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Among bone cells, osteocytes are the most abundant, but also the most challenging to study because they are located inside a dense mineralized matrix. Due to their involvement in bone homeostasis, diverse tools are needed to understand their roles in bone physiology and pathology. This work was aimed at establishing a laser-assisted microdissection protocol to isolate osteocytes and analyze their gene expressions. The goal was to overcome the limitations of the technique currently most used: RNA extraction from the whole bone. To perform laser microdissection and subsequent gene expression analysis, the five main steps of the protocol have been adapted for the bone tissue. After testing many parameters, we found that the best options were (1) take unfixed snap-frozen tissue, (2) cryosection with a supported tape system to improve the tissue morphology if necessary, (3) microdissect regions of interest, and (4) recover the bone pieces by catapulting, if feasible, or by gravity. Finally, RNA extraction (5) was the most efficient with a precipitation method and allowed quantifying the expression of well described osteocyte genes (Gja1/Cx43, Phex, Pdpn, Dmp1, Sost). This work describes two protocols optimized for femur and calvaria and gives an overview of the many optimization options that one could try when facing difficulties with laser microdissection.

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As a highly dynamic tissue, bone is continuously rebuilt throughout life. Both bone formation by osteoblasts and bone resorption by osteoclasts constitute bone reconstruction homeostasis. The equilibrium of bone homeostasis is governed by many complicated signaling pathways that weave together to form an intricate network. These pathways coordinate the meticulous processes of bone formation and resorption, ensuring the structural integrity and dynamic vitality of the skeletal system. Dysregulation of the bone homeostatic regulatory signaling network contributes to the development and progression of many skeletal diseases. Significantly, imbalanced bone homeostasis further disrupts the signaling network and triggers a cascade reaction that exacerbates disease progression and engenders a deleterious cycle. Here, we summarize the influence of signaling pathways on bone homeostasis, elucidating the interplay and crosstalk among them. Additionally, we review the mechanisms underpinning bone homeostatic imbalances across diverse disease landscapes, highlighting current and prospective therapeutic targets and clinical drugs. We hope that this review will contribute to a holistic understanding of the signaling pathways and molecular mechanisms sustaining bone homeostasis, which are promising to contribute to further research on bone homeostasis and shed light on the development of targeted drugs.

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BACKGROUND: When the natural process of bone remodeling is disturbed, the need arises for a stimulant material in order to enhance the formation of a new healthy and strong osseous tissue to replace the damaged one. Recent studies have reported synthetic biomaterials to be a very good option for supporting bone regeneration. STUDY DESIGN: Narrative review. OBJECTIVE: This review aims to provide a brief presentation of two of the most recently developed synthetic biomaterials, i.e. calcium phosphate cements and synthetic composites, that are currently being used in bone regeneration with promising results. METHODS: Literature searches using broad terms such as "bone regeneration," "biomaterials," "synthetic composites" and "calcium phosphate cements" were performed using PubMed. The osteal cells state of the art was explored by searching topic-specific full text keywords using Google Scholar. CONCLUSIONS: Synthetic polymers such as PCL (poly-ε-caprolactone) and PLGA (poly lactic-co-glycolic acid) can improve the effectiveness of biomaterials like HA (hydroxyapatite) and BG (bioglass). Calcium phosphate, although being a suitable material for stimulating bone regeneration, needs an adjuvant in order to be effective in larger bone defects.

