Osteoblast-specific down-regulation of NLRP3 inflammasome by aptamer-functionalized liposome nanoparticles improves bone quality in postmenopausal osteoporosis rats.

Rationale: NLRP3 inflammasome is critical in the development and progression of many metabolic diseases driven by chronic inflammation, but its effect on the pathology of postmenopausal osteoporosis (PMOP) remains poorly understood. Methods: We here firstly examined the levels of NLRP3 inflammasome in PMOP patients by ELISA. Then we investigated the possible mechanisms underlying the effect of NLRP3 inflammasome on PMOP by RNA sequencing of osteoblasts treated with NLRP3 siRNA and qPCR. Lastly, we accessed the effect of decreased NLRP3 levels on ovariectomized (OVX) rats. To specifically deliver NLRP3 siRNA to osteoblasts, we constructed NLRP3 siRNA wrapping osteoblast-specific aptamer (CH6)-functionalized lipid nanoparticles (termed as CH6-LNPs-siNLRP3). Results: We found that the levels of NLRP3 inflammasome were significantly increased in patients with PMOP, and were negatively correlated with estradiol levels. NLRP3 knock-down influenced signal pathways including immune system process, interferon signal pathway. Notably, of the top ten up-regulated genes in NLRP3-reduced osteoblasts, nine genes (except Mx2) were enriched in immune system process, and five genes were related to interferon signal pathway. The in vitro results showed that CH6-LNPs-siNLRP3 was relatively uniform with a dimeter of 96.64 ± 16.83 nm and zeta potential of 38.37 ± 1.86 mV. CH6-LNPs-siNLRP3 did not show obvious cytotoxicity and selectively delivered siRNA to bone tissue. Moreover, CH6-LNPs-siNLRP3 stimulated osteoblast differentiation by activating ALP and enhancing osteoblast matrix mineralization. When administrated to OVX rats, CH6-LNPs-siNLRP3 promoted bone formation and bone mass, improved bone microarchitecture and mechanical properties by decreasing the levels of NLRP3, IL-1ß and IL-18 and increasing the levels of OCN and Runx2. Conclusion: NLRP3 inflammasome may be a new biomarker for PMOP diagnosis and plays a key role in the pathology of PMOP. CH6-LNPs-siNLRP3 has potential application for the treatment of PMOP.

RhoA/ROCK-TAZ Axis regulates bone formation within calvarial trans-sutural distraction osteogenesis.

BACKGROUND: Craniofacial skeletal deformities can be addressed by applying tensile force to sutures to prompt sutural bone formation. The intricate process of mechanical modulation in craniofacial sutures involves complex biomechanical signal transduction. The small GTPase Ras homolog gene family member A (RhoA) functions as a key mechanotransduction protein, orchestrating the dynamic assembly of the cytoskeleton by activating the Rho-associated coiled-coil containing protein kinase (ROCK). Transcriptional coactivator with PDZ-binding motif (TAZ) serves as a crucial mediator in the regulation of genes and the orchestration of biological functions within the mechanotransduction signaling pathway. However, the role of RhoA/ROCK-TAZ in trans-sutural distraction osteogenesis has not been reported. METHODS: We utilized pre-osteoblast-specific RhoA deletion mice to establish an in vivo calvarial trans-sutural distraction model and an in vitro mechanical stretch model for pre-osteoblasts isolated from neonatal mice. Micro-CT and histological staining were utilized to detect the formation of new bone in the sagittal suture of the skull as well as the activation of RhoA, Osterix and TAZ. The activation of ROCK-limk-cofilin and the nuclear translocation of TAZ in pre-osteoblasts under mechanical tension were detected through Western blot, qRT-PCR, and immunofluorescence. RESULTS: The osteogenic differentiation of pre-osteoblasts was facilitated by mechanical tension through the activation of RhoA and Rho-associated kinase (ROCK), while ablation of RhoA impaired osteogenesis by inhibiting pre-osteoblast differentiation after suture expansion. Furthermore, inhibiting RhoA expression could block tensile-stimulated nuclear translocation of TAZ by preventing F-actin assembly through ROCK-LIM-domain kinase (LIMK)-cofilin pathway. In addition, the TAZ agonist TM-25659 could attenuate impaired osteogenesis caused by ablation of RhoA in pre-osteoblasts by increasing TAZ nuclear accumulation. CONCLUSIONS: This study demonstrates that mechanical stretching promotes the osteogenic differentiation of pre-osteoblasts in trans-sutural distraction osteogenesis, and this process is mediated by the RhoA/ROCK-TAZ signaling axis. Overall, our results may provide an insight for potential treatment strategies for craniosynostosis patients through trans-sutural distraction osteogenesis.

