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Femur fracture leads to loss of bone at uninjured skeletal sites, which may increase risk of subsequent fracture. Osteocytes, the most abundant bone cells, can directly resorb bone matrix and regulate osteoclast and osteoblast activity, but their role in systemic bone loss after fracture remains poorly understood. In this study we used a transgenic (TG+) mouse model that overexpresses human B-cell lymphoma 2 (BCL-2) in osteoblasts and osteocytes. This causes enhanced osteoblast proliferation, followed by disruption in lacunar-canalicular connectivity and massive osteocyte death by 10 wk of age. We hypothesized that reduced viable osteocyte density would decrease the magnitude of systemic bone loss after femur fracture, reduce perilacunar remodeling, and alter callus formation. Bone remodeling was assessed using serum biomarkers of bone formation and resorption at 5 d post-fracture. We used micro-computed tomography, high resolution x-ray microscopy, mechanical testing, and Raman spectroscopy to quantify the magnitude of systemic bone loss, as well as changes in osteocyte lacunar volume, bone strength, and bone composition 2 wk post-fracture. Fracture was associated with a reduction in circulating markers of bone resorption in non-transgenic (TG-) animals. TG+ mice exhibited high bone mass in the limbs, greater cortical elastic modulus and reduced post-yield displacement. After fracture, TG+ mice lost less trabecular bone than TG- mice, but conversely TG+ mice exhibited trends toward a lower yield point and reduced femoral cortical thickness after fracture, though these were not statistically significant. Lacunar density was greater in TG+ mice, but fracture did not alter lacunar volume in TG+ or TG- mice. These findings suggest that osteocytes potentially play a significant role in the post-traumatic systemic response to fracture, though the effects differ between trabecular and cortical bone.

Contrary to the popular perception that bone is merely a structural organ, decades of research has established its functional importance in whole-body metabolism. Osteocytes, which comprise >80% of all bone cells, were also initially thought to be terminally differentiated dormant cells lacking metabolic functions. New research, however, is increasingly providing evidence that osteocytes not only play a role in the structural integrity of bone but also have secretory functions that regulate other bone cells and other organs, including skeletal muscle, the structural-mechanical neighbor of the bone, via paracrine and endocrine pathways. However, interpretations of the publicly available preclinical and clinical data pertaining to the factors secreted by osteocytes and their functions in the musculoskeletal system largely fail to reach a consensus. This review aimed to objectively collate all information available in the public domain for efficient access by researchers in the field. We strongly believe that this review will assist researchers attempting the unbiased design of therapeutic strategies for musculoskeletal disorders. [ABSTRACT FROM AUTHOR]

Renal osteodystrophy (ROD) leads to increased fractures, potentially due to underlying low bone turnover in chronic kidney disease (CKD). We hypothesized that indoxyl sulfate (IS), a circulating toxin elevated in CKD and a ligand for the aryl hydrocarbon receptor (AhR), may target the osteocytes leading to bone cell uncoupling in ROD. The IDG-SW3 osteocytes were cultured for 14 days (early) and 35 days (mature osteocytes) and incubated with 500 μM of IS after dose finding studies to confirm AhR activation. Long-term incubation of IS for 14 days led to decreased expression of Tnfsf11/Tnfrsf11b ratio (RANKL/OPG), which would increase osteoclast activity, and increased expression of Wnt inhibitors Sost and Dkk1, which would decrease bone formation in addition to decreased mineralization and alkaline phosphatase (ALP) activity. When osteocytes were incubated with IS and the AhR translocation inhibitor CH223191, mineralization and ALP activity were restored. However, the Tnfsf11/Tnfrsf11b ratio and Sost, Dkk1 expression were not altered compared with IS alone, suggesting more complex signaling. In both early and mature osteocytes, co-culture with parathyroid hormone (PTH) and IS reversed the IS-induced upregulation of Sost and Dkk1, and IS enhanced the PTH-induced increase of the Tnfsf11/Tnfrsf11b ratio. Co-culture of IS with PTH additively enhanced the AhR activity assessed by Cyp1a1 and Cyp1b1 expression. In summary, IS in the absence of PTH increased osteocyte messenger RNA (mRNA) Wnt inhibitor expression in both early and mature osteocytes, decreased mRNA expression ofTnfsf11/Tnfrsf11b ratio and decreased mineralization in early osteocytes. These changes would lead to decreased resorption and formation resulting in low bone remodeling. These data suggest IS may be important in the underlying low turnover bone disease observed in CKD when PTH is not elevated. In addition, when PTH is elevated, IS interacts to further increase Tnfsf11/Tnfrsf11b ratio for osteoclast activity in both early and mature osteocytes, which would worsen bone resorption. [ABSTRACT FROM AUTHOR]

ABSTRACT As the aging population continues to grow, the incidence of osteoporotic fractures increases and is compounded by our lack of therapeutic strategies that increase bone formation. Although exercise and physical activity play a key role in maintaining bone mass throughout our lives, the loads and exertion required to elicit an anabolic response becomes exceedingly difficult to achieve with age. Based on previous work, the P2Y2 receptor offers a unique therapeutic target to increasing bone mass by modifying the mechanotransduction. Others have also shown P2Y2 to have a negative effect on osteoblast function. However, the extent to which inhibiting P2Y2 pharmaceutically improves bone mass or the mechanotransduction of bone remains unknown. Our central hypothesis for this study states that inhibiting P2Y2 activity can enhance the anabolic response to loading in an aging population. To test this hypothesis, the anabolic response to exercise was examined by treating adult mice, which typically display a minimal response, with the P2Y2 inhibitor AR‐C118925XX (ARC). Our findings from this study demonstrate that ARC treatment of adult mice increases periosteal bone formation in response to exercise. The enhanced response to exercise was characterized by a reduction in osteocytes' induction of osteoclast activity. Endocortical bone formation also increased with treatment independently of exercise, providing gains in mechanical strength and tissue level properties. Overall, inhibiting P2Y2 activation has a beneficial effect on bone formation and the anabolic response to loading, namely by limiting osteoclast activation. [ABSTRACT FROM AUTHOR]

Periimplantitis represents an inflammatory disease of the soft and hard tissues surrounding the osseointegrated dental implant, triggering progressive damage to the alveolar bone. Cumulative data have revealed that periimplantitis plays a crucial part in implant failure. Due to the strategic roles of autophagy and its upstream coordinator, the AKT/mTOR pathway, in inflammatory responses, the crosstalk between them in the context of periimplantitis should become a key research target, as it opens up an area of interesting data with clinical significance. Therefore, in this article, we aimed to briefly review the existing data concerning the complex roles played by ROS in the interplay between the AKT/mTOR signaling pathway and autophagy in periimplantitis, in each of the main cell types involved in periimplantitis pathogenesis and evolution. Knowing how to modulate specifically the autophagic machinery in each of the cellular types involved in the healing and osseointegration steps post implant surgery can help the clinician to make the most appropriate post-surgery decisions. These decisions might be crucial in order to prevent the occurrence of periimplantitis and ensure the proper conditions for effective osseointegration, depending on patients' clinical particularities. [ABSTRACT FROM AUTHOR]