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We have studied the effects of individual and combined treatment of insulin (I) and naringin (NAR) on the bone structure and biomechanical properties of femurs from streptozotocin (STZ)-induced diabetic rats. Male Wistar rats were divided into five groups: (1) controls, (2) STZ-induced diabetic rats, (3) STZ-induced diabetic rats treated with I, (4) STZ-induced diabetic rats treated with NAR, and (5) STZ-induced diabetic rats treated with I + NAR. Bone mineral density (BMD), bone histomorphometry, biomechanical testing, and bone biomarker expressions were accomplished in femur of all animals, as well as serum biochemical analyses. The combined treatment of I + NAR increased the body weight and the femur BMD from STZ-induced diabetic rats. The bone biomechanical properties and the bone morphology of the femurs from STZ-induced diabetic rats were also improved by the combined treatment. The increased number of osteoclasts in STZ-induced diabetic rats was partially prevented by I, NAR, or I + NAR. NAR or I + NAR completely blocked the decrease in the number of osteocalcin (+) cells in the femur from STZ-induced diabetic rats. RUNX family transcription factor 2 immunostaining was much lower in STZ-induced diabetic rats than in control animals; the combination of I + NAR totally blocked this effect. The combined treatment not only ameliorated bone quality and function, but also normalized the variables related to glucose metabolism. Therefore, the combination of I + NAR might be a better therapeutic strategy than the individual I or NAR administration to reduce bone complications in diabetic patients.

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Additive manufacturing (3D printing) has been deployed across multiple platforms to fabricate bioengineered tissues. We demonstrate the use of a Thermal Inkjet Pipette System (TIPS) for targeted delivery of cells onto manufactured substrates to design bio-bandages. Two cell lines - HEK 293 (kidney) and K7M2 wt (bone) - were applied using TIPS. We demonstrate a novel means for targeted cell delivery to a hydrogel support structure. These cell/support constructs (bio-bandages) had a high viability for survival and growth over extended periods. Combining a flexible biosupport with application of cells via TIPS printing now for the first time allows for custom cell substrate constructs with various densities to be deployed for regenerative medicine applications.

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Isoflavones and probiotics are promising nutrients for bone health, and magnesium (Mg) is essential for bone metabolism. This study aimed to determine the effects of daidzein, genistein and Lactobacillus acidophilus on the Mg status of healthy female rats. Forty-eight rats were randomly assigned to six groups, with the control group receiving a standard diet (AIN 93M). The remaining groups were fed the same diet with added ingredients such as tempeh flour; soy flour; pure daidzein and genistein; L. acidophilus or a combination of daidzein, genistein, and L. acidophilus. Tissue samples were collected after the eight-week intervention, and Mg concentrations were analysed using flame atomic absorption spectrometry. Myeloid and erythroid cells were determined using the haematoxylin and eosin bone staining method. Statistical analyses were conducted using one-way ANOVA with Tukey's post hoc test and Pearson's correlation coefficient. The threshold for significance was p < 0.05. Compared with the control group, adding tempeh to the diet of rats resulted in significant changes in Mg concentrations in various tissues, with a decrease in the kidneys and an increase in the fur. Although not statistically significant compared to the control group, the tempeh group showed increased Mg concentrations in the femur and spleen. The myeloid-to-erythroid cell ratio did not differ significantly among groups, but all intervention groups showed higher ratios than the control group. A strong negative correlation was observed between Mg concentrations in the kidneys and fur. Conversely, a positive correlation was identified between Mg concentrations in the pancreas and fur. Daily consumption of tempeh may improve Mg status in the organism. Intake of pure daidzein, genistein, or probiotic seems to have no effect on Mg concentrations in healthy rats.

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This review focuses on understanding the macroscopic and microscopic characteristics of bone tissue and reviews current knowledge of its physiology. It explores how these features intricately collaborate to maintain the balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption, which plays a pivotal role in shaping not only our physical framework but also overall health. In this work, a comprehensive exploration of microscopic and macroscopic features of bone tissue is presented.

