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Adipocyte enhancer‐binding protein 1 (AEBP1) is closely implicated in osteoblastic differentiation and bone fracture; this research aimed to investigate the effect of AEBP1 on restoring osteoblastic differentiation under dexamethasone (Dex) treatment, and its interaction with the phosphatidylinositol 3‐kinase (PI3K)/protein kinase B (AKT) pathway. Pre‐osteoblastic MC3T3‐E1 cells were cultured in osteogenic medium and treated by Dex to mimic steroid‐induced osteonecrosis cellular model. They were then further transfected with control or AEBP1‐overexpressed lentiviral vectors. Finally, cells were treated with the PI3K inhibitor LY294002, with or without AEBP1‐overexpressed lentiviral vectors. AEBP1 expression showed a downward trend in MC3T3‐E1 cells under Dex treatment in a dose‐dependent manner. AEBP1‐overexpressed lentiviral vectors increased relative cell viability, alkaline phosphatase (ALP) staining, Alizarin red staining and osteoblastic differentiation markers including osteocalcin (OCN), osteopontin (OPN), collagen type I alpha 1 (COL1A1), runt‐related transcription factor 2 (RUNX2) and bone morphogenetic protein 2 (BMP2), but decreased cell apoptosis rate in MC3T3‐E1 cells under Dex treatment; besides, AEBP1‐overexpressed lentiviral vectors positively regulated p‐PI3K and p‐AKT expressions. Furthermore, LY294002 treatment decreased relative cell viability, Alizarin red staining, osteoblastic differentiation markers including OCN, OPN, RUNX2 and BMP, increased cell apoptosis rate and did not affect ALP staining in MC3T3‐E1 cells under Dex treatment; meanwhile, LY294002 treatment weakened the effect of AEBP1 overexpression vectors on the above cell functions. AEBP1 restores osteoblastic differentiation under Dex treatment by activating the PI3K/AKT pathway. [ABSTRACT FROM AUTHOR]

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Type 2 diabetic osteoporosis (T2DOP) is a skeletal metabolic syndrome characterized by impaired bone remodeling due to type 2 diabetes mellitus, and there are drawbacks in the present treatment. Osteoking (OK) is widely used for treating fractures and femoral head necrosis. However, OK is seldom reported in the field of T2DOP, and its role and mechanism of action need to be elucidated. Consequently, this study investigated whether OK improves bone remodeling and the mechanisms of diabetes‐induced injury. We used db/db mice as a T2DOP model and stimulated MC3T3‐E1 cells (osteoblast cell line) with high glucose (HG, 50 mM) and advanced glycation end products (AGEs, 100 µg/mL), respectively. The effect of OK on T2DOP was assessed using a combined 3‐point mechanical bending test, hematoxylin and eosin staining, and enzyme‐linked immunosorbent assay. The effect of OK on enhancing MC3T3‐E1 cell differentiation and mineralization under HG and AGEs conditions was assessed by an alkaline phosphatase activity assay and alizarin red S staining. The AGEs/insulin‐like growth factor‐1(IGF‐1)/β‐catenin/osteoprotegerin (OPG) pathway‐associated protein levels were assayed by western blot analysis and immunohistochemical staining. We found that OK reduced hyperglycemia, attenuated bone damage, repaired bone remodeling, increased tibial and femoral IGF‐1, β‐catenin, and OPG expression, and decreased receptor activator of nuclear kappa B ligand and receptor activator of nuclear kappa B expression in db/db mice. Moreover, OK promoted the differentiation and mineralization of MC3T3‐E1 cells under HG and AGEs conditions, respectively, and regulated the levels of AGEs/IGF‐1/β‐catenin/OPG pathway‐associated proteins. In conclusion, our results suggest that OK may lower blood glucose, alleviate bone damage, and attenuate T2DOP, in part through activation of the AGEs/IGF‐1/β‐catenin/OPG pathway. [ABSTRACT FROM AUTHOR]

This study examined changes in the degree of hydrolysis, peptide yield, digestibility, and hydrolyzed amino acid content during the simulated gastrointestinal digestion of Antarctic krill protein. Furthermore, the in vitro proosteogenic activity of different concentrations of the lyophilized residue from the supernatant of the digestion product at the optimal digestion time was evaluated. The results showed that the hydrolysis degree, peptide yield, digestibility, and hydrolyzed amino acid content in the supernatant from Antarctic krill protein digestion increased significantly with digestion time, reaching an equilibrium after 120 min of intestinal digestion, with a hydrolysis degree of 42.67%, a peptide yield of 35.30% and a digestibility of 82.21%. The supernatants at all digestion time points enhanced the osteogenic activity of mouse MC3T3-E1 osteoblasts, the one obtained after 120 min of intestinal digestion being the most effective. Living/dead cell staining, alkaline phosphatase (ALP) activity assay and alizarin red staining confirmed that the supernatant from intestinal digestion for 120 min had good cellular compatibility, promoted osteoblast differentiation and mineralization and consequently regulated its osteogenic activity by enhancing ALP activity and intracellular calcium deposition. The results of this study provide a theoretical basis for the high-value application of Antarctic krill protein.

Recent studies have confirmed that melatonin and N6-methyladenosine (m6A) modification can influence bone cell differentiation and bone formation. Melatonin can also regulate a variety of biological processes through m6A modification. Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1) serves as a reader of m6A modification. In this study, we used the hindlimb unloading model as an animal model of bone loss induced by simulated microgravity and used 2D clinorotation to simulate a microgravity environment for cells on the ground. We found that hnRNPA2B1 was downregulated both in vitro and in vivo during simulated microgravity. Further investigations showed that hnRNPA2B1 could promote osteoblast differentiation and that overexpression of hnRNPA2B1 attenuated the suppression of osteoblast differentiation induced by simulated microgravity. We also discovered that melatonin could promote the expression of hnRNPA2B1 under simulated microgravity. Moreover, we found that promotion of osteoblast differentiation by melatonin was partially dependent on hnRNPA2B1. Therefore, this research revealed, for the first time, the role of the melatonin/hnRNPA2B1 axis in osteoblast differentiation under simulated microgravity. Targeting this axis may be a potential protective strategy against microgravity-induced bone loss and osteoporosis.

Highlights SPI1 and SMAD5 are upregulated during MC3T3-E1 cell osteogenic differentiation. SPI1 enhances SMAD5 transcription. Icariin increases SMAD5 expression by upregulating SPI1. The SPI1/SMAD5 axis underlies the promoting effect of icariin on MC3T3-E1 cell osteogenic differentiation.

Highlights: SPI1 and SMAD5 are upregulated during MC3T3-E1 cell osteogenic differentiation. SPI1 enhances SMAD5 transcription. Icariin increases SMAD5 expression by upregulating SPI1. The SPI1/SMAD5 axis underlies the promoting effect of icariin on MC3T3-E1 cell osteogenic differentiation. Background: Dysregulation of osteogenic differentiation is a crucial event during osteoporosis. The bioactive phytochemical icariin has become an anti-osteoporosis candidate. Here, we elucidated the mechanisms underlying the promoting function of icariin in osteogenic differentiation. Methods: Murine pre-osteoblast MC3T3-E1 cells were stimulated with dexamethasone (DEX) to induce osteogenic differentiation, which was evaluated by an Alizarin Red staining assay and ALP activity measurement. The mRNA amounts of SPI1 and SMAD5 were detected by real-time quantitative PCR. Expression analysis of proteins, including osteogenic markers (OPN, OCN and RUNX2) and autophagy-associated proteins (LC3, Beclin-1, and ATG5), was performed by immunoblotting. The binding of SPI1 and the SMAD5 promoter was predicted by the Jaspar2024 algorithm and confirmed by chromatin immunoprecipitation (ChIP) experiments. The regulation of SPI1 in SMAD5 was examined by luciferase assays. Results: During osteogenic differentiation of MC3T3-E1 cells, SPI1 and SMAD5 were upregulated. Functionally, SPI1 overexpression enhanced autophagy and osteogenic differentiation of MC3T3-E1 cells, while SMAD5 downregulation exhibited opposite effects. Mechanistically, SPI1 could enhance SMAD5 transcription and expression. Downregulation of SMAD5 also reversed SPI1 overexpression-induced autophagy and osteogenic differentiation in MC3T3-E1 cells. In MC3T3-E1 cells under DEX stimulation, icariin increased SMAD5 expression by upregulating SPI1. Furthermore, icariin could attenuate SPI1 depletion-imposed inhibition of autophagy and osteogenic differentiation of MC3T3-E1 cells. Conclusion: Our findings demonstrate that the SPI1/SMAD5 cascade, with the ability to enhance osteogenic differentiation, underlies the promoting effect of icariin on osteogenic differentiation of MC3T3-E1 cells. In dexamethasone-stimulated murine pre-osteoblast MC3T3-E1 cells, icariin upregulates SPI1 and thus enhances SMAD5 transcription, leading to enhanced autophagy and osteogenic differentiation of MC3T3-E1 cells. [ABSTRACT FROM AUTHOR]