Osteoporosis (OP) is the most prevalent metabolic bone disease and an important postmenopausal consequence. This study aimed to investigate the effects of morin, a flavonoid with beneficial properties, on ovariectomy‐induced OP. Animals were ovariectomized (OVX) and treated with different doses of morin (15, 30, and 45 mg/kg/day) or estradiol (10 μg/kg/day) for 10 weeks by gavage. Then bone histo‐stereology, bone‐related biochemical indicators, and gene and protein levels of autophagy and apoptosis‐related markers were analyzed. In comparison to controls, OVX significantly decreased the number of osteoblasts (5.78 × 106 vs. 1.66 × 106) and osteocytes (32.55 × 106 vs. 11.92 × 106), whereas increasing the number of osteoclasts (83.38 × 103 vs. 392.1 × 103). Moreover, OVX caused a remarkable decrease in bone structures and Ca, P, and estradiol levels while increasing ALP and OC (p < 0.001). The administration of 45 mg/kg/day morin restored the effects of OP on bone histomorphology and biochemical markers (p < 0.05). Further studies revealed that morin caused a 7.1% and 36.6% decrease in the bone level of LC3 and BECN1 proteins, respectively, compared to the OVX group. Also, morin caused a significant decrease of 47.4% in the CASP3 level and a significant increase of 23.6% in the BCL‐2 level compared to OVX animals (p < 0.001). The present findings showed that morin is potentially able to improve the bone‐related histomorphological and biochemical changes caused by osteoporosis, which is probably attributed to the suppression of apoptosis‐ and autophagy‐caused cell death. [ABSTRACT FROM AUTHOR]

Osteocytes, situated within the lacunocanalicular network (LCN) of the bone matrix, play crucial roles in sensing mechanical signals and orchestrating bone adaptive responses. Alterations in LCN structure could significantly modify the fluid dynamic microenvironment of osteocytes, thereby influencing bone mechanoresponses (BMRs). However, a comprehensive understanding of this tissue remains elusive. In this study, a multi-scale model of whole bone-LCN was developed to systematically investigate the effects of lacunocanalicular morphology (lacunar volume [Lc.V] and canalicular area [Ca.S]) and network architecture (lacunar density [Lc.ρ] and canalicular density [Ca.ρ]) on fluid shear stress (FSS) within the LCN and BMR predicted by fluid flow. Furthermore, the relationships between fluid flow within the LCN and BMRs were examined in two specific scenarios: aging and lactation. Results demonstrated that changes in lacunocanalicular morphology (Lc.V and Ca.S) primarily influenced the intensity of fluid flow, while alterations in the LCN (Lc.ρ and Ca.ρ) largely affected the distribution of fluid flow. Increases in Lc.V or decreases in Ca.S increased FSS, whereas decreases in Lc.ρ or increases in Ca.ρ reduced FSS. Compared with other structural parameters, alterations in Ca.ρ had the greatest effect on FSS, while BMR primarily depended on changes in Lc.V and Ca.S. In agreement with experimental observations, aging- or lactation-induced changes in LCN structure (and fluid dynamics) were associated with reduced (−50%) or increased (+20%) bone responses to mechanical loading, respectively. These findings suggest that modifications in lacunocanalicular morphology and network architecture can substantially impact the fluid dynamic microenvironment for mechanosensing osteocytes and, consequently, BMRs. [ABSTRACT FROM AUTHOR]

Osteocyte senescence is associated with skeletal dysfunction, but how to prevent bone loss and find the effective therapeutic targets is a potential scientific concern. Cadmium (Cd) is a widespread environmental contaminant that causes substantial bone damage in both animals and humans. Oligomeric proanthocyanidins (OPC) are naturally polyphenolic substances found in various plants and demonstrate significant anti-senescence potential. Here, we investigated the protective effects of OPC against Cd-induced senescence of osteocytes and identify potential regulatory mechanisms. OPC alleviated Cd-induced senescence of osteocytes by attenuating cell cycle arrest, reducing ROS accumulation, and suppressing pro-inflammatory responses in vitro. Furthermore, OPC effectively prevented the Cd-induced breakdown of dendritic synapses in osteocytes in vitro. Correspondingly, OPC ameliorated Cd-induced damage of osteocytes through anti-senescence activity in vivo. Taken together, our results establish OPC as a promising therapeutic agent that ameliorates Cd-induced osteocyte senescence by mitigating oxidative stress and inflammatory responses. [ABSTRACT FROM AUTHOR]

Poor bone quality is a major factor in skeletal fragility in elderly individuals. The molecular mechanisms that establish and maintain bone quality, independent of bone mass, are unknown but are thought to be primarily determined by osteocytes. We hypothesize that the age-related decline in bone quality results from the suppression of osteocyte perilacunar/canalicular remodeling (PLR), which maintains bone material properties. We examined bones from young and aged mice with osteocyte-intrinsic repression of TGFβ signaling (TβRIIocy-/-) that suppresses PLR. The control aged bone displayed decreased TGFβ signaling and PLR, but aging did not worsen the existing PLR suppression in male TβRIIocy-/- bone. This relationship impacted the behavior of collagen material at the nanoscale and tissue scale in macromechanical tests. The effects of age on bone mass, density, and mineral material behavior were independent of osteocytic TGFβ. We determined that the decline in bone quality with age arises from the loss of osteocyte function and the loss of TGFβ-dependent maintenance of collagen integrity.

Osteocytes locally remodel their surrounding tissue through perilacunar canalicular remodeling (PLR). During lactation, osteocytes remove minerals to satisfy the metabolic demand, resulting in increased lacunar volume, quantifiable with synchrotron X-ray radiation micro-tomography (SRµCT). Although the effects of lactation on PLR are well-studied, it remains unclear whether PLR occurs uniformly throughout the bone and what mechanisms prevent PLR from undermining bone quality. We used SRµCT imaging to conduct an in-depth spatial analysis of the impact of lactation and osteocyte-intrinsic MMP13 deletion on PLR in murine bone. We found larger lacunae undergoing PLR are located near canals in the mid-cortex or endosteum. We show lactation-induced hypomineralization occurs 14 µm away from lacunar edges, past a hypermineralized barrier. Our findings reveal that osteocyte-intrinsic MMP13 is crucial for lactation-induced PLR near lacunae in the mid-cortex but not for whole-bone resorption. This research highlights the spatial control of PLR on mineral distribution during lactation.

Abstract Background Glucocorticoid-associated osteonecrosis of the femoral head (GA-ONFH) is a progressive bone disorder which frequently results in femoral head collapse and hip joint dysfunction. Sclerostin (SOST) is principally secreted by osteocytes in bone and plays an important role in bone homeostasis and homeostasis of skeletal integrity. Our previous study reported that short‐term use of glucocorticoid increased serum sclerostin levels. Here this study is aimed to identify whether sclerostin played an essential role in the occurrence and development of GA-ONFH. Methods Glucocorticoid-induced osteonecrosis of femoral head (ARCO stage II) samples were collected and sclerostin staining was conducted. Osteocyte cell line Ocy454, MC3T3-E1 and endothelial cells was used. MC3T3-E1 or endothelial cells were co-cultured with Ocy454 or SOST-silencing Ocy454 in presence of dexamethasone to mimic the crosstalk of various cells in the bone niche. GA-ONFH rat model and SOST knockout model was built to better understand the phenomenon in vivo. Results Sclerostin was highly concentrated in osteonecrosis patient sample in the necrotic area. Co-culture with osteocytes aggravated the inhibition of dexamethasone on MC3T3-E1 and endothelial cells. Sclerostin derived from osteocytes impaired osteogenesis and angiogenesis via inhibiting the Wnt pathway. In GA-ONFH rat model, SOST knockout ameliorated the incidence of osteonecrosis and improved bone metabolism compared with the wild type group through histological, immunohistochemical and bone metabolic analyses. Conclusion Sclerostin contribute to pathologic process of GA-ONFH by impairing osteogenesis and angiogenesis.