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Bone metabolism is a complex process which is influenced by the activity of bone cells (e.g., osteocytes, osteoblasts, osteoclasts); the effect of some specific biomarkers (e.g., parathyroid hormone, vitamin D, alkaline phosphatase, osteocalcin, osteopontin, osteoprotegerin, osterix, RANKL, Runx2); and the characteristic signaling pathways (e.g., RANKL/RANK, Wnt/ß, Notch, BMP, SMAD). Some phytochemical compounds-such as flavonoids, tannins, polyphenols, anthocyanins, terpenoids, polysaccharides, alkaloids and others-presented a beneficial and stimulating effect in the bone regeneration process due to the pro-estrogenic activity, the antioxidant and the anti-inflammatory effect and modulation of bone signaling pathways. Lately, nanomedicine has emerged as an innovative concept for new treatments in bone-related pathologies envisaged through the incorporation of medicinal substances in nanometric systems for oral or local administration, as well as in nanostructured scaffolds with huge potential in bone tissue engineering.

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Zebrafish cultured scales have been used effectively to study cellular and molecular responses of bone cells. In order to expose zebrafish scales to simulated microgravity (SMG) and/or vibration, we first determined via apoptosis staining whether cells of the scale survive in culture for two days and hence, we restricted our analyses to two-day durations. Next, we measured the effects of SMG and vibration on cell death, osteoclast tartrate-resistant acid phosphatase, and osteoblast alkaline phosphatase activity and on the number of Runx2a positive cells. We found that during the SMG treatment, osteoclast tartrate-resistant acid phosphatase activity increased on average, while the number of Runx2a positive cells decreased significantly. In contrast, SMG exposure caused a decrease in osteoblast activity. The vibration treatment showed an increase, on average, in the osteoblast alkaline phosphatase activity. This study demonstrates the effect of SMG and vibration on zebrafish scales and the effects of SMG on bone cells. We also show that zebrafish scales can be used to examine the effects of SMG on bone maintenance.

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Since our discovery in 2013 that genetic defects in PLS3 lead to bone fragility, the mechanistic details of this process have remained obscure. It has been established that PLS3 variants cause syndromic and nonsyndromic osteoporosis as well as osteoarthritis. PLS3 codes for an actin-bundling protein with a broad pattern of expression. As such, it is puzzling how PLS3 specifically leads to bone-related disease presentation. Our review aims to summarize the current state of knowledge regarding the function of PLS3 in the predominant cell types in the bone tissue, the osteocytes, osteoblasts and osteoclasts. This is related to the role of PLS3 in regulating mechanotransduction, calcium regulation, vesicle trafficking, cell differentiation and mineralization as part of the complex bone pathology presented by PLS3 defects. Considering the consequences of PLS3 defects on multiple aspects of bone tissue metabolism, our review motivates the study of its mechanism in bone diseases which can potentially help in the design of suitable therapy.

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Isoflavones and probiotics have shown the therapeutic potential to alter calcium absorption and bone cell metabolism. This study sought to ascertain the effect of isoflavones and probiotics on calcium status and bone health in healthy female rats. Forty-eight adult female Wistar rats were grouped and fed: a standard diet (control); and standard diets with tempeh; soy; daidzein and genistein; Lactobacillus acidophilus; and a combination of daidzein, genistein, and L. acidophilus. The biochemical serum parameters, such as alanine transaminase, aspartate transaminase, glucose, and triacylglycerol concentrations, were measured, and calcium contents in tissues were determined. After staining the bone with hematoxylin and eosin, the number of osteoblasts, osteocytes, and the percentage of bone marrow adipocytes were counted. Compared with the control group, the soy group showed a significantly lower triacylglycerol concentration. The L. acidophilus group considerably increased the calcium content in the femoral bone. The daidzein and genistein, L. acidophilus, and a combination of daidzein, genistein, and L. acidophilus groups showed significantly lower calcium contents in the heart and kidneys. The daidzein and genistein group significantly enhanced the number of osteoblasts and osteocytes. A substantial inverse correlation was observed between calcium contents in kidneys and osteoblasts. In conclusion, the combination of daidzein, genistein, and L. acidophilus may improve bone calcium concentrations and bone cells. However, no synergistic effect between isoflavones and probiotics was detected in this study.