Recently, interest in fish byproduct-derived bioactive components as potential ingredients for treating and preventing osteoporosis is increasing. In this study, we investigated the antiosteoporotic effects of mackerel Scomber japonicus extracts in MC3T3-E1 preosteoblasts in vitro and ovariectomized rats in vivo. Scomber japonicus neutrase-assisted protein hydrolysate (SJNH) treatment enhanced alkaline phosphatase (ALP) activity and osteoprotegerin (OPG) level, and attenuated receptor activator of nuclear factor κB ligand (RANKL) level in MC3T3-E1 cells. In an osteoporotic animal model, ovariectomized (OVX) rats were administered SJNH (200 mg/kg/day) for 8 weeks. SJNH prevented OVX-induced bone mineral density (BMD) loss and restored bone structural compartment loss based on micro-computed tomography (CT). In addition, SJNH administration normalized bone remodeling by regulating bone regeneration and resorption-related proteins such as ALP, osteocalcin (OC), and tartrate-resistant acid phosphatase 5b (TRACP-5b) as well as calcium contents in serum. Taken together, our findings suggest that SJNH can promote antiosteoporotic effects both in vitro and in vivo. Thus, SJNH might be a potent therapeutic agent or functional food ingredient for preventing osteoporosis. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the effect and the mechanism of recombinant human fibroblast growth factor 18 (rhFGF18) on postmenopausal osteoporosis. The effect of rhFGF18 on the proliferation and apoptosis of osteoblasts and the mechanism underlying such an effect was evaluated using an oxidative stress model of the MC3T3-E1 cell line. Furthermore, ovariectomy was performed on ICR mice to imitate estrogen-deficiency postmenopausal osteoporosis. Bone metabolism and bone morphological parameters in the ovariectomized (OVX) mice were evaluated. The results obtained from the cell model showed that FGF18 promoted MC3T3-E1 cell proliferation by activating the extracellular signal-regulated kinase (ERK) and p38 instead of c-Jun N-terminal kinase (JNK). FGF18 also prevented cells from damage inflicted by oxidative stress via inhibition of apoptosis. After FGF18 administration, the expression level of anti-apoptotic protein Bcl-2 in the mice was upregulated, whereas those of the pro-apoptotic proteins Bax and caspase-3 were downregulated. Administering FGF18 also improved bone metabolism and bone morphological parameters in OVX mice. FGF18 could effectively prevent bone loss in OVX mice by enhancing osteoblastogenesis and protecting osteoblasts from oxidative stress-induced apoptosis. [ABSTRACT FROM AUTHOR]

Natural macromolecules like alginate and gelatin are employed to create medication delivery systems that are both safe and effective. Zirconium nanoparticles (ZrO2 NPs) have been proposed as a means of enhancing the alginate-gelatin hydrogel's physical and biological properties. This study combines the synthesis of the biopolymers gelatin and alginate nanofibers with nanoparticles of zirconium oxide (GA/NF– ZrO2 NPs). UV, XRD, FTIR, and SEM were used to characterize the synthesized nanofibers. The expression of osteogenic genes was analyzed by western blotting and qualitative real-time polymerase chain reaction (qRT-PCR). Based on our findings, MC3T3-E1 cells are performed for cell viability, apoptosis and reactive oxygen species production by GA/NF– ZrO2 NPs through the Wnt/β-catenin signaling pathway. Cell migration was accelerated at 75 μg/mL concentration after 24 h of damage in a scratch wound healing assay. Proliferation of the MC3T3-E1 cell line was also detected. GA/NF–ZrO2 NPs influenced the osteogenic variation of MC3T3-E1 cells by inducing autophagy. Furthermore, the impact of obstruction on the temporomandibular joint (TMJ) is a subject of ongoing discussion and analysis within the context of animal models. Coordinated GA/NF–ZrO2 NPs on biomaterial nanofibers could be used to introduce physical signals for modifying MC3T3-E1 responds for orthodontic engineering constructs.

Incorporating nanoparticles into scaffolds with regenerative potential is a promissory strategy to provide them with antimicrobial activity. Bioceramics, such as β-tricalcium phosphate (β-TCP) and bioactive glasses (BGs), stand out among synthetic materials for bone regeneration. In this context, we report the incorporation of zinc oxide (ZnO) and copper oxide/copper nitrate (CuO/Cu2H3NO5) nanoparticles onto the surface of the β-TCP/BG scaffolds. This report addresses the physicochemical characterization of the scaffolds, their antimicrobial activity, and their response to MC3T3-E1 cells. Our findings show that the incorporation of both nanoparticles effectively inhibited S. aureus growth, including its biofilm formation. While the presence of the nanoparticles initially decreased MC3T3-E1 cell viability, cell proliferation improved with prolonged incubation. Overall, the β-TCP/BG_Zn and β-TCP/BG_Cu scaffolds showed an early antimicrobial response, aiding infection eradication, while also supporting cell proliferation over time.

Summary: This study aimed to recover protein hydrolysate from chicken bone by steam explosion and investigate its calcium‐binding capacity and potential pro‐osteogenic activity. The results showed that the recovery rate was significantly affected by pressure, time, and filling ratio. The molecular weight distribution and amino acid composition of the protein hydrolysate were also analysed. Furthermore, the protein hydrolysate extracted at 12 min, 1.8 MPa and 40% filling ratio showed well calcium‐binding capacity, and the peptides‐calcium chelate showed good stability at different temperatures. The cell experiment showed that the peptides‐calcium chelate can significantly improve the osteogenic activity of MC3T3‐E1 cells, including the proliferation, differentiation, and mineralisation. In conclusion, the steam explosion was an effective strategy to recover protein hydrolysate with well calcium‐binding capacity and potential pro‐osteogenic activity from chicken bone. This study provided new insights and theoretical bases for the application of steam explosion in extracting protein hydrolysate to improve bone health. [ABSTRACT FROM AUTHOR]

AbstractContext Morroniside (MOR) possesses antiosteoporosis (OP) effects, but its molecular target and relevant mechanisms remain unknown.Objective We investigated the effects of MOR on glucocorticoid-induced OP and osteoblastogenesis and its underlying mechanisms.Materials and methods The effects of MOR (10–100 μM) on the proliferation and differentiation of MC3T3-E1 cells were studied in vitro. The glucocorticoid-induced zebrafish OP model was treated with 10, 20 and 40 μM MOR for five days to evaluate its effects on vertebral bone density and related osteogenic markers. In addition, molecular targets prediction and molecular docking analysis were carried out to explore the binding interactions of MOR with the target proteins.Results In cultured MC3T3-E1 cells, 20 μM MOR significantly increased cell viability (1.64 ± 0.12 vs. 0.95 ± 0.16; p

This study explores the efficacy of a vancomycin-loaded silk fibroin/calcium phosphate/methylcellulose-based in situ thermosensitive hydrogel (VM-SF/CaP/MC) in promoting the osteogenic differentiation of preosteoblast cells. Three VM-SF/CaP/MC formulations with varying low (L) and high (H) concentrations of silk fibroin (SF) and calcium phosphate (CaP) were prepared: VM-HSF/LCaP/MC, VM-LSF/HCaP/MC, and VM-HSF/HCaP/MC. These hydrogels significantly enhanced MC3T3-E1 cell migration and proliferation in a dose- and time-dependent manner, achieving complete cell migration within 48 h. In addition, they significantly promoted alkaline phosphatase activity, collagen content, and mineralization in MC3T3-E1 cells, indicating their potential for osteogenesis. Among the hydrogel formulations, the VM-HSF/HCaP/MC hydrogel, with high SF and CaP content, demonstrated superior potential in promoting the osteogenic differentiation of MC3T3-E1 cells. It exhibited the highest ALP activity (11.13 ± 0.91 U/mg protein) over 14 days, along with increased collagen content (54.00 ± 1.71 µg/mg protein) and mineralization (15.79 ± 1.48 mM) over 35 days. Therefore, this formulation showed a promising candidate for clinical application in localized bone regeneration, particularly in treating osteomyelitis.

