Osteoblastic Cell Line MC3T3-E1 Research Articles

Abstract P5-12-04: Osteogenic cocktail induces calcifications in human breast cancer cell line via placental alkaline phosphatase expression

Abstract Breast cancer is frequently characterized by calcifications in mammography. Although breast calcifications were previously thought to be produced passively as the final stage of cell regression, recent studies have shown active production. In another context, vascular smooth muscle cells have been found to produce vascular calcifications by acquiring osteoblast-like characteristics. The same mechanism has been suggested to account for breast calcifications. A report showed that breast calcifications are clearly generated by the mouse breast cancer cell line 4T1 through osteoblast-like characteristics. However, the details underlying such a mechanism of mammary calcification is unknown. Understanding this mechanism will improve diagnostic accuracy for breast cancer. We induced calcifications using osteogenic cocktail on the human breast cancer cell line MDA-MB-231. Our hypothesis was that breast cancer calcifications are generated in a process similar to that of osteoblast differentiation. By a comprehensive analysis of transcription compared with osteoblastic cell line MC3T3-E1 in a microarray, we showed that placental alkaline phosphatase (PLAP) is involved in producing calcifications through PI3K-Akt signaling pathways. Herein, we demonstrated that calcifications occur and that alkaline phosphatase enzyme activity increases in MDA-MB-231 cells cultured using an osteogenic cocktail-containing medium. Microarray transcript analysis showed that the PI3K-Akt signaling pathway was significantly involved, with recruitment of placental alkaline phosphatase. Calcifications and alkaline phosphatase enzyme activity were suppressed by silencing placental alkaline phosphatase using a small interfering RNA. Inhibition of the PI3K-Akt signaling pathway suppressed phospho-c-Jun and placental alkaline phosphatase and resulted in absence of calcifications. These findings reveal that breast cancer cells acquire alkaline phosphatase enzyme activity via placental alkaline phosphatase expression and suggest that breast calcification formation is closely associated with the PI3K-Akt signaling pathway. Our results suggest that PLAP produces ALP enzyme activity required for calcification in MDA-MB-231 cells. PLAP is clinically expressed in some breast cancers. A report showed that dexamethasone induces PLAP enzyme activity and mRNA in the human breast cancer BC-M1 cell line. Therefore, dexamethasone in osteogenic cocktail may be associated with PLAP expression in the MDA-MB-231 cells. On the other hand, c-Jun binds to the promoter region of ALPP in MDA-MB-231, and we showed that c-Jun phosphorylation appears in MDA-MB-231 cells cultured using osteogenic cocktail. We suggest that PLAP transcript would be activated when phospho-c-Jun enters the nucleus and binds to the promoter region of PLAP. Our proposed mechanism in breast calcifications is that osteogenic cocktail induces c-Jun phosphorylation via PI3K-Akt signaling pathway and that PLAP transcription would be activated when phospho-c-Jun enters the nucleus and binds to the promoter region of PLAP. Future research might assess whether increased PLAP levels are the main driver of intracellular calcifications in breast cancer, as well as whether the PI3K-Akt signaling pathway has a similar effect in vivo that we demonstrated to cause calcifications in vitro. Citation Format: Atsushi Fushimi, Hiroshi Takeyama, Toshiaki Tachibana, Yoshinobu Manome. Osteogenic cocktail induces calcifications in human breast cancer cell line via placental alkaline phosphatase expression [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-12-04.

Synthesis and Characterization of Sol-Gel Derived Strontium Doped S53P4 Bioglass

This study concerns the evaluation of the bioactivity and cells response of strontium (Sr) doped sol-gel derived S53P4 bioglass due to Sr induced osteoblast. Moreover it prevents in-vitro osteoclastic activity and is clinically used as osteoporosis treatment. The different amount of Sr was doped into the S53P4 bioglass formulation (53.82%SiO2-1.72%P2O5-22.64%Na2O-(21.76-x)%CaO-x%SrO) (x=0, 3 and 5 mol %) and synthesized via sol-gel method. These samples were denoted as 0Sr, 3Sr and 5Sr respectively. After soaking in Hank's balanced salt solution (HBSS) for 7 and 14 days, the apatite formation was examined using X-ray powder diffraction (XRD) and scanning electron microscope (SEM) techniques. Proliferation and alkaline phosphatase activity were evaluated using osteoblastic cell line MC3T3-E1. The XRD and SEM findings confirmed the hydroxyapatite (HA) structure on the bioglass surface after soaking. More intense HA peaks were observed in 3Sr specimen on 7 day while in 5Sr specimen on 14 day. Meanwhile, 3Sr specimen showed the highest cells proliferation and ‌ significant difference in alkaline phosphatase (ALP) activity than 0Sr and 5Sr. As a result, this finding indicates that S53P4 bioglass with 3 mol % SrO (3Sr) is a good candidate for bone tissue engineering because it allows for optimum cell proliferation and ALP activity while also having a high bioactivity efficiency.

