Osteoblastic MC3T3-E1 Cells Research Articles

Osteoprotegerin secretion and its inhibition by RANKL in osteoblastic cells visualized using bioluminescence imaging

Bone remodeling is regulated by the interaction between receptor activator of nuclear factor kappa-B ligand (RANKL) and its receptor RANK on osteoblasts and osteoclasts, respectively. Osteoprotegerin (OPG) is secreted from osteoblasts and inhibits osteoclast differentiation by acting as a decoy receptor for RANKL. Despite its importance, the mechanism underlying the secretion of OPG remains poorly understood. Here, we applied a method of video-rate bioluminescence imaging using a fusion protein with Gaussia luciferase (GLase) and visualized the secretion of OPG from living mouse osteoblastic MC3T3-E1 cells. The bioluminescence imaging revealed that the secretion of OPG fused to GLase (OPG-GLase) occurred frequently and widely across the cell surface. Notably, co-expression of RANKL significantly reduced the secretion of OPG-GLase, indicating an inhibitory role of RANKL on OPG secretion within cells. Further imaging and biochemical analyses using deletion mutants of OPG and RANKL, as well as RANKL mutants that cause autosomal recessive osteopetrosis, demonstrated the essential role of protein-protein interaction between OPG and RANKL in the inhibition of OPG secretion. Treatment with proteasome inhibitors resulted in increased levels of OPG in both culture medium and cell lysates. However, the fold-increase of OPG was similar regardless of the presence or absence of RANKL, suggesting that the regulation of OPG secretion by RANKL is independent of proteasome activity. This report visualized the secretion of OPG from living cells and provided evidence for a novel intracellular inhibitory effect of RANKL on OPG secretion.

Fabrication and in vitro biological properties of hydroxyapatite-sodium potassium niobate-barium titanate piezoelectric bioceramics

The utilization of piezoelectric materials in bone implants is appealing due to the inherent piezoelectric property of natural bone. The intrinsic electrical characteristics of piezoelectric biomaterials enhance antibacterial activities, biocompatibility, and bioactivity properties. This study delves into investigating the antibacterial properties, biocompatibility, and bioactivity of three-component biocomposites: sodium potassium niobate (KNN)-barium titanate (BT)-hydroxyapatite (HA). The combination of sodium potassium niobate and barium titanate, possessing suitable piezoelectric properties, with hydroxyapatite, known for its favorable biological properties, enhances the requisite properties for a bone implant. Among the various compositions studied, the combination comprising 70 wt% of the piezoelectric component (KNN-BT) and 30 wt% hydroxyapatite, labeled as 30HKB, emerged as the most optimal blend in terms of density, morphotropic phase boundary, and other biological tests conducted. Following polarization, the antibacterial efficacy of 30HKB against S. aureus bacteria cells increased by 61 %. Furthermore, the growth and adhesion of MC3T3-E1 osteoblast cells suggest enhanced biocompatibility of the 30HKB composite attributed to surface polarization. The surface charges generated by polarization facilitated the absorption of Ca2+ ions, as well as the interaction of HPO4 - and OH- ions with the precipitated Ca2+ ions, leading to the formation of the CaP layer. Hence, polarized piezoelectric ceramics exhibit heightened bioactivity compared to their non-polarized counterparts.

O-GlcNAcylation regulates osteoblast differentiation through the morphological changes in mitochondria, cytoskeleton, and endoplasmic reticulum.