Three-dimensional cultures are widely used to study bone and cartilage. These models often focus on the interaction between osteoblasts and osteoclasts or osteoblasts and chondrocytes. A culture of osteoblasts, osteoclasts and chondrocytes would represent the cells that interact in the joint and a model with these cells could be used to study many diseases that affect the joints. The goal of this study was to develop 3D bone-cartilage interface (3D-BCI) that included osteoblasts, osteocytes, osteoclasts, and cartilage. Fluorescently tagged cell lines were developed to assess the interactions as cells differentiate to form bone and cartilage. Mouse cell line, MC3T3, was labeled with a nuclear GFP tag and differentiated into osteoblasts and osteocytes in Matrigel. Raw264.7 cells transfected with a red cytoplasmic tag were added to the system and differentiated with the MC3T3 cells to form osteoclasts. A new method was developed to differentiate chondrocyte cell line ATDC5 in a cartilage spheroid, and the ATDC5 spheroid was added to the MC3T3 and Raw264.7 cell model. We used an Incucyte and functional analysis to assess the cells throughout the differentiation process. The 3D-BCI model was found to be positive for TRAP, ALP, Alizarin red and Alcian blue staining to confirm osteoblastogenesis, osteoclastogenesis, and cartilage formation. Gene expression confirmed differentiation of cells based on increased expression of osteoblast markers: Alpl , Bglap , Col1A2 , and Runx 2 , cartilage markers: Acan , Col2A1 , Plod2 , and osteoclast markers: Acp5 , Rank and Ctsk. Based on staining, protein expression and gene expression results, we conclude that we successfully developed a mouse model with a 3D bone-cartilage interface. [ABSTRACT FROM AUTHOR]

The bone-derived hormone FGF23, primarily secreted by osteocytes, is a major player in the regulation of phosphate homeostasis. It becomes upregulated by increased circulating phosphate concentration, e.g. due to elevations in phosphorus intake (P-In) or alterations in habitual dietary acid load. The present study aimed to investigate whether long-term endogenous acid production or a habitual high phosphorus intake during childhood and adolescence may be prospectively related with altered adult levels of FGF23 and the FGF23-related metabolite α-klotho. Urinary phosphate excretion (PO4-Ex), net acid excretion (NAE), and potential renal acid load (uPRAL) were analyzed in 24-h urine samples (n = 3369) collected from 343 healthy 3–17 years old participants of the DONALD Study (Dortmund, Germany) to assess, biomarker-based, P-In and habitual dietary acid load. Circulating FGF23, α-klotho, and further blood parameters were additionally examined in young adulthood. Individual means of standard-deviation-scores were calculated for 24-h urinary biomarker excretions and anthropometrics longitudinally determined between ages 3–17 years. Multivariable linear regression was used to analyze the prospective relations of pre-adulthood PO4-Ex, NAE, and uPRAL with the adulthood outcomes FGF23 and α-klotho. After adjusting for growth period-related covariates and adulthood confounders only for P-In during growth, i.e., PO4-Ex, but not for NAE and uPRAL, a significant positive association (p = 0.03) with FGF23 and an inverse trend (p = 0.10) with the FGF23-α-klotho ratio were observed. Neither PO4-Ex, nor NAE or uPRAL were associated with soluble α-klotho levels in adulthood. The prospective relationships of long-term assessed 24-h phosphaturia and habitual dietary acid load during growth with adult circulating, phosphate-adjusted FGF23 strongly suggest that children´s habitually higher P-In does unfavorably affect adult FGF23–α-klotho axis. [ABSTRACT FROM AUTHOR]

Osteocytes' response to dynamic loading plays a crucial role in regulating the bone mass but quickly becomes saturated such that downstream induction of bone formation plateaus. The underlying mechanisms that downregulate osteocytes' sensitivity and overall response to loading remain unknown. In other cell types, purinergic signaling through the P2Y2 receptor has the potential to downregulate the sensitivity to loading by modifying cell stiffness through actin polymerization and cytoskeleton organization. Herein, we examined the role of P2Y2 activation in regulating osteocytes' mechanotransduction using a P2Y2 knockout cell line alongside conditional knockout mice. Our findings demonstrate that the absence of P2Y2 expression in MLO-Y4 cells prevents actin polymerization while increasing the sensitivity to fluid flow-induced shear stress. Deleting osteocytes' P2Y2 expression in conditional-knockout mice enabled bone formation to increase when increasing the duration of exercise. Overall, P2Y2 activation under loading produces a negative feedback loop, limiting osteocytes' response to continuous loading by shifting the sensitivity to mechanical strain through actin stress fiber formation. [ABSTRACT FROM AUTHOR]

Different gravity fields have important effects on the structural morphology of bone. The fluid flow caused by loadings in the bone lacunar-canalicular system (LCS), converts mechanical signals into biological signals and regulates bone reconstruction by affecting effector cells, which ensures the efficient transport of signaling molecules, nutrients, and waste products. In this study, the fluid flow and mass transfer effects of bone lacunar-canalicular system at multi-scale were firstly investigated, and a three-dimensional axisymmetric fluid-solid coupled finite element model of the LCS within three continuous osteocytes was established. The changes in fluid pressure field, flow velocity field, and fluid shear force variation on the surface of osteocytes within the LCS were studied comparatively under different gravitational fields (0 G, 1 G, 5 G), frequencies (1 Hz, 1.5 Hz, 2 Hz) and forms of cyclic compressive loading. The results showed that different frequencies represented different exercise intensities, suggesting that high-intensity exercise may accelerate the fluid flow rate within the LCS and enhance osteocytes activity. Hypergravity enhanced the transport of solute molecules, nutrients, and signaling molecules within the LCS. Conversely, the mass transfer in the LCS may be inhibited under microgravity, which may cause bone loss and eventually lead to the onset of osteoporosis. This investigation provides theoretical guidance for rehabilitative training against osteoporosis. [ABSTRACT FROM AUTHOR]

Low-intensity pulsed ultrasound (LIPUS) is a potential effective means for the prevention and treatment of disuse osteoporosis. In this paper, the effect of LIPUS exposure on the mechanical properties distribution of the osteocyte system (osteocyte body contains nucleus, osteocyte process, and primary cilia) is simulated. The results demonstrate that the mechanical micro-environment of the osteocyte is significantly improved by ultrasound exposure, and the mean von Mises stress of the osteocyte system increases linearly with the excitation sound pressure amplitude. The mechanical effect of LIPUS on osteocytes is enhanced by the stress amplification mechanism of the primary cilia and osteocyte processes. [ABSTRACT FROM AUTHOR]

The lacunocanalicular network (LCN) is an intricate arrangement of cavities (lacunae) and channels (canaliculi), which permeates the mineralized bone matrix. In its porosity, the LCN accommodates the cell network of osteocytes. These two nested networks are attributed a variety of essential functions including transport, signaling, and mechanosensitivity due to load-induced fluid flow through the LCN. For a more quantitative assessment of the networks' function, the three-dimensional architecture has to be known. For this reason, we aimed (i) to quantitatively characterize spatial heterogeneities of the LCN in whole mouse tibial cross-sections of BALB/c mice and (ii) to analyze differences in LCN architecture by comparison with another commonly used inbred mouse strain, the C57BL/6 mouse. Both tibiae of five BALB/c mice (female, 26-week-old) were stained using rhodamine 6G and whole tibiae cross-sections were imaged using confocal laser scanning microscopy. Using image analysis, the LCN was quantified in terms of density and connectivity and lacunar parameters, such as lacunar degree, volume, and shape. In the same tibial cross-sections, the calcium content was measured using quantitative backscattered electron imaging (qBEI). A structural analysis of the LCN properties showed that spatially denser parts of the LCN are mainly due to a higher density of branching points in the network. While a high intra-individual variability of network density was detected within the cortex, the inter-individual variability between different mice was low. In comparison to C57BL/6J mice, BALB/c mice showed a distinct lower canalicular density. This reduced network was already detectable on a local network level with fewer canaliculi emanating from lacunae. Spatial correlation with qBEI images demonstrated that bone modeling resulted in disruptions in the network architecture. The spatial heterogeneity and differences in density of the LCN likely affects the fluid flow within the network and therefore bone's mechanoresponse to loading. [ABSTRACT FROM AUTHOR]