Objective: The therapeutic efficacy and molecular mechanisms of traditional Chinese medicines (TCMs), such as Liuwei Dihuang pills (LWDH pills), in treating osteoporosis (OP) remain an area of active research and interest in modern medicine. This study investigated the mechanistic underpinnings of LWDH pills in the treatment of OP based on network pharmacology, bioinformatics, and in vitro experiments. Methods: The active ingredients and targets of LWDH pills were retrieved through the TCMSP database. OP-related targets were identified using the CTD, GeneCards, and DisGeNET databases. The STRING platform was employed to construct a protein–protein interaction (PPI) network, and core targets for LWDH pills in treating OP were identified. The GO functional and KEGG pathway enrichment analyses for potential targets were performed using the R package "clusterProfiler". A "drug–target" network diagram was created using Cytoscape 3.7.1 software. The viability of MC3T3-E1 cells was evaluated using the CCK-8 method after treatment with various concentrations (1.25%, 2.5%, 5%, and 10%) of LWDH pill-medicated serum for 24, 48, and 72 h. Following a 48 h treatment of MC3T3-E1 cells with LWDH pill-medicated serum, the protein levels of collagen Ⅰ, RUNX2, Wnt3, and β-catenin were quantified using the Western blot analysis, and the activity of alkaline phosphatase (ALP) was measured. Results: A total of 197 putative targets for LWDH pills for OP treatment were pinpointed, from which 20 core targets were singled out, including TP53, JUN, TNF, CTNNB1 (β-catenin), and GSK3B. The putative targets were predominantly involved in signaling pathways such as the Wnt signaling pathway, the MAPK signaling pathway, and the PI3K-Akt signaling pathway. The intervention with LWDH pill-medicated serum for 24, 48, and 72 h did not result in any notable alterations in the cell viability of MC3T3-E1 cells relative to the control group (all p > 0.05). Significant upregulation in protein levels of collagen Ⅰ, RUNX2, Wnt3, and β-catenin in MC3T3-E1 cells was observed in response to the treatment with 2.5%, 5%, and 10% of LWDH pill-medicated serum in comparison to that with the 10% rabbit serum group (all p < 0.05). Furthermore, the intervention with LWDH pill-medicated serum resulted in the formation of red calcified nodules in MC3T3-E1 cells, as indicated by ARS staining. Conclusions: LWDH pills may upregulate the Wnt/β-catenin signaling pathway to elevate the expression of osteogenic differentiation proteins, including collagen Ⅰ and RUNX2, and to increase the ALP activity in MC3T3-E1 cells for the treatment of OP. [ABSTRACT FROM AUTHOR]

Morroniside (MOR) possesses antiosteoporosis (OP) effects, but its molecular target and relevant mechanisms remain unknown. We investigated the effects of MOR on glucocorticoid-induced OP and osteoblastogenesis and its underlying mechanisms. The effects of MOR (10–100 μM) on the proliferation and differentiation of MC3T3-E1 cells were studied in vitro. The glucocorticoid-induced zebrafish OP model was treated with 10, 20 and 40 μM MOR for five days to evaluate its effects on vertebral bone density and related osteogenic markers. In addition, molecular targets prediction and molecular docking analysis were carried out to explore the binding interactions of MOR with the target proteins. In cultured MC3T3-E1 cells, 20 μM MOR significantly increased cell viability (1.64 ± 0.12 vs. 0.95 ± 0.16; p < 0.01) and cell differentiation (1.57 ± 0.01 vs. 1.00 ± 0.04; p < 0.01) compared to the control group. MOR treatment significantly ameliorated vertebral bone loss in the glucocorticoid-induced OP zebrafish model (0.86 ± 0.02 vs. 0.40 ± 0.03; p < 0.01) and restored the expression of osteoblast-specific markers, including ALP, Runx2 and Col-І. Ligand-based target prediction and molecular docking revealed the binding interaction between MOR and the glucose pockets in sodium-glucose cotransporter 2 (SGLT2). These findings demonstrated that MOR treatment promoted osteoblastogenesis and ameliorated glucocorticoid-induced OP by targeting SGLT2, which may provide therapeutic potential in managing glucocorticoid-induced OP. [ABSTRACT FROM AUTHOR]

Background: Recently, the antioxidant properties of the natural compound, selenomethionine (Se-Met), have been recognized. However, its effect on the osteogenic mineralization of the Wnt/β-Catenin pathway under conditions of oxidative stress and inflammation remain unclear. Methods: This study utilized tert-butyl hydroperoxide (TBHP) to simulate oxidative stress and inflammation. Se-Met was then subsequently used to inhibit these effects in vitro. Results: TBHP induces oxidative stress and inflammatory responses by increasing the expression of reactive oxygen species and NLRP3, whereas decreasing the expression of GPX4, thereby inhibiting the viability of MC3T3-E1 cells. TBHP further promotes lipid peroxidation and damages the ultrastructure of mitochondria. Furthermore, TBHP inhibits the expression levels of β-Catenin, thereby reducing the activity of the Wnt pathway, which in turn suppresses the osteogenic differentiation and mineralization capacity. Importantly, Se-Met significantly alters the aforementioned responses to enhance expression levels of Wnt pathway-related proteins and improving the osteogenic differentiation and mineralization capacity of the cells. Conclusion: Se-Met enhances antioxidant and anti-inflammatory responses in MC3T3-E1 cells via the Wnt/β-Catenin signaling pathway to promote osteogenesis. Thus, Se-Met plays a crucial role in the field of bone homeostasis, and presents an opportunity for the future development of novel drugs for treating osteoporosis and maintaining bone stability. However, further detailed preclinical animal studies are required to generate solid and reliable data to aid this development.

Phenolic compounds are promising agents for the prevention of osteoporosis. 5-(3′,4′-dihydroxyphenyl)-γ-valerolactone (DHPV) is the major microbiota metabolite of the flavan-3-ols phenolic compound. Herein, we aimed to investigate the potential mechanisms underlying the effects of DHPV on an osteoblast cell model with H 2 O 2 -induced oxidative injury. The MC3T3-E1 cell cultured with H 2 O 2 was used as an oxidative injury model after pretreating with DHPV. Pretreatment with DHPV significantly attenuated cell viability decline, enhanced the activity of alkaline phosphatase and mineralization capacity in MC3T3-E1 cells. Reduced reactive oxygen species (ROS) and malondialdehyde (MDA) levels as well as increased in mitochondrial membrane potential and superoxide dismutase (SOD) activities indicated that DHPV affected both the oxidative and antioxidative processes in the cells. DHPV administration increased the LC3-II/I ratio and Beclin-1 protein levels, thereby promoting autophagy, which perhaps contributes to ROS elimination. However, the inhibition of Sirtuin 1 (SIRT1) by SIRT1 small interfering RNA reduced the protective effect of DHPV or SRT1720, as revealed by the increased ROS and MDA levels and decreased SOD, LC3-II/I ratio and Beclin-1 levels. DHPV may promote autophagy and reduce oxidative stress through the SIRT1-mediated pathway, thereby protecting MC3T3-E1 cells from H 2 O 2 -induced oxidative damage. [Display omitted] • DHPV improved autophagy and inhibited oxidative stress in H 2 O 2 -treated MC3T3-E1 cells. • DHPV promoted osteoblast differentiation of H 2 O 2 -induced MC3T3-E1 cells via stimulating SIRT1-mediated autophagy pathway. • DHPV hold promise as a novel protective agent for osteoporosis. [ABSTRACT FROM AUTHOR]

Sword bean (SB) contains various phytochemicals, such as flavonoids, tannins, saponins, and terpenoids. Although the evaluation of its potential functions, including antioxidant, anti-obesity, anti-inflammatory, liver protection, and antiangiogenic activities, has been widely reported, research on their use in osteoporosis prevention is insufficient. Furthermore, while various studies are conducted on SB, research on sword bean pods (SBP) is not yet active, and little is known about it. Therefore, this study investigated the effects of promoting osteoblast differentiation of MC3T3-E1 cells using SB and SBP extracts and their mechanisms. We show that SBP extracts increase osteoblast proliferation, mineralization-activated alkaline phosphatase (ALP), and collagen synthesis activities. Additionally, treatment with SBP extract increased the expression of markers related to osteoblast differentiation, such as ALP, SPARC, RUNX2, COL-I, BMP2, OCN, and OPN. It was confirmed that SBP induces differentiation by activating the BMP2/SMAD/RUNX2 pathway. We also show that SBP is more effective than SB, and SBP may be useful in assimilating bone minerals and preventing osteoporosis. [ABSTRACT FROM AUTHOR]

BACKGROUND: Calcium phosphates including β-tricalcium phosphate (β-TCP) and hydroxyapatite (HAp) have been widely used for bone regeneration application because of their high osteoconductive activities. In addition, various kinds of inorganic ions enhance differentiation, proliferation, and mineralization of osteoblasts. However, information about the effects of silver-doped β-TCP [β-TCP (Ag)] and HAp [HAp (Ag)] particles on osteogenic differentiation is not available yet. OBJECTIVE: We focused on the impact of β-TCP (Ag) and HAp (Ag) particles on the osteogenic differentiation of MC3T3-E1 osteoblast precursor cells. METHODS: MC3T3-E1 osteoblast precursor cells were pre-treated by β-TCP (Ag) or HAp (Ag). And then the medium was changed to differentiation medium. Subsequently, osteoblast differentiation-related markers were determined. RESULTS: We found that treatment with β-TCP (Ag) or HAp (Ag) particles increased alkaline phosphatase activity in MC3T3-E1 cells. Expression of osteoblast differentiation-related genes also increased after treatment with β-TCP (Ag) or HAp (Ag) particles, a response thought to be regulated by zinc finger-containing transcription factor osterix. The ratio of the receptor activator of nuclear factor kappa-B ligand (RANKL) to osteoprotegerin (OPG) was decreased by β-TCP (Ag) and HAp (Ag) particles. CONCLUSION: Silver doping of β-TCP and HAp particles is effective for bone regeneration. [ABSTRACT FROM AUTHOR]

