Osteoporosis(OP) is a senile bone disease characterized by an imbalance between bone remodeling and bone formation. Targeting pathogenesis of kidney deficiency, spleen deficiency, and blood stasis, Bushen Jianpi Huoxue Decoction has a significant effect on the treatment of OP by tonifying kidney, invigorating spleen, and activating blood circulation. MicroRNA(miRNA) and the anti-apoptotic protein B-cell lymphoma-2-like protein 1(BCL2L1) are closely related to bone cell metabolism. Therefore, in this study, the binding of miR-140-5p to BCL2L1 was detected by dual luciferase assay and polymerase chain reaction(PCR). After silencing or overexpressing miR-140-5p, the apoptosis, autophagy, and osteogenic function of human fetal osteoblast cell line 1.19(HFOB1.19) were observed by flow cytometry and Western blot. Bushen Jianpi Huoxue Decoction-containing serum was prepared by intragastric administration of Bushen Jianpi Huoxue Decoction in rats. Different concentrations of Bushen Jianpi Huoxue Decoction-containing serum were used to treat HFOB1.19 with or without miR-140-5p mimic. The expression of osteogenic proteins in each group was observed, and the role of miR-140-5p/BCL2L1 in apoptosis and autophagy of HFOB1.19 was studied, along with the effect of Bushen Jianpi Huoxue Decoction on these processes. As indicated by the dual luciferase assay, miR-140-5p bound to BCL2L1. Flow cytometry and Western blot showed that miR-140-5p promoted apoptosis and inhibited autophagy in HFOB1.19. After intervention with high, medium, and low doses of Bushen Jianpi Huoxue Decoction-medicated serum, compared with the miR-140-5p NC group, the expression of osteocalcin(OCN), osteopontin(OPN), Runt-related transcription factor 2(RUNX2), and transforming growth factor beta 1(TGF-β1) decreased in the miR-140-5p mimic group, while the expression of bone morphogenetic protein 2(BMP2) showed no significant difference under high-dose intervention. Therefore, miR-140-5p/BCL2L1 can promote apoptosis and inhibit autophagy in HFOB1.19. Bushen Jianpi Huoxue Decoction can affect the osteogenic effect of miR-140-5p through BMP2.
MicroRNAs/metabolism* ; Autophagy/drug effects* ; Apoptosis/drug effects* ; Humans ; Drugs, Chinese Herbal/administration & dosage* ; Animals ; Cell Line ; bcl-X Protein/metabolism* ; Osteoblasts/metabolism* ; Rats ; Osteoporosis/physiopathology* ; Male ; Rats, Sprague-Dawley ; Osteogenesis/drug effects*

This study investigated the mechanism of autophagy in the differentiation processes of MC3T3-E1 cells under osteogenic induction(physiological) and alcohol(AL) intervention(pathological), as well as the mechanism by which icariin(ICA) affected osteogenic differentiation of MC3T3-E1 cells under the pathological condition of AL intervention. Osteogenic mineralized nodule staining confirmed that the cells could differentiate into osteoblasts. After determining the appropriate concentrations of AL and ICA using the CCK-8 assay, seven groups were set up in this study: complete medium(CM) group, osteogenic induction medium(OIM) group, OIM+0.25 mol·L~(-1) AL group, OIM+0.25 mol·L~(-1) AL+1×10~(-8) mol·L~(-1) ICA group, OIM+0.25 mol·L~(-1) AL+1×10~(-7) mol·L~(-1) ICA group, OIM+0.25 mol·L~(-1) AL+1×10~(-6) mol·L~(-1) ICA group, and OIM+0.25 mol·L~(-1) AL+1×10~(-5) mol·L~(-1) ICA group, with a culture period of 7 days. Alkaline phosphatase(ALP) staining was used to detect the relative ALP area. Western blot and RT-qPCR were employed to analyze the expression of osteogenesis-and autophagy-related proteins and mRNAs. Reactive oxygen species(ROS) staining was used to detect ROS levels, and apoptosis was assessed through mitochondrial membrane potential assays. The results showed that ICA increased the relative ALP area that had been reduced by AL intervention. AL down-regulated the expression levels of Wnt family member 1(Wnt1), along with the osteogenesis-related mRNAs Wnt1, β-catenin, Runt-related transcription factor 2(Runx2), osteoprotegerin(OPG), and ALP, thereby inhibiting osteogenic differentiation. ICA up-regulated the expression levels of the osteogenesis-related proteins and mRNAs that had been inhibited by AL, promoting osteogenic differentiation. AL inhibited typical autophagy, while ICA regulated Rubicon to suppress LC3-associated phagocytosis(LAP) and promote typical autophagy. ICA also reduced the ROS levels that were elevated by AL and decreased the apoptosis of osteoblasts induced by AL intervention. In conclusion, ICA can regulate Rubicon to inhibit LAP, promote typical autophagy, eliminate ROS, reduce apoptosis, and ultimately enhance the osteogenic differentiation of MC3T3-E1 cells under the pathological condition of AL intervention by modulating the Wnt/β-catenin signaling pathway.