Poor bone quality is a critical factor associated with an increased risk of complications after total hip arthroplasty (THA). However, no consistent recommendations have yet been established for assessing indicators of bone quality preoperatively. Thus, it remains unclear which preoperatively available and readily accessible parameters are most closely associated with femoral bone quality. Here, we obtained femoral neck specimens from 50 patients undergoing THA. Preoperative Dual-energy X-ray absorptiometry (DXA) scans, pelvic radiographs, and laboratory parameters were analyzed. In the obtained specimens, bone microstructure was assessed using micro-CT and histomorphometry. Additionally, matrix mineralization and osteocyte lacunar morphology were evaluated using quantitative backscattered electron imaging. Our analysis revealed that DXA-derived T-scores correlated with trabecular microstructure. Furthermore, radiographic indices and body mass index correlated differentially with aspects of bone quality in women and men. Contrary to previous observations, no correlation was found between serum vitamin D levels and osteoid indices, nor between clinical parameters and matrix mineralization. Age was strongly associated with the number of mineralized osteocyte lacunae, a factor that appeared to be independent of sex. Taken together, our study demonstrates that no single preoperatively available parameter exhibits a strong and consistent association with femoral bone quality. However, DXA remains a reliable preoperative measure for determining the trabecular microstructure of the femoral neck. In clinical practice, surgeons should adopt an individualized approach to preoperative assessments by considering age, sex, BMI, and radiographic indices to enhance their insight into femoral bone quality, particularly when DXA is unavailable. [ABSTRACT FROM AUTHOR]

Bone is a three‐dimensional (3D) highly dynamic tissue under constant remodeling. Commonly used tools to investigate bone biology require sample digestion for biomolecule extraction or provide only two‐dimensional (2D) spatial information. There is a need for 3D tools to investigate spatially preserved biomarker expression in osteocytes. In this work, we present a new method, WISH‐BONE, to label osteocyte messenger RNA (mRNA) and protein in whole‐mount mouse bone. For mRNA labeling, we used hybridization chain reaction‐fluorescence in situ hybridization (HCR‐FISH) to label genes of interest in osteocytes. For protein labeling, samples were preserved using an epoxy‐based solution that protects tissue structure and biomolecular components. Then an enzymatic matrix permeabilization step was performed to enable antibody penetration. Immunostaining was used to label various proteins involved in bone homeostasis. We also demonstrate the use of customized fluorescent nanobodies to target and label proteins in the cortical bone (CB). However, the relatively dim signal observed from nanobodies' staining limited detection. mRNA and protein labeling were performed in separate samples. In this study, we share protocols, highlight opportunities, and identify the challenges of this novel 3D labeling method. They are the first protocols for whole‐mount osteocyte 3D labeling of mRNA and protein in mature mouse bones. WISH‐BONE will allow the investigation of molecular signaling in bone cells in their 3D environment and could be applied to various bone‐related fields of research. [ABSTRACT FROM AUTHOR]

The phosphorites of the Quercy from SouthWest France are well known for fossils preserved in 3D with phosphatized soft-tissues. Given that phosphatization is known to favor fine cellular preservation, the present study delves into the histological analysis of white and brown bones of Cainotheriidae (Artiodactyla) recently excavated from the DAM1 site near Caylus. Microscopy revealed that the white bones were completely filled with bacterial erosions, while the brown bones showed a pristine histology and intralacunar content resembling fossilized osteocytes in some areas. After decalcification, a brown bone revealed an abundance of blood vessel-like structures, innumerable osteocyte-like structures with canaliculi and a few chondrocyte-like structures, while a white bone revealed only blood vessel-like structures that looked eaten away. All the data combined suggest the brown bones were shielded from bacterial attacks and were filled with fossilized organic matter and original biological structures. The data taken all together do not support that these structures are casts, but indeed original and endogenous cells. This study encourages further histochemical and mineralogical analyses on Quercy fossils and the unique taphonomy of DAM1 to better understand fossilization processes and their impact on the color of bones, the chemistry of skeletal tissues, soft tissues, and cells. [ABSTRACT FROM AUTHOR]

Objective: This study aims to evaluate the protective role of zinc supplementation against bone damage induced by a highsalt diet, with a specific focus on osteocyte apoptosis in rats. Methods: This was an investigational study conducted at the Army Medical College, Rawalpindi, Pakistan, over eight weeks. Thirty female Sprague-Dawley rats were randomly divided into three groups: control (Group C), high-salt diet (Group A), and high-salt diet with zinc supplementation (Group B). Group C received a standard diet, Group A received a diet with 8% sodium chloride, and Group B received the high-salt diet plus zinc supplementation at 50 mg/kg body weight daily via oral gavage. After eight weeks, femurs were harvested, processed, and stained with hematoxylin and eosin. Osteocyte apoptosis was assessed by counting empty lacunae and apoptotic bodies under light microscopy. Results: The high-salt diet group (Group A) exhibited a significantly higher density of apoptotic osteocytes compared to the control group (mean ± SD: 2.3166 ± 0.820 vs. 1.3666 ± 0.431 per unit area; p=0.0005). Zinc supplementation in Group B resulted in a significant reduction in osteocyte apoptosis compared to both the high-salt diet group (mean ± SD: 1.7000 ± 0.492 per unit area; p=0.0087) and the control group (p=0.0009). These findings indicate that zinc supplementation effectively reduces osteocyte apoptosis caused by high salt intake. Conclusion: Zinc supplementation significantly mitigates the harmful effects of a high-salt diet on bone health by reducing osteocyte apoptosis. This suggests its potential as a therapeutic intervention to counteract salt-induced bone damage and prevent related diseases such as osteoporosis. [ABSTRACT FROM AUTHOR]

Cortical bone loss is intricately associated with ageing and coincides with iron accumulation. The precise role of ferroptosis, characterized by iron overload and lipid peroxidation, in senescent osteocytes remains elusive. We found that ferroptosis was a crucial mode of osteocyte death in cortical bone during ageing. Using a single‐cell transcriptome analysis, we identified activating transcription factor 3 (ATF3) as a critical driver of osteocyte ferroptosis. Elevated ATF3 expression in senescent osteocytes promotes iron uptake by upregulating transferrin receptor 1 while simultaneously inhibiting solute carrier family 7‐member 11‐mediated cystine import. This process leads to an iron overload and lipid peroxidation, culminating in ferroptosis. Importantly, ATF3 inhibition in aged mice effectively alleviated ferroptosis in the cortical bone and mitigated cortical bone mass loss. Taken together, our findings establish a pivotal role of ferroptosis in cortical bone loss in older adults, providing promising prevention and treatment strategies for osteoporosis and fractures. [ABSTRACT FROM AUTHOR]