Background: We investigated the toxicity and biocompatibility of a novel Mg-3Nd-1Gd-0.3Sr-0.2Zn-0.4Zr (abbreviated to Mg-Nd-Gd-Sr) alloy in the osteoblastic cell line MC3T3-E1 as osteoblasts play an important role in bone repair and remodeling. Methods: We used cytotoxicity tests and apoptosis to investigate the effects of the Mg-Nd-Gd-Sr alloy on osteoblastic cells. Cell bioactivity, cell adhesion, cell proliferation, mineralization, ALP activity, and expression of BMP-2 and OPG by osteoblastic cells were also used to investigate the biocompatibility of Mg-Nd-Gd-Sr alloy. Results: The results showed that the Mg-Nd-Gd-Sr alloy had no obvious cytotoxicity, and did not induce apoptosis to MC3T3-E1 cells. Compared with the control group, the number of adherent cells within 12 h was increased significantly in each experimental group (P < 0.05); the OD value of MC3T3-E1 cells was increased significantly in each experimental group on days 1 and 3 of culture (P < 0.05); the number of mineralized nodules formed in each experimental group was significantly increased (P < 0.05), and ALP activity was significantly increased in each experimental group (P < 0.05). RT-PCR results showed that the mRNA expression of BMP-2 and OPG was significantly higher in each experimental group compared with the control group (P < 0.05). Western blotting showed that the Mg-Nd-Gd-Sr alloy extract significantly increased the protein expression of BMP-2 and OPG compared with the control group (P < 0.05). Conclusions: Our data indicated that the novel Mg-Nd-Gd-Sr-Zn-Zr alloy had no obvious cytotoxic effects, and did not cause apoptosis to MC3T3-E1 cells; meanwhile it promoted cell adhesion, cell proliferation, mineralization, and ALP activity of osteoblasts. During this process, there was an increase in the expressions of BMP-2 and OPG mRNAs and proteins. [ABSTRACT FROM AUTHOR]

Purpose: To prepare a calcium supplement for efficient absorption and utilization in the human body and to explore its effect on the osteogenic activity of MC3T3-E1 cells. Methods: Bovine bone peptide was chelated with calcium, and the optimal reaction conditions were determined by response surface methodology (RSM). The structural characteristics and stability of peptide-calcium chelate were studied by infrared spectroscopy, X-ray diffraction and atomic absorption spectrometry. The effects of bovine bone peptide-calcium chelate on the proliferation, differentiation and mineralization of MC3T3-E1 cells were investigated. Results: The optimum conditions for the preparation of bovine bone peptide calcium chelate were peptide/calcium mass ratio 2.97, temperature 62.54 ℃ and pH 9.06. Under these conditions, the calcium chelating capacity was 55.65 μg/mg. The results of spectral analysis showed that amino nitrogen, hydroxyl oxygen and carboxyl oxygen were the major calcium-chelating sites. Bovine bone peptide could form coordinate bonds with calcium, and the molecular mass of the peptide increased after chelation with calcium. In addition, the stability of bovine bone peptide-calcium chelate was little affected by temperature, but sensitive to pH. The peptide-calcium chelate could significantly promote the proliferation, differentiation and mineralization of MC3T3-E1 cells. Conclusion: Bovine bone peptide-calcium chelate has the potential to promote the osteogenic activity of MC3T3-E1 cells.

AIM: To investigate the effect of microRNA-210-3p（ miR-210-3p） on osteogenic differentiation of mouse osteoblastic cell line MC3T3-E1, and to explore its mechanism related to autophagy. METHODS: （ 1） Lipofection was used to construct MC3T3-E1 cell model with overexpression or silencing of miR-210-3p, and negative control （NC） mimic group, miR-210-3p mimic group, NC inhibitor group and miR-210-3p inhibitor group were set up, Western blot, RT-qPCR and immunofluorescence were used to detect the changes of osteogenic differentiation- and autophagy-related factors in MC3T3-E1 cells, and alizarin red staining was used to observe the mineralized nodules. （2） Rapamycin （RAPA） or 3-methyladenine （3-MA） was used to construct autophagy activation or inhibition model, and control group, RAPA group and 3-MA group were set up. Western blot, RT-qPCR and immunofluorescence were used to detect the changes of osteogenic differentiation factors in MC3T3-E1 cells after autophagy activation or inhibition, and alizarin red staining was used to observe the mineralized nodules. （3） The cells were divided into NC mimic group, NC mimic+RAPA group and miR-210-3p mimic+RAPA group, and Western blot and RT-qPCR were used to detect the changes of osteogenic differentiation factors in each group. RESULTS: （1） Compared with NC mimic group, the mRNA and protein expression levels of osteogenic differentiation factors were increased in miR-210-3p mimic group （P<0. 05）, the immunofluorescence intensity of Runx2 was increased in the cells, the number of mineralized nodules was increased, and the level of autophagy was decreased （P<0. 05）. Compared with NC inhibitor group, the mRNA and protein expression levels of osteogenic differentiation factors were decreased in miR-210-3p inhibitor group（ P<0. 05）, the immunofluorescent intensity of Runx2 was decreased in the cells, the number of mineralized nodules was reduced, and the level of autophagy was increased in the miR-210-3p inhibitor group （P<0. 05）. （2） Compared with control group, the mRNA and protein expression levels of osteogenic differentiation factors were increased in 3-MA group（ P<0. 05）, and the immunofluorescence intensity of Runx2 was increased in the cells. By contrast, the mRNA and protein expression levels of osteogenic differentiation factors were decreased in RAPA group（ P<0. 05）, and the immunofluorescence intensity of Runx2 was decreased in the cells.（ 3） Overexpression of miR-210-3p reversed the inhibitory effect of RAPA on osteogenic differentiation of MC3T3-E1 cells （P< 0. 01）. CONCLUSION: miR-210-3p promotes osteogenic differentiation of MC3T3-E1 cells by inhibiting autophagy. [ABSTRACT FROM AUTHOR]

Abstract Background Deer antler is a traditional Chinese medicine with the function of tonifying kidney and strengthening bone, which is often used to treat orthopedic diseases. Methods Eight-week-old C57BL/6 mice were used as the fixation model of open tibial fracture with intramedullary nail. The mice were treated with deer antler extract (DAE) or PBS by oral gavage once daily. The tibial fracture samples were collected and performed to the tissue analysis, including X-ray, micro-CT, histology, qRT-PCR, immunohistochemistry. MC3T3-E1 cells were used to detect the effect of deer antler extract on ability of cell proliferation and migration by CCK-8 assay and cell scratch test. Results Imaging and micro-CT showed that DAE could promote the healing of tibial fracture in mice, and histological analysis showed that DAE could promote the transformation of cartilage callus to bone callus in fracture area. The results of qRT-PCR and immunohistochemistry showed that DAE could promote intrachondral ossification in fracture zone and the mechanism of promoting fracture healing may be related to the activation of BMP-2/SMAD4 signaling pathway. In the cytological experiment of DAE, it can be found that DAE promoted the proliferation of MC3T3-E1 cells and the migration of MC3T3-E1 cells at a certain concentration, which is also related to the promotion of fracture healing by DAE. Conclusion DAE can promote fracture healing by activating BMP-2/SMAD4 signaling pathway. DAE has the potential to be used in clinic as an important means of promoting fracture healing.