To explore the potential mechanisms which O-linked-N-acetylglucosaminylation (O-GlcNAcylation) regulates osteogenesis, a publicly RNA-seq dataset was re-analyzed with literature-mining and showed the primary targets of O-GlcNAcylation in osteoblasts are mitochondria/cytoskeleton. Although the O-GlcNAcylation-regulated mitochondria/cytoskeleton has been extensively studied, its specific role during osteogenesis remains unclear. To address this, we knocked out Ogt (Ogt-KO) in MC3T3-E1 osteoblastic cells. Then, significantly reduced osteoblast differentiation, motility, proliferation, mitochondria-endoplasmic reticulum (Mito-ER) coupling, volume of ER, nuclear tubulins, and oxygen metabolism were observed in Ogt-KO cells. Through artificial intelligence (AI)-predicted cellular structures, the time-lapse live cells imaging with reactive-oxygen-species/hypoxia staining showed that lower cell proliferation and altered oxygen metabolism in the Ogt-KO cells were correlated with the Mito-ER coupling. Bioinformatics analysis, combined with correlated mRNA and protein expression, suggested that Ezh2 and its downstream targets (Opa1, Gsk3a, Wnt3a, Hif1a, and Hspa9) may be involved in O-GlcNAcylation-regulated Mito-ER coupling, ultimately impacting osteoblast differentiation. In conclusion, our findings indicate that O-GlcNAcylation-regulated osteoblast differentiation is linked to morphological changes in mitochondria, cytoskeleton, and ER, with Ezh2 potentially playing a crucial role.

7050 Effect of Spironolactone On Reducing Sugar-Induced Cellular Damage In MC3T3-E1 Osteoblastic Cells

Abstract Disclosure: S. Yun: None. H. Sang: None. S. Park: None. K. Suh: None. H. Kim: None. S. Chin: None. Background: In diabetes, prolonged hyperglycemia causes oxidative stress, resulting in pancreatic beta cells' dysfunction and diabetic complications. Oxidative stress can inhibit osteoblast differentiation and induce dysfunction and apoptosis. Spironolactone is an aldosterone antagonist used to treat hypertension and heart failure. In this study, we observed whether spironolactone can reduce osteoblast cytotoxicity and differentiation caused by oxidative stress induced by 2-deoxy-d-ribose (dRib). Methods: MC3T3-E1 cells were treated with spironolactone (0-100 uM) in the presence of 15 mM dRib. The cytotoxicity (lactate dehydrogenase, LDH) and differentiation (collagen content, alkaline phosphatase [ALP] activity, and mineralization) of osteoblasts, inflammatory cytokines (tumor necrosis factor-α [TNF-α], interleukin [IL]-6), oxidative stress (reactive oxygen species [ROS], mitochondrial superoxide), endoplasmic reticulum (ER) stress (activating transcription factor 6 [ATF-6], inositol-requiring 1 [IRE1]), mitochondrial function (mitochondrial membrane potential [MMP], adenosine triphosphate [ATP]), glyoxalase I activity, and glutathione (GSH) were measured. Results: Pretreatment of MC3T3-E1 osteoblastic cells with spironolactone prevented the dRib-induced cytotoxicity of osteoblasts and promoted differentiation of osteoblasts suppressed by dRib. Spironolactone significantly reduced dRib-induced inflammatory cytokines, ROS, mitochondrial superoxide, and ER stress. dRib-induced mitochondrial dysfunction was significantly improved by treatment with spironolactone. dRib is detoxified by the glyoxalase system. Pretreatment with spironolactone also increased the level of reduced GSH and the activity of glyoxalase I in dRib. Conclusion: Spironolactone could reduce dRib-induced cytotoxicity and promote differentiation in MC3T3-E1 osteoblastic cells by reducing oxidative stress and inflammatory cytokines, increasing mitochondrial biogenesis, and detoxifying dRib. These results suggest that spironolactone can be useful in the prevention and treatment of diabetic bone disease. Presentation: 6/1/2024

A biodegradable Fe–0.6Se alloy with superior strength and effective antibacterial and antitumor capabilities for orthopedic applications