LINE-1 RNA triggers matrix formation in bone cells via a PKR-mediated inflammatory response.

Transposable elements (TEs) are mobile genetic modules of viral derivation that have been co-opted to become modulators of mammalian gene expression. TEs are a major source of endogenous dsRNAs, signaling molecules able to coordinate inflammatory responses in various physiological processes. Here, we provide evidence for a positive involvement of TEs in inflammation-driven bone repair and mineralization. In newly fractured mice bone, we observed an early transient upregulation of repeats occurring concurrently with the initiation of the inflammatory stage. In human bone biopsies, analysis revealed a significant correlation between repeats expression, mechanical stress and bone mineral density. We investigated a potential link between LINE-1 (L1) expression and bone mineralization by delivering a synthetic L1 RNA to osteoporotic patient-derived mesenchymal stem cells and observed a dsRNA-triggered protein kinase (PKR)-mediated stress response that led to strongly increased mineralization. This response was associated with a strong and transient inflammation, accompanied by a global translation attenuation induced by eIF2α phosphorylation. We demonstrated that L1 transfection reshaped the secretory profile of osteoblasts, triggering a paracrine activity that stimulated the mineralization of recipient cells.

Melatonin prevents cadmium-induced osteoporosis by affecting the osteoblast and osteoclast differentiation and pyroptosis in duck.

Cadmium (Cd), is a highly toxic environmental pollutant, which seriously threatens the health of poultry and humans. The occurrence of osteoporosis is the main manifestation of cadmium toxicity. Pyroptosis plays an important role in the development of osteoporosis. Melatonin has been shown to affect preserving bone health. However, the underlying mechanism has not been elucidated. In the present study, these functions of melatonin have been investigated in duck bone tissue and osteoblast during cadmium exposure. In vivo, the studies suggest that melatonin protects against cadmium-induced duck osteoporosis by improving the osteogenesis function, inhibiting bone resorption, and suppressing the occurrence of pyroptosis. In vitro, the findings demonstrated that melatonin alleviated the inhibition effect of cadmium on duck bone marrow-derived mesenchymal stem cells (BMSC) osteogenic differentiation, and suppressed the cadmium-induced osteoclast differentiation. In addition, we also found that melatonin prevents cytokines release of lactate dehydrogenase (LDH), interleukin-18 (IL-18), and interleukin-1ß (IL-1ß) by cadmium-induced, and reduces the expression of n-terminal Gasdermin D (N-GSDMD), alleviates the osteoblast death rate. In short, melatonin as a potential therapeutic agent has bright prospects in cadmium-induced bone toxicity.

Osteoclast-derived coupling factors: origins and state-of-play Louis V Avioli Lecture, ASBMR 2023.

Coupling, the mechanism that controls the sequence of events in bone remodelling, is a fundamental theory for understanding the way the skeleton changes throughout life. This review is an adapted version of the Louis V Avioli lecture, delivered at the Annual Scientific Meeting of the American Society of Bone and Mineral Research. It outlines the history of the coupling concept and details how coupling occurs within trabecular and cortical bone and describes its multiple contexts and the many mechanisms suggested to couple bone forming osteoblasts to the prior action of osteoclasts on the same bone surface. These mechanisms include signals produced at each stage of the remodelling sequence (resorption, reversal, and formation), such as factors released by osteoclasts through their resorptive action and through protein synthesis, molecules deposited in the cement line during the reversal phase, and potentially signals from osteocytes within the local bone environment. The review highlights two examples of coupling factors (Cardiotrophin 1 and EphrinB2:EphB4) to illustrate the limited data available, and the need to integrate both the many functions of these factors within the basic multicellular unit (BMU), and the multiple origins of these factors, including other cell types present during the remodelling sequence (such as osteocytes, macrophages, endothelial cells, and T-cells).

Coupling is a fundamental process by which bone resorbing cells (osteoclasts) are followed by bone forming cells (osteoblasts) on the same surface during the process of bone remodelling. This review outlines the history, basic concepts, and mechanisms proposed, and suggests directions for further research into the way this sequence of events in controlled in bone maintenance, development, and healing.

Deletion of the transcription factor EBF1 in perivascular stroma disrupts skeletal homeostasis and precipitates premature aging of the marrow microenvironment.