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OBJECTIVE: Contagious ecthyma is a severe and highly contagious disease caused by an orf virus (ORFV). The virus is responsible for substantial economic losses in the goat industry and threatens humans. We previously determined the role of ORFV129 protein, one of the five ankyrin-repeat proteins coded by the orf genome, in suppressing the transcription of pro-inflammatory cytokines IL-6, IL-1ß and IFN-γ. In the present study, we identified 14 cellular proteins (complement C1q binding protein [C1QBP], MCM7, EIF5A, PKM, SLC6A, TSPAN6, ATP6AP2, GPS1, MMADHC, HSPB6, SLC35B1, MTF1, P3H4, and IL15RA) that interact with ORFV129 using a yeast two-hybrid system in goat turbinate bone cells (GFTCs). The interaction between ORFV129 and (C1QBP), an immune-related protein, was confirmed using immunofluorescence co-localization and co-immunoprecipitation assays. C1QBP overexpression inhibited ORFV replication, whereas the knockdown of C1QBP promoted ORFV replication in GFTCs. Furthermore, ORFV or ORFV129 increased C1QBP expression in GFTCs, indicated that ORFV129-C1QBP interaction might contribute to the ORFV-induced host immune process. In addition, our research showed that ORFV increased the expression of ORFV129, cytokine IL-6, IL-1ß and IFN-γ. C1QBP overexpression induced IFN-γ production and reduced IL-6 and IL-1ß production. Conversely, C1QBP knockdown induced IL-1ß production and reduced IFN-γ and IL-1ß production. Moreover, augmentation of ORFV129 expression enhanced the inhibition of the secretion of cytokines IL-6, IL-1ß, and IFN-γ induced by the altered expression of C1QBP. These findings suggest different downstream pathways might be involved in regulating different cytokines induced by ORFV129 expression in GFTCs.

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Recently, there has been an increasing focus on cellular morphology and mechanical behavior in order to gain a better understanding of the modulation of cell malignancy. This study used uniaxial-stretching technology to select a mechanical regimen able to elevate SAOS-2 cell migration, which is crucial in osteosarcoma cell pathology. Using confocal and atomic force microscopy, we demonstrated that a 24 h 0.5% cyclic elongation applied at 1 Hz induces morphological changes in cells. Following mechanical stimulation, the cell area enlarged, developing a more elongated shape, which disrupted the initial nuclear-to-cytoplasm ratio. The peripheral cell surface also increased its roughness. Cell-based biochemical assays and real-time PCR quantification showed that these morphologically induced changes are unrelated to the osteoblastic differentiative grade. Interestingly, two essential cell-motility properties in the modulation of the metastatic process changed following the 24 h 1 Hz mechanical stimulation. These were cell adhesion and cell migration, which, in fact, were dampened and enhanced, respectively. Notably, our results showed that the stretch-induced up-regulation of cell motility occurs through a mechanism that does not depend on matrix metalloproteinase (MMP) activity, while the inhibition of ion-stretch channels could counteract it. Overall, our results suggest that further research on mechanobiology could represent an alternative approach for the identification of novel molecular targets of osteosarcoma cell malignancy.

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The main target of bone tissue engineering is to design biomaterials that support bone regeneration and vascularization. Nanostructured membranes of (MMA)1-co-(HEMA)1/(MA)3-co-(HEA)2 loaded with 5% wt of SiO2-nanoparticles (Si-M) were doped with zinc (Zn-Si-M) or doxycycline (Dox-Si-M). Critical bone defects were effectuated on six New Zealand-bred rabbit skulls and then they were covered with the membranes. After six weeks, a histological analysis (toluidine blue technique) was employed to determine bone cell population as osteoblasts, osteoclasts, osteocytes, M1 and M2 macrophages and vasculature. Membranes covering the bone defect determined a higher count of bone cells and blood vessels than in the sham group at the top regions of the defect. Pro-inflammatory M1 appeared in a higher number in the top regions than in the bottom regions, when Si-M and Dox-Si-M were used. Samples treated with Dox-Si-M showed a higher amount of anti-inflammatory and pro-regenerative M2 macrophages. The M1/M2 ratio obtained its lowest value in the absence of membranes. On the top regions, osteoblasts were more abundant when using Si-M and Zn-Si-M. Osteoclasts were equally distributed at the central and lateral regions. The sham group and samples treated with Zn-Si-M attained a higher number of osteocytes at the top regions. A preferential osteoconductive, osteoinductive and angiogenic clinical environment was created in the vicinity of the membrane placed on critical bone defects.