For a better understanding of living tissues and materials, it is essential to study the intricate spatial relationship between cells and their surrounding tissue on the nanoscale, with a need for 3D, high‐resolution imaging techniques. In the case of bone, focused ion beam‐scanning electron microscopy (FIB‐SEM) operated in the backscattered electron (BSE) mode proves to be a suitable method to image mineralized areas with a nominal resolution of 5 nm. However, as clinically relevant samples are often resin‐embedded, the lack of atomic number (Z) contrast makes it difficult to distinguish the embedding material from unmineralized parts of the tissue, such as osteoid, in BSE images. Staining embedded samples with iodine vapor has been shown to be effective in revealing osteoid microstructure by 2D BSE imaging. Based on this idea, an iodine (Z = 53) staining protocol is developed for 3D imaging with FIB‐SEM, investigating how the amount of iodine and exposure time influences the imaging outcome. Bone samples stained with this protocol also remain compatible with confocal laser scanning microscopy to visualize the lacunocanalicular network. The proposed protocol can be applied for 3D imaging of tissues exhibiting mineralized and nonmineralized regions to study physiological and pathological biomineralization. [ABSTRACT FROM AUTHOR]

Macrophages and osteocytes are important regulators of inflammation, osteogenesis and osteoclastogenesis. However, their interactions under adverse conditions, such as biomaterial-associated infection (BAI) are not fully understood. We aimed to elucidate how factors released from macrophages modulate osteocyte responses in an in vitro indirect 3D co-culture model. Human monocyte-derived macrophages were cultured on etched titanium disks and activated with either IL-4 cytokine (anti-inflammatory M2 phenotype) or Staphylococcus aureus secreted virulence factors to simulate BAI (pro-inflammatory M1 phenotype). Primary osteocytes in collagen gels were then stimulated with conditioned media (CM) from these macrophages. The osteocyte response was analyzed by gene expression, protein secretion, and immunostaining. M1 phenotype macrophages were confirmed by IL-1β and TNF-α secretion, and M2 macrophages by ARG-1 and MRC-1. Osteocytes receiving M1 CM revealed bone inhibitory effects, denoted by reduced secretion of bone formation osteocalcin (BGLAP) and increased secretion of the bone inhibitory sclerostin (SOST). These osteocytes also downregulated the pro-mineralization gene PHEX and upregulated the anti-mineralization gene MEPE. Additionally, exhibited pro-osteoclastic potential by upregulating pro-osteoclastic gene RANKL expression. Nonetheless, M1-stimulated osteocytes expressed a higher level of the potent pro-osteogenic factor BMP-2 in parallel with the downregulation of the bone inhibitor genes DKK1 and SOST , suggesting a compensatory feedback mechanisms. Conversely, M2-stimulated osteocytes mainly upregulated anti-osteoclastic gene OPG expression, suggesting an anti-catabolic effect. Altogether, our findings demonstrate a strong communication between M1 macrophages and osteocytes under M1 (BAI)-simulated conditions, suggesting that the BAI adverse effects on osteoblastic and osteoclastic processes in vitro are partly mediated via this communication. Biomaterial-associated infections are major challenges and the underlying mechanisms in the cellular interactions are missing, especially among the major cells from the inflammatory side (macrophages as the key cell in bacterial clearance) and the regenerative side (osteocyte as main regulator of bone). We evaluated the effect of macrophage polarization driven by the stimulation with bacterial virulence factors on the osteocyte function using an indirect co-culture model, hence mimicking the scenario of a biomaterial-associated infection. The results suggest that at least part of the adverse effects of biomaterial associated infection on osteoblastic and osteoclastic processes in vitro are mediated via macrophage-to-osteocyte communication. [Display omitted] [ABSTRACT FROM AUTHOR]

Objectives: The incorporation of retromolar bone grafts used for alveolar ridge augmentation is not well understood. This prospective observational study aims to supply histomorphometrical data from bone graft biopsies taken at the time of retrieval and after a 3‐month healing period using patient‐matched biopsies. Materials and Methods: In 17 patients, trephine biopsies of the graft were acquired at the time of graft retrieval and after a 3‐month healing period. The biopsies were compared histomorphometrically regarding the number of osteocytes, appearance of osteocyte lacunae, quantity, surface area, and activity of the Haversian canals. Results: All grafts appeared clinically stable after screw removal and 17 implants were placed. Histomorphometric analysis revealed no significant difference in the number of osteocytes (p =.413), osteocyte lacunae (p =.611), the ratio of filled/empty osteocyte lacunae (p =.467) and active Haversian canals (p =.495) between the biopsies retrieved after a 3‐months healing period with those at the time of grafting. The only significant difference was noted in the mean surface area of the Haversian canals (p =.002). Specifically, the grafts post 3‐month healing showed a significantly larger mean area (0.069 mm2) compared to the time of grafting (0.029 mm2). Conclusion: This study demonstrates, compared to other data, a high rate of vital structures in retromolar bone block grafts after 3 months of healing, exhibiting the same histological features in comparison to the biopsies from the native alveolar ridge. Standard histomorphometrical parameters, e.g., the amount of filled or empty osteocyte lacunae for the description of the vitality of the graft need to be reappraised. [ABSTRACT FROM AUTHOR]

Bone minerals may be more complex than the prevailing opinion suggests. Understanding its biomaterial properties in health and disease may address fundamental geo/biomorphological ambiguities recurrent within its calcified cancellous hierarchy of macro-, micro-, and nano-skeletal networks. (i) There is evidence that the outer mineral macroskeleton of interconnected trabeculae (150 µm diameter) is modulated according to axes of tensile stress by permeating arrays of periosteal Sharpey's fibres (collagen type III/VI, 5–25 µm thick) studded with tenascin organiser protein. (ii) Its substructural mineral microskeleton is a reticulation of bridged and deformable calcium phosphate/carbonate microspheres (about 1 µm diameter). These organically enshrouded (e.g., bone sialoprotein, osteocalcin, osteopontin) objects, configured by the adhesive organiser protein fibronectin and tempered by trace elements (e.g., Si, Mg, Fe, Al), display differential histochemistry (e.g., acid phosphatase, carbonic anhydrase) and anomalous traits (tetracycline binding, gram-positive microbial staining and nucleic acid staining affinity). The calcified microspheres are intracellular fabrications of osteocyte cohorts developed within "switched on" Golgi cisternae prior to aggregation at the extracellular calcification front in chains and looped assemblies. (iii) Within each microsphere, a less dense centre is encircled by a mineral nanoskeleton of beaded filaments (5 nm in diameter) transmutable in electron density, with a trait for lateral fusion into ladder-like struts, stays and senescent fenestrated plates, constituting domains of microparticle slip and crystal fracture. The evidence suggests a bone mineral biosystem of integrated complexity within which a particulate assemblage at the animate: inanimate calcification front resembles a colonial construct of prokaryote-like, Golgi-fabricated objects calcified with phosphate and harbouring a resident biochemistry. A self-contained "Petrified Microbiome" is proposed to be orchestrated according to a biodynamic primordial paradigm. [ABSTRACT FROM AUTHOR]

