BACKGROUND: Osteoporosis (OP) is a high-incidence bone disease that is prone to osteoporotic fractures (OF), so it has attracted widespread attention.
OBJECTIVE: This study investigated the specific expression and role of miR-331 in patients with OP and OF. The findings have profound implications for the clinical prevention and treatment of these conditions.
METHODS: The study included 60 OP patients, 46 OF patients, and 40 healthy controls. The expression level of miR-331-3p was detected using RT-qPCR. BMP2 was used to stimulate differentiation in MC3T3-E1 cells. After induction, the expression activity of osteogenic differentiation-related gene markers was detected using RT-qPCR. The target gene analysis was conducted using a luciferase reporter assay.
RESULTS: The levels of miR-331-3p were significantly elevated, while NRP2 levels were significantly reduced in OF patients. Post-surgery, miR-331-3p levels decreased over time. MiR-331-3p was found to negatively regulate the luciferase activity of NPR2 in MC3T3-E1 cells. Furthermore, overexpression of miR-331-3p inhibited cell proliferation and decreased the levels of osteoblast differentiation markers.
CONCLUSIONS: The up-regulation of miR-331-3p can promote OP and might also encourage the occurrence of OF by regulating NRP2. However, this needs further verification.

OBJECTIVE: Increasing ratio of bone fragility, and susceptibility to fractures constitutes a major health problem worldwide. Therefore, we aimed to identify new compounds with a potential to increase proliferation and differentiation of osteoblasts.
METHODS: Cellular and molecular assays, such as ALP activity, alizarin staining, and flow cytometry were employed to check the effect of TMF on osteogenesis. Moreover, gene expression analysis of certain important genes and transcriptional factors was also performed.
RESULTS: Our findings report for the first time that 7,3\',4\'-trimethoxyflavone is capable of enhancing proliferation, and differentiation in osteoblast cells. Results from flow cytometry analysis also indicated that TMF increases the number of cells in S-phase. Furthermore, treatment with TMF altered the expression of osteogenic genes, OCN and Axin-2 indicating possible activation of Wnt signaling pathway.
CONCLUSIONS: Taken together, this study identified that 7,3\',4\'-trimethoxyflavone has the potential to enhance osteoblast proliferation and differentiation, possibly due to the activation of Wnt/β-catenin pathway. Thus, demonstrating TMF as naturally occurring agent to promote osteogenesis and prevention of bone fragility, and related disorders.

BACKGROUND: Fragility fracture is common in the elderly. Osteoblast differentiation is essential for bone healing and regeneration. Expression pattern of long non-coding RNA MIAT during fracture healing was examined, and its role in osteoblast differentiation was investigated.
METHODS: 90 women with simple osteoporosis and 90 women with fragility fractures were included. Another 90 age-matched women were set as the control group. mRNA levels were tested using RT-qPCR. Cell viability was detected via CCK-8, and osteoblastic biomarkers, including ALP, OCN, Collagen I, and RUNX2 were tested via ELISA. The downstream miRNAs and genes targeted by MIAT were predicted by bioinformatics analysis, whose functions and pathways were annotated via GO and KEGG analysis.
RESULTS: Serum MIAT was upregulated in osteoporosis women with high accuracy of diagnostic efficacy. Serum MIAT was even elevated in the fragility fracture group, but decreased in a time manner after operation. MIAT knockdown promoted osteogenic proliferation and differentiation of MC3T3-E1, but the influences were reversed by miR-181a-5p inhibitor. A total of 137 overlapping target genes of miR-181a-5p were predicted based on the miRDB, TargetScan and microT datasets, which were mainly enriched for terms related to signaling pathways regulating pluripotency of stem cells, cellular senescence, and osteoclast differentiation.
CONCLUSIONS: LncRNA MIAT serves as a promising biomarker for osteoporosis, and promotes osteogenic differentiation via targeting miR-181a-5p.

Estrogen plays an important role in osteoporosis prevention. We herein report the possible novel signaling pathway of 17β-estradiol (E2) in the matrix mineralization of MC3T3-E1, an osteoblast-like cell line. In the culture media-containing stripped serum, in which small lipophilic molecules such as steroid hormones including E2 were depleted, matrix mineralization was significantly reduced. However, the E2 treatment induced this. The E2 effects were suppressed by ICI182,780, the estrogen receptor (ER)α, and the ERβ antagonist, as well as their mRNA knockdown, whereas Raloxifene, an inhibitor of estrogen-induced transcription, and G15, a G-protein-coupled estrogen receptor (GPER) 1 inhibitor, had little or no effect. Furthermore, the E2-activated matrix mineralization was disrupted by PMA, a PKC activator, and SB202190, a p38 MAPK inhibitor, but not by wortmannin, a PI3K inhibitor. Matrix mineralization was also induced by the culture media from the E2-stimulated cell culture. This effect was hindered by PMA or heat treatment, but not by SB202190. These results indicate that E2 activates the p38 MAPK pathway via ERs independently from actions in the nucleus. Such activation may cause the secretion of certain signaling molecule(s), which inhibit the PKC pathway. Our study provides a novel pathway of E2 action that could be a therapeutic target to activate matrix mineralization under various diseases, including osteoporosis.