Direct effects of octreotide on osteoblast cell proliferation and function.

Octreotide (OCT) is a first-generation somatostatin analog (SSA) used in the treatment of acromegaly and neuroendocrine tumors (NETs). In both diseases, OCT interacts with somatostatin receptors 2 and 5 (SSTR2 and SSTR5), inhibiting hormone hypersecretion and cell proliferation. Skeletal health is an important clinical concern inacromegaly and NETs, since acromegalic osteopathy and NET bone metastasis occur in a remarkable number of patients. While OCT's effect on NET and pituitarycells has been extensively investigated, its direct action on bone cells remains unknown. Here, we investigated OCT direct effects on cell proliferation, differentiation, mineralization, and chemoattractant capacity of murine primary osteoblasts and osteoblast cell line MC3T3-E1. OCT inhibited osteoblasts and MC3T3-E1 cell proliferation (-30 ± 16%, and -22 ± 4%, both p < 0.05vs control) and increased MC3T3-E1 cell apoptosis (+ 76 ± 32%, p < 0.05 vs control). The anti-proliferative action of OCT was mediated by SSTR2 and SSTR5 in MC3T3-E1, while its pro-apoptotic effect was abrogated in SSTR2-silenced cells. The analysis of genes related to the early and late phases of osteoblast differentiation showed that OCT did not affect Alp, Runx2, Bglap, Spp1, and Sost levels in MC3T3-E1 cells. Similarly, OCT did not affect ALP activity, mineralization, and osteoclastogenic induction. Finally, Vegfa expression decreased in OCT-treated MC3T3-E1 cells and OCT inhibited pancreatic NET cell migration toward the osteoblast-conditioned medium. This study provides the first evidence of the direct action of OCT on osteoblasts which may have clinically relevant implications for the management of skeletal health in subjects with acromegaly and metastatic NETs.

Bone Formation Ability and Cell Viability Enhancement of MC3T3-E1 Cells by Ferrostatin-1 a Ferroptosis Inhibitor of Cancer Cells.

Recently, ferroptosis has gained scientists’ attention as an iron-related regulated necrosis. However, not many reports have investigated the effect of ferroptosis on bone. Therefore, with the present study, we assessed the effect of ferroptosis inhibition using ferrostatin-1 on the MC3T3-E1 pre-osteoblast cell. Cell images, cell viability, alkaline phosphatase activity test, alizarin red staining, and RUNX2 gene expression using real-time PCR were applied to investigate the effects of ferrostatin and erastin on MC3T3-E1 osteoblast cells. Erastin was used as a well-known ferroptosis inducer reagent. Erastin with different concentrations ranging from 0 to 50 µmol/L was used for inducing cell death. The 25 µmol/L erastin led to controllable partial cell death on osteoblast cells. Ferrostatin-1 with 0 to 40 µmol/L was used for cell doping and cell death inhibition effect. Ferrostatin-1 also displayed a recovery effect on the samples, which had already received the partially artificial cell death by erastin. Cell differentiation, alizarin red staining, and RUNX2 gene expression confirmed the promotion of the bone formation ability effect of ferrostatin-1 on osteoblast cells. The objective of this study was to assess ferrostatin-1’s effect on the MC3T3-E1 osteoblast cell line based on its ferroptosis inhibitory property.

Clonal osteoblastic cell lines with CRISPR/Cas9-mediated ablation of Pit1 or Pit2 show enhanced mineralization despite reduced osteogenic gene expression.

Multiple actions of extracellular Pi on the skeletal cells are likely to be partly mediated by type III sodium/phosphate (Na+/Pi) cotransporters Pit1 and Pit2, although the details are not fully understood. In the current study, to determine the roles of Pit1 and Pit2 in osteoblasts, we generated Pit1-knockout (KO) and Pit2-KO osteoblastic cells by applying CRISPR/Cas9 genome editing to an osteoblastic cell line MC3T3-E1 subclone 4. The extracellular Pi level was increased in the Pit1-KO and Pit2-KO clones due to the reduced Pi uptake. Interestingly, in vitro mineralization was accelerated in the Pit1-KO and Pit2-KO clones, although the induction of the expression of osteogenic marker genes was suppressed. In the cells before mineralization, extracellular levels of pyrophosphate (PPi) and adenosine triphosphate (ATP) were increased in the Pit1-KO and Pit2-KO clones, which might be attributable to the reduced expression and activity of tissue-nonspecific alkaline phosphatase (TNSALP). A 24-h treatment with high Pi reduced the expression and activity of TNSALP, suggesting that the suppression of TNSALP in the Pit1-KO and Pit2-KO clones was caused by the increased availability of extracellular Pi. Lentiviral gene transfer of Pit1 and Pit2 restored the changes observed in Pit1-KO and Pit2-KO clones, respectively. The expressions of P2Y2 and P2X7 which encode receptors for extracellular ATP were altered in the Pit1-KO and Pit2-KO clones, suggesting an influence on purinergic signaling. In mineralized cells after long-term culture, intracellular levels of PPi and ATP were higher in the Pit1-KO and Pit2-KO clones. Taken together, ablation of Pit1 or Pit2 in this osteoblastic cell model led to accelerated mineralization, suppressed TNSALP and altered the levels of extracellular and intracellular PPi and ATP, which might be partly mediated by changes in the availability of extracellular Pi.