Autophagy/drug effects* ; Animals ; Osteogenesis/drug effects* ; Mice ; Cell Differentiation/drug effects* ; Osteoblasts/metabolism* ; Ethanol/pharmacology* ; Flavonoids/pharmacology* ; Cell Line ; Reactive Oxygen Species/metabolism* ; Drugs, Chinese Herbal/pharmacology*

Guided by the theory of "the kidney storing essence, governing the bones, and producing marrow", this study explored the mechanism of icariin(ICA) in regulating the osteogenic differentiation of rat bone mesenchymal stem cells(BMSCs) through caveolin-1(Cav1) via in vitro and in vivo experiments, aiming to provide a theoretical basis for the prevention and treatment of postmenopausal osteoporosis with traditional Chinese medicine(TCM). Primary cells were obtained from 4-week-old female SD rats using the whole bone marrow adherent method. Flow cytometry was used to detect the expression of surface markers CD29, CD90, CD11b, and CD45. The potential for osteogenic and adipogenic differentiation was assessed. The effect of ICA on cell viability was determined using the CCK-8 assay, and the impact of ICA on the formation of mineralized nodules was verified by alizarin red staining. A stable Cav1-silenced cell line was constructed using lentivirus. The effect of Cav1 silencing on osteogenic differentiation was observed via alizarin red staining. Western blot analysis was conducted to detect the expression of Cav1, Hippo/TAZ, and osteogenic markers such as Runt-related transcription factor 2(RUNX2) and alkaline phosphatase(ALP). The results showed that primary cells were successfully obtained using the whole bone marrow adherent method, positively expressing surface markers of rat BMSCs and possessing the potential for both osteogenic and adipogenic differentiation. The CCK-8 assay and alizarin red staining results indicated that 1×10~(-7) mol·L~(-1) was the optimal concentration of ICA for intervention in this experiment(P<0.05). During osteogenic induction, ICA inhibited Cav1 expression(P<0.05) while promoting TAZ expression(P<0.05). Alizarin red staining demonstrated that Cav1 silencing significantly promoted the osteogenic differentiation of BMSCs. After ICA intervention, TAZ expression was activated, and the expression of osteogenic markers ALP and RUNX2 was increased. In conclusion, Cav1 silencing significantly promotes the osteogenic differentiation of BMSCs, and ICA promotes this differentiation by inhibiting Cav1 and regulating the Hippo/TAZ signaling pathway.