Early B cell factor 1 (EBF1) is a transcription factor expressed by multiple lineages of stromal cells within the bone marrow. While cultures of Ebf1-deficient cells have been demonstrated to have impaired differentiation into either the osteoblast or adipogenic lineage in vitro by several groups, in vivo there has been a nominal consequence of the loss of EBF1 on skeletal development. In this study we used Prx-cre driven deletion of Ebf1 to eliminate EBF1 from the entire mesenchymal lineage of the skeleton and resolve this discrepancy. We report here that EBF1 is expressed primarily in the Mesenchymal Stem and Progenitor Cell (MSPC)-Adipo, MSPC-Osteo, and the Early Mesenchymal Progenitors, and that loss of EBF1 has a plethora of consequences to maintenance of the skeleton throughout adulthood. Stroma from the Prx-cre;Ebf1fl/fl bones had impaired osteogenic differentiation, an age-dependent loss of CFU-F, and elevated senescence accompanying Ebf1-deletion. New bone formation was reduced after 3 months, and resulted in a quiescent bone environment with fewer osteoblasts and an accompanied reduction in osteoclast-mediated remodeling. Consequently, bones were less ductile at a younger age, and deletion of EBF1 dramatically impaired fracture repair. Disruption of EBF1 in perivascular populations also rearranged the vascular network within these bones and disrupted cytokine signaling from key hematopoietic niches resulting in anemia, reductions in B cells, and myeloid skewing of marrow hematopoietic lineages. Mechanistically we observed disrupted BMP signaling within Ebf1-deficient progenitors with reduced SMAD1-phosphorylation, and elevated secretion of the soluble BMP-inhibitor Gremlin from the MSPC-Adipo cells. Ebf1-deficient progenitors also exhibited posttranslational suppression of glucocorticoid receptor expression. Together, these results suggest that EBF1 signaling is required for mesenchymal progenitor mobilization to maintain the adult skeleton, and that the primary action of EBF1 in the early mesenchymal lineage is to promote proliferation, and differentiation of these perivascular cells to sustain a healthy tissue.

Bone Fracture Healing under the Intervention of a Stretchable Ultrasound Array.

Ultrasound treatment has been recognized as an effective and noninvasive approach to promote fracture healing. However, traditional rigid ultrasound probe is bulky, requiring cumbersome manual operations and inducing unfavorable side effects when functioning, which precludes the wide application of ultrasound in bone fracture healing. Here, we report a stretchable ultrasound array for bone fracture healing, which features high-performance 1-3 piezoelectric composites as transducers, stretchable multilayered serpentine metal films in a bridge-island pattern as electrical interconnects, soft elastomeric membranes as encapsulations, and polydimethylsiloxane (PDMS) with low curing agent ratio as adhesive layers. The resulting ultrasound array offers the benefits of large stretchability for easy skin integration and effective affecting region for simple skin alignment with good electromechanical performance. Experimental investigations of the stretchable ultrasound array on the delayed union model in femoral shafts of rats show that the callus growth is more active in the second week of treatment and the fracture site is completely osseous healed in the sixth week of treatment. Various bone quality indicators (e.g., bone modulus, bone mineral density, bone tissue/total tissue volume, and trabecular bone thickness) could be enhanced with the intervention of a stretchable ultrasound array. Histological and immunohistochemical examinations indicate that ultrasound promotes osteoblast differentiation, bone formation, and remodeling by promoting the expression of osteopontin (OPN) and runt-related transcription factor 2 (RUNX2). This work provides an effective tool for bone fracture healing in a simple and convenient manner and creates engineering opportunities for applying ultrasound in medical applications.

Tailoring zirconia surface topography via femtosecond laser-induced nanoscale features: effects on osteoblast cells and antibacterial properties.

The performance and long-term durability of dental implants hinge on the quality of bone integration and their resistance to bacteria. This research aims to introduce a surface modification strategy for zirconia implants utilizing femtosecond laser ablation techniques, exploring their impact on osteoblast cell behavior and bacterial performance, as well as the integral factors influencing the soft tissue quality surrounding dental implants. Ultrafast lasers were employed to craft nanoscale groove geometries on zirconia surfaces, with thorough analyses conducted using x-ray diffraction, scanning electron microscopy, atomic force microscopy, and water contact angle measurements. The study evaluated the response of human fetal osteoblastic cell lines to textured zirconia ceramics by assessing alkaline phosphatase activity, collagen I, and interleukin 1ßsecretion over a 7 day period. Additionally, the antibacterial behavior of the textured surfaces was investigated usingFusobacterium nucleatum, a common culprit in infections associated with dental implants. Ciprofloxacin (CIP), a widely used antibacterial antibiotic, was loaded onto zirconia ceramic surfaces. The results of this study unveiled a substantial reduction in bacterial adhesion on textured zirconia surfaces. The fine biocompatibility of these surfaces was confirmed through the MTT assay and observations of cell morphology. Moreover, the human fetal osteoblastic cell line exhibited extensive spreading and secreted elevated levels of collagen I and interleukin 1ßin the modified samples. Drug release evaluations demonstrated sustained CIP release through a diffusion mechanism, showcasing excellent antibacterial activity against pathogenic bacteria, includingStreptococcus mutans, Pseudomonas aeruginosa, andEscherichia coli.