Iron–selenium (Fe–Se) alloys have potential as attractive biodegradable bone–implant materials, given the antitumor properties of Se in cancer prevention and therapy. However, the fabrication of Fe–Se alloys is challenging due to the volatility of elemental Se and the significantly different melting points of Se and Fe. In this study, we successfully fabricated Fe–xSe (x = 0.2, 0.4, 0.6, 0.8, and 1 wt.%) alloys using suction casting, with FeSe compounds as the Se source. The microstructures, tensile properties, corrosion behavior, biocompatibility, antibacterial ability, and antitumor properties of the Fe–Se alloys were evaluated. The microstructures of the Fe–Se alloys were composed of α–Fe and FeSe phases. Among the Fe–Se alloys, Fe–0.6Se showed the best combination of tensile properties, with a yield strength of 1096.5 ± 7.2 MPa, an ultimate tensile strength of 1271.6 ± 6.3 MPa, and a fracture strain of 15.6 ± 3.3 %, and a degradation rate of 56.9 ± 0.4 μm/year. Moreover, the Fe–0.6Se alloy showed superb antibacterial ability against S. aureus, antitumor activity against 143B osteosarcoma cells, and osteogenicity and biocompatibility toward pre–osteoblast MC3T3–E1 cells. In summary, adding 0.2–1.0 wt.% Se to Fe does not affect the growth of healthy cells but effectively inhibits the growth and reproduction of tumor cells, and the Fe–0.6Se alloy is promising for orthopedic applications owing to its unique combination of mechanical and biofunctional properties. Statement of significanceThis work reports on Fe-xSe (x = 0.2, 0.4, 0.6, 0.8, and 1 wt.%) alloys fabricated using suction casting. The microstructures of the Fe–Se alloys were composed of α-Fe and FeSe phases. Among the Fe–Se alloys, the Fe-0.6Se showed the best combination of tensile properties, with a yield strength of 1058.6 ± 3.9 MPa, an ultimate tensile strength of 1134.1 ± 2.9 MPa, and a fracture strain of 16.8 ± 1.5 %, and a degradation rate of 56.9 ± 0.4 μm/year. Moreover, the Fe-0.6Se alloy showed superb antibacterial ability against S. aureus, antitumor activity against 143B osteosarcoma cells, and significant osteogenic ability and biocompatibility toward pre-osteoblast MC3T3-E1 cells. In summary, the Fe-0.6Se alloy is promising for orthopedic applications owing to its unique combination of mechanical and biofunctional properties.

Bioactive matrix scaffold of oxidized sacchachitin via green catalyst of siliacat tempo for enhancing bone regeneration

Bone regeneration remains a critical challenge in orthopedic medicine. Extracellular matrix proteins play important roles in bionic mineralization and osteogenesis. Sacchachitin nanofibers (SCNFs) oxidized via TEMPO have shown great potential as biomaterial scaffolds due to the introduction of carboxylic groups that mimic the natural extracellular matrix. However, TEMPO is harmful to the environment, which calls for greener and sustainable alternatives. Therefore, this study explored the green fabrication of oxidized-SCNFs through SiliaCat-TEMPO (STEMPO) as a renewable catalyst with various amount of oxidation agents to evaluate their bone regeneration ability. We developed an optimal STEMPO oxidation process, enabling efficient removal of most STEMPO particles through centrifugation with minimal residue. The oxidation efficiency of recovered STEMPO particles remained comparable to fresh ones. STEMPO-oxidized SCNFs, oxidized at increasing NaClO levels, showed an increase in carboxylate content, from 0.1360 mmol/g to 3.2415 mmol/g. Additionally, STEMPO-oxidized SCNFs exhibited excellent biocompatibility with MC3T3-E1 osteoblastic cells, facilitating calcium precipitation, cell migration, and osteogenic differentiation. Mixed composites of β-tricalcium phosphate (BCP) and STEMPO-oxidized SCNFs with higher oxidation levels further enhanced osteogenic differentiation. In vivo studies using a rat femur defect model showed that SCNFs at the highest oxidation level (ST100SC) significantly promoted bone regeneration to full recovery, achieving a bone volume to total tissue volume (BV/TV) ratio of 75.6 %, compared to 48.3 % in the control. These results demonstrated that STEMPO-oxidized SCNFs with higher oxidation levels can be used as a bioactive matrix scaffold to achieve full recovery of bone regeneration.