Animals ; Mesenchymal Stem Cells/metabolism* ; Caveolin 1/genetics* ; Osteogenesis/drug effects* ; Rats, Sprague-Dawley ; Rats ; Cell Differentiation/drug effects* ; Female ; Signal Transduction/drug effects* ; Flavonoids/administration & dosage* ; Protein Serine-Threonine Kinases/genetics* ; Drugs, Chinese Herbal/pharmacology* ; Cells, Cultured ; Humans

This paper aimed to explore the effects of Duzhong Decoction(DZD)-containing serum on the proliferation and osteoblast differentiation of MC3T3-E1 cells through L-type voltage-gated calcium channels(L-VGCCs). L-VGCCs inhibitors, nifedipine and verapamil, were used to block L-VGCCs in osteoblasts. MC3T3-E1 cells were divided into a control group, a low-dose DZD-containing serum(L-DZD) group, a medium-dose DZD-containing serum(M-DZD) group, a high-dose DZD-containing serum(H-DZD) group, a nifedipine group, a H-DZD + nifedipine group, verapamil group, and a H-DZD + verapamil group. The CCK-8 method was used for cell proliferation analysis, alkaline phosphatase(ALP) assay kits for intracellular ALP activity measurement, Western blot for protein expression level in cells, real-time fluorescence quantitative PCR technology for intracellular mRNA expression level determination, fluorescence spectrophotometer for free Ca~(2+) concentration determination in osteoblasts, and alizarin red staining(ARS) for mineralized nodule formation in osteoblasts. The experimental results show that compared to the control group, DZD groups can promote MC3T3-E1 cell proliferation, ALP activity, and mineralized nodule formation, increase intracellular Ca~(2+) concentrations, and upregulate the protein expression of bone morphogenetic protein 2(BMP2), collagen Ⅰ(COL1), α2 subunit protein of L-VGCCs(L-VGCCα2), and the mRNA expression of Runt-related transcription factor 2(RUNX2), and BMP2. After blocking L-VGCCs with nifedipine and verapamil, the intervention effects of DZD-containing serum were inhibited to varying degrees. Both nifedipine and verapamil could inhibit ALP activity, reduce mineralized nodule areas, and downregulate the expression of bone formation-related proteins. Moreover, the effects of DZD-containing serum on increasing MC3T3-E1 cell proliferation, osteoblast differentiation, and Ca~(2+) concentrations, upregulating the mRNA expression of osteoprotegerin(OPG) and protein expression of phosphorylated protein kinase B(p-Akt) and phosphorylated forkhead box protein O1(p-FOXO1), and upregulating phosphatase and tensin homolog(PTEN) expression were reversed by nifedipine. The results indicate that DZD-containing serum can increase the Ca~(2+) concentration in MC3T3-E1 cells to promote bone formation, which may be mediated by L-VGCCs and the PTEN/Akt/FoxO1 signaling pathway, providing a new perspective on the mechanism of DZD in treating osteoporosis.
Animals ; Osteoblasts/metabolism* ; Cell Proliferation/drug effects* ; Cell Differentiation/drug effects* ; Mice ; Drugs, Chinese Herbal/pharmacology* ; Calcium Channels, L-Type/genetics* ; Alkaline Phosphatase/genetics* ; Serum/chemistry* ; Cell Line ; Osteogenesis/drug effects* ; Bone Morphogenetic Protein 2/genetics*

Osteoporotic fractures(OPF) refer to the fractures caused by minor violence in the state of osteoporosis, seriously threatening the life and health of elderly patients. Drug and surgical therapies have limitations such as single targets, diverse adverse reactions, and poor prognosis. Kidney-tonifying traditional Chinese medicine(TCM) has good potential in the treatment of OPF. TCM can promote the healing of OPF by promoting angiogenesis in the early stage of bone healing, promoting osteogenic differentiation of bone marrow mesenchymal stem cells in the stage of bone repair, maintaining the balance of osteogenic and osteoclastic system in the stage of bone remodeling, and regulating the oxidative stress responses throughout the process of OPF healing. TCM can alleviate the pathological state of osteoporosis and promote fracture healing in OPF patients via multiple pathways and targets, demonstrating the advantages and potential of biphasic regulation.
Humans ; Drugs, Chinese Herbal/therapeutic use* ; Osteoporotic Fractures/metabolism* ; Animals ; Fracture Healing/drug effects* ; Medicine, Chinese Traditional ; Kidney/metabolism* ; Osteogenesis/drug effects*

OBJECTIVE: To summarize the latest research progress of graphene and its derivatives (GDs) in bone repair. METHODS: The relevant research literature at home and abroad in recent years was extensively accessed. The properties of GDs in bone repair materials, including mechanical properties, electrical conductivity, and antibacterial properties, were systematically summarized, and the unique advantages of GDs in material preparation, functionalization, and application, as well as the contributions and challenges to bone tissue engineering, were discussed. RESULTS: The application of GDs in bone repair materials has broad prospects, and the functionalization and modification technology effectively improve the osteogenic activity and material properties of GDs. GDs can induce osteogenic differentiation of stem cells through specific signaling pathways and promote osteogenic activity through immunomodulatory mechanisms. In addition, the parameters of GDs have significant effects on the cytotoxicity and degradation behavior. CONCLUSION GDs has great potential in the field of bone repair because of its excellent physical and chemical properties and biological properties. However, the cytotoxicity, biodegradability, and functionalization strategies of GDs still need to be further studied in order to achieve a wider application in the field of bone tissue engineering.