Halofuginone Inhibits Osteoclastogenesis and Enhances Osteoblastogenesis by Regulating Th17/Treg Cell Balance in Multiple Myeloma Mice with Bone Lesions.

Evidences shows that T helper 17 (Th17) and regulatory T (Treg) cells imbalance plays a critical role in bone lesions of MM patients. Therefore, regulating the Th17/Treg imbalance may be beneficial for bone lesions in MM. Ten MM mice complicated with bone lesions were established and divided into the halofuginone (HF) group and the PBS group. After treatment, tibia and fibula from both groups were scanned by micro-CT. Osteoclasts and osteoblasts were validated by histochemical staining and ELISA. Th17 and Treg cells were tested by flow cytometry. The correlations between Th17/Treg cell ratio and osteoclasts, osteoblasts and bone remodeling were analyzed using the Spearman relative analysis. After treatment, mice in the HF group had an increase in trabecular bone volume fraction and thickened cortex, but a decrease in trabecular separation compared to mice in the PBS group.Tartrate-resistant acid phosphase (TRAP) + osteoclasts and its biomarker TRACP5b in serum were reduced, while alkaline phosphatase (ALP) + osteoblasts and its biomarker N-terminal propeptide of type 1precollagen (P1NP) in serum were accreted in the HF group. Th17/Treg cell ratio in halofuginone-treated mice was 0.85 ± 0.05, and was significantly lower than that in PBS-treated mice, which was 1.51 ± 0.03. In addition, it showed that the Th17/Treg cell ratio was significantly and positively associated with osteoclasts, but was significantly and negatively associated with osteoblasts and bone remodeling. Halofuginone plays a critical role in the amelioration bone lesions in MM, as it can inhibit osteoclastogenesis and enhance osteoblastogenesis by regulating the Th17/Treg cell balance. Supplementary Information: The online version contains supplementary material available at 10.1007/s12288-024-01756-4.

Blackcurrant extract promotes differentiation of MC3T3­E1 pre­osteoblasts.

Osteoporosis risk increases in menopausal individuals owing to the decrease in estrogen secretion. Blackcurrant extract (BCE) ameliorates osteoporosis; however, the underlying mechanisms are unclear. Furthermore, although BCE has phytoestrogenic activity, its effects on osteoblasts are unknown. In the present study, we investigated BCE-mediated attenuation of osteoporosis using mouse MC3T3-E1 pre-osteoblasts, with a focus on osteogenesis. After treating MC3T3-E1 cells with BCE for 48 h, cell proliferation was assessed using Cell Counting Kit-8. Levels of osteoblast differentiation markers, namely alkaline phosphatase activity and total collagen content in the cells, were evaluated after 3 and 14 days of BCE treatment, respectively. The expression of genes encoding osteoblast differentiation markers, including collagen type I (Col-I), alkaline phosphatase (Alp), bone γ-carboxyglutamate protein (Bglap), and runt-related transcription factor 2 (Runx2), was evaluated using reverse transcription-quantitative polymerase chain reaction. Mineralization of the cells was evaluated using Alizarin Red staining. Femoral tissues of ovariectomized (OVX) rats with or without 3% BCE were stained using ALP to evaluate osteogenic differentiation in femoral tissue. After treating MC3T3-E1 cells with BCE, cell proliferation had increased. BCE treatment increased Alp activity and total collagen content. Moreover, the expression of Col-I, Alp, Bglap, and Runx2 increased in BCE-treated cells. Furthermore, when MC3T3-E1 cells were treated with BCE for 21 days, the levels of calcified nodules increased. Alp staining intensity was stronger in the epiphyses on femoral tissue of OVX rats treated with 3% BCE than in those of untreated OVX rats. The results suggest that BCE may promote osteogenesis by inducing osteoblast differentiation.

Direct activation of PI3K in osteoblasts and osteocytes strengthens murine bone through sex-specific actions on cortical surfaces.