Osteoporosis is primarily associated with bone loss, but changes in bone tissue matrix composition and osteocyte mechanotransduction have also been identified. However, the molecular mechanisms underlying these changes and their relation to bone loss are not fully understood. The objectives of this study were to (1) conduct comprehensive temporal gene expression analyses on cortical bone tissue from ovariectomized rats, with a specific focus on genes known to govern matrix degradation, matrix production, and mechanotransduction, and (2) correlate these findings with bone mass, trabecular and cortical microarchitecture, and mineral and matrix composition. Microarray data revealed 35 differentially expressed genes in the cortical bone tissue of the ovariectomized cohort. We report that catabolic gene expression abates after the initial accelerated bone loss period, which occurs within the first 4 wk of estrogen deficiency. However, in long-term estrogen deficiency, we report increased expression of genes associated with extracellular matrix deposition (Spp1, COL1A1, COL1A2, OCN) and mechanotransduction (Cx43) compared with age-matched controls and short-term estrogen deficiency. These changes coincided with increased heterogeneity of mineral-to-matrix ratio and collagen maturity, to which extracellular matrix markers COL1A1 and COL1A2 were positively correlated. Interestingly, mineral heterogeneity and collagen maturity, exhibited a negative correlation with PHEX and IFT88, associated with mechanosensory cilia formation and Hedgehog (Hh) signaling. This study provides the first insight into the underlying mechanisms governing secondary mineralization and heterogeneity of matrix composition of bone tissue in long-term estrogen deficiency. We propose that altered mechanobiological responses in long-term estrogen deficiency may play a role in these changes. Graphical Abstract [ABSTRACT FROM AUTHOR]

Myeloma bone disease (MBD) affects ~90% of multiple myeloma patients, but current treatment options are suboptimal. Therefore, to successfully develop new therapies or optimize current ones, we must improve our fundamental knowledge of how myeloma affects bone microstructure and function. Here, we have investigated the osteocyte lacuno-canalicular network (LCN) in MBD, as bone porosity affects bone quality and resilience. We used the syngeneic 5TGM1-C57BL-Kalwrij and the xenograft U266-NSG models at end stage and compared them to healthy controls (naïve). Micro-computed tomography (μCT) and histomorphometry indicated the 5TGM1 and U266 models developed mild and extensive MBD, respectively, with the U266 model producing large osteolytic lesions. High-resolution synchrotron micro-CT (SR-μCT) revealed significant osteocyte lacunae changes in U266 bones but not 5TGM1, with a reduction in lacunae number and sphericity, and an increase in lacunae volume compared with naïve. Canalicular length, visualized using histological Ploton silver staining, appeared significantly shorter in 5TGM1 and U266 bones compared with naïve. Canalicular area as a proportion of the bone was also decreased by 24.2% in the U266 model. We observed significant upregulation of genes implicated in peri-lacunar remodeling (PLR), but immunohistochemistry confirmed that the osteocyte-specific protein sclerostin, a known driver of PLR, was unchanged between MBD and naïve bones. In summary, we have demonstrated evidence of PLR and altered organization of the osteocyte LCN in MBD mouse models. The next step would be to further understand the drivers and implications of PLR in MBD, and whether treatments to manipulate PLR and the LCN may improve patient outcomes. Lay Summary: Multiple myeloma is a blood cancer that causes bone damage in ~90% of patients. Currently, treatment options for myeloma bone disease (MBD) are suboptimal, leaving patients with chronic pain and increased fracture risk. Therefore, it is important that we improve our fundamental knowledge of how myeloma causes bone damage. With the development of more powerful imaging technologies, this allows us to visualize bone at the submicron level. Here, we assessed bones from two murine models of MBD (termed 5TGM1 and U266) and compared them to bones from healthy control mice (naïve). Using high-resolution imaging (SR-μCT), we found structural changes in the pores where the most abundant bone cells (osteocytes) reside, and that the orientation of their signaling network (lacuno-canalicular network, LCN) is altered in MBD compared with healthy bones. We also found differences in gene expression of key molecules in osteocytes from bones with MBD compared with healthy bones and identified a potential mechanism leading to these changes. The next step would be to use this knowledge to determine how myeloma treatments can affect the osteocyte LCN, as this may allow informed treatment decisions to be made, potentially reducing fracture risk and improving outcomes for patients with MBD. Graphical Abstract [ABSTRACT FROM AUTHOR]

Bone fractures and bone loss represent significant global health challenges, with their incidence rising due to an aging population. Despite autologous bone grafts remain the gold standard for treatment, challenges such as limited bone availability, immune reactions, and the risk of infectious disease transmission have driven the search for alternative cell-based therapies for bone regeneration. Stem cells derived from oral tissues and umbilical cord mesenchymal stem cells (MSCs) have shown potential in both preclinical and clinical studies for bone tissue regeneration. However, their limited differentiation capacity and wound healing abilities necessitate the exploration of alternative cell sources. In this study, we generated induced pluripotent stem cells (iPSCs) using a safe, nonviral and mRNA-based approach from human periodontal ligament fibroblasts (PDLF), an easily accessible cell source. These iPSCs were subsequently differentiated into MSCs, referred to as induced MSCs (iMSCs). The resulting iMSCs were homogeneous, highly proliferative, and possessed anti-inflammatory properties, suggesting their potential as a superior alternative to traditional MSCs for regenerative therapy. These iMSCs demonstrated trilineage differentiation potential, giving rise to osteocytes, chondrocytes, and adipocytes. The iMSC-derived osteocytes (iOSTs) were homogeneous, patient-specific and showed excellent attachment and growth on commercial collagen-based membranes, highlighting their suitability for bone tissue regeneration applications. Given their promising characteristics compared to traditional MSCs, PDLF-derived iMSCs are strong candidates for future clinical studies in bone regeneration and other regenerative dental therapies.

Background: Mobe (Artocarpus lakoocha) is recognized for its potential in accelerating wound healing, attributed to its secondary metabolites. However, its impact on hard tissue healing on post- extraction tooth sockets has been underexplored. Purpose: This study aims to analyze the effects of 3% Mobe leaf extract gel on the number of fibroblasts, osteoblasts, osteocytes and post-extraction sockets in Wistar rats. Methods: Thirty-two male Wistar rats had their left mandibular incisors extracted and were divided into eight groups. Mobe leaf extract gel was applied to Group I-IV and Aloclair® gel was applied to Group V-VIII for 14 days, twice a day. Residual socket volume (RSV) and fibroblast counts were measured on days 3, 7, and 14, while osteoblast and osteocyte counts were assessed on days 7, 14, and 28 post extraction. The RSV data were analyzed using repeated measures analysis of variance (ANOVA) and post-hoc least significant difference (LSD) test, while fibroblasts, osteoblasts, and osteocytes counts were analyzed with one-way ANOVA and post-hoc LSD tests. Results: RSV decreased significantly in both groups on day 14 (p=0.001 and p=0.002); however, the RSV was lower on the Mobe group. The fibroblast counts were higher in the Mobe group (p=0.001), and there was a significant difference in the mean number of osteoblasts and osteocytes in the Mobe group (p= 0.043 and p=0.008). Conclusion: The study concludes that 3% Mobe leaf gel extract is better than Aloclair® in accelerating socket healing mainly due to increased proliferation of fibroblasts, osteoblasts, and osteocytes.