Graphite/pharmacology* ; Tissue Engineering/methods* ; Humans ; Osteogenesis/drug effects* ; Biocompatible Materials/pharmacology* ; Bone Regeneration ; Tissue Scaffolds/chemistry* ; Cell Differentiation ; Bone and Bones ; Bone Substitutes/chemistry* ; Animals

OBJECTIVE: To investigate the effects of sodium valproate (VPA) in inhibiting Erastin-induced ferroptosis in bone marrow mesenchymal stem cells (BMSCs) and its underlying mechanisms. METHODS: BMSCs were isolated from bone marrow of 8-week-old Spragur Dawley rats and identified [cell surface antigens CD90, CD44, and CD45 were analyzed by flow cytometry, and osteogenic and adipogenic differentiation abilities were assessed by alizarin red S (ARS) and oil red O staining, respectively]. Cells of passage 3 were used for the Erastin-induced ferroptosis model, with different concentrations of VPA for intervention. The optimal drug concentration was determined using the cell counting kit 8 assay. The experiment was divided into 4 groups: group A, cells were cultured in osteogenic induction medium for 24 hours; group B, cells were cultured in osteogenic induction medium containing optimal concentration Erastin for 24 hours; group C, cells were cultured in osteogenic induction medium containing optimal concentration Erastin and VPA for 24 hours; group D, cells were cultured in osteogenic induction medium containing optimal concentration Erastin and VPA, and 8 μmol/L EX527 for 24 hours. The mitochondrial state of the cells was evaluated, including the levels of malondialdehyde (MDA), glutathione (GSH), and reactive oxygen species (ROS). Osteogenic capacity was assessed by alkaline phosphatase (ALP) activity and ARS staining. Western blot analysis was performed to detect the expressions of osteogenic-related proteins [Runt-related transcription factor 2 (RUNX2) and osteopontin (OPN)], ferroptosis-related proteins [glutathione peroxidase 4 (GPX4), ferritin heavy chain 1 (FTH1), and solute carrier family 7 member 11 (SLC7A11)], and pathway-related proteins [adenosine monophosphate-activated protein kinase (AMPK) and Sirtuin 1 (SIRT1)]. RESULTS: The cultured cells were identified as BMSCs. VPA inhibited Erastin-induced ferroptosis and the decline of osteogenic ability in BMSCs, acting through the activation of the AMPK/SIRT1 pathway. VPA significantly reduced the levels of ROS and MDA in Erastin-treated BMSCs and significantly increased GSH levels. Additionally, the expression levels of ferroptosis-related proteins (GPX4, FTH1, and SLC7A11) significantly decreased. VPA also upregulated the expressions of osteogenic-related proteins (RUNX2 and OPN), enhanced mineralization and osteogenic differentiation, and increased the expressions of pathway-related proteins (AMPK and SIRT1). These effects could be reversed by the SIRT1 inhibitor EX527. CONCLUSION VPA inhibits ferroptosis in BMSCs through the AMPK/SIRT1 axis and promotes osteogenesis.
Mesenchymal Stem Cells/metabolism* ; Ferroptosis/drug effects* ; Animals ; Valproic Acid/pharmacology* ; Rats ; Rats, Sprague-Dawley ; Sirtuin 1/metabolism* ; Cell Differentiation/drug effects* ; Cells, Cultured ; AMP-Activated Protein Kinases/metabolism* ; Osteogenesis/drug effects* ; Piperazines/pharmacology* ; Bone Marrow Cells/cytology* ; Reactive Oxygen Species/metabolism* ; Signal Transduction/drug effects*

OBJECTIVE: To review the research progress of strontium (Sr) modified β-tricalcium phosphate composite biomaterials (SrTCP) promoting osteogenesis through immune regulation, and provides reference and theoretical support for the further development and research of SrTCP bone repair materials in bone tissue engineering in the future. METHODS: The literature about SrTCP promoting osteogenesis through immune regulation at home and abroad in recent years was extensively reviewed, and the preparation methods, immune mechanism and application of promoting osteogenesis were summarized and analyzed. RESULTS: The preparation methods of SrTCP include solid-state reaction sintering method, solution combustion quenching method, direct doping method, ion substitution method, etc. SrTCP has immune regulatory effects, which can play an immune regulatory role in inducing macrophage polarization, inducing angiogenesis and anti oxidative stress to promote osteogenesis. CONCLUSION At present, studies have shown that SrTCP can promote bone defect repair through immune regulation. Subsequent studies can start from the control of the optimal repair concentration and release rate of Sr, and further clarify the specific mechanism of SrTCP in promoting angiogenesis and anti oxidative stress, which is helpful to develop new materials for bone defect repair.