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Objective: The therapeutic efficacy and molecular mechanisms of traditional Chinese medicines (TCMs), such as Liuwei Dihuang pills (LWDH pills), in treating osteoporosis (OP) remain an area of active research and interest in modern medicine. This study investigated the mechanistic underpinnings of LWDH pills in the treatment of OP based on network pharmacology, bioinformatics, and in vitro experiments. Methods: The active ingredients and targets of LWDH pills were retrieved through the TCMSP database. OP-related targets were identified using the CTD, GeneCards, and DisGeNET databases. The STRING platform was employed to construct a protein–protein interaction (PPI) network, and core targets for LWDH pills in treating OP were identified. The GO functional and KEGG pathway enrichment analyses for potential targets were performed using the R package “clusterProfiler”. A “drug–target” network diagram was created using Cytoscape 3.7.1 software. The viability of MC3T3-E1 cells was evaluated using the CCK-8 method after treatment with various concentrations (1.25%, 2.5%, 5%, and 10%) of LWDH pill-medicated serum for 24, 48, and 72 h. Following a 48 h treatment of MC3T3-E1 cells with LWDH pill-medicated serum, the protein levels of collagen Ⅰ, RUNX2, Wnt3, and β-catenin were quantified using the Western blot analysis, and the activity of alkaline phosphatase (ALP) was measured. Results: A total of 197 putative targets for LWDH pills for OP treatment were pinpointed, from which 20 core targets were singled out, including TP53, JUN, TNF, CTNNB1 (β-catenin), and GSK3B. The putative targets were predominantly involved in signaling pathways such as the Wnt signaling pathway, the MAPK signaling pathway, and the PI3K-Akt signaling pathway. The intervention with LWDH pill-medicated serum for 24, 48, and 72 h did not result in any notable alterations in the cell viability of MC3T3-E1 cells relative to the control group (all p > 0.05). Significant upregulation in protein levels of collagen Ⅰ, RUNX2, Wnt3, and β-catenin in MC3T3-E1 cells was observed in response to the treatment with 2.5%, 5%, and 10% of LWDH pill-medicated serum in comparison to that with the 10% rabbit serum group (all p < 0.05). Furthermore, the intervention with LWDH pill-medicated serum resulted in the formation of red calcified nodules in MC3T3-E1 cells, as indicated by ARS staining. Conclusions: LWDH pills may upregulate the Wnt/β-catenin signaling pathway to elevate the expression of osteogenic differentiation proteins, including collagen Ⅰ and RUNX2, and to increase the ALP activity in MC3T3-E1 cells for the treatment of OP.

Progressive aging harms bone tissue structure and function and, thus, requires effective therapies focusing on permanent tissue regeneration rather than partial cure, beginning with regenerative medicine. Due to advances in tissue engineering, stimulating osteogenesis with biomimetic nanoparticles to create a regenerative niche has gained attention for its efficacy and cost-effectiveness. In particular, hydroxyapatite (HAP, Ca10(PO4)6(OH)2) has gained significant interest in orthopedic applications as a major inorganic mineral of native bone. Recently, magnetic nanoparticles (MNPs) have also been noted for their multifunctional potential for hyperthermia, MRI contrast agents, drug delivery, and mechanosensitive receptor manipulation to induce cell differentiation, etc. Thus, the present study synthesizes HAP-decorated MNPs (MHAP NPs) via the wet chemical co-precipitation method. Synthesized MHAP NPs were evaluated against the preosteoblast MC3T3-E1 cells towards concentration-dependent cytotoxicity, proliferation, morphology staining, ROS generation, and osteogenic differentiation. The result evidenced that MHAP NPs concentration up to 10 µg/mL was non-toxic even with the time-dependent proliferation studies. As nanoparticle concentration increased, FACS apoptosis assay and ROS data showed a significant rise in apoptosis and ROS generation. The MC3T3-E1 cells cocultured with 5 µg/mL MHAP NPs showed significant osteogenic differentiation potential. Thus, MHAP NPs synthesized with simple wet chemistry could be employed in bone regenerative therapy. [ABSTRACT FROM AUTHOR]

Osteoplastic precursors are critical for fracture repair and bone homeostasis maintenance. Cerium oxide nanoparticles (CeO2 NPs) can promote the osteogenic differentiation of mesenchymal stem cells and secrete vascular endothelial growth factors. However, little is known about its role in precursor osteoblasts; therefore, we further investigated the effect and mechanism of CeO2 NPs in precursor osteoblasts. Cell counting kit-8 analysis was utilized to detect the toxicity of CeO2 NPs on MC3T3-E1 mouse osteogenic precursor cells. Then, alizarin red S staining was employed to assess the degree of extracellular matrix mineralization, and quantitative real-time polymerase chain reaction analysis was performed to measure the levels of osteogenesis-related genes. To identify differentially expressed genes, mRNA-sequencing was performed. Subsequently, GO and KEGG analyses were deployed to identify the major downstream pathways, whereas Western blot was used for verification. CeO2 NPs significantly enhanced the ability of MC3T3-E1 precursor osteoblasts to enhance matrix mineralization and increased the expression of osteogenic genes such as runt-related transcription factor 2, collagen Iα1, and osteocalcin. Pathway analysis revealed that CeO2 NPs enhanced the nuclear translocation of β-catenin and activated the Wnt pathway by promoting family with sequence similarity 53 member B/simplet expression, while Western blot analysis indicated the same results. After using a Wnt pathway inhibitor (KYA1797K), the simulative effect of CeO2 NPs was abolished. This study revealed that CeO2 NPs promoted MC3T3-E1 precursor osteoblast differentiation by activating the Wnt pathway. [ABSTRACT FROM AUTHOR]

Zinc (Zn) has been reported to mediate leptin secretion, and thus leptin can be an important candidate molecule linking Zn with bone formation. The present study investigated whether zinc deficiency induces leptin secretion by activating a JAK2/STAT3 signaling pathway and leads to osteoblastic apoptosis. MC3T3-E1 cells were incubated for 24 h in normal osteogenic differentiation medium (OSM) or OSM treated with either 1 μM (Low Zn) or 15 μM (High Zn) of ZnCl2 containing 5 μM TPEN (Zn chelator). Our results demonstrated that low Zn stimulated extracellular leptin secretion and increased mRNA and protein expression of leptin in osteoblastic MC3T3-E1 cells. The OB-Rb (long isoform of leptin receptor) expressions were also elevated in osteoblasts under depletion of Zn. Leptin-signaling proteins, JAK2 and p-JAK2 in the cytosol of low Zn osteoblast conveyed leptin signaling, which ultimately induced higher p-STAT3 expression in the nucleus. Apoptotic effects of JAK2/STAT3 pathway were shown by increased caspase-3 in low Zn osteoblasts as well as apoptotic morphological features observed by TEM. Together, these data suggest that low Zn modulates leptin secretion by activating JAK2/STAT3 signaling pathway and induces apoptosis of osteoblastic MC3T3-E1 cells. [ABSTRACT FROM AUTHOR]

Objective To investigate the effects and its mechanism of isoflavones extracted from chickpea sprouts (ICS) on the proliferation and the differentiation of the osteogenic precursor MC3T3-E1 cells. Methods CCK-8 was used to detect the proliferation of MC3T3-E1 cells at different concentrations of ICS (0, 0. 1, 0. 5, 1, 5, 10, 50, 100 mg/L) at 24, 48, 72 h. Different concentrations of ICS (0, 0.5, 1195 mg/L) were added into the osteogenic induction medium to observe its effects on the osteogenic differentiation: Alkaline phosphatase staining (ALP) assay was performed to observe the enzyme activities after 7-day induction; Alizarin red staining was performed on the 21st day to observe the calcium deposition; RT-qPCR was used to detect the mRNA expression levels of runt-related transcription factor 2 (RUNX2), osteocalcin (OCN) and type 1 collagen (COL1) in each group on the 7th day of induction; Western Blot was used to detect the expression levels of RUNX2 and COL1 on the 7th day of induction, and estrogen receptor (ER) inhibitor ICI 182, 780 (1 μmol/L) was also added to detect the effect on the protein expression of the osteogenic differentiation-related proteins RUNX2 and COL1. Results ICS promoted cell proliferation in a certain concentration range (1-10 mg/L), and had some toxicity to cells when the concentration was greater than 10 mg/L (P< 0. 05). Compared with the control group (0 mg/L), ICS at concentrations of 0. 5,1,5 mg/L could promote alkaline phosphatase activity, with significant differences in a dose-dependent manner; ICS could promote calcium deposition, with a significant increase in the number of calcium nodules with increasing dose, with statistical differences; ICS could up-regulate the mRNA transcription levels of osteogenic differentiation-related genes RUNX2, OCN and COLT (P<0. 05), and promote the expression of osteogenic differentiation-related proteins COL1 and RUNX2 (P<0. 05). The estrogen receptor inhibitor ICI 182 780 inhibited the promoting effect of ICS on osteogenic differentiation-related proteins COL1 and RUNX2 (P < 0. 05). Conclusion ICS can promote the proliferation of MC3T3-E1 osteogenic precursor cells in mice, and improve the osteogenic differentiation of MC3T3-E1 cells by up-regulating the expression levels of osteogenic differentiation-related genes and proteins. The use of estrogen receptor inhibitor ICI 182,780 can inhibit the osteogenic differentiation of ICS. It is speculated that ICS promotes osteogenic differentiation through ER-related pathway. [ABSTRACT FROM AUTHOR]