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BACKGROUND: ß-Arrestin 2 (ß-arr2) binds activated parathyroid hormone (PTH) receptors stimulating internalization. PTH stimulates both anabolic and catabolic effect on bone depending on the way it is administered. Intermittent PTH stimulation increases trabecular bone formation in mice, but this is decreased in mice lacking ß-arr 2, suggesting a role for ß-arr 2 in the anabolic effects of PTH. The role of ß-arr 2 in the catabolic effects of continuous PTH (cPTH) treatment is not known. OBJECTIVE: To assess the effects of cPTH administration on bone in mice lacking ß-arr 2 compared to wild-type (WT). METHODS: Groups of male and female WT or ß-arr2 knockout (KO) mice were administered either PTH or phosphate-buffered saline by osmotic pumps for 2 weeks. Following treatment, serum calcium and phosphate levels were measured, bone structure and mineral density were measured by microcomputed tomography, and bone cells measured by static and dynamic histomorphometry. RESULTS: ß-arr2 KO had no effects on skeletal development in mice of either sex. PTH treatment caused hypercalcemia and hypophosphatemia and decreased trabecular and cortical bone only in male WT mice. ß-arr2 KO in male mice completely abrogated the effects of PTH on bone, while in female ß-arr2 KO mice, PTH treatment increased trabecular bone with no effects on cortical bone. CONCLUSIONS: These results demonstrate a profound sex effect on skeletal responses to cPTH treatment, suggesting a protective effect of estrogen on bone loss. ß-arr2 plays a role in restraining the anabolic effects of PTH in both male and female mice.

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Glucocorticoid-induced osteoporosis (GIOP) is considered the third type of osteoporosis and is accompanied by high morbidity and mortality. Long-term usage of glucocorticoids (GCs) causes worsened bone quality and low bone mass via their effects on bone cells. Currently, there are various clinical pharmacological treatments to regulate bone mass and skeletal health. Pulsed electromagnetic fields (PEMFs) are applied to treat patients suffering from delayed fracture healing and non-unions. PEMFs may be considered a potential and side-effect-free therapy for GIOP. PEMFs inhibit osteoclastogenesis, stimulate osteoblastogenesis, and affect the activity of bone marrow mesenchymal stem cells (BMSCs), osteocytes and blood vessels, ultimately leading to the retention of bone mass and strength. However, the underlying signaling pathways via which PEMFs influence GIOP remain unclear. This review attempts to summarize the underlying cellular mechanisms of GIOP. Furthermore, recent advances showing that PEMFs affect bone cells are discussed. Finally, we discuss the possibility of using PEMFs as therapy for GIOP.

