Resveratrol Alleviates Osteoporosis by Promoting Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells via SITR1/PI3K/AKT Pathway / El Resveratrol Alivia la Osteoporosis al Promover la Diferenciación Osteogénica de las Células Madre Mesenquimales de la Médula Ósea a Través de la Vía SITR1/PI3K/AKT

SUMMARY: Senile osteoporosis is mainly caused by reduced osteoblast differentiation and has become the leading cause of fractures in the elderly worldwide. Natural organics are emerging as a potential option for the prevention and treatment of osteoporosis. This study was designed to study the effect of resveratrol on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in osteoporosis mice. A mouse model of osteoporosis was established by subcutaneous injection of dexamethasone and treated with resveratrol administered by gavage. In vivo and in vitro, we used western blot to detect protein expression, and evaluated osteogenic differentiation of BMSCs by detecting the expression of osteogenic differentiation related proteins, calcium deposition, ALP activity and osteocalcin content. Resveratrol treatment significantly increased the body weight of mice, the level of serum Ca2+, 25(OH)D and osteocalcin, ration of bone weight, bone volume/total volume, trabecular thickness, trabecular number, trabecular spacing and cortical thickness in osteoporosis mice. In BMSCs of osteoporosis mice, resveratrol treatment significantly increased the expression of Runx2, osterix (OSX) and osteocalcin (OCN) protein, the level of calcium deposition, ALP activity and osteocalcin content. In addition, resveratrol treatment also significantly increased the expression of SIRT1, p-PI3K / PI3K and p-AKT / AKT in BMSCs of osteoporosis mice. In vitro, resveratrol increased the expression of SIRT1, p-PI3K / PI3K and p-AKT / AKT, Runx2, OSX and OCN protein, the level of calcium deposition, ALP activity and osteocalcin content in BMSCs in a concentration-dependent manner, while SIRT1 knockdown significantly reversed the effect of resveratrol. Resveratrol can attenuate osteoporosis by promoting osteogenic differentiation of bone marrow mesenchymal stem cells, and the mechanism may be related to the regulation of SIRT1/PI3K/AKT pathway.

La osteoporosis senil es causada principalmente por una diferenciación reducida de osteoblastos y se ha convertido en la principal causa de fracturas en las personas mayores en todo el mundo. Los productos orgánicos naturales están surgiendo como una opción potencial para la prevención y el tratamiento de la osteoporosis. Este estudio fue diseñado para estudiar el efecto del resveratrol en la diferenciación osteogénica de las células madre mesenquimales de la médula ósea (BMSC) en ratones con osteoporosis. Se estableció un modelo de osteoporosis en ratones mediante inyección subcutánea de dexametasona y se trató con resveratrol administrado por sonda. In vivo e in vitro, utilizamos Western blot para detectar la expresión de proteínas y evaluamos la diferenciación osteogénica de BMSC detectando la expresión de proteínas relacionadas con la diferenciación osteogénica, la deposición de calcio, la actividad de ALP y el contenido de osteocalcina. El tratamiento con resveratrol aumentó significativamente el peso corporal de los ratones, el nivel sérico de Ca2+, 25(OH)D y osteocalcina, la proporción de peso óseo, el volumen óseo/ volumen total, el espesor trabecular, el número trabecular, el espaciado trabecular y el espesor cortical en ratones con osteoporosis. En BMSC de ratones con osteoporosis, el tratamiento con resveratrol aumentó significativamente la expresión de las proteínas Runx2, osterix (OSX) y osteocalcina (OCN), el nivel de deposición de calcio, la actividad de ALP y el contenido de osteocalcina. Además, el tratamiento con resveratrol también aumentó significativamente la expresión de SIRT1, p-PI3K/PI3K y p-AKT/AKT en BMSC de ratones con osteoporosis. In vitro, el resveratrol aumentó la expresión de las proteínas SIRT1, p-PI3K/PI3K y p- AKT/AKT, Runx2, OSX y OCN, el nivel de deposición de calcio, la actividad de ALP y el contenido de osteocalcina en BMSC de manera dependiente de la concentración, mientras que La caída de SIRT1 revirtió significativamente el efecto del resveratrol. El resveratrol puede atenuar la osteoporosis al promover la diferenciación osteogénica de las células madre mesenquimales de la médula ósea, y el mecanismo puede estar relacionado con la regulación de la vía SIRT1/PI3K/AKT.