MiR-193a-3p Promotes Fracture Healing via Targeting PTEN Gene.

The aim of this study was to investigate the role and potential mechanism of miR-193a-3p in fracture healing. The 70 fragility fracture patients and 45 healthy controls were enrolled in this study. Quantitative real-time PCR (qRT-PCR) was used for the measurement of the expression levels of miR-193a-3p and PTEN. MTT assay and flow cytometry were used to detect cell viability and apoptosis in the mouse osteoblastic cell line MC3T3-E1. Luciferase reporter assay was performed to confirm the correlation of miR-193a-3p with PTEN. The serum expression level of miR-193a-3p showed no significant change in fracture patients 7days after fixation treatment, but over time, there was a significant decrease in the expression at 14days and 21days after treatment (P < 0.01). Overexpression of miR-193a-3p significantly enhanced cell viability and inhibited cell apoptosis in MC3T3-E1 cells (P < 0.001). Serum PTEN level in fracture patients was increased gradually during the fracture healing process (P < 0.01). PTEN was demonstrated to be a target gene of miR-9-5p and reversed the effect of miR-193a-3p on cell viability and apoptosis (P < 0.001). miR-193a-3p promoted fracture healing via regulating PTEN and may serve as a novel potential target for enhancing bone repair of fragility fracture.

Strontium and selenium doped bioceramics incorporated polyacrylamide-carboxymethylcellulose hydrogel scaffolds: mimicking key features of bone regeneration

ABSTRACT Polyacrylamide-carboxymethylcellulose hydrogels lack mechanical strength, uncontrolled rate of degradation, and poor osteogenic properties which can be improved by the incorporation of bioceramics. Strontium and selenium doped bioceramics xSrO.(43-x)CaO.40SiO2.(12-y)P2O5.ySeO3.5MgO (where x = 6 mol%, y = 3 mol%) incorporated polyacrylamide-carboxymethylcellulose hydrogels were synthesized by free-radical polymerization. Above mentioned bioceramics were prepared by the sol-gel route. XRD analysis indicated an increase in crystallinity with the increment of bioceramics content. With the incorporation of bioceramics, the apatite phase formation was observed on 1st day of immersion in simulated body fluid at pH 7.4. Strontium and selenium doped bioceramics impregnated hydrogels exhibited an inhibitory effect on human osteosarcoma MG63 cell line. Moreover, bioceramics incorporated hydrogels provide a conducive environment for MC3T3-E1 osteoblast cell line proliferation, adhesion and also exhibited good alkaline phosphatase activity. Synthesized hydrogels indicated special osteogenic properties and can be potentially utilized in the hard tissue regeneration as well as for recovery of bone cancer patients.

Decitabine Inhibits Bone Resorption in Periodontitis by Upregulating Anti-Inflammatory Cytokines and Suppressing Osteoclastogenesis.

DNA methylation controls several inflammatory genes affecting bone homeostasis. Hitherto, inhibition of DNA methylation in vivo in the context of periodontitis and osteoclastogenesis has not been attempted. Ligature-induced periodontitis in C57BL/6J mice was induced by placing ligature for five days with Decitabine (5-aza-2′-deoxycytidine) (1 mg/kg/day) or vehicle treatment. We evaluated bone resorption, osteoclast differentiation by tartrate-resistant acid phosphatase (TRAP) and mRNA expression of anti-inflammatory molecules using cluster differentiation 14 positive (CD14+) monocytes from human peripheral blood. Our data showed that decitabine inhibited bone loss and osteoclast differentiation experimental periodontitis, and suppressed osteoclast CD14+ human monocytes; and conversely, that it increased bone mineralization in osteoblastic cell line MC3T3-E1 in a concentration-dependent manner. In addition to increasing IL10 (interleukin-10), TGFB (transforming growth factor beta-1) in CD14+ monocytes, decitabine upregulated KLF2 (Krüppel-like factor-2) expression. Overexpression of KLF2 protein enhanced the transcription of IL10 and TGFB. On the contrary, site-directed mutagenesis of KLF2 binding site in IL10 and TFGB abrogated luciferase activity in HEK293T cells. Decitabine reduces bone loss in a mouse model of periodontitis by inhibiting osteoclastogenesis through the upregulation of anti-inflammatory cytokines via KLF2 dependent mechanisms. DNA methyltransferase inhibitors merit further investigation as a possible novel therapy for periodontitis.