Mubong-derived Nanovesicles Regulate Differentiation and Mineralization in Osteoblastic MC3T3-E1 Cells

Mubong-derived Nanovesicles Regulate Differentiation and Mineralization in Osteoblastic MC3T3-E1 Cells

Suppression of bone resorption in ovariectomized mice using estrogen-immobilized polyphosphodiesters

Estrogens play a crucial role in bone remodeling. In this study, we synthesized two types of bone-targeted polyphosphodiesters (PPDEs): poly(diethyl sodium phosphate) (PEP•Na) and β-estradiol 17-valerate terminated PEP•Na (E2V-PEP•Na), and determined the inhibitory effect of the terminal estrogen on ovariectomy-induced bone resorption. We first assessed the impact of these polymers on the differentiation of osteoblasts and osteoclasts in vitro. Both PPDEs effectively promoted the differentiation of mouse osteoblastic MC3T3-E1 cells, with E2V-PEP•Na significantly enhancing the mineralization of these cells. Conversely, the density of osteoclasts formed from bone marrow mononuclear cells (BMNCs) significantly decreased when exposed to E2V-PEP•Na. Additionally, the formation of resorption pits on calcium phosphate-coated plates was reduced after treating BMNCs with both PPDEs. We then investigated the role of these polymers in treating osteoporosis using ovariectomized (OVX) mice. PEP•Na or E2V-PEP•Na was separately injected into the tail vein of the mice three times at 2-week intervals, starting 1 day after OVX treatment. Two weeks after the final injection, we observed that E2V-PEP•Na was distributed in bone tissue in a dose-dependent manner. The injection of E2V-PEP•Na significantly reduced bone resorption, and all bone parameters of OVX mice treated with E2V-PEP•Na were superior to those of sham mice. In contrast, PEP•Na did not affect bone mineral metabolism. In conclusion, our study is the first to demonstrate that E2V-PEP•Na can inhibit bone resorption in vivo, offering a promising new polymeric drug for preventing osteoporosis.

Biological activity of tantalum oxide coating doped with calcium and strontium by micro-arc oxidation under osteoimmunomodulation

Tantalum is widely used in clinics due to its excellent properties. However, its high density and elastic modulus constrain its application as a large implant. The new porous tantalum material addresses the issue of incompatibility between the implant and the human body. Nevertheless, it still exhibits biological inertia and limited bone-bonding ability; therefore, it is necessary to modify its surface. At present, some surface modification methods have inconsistent results in vitro and in vivo. To solve this problem, this study described the preparation of a ceramic coating with a "trabeculae-like" structure on the surface of pure tantalum using micro-arc oxidation (MAO). Calcium and strontium ions were introduced by adding acetate to the electrolyte. The effects of incorporating bioactive ions Ca2+ and Sr2+ into the electrolyte on the coating's morphology, coating thickness, phase composition, roughness, hydrophilicity, and the activity of MC3T3-E1 osteoblast cells under osteoimmunomodulation were investigated. The results demonstrated that the introduction of bioactive ions did not change the porous morphology of the coating; instead, it led to an increase in coating thickness, enhancement of surface roughness and hydrophilicity, as well as promotion of osteoblast spreading, proliferation, and ALP activity. Moreover, it regulated the phenotypic polarization of RAW264.7 macrophages to augment osteoblast proliferation, spreading, and ALP activity, implying that the coating has superior osteogenic potential under osteoimmunomodulation.

Effect of rare earth elements Ce and Yb on the in vitro properties of biodegradable Zn alloys

Biodegradable pure Zn and zinc alloys have received much attention in recent years, but the mechanical properties, corrosion behavior and biocompatibility of the previous reported pure zinc and zinc alloys do not fully meet the clinical requirements. Alloying and deformation technology are widely used in improving the properties of biodegradable pure Zn and zinc alloys. In this work, we have developed two new types of binary zinc alloys with the two rare earth elements (REE) cerium (Ce) and ytterbium (Yb). The effects of Ce and Yb on the microstructure, mechanical properties, corrosion behavior, in vitro biocompatibility of biodegradable Zn-xCe and Zn-xYb (x = 0.1, 0.2, 0.5, 1.0 wt %) were systematically investigated. The ultimate tensile strength, yield strength and elongation of Zn-xCe and Zn-xYb alloys are significantly improved compared with pure Zn. The Zn-xCe and Zn-xYb alloys have superior antibacterial properties The osteoblast MC3T3-E1 cells exhibited excellent cell activity with polygonal or fusiform extension and adhesion cultured in the series of Zn-xCe and Zn-xYb alloys’ extract solutions, indicating the good in vitro biocompatibility of Zn-xCe and Zn-xYb alloys. This work demonstrated that the Zn-xCe and Zn-xYb binary alloys have great potential as new biodegradable metals for future biomedical implants and devices.