BACKGROUND: Postmenopausal osteoporosis (PMOP) is a systemic bone disease characterized by low bone mass and microstructural damage. Morinda Officinalis (MO) contains various components with anti-PMOP activities. Morinda Officinalis-derived extracellular vesicle-like particles (MOEVLPs) are new active components isolated from MO, and no relevant studies have investigated their anti-osteoporosis effect and mechanism.
OBJECTIVE: To investigate the alleviating effect of MOEVLPs on PMOP and the underlying mechanism.
METHODS: Differential centrifugation and ultracentrifugation were used to isolate MOEVLPs from MO. Transmission electron microscopy (TEM), flow nano analyzer, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), agarose gel electrophoresis, and thin-layer chromatography were employed to characterize MOEVLPs. PMOP mouse models were utilized to examine the anti-PMOP effect of MOEVLPs. H&E and immunohistochemical staining were used for drug safety and osteogenic effect assessment. Mouse embryo osteoblast precursor cells (MC3T3-E1) were used in vitro experiments. CCK-8 kit, alizarin red staining, proteomic, bioinformatic analyses, and western blot were used to explore the mechanism of MOEVLPs.
RESULTS: In this study, MOEVLPs from MO were successfully isolated and characterized. Animal experiments demonstrated that MOEVLPs exhibited specific femur targeting, were non-toxic to the heart, liver, spleen, lung, kidney, and aorta, and possessed anti-PMOP properties. The ability of MOEVLPs to strengthen bone formation was better than that of alendronate. In vitro experiments, results revealed that MOEVLPs did not significantly enhance osteogenic differentiation in MC3T3-E1 cells. Instead, MOEVLPs promoted the proliferation of MC3T3-E1 cells. Proteomic and bioinformatic analyses suggested that the proliferative effect of MOEVLPs was closely associated with the mitogen-activated protein kinase (MAPK) signaling pathway, particularly the altered expression of cAMP response element-binding protein (CREB) and ribosomal S6 kinase 1 (RSK1). Western blot results further confirmed these findings.
CONCLUSIONS: Our studies successfully isolated high-quality MOEVLPs and demonstrated that MOEVLPs can alleviate PMOP by promoting osteoblast proliferation through the MAPK pathway. MOEVLPs have the potential to become a novel and natural anti-PMOP drug.

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.

The purpose of this study was to investigate the mechanism by which resveratrol (Res) inhibits apoptosis and promotes proliferation and differentiation of pre-osteoblastic MC3T3-E1 cells, laying the groundwork for the treatment of osteoporosis (OP). The TCMSP database was used to find the gene targets for Res. The GeneCards database acquire the gene targets for OP. After discovering the potential target genes, GO, KEGG, and Reactome enrichment analysis were conducted. Verifying the major proteins involved in apoptosis can bind to Res using molecular docking. CCK8 measured the proliferative activity of mouse pre-osteoblasts in every group following Res intervention. Alkaline phosphatase staining (ALP) and alizarin red staining to measure the ability of osteogenic differentiation. RT-qPCR to determine the expression levels of Runx2 and OPG genes for osteogenic differentiation ability of cells. Western blot to measure the degree of apoptosis-related protein activity in each group following Res intervention. The biological processes investigated for GO of Res therapeutic OP involved in cytokine-mediated signaling pathway, negative regulation of apoptotic process, Aging, extrinsic apoptotic signaling pathway in absence of ligand, according to potential therapeutic target enrichment study. Apoptosis, FoxO signaling pathway, and TNF signaling pathway are the primary KEGG signaling pathways. Recactome pathways are primarily engaged in Programmed Cell Death, Apoptosis, Intrinsic Apoptotic Pathway, and Caspase activation via extrinsic apoptotic signaling pathways. This research established a new approach for Res treatment of OP by demonstrating how Res controls the apoptosis-related proteins TNF, IL6, and CASP3 to suppress osteoblast death and increase osteoclastogenesis.