Melatonin Inhibits Glucose-Induced Apoptosis in Osteoblastic Cell Line Through PERK-eIF2α-ATF4 Pathway.

Osteoporosis is a common disease resulting in deteriorated microarchitecture and decreased bone mass. In type 2 diabetes patients, the incidence of osteoporosis is significantly higher accompanied by increased apoptosis of osteoblasts. In this study, using the osteoblastic cell line MC3T3-E1, we show that high glucose reduces cell viability and induces apoptosis. Also, high glucose leads to endoplasmic reticulum (ER) stress (ERS) via an increase in calcium flux and upregulation of the ER chaperone binding immunoglobulin protein (BiP). Moreover, it induces post-translational activation of eukaryotic initiation factor 2 alpha (eIF2α) which functions downstream of PKR-like ER kinase (PERK). This subsequently leads to post-translational activation of the transcription factor 4 (ATF4) and upregulation of C/EBP-homologous protein (CHOP) which is an ER stress-induced regulator of apoptosis, as well as downstream effectors DNAJC3, HYOU1, and CALR. Interestingly, melatonin treatment significantly alleviates the high-glucose induced changes in cell growth, apoptosis, and calcium influx by inhibiting the PERK-eIF2α-ATF4-CHOP signaling pathway. Additionally, the MC3T3-E1 cells engineered to express a phosphodead eIF2α mutant did not show high glucose induced ER stress, confirming that melatonin protects osteoblasts against high-glucose induced changes by decreasing ER-stress induced apoptosis by impacting the PERK-eIF2α-ATF4-CHOP signaling pathway. The protective of melatonin against high glucose-induced ER stress and apoptosis was attenuated when the cells were pre-treated with a melatonin receptor antagonist, indicating that the effect of melatonin was mediated via the melatonin receptors in this context. These findings lay the provide mechanistic insights of melatonin’s protective action on osteoblasts and will be potentially be useful in ongoing pre-clinical and clinical studies to evaluate melatonin as a therapeutic option for diabetic osteoporosis.

Caspase-12 Is Present During Craniofacial Development and Participates in Regulation of Osteogenic Markers.

Caspases are evolutionary conserved proteases traditionally known as participating in apoptosis and inflammation but recently discovered also in association with other processes such as proliferation or differentiation. This investigation focuses on caspase-12, ranked among inflammatory caspases but displaying other, not yet defined functions. A screening analysis pointed to statistically significant (P < 0.001) increase in expression of caspase-12 in a decisive period of mandibular bone formation when the original mesenchymal condensation turns into vascularized bone tissue. Immunofluorescence analysis confirmed the presence of caspase-12 protein in osteoblasts. Therefore, the osteoblastic cell line MC3T3-E1 was challenged to investigate any impact of caspase-12 on the osteogenic pathways. Pharmacological inhibition of caspase-12 in MC3T3-E1 cells caused a statistically significant decrease in expression of some major osteogenic genes, including those for alkaline phosphatase, osteocalcin and Phex. This downregulation was further confirmed by an alkaline phosphatase activity assay and by a siRNA inhibition approach. Altogether, this study demonstrates caspase-12 expression and points to its unknown physiological engagement in bone cells during the course of craniofacial development.

Cytotoxicity assay using a human pluripotent stem cell-derived cranial neural crest cell model.

Cleft lip and palate are the most common congenital abnormalities that occur early in pregnancy. The majority of cranial mesenchyme is derived from cranial neural crest cells that differentiate into odontoblasts, cartilage, craniofacial bone, and connective tissue. A subset of these cells differentiates into cranial ganglia. We have previously reported an induction protocol of cranial neural crest cell-like cells from human pluripotent stem cells. This study tested detection of the cytotoxic sensitivities of dental materials, including titanium ions, palladium ions, and hydroxyethyl methacrylate, on the cell viability of induced cranial neural crest cell-like cells (iNC-LCs) derived from Tic human induced pluripotent stem cell (hiPSC) line. Further, the sensitivity was compared with those of human fetal lung fibroblastic cell line MRC-5, which is origin of Tic hiPSC, and osteoblastic cell line MC3T3-E1 which was derived from mouse calvaria. The results suggested that this cell-based assay system using iNC-LCs is a potential method for in vitro screening as an alternative to animal testing to predict toxic effects of dental materials on early craniofacial development.