Intracellular phosphoinositide 3-kinase (PI3K) signaling is activated by multiple bone-active receptors. Genetic mutations activating PI3K signaling are associated with clinical syndromes of tissue overgrowth in multiple organs, often including the skeleton. Bone formation is increased by removing the PI3K inhibitor PTEN, but the effect of direct PI3K in the osteoblast lineage has not been reported. We introduced a known gain-of-function mutation in Pik3ca, the gene encoding the p110α catalytic subunit of PI3K, in osteocytes and late osteoblasts using the dentin matrix protein-1 Cre (Dmp1Cre) mouse and assessed the skeletal phenotype. Femur shape was grossly normal, but cortical thickness was significantly greater in both male and female Dmp1Cre.Pik3caH1047R mice, leading to almost doubled bone strength at 12 weeks of age. Both sexes had smaller marrow areas from 6 weeks of age. Female mice also exhibited greater cross sectional area, which continued to increase until 24 weeks of age, resulting in a further increase in bone strength. While both male and female mice had increased endocortical mineralizing surface, only female mice had increased periosteal mineralizing surface. The bone formed in the Dmp1Cre.Pik3caH1047R mice showed no increase in intracortical remodeling nor any defect in cortical bone consolidation. In contrast, on both endocortical and periosteal surfaces, there was a greater extent of lamellar bone formation with highly organized osteocyte networks extending along the entire surface at a greater thickness than in control mice. In conclusion, direct activation of PI3Kα in cells targeted by Dmp1Cre leads to high cortical bone mass and strength with abundant lamellar cortical bone in female and male mice with no increase in intracortical remodeling. This differs from the effect of PTEN deletion in the same cells, suggesting that activating PI3Kα in osteoblasts and osteocytes may be a more suitable target to promote formation of lamellar bone.

Patients with genetic activation of an enzyme called phosphoinositide-3 kinase (PI3K) have tissue overgrowth syndromes, where parts of the body become enlarged, sometimes including the skeleton. There are two types of mutations that cause these problems: one that directly causes the PI3K enzyme to be more active, or one that removes the normal brake on PI3K signaling (called PTEN). We studied the effect of directly activating PI3K enzyme specifically in osteoblasts (the cells that form bone) and osteocytes (osteoblasts that make a network inside the bone tissue itself). We found mice with these mutations formed normally shaped bones that were very strong because the outer shell was thicker than usual. In both male and female mice, it became thicker on the inside of the shell, but in female mice it also became thicker on the outside, making the bones even stronger over time. The new bone was well-organized bone, which likely helped make the increase in bone strength so profound. This is very different to what has previously been shown in mice with the other type of mutation in their bone forming cells; those mice had a shell that contained many large holes (pores). This indicates that directly stimulating PI3K enzyme is more beneficial for bone than removing the PTEN brake.

Isopsoralen Improves Glucocorticoid-induced Osteoporosis by Regulating Purine Metabolism and Promoting cGMP/PKG Pathway-mediated Osteoblast Differentiation.

BACKGROUND: The effects of Isopsoralen (ISO) in promoting osteoblast differentiation and inhibiting osteoclast formation are well-established, but the mechanism underlying ISO's improvement of Glucocorticoid- Induced Osteoporosis (GIOP) by regulating metabolism remains unclear. METHODS: This study aims to elucidate the mechanism of ISO treatment for GIOP through non-targeted metabolomics based on ISO's efficacy in GIOP. Initially, we established a GIOP female mouse model and assessed ISO's therapeutic effects using micro-CT detection, biomechanical testing, serum calcium (Ca), and phosphorus (P) level detection, along with histological analyses using hematoxylin and eosin (HE), Masson, and tartrate-resistant acidic phosphatase (TRAP) staining. Subsequently, non-targeted metabolomics was employed to investigate ISO's impact on serum metabolites in GIOP mice. RT-qPCR and Western blot analyses were conducted to measure the levels of enzymes associated with these metabolites. Building on the metabolomic results, we explored the effects of ISO on the cyclic Guanosine Monophosphate (cGMP)/Protein Kinase G (PKG) pathway and its role in mediating osteoblast differentiation. RESULTS: Our findings demonstrate that ISO intervention effectively enhances the bone microarchitecture and strength of GIOP mice. It mitigates pathological damage, such as structural damage in bone trabeculae, reduced collagen fibers, and increased osteoclasts, while improving serum Ca and P levels in GIOP mice. Non-- targeted metabolomics revealed purine metabolism as a common pathway between the Control and GIOP groups, as well as between the ISO high-dose (ISOH) group and the GIOP group. ISO intervention upregulated inosine and adenosine levels, downregulated guanosine monophosphate levels, increased Adenosine Deaminase (ADA) expression, and decreased cGMP-specific 3',5'-cyclic phosphodiesterase (PDE5) expression. Additionally, ISO intervention elevated serum cGMP levels, upregulated PKGI and PKGII expression in bone tissues, as well as the expression of Runt-related transcription factor 2 (Runx2) and Osterix, and increased serum Alkaline Phosphatase (ALP) activity. CONCLUSION: In summary, ISO was able to enhance the bone microstructure and bone strength of GIOP mice and improve their Ca, P, and ALP levels, which may be related to ISO's regulation of purine metabolism and promotion of osteoblast differentiation mediated by the cGMP/PKG pathway. This suggests that ISO is a potential drug for treating GIOP. However, further research is still needed to explore the specific targets and clinical applications of ISO.