Mechanism of Chir99021 regulating Wnt / β⁃catenin signaling pathway inhibiting osteogenic differentiation of rat dental pulp stem cells / 解剖学报

Objective To explore the effect and mechanism of Chir99021 on osteogenic differentiation of rat dental pulp stem cells. Methods Primary rat dental pulp stem cells were isolated from rat dental pulp and verified by fluorescence immunoassay. Different concentrations of Chir99021 were set, and the cell proliferation was detected by CCK⁃8 to select the optimal concentration. Osteogenic differentiation was detected by alizarin red staining. The expression of osteogenic differentiation related genes and proteins recombinant wingless type MMTV integration site famity member 1 (Wnt1), Wnt3a and Wnt3a β⁃expression of catenin, axis inhibition protein 2(Axin 2), dentin sialophosphoprotein(OCN) and dentin matrix acidic phosphoprotein 1(DMP1) was detected by Real⁃time PCR and Western blotting. Results The positive expression of dentin sialophosphoprotein (DSPP) and vimentin indicated that rat dental pulp stem cells were successfully isolated. After osteogenic induction of rat dental pulp stem cells, calcium deposits significantly increased with the addition of glycogen synthase kinase⁃3β(GSK⁃3β) inhibitor Chir99021, calcium deposits were significanted reduced. After osteogenic differentiation of rat dental pulp stem cells, the expression of Wnt1, Wnt3a, β⁃catenin, Axin2, OCN and DMP1 increased, while the expression of Wnt1, Axin2, OCN and DMP1 decreased with the addition of Chir99021. Conclusion Chir99021 can inhibit the osteogenic differentiation of rat dental pulp stem cells after 7 days of induction.

Mechanism of miRNA Intervention in Osteoporosis and Intervention Effect of Traditional Chinese Medicine： A Review / 中国实验方剂学杂志

Osteoporosis （OP） is a skeletal metabolic disease characterized by bone loss and destruction of bone microstructure. Changes in estrogen levels are not the only pathogenic factors for the occurrence and development of OP. MicroRNA （miRNA） plays an important regulatory role in cells. The complementary sequences of miRNA and targeted mRNA combine to inhibit the expression of targeted mRNA through post-transcriptional regulation， forming a complex regulatory network. Research suggests that miRNA is closely related to the occurrence and development of various diseases， including inflammatory diseases， metabolic diseases， and cancer. Targeted mRNA participates in post-transcriptional gene expression regulation in OP， mainly regulating the balance among bone construction， bone resorption， and osteoblast differentiation. Therefore， miRNA-based gene therapy is a rapidly developing disease treatment strategy. Traditional Chinese medicine can improve bone metabolism by intervening in miRNA differential expression to target and regulate osteogenic/osteoclast differentiation. This article summarized the targeting effects of miRNAs in physiological and developmental processes such as bone cell proliferation， differentiation， survival， and apoptosis， reviewed and classified their mechanisms of action and targets， and sorted out the current treatment methods of traditional Chinese medicine for preventing and treating OP and drugs that exert bone protective functions through miRNAs. This review is expected to provide theoretical reference and research guidance for future research on OP treatment by regulating miRNA.

Impact of lithocholic acid on the osteogenic and adipogenic differentiation balance of bone marrow mesenchymal stem cells / 中国修复重建外科杂志

OBJECTIVE@#To Investigate the effects of lithocholic acid (LCA) on the balance between osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).@*METHODS@#Twelve 10-week-old SPF C57BL/6J female mice were randomly divided into an experimental group (undergoing bilateral ovariectomy) and a control group (only removing the same volume of adipose tissue around the ovaries), with 6 mice in each group. The body mass was measured every week after operation. After 4 weeks post-surgery, the weight of mouse uterus was measured, femur specimens of the mice were taken for micro-CT scanning and three-dimensional reconstruction to analyze changes in bone mass. Tibia specimens were taken for HE staining to calculate the number and area of bone marrow adipocytes in the marrow cavity area. ELISA was used to detect the expression of bone turnover markers in the serum. Liver samples were subjected to real-time fluorescence quantitative PCR (RT-qPCR) to detect the expression of key genes related to bile acid metabolism, including cyp7a1, cyp7b1, cyp8b1, and cyp27a1. BMSCs were isolated by centrifugation from 2 C57BL/6J female mice (10-week-old). The third-generation cells were exposed to 0, 1, 10, and 100 μmol/L LCA, following which cell viability was evaluated using the cell counting kit 8 assay. Subsequently, alkaline phosphatase (ALP) staining and oil red O staining were conducted after 7 days of osteogenic and adipogenic induction. RT-qPCR was employed to analyze the expressions of osteogenic-related genes, namely ALP, Runt-related transcription factor 2 (Runx2), and osteocalcin (OCN), as well as adipogenic-related genes including Adiponectin (Adipoq), fatty acid binding protein 4 (FABP4), and peroxisome proliferator-activated receptor γ (PPARγ).@*RESULTS@#Compared with the control group, the body mass of the mice in the experimental group increased, the uterus atrophied, the bone mass decreased, the bone marrow fat expanded, and the bone metabolism showed a high bone turnover state. RT-qPCR showed that the expressions of cyp7a1, cyp8b1, and cyp27a1, which were related to the key enzymes of bile acid metabolism in the liver, decreased significantly ( P<0.05), while the expression of cyp7b1 had no significant difference ( P>0.05). Intervention with LCA at concentrations of 1, 10, and 100 μmol/L did not demonstrate any apparent toxic effects on BMSCs. Furthermore, LCA inhibited the expressions of osteogenic-related genes (ALP, Runx2, and OCN) in a dose-dependent manner, resulting in a reduction in ALP staining positive area. Concurrently, LCA promoted the expressions of adipogenic-related genes (Adipoq, FABP4, and PPARγ), and an increase in oil red O staining positive area.@*CONCLUSION@#After menopause, the metabolism of bile acids is altered, and secondary bile acid LCA interferes with the balance of osteogenic and adipogenic differentiation of BMSCs, thereby affecting bone remodelling.