New cannabinoid receptor antagonists as pharmacological tool

Synthesis and pharmacological evaluation of a new series of cannabinoid receptor antagonists of indazole ether derivatives have been performed. Pharmacological evaluation includes radioligand binding assays with [3H]-CP55940 for CB1 and CB2 receptors and functional activity for cannabinoid receptors on isolated tissue. In addition, functional activity of the two synthetic cannabinoids antagonists 18 (PGN36) and 17 (PGN38) were carried out in the osteoblastic cell line MC3T3-E1 that is able to express CB2R upon osteogenic conditions. Both antagonists abolished the increase in collagen type I gene expression by the well-known inducer of bone activity, the HU308 agonist. The results of pharmacological tests have revealed that four of these derivatives behave as CB2R cannabinoid antagonists. In particular, the compounds 17 (PGN38) and 18 (PGN36) highlight as promising candidates as pharmacological tools.

Cytotoxicity, Oxidative Stress, and Autophagy Effects of Tantalum Nanoparticles on MC3T3-E1 Mouse Osteoblasts.

As a bone implant material, porous tantalum (Ta) has better corrosion resistance and more suitable elastic modulus than titanium. Surface nanomodification can accelerate the integration of Ta implants with bone tissue, which has broad application prospects in the field of dental implantology. Due to mechanical stress and load wear, nanoscale Ta fragments are inevitably exfoliated from the implant surface and brought into direct contact with osteoblasts surrounding the implant. These wear fragments may affect the biological characteristics of osteoblasts and thus the stability of implants. To date, the interaction of nanoscale Ta fragments with osteoblasts has not been clearly investigated. In the current study, we used the mouse osteoblast cell line MC3T3-E1 to explore the effects of Ta nanoparticles (Ta-NPs) on the cytotoxicity, oxidative stress and autophagy of osteoblasts. We found that a low concentration (12.5 μg/mL) of Ta-NPs can promote the proliferation of osteoblasts, while the Ta-NPs began to induce a decrease in cell viability at concentrations ≥25 μg/mL. Increased cell mortality, reactive oxygen species (ROS) production and decreased mitochondrial membrane potential (MMP) occurred in a dose-dependent manner after Ta-NP treatment. Moreover, with Ta-NP stimulation, the ratio of LC3-II/LC3-I increased, and the level of p62 protein was reduced. However, the degradation of p62 was not continuously increased when the concentration of Ta-NPs was ≥25 μg/mL. These results indicate that Ta-NPs induced osteoblast damage via oxidative stress. Autophagy activation may be a key factor in the cellular response to Ta-NP toxicity and could have an important impact on determining the survival or death of osteoblasts.

Curculigoside Protects against Excess-Iron-Induced Bone Loss by Attenuating Akt-FoxO1-Dependent Oxidative Damage to Mice and Osteoblastic MC3T3-E1 Cells.

Summary The present investigation found that curculigoside (CUR) can prevent excess-iron-induced bone loss in mice and cells through antioxidation and inhibiting excess-iron-induced phosphorylation of the Akt-FoxO1 pathway. CUR can attenuate the decreasing of cell viability, enhance autophagy, potentiate the antioxidant effect, and reduce apoptosis in MC3T3-E1 cells treated with excess iron through regulating the expression of FoxO1 target gene. Introduction Oxidative stress induced by iron overload is an important factor involved in primary osteoporosis disease and iron overload-related diseases. Curculigoside (CUR), a phenolic glycoside found abundantly in Curculigo orchioides Gaertn., has been demonstrated to possess antioxidant and antiosteoporotic properties. The aim of the present study is to explore the underlying molecular mechanism of CUR on excess-iron-induced bone loss in mice and osteoblastic MC3T3-E1 cells. Methods An iron-overload mice model was used to study the protective effects of CUR on bone loss induced by oxidative stress. Serum bone metabolism markers and antioxidant enzymes were also measured. To explore the antioxidant mechanism of CUR, the MC3T3-E1 osteoblastic cell line was used. Results In vivo studies showed that BMD and microarchitectural parameters were improved after a 3-month administration of CUR. CUR improved the biochemical parameters related to bone metabolism and the expressions of Runx2, OCN, and type 1 collagen and increased the formation of bone-mineralized nodules in vitro. CUR also inhibited ROS generation and increased the activities of antioxidant enzymes both in vivo and in vitro treated with excess iron. CUR can upregulate the level of FoxO1 and Nrf2, downregulate the level of p53 and the phosphorylation level of FoxO1, improve nuclear translocation of FoxO1, probably by inhibiting the IGFR/AKT signaling pathway, then increased cell viability and autophagy, and reduced apoptosis of MC3T3-E1 cells treated with excess iron by regulating the expression of FoxO1 target genes MnSOD, Gadd45a, Bim, FasL, and Rab7. Conclusions These results demonstrated that CUR was able to alleviate bone loss induced by oxidative stress resulting from iron overload, suggesting its potential use for the treatment of primary osteoporosis and bone loss in iron-overload-related diseases.