Yunnan Baiyao Might Mitigate Periodontitis Bone Destruction by Inhibiting Autophagy and Promoting Osteoblast Differentiation in vivo, ex vivo and in vitro.

Periodontitis is an inflammatory disease that eventually destroys tooth-supporting tissue. Yunnan Baiyao (YNBY), a traditional Chinese medicine compound with haemostatic and anti-inflammatory properties has shown therapeutic potential in several diseases. Our previous study revealed that YNBY suppressed osteoclast differentiation in periodontitis. The purpose of this study is to investigate the influences of YNBY on osteoblasts and explore its potential mechanisms. A rat periodontitis model was established by ligation of maxillary second molars. After the end of modelling, histopathological observation by hematoxylin-eosin (HE) staining and Masson trichrome staining, detection of bone resorption by Micro-CT scanning, detection of osteoclasts by tartrate-resistant acid phosphatase (TRAP) staining, expression of osteocalcin (OCN) and microtubule-associated protein 1 light chain 3 (LC3) by immunohistochemistry. Lipopolysaccharides was used to irritate MC3T3-E1 osteoblastic cells and ex vivo calvarial organ as an in vitro model of inflammation. CCK-8 assay was performed to examine the toxicity of YNBY to MC3T3-E1 osteoblastic cells. Osteogenesis was assessed with alizarin red staining, immunofluorescence staining, Western blot and immunohistochemical staining. Transmission electron microscopy, fluorescent double staining, Western blot and immunohistochemical staining were employed to detect autophagy. Histological and micro-CT analyses revealed that YNBY gavage reduced bone loss caused by experimental periodontitis and upregulated osteogenic proteins in vivo. YNBY attenuated the production of autophagy-related proteins in periodontitis rats. Additionally, YNBY promoted osteogenesis by inhibiting inflammation-induced autophagy in vitro. Furthermore, YNBY suppressed LPS-mediated bone resorption and promoted the production of osteoblast-related proteins in inflamed calvarial tissues ex vivo. This study demonstrated, through in vivo, in vitro and ex vivo experiments, that YNBY promoted osteoblast differentiation by suppressing autophagy, which markedly alleviated bone destruction caused by periodontitis.

Caspase-9 Is a Positive Regulator of Osteoblastic Cell Migration Identified by diaPASEF Proteomics.

Caspase-9 is traditionally considered the initiator caspase of the intrinsic apoptotic pathway. In the past decade, however, other functions beyond initiation/execution of cell death have been described including cell type-dependent regulation of proliferation, differentiation/maturation, mitochondrial, and endosomal/lysosomal homeostasis. As previous studies revealed nonapoptotic functions of caspases in osteogenesis and bone homeostasis, this study was performed to identify proteins and pathways deregulated by knockout of caspase-9 in mouse MC3T3-E1 osteoblasts. Data-independent acquisition-parallel accumulation serial fragmentation (diaPASEF) proteomics was used to compare protein profiles of control and caspase-9 knockout cells. A total of 7669 protein groups were quantified, and 283 upregulated/141 downregulated protein groups were associated with the caspase-9 knockout phenotype. The deregulated proteins were mainly enriched for those associated with cell migration and motility and DNA replication/repair. Altered migration was confirmed in MC3T3-E1 cells with the genetic and pharmacological inhibition of caspase-9. ABHD2, an established regulator of cell migration, was identified as a possible substrate of caspase-9. We conclude that caspase-9 acts as a modulator of osteoblastic MC3T3-E1 cell migration and, therefore, may be involved in bone remodeling and fracture repair.