Calcium Phosphates/pharmacology* ; Strontium/pharmacology* ; Biocompatible Materials/pharmacology* ; Humans ; Osteogenesis/drug effects* ; Tissue Engineering/methods* ; Bone Substitutes/pharmacology* ; Bone Regeneration/drug effects* ; Animals ; Tissue Scaffolds/chemistry* ; Neovascularization, Physiologic/drug effects* ; Macrophages/immunology*

OBJECTIVE: To investigate the antioxidant and osteogenic induction capabilities of calcium phosphate nanoflowers (hereinafter referred to as nanoflowers) in vitro at different concentrations. METHODS: Nanoflowers were prepared using gelatin, tripolyphosphate, and calcium chloride. Their morphology, microstructure, elemental composition and distribution, diameter, and molecular constitution were characterized using scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, and energy-dispersive spectroscopy. Femurs and tibias were harvested from twelve 4-week-old Sprague Dawley rats, and bone marrow mesenchymal stem cells (BMSCs) were isolated and cultured using the whole bone marrow adherent method, followed by passaging. The third passage cells were identified as stem cells by flow cytometry and then co-cultured with nanoflowers at concentrations of 0, 0.4, 0.8, 1.2, 1.6, 2.0, 2.4, 2.8, 3.2, and 3.6 mg/mL. Cell counting kit 8 (CCK-8) assay was performed to screen for the optimal concentration that demonstrated the best cell viability, which was subsequently used as the experimental concentration for further studies. After co-culturing BMSCs with the screened concentration of nanoflowers, the biocompatibility of the nanoflowers was verified through live/dead cell staining, scratch assay, and cytoskeleton staining. The antioxidant capacity was assessed by using reactive oxygen species (ROS) fluorescence staining. The in vitro osteoinductive ability was evaluated via alkaline phosphatase (ALP) staining, alizarin red staining, and immunofluorescence staining of osteocalcin (OCN) and Runt-related transcription factor 2 (RUNX2). All the above indicators were compared with the control group of normally cultured BMSCs without the addition of nanoflowers. RESULTS: Scanning electron microscopy revealed that the prepared nanoflowers exhibited a flower-like structure; transmission electron microscopy scans discovered that the nanoflowers possessed a multi-layered structure, and high-magnification images displayed continuous atomic arrangements, with the nanoflower diameter measuring (2.00±0.25) μm; energy-dispersive spectroscopy indicated that the nanoflowers contained elements such as C, N, O, P, and Ca, which were uniformly distributed across the flower region; Fourier transform infrared spectroscopy analyzed the absorption peaks of each component, demonstrating the successful preparation of the nanoflowers. Through CCK-8 screening, the concentrations of 0.8, 1.2, and 1.6 mg/mL were selected for subsequent experiments. The live/dead cell staining showed that nanoflowers at different concentrations exhibited good cell compatibility, with the 1.2 mg/mL concentration being the best (P<0.05). The scratch assay results indicated that the cell migration ability in the 1.2 mg/mL group was superior to the other groups (P<0.05). The cytoskeleton staining revealed that the cell morphology was well-extended in all concentration groups, with no significant difference compared to the control group. The ROS fluorescence staining demonstrated that the ROS fluorescence in all concentration groups decreased compared to the control group after lipopolysaccharide induction (P<0.05), with the 1.2 mg/mL group showing the weakest fluorescence. The ALP staining showed blue-purple nodular deposits around the cells in all groups, with the 1.2 mg/mL group being significantly more prominent. The alizarin red staining displayed orange-red mineralized nodules around the cells in all groups, with the 1.2 mg/mL group having more and denser nodules. The immunofluorescence staining revealed that the expressions of RUNX2 and OCN proteins in all concentration groups increased compared to the control group, with the 1.2 mg/mL group showing the strongest protein expression (P<0.05). CONCLUSION The study successfully prepares nanoflowers, among which the 1.2 mg/mL nanoflowers exhibits excellent cell compatibility, antioxidant properties, and osteogenic induction capability, demonstrating their potential as an artificial bone substitute material.