Investigation of Pyrophosphates KYP2O7Co-Doped with Lanthanide Ions Useful for Theranostics.

Diphosphate compounds (KYP2O7) co-doped with Yb3+ and Er3+ ions were obtained by one step urea assisted combustion synthesis. The experimental parameters of synthesis were optimized using an experimental design approach related to co-dopants concentration and heattreatment as well as annealing time. The obtained materials were studied with theinitial requirements showing appropriate morphological (X-Ray Diffraction (XRD), Scanning Electron Microscopy (SEM)) and spectroscopic properties (emission, luminescence kinetics). Moreover, the effect of Er3+ and Yb3+ ions doped KYP2O7 on morphology, proliferative and metabolic activity and apoptosis in MC3T3-E1 osteoblast cell line and 4B12osteoclasts cell line was investigated. Furthermore, the expression of the common pro-osteogenic markers in MC3T3-E1 osteoblast as well as osteoclastogenesis related markers in 4B12 osteoclasts was evaluated. The extensive in vitro studies showed that KYP2O7 doped with 1 mol% Er3+ and 20 mol% Yb3+ ions positively affected the MC3T3-E1 and 4B12 cells activity without triggering their apoptosis. Moreover, it was shown that an activation of mTOR and Pi3k signaling pathways with 1 mol% Er3+, 20 mol% Yb3+: KYP2O7 can promote the MC3T3-E1 cells expression of late osteogenic markers including RUNX and BMP-2. The obtained data shed a promising light for KYP2O7 doped with Er3+ and Yb3+ ions as a potential factors improving bone fracture healing as well as in bioimaging (so-called in theranostics).

Anti-Osteoporotic Effects of Combined Extract of Lycii Radicis Cortex and Achyranthes japonica in Osteoblast and Osteoclast Cells and Ovariectomized Mice.

Osteoporosis is characterized by low bone density and quality with high risk of bone fracture. Here, we investigated anti-osteoporotic effects of natural plants (Lycii Radicis Cortex (LRC) and Achyranthes japonica (AJ)) in osteoblast and osteoclast cells in vitro and ovariectomized mice in vivo. Combined LRC and AJ enhanced osteoblast differentiation and mineralized bone-forming osteoblasts by the up-regulation of bone metabolic markers (Alpl, Runx2 and Bglap) in the osteoblastic cell line MC3T3-E1. However, LRC and AJ inhibited osteoclast differentiation of monocytes isolated from mouse bone marrow. In vivo experiments showed that treatment of LRC+AJ extract prevented OVX-induced trabecular bone loss and osteoclastogenesis in an osteoporotic animal model. These results suggest that LRC+AJ extract may be a good therapeutic agent for the treatment and prevention of osteoporotic bone loss.

MicroRNA-495 downregulates AQP1 and facilitates proliferation and differentiation of osteoblasts in mice with tibial fracture through activation of p38 MAPK signaling pathway

Osteoblasts are implicated in the building of the vertebrate skeleton. The current study aimed to investigate the role of microRNA-495 (miR-495) in the osteoblasts of mice with tibial fractures and the underlying mechanism involving in aquaporin-1 (AQP1) and the p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway. Initially, a microarray-based analysis was performed to screen the differentially expressed genes and miRNAs associated with tibial fracture. Following the establishment of a tibial fracture mouse model, the positive rate of the AQP1 protein in the fracture tissue was detected by immunohistochemistry (IHC). Next, to verify the binding site between miR-495 on AQP1, bioinformatics data were employed in addition to the application of a dual-luciferase reporter gene assay. The osteoblast cell line MC3T3-E1 was treated with miR-495 mimic, miR-495 inhibitor and Anisomycin to explore the potent effects of miR-495 on proliferation and differentiation of osteoblasts in mice with tibial fracture. The expression of miR-495, AQP1, p38 MAPK, PCNA, Cyclin D1, OCN, and OPN was subsequently evaluated by RT-qPCR and Western blot analysis. Cell viability, the number of calcium nodules and alkaline phosphatase (ALP) activity were detected by MTT assay, alizarin red staining, and ALP activity assay, respectively. Our results revealed that miR-495 was down-regulated while AQP1 was up-regulated in the mice with tibial fractures. AQP1 was verified as a target gene of miR-495. When the cells were treated with overexpressed miR-495 or activated p38 MAPK signaling pathway, elevated expression of PCNA, Cyclin, D1, OCN, and OPN along with an increased amount of calcium nodules, higher cell viability, and enhanced ALP activity was detected, while the expression of AQP1 was reduced. Collectively, the key findings of the present study support the notion that overexpressed miR-495 may activate the p38 MAPK signaling pathway to inhibit AQP1 and to promote the proliferation and differentiation of osteoblasts in mice with tibial fracture.