Scratch resistance and in-vitro biocompatibility of plasma-sprayed baghdadite coatings reinforced with carbon nanotubes

Hydroxyapatite (HA, Ca10(PO4)6(OH)2) coatings, commonly used for metallic implants, have limitations such as excessive brittleness, low fracture toughness, inadequate wear resistance, and slow osseointegration properties. In contrast, baghdadite (BAG, Ca3ZrSi2O9), a calcium zirconium silicate-based bioceramic, exhibits outstanding biological properties, to promote cell proliferation and differentiation of human osteoblasts along with adequate mechanical strength. In this study, plasma-sprayed HA, BAG, and carbon nanotubes (CNT, 1 wt%, and 2 wt%) reinforced BAG coatings are deposited on titanium (Ti) substrate to enhance its mechanical as well as biological properties. The microhardness values of CNT-reinforced BAG coatings increase due to a decrease in porosity and the retention of CNT inside the BAG matrix. Progressive loading scratch tests are performed, revealing a reduction in wear volume loss from 15.051 mm3 to 12.574 mm3 and scratch rate from 43.262 mm3N−1 m−1 to 36.172 mm3N−1 m−1 with the addition of 2 wt% CNT in BAG coatings. The scratch hardness test results demonstrated that introducing CNT into BAG coating significantly enhances its performance, showing a remarkable 38.7 % improvement. In-vitro cell culture studies indicate excellent cell adhesion, growth, and proliferation of MC3T3-E1 osteoblast cells on the surfaces of the CNT-reinforced BAG coatings.

Identification of the murine osteoblastic cell MC3T3-E1 as a permissive cell line in response to lumpy skin disease virus

Lumpy skin disease virus (LSDV) is a rapidly emerging pathogen in China. Screening suitable cells for LSDV replication is vital for future research on pathogenic mechanisms and vaccine development. Previous comparative studies have identified that the rodent-derived BHK21 is a highly susceptible cell model to LSDV infection. Using western blot, indirect immune-fluorescence assay, flow cytometry, and transmission electron microscopy methods, this study is the first to identify the murine osteoblastic cell line MC3T3-E1 as a novel permissive cell model for LSDV infection. The establishment of MC3T3-E1 as a suitable infectious cell model enhances our understanding of the species range and cell types of the permissive cells and nonpermissive that support LSDV replication. It is helpful to accelerate future research on the pathogenesis, clinical application, and vaccine development of LSDV.

Sol-gel derived bioactive glasses containing boron and strontium: Bioactivity, biocompatibility, and antibacterial properties

This study was aimed at the biological characterization of bioactive glasses in the systems of (60-x) SiO2–36CaO–4P2O5–xB2O3, and (60-x) SiO2–31CaO–4P2O5–5SrO-xB2O3 (x = 0, 1, 5, 10 and 15 mol%) synthesized through sol-gel technique. The influence of boron and strontium addition on in vivo bioactivity, antibacterial efficiency, and cell viability was studied. The morphological and structural assessments indicated that the bioactive glasses with chemical compositions of 50SiO2–31CaO–4P2O5–5SrO-10B2O3 (BG-5S10B), and 45SiO2–31CaO–4P2O5–5SrO-15B2O3 (BG-5S15B) possessed the highest bioactivity, owing to faster ion release and superior ability to induce apatite precipitation. The findings of the cell viability assay highlighted significant improvements in the proliferation and differentiation of osteoblastic MC3T3-E1 cells through the incorporation of strontium and boron (up to 10 %). Notably, the BG-5S10B sample exhibited exceptional biocompatibility and substantial antibacterial effectiveness on both E. coli and S. aureus. A combination of favorable biological properties makes BG-5S10B a highly promising option for use in bone tissue engineering applications.