Chinese Medicine Regulates Signaling Pathways Related to Proliferation and Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells： A Review / 中国实验方剂学杂志

Bone marrow mesenchymal stem cells （BMSCs） are derived from stem cells isolated from bone marrow and have the potential for multidirectional differentiation and self-renewal. Under certain conditions， BMSCs can be induced to differentiate into osteoblast （OB）， chondrocyte， adipocyte， fibroblast， etc. BMSCs play an important role in maintaining the stability of bone structure and balancing bone metabolism. Promoting the proliferation of BMSCs and inducing their differentiation into OB of great significance for the clinical prevention and treatment of osteoporosis， bone defects， fracture healing， and other diseases. Because the proliferation and osteogenic differentiation of BMSCs are complex processes controlled by multiple genes and regulated by multiple signal transduction pathways， traditional Chinese medicine （TCM） happens to have the advantages of multi-bioactive component， multi-target， and multi-pathway synergism， which can affect the proliferation and differentiation of BMSCs through multiple channels and induce the proliferation of BMSCs. The transcription and expression of genes related to osteogenesis can be enhanced to promote the differentiation of BMSCs into OB， so as to achieve the purpose of preventing and treating osteoporosis， bone defects， and other bone diseases. Based on the literature on the intervention of TCM monomers and compounds in the proliferation and osteogenic differentiation of BMSCs， this study reviewed TCM monomers and compounds in promoting the proliferation and osteogenic differentiation of BMSCs by regulating secreted glycoprotein （Wnt）， neurogenic locus notch homolog protein （Notch）， mitogen-activated protein kinase （MAPK）， phosphatidylinositol-3 kinase （PI3K） /protein kinase B （Akt）， bone morphogenetic protein （BMP）/Smad， Janus kinase （JAK）/signal transducer and activator of transcription protein （STAT）， osteoprotegerin （OPG）/receptor activator of nuclear factor-kappa B （RANK）/RANK ligand （RANKL）， and other signaling pathways to provide new ideas for the research and clinical application of Chinese medicine in the prevention and treatment of orthopedic diseases.

Red light-emitting diode light mediated by the KEAP1-NRF2/HO-1 pathway induces osteogenic differentiation and attenuates the oxidative stress damage of human periodontal ligament stem cells induced by high glucose / 口腔疾病防治