P18. Treatment of osteoporotic vertebral fractures/defect in rats by naringin/hydroxyapatite/silk fibroin composite scaffolds in regulation of osteogenic differentiation, angiogenesis and inflammation

BACKGROUND CONTEXT Biomaterial scaffolds, as one of the three major elements of regenerative medicine, play an important role in bone remodeling and are therefore commonly used in the treatment of osteoporotic vertebral fractures (OVF). Among many biological materials, hydroxyapatite has attracted much attention in the research of bone defect regeneration due to its better bone conduction characteristics and mineralization processes and structures similar to bone tissue. Silk fibroin is a kind of natural biopolymer with high mechanical strength, controlled biodegradability, good biocompatibility and easy handling. The use of modern science and technology to make silk fibroin and hydroxyapatite into a composite scaffold can not only integrate the advantages of the two, but also avoid the disadvantages of low load bearing pressure, rejection reaction and non-degradability of traditional materials, which is an ideal, new tissue engineering material for OVF treatment. In recent years, traditional Chinese medicine has shown its unique advantages in the prevention and treatment of OVF. Traditional Chinese medicine has the effect of strengthening kidney and strengthening bone, promoting blood circulation and removing blood stasis. It is often used to promote fracture healing in clinic. Naringin is the main active ingredient of Rhizoma Drynariae, which can promote bone formation, angiogenesis, and inhibit inflammation. The combination of naringin, hydroxyapatite and silk fibroin as a new composite scaffold may have a synergistic effect and provide new insight on osteoporotic vertebral fractures/bone defect. PURPOSE To investigate the therapeutic effect and molecular mechanism of a new scaffold with naringin /hydroxyapatite/silk fibroin (NG/GMs/HA/SF) on vertebral fractures in osteoporotic rats. METHODS Loaded granules of naringin microspheres (NG/GMs), silk fibroin scaffolds (SF), hydroxyapatite/silk fibroin composite scaffolds (HA/SF), and granule naringin /hydroxyapatite/silk fibroin composite scaffold (NG//HA/SF) and loaded granule naringenin microsphere/hydroxyapatite/silk fibroin composite scaffold (NG/GMs/HA/SF) were conducted. The microscope was used to observe, calculate the volume of GMs and swelling ratio. X-ray diffraction (XRD) was used to analyse HA structure characterization; Fourier transform infrared spectroscopy was used to detect each scaffold structure. Scanning electron microscopy was used to analyse the surface structure, pore size and biocompatibility of each scaffold. Scanning calorimetry (DSC) was used to analyze the thermal properties and stability of each scaffold. UV spectrophotometer was used to detect the drug release rate in vitro. Trypan blue staining was used to detect the activity of MC3T3-E1 osteoblast cell line in the scaffold. We co-cultured bone marrow derived-mesenchymal stem cells (BMSCs) and scaffolds. And then ALP staining was used to detect the osteogenic differentiation potential of BMSCs, alizarin red staining used to detect mineralization ability, qRT-PCR used to detect the expression levels of osteogenic markers OCN, BMP-2 and Runx2 mRNA; AKP reagent used to detect osteoblast activity, Western-blot used to detect OCN, BMP-2, Runx2 protein expression. The rats experimented ovariectomize surgery to construct postmenopausal osteoporosis model, and next osteoporosis model was successfully used to construct lumbar vertebrae bone defect model, and then the scaffold was filled in bone Defects. X-ray was for the detection of bone defect formation, HE staining, Safranin solid staining and Masson staining for detection of bone defect morphological changes. Micro-CT examined three-dimensional structure and bone structure parameters of bone defect. Serum estrogen, CTSK, OCN/BGP, AKP levels were detected by ELISA. qRT-PCR, immunohistochemistry and Western-blot was used to detect osteoclast markers (OCN, Runx2, BMP2, CTSK), Collagen II), angiogenesis markers (VEGFA) and inflammatory markers (IK-6, TNF-a, NF-kB) mRNA and protein expression. RESULTS NG/GMs, SF, HA/SF, NG//HA/SF, NG/GMs/HA/SF scaffolds were successfully constructed, and the scaffolds had good biocompatibility and sustained drug release rate. The loaded granule naringenin microsphere/hydroxyapatite/silk fibroin composite scaffold can promote osteogenic differentiation and mineralization of BMSCs, and the scaffold can up-regulate the mRNA and protein expression of OCN, BMP-2 and Runx2. After 4 months, the X-ray and micro-CT three-dimensional reconstruction results showed that there was no obvious healing of the bone defect in the blank group, and the bone defect in the SF, HA/SF, NG/HA/SF group was partially healed, and the NG/GMs/HA/SF group has basically healed whose healing effect was better than that of SF, HA/SF, NG/HA/SF group. In the NG/GMs/HA/SF group, HE staining, Safranin solid staining and Masson staining showed more osteogenesis area. Futhermore, serological osteogenic indicators, molecular biology, osteogenesis, angiogenesis, inflammation-related genes and protein up-regulated. CONCLUSIONS The NG/GMs/HA/SF composite scaffolds may be used to treat osteoporotic vertebral fractures in rats by regulating the “osteogenesis differentiation-angiogenesis-inflammation” homeostasis. FDA DEVICE/DRUG STATUS Unavailable from authors at time of publication.

