend in the area covered by TRAP+ osteoclasts (24% vs. 19.4%, p = 0.06). Most interestingly, upon treatment with the ROCK-II inhibitor, RANKL/OPG and M-CSF gene expression by estrogen-deficient osteocytes were downregulated. Yet, this inhibition of the pro-osteoclastogenic factors by osteocytes did not ultimately reduce the differentiation of osteoclast precursors. Indeed, TRAP and CTSK gene expressions in osteoclast precursors were upregulated, and there was an increased trend for osteoclast area (30.4% vs. 24%, p = 0.07), which may have been influenced by static osteoblasts (MC3T3-E1) that were included in the niche. We conclude that ROCK-II inhibition can attenuate bone loss driven by osteocytes during estrogen deficiency.

Ultrasound stimulation is thought to influence bone remodelling process. But recently, the efficiency of ultrasound therapy for bone healing has been questioned. Despite an extensive literature describing the positive effect of ultrasound on bone regeneration-cell cultures, animal models, clinical studies-there are more and more reviews denouncing the inefficiency of clinical devices based on low-intensity pulsed ultrasound stimulation (LIPUS) of the bone healing. One of the reasons to cause controversy comes from the persistent misunderstanding of the underlying physical and biological mechanisms of ultrasound stimulation of bone repair. As ultrasonic waves are mechanical waves, the process to be studied is the one of the mechanotransduction. Previous studies on the bone mechanotransduction have demonstrated the key role of the osteocytes in bone mechano-sensing. Osteocytes are bone cells ubiquitous inside the bone matrix; they are immersed in the interstitial fluid (IF) inside the lacuno-canalicular network (LCN). They are considered as particularly sensitive to a particular type of mechanical stress: wall shear stress on osteocytes due to the IF flow in the LCN. Inspired from these findings and observations, the present work investigates the effect of LIPUS on the cortical bone from the tissue to the osteocytes, considering that the impact of the ultrasound stimulation applied at the tissue scale is related to the mechanical stress experimented by the bone cells. To do that simulations based on the finite element method are carried out in the commercial software Comsol Multiphysics to assess the wall shear stress levels induced by the LIPUS on the osteocytes. Two formulations of the wall shear stress were investigated based on two IF flow models inside the LCN and associated with two different values of the LCN permeability. The wall shear stress estimate is very different depending on the assumption considered. One of these two models provides wall shear stress values in accordance with previous works published on bone mechanotransduction. This study presents the preliminary results of a computational model that could provide keys to understanding the underlying mechanisms of the LIPUS.

Osteocyte apoptosis has been associated with a number of clinical conditions in bone and with the targeted turnover of specific skeletal areas. There has been great interest in the identification of the mechanisms by which apoptosis is regulated in bone and in the biological role that this process plays in bone metabolism and associated bone disease or loss of structural integrity. Here we describe several methods for the detection of apoptosis in bone sections and in bone cell cultures.

Ischemic injuries and local hypoxia can result in osteocytes dysfunction and play a key role in the pathogenesis of avascular osteonecrosis. Conventional imaging techniques including magnetic resonance imaging (MRI) and computed tomography (CT) can reveal structural and functional changes within bony anatomy; however, characterization of osteocyte behavioral dynamics in the setting of osteonecrosis at the single cell resolution is limited. Here, we demonstrate an optical approach to study real-time osteocyte functions in vivo. Using nicotinamide adenine dinucleotide (NADH) as a biomarker for metabolic dynamics in osteocytes, we showed that NADH level within osteocytes transiently increase significantly after local ischemia through non-invasive photo-induced thrombosis of afferent arterioles followed by a steady decline. Our study presents a non-invasive optical approach to study osteocyte behavior through the modulation of local environmental conditions. Thus it provides a powerful toolkit to study cellular processes involved in bone pathologies in vivo.