Objective@# To explore the effects of red LED light mediated by the Kelch-like ECH-associated protein 1-nuclear factor erythroid 2-related factor 2/heme oxygenase-1 (KEAP1-NRF2/HO-1) pathway on osteogenic differentiation and oxidative stress damage of human periodontal ligament stem cells (hPDLSCs) induced by high glucose, which provides a basis for the application of red light-emitting diode (LED) light in cell antioxidative damage.@*Methods@#hPDLSCs were identified by flow cytometric analysis, alkaline phosphatase (ALP) staining and Alizarin red-S staining; hPDLSCs were pretreated in a high glucose environment for 48 hours and irradiated with 1, 3, or 5 J/cm2 red LED light. A CCK-8 assay was performed to choose the radiant exposure that had the strongest effect on promoting the cell proliferation rate for subsequent experiments. hPDLSCs were divided into a control group, a high glucose group and a high glucose+light exposure group. ALP staining, ALP activity, Alizarin red-S staining and quantitative calcified nodules were used to detect the osteogenic differentiation of hPDLSCs; qRT-PCR and Western blot were used to detect the gene and protein expression levels of ALP, runt-related transcription factor 2 (RUNX2) and osterix (OSX); the relative mRNA expression levels of antioxidant enzyme-related genes superoxide dismutase 2 (SOD2) and catalase (CAT) in hPDLSCs were detected by qRT-PCR; reactive oxygen species (ROS) levels were detected by fluorescence microscopy and flow cytometry; the tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) levels in cell supernatants were detected by ELISA; the NRF2-specific inhibitor ML385 was used to inhibit the NRF2 pathway; ALP staining and ALP activity were used to detect the markers of early osteogenic differentiation; qRT-PCR was used to detect the gene expression of ALP, RUNX2 and OSX; and the protein expression levels of KEAP1, NRF2 and HO-1 were detected by Western blot.@*Results @# Identified, and irradiant exposure of 5 J/cm2 was chosen for subsequent experiments. Red LED light irradiation (5 J/cm2) improved the osteogenic differentiation of hPDLSCs induced by high glucose (P<0.05), increased the mRNA and protein levels of ALP, RUNX2 and OSX (P<0.05), upregulated the mRNA expression levels of SOD2 and CAT (P<0.05), reduced the levels of ROS (P<0.05), and reduced TNF-α and IL-1β levels in the cell supernatants (P<0.05). When ML385 was added to inhibit the NRF2 pathway, the ALP activity of cells was decreased (P<0.05); the gene expression levels of ALP, RUNX2 and OSX were downregulated (P<0.05); the protein level of KEAP1 was upregulated (P<0.05); and the protein levels of NRF2 and HO-1 were downregulated (P<0.05)@*Conclusion@#Red LED light may promote the proliferation and osteoblastic differentiation of hPDLSCs induced by high glucose through the KEAP1-NRF2/HO-1 pathway and reduce the oxidative stress damage to hPDLSCs induced by high glucose.

Study on the role and mechanism of osteogenesis induced by advanced platelet-rich fibrin and β-tricalcium phosphate complex / 口腔疾病防治

Objective @#To investigate the role and mechanism of bone formation caused by the ratio of advanced platelet-rich fibrin (A-PRF) and β-tricalcium phosphate (β-TCP) in rabbit femur defect model, which provides a new idea for clinical treatment of bone defect.@*Methods @#Twenty-four New Zealand white rabbits were divided into model group, 1∶1 complex group (A-PRF∶β-TCP=1∶1), 2∶1 complex group (A-PRF∶β- TCP=2∶1) and 4∶1 complex group (A-PRF∶β- TCP=4∶1）, with 6 rabbits in each group. Femoral defect models were constructed in each group. In the composite group, the bone defect was filled with composite material, while in the model group, no material was filled. After 8 weeks, the animals were euthanized and specimens were collected. Bone mineral density (BMD), bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.SP) and trabecular number (Tb.N) in femoral defect tissue were measured by micro-CT and photographed. Hematoxylin - eosin staining was used to detect the pathological changes of new bone tissue. The morphological changes of the new bone tissue were observed by scanning electron microscopy. Determination of phospho-mitogen activated protein kinase p38 (p-p38MAPK), CCAAT/enhancer binding protein homologous protein (CHOP) and phospho-cysteine aspartic protease-3 (p-Caspase3) in newborn femur by ELISA. The mRNA expressions of osteoprotegerin (OPG), bone morphogenetic protein-2 (BMP-2), receptor activator of nuclear factor kappa-B ligand (RANKL) and p38MAPK were detected by real-time quantitative PCR. The expression of OPG, BMP-2, RANKL, p-p38MAPK and p-Caspase3 protein in the new bone tissue was observed by immunohistochemistry. @*Results @#In the model group, bone formation in the femoral defect area was slow and osteogenic quality was poor. Compared with the model group, the bone formation and neocapillaries of femoral defect area in the complex group was good, BMD, BV.TV, Tb.Th, Tb.N were increased, and Tb.Sp were decreased, the expressions of p-p38MAPK, CHOP and p-Caspase3 were decreased, and the mRNA and protein expressions of OPG and BMP-2 were increased. The mRNA expression of RANKL and p38MAPK was decreased. Apoptosis in new bone tissue of each group showed the lowest apoptosis rate in samples of the 2∶1 complex group (P<0.05); A-PRF: β-TCP=2∶1 ratio has the best osteogenic effect. @*Conclusion@#The complex composed of A-PRF and β-TCP can promote the expression of OPG, inhibit the expression of RANKL and phosphorylation of p38MAPK, reduce the apoptosis of new bone tissue cells, and promote osteogenic differentiation.