RANKL-mediated osteoclast formation is required for bone loss in a murine model of Staphylococcus aureus osteomyelitis.

Staphylococcus aureus osteomyelitis leads to extensive bone destruction. Osteoclasts are bone resorbing cells that are often increased in bone infected with S. aureus. The cytokine RANKL is essential for osteoclast formation under physiological conditions but in vitro evidence suggests that inflammatory cytokines may by-pass the requirement for RANKL. The goal of this study was to determine whether RANKL-dependent osteoclast formation is essential for the bone loss that occurs in a murine model of S. aureus osteomyelitis. To this end, humanized-RANKL mice were infected by direct inoculation of S. aureus into a unicortical defect in the femur. Mice were treated with vehicle or denosumab, a human monoclonal antibody that inhibits RANKL, both before and during a 14-day infection period. The severe cortical bone destruction caused by infection was completely prevented by denosumab administration even though the bacterial burden in the femur was not affected. Osteoclasts were abundant near the inoculation site in vehicle-treated mice but absent in denosumab-treated mice. In situ hybridization demonstrated that S. aureus infection potently stimulated RANKL expression in bone marrow stromal cells. The extensive reactive bone formation that occurs in this osteomyelitis model was also reduced by denosumab administration. Lastly, there was a notable lack of osteoblasts near the infection site suggesting that the normal coupling of bone formation to bone resorption was disrupted by S. aureus infection. These results demonstrate that RANKL-mediated osteoclast formation is required for the bone loss that occurs in S. aureus infection and suggest that disruption of the coupling of bone formation to bone resorption may also contribute to bone loss in this condition.

Roles of Sp7 in osteoblasts for the proliferation, differentiation, and osteocyte process formation.

Background: Zinc finger-containing transcription factor Osterix/Specificity protein-7 (Sp7) is an essential transcription factor for osteoblast differentiation. However, its functions in differentiated osteoblasts remain unclear and the effects of osteoblast-specific Sp7 deletion on osteocytes have not been sufficiently studied. Methods: Sp7 floxneo/floxneo mice, in which Sp7 expression was 30 % of that in wild-type mice because of disturbed splicing by neo gene insertion, and osteoblast-specific knockout (Sp7 fl/fl;Col1a1-Cre) mice using 2.3-kb Col1a1 enhanced green fluorescent protein (EGFP)-Cre were examined by micro-computed tomography (micro-CT), bone histomorphometry, serum markers, and histological analyses. The expression of osteoblast and osteocyte marker genes was examined by real-time reverse transcription (RT)-PCR analysis. Osteoblastogenesis, osteoclastogenesis, and regulation of the expression of collagen type I alpha 1 chain (Col1a1) were examined in primary osteoblasts. Results: Femoral trabecular bone volume was higher in female Sp7 floxneo/floxneo and Sp7 fl/fl;Col1a1-Cre mice than in the respective controls, but not in males. Bromodeoxyuridine (BrdU)-positive osteoblastic cells were increased in male Sp7 fl/fl;Col1a1-Cre mice, and osteoblast number and the bone formation rate were increased in tibial trabecular bone in female Sp7 fl/fl;Col1a1-Cre mice, although osteoblast maturation was inhibited in female Sp7 fl/fl;Col1a1-Cre mice as shown by the increased expression of an immature osteoblast marker gene, secreted phosphoprotein 1 (Spp1), and reduced expression of a mature osteoblast marker gene, bone gamma-carboxyglutamate protein/bone gamma-carboxyglutamate protein 2 (Bglap/Bglap2). Furthermore, alkaline phosphatase activity was increased but mineralization was reduced in the culture of primary osteoblasts from Sp7 fl/fl;Col1a1-Cre mice. Therefore, the accumulated immature osteoblasts in Sp7 fl/fl;Col1a1-Cre mice was likely compensated for the inhibition of osteoblast maturation at different levels in males and females. Vertebral trabecular bone volume was lower in both male and female Sp7 fl/fl;Col1a1-Cre mice than in the controls and the osteoblast parameters and bone formation rate in females were lower in Sp7 fl/fl;Col1a1-Cre mice than in Sp7 fl/fl mice, suggesting differential regulatory mechanisms in long bones and vertebrae. The femoral cortical bone was thin and porous in Sp7 floxneo/floxneo and Sp7 fl/fl;Col1a1-Cre mice of both sexes, the number of canaliculi was reduced, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL)-positive lacunae and the osteoclasts were increased, whereas the bone formation rate was similar in Sp7 fl/fl;Col1a1-Cre and Sp7 fl/fl mice. The serum levels of total procollagen type 1 N-terminal propeptide (P1NP), a marker for bone formation, were similar, while those of tartrate-resistant acid phosphatase 5b (TRAP5b), a marker for bone resorption, were higher in Sp7 fl/fl;Col1a1-Cre mice. Osteoblasts were less cuboidal, the expression of Col1a1 and Col1a1-EGFP-Cre was lower in Sp7 fl/fl;Col1a1-Cre mice, and overexpression of Sp7 induced Col1a1 expression. Conclusions: Our studies indicated that Sp7 inhibits the proliferation of immature osteoblasts, induces osteoblast maturation and Col1a1 expression, and is required for osteocytes to acquire a sufficient number of processes for their survival, which prevents cortical porosity. The translational potential of this article: This study clarified the roles of Sp7 in differentiated osteoblasts in proliferarion, maturation, Col1a1 expression, and osteocyte process formation, which are required for targeting SP7 in the development of therapies for osteoporosis.