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Bone remodeling is regulated by the interaction between different cells and tissues across many spatial and temporal scales. Notably, in silico models are regarded as powerful tools to further understand the signaling pathways that regulate this intricate spatial cellular interplay. To this end, we have established a 3D multiscale micro-multiphysics agent-based (micro-MPA) in silico model of trabecular bone remodeling using longitudinal in vivo data from the sixth caudal vertebra (CV6) of PolgA(D257A/D257A) mice, a mouse model of premature aging. Our in silico model includes a variety of cells as single agents and receptor-ligand kinetics, mechanomics, diffusion and decay of cytokines which regulate the cells' behavior. We highlighted its capabilities by simulating trabecular bone remodeling in the CV6 of five mice over 4 weeks and we evaluated the static and dynamic morphometry of the trabecular bone microarchitecture. Based on the progression of the average trabecular bone volume fraction (BV/TV), we identified a configuration of the model parameters to simulate homeostatic trabecular bone remodeling, here named basal. Crucially, we also produced anabolic, anti-anabolic, catabolic and anti-catabolic responses with an increase or decrease by one standard deviation in the levels of osteoprotegerin (OPG), receptor activator of nuclear factor kB ligand (RANKL), and sclerostin (Scl) produced by the osteocytes. Our results showed that changes in the levels of OPG and RANKL were positively and negatively correlated with the BV/TV values after 4 weeks in comparison to basal levels, respectively. Conversely, changes in Scl levels produced small fluctuations in BV/TV in comparison to the basal state. From these results, Scl was deemed to be the main driver of equilibrium while RANKL and OPG were shown to be involved in changes in bone volume fraction with potential relevance for age-related bone features. Ultimately, this micro-MPA model provides valuable insights into how cells respond to their local mechanical environment and can help to identify critical pathways affected by degenerative conditions and ageing.

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The goal of bone tissue engineering is to build artificial bone tissue with properties that closely resemble human bone and thereby support the optimal integration of the constructs (biografts) into the body. The development of tissues in 3D scaffolds includes several complex steps that need to be optimized and monitored. In particular, cell-material interaction during seeding, cell proliferation and cell differentiation within the scaffold pores play a key role. In this work, we seeded two types of 3D-printed scaffolds with pre-osteoblastic MC3T3-E1 cells, proliferated and differentiated the cells, before testing and adapting different assays and imaging methods to monitor these processes. Alpha-TCP/HA (α-TCP with low calcium hydroxyapatite) and baghdadite (Ca3ZrSi2O9) scaffolds were used, which had comparable porosity (~50%) and pore sizes (~300-400 µm). Cell adhesion to both scaffolds showed ~95% seeding efficiency. Cell proliferation tests provided characteristic progression curves over time and increased values for α-TCP/HA. Transmitted light imaging displayed a homogeneous population of scaffold pores and allowed us to track their opening state for the supply of the inner scaffold regions by diffusion. Fluorescence labeling enabled us to image the arrangement and morphology of the cells within the pores. During three weeks of osteogenesis, ALP activity increased sharply in both scaffolds, but was again markedly increased in α-TCP/HA scaffolds. Multiphoton SHG and autofluorescence imaging were used to investigate the distribution, morphology, and arrangement of cells; collagen-I fiber networks; and hydroxyapatite crystals. The collagen-I networks became denser and more structured during osteogenic differentiation and appeared comparable in both scaffolds. However, imaging of the HA crystals showed a different morphology between the two scaffolds and appeared to arrange in the α-TCP/HA scaffolds along collagen-I fibers. ALP activity and SHG imaging indicated a pronounced osteo-inductive effect of baghdadite. This study describes a series of methods, in particular multiphoton imaging and complementary biochemical assays, to validly measure and track the development of bone tissue in 3D scaffolds. The results contribute to the understanding of cell colonization, growth, and differentiation, emphasizing the importance of optimal media supply of the inner scaffold regions.

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Bone regeneration has gained attention in the biomedical field, which has led to the development of materials and synthesis methods meant to improve osseointegration and cellular bone activity. The properties of hydroxyapatite, a type of calcium phosphate, have been researched to determine its advantages for bone tissue engineering, particularly its biocompatibility and ability to interact with bone cells. Recently, the advantages of utilizing nanomolecules of hydroxyapatite, combined with various substances, in order to enhance and combine their characteristics, have been reported in the literature. This review will outline the cellular and molecular roles of hydroxypatite, its interactions with bone cells, and its nano-combinations with various ions and natural products and their effects on bone growth, development, and bone repair.