Effects of lncRNA DANCR on proliferation and differentiation of osteoblasts by regulating the Wnt/β-catenin pathway.

To measure the expression level of long non-coding ribonucleic acids (lncRNAs) differentiation antagonizing non-protein coding RNA (DANCR) in serum of patients with fracture and investigate its influences on the proliferation and differentiation of osteoblasts. Serum samples were collected from 44 fracture patients treated in our hospital and 24 healthy people receiving physical examination in our hospital. Then, reverse transcription-polymerase chain reaction (RT-PCR) technique was used to detect the expression of lncRNA DANCR in the serum of patients with fracture and healthy subjects. MC3T3-E1 mouse osteoblast cell line with stably-knocked out DANCR was further established using small interfering RNAs (siRNAs), and the effect of DANCR knockout on the proliferation of osteoblasts was determined using cell counting kit-8 (CCK-8). At the same time, 5-Ethynyl-2'-deoxyuridine (EdU) staining assay was performed to detect the percentage of EdU-positive cells in osteoblasts in control group and DANCR knockout group. In addition, the mRNA levels of differentiation-related genes including Runt-related transcription factor 2 (Runx2), Collagen1α1, osteocalcin (OC) and osterix (OSX) were detected via RT-PCR, and the protein level of Runx2 was measured through Western blotting. Moreover, osteoblasts were cultured with osteogenic medium for 14 d, and then alizarin red staining and alkaline phosphatase (ALP) staining assays were carried out to examine the differentiation of these osteoblasts. Lastly, Western blotting technique was employed to analyze the expression of the Wnt/β-catenin signaling pathway. The expression of lncRNA DANCR was significantly increased in the serum of fracture patients (p<0.05). The results of in-vitro cell experiments showed that the intervention of DANCR with siRNA was able to clearly promote the proliferation and differentiation of MC3T3-E1 osteoblast cell line. According to the results of Western blotting, DANCR promoted the apoptosis and proliferation, which was mediated by the activated Wnt/β-catenin signaling pathway in osteoblasts. LncRNA DANCR inhibition can facilitate the proliferation and differentiation of osteoblasts by activating the Wnt/β-catenin signaling pathway in osteoblasts. Therefore, DANCR is expected to be a new target promoting fracture healing.

A comparatively study of menaquinone-7 isolated from Cheonggukjang with vitamin K1 and menaquinone-4 on osteoblastic cells differentiation and mineralization

The effect of menaquinone-7 isolated from cheonggukjang was comparatively investigated with vitamin K1 and menaquinone-4 on cell differentiation and mineralization of the osteoblastic cell line MC3T3-E1. Results indicated that all vitamin K species significantly increased MC3T3-E1 cell proliferation, cellular alkaline phosphatase activity, osteocalcin synthesis, and calcium deposition in a dose-dependent manner. Menaquinone-4 and menaquinone-7 had more potent effects on calcium deposition than vitamin K1, and their effects were only partly reduced by warfarin (γ-carboxylation inhibitor) treatment, while warfarin abolished the induction activity of vitamin K1 on calcification. This suggests that vitamin K1 and K2 (menaquinone-4 & menaquinone-7) may have different mechanisms in stimulating osteoblast mineralization. In addition, the mRNA expression ratio of osteoprotegerin and the receptor activator of nuclear factor-kB ligand was also dramatically increased by treatment with vitamin K1 (62%), menaquinone-4 (247%), and menaquinone-7 (329%), suggesting that vitamin K may suppress the formation of osteoclast by up-regulating the ratio of osteoprotegerin/receptor activator of nuclear factor-kB ligand in osteoblasts. These results provide compelling evidence that vitamin K1, menaquinone-4, and menaquinone-7 all can promote bone health, which might be associated with elevations in the osteoprotegerin/receptor activator of nuclear factor-kB ligand ratio.