Animals ; Osteogenesis/drug effects* ; Mesenchymal Stem Cells/drug effects* ; Calcium Phosphates/pharmacology* ; Rats, Sprague-Dawley ; Rats ; Antioxidants/chemistry* ; Cells, Cultured ; Cell Differentiation/drug effects* ; Nanostructures/chemistry* ; Tissue Engineering/methods* ; Bone Marrow Cells/cytology* ; Coculture Techniques ; Tissue Scaffolds/chemistry* ; Male ; Biocompatible Materials/chemistry* ; Cell Survival ; Core Binding Factor Alpha 1 Subunit/metabolism* ; Cell Proliferation

OBJECTIVE: To explore the mechanism by which ω-3 polyunsaturated fatty acids (hereinafter referred to as "ω-3") exert antioxidant stress protection and promote osteogenic differentiation in MC3T3-E1 cells, and to reveal the relationship between ω-3 and the key antioxidant stress pathway involving nuclear factor E2-related factor 2 (Nrf2) and NAD (P) H quinone oxidoreductase 1 (NQO1) in MC3T3-E1 cells. METHODS: The optimal concentration of H ₂O ₂ (used to establish the oxidative stress model of MC3T3-E1 cells in vitro) and the optimal intervention concentrations of ω-3 were screened by cell counting kit 8. MC3T3-E1 cells were divided into blank control group, oxidative stress group (H ₂O ₂), low-dose ω-3 group (H ₂O ₂+low-dose ω-3), and high-dose ω-3 group (H ₂O ₂+high-dose ω-3). After osteoblastic differentiation for 7 or 14 days, the intracellular reactive oxygen species (ROS) level was measured by fluorescence staining and flow cytometry, and the mitochondrial morphological changes were observed by biological transmission electron microscope; the expression levels of Nrf2, NQO1, heme oxygenase 1 (HO-1), Mitofusin 1 (Mfn1), and Mfn2 were detected by Western blot to evaluate the cells' antioxidant stress capacity; the expression levels of Runt-related transcription factor 2 (RUNX2) and osteocalcin (OCN) were detected by immunofluorescence staining and Western blot; osteogenic potential of MC3T3-E1 cells was evaluated by alkaline phosphatase (ALP) staining and alizarin red staining. RESULTS: Compared with the oxidative stress group, the content of ROS in the low and high dose ω-3 groups significantly decreased, and the protein expressions of Nrf2, NQO1, and HO-1 significantly increased ( P<0.05). At the same time, the mitochondrial morphology of MC3T3-E1 cells improved, and the expressions of mitochondrial morphology-related proteins Mfn1 and Mfn2 significantly increased ( P<0.05). ALP staining and alizarin red staining showed that the low-dose and high-dose ω-3 groups showed stronger osteogenic ability, and the expressions of osteogenesis-related proteins RUNX2 and OCN significantly increased ( P<0.05). And the above results showed a dose-dependence in the two ω-3 treatment groups ( P<0.05). CONCLUSION ω-3 can enhance the antioxidant capacity of MC3T3-E1 cells under oxidative stress conditions and upregulate their osteogenic activity, possibly through the Nrf2/NQO1 signaling pathway.
Oxidative Stress/drug effects* ; NF-E2-Related Factor 2/metabolism* ; NAD(P)H Dehydrogenase (Quinone)/metabolism* ; Animals ; Mice ; Osteogenesis/drug effects* ; Cell Differentiation/drug effects* ; Fatty Acids, Omega-3/pharmacology* ; Signal Transduction/drug effects* ; Osteoblasts/drug effects* ; Reactive Oxygen Species/metabolism* ; Cell Line ; Hydrogen Peroxide/pharmacology* ; Core Binding Factor Alpha 1 Subunit/metabolism* ; Antioxidants/pharmacology* ; Heme Oxygenase-1/metabolism*