This study aimed to screen for a novel osteogenic peptide based on the calcium-sensing receptor (CaSR) and explore its molecular mechanism and gastrointestinal stability. In this study, a novel osteogenic peptide (Phe-Ser-Gly-Leu, FSGL) derived from bovine bone collagen hydrolysate was successfully screened by molecular docking and synthesised by solid phase peptide synthesis for further analysis. Cell experiments showed that FSGL significantly enhanced the osteogenic activity of MC3T3-E1 cells by acting on CaSR, including proliferation (152.53%), differentiation, and mineralization. Molecular docking and molecular dynamics further demonstrated that FSGL was a potential allosteric activator of CaSR, that turned on the activation switch of CaSR by closing the Venus flytrap (VFT) domain and driving the two protein chains in the VFT domain to easily form dimers. In addition, 96.03% of the novel osteogenic peptide FSGL was stable during gastrointestinal digestion. Therefore, FSGL showed substantial potential for enhancing the osteogenic activity of osteoblasts. This study provided new insights for the application of CaSR in the targeted screening of osteogenic peptides to improve bone health. [Display omitted] • The novel osteogenic peptide FSGL was successfully screened based on CaSR. • The FSGL significantly improved the osteogenic activity of MC3T3-E1 cells. • The molecular mechanism of CaSR activation by FSGL was explored. • The stability of FSGL during gastrointestinal digestion was investigated. [ABSTRACT FROM AUTHOR]

Abstract Background The incidence of osteoporosis and osteoporotic fractures is increasing every year. Traditional Chinese Medicine (TCM) can shed new light on the treatment of osteoporosis. This study aimed to explore the role and mechanism of paeoniflorin in promoting osteogenic differentiation of an osteoblast precursor cell line (MC3T3-E1). Methods MC3T3-E1 cells were cultured in osteogenic induction medium (OIM) and OIM combined with different concentrations of paeoniflorin. The optimal dose of paeoniflorin was assessed by a cell counting kit-8 (CCK-8) assay. Then, alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining were performed to assess the osteogenic capacity of paeoniflorin. The transcription of osteogenic genes and the expression of osteogenic proteins were assessed by RT-PCR and Western blotting, respectively. The transcription of Wnt/β-catenin signaling pathway genes and proteins was assessed by RT-PCR and Western blotting, respectively. Finally, Dickkopf-1 (DKK-1), a Wnt/β-catenin signaling pathway inhibitor, was used to identify whether the Wnt/β-catenin signaling pathway was involved in the osteogenic differentiation of paeoniflorin. Osteoclastogenesis in RAW264.7 cells was identified by tartrate-resistant acid phosphatase (TRAP) staining. Results At concentrations ranging from 0.1 to 100 μM, paeoniflorin was not cytotoxic to MC3T3-E1 cells. Paeoniflorin significantly increased the osteogenic differentiation of MC3T3-E1 cells in a dose-dependent manner. Moreover, paeoniflorin significantly increased osteogenic differentiation gene and protein expression. Through bioinformatic analysis, paeoniflorin-affected genes were found to be involved in different signaling pathways, such as the Wnt/β-catenin signaling pathway. Paeoniflorin enhanced β-catenin and CyclinD1 expression compared with that of the control groups. DKK-1 partially reversed the promoting effects of paeoniflorin in promoting osteogenic differentiation of MC3T3-E1 cells. Moreover, paeoniflorin inhibited the osteoclastogenesis of RAW264.7 cells. Conclusion Paeoniflorin promotes osteogenic differentiation in MC3T3-E1 cells by regulating the Wnt/β-catenin pathway. Paeoniflorin is a potential therapeutic agent for the treatment of osteoporosis.

This study aimed to produce and characterize of a powder mixture from tilapia (Oreochromis niloticus) bone resulting from the addition to hydroxyapatite (HA) of various acids (lactic acid, citric acid, and malic acid). Structured hydroxyapatite lactate compound (SHA-Lac) and SHA-citrate malate compound (SHA-CiMa) were water-soluble and Ca and P were the most abundant minerals contained in those compounds had main elements in Ca and P. FT-IR indicated that COOH played an important role for Ca2+ binding. XRD patterns indicated these compounds were amorphous crystal. For bioavailability on preosteoblast MC3T3-E1 cells, all samples at 100 μg/mL showed no cytotoxicity, no change in morphology, and no cell apoptosis, with the percentage of cell viability higher than 80%. For ability of mineral deposition at 7, 14, and 21 days of culture, all samples significantly increased the mineralization of MC3T3-E1osteoblast cells, thereby resulting in rising of the proliferation and differentiation during the early stages.

Bone metastatic cancer-secreted extracellular factors are capable of modifying the bone microenvironment through interacting with bone cells, including osteoblasts. Reticulum ribosome-binding protein 1 (RRBP1) is substantially expressed in certain bone metastatic cancer cells. This study was undertaken to determine whether RRBP1 from bone metastatic cancer cells affects the osteoblastic phenotype expression. Breast and prostate cancer cells, MDA-MB-231 and PC3, were cultured, respectively, followed by collecting conditioned mediums (CMs) and identifying the abundance of RRBP1 in CMs using LC-MS/MS. MC3T3-E1 cells were cultured with a mixed medium (including CMs from shRRBP1-transduced two-type cancer cells) with or without endoplasmic reticulum (ER) stress inhibitor 4-PBA, followed by measuring the levels of osteoblastic phenotype expression and biomarkers of ER stress using western blotting, qPCR, and ARS staining, respectively. Similar experiments were performed in shRrbp1-transduced MC3T3-E1 cells cultured with a mixed medium (including CMs from the two-type cancer cells). Bone formation parameters were measured in the tibia of nude mice injected with shRRBP1-transduced two-type cancer cells using micro-CT analysis. These results showed that RRBP1 is the sole shared high-abundance protein in CMs from the two-type cancer cells, involving osteoblast differentiation. CMs from shRRBP1-transduced two-type cells boosted the osteoblastic phenotype expression partially through increasing ER stress. CMs from the two-type cancer cells partially offset the similar alterations induced by shRrbp1 in MC3T3-E1 cells. Injection with shRRBP1-transduced two-type cells ameliorated the bone lesions in nude mice. Therefore, RRBP1 depletion of bone metastatic cancer enhanced the osteoblastic phenotype expression, suggesting a role of RRBP1 in the bone microenvironment.

Background: Deer antler is a traditional Chinese medicine with the function of tonifying kidney and strengthening bone, which is often used to treat orthopedic diseases. Methods: Eight-week-old C57BL/6 mice were used as the fixation model of open tibial fracture with intramedullary nail. The mice were treated with deer antler extract (DAE) or PBS by oral gavage once daily. The tibial fracture samples were collected and performed to the tissue analysis, including X-ray, micro-CT, histology, qRT-PCR, immunohistochemistry. MC3T3-E1 cells were used to detect the effect of deer antler extract on ability of cell proliferation and migration by CCK-8 assay and cell scratch test. Results: Imaging and micro-CT showed that DAE could promote the healing of tibial fracture in mice, and histological analysis showed that DAE could promote the transformation of cartilage callus to bone callus in fracture area. The results of qRT-PCR and immunohistochemistry showed that DAE could promote intrachondral ossification in fracture zone and the mechanism of promoting fracture healing may be related to the activation of BMP-2/SMAD4 signaling pathway. In the cytological experiment of DAE, it can be found that DAE promoted the proliferation of MC3T3-E1 cells and the migration of MC3T3-E1 cells at a certain concentration, which is also related to the promotion of fracture healing by DAE. Conclusion: DAE can promote fracture healing by activating BMP-2/SMAD4 signaling pathway. DAE has the potential to be used in clinic as an important means of promoting fracture healing. [ABSTRACT FROM AUTHOR]

Periodontitis is an excessive inflammatory event in tooth-supporting tissues and can cause tooth loss.We used erythropoietin (EPO), which has been reported to play an important role in bone healing and modulation of angiogenesis, as a therapeutic agent in vivo and in vitro experimental models to analyze its effect on periodontitis. First, EPO was applied to in vitro MC3T3-E1 cells and human periodontal ligament fibroblast (hPDLF) cells to examine its function in altered cellular events and gene expression patterns. In vitro cultivation of MC3T3-E1 and hPDLF cells with 10 IU/ml EPO at 24 and 48 h showed an obvious increase in cell proliferation. Interestingly, EPO treatment altered the expression of osteogenesis-related molecules, including alkaline phosphatase (ALP), bone morphogenetic protein-2 (BMP-2), and osteocalcin (OC) in MC3T3-E1 cells but not in hPDLF cells. In particular, MC3T3-E1 cells showed increased expression of ALP, BMP-2, and OC on day 5, while hPDLF cells showed increased expression of BMP-2, and OC on day 14. Based on the in vitro examination, we evaluated the effect of EPO on bone formation using an experimentally- induced animal periodontitis model. After the induction of periodontitis in the maxillary left second M, 10 IU/ml of EPO was locally applied to the extraction tooth sockets. Histomorphological examination using Masson's trichrome (MTC) staining showed facilitated bone formation in the EPO-treated groups after 14 days. Similarly, stronger positive reactions against vascular endothelial growth factor (VEGF), cluster of differentiation 31 (CD31), runt-related transcription factor 2 (RUNX2), and osteocalcin (OC) were detected in the EPO-treated group compared to the control. Meanwhile, myeloperoxidase, an inflammatorymarker, was decreased in the EPO-treated group on days 1 and 5. Overall, EPOfacilitates bone healing and regeneration through altered signaling regulation and modulation of inflammation in the osteoblast cell lineage and to a lesser extent in hPDLF cells. [ABSTRACT FROM AUTHOR]