The impact of copper on bone metabolism.

Copper is an essential trace element for the human body. Abnormalities in copper metabolism can lead to bone defects, mainly by directly affecting the viability of osteoblasts and osteoclasts and their bone remodeling function, or indirectly regulating bone metabolism by influencing enzyme activities as cofactors. Copper ions released from biological materials can affect osteoblasts and osteoclasts, either directly or indirectly by modulating the inflammatory response, oxidative stress, and rapamycin signaling. This review presents an overview of recent progress in the impact of copper on bone metabolism. Translational potential of this article: The impact of copper on bone metabolism can provide insights into clinical application of copper-containing supplements and biomaterials.

Tmem119 deficiency delays bone repair in mice.

Tmem119 was identified as a bone anabolic factor in osteoblasts, however the roles of Tmem119 on bone repair have remained unknown. Therefore, we herein investigated the roles of Tmem119 on bone repair by examining the bone repair process after a femoral bone defect using Tmem119-deficient mice. In Tmem119-deficient mice, bone repair after a femoral bone defect was significantly delayed 10 and 14 days after bone injury in female and male mice with 3-dimensional micro-computed tomography analyses, respectively. The number of alkaline phosphatase-positive cells at the damaged sites was significantly decreased 7 days after bone injury in Tmem119-deficient mice, although the number of Osterix-positive cells was not significantly different 4 days after bone injury. The number of tartrate-resistant acid phosphatase-positive multinucleated cells as well as the number and luminal area of CD31-positive vessels at the damaged sites were not significantly different between Tmem119-deficient and wild-type mice. The present study first showed that Tmem119 deficiency delayed bone repair partly through a decrease in the osteoblastic bone formation of differentiated osteoblasts.

Nano-ZnO-modified hydroxyapatite whiskers with enhanced osteoinductivity for bone defect repair.

Hydroxyapatite (HA) whisker (HAw) represents a distinct form of HA characterized by its high aspect ratio, offering significant potential for enhancing the mechanical properties of bone tissue engineering scaffolds. However, the limited osteoinductivity of HAw hampers its widespread application. In this investigation, we observed HAw-punctured osteoblast membranes and infiltrated the cell body, resulting in mechanical damage to cells that adversely impacted osteoblast proliferation and differentiation. To address this challenge, we developed nano-zinc oxide particle-modified HAw (nano-ZnO/HAw). Acting as a reinforcing and toughening agent, nano-ZnO/HAw augmented the compressive strength and ductility of the matrix materials. At the same time, the surface modification with nano-ZnO particles improved osteoblast differentiation by reducing the mechanical damage from HAw to cells and releasing zinc ion, the two aspects collectively promoted the osteoinductivity of HAw. Encouragingly, the osteoinductive potential of 5% nano-ZnO/HAw and 10% nano-ZnO/HAw was validated in relevant rat models, demonstrating the efficacy of this approach in promoting new bone formation in vivo. Our findings underscore the role of nano-ZnO particle surface modification in enhancing the osteoinductivity of HAw from a physical standpoint, offering valuable insights into the development of bone substitutes with favorable osteoinductive properties while simultaneously bolstering matrix material strength and toughness.