Cyclin A1 (CCNA1) inhibits osteoporosis by suppressing transforming growth factor-beta (TGF-beta) pathway in osteoblasts

BackgroundOsteoporosis is a genetic disease caused by the imbalance between osteoblast-led bone formation and osteoclast-induced bone resorption. However, further gene-related pathogenesis remains to be elucidated.MethodsThe aberrant expressed genes in osteoporosis was identified by analyzing the microarray profile GSE100609. Serum samples of patients with osteoporosis and normal group were collected, and the mRNA expression of candidate genes was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The mouse cranial osteoblast MC3T3-E1 cells were treated with dexamethasone (DEX) to mimic osteoporosis in vitro. Alizarin Red staining and alkaline phosphatase (ALP) staining methods were combined to measure matrix mineralization deposition of MC3T3-E1 cells. Meanwhile, the expression of osteogenesis related genes including alkaline phosphatase (ALP), osteocalcin (OCN), osteopontin (OPN), Osterix, and bone morphogenetic protein 2 (BMP2) were evaluated by qRT-PCR and western blotting methods. Then the effects of candidate genes on regulating impede bone loss caused by ovariectomy (OVX) in mice were studied.ResultsCyclin A1 (CCNA1) was found to be significantly upregulated in serum of osteoporosis patients and the osteoporosis model cells, which was in line with the bioinformatic analysis. The osteogenic differentiation ability of MC3T3-E1 cells was inhibited by DEX treatment, which was manifested by decreased Alizarin Red staining intensity, ALP staining intensity, and expression levels of ALP, OCN, OPN, Osterix, and BMP2. The effects of CCNA1 inhibition on regulating osteogenesis were opposite to that of DEX. Then, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis demonstrated that genes negatively associated with CCNA1 were enriched in the TGF-beta signaling pathway. Inhibitor of TGF-beta signaling pathway partly reversed osteogenesis induced by suppressed CCNA1. Furthermore, suppressed CCNA1 relieved bone mass of OVX mice in vivo.ConclusionDownregulation of CCNA1 could activate TGF-beta signaling pathway and promote bone formation, thus playing a role in treatment of osteoporosis.

Scallop mantle-derived exosomes promote osteoblast formation and alleviate osteoporosis in mice

Osteoporosis, the most prevalent metabolic bone disease, has emerged as a serious public health concern. Safe and effective therapeutic interventions for osteoporosis are urgently needed. Here, we demonstrated that scallop mantle-derived exosomes (SMEs) promoted osteogenesis and prevented osteoporosis in ovariectomy-induced osteoporotic mice. Specifically, we successfully isolated SMEs with typical exosome morphology and desirable particle size. SMEs were found to contain abundant functional lipids, metabolites, and osteogenic-related proteins. Cell experiments revealed that SMEs could be internalized by osteoblastic MC3T3-E1 cells and promoted the proliferation, differentiation, and mineralization of MC3T3-E1 cells. In addition, SMEs survived the hostile gastrointestinal conditions and accumulated in the bone of mice after oral administration. Further mice investigations demonstrated that orally administered SMEs efficiently alleviated the bone loss, and improved the bone microstructure and biochemical properties in an ovariectomy-induced osteoporosis mouse model. Overall, these results reveal the osteogenesis effect of dietary SMEs, suggesting the potential of SMEs as an alternative or adjunct therapeutic agent in preventing or ameliorating osteoporosis.

Zinc Deficiency Promotes Calcification in Vascular Smooth Muscle Cells Independent of Alkaline Phosphatase Action and Partly Impacted by Pit1 Upregulation.