Osteoporosis is a serious threat to human life. Guben Zenggu Granule is an empirical prescription for clinical treatment of osteoporosis. MC3T3-E1 cells are mouse osteogenic precursor cells with osteogenic differentiation, and are classic cells for studying bone metabolism and osteogenic mechanism, as well as mechanical stimulation sensitive cells. Therefore, it can be inferred that Guben Zenggu granule can repair MC3T3-E1 cells under continuous static pressure overload. This study aims to through the network of pharmacology and gene sequencing method, reveal thrift increase bone particles under the condition of continuous static pressure overload on osteogenesis mechanism of MC3T3-E1 cells. In the process of analysis, from a variety of 98 compounds was predicted in the database, a collection of 474 goals, a total of 29,164 difference between two groups of genes. Then, construction of composite targets between cells and predict targets and protein - protein interaction networks, and through the cluster analysis to further explore the relationship between the target. In addition, linkages between target proteins and cells were further identified using Gene Ontology (GO) and Pathways (KEGG Pathway). Finally, the repair effect of Guben Zenggu granule on MC3T3-E1 cells under continuous static pressure overload was verified through experiments, so as to accurately explain the pharmacodynamic mechanism of Traditional Chinese medicine. [ABSTRACT FROM AUTHOR]

Objective To investigate the effect of microRNA (miR) - 196a on osteogenic differentiation of MC3T3-El cells by target regulation of his tone deacetylase 9 (HDAC9). Methods MC3T3-El cells were divided into control group (Cont) group, induction group, miR-196a-mimics-NC group, miR-196a-mimics group, miR-196a-inhibitor-NC group, miR-196a-inhibitor group, miR-196a-mimics+pCMV-HDAC9-NC group, and miR-196a-mimics+pCMV-HDAC9 group. Osteogenic induction was performed after transfection according to the group. Quantitative fluorescent PCR was performed to measure the expression of miR-196a and HDAC9 in MC3T3-El cells. Alkaline phosphatase (ALP) activity was measured with a commercial kit. Alizarin red staining was performed to observe the degree of mineralization. Western blotting was performed to measure the expressions of HDAC9, ALP, Runt-related transcription factor 2 (RUNX2), collagen I (COLl), osteopontin (OPN), Histone H3, and Histone H3 (acetyl K9, K 14 and K23). Results Compared to those in the Cont group, the expression of miR-196a, the expressions of ALP, Runx2, COLl, and OPN proteins, ALP activity, mineralization degree, and the acetylation levels of Histone H3 K9, K14, and K23 sites in MC3T3-E I cells in the induction group increased (P < 0. 05), but the expression of HDAC9 mRNA and protein decreased (P < 0. 05). Transfection of miR-196a-mimics was able to significantly increase miR-196a expression, to decrease HDAC9 expression, and to increase ALP, Runx2, COL 1, and OPN protein expression, ALP activity, mineralization degree, and Histone H3 acetylation. Transfection of miR-196a-inhibitor had the opposite effect. MiR-196a was able to target down-regulation of HDAC9 expression. Overexpression of HDAC9 was able to partially reverse the promoting effect of miR-196a mimics on osteogenic differentiation of MC3T3-E I cells. Conclusion MiR-196a target down-regulates HDAC9 expression, increases hi stone acetylation level, and promotes osteogenic differentiation of MC3T3-El cells. [ABSTRACT FROM AUTHOR]

The aim of this study was to determine the osteogenic activity and mechanism of soybean peptide VVELLKAFEEKF (SOP) and the potential relationship between SOP and transforming growth factor-β1 (TGF-β1). The results show that SOP promotes MC3T3-E1 cell proliferation by altering cell progression. SOP induced cell differentiation and mineralization in a dose-dependent manner at 0.7-7 μM. Moreover, SOP stimulates osteoblast differentiation, which may be achieved through the activation of p38-MAPK and Smad2/3 signaling pathways. Furthermore, treatment with a TβRI inhibitor (SB525334) inhibited the phosphorylation levels of p38 and Smad2/3, which indicates the involvement of TβRI in the process of osteoblast differentiation caused by SOP. Besides, in non-FBS-cultured MC3T3-E1 cells, SOP and TGF-β1 promoted the phosphorylation of Smad2/3 and alkaline phosphatase (ALP) activity, but the effect was lost when SOP was incubated separately, indicating that SOP stimulated osteoblast differentiation by promoting TGF-β1 activity. In vivo , SOP significantly restores bone mineral density loss and behavioral deficits in a model of glucocorticoid-induced osteoporosis (GIOP) in zebrafish. These results suggest that SOP may have the function of promoting bone remodeling and may be used as a potential active factor for functional food development to prevent osteoporosis.

Recent studies have confirmed that melatonin and N6-methyladenosine (m6A) modification can influence bone cell differentiation and bone formation. Melatonin can also regulate a variety of biological processes through m6A modification. Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1) serves as a reader of m6A modification. In this study, we used the hindlimb unloading model as an animal model of bone loss induced by simulated microgravity and used 2D clinorotation to simulate a microgravity environment for cells on the ground. We found that hnRNPA2B1 was downregulated both in vitro and in vivo during simulated microgravity. Further investigations showed that hnRNPA2B1 could promote osteoblast differentiation and that overexpression of hnRNPA2B1 attenuated the suppression of osteoblast differentiation induced by simulated microgravity. We also discovered that melatonin could promote the expression of hnRNPA2B1 under simulated microgravity. Moreover, we found that promotion of osteoblast differentiation by melatonin was partially dependent on hnRNPA2B1. Therefore, this research revealed, for the first time, the role of the melatonin/hnRNPA2B1 axis in osteoblast differentiation under simulated microgravity. Targeting this axis may be a potential protective strategy against microgravity-induced bone loss and osteoporosis.

Azithromycin displays immunomodulatory and anti-inflammatory effects in addition to broad-spectrum antimicrobial activity and is used to treat inflammatory diseases, including respiratory and odontogenic infections. Few studies have reported the effect of azithromycin therapy on bone remodeling processes. The aim of this study was to examine the effects of azithromycin on the osteogenic function of osteoblasts using osteoblast-like MC3T3-E1 cells. Cells were cultured in the presence of 0, 0.1, 1, and 10 µg/mL azithromycin, and cell proliferation and alkaline phosphatase (ALPase) activity were determined. In vitro mineralized nodule formation was detected with alizarin red staining. The expression of collagenous and non-collagenous bone matrix protein was determined using real-time PCR or enzyme-linked immunosorbent assays. In cells cultured with 10 µg/mL azithromycin, the ALPase activity and mineralized nodule formation decreased, while the type I collagen, bone sialoprotein, osteocalcin, and osteopontin mRNA expression as well as osteopontin and phosphorylated osteopontin levels increased. These results suggest that a high azithromycin concentration (10 µg/mL) suppresses mineralized nodule formation by decreasing ALPase activity and increasing osteopontin production, whereas low concentrations (≤l.0 µg/mL) have no effect on osteogenic function in osteoblastic MC3T3-E1 cells.

Abstract Background Osteoporosis is a widespread chronic disease characterized by low bone density. There is currently no gold standard treatment for osteoporosis. The aim of this study was to explore the role and mechanism of Astragaloside on osteogenic differentiation of MC3T3-E1 cells. Methods MC3T3-E1 cells were divided into control and different dose of Astragaloside (10, 20, 40, 50, and 60 μg/ml). Then, ALP and ARS staining were performed to identify the effects of Astragaloside for early and late osteogenic capacity of MC3T3-E1 cells, respectively. Real-time PCR and western blot were performed to assess the ALP, OCN, and OSX expression. PI3K/Akt signaling pathway molecules were then assessed by Western blot. Finally, PI3K inhibitor, LY294002, was implemented to assess the mechanism of Astragaloside in promoting osteogenic differentiation of MC3T3-E1 cells. Results Astragaloside significantly increased the cell viability than the control group. Moreover, Astragaloside enhanced the ALP activity and calcium deposition than the control groups. Compared with the control group, Astragaloside increased the ALP, OCN, and OSX expression in a dose-response manner. Western blot assay further confirmed the real-time PCR results. Astragaloside could significantly increase the p-PI3K and p-Akt expression than the control group. LY294002 partially reversed the promotion effects of Astragaloside on osteogenic differentiation of MC3T3-E1 cells. LY294002 partially reversed the promotion effects of Astragaloside on ALP, OCN, and OSX of MC3T3-E1 cells. Conclusion The present study suggested that Astragaloside promoted osteogenic differentiation of MC3T3-E1 cells through regulating PI3K/Akt signaling pathway.