Mastering new and efficient ways to obtain successful drug delivery systems (DDS) with controlled release became a paramount quest in the scientific community. Increase of malignant bone tumors and the necessity to optimize an approach of localized drug delivery require research to be even more intensified. Octacalcium phosphate (OCP), with a number of advantages over current counterparts is extensively used in bone engineering. The aim of the present research was to synthesize bioactive and biocompatible doxorubicin (DOX) containing OCP particles. DOX-OCP was successfully obtained in situ in an exhaustive range of added drug (1-20 wt%, theoretical loading). Based on XRD, above 10 wt% of DOX, OCP formation was inhibited and the obtained product was low crystalline α-TCP. In-vitro drug release was performed in pH 7.4 and 6.0. In both pH environments DOX had a continuous release over six weeks. However, the initial drug burst for pH 7.4, in the first 24 h, ranged from 15.9 ± 1.3 % to 33.5 ± 12 % and for pH 6.0 23.7 ± 1.5 % to 36.2 ± 12 %.The DOX-OCP exhibited an inhibitory effect on viability of osteosarcoma cell lines MG63, U2OS and HOS. In contrast, MC3T3-E1 cells (IC50 > 0.062 µM) displayed increased viability and proliferation from 3rd to 7th day. Testing of the DDS on ferroptotic markers (CHAC1, ACSL4 and PTGS2) showed that OCP-DOX does not induce ferroptotic cell death. Moreover, the evaluation of protein levels of cleaved PARP, by western blotting analysis, corroborated that apoptosis is the main pathway of programmed cell death in osteosarcoma cells induced by DOX-OCP.

In the study, the fabrication of superparamagnetic-fluorescent bioactive glasses in the form of the particle, nanofiber, and 3D scaffolds was performed by including maghemite (γ-Fe2O3) nanoparticles and photoluminescent rare earth element ions (Eu3+, Gd3+, and Yb3+) using sol-gel, electrospinning, and robocasting techniques, respectively. The in vitro cytotoxicity of the magnetic-fluorescent bioactive glasses on osteosarcoma SaOS-2, pre-osteoblast MC3T3-E1, and BJ fibroblast cells, as well as their hemolytic activity and sorafenib tosylate loading and release behavior, were investigated. The cytotoxicity of the bioactive glass samples was tested using the MTT assay. Additionally, the alkaline phosphatase activity of the studied glasses was examined as a function of time. The mineralization behavior of the pre-osteoblast cell-seeded glass samples was analyzed using Alizarin red S staining. Results revealed that the in vitro cytotoxicity of the studied bioactive glasses in the form of particles and nanofibers depended on the sample concentration, whereas in the case of the 3D scaffolds, no cytotoxic response was observed on the osteosarcoma, pre-osteoblast, and fibroblast cells. Similarly, particle and nanofiber-based glass samples induced dose-dependent hemolysis on red blood cells. Drug loading rates were much lower for the 3D scaffolds compared to the particle and nanofiber-based samples. Drug release rates ranged from 25 % to 90 %, depending on the bioactive glass morphology and the pH of the release medium. It was concluded that the studied bioactive glasses have the potential to be used in tissue engineering applications and cancer therapy.

Stimulating bone formation potentially suggests therapeutics for orthopedic diseases including osteoporosis and osteoarthritis. Osteoblasts are key to bone remodeling because they act as the only bone-forming cells. miR-877-5p has a chondrocyte-improving function in osteoarthritis, but its effect on osteoblast differentiation is unknown. Here, miR-877-5p-mediated osteoblast differentiation was studied. Real-time reverse transcriptase-polymerase chain reaction was performed to measure miR-877-5p expression during the osteogenic differentiation of MC3T3-E1 cells. Osteoblast markers, including alkaline phosphatase (ALP), collagen type I a1 chain, and osteopontin, were measured and detected by alizarin red staining and ALP staining. Potential targets of miR-877-5p were predicted from three different algorithms: starBase ( http://starbase.sysu.edu.cn/ ), PITA ( http://genie.weizmann.ac.il/pubs/mir07/mir07_data.html ), and miRanda ( http://www.microrna.org/microrna/home.do ). It was further verified by dual luciferase reporter gene assay. The experimental results found that miR-877-5p was upregulated during the osteogenic differentiation of MC3T3-E1 cells. Overexpression of miR-877-5p promoted osteogenic differentiation, which was characterized by increased cell mineralization, ALP activity, and osteogenesis-related gene expression. Knockdown of miR-877-5p produced the opposite result. Dual luciferase reporter gene assay showed that miR-877-5p directly targeted eukaryotic translation initiation factor 4γ2 (EIF4G2). Overexpression of EIF4G2 inhibited osteogenic differentiation and reversed the promoting effect of overexpression of miR-135-5p on osteogenic differentiation. These results indicate that miR-877-5p might have a therapeutic application related to its promotion of bone formation through targeting EIF4G2.