Epigallocatechin gallate protects MC3T3-E1 cells from cadmium-induced apoptosis and dysfunction via modulating PI3K/AKT/mTOR and Nrf2/HO-1 pathways.

Epigallocatechin gallate (EGCG), an active constituent of tea, is recognized for its anticancer and anti-inflammatory properties. However, the specific mechanism by which EGCG protects osteoblasts from cadmium-induced damage remains incompletely understood. Here, the action of EGCG was investigated by exposing MC3T3-E1 osteoblasts to EGCG and CdCl2 and examining their growth, apoptosis, and differentiation. It was found that EGCG promoted the viability of cadmium-exposed MC3T3-E1 cells, mitigated apoptosis, and promoted both maturation and mineralization. Additionally, CdCl2 has been reported to inhibit both the phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) and nuclear factor erythroid 2-related factor 2/heme oxygenase-1(Nrf2/HO-1) signaling pathways. EGCG treatment attenuated cadmium-induced apoptosis in osteoblasts and restored their function by upregulating both signaling pathways. The findings provide compelling evidence for EGCG's role in attenuating cadmium-induced osteoblast apoptosis and dysfunction through activating the PI3K/AKT/mTOR and Nrf2/HO-1 pathways. This suggests the potential of using EGCG for treating cadmium-induced osteoblast dysfunction.

Effects of cosmos caudatus (Kenikir) antioxidant properties on bone metabolism marker in rat.

Cosmos caudatus leaves are one of around 7500 types of plants that are known to have herbal or medicinal plant properties in Indonesia. This research determines the effectiveness of Cosmos caudatus as an antioxidant agent against cells, biomolecules, and bone density. Forty-three male rat aged 3-4 months were divided into four groups.Group P0 was only given distilled water. Group P1 was given kenikir leaf extract at a dose of 0.91 mg/kg. Group P2 was given kenikir leaf extract at a dose of 1.82 mg/kg. And group P3 was given kenikir leaf extract at 3.64 mg/kg ad libitum once a day for 28 days. The highest average SOD level was in the 1.82 mg/bb P2 conversion dose group (1.09 ± 1.76). The lowest mean CTX level was in the P2 group (8.30 ± 1.10). There was a significant increase in mean trabecular bone in the P2 group (43.33 ± 5.32). The number of osteoblast cells increased significantly at P2 (103.94 (SD 38.14)). The number of osteoclasts decreased from the control group (P0) to 0.60 (SD 0.43) at P2. Indicate that the Cosmos caudatus extract may have advantages as an antioxidant support agent for bone metabolism.

Randomized split-mouth clinical trial comparing osteoblastic activity and osteogenic potential of autogenous particle harvesting during implant surgery without irrigation and with irrigation.

OBJECTIVE: To compare the osteoblastic activity and osteogenic potential of autogenous particle harvesting during implant surgery using low-speed drilling without irrigation and high-speed drilling with irrigation. MATERIALS AND METHODS: Thirty patients with bilateral missing teeth of 3.6 and 4.6 were randomized into two groups (Group 1: low-speed drilling without irrigation and Group 2: high-speed drilling with irrigation) and 60 single dental implants were placed. The temperature at the tip of each drill was recorded and the harvested bone was weighed; particle size and Ca and P levels were also analyzed. After osteoblast culture, cell viability, cell cycle assay, cell migration, vascular endothelial growth factor (VEGF) concentration, and mineralized nodule formation were assessed. RESULTS: Although the temperature of the drills was slightly higher in Group 1, no statistically significant differences were observed (p ≤ 0.05); however, the amount of harvested bone was higher (p < 0.001) and the size of the particles was higher (p = 0.019). In relation to osteoblastic activity and osteogenic potential, higher cell proliferation, higher number of cells in G2/M and S phases, higher cell migration capacity, higher VEGF concentration, and higher amount of mineralized nodule formation were observed in Group 1. CONCLUSIONS: Low-speed drilling without irrigation does not result in a significant increase in bone temperature compared to conventional drilling. However, a greater amount of bone is obtained; in addition, osteoblastic activity and osteogenic potential are higher with this technique, but further clinical studies are necessary.