Phosphate homeostasis is vital for many biological processes and disruptions in circulating levels can be detrimental. While the mechanisms behind FGF23 regulation have been regularly studied, the role of extracellular phosphate sensing and its impact on fibroblast growth factor 23 (FGF23) expression remains unclear. This study aimed to investigate the involvement of reactive oxygen species (ROS), silent information regulator 1 (SIRT1), and Hairy and Enhancer of Split-1 (HES1) in regulating FGF23 in FGF23 expressing MC3T3-E1 cells. MC3T3-E1 cells treated with β-glycerophosphate (BGP) resulted in increased Fgf23 expression. Inhibition of ROS formation by inhibition of NADPH oxidase, which is essential for ROS production, did not affect this response to BGP, suggesting ROS is not involved in this process. Moreover, treatment with tert-butyl hydroperoxide (TBHP), a ROS-inducing agent, did not increase Fgf23 expression. This suggests that ROS machinery is not involved in FGF23 stimulation as previously suggested. Nonetheless, inhibition of SIRT1 using Ex527 eliminated the Fgf23 response to BGP, indicating its involvement in FGF23 regulation after BGP treatment. Indeed, activation of SIRT1 using SRT1720 increased Fgf23 expression. Moreover, transcription factor Hes1 was upregulated by BGP treatment, which was diminished when cells were treated with Ex527 implying it is also regulated through SIRT1. These findings suggest the existence of an upstream SIRT1-HES1 axis in the regulation of FGF23 by phosphate, though we were unable to find a role for ROS in this process. Further research should provide insights into phosphate homeostasis and potential therapeutic targets for phosphate-related disorders. [ABSTRACT FROM AUTHOR]

Osteocytes perceive and process mechanical stimuli in the lacuno-canalicular network in bone. As a result, they secrete signaling molecules that mediate bone formation and resorption. To date, few three-dimensional (3D) models exist to study the response of mature osteocytes to biophysical stimuli that mimic fluid shear stress and substrate strain in a mineralized, biomimetic bone-like environment. Here we established a biomimetic 3D bone model by utilizing a state-of-art perfusion bioreactor platform where immortomouse/Dmp1-GFP-derived osteoblastic IDG-SW3 cells were differentiated into mature osteocytes. We evaluated proliferation and differentiation properties of the cells on 3D microporous scaffolds of decellularized bone (dBone), poly(L-lactide-co-trimethylene carbonate) lactide (LTMC), and beta-tricalcium phosphate (β-TCP) under physiological fluid flow conditions over 21 days. Osteocyte viability and proliferation were similar on the scaffolds with equal distribution of IDG-SW3 cells on dBone and LTMC scaffolds. After seven days, the differentiation marker alkaline phosphatase (Alpl), dentin matrix acidic phosphoprotein 1 (Dmp1), and sclerostin (Sost) were significantly upregulated in IDG-SW3 cells (p = 0.05) on LTMC scaffolds under fluid flow conditions at 1.7 ml/min, indicating rapid and efficient maturation into osteocytes. Osteocytes responded by inducing the mechanoresponsive genes FBJ osteosarcoma oncogene (Fos) and prostaglandin-endoperoxide synthase 2 (Ptgs2) under perfusion and dynamic compressive loading at 1 Hz with 5 % strain. Together, we successfully created a 3D biomimetic platform as a robust tool to evaluate osteocyte differentiation and mechanobiology in vitro while recapitulating in vivo mechanical cues such as fluid flow within the lacuno-canalicular network. This study highlights the importance of creating a three-dimensional (3D) in vitro model to study osteocyte differentiation and mechanobiology, as cellular functions are limited in two-dimensional (2D) models lacking in vivo tissue organization. By using a perfusion bioreactor platform, physiological conditions of fluid flow and compressive loading were mimicked to which osteocytes are exposed in vivo. Microporous poly(L-lactide-co-trimethylene carbonate) lactide (LTMC) scaffolds in 3D are identified as a valuable tool to create a favorable environment for osteocyte differentiation and to enable mechanical stimulation of osteocytes by perfusion and compressive loading. The LTMC platform imitates the mechanical bone environment of osteocytes, allowing the analysis of the interaction with other cell types in bone under in vivo biophysical stimuli. [Display omitted] [ABSTRACT FROM AUTHOR]

Preclinical models (typically ovariectomized rats and genetically altered mice) have underpinned much of what we know about skeletal biology. They have been pivotal for developing therapies for osteoporosis and monogenic skeletal conditions, including osteogenesis imperfecta, achondroplasia, hypophosphatasia, and craniodysplasias. Further therapeutic advances, particularly to improve cortical strength, require improved understanding and more rigorous use and reporting. We describe here how trabecular and cortical bone structure develop, are maintained, and degenerate with aging in mice, rats, and humans, and how cortical bone structure is changed in some preclinical models of endocrine conditions (eg, postmenopausal osteoporosis, chronic kidney disease, hyperparathyroidism, diabetes). We provide examples of preclinical models used to identify and test current therapies for osteoporosis, and discuss common concerns raised when comparing rodent preclinical models to the human skeleton. We focus especially on cortical bone, because it differs between small and larger mammals in its organizational structure. We discuss mechanisms common to mouse and human controlling cortical bone strength and structure, including recent examples revealing genetic contributors to cortical porosity and osteocyte network configurations during growth, maturity, and aging. We conclude with guidelines for clear reporting on mouse models with a goal for better consistency in the use and interpretation of these models. [ABSTRACT FROM AUTHOR]

The petrous bone generally preserves ancient DNA better than other fossil bones. One reason for this is that the inner layer of the petrous bone of pigs and humans contains about three times as many osteocytes as other bones, and hence more DNA. A FIB-SEM study of modern pig petrous bones showed that the 3D structure of the thin inner layer is typical of woven bone that forms in the fetus, whereas the thicker outer layer has a lamellar structure. The lamellar structure is common in mammalian bones. Here we study human petrous bones that are about 2500 years old, obtained from three Phoenician sites in Sicily, Italy. A detailed FIB-SEM study of two of these bones, one well preserved and the other poorly preserved, shows that the 3D bone type structure of the human petrous inner layer is woven bone, and the outer layer is lamellar bone. These are the same bone type structures found in pig petrous bones. Furthermore, by comparing nine differently preserved petrous bones from the same archaeological region and age, we show that their collagen contents vary widely, implying that organic material can be significantly altered during diagenesis. The mineral crystals are better preserved and hence less crystalline in the inner layers compared to the outer layers. We therefore infer that the best-preserved DNA in fossil petrous bones should be found in the thin inner layers immediately adjacent to the otic cavity where much more DNA is initially present and the mineral phase tends to be better preserved. [ABSTRACT FROM AUTHOR]

To ensure the prevention of thermal trauma and tissue necrosis during bone drilling in surgical procedures, it is crucial to maintain temperatures below the time- and temperature-dependent threshold of 50 °C for 30 s. However, the absence of a current standard for assessing temperatures attained during bone drilling poses a challenge when comparing findings across different studies. This article aims to address this issue by introducing a standardized testing method for acquiring thermal data during experimental bone drilling. The method requires the use of three controlled variables: infrared thermography, standard bone blocks, and a regulated drilling procedure involving a drill press with irrigation that simulates a surgeon. By utilizing this setup, we can obtain temperature data that can be effectively applied in the evaluation of other variables, such as surgical techniques or drill bit design, and translate the data into bone damage/clinical outcomes. Two surgical drill bits (2.0 mm-diameter twist drill bit and 3.3 mm-diameter multi-step drill bit) are compared using this experimental protocol. The results show the 2.0 mm bit reached significantly higher temperatures compared to the 3.3 mm bit when preparing an osteotomy (p < 0.05). The 2.0 mm drill bit reached temperatures over 100 °C while the 3.3 mm drill bit did not exceed 50 °C. [ABSTRACT FROM AUTHOR]