Cyanidin-3-O-glucoside (C3G) is the most widely distributed anthocyanin and it can reportedly reduce the risk of osteoporosis, but the molecular mechanism by which C3G promotes bone formation is poorly understood. In the current study, RNA sequencing (RNA-seq) was used to investigate the mechanism of action of C3G in osteogenesis. MC3T3-E1 mouse osteoblasts were divided into a C3G (100 μmol/L)-treated group and a vehicle-treated control group, and differentially expressed genes (DEGs) in groups were evaluated via RNA-seq analysis. The functions of the DEGs were evaluated by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, and the genes were validated by quantitative real-time PCR. The RNA-seq analysis identified 34 genes that were upregulated in C3G-treated cells compared to vehicle-treated cells, and 17 that were downregulated GO and KEGG pathway analyses indicated that these genes were highly enriched in functions related to lysosomes and glycolipid biosynthesis, among others. The differential expression of ATPase H+-transporting V0 subunit C (Atp6v0c), chemokine (C-X3-C motif) ligand 1 (Cx3cl1), and lymphocyte antigen 6 complex, locus A (Ly6a) genes was validated by quantitative real-time-PCR. Because these genes have been previously implicated in osteoporosis, they are potential target genes of C3G action in MC3T3-E1 cells. These results provide molecular level evidence for the therapeutic potential of C3G in the treatment of osteoporosis and other disorders of bone metabolism.

Osteoporosis is an age-related disorder with enormous public health burden with an urgent need for better diagnosis and treatment. Herein, we have investigated the potential role of jatrorrhizine, a plant bioactive, in osteoporosis since it is known to exhibit a wide array of biologic and pharmacologic activities. Using MC3T3-E1 cells, a clonal murine cell line of immature osteoblasts derived from mice, we examined the effect of jatrorrhizine on various parameters of cell function. While jatrorrhizine exhibited no effect on MC3T3-E1 cell viability, it caused reversal of dexamethasone-induced suppression of calcium deposition and alkaline phosphatase activity, elevation of osteogenic differentiation related proteins, and decrease in the level of histone deacetylase 4. These observations led us to conclude that jatrorrhizine ameliorates osteogenic differentiation in dexamethasone treated MC3T3-E1 cells via histone deacetylase 4 suggesting its potential application in the treatment of osteoporosis. [ABSTRACT FROM AUTHOR]

Peri-implantitis is a pathological condition involving tissues around dental implants that are characterized by inflammation of the peri-implant mucosa and progressive loss of supporting bone. We found that the antimicrobial peptide KR-12-3 (KRIVKWIKKFLR) derived from LL-37 had antibacterial properties against Streptococcus gordonii. The purpose of this study was to evaluate its antibacterial and anti-inflammatory activities and its underlying mechanisms. We evaluated the antibacterial activities of antimicrobial peptides in planktonic and biofilm states by measuring their minimum inhibitory concentration, minimum bactericidal concentration, and biofilm susceptibility. The effects of antimicrobial peptides on the production of IL-6 and IL-8 in LPS-stimulated RAW264.7 cells were detected by enzyme-linked immunosorbent assay and other experiments, and their toxicity to MC3T3-E1 cells was also studied. While maintaining low cytotoxicity, KR-12-3 exhibited growth inhibitory effects on S. gordonii in planktonic and biofilm states. Lower concentrations of KR-12-3 treatment reduced the production of inflammatory cytokines in LPS-stimulated RAW264.8 cells. The mechanisms underlying the inhibition of biofilm formation and anti-inflammatory effects have been associated with the low expression of related genes. KR-12-3 may be used to develop an antibacterial, anti-infective, and anti-inflammatory drugs for peri-implantitis. [ABSTRACT FROM AUTHOR]

Abstract Background Iron overload can promote the development of osteoporosis by inducing apoptosis in osteoblasts. However, the mechanism by which miRNAs regulate apoptosis in osteoblasts under iron overload has not been elucidated. Method The miRNA expression profile in MC3T3-E1 cells under iron overload was detected by next generation sequencing. qRT-PCR was used to determine the expression of miR-3074-5p in MC3T3-E1 cells under iron overload. The proliferation of MC3T3-E1 cells was tested using CCK-8 assays, and apoptosis was measured using flow cytometry. The miRanda and TargetScan databases were used to predict the target genes of miR-3074-5p. Interaction between miR-3074-5p and the potential target gene was validated by qRT-PCR, luciferase reporter assay and western blotting. Results We found that iron overload decreased the cell viability and induced apoptosis of MC3T3-E1 cells. The results of next generation sequencing analysis showed that miR-3074-5p expression was significantly increased in MC3T3-E1 cells under iron overload conditions, which was confirmed by further experiments. The inhibition of miR-3074-5p attenuated the apoptosis of iron-overloaded MC3T3-E1 cells. Furthermore, the expression of Smad4 was decreased and was inversely correlated with miR-3074-5p expression, and overexpression of Smad4 partially reversed the viability inhibition of iron-overloaded MC3T3-E1 cells by relieving the suppression of ERK, AKT, and Stat3 phosphorylation, suggesting its regulatory role in the viability inhibition of iron-overloaded MC3T3-E1 cells. The luciferase reporter assay results showed that Smad4 was the target gene of miR-3074-5p. Conclusion miR-3074-5p functions as an apoptosis promoter in iron-overloaded MC3T3-E1 cells by directly targeting Smad4.

Hualei Song,1,&ast; Yuntao Zhang,2,&ast; Zihan Zhang,2,&ast; Shijiang Xiong,3 Xiangrui Ma,2 Yourui Li2 1Department of Laboratory, Binzhou Medical University Hospital, Binzhou, 256603, People’s Republic of China; 2Department of Stomatology, Binzhou Medical University Hospital, Binzhou, 256603, People’s Republic of China; 3Shandong Provincial Key Laboratory of Oral Tissue Regeneration, School of Stomatology, Shandong University, Jinan, 250012, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Yourui LiDepartment of Stomatology, Binzhou Medical University Hospital, 661#, 2nd Huanghe Road, Binzhou, 256603, Shangdong Province, People’s Republic of ChinaTel +86 543 3256715Email carylee37@qq.comBackground: As commonly bone defect is a disease of jaw that can seriously affect implant restoration, the bioactive scaffold can be used as potential systems to provide effective repair for bone defect.Purpose: A osteoinductive bone tissue engineering scaffold has been prepared in order to explore the effect of bioactive materials on bone tissue engineering.Methods: In this study, NELL-1 nanoparticles (Chi/NNP) and nano hydroxyapatite were incorporated in composite scaffolds by electrospinning and characterized using TEM, SEM, contact angle, tensile tests and in vitro drug release. In vitro biological activities such as MC3T3-E1 cell attachment, proliferation and osteogenic activity were studied.Results: With the addition of nHA and nanoparticles, the fiber diameter of PCL/BNPs group, PCL/NNPs group and PCL/nHA/NNPs group was significantly increased. Moreover, the hydrophilic hydroxyl group and amino group presented in nHA and nanoparticles had improved the hydrophilicity of the composite fibers. The composite electrospun containing Chi/NNPs can form a double protective barrier which can effectively prolong the release time of NELL-1 growth factor. In addition, the hydroxyapatite/NELL-1 nanoparticles electrospun fibers can promote attachment, proliferation, differentiation of MC3T3-E1 cells and good cytocompatibility, indicating better ability of inducing osteogenic differentiation.Conclusion: A multi-functional PCL/nHA/NNPs composite fiber with long-term bioactivity and osteoinductivity was successfully prepared by electrospinning. This potential composite could be used as scaffolds in bone tissue engineering application after in vivo studies.Keywords: polycaprolactone, nanoscaffold, MC3T3-E1 cells, osteogenic activity

Orthopedic hybrid implants combining both titanium (Ti) and magnesium (Mg) have gained wide attraction nowadays. However, it still remains a huge challenge in the fabrication of Mg-Ti composites because of the different temperatures of Ti melting point and pure Mg volatilization point. In this study, we successfully fabricated a new Mg-Ti composite with bi-continuous interpenetrating phase architecture by infiltrating Mg melt into Ti scaffolds, which were prepared by 3D printing and subsequent acid treatment. We attempted to understand the 7-day degradation process of the Mg-Ti composite and examine the different Mg2+ concentration composite impacts on the MC3T3-E1 cells, including toxicity, morphology, apoptosis, and osteogenic activity. CCK-8 results indicated cytotoxicity and absence of the Mg-Ti composite during 7-day degradation. Moreover, the composite significantly improved the morphology, reduced the apoptosis rate, and enhanced the osteogenic activity of MC3T3-E1 cells. The favorable impacts might be attributed to the appropriate Mg2+ concentration of the extracts. The results on varying Mg2+ concentration tests indicated that Mg2+ showed no cell adverse effect under 10-mM concentration. The 8-mM group exhibited the best cell morphology, minimum apoptosis rate, and maximum osteogenic activity. This work may open a new perspective on the development and biomedical applications for Mg-Ti composites.