Inorganic phosphate (Pi) is a critical determinant of calcification, and its concentration is regulated by alkaline phosphatase (ALP) and Pit1. ALP is a key regulator of osteogenic calcification and acts by modulating local inorganic phosphate (Pi) concentrations through hydrolyzing pyrophosphate in the extracellular matrix (ECM). Pit1, a sodium-dependent phosphate transporter, regulates calcification via facilitating phosphate uptake within the cells. To investigate whether zinc differentially regulates osteoblastic and vascular calcifications, we examined ALP activity and Pit1 in osteoblastic and vascular smooth muscle cells (VSMCs). Our findings demonstrate that calcification in osteoblastic MC3T3-E1 cells is decreased via diminished ALP action under zinc deficiency. In contrast, zinc-deficiency-induced calcification in VSMCs is independent of ALP action, as demonstrated by very weak ALP activity and expression in calcified VSMCs. In zinc-deficient A7r5 VSMC, P accumulation increased with increasing Na phosphate concentration (3-7 mM) but not with β-GP treatment, which requires ALP activity to generate Pi. Ca deposition also increased with Na phosphate in a dose-dependent manner; in contrast, β-GP did not affect Ca deposition. In osteoblastic cells, Pit1 expression was not affected by zinc treatments. In contrast, Pit1 expression is highly upregulated in A7r5 VSMC under zinc deficiency. Using phosphonoformic acid, a competitive inhibitor of Pit1, we showed that calcification is inhibited in both A7r5 and MC3T3-E1 cells, indicating a requirement for Pit1 in both calcifications. Moreover, the downregulation of VSMC markers under zinc deficiency was restored by blocking Pit1. Taken together, our results imply that zinc-deficiency-induced calcification in VSMC is independent of ALP action in contrast to osteoblastic calcification. Moreover, Pit1 expression in VSMCs is a target for zinc deficiency and may mediate the inhibition of VSMC marker expression under zinc deficiency.

The marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol prevents TNF-α-mediated impairment of mineralization in mouse osteoblastic MC3T3-E1 cells: Impact of macrophage activation

The phenolic antioxidant 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA), found in the Pacific oyster Crassostrea gigas, is a superior peroxyl radical scavenger compared to other materials, including Trolox. DHMBA may play an important role in the prevention of health disorders. This study elucidates whether DHMBA prevents the impairment of mineralization of mouse osteoblastic MC3T3-E1 cells under inflammatory conditions by using mouse macrophage RAW264.7 cells in vitro. Culturing with DHMBA (1–100 μM) did not affect the proliferation and death of MC3T3-E1 cells. DHMBA stimulated osteoblastic mineralization. DHMBA blocked the decrease in mineralization of MC3T3-E1 cells caused by culture with the inflammatory cytokine TNF-α. DHMBA inhibited the production of TNF-α by stimulation with lipopolysaccharide (LPS) in RAW264.7 cells. The growth of MC3T3-E1 cells was suppressed by coculture with macrophages under LPS stimulation through the crosstalk of both cells. Interestingly, the growth of MC3T3-E1 cells was suppressed by culturing with the conditioned medium obtained by culturing macrophages with LPS. The effect of the conditioned medium was blocked by the presence of DHMBA or Bay 11–7082, an inhibitor of the TNF-α pathway. The blocking effect of DHMBA was not further enhanced in the presence of Bay 11–7082. Mechanistically, DHMBA was found to decrease the levels of NF-κB p65 and the activity of NF-κB reporter expression in MC3T3-E1 cells. DHMBA was shown to prevent the impairment of osteoblastic mineralization via TNF-α signaling involved in macrophage activation in the bone marrow microenvironment. This study may provide a novel strategy for the therapy of osteoblastic impairment.

Effect of silver/zinc co-dopant on enhancing cytotoxicity and antibacterial property of spray pyrolyzed bioactive glasses

ABSTRACT Owing to the advancement of bioactive materials, bioactive glasses (BGs) are progressively being recognized as promising choices in fields of bone and tissue engineering with their superior properties such as bioactivity and biocompatibility. However, their applications are limited by the fact that the material itself does not possess antibacterial properties. To address this limitation, antibacterial BGs with silver-zinc co-dopant were proposed and successfully synthesized via the one-step spray pyrolysis technique in this work. The phase composition, chemical composition, surface morphology, and nanoscale structures of the obtained BG specimens were characterized using X-ray diffraction, energy dispersive spectroscopy, scanning electron microscopy, and transmission electron microscopy. Moreover, in-vitro bioactivity was assessed through immersion of the SBF solution, while the in-vitro antibacterial property was examined using Escherichia coli bacteria. Meanwhile, the viability of MC3T3-E1 osteoblast cells was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Finally, the results showed positive effects of zinc addition, and the influence of silver and zinc dopants on the in-vitro bioactivity, antibacterial property, and cell viability were discussed.