Hypercholesterolaemia is a systemic metabolic disease, but the role of organs other than liver in cholesterol metabolism is unappreciated. The phenotypic characterization of the Tsc1Dmp1 mice reveal that genetic depletion of tuberous sclerosis complex 1 (TSC1) in osteocytes/osteoblasts (Dmp1‐Cre) triggers progressive increase in serum cholesterol level. The resulting cholesterol metabolic dysregulation is shown to be associated with upregulation and elevation of serum amyloid A3 (SAA3), a lipid metabolism related factor, in the bone and serum respectively. SAA3, elicited from the bone, bound to toll‐like receptor 4 (TLR4) on hepatocytes to phosphorylate c‐Jun, and caused impeded conversion of cholesterol to bile acids via suppression on cholesterol 7 α‐hydroxylase (Cyp7a1) expression. Ablation of Saa3 in Tsc1Dmp1 mice prevented the CYP7A1 reduction in liver and cholesterol elevation in serum. These results expand the understanding of bone function and hepatic regulation of cholesterol metabolism and uncover a potential therapeutic use of pharmacological modulation of SAA3 in hypercholesterolaemia. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is a major causative pathogen of osteomyelitis. Intracellular infections of resident bone cells including osteocytes can persist despite goldstandard clinical intervention. The mechanisms by which intracellular S. aureus evades antibiotic therapy are unknown. In this study, we utilised an in vitro S. aureus infection model of human osteocytes to investigate whether antibioticmediated dysregulation of autophagy contributes to this phenomenon. Infected or non-infected osteocyte-like cells were exposed to combinations of rifampicin, vancomycin, and modulators of autophagy. Intracellular bacterial growth characteristics were assessed using colony-forming unit (CFU) analysis, viable bacterial DNA abundance, and the rate of escape into antibiotic-free medium, together with measures of autophagic flux. Rifampicin, alone or in combination with vancomycin, caused a rapid decrease in the culturability of intracellular bacteria, concomitant with stable or increased absolute bacterial DNA levels. Both antibiotics significantly inhibited autophagic flux. However, modulation of autophagic flux did not affect viable bacterial DNA levels. In summary, autophagy was shown to be a factor in the host--pathogen relationship in this model, as itsmodulation affected the growth state of intracellular S. aureus with respect to both their culturability and propensity to escape the intracellular niche. While rifampicin and vancomycin treatments moderately suppressed autophagic flux acutely, this did not explain the paradoxical response of antibiotic treatment in decreasing S. aureus culturability whilst failing to clear bacterial DNA and hence intracellular bacterial load. Thus, offtarget effects of rifampicin and vancomycin on autophagic flux in osteocyte-like cells could not explain the persistent S. aureus infection in these cells. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSCs) of placental origin hold great promise in tissue engineering and regenerative medicine for diseases affecting cartilage and bone. However, their utility has been limited by their tendency to undergo premature senescence and phenotypic drift into adipocytes. This study aimed to explore the potential involvement of a specific subset of aging and antiaging genes by measuring their expression prior to and following in vitro-induced differentiation of placental MSCs into chondrocytes and osteoblasts as opposed to adipocytes. The targeted genes of interest included the various LMNA/C transcript variants (lamin A, lamin C, and lamin A∆10), sirtuin 7 (SIRT7), and SM22α, along with the classic aging markers plasminogen activator inhibitor 1 (PAI-1), p53, and p16INK4a. MSCs were isolated from the decidua basalis of human term placentas, expanded, and then analyzed for phenotypic properties by flow cytometry and evaluated for colony-forming efficiency. The cells were then induced to differentiate in vitro into chondrocytes, osteocytes, and adipocytes following established protocols. The mRNA expression of the targeted genes was measured by RT-qPCR in the undifferentiated cells and those fully differentiated into the three cellular lineages. Compared to undifferentiated cells, the differentiated chondrocytes demonstrated decreased expression of SIRT7, along with decreased PAI-1, lamin A, and SM22α expression, but the expression of p16INK4a and p53 increased, suggesting their tendency to undergo premature senescence. Interestingly, the cells maintained the expression of lamin C, which indicates that it is the primary lamin variant influencing the mechanoelastic properties of the differentiated cells. Notably, the expression of all targeted genes did not differ from the undifferentiated cells following osteogenic differentiation. On the other hand, the differentiation of the cells into adipocytes was associated with decreased expression of lamin A and PAI-1. The distinct patterns of expression of aging and antiaging genes following in vitro-induced differentiation of MSCs into chondrocytes, osteocytes, and adipocytes potentially reflect specific roles for these genes during and following differentiation in the fully functional cells. Understanding these roles and the network of signaling molecules involved can open opportunities to improve the handling and utility of MSCs as cellular precursors for the treatment of cartilage and bone diseases. [ABSTRACT FROM AUTHOR]

Purpose: This scoping review presents the preclinical and clinical data on the effects of high-dose radiation therapy (RT) on bone structure and function. Materials and Methods: An extensive PubMed search was performed for the relevant questions. The data were then synthesized into a comprehensive summary of the available relevant in-vitro, preclinical and clinical literature. Results: In-vitro studies of high-dose RT on cell cultures show considerable damage in the viability and functional capacity of the primary cells of the bones; the osteoclasts, the osteoblasts, and the osteocytes. In-vivo animal models show that high-dose RT induces significant morphological changes to the bone, inhibits the ability of bone to repair damage, and increases the fragility of the bone. Clinical data show that there is an increasing risk over time of damage to the bone, such as fractures, after high-dose RT. Conclusion: These findings suggest that there may be a limit to the safe dose for single-fraction RT, and the long-term consequences of high-dose RT for the patients must be considered. [ABSTRACT FROM AUTHOR]

An osteocyte is a bone cell situated inside a hard bone matrix in an interstice (lacuna). It has many dendritic structures called cellular processes that radiate outward from the cell through the bone matrix via cylindrical openings (canaliculi). Osteocytes can sense stress and strain applied by the interstitial fluid flow and respond by releasing biochemical signals that regulate bone remodeling. In vitro experiments have suggested that the stress and strain typically experienced at the macroscale tissue level have to be amplified 10× in order for osteocytes to have a significant response in vivo. This stress and strain amplification mechanism is not yet well understood. Previous studies suggest that the processes are the primary sites for mechanosensation thanks to the tethering elements that attach the process membrane to the canalicular wall. However, there are other potential factors which may also contribute to stress and strain amplification, such as canalicular wall geometry and osteocyte-associated proteins in the interstitial space called pericellular matrix. In this work, we perform computational studies to study how canalicular wall roughness affects stress and strain amplification. Our major finding is that the wall roughness induces significantly greater wall shear stress (WSS) on the process when the wall roughness increases flow resistance; and the roughness has relatively smaller influence on the WSS when the resistance remains the same. [ABSTRACT FROM AUTHOR]

Low back pain derived from intervertebral disc (IVD) degeneration is a debilitating spinal condition that, despite its prevalence, does not have any intermediary guidelines for pharmacological treatment between palliative care and invasive surgery. The development of treatments for the IVD is complicated by the variety of resident cell types needed to maintain the regionally distinct structural properties of the IVD that permit the safe, complex motions of the spine. Osteoporosis of the spine increases the risk of vertebral bone fracture that can increase the incidence of back pain. Fortunately, there are a variety of pharmacological treatments for osteoporosis that target osteoblasts, osteoclasts and/or osteocytes to build bone and prevent vertebral fracture. Of particular note, clinical and preclinical studies suggest that commonly prescribed osteoporosis drugs like bisphosphonates, intermittent parathyroid hormone, anti-sclerostin antibody, selective estrogen receptor modulators and anti-receptor activator of nuclear factor-kappa B ligand inhibitor denosumab may also relieve back pain. Here, we cite clinical and preclinical studies and include unpublished data to support the argument that a subset of these therapeutics for osteoporosis may alleviate low back pain by also targeting the IVD. [ABSTRACT FROM AUTHOR]