Mechanism of microRNA-30d-5p regulating osteogenic differentiation of bone marrow stromal cells through glucose-regulated 78 / 解剖学报

Objective To investigate the effect of microRNA(miR)-30d-5p on osteogenic differentiation and apoptosis of bone marrow stromal cells and its mechanism. Methods Bone marrow stromal cells were divided into miR-30d-5p overexpression negative control group, miR-30d-5p overexpression group, miR-30d-5p inhibition negative control group and miR-30d-5p inhibition group. Alkaline phosphatase (ALP) staining was used to identify osteogenesis, alizarin red staining was used to detect calcium nodules precipitation, and TUNEL was used to detect apoptosis. mRNA and protein expression levels were detected by Real-time PCR and Western blotting, and the potential binding sites of miR-30d-5p were predicted by the bioinformatics analysis website Targetscan 7.1. Results After miR-30d-5p overexpression, osteogenic differentiation ability, and mineralization ability of the cells decreased (P<0.05), while apoptosis level increased (P< 0.05). The expression of glucoregulatory protein 78 (GRP78) and osteogenic specific transcription factor Runt related transcription factor 2(RUNX2) decreased significantly (P<0.05). However, miR-30d-5p inhibitor-treated the cells with increased osteogenic differentiation and mineralization ability (P < 0.05), and apoptosis level decreased (P < 0.05). GRP78 and RUNX2 protein levels increased (P<0.05). The miR-30d-5p binding site was located at 142-148 bp of the 3'UTR of the GRP78 gene. Conclusion MiR-30d-5p inhibits osteogenic differentiation and promotes apoptosis of bone marrow stromal cells by down-regulating the expression of GRP78 protein.

Mechanism of miR-26a-5p/cAMP response element binding protein 1 molecular axis regulating osteogenic differentiation of adipose-derived mesenchymal stem cells / 中国修复重建外科杂志

OBJECTIVE@#To investigate the regulatory effects of miR-26a-5p on the osteogenic differentiation of adipose-derived mesenchymal stem cells (ADSCs) by regulating cAMP response element binding protein 1 (CREB1).@*METHODS@#The adipose tissues of four 3-4 weeks old female C57BL/6 mice were collected and the cells were isolated and cultured by digestion separation method. After morphological observation and identification by flow cytometry, the 3rd-generation cells were subjected to osteogenic differentiation induction. At 0, 3, 7, and 14 days after osteogenic differentiation induction, the calcium deposition was observed by alizarin red staining, ALP activity was detected, miR- 26a-5p and CREB1 mRNA expressions were examined by real-time fluorescence quantitative PCR, and CREB1 protein and its phosphorylation (phospho-CREB1, p-CREB1) level were measured by Western blot. After the binding sites between miR-26a-5p and CREB1 was predicted by the starBase database, HEK-293T cells were used to conduct a dual-luciferase reporter gene experiment to verify the targeting relationship (represented as luciferase activity after 48 hours of culture). Finally, miR-26a-p inhibitor (experimental group) and the corresponding negative control (control group) were transfected into ADSCs. Alizarin red staining, ALP activity, real-time fluorescent quantitative PCR (miR-26a-5p) and Western blot [CREB1, p-CREB1, Runt-related transcription factor 2 (RUNX2), and osteocalcin (OCN)] were performed at 7 and 14 days after osteogenic induction culture.@*RESULTS@#The cultured cells were identified as ADSCs. With the prolongation of osteogenic induction culture, the number of calcified nodules and ALP activity significantly increased ( P<0.05). The relative expression of miR-26a-5p in the cells gradually decreased, while the relative expressions of CREB1 mRNA and protein, as well as the relative expression of p-CREB1 protein were increased. The differences were significant between 7, 14 days and 0 day ( P<0.05). There was no significant difference in p-CREB1/CREB1 between different time points ( P>0.05). The starBase database predicted that miR-26a-5p and CREB1 had targeted binding sequences, and the dual-luciferase reporter gene experiment revealed that overexpression of miR-26a-5p significantly suppressed CREB1 wild-type luciferase activity ( P<0.05). After 7 and 14 days of osteogenic induction, compared with the control group, the number of calcified nodules, ALP activity, and relative expressions of CREB1, p-CREB1, OCN, and RUNX2 proteins in the experimental group significantly increased ( P<0.05). There was no significant difference in p-CREB1/CREB1 between the two groups ( P>0.05).@*CONCLUSION@#Knocking down miR-26a-5p promoted the osteogenic differentiation of ADSCs by up-regulating CREB1 and its phosphorylation.

Effect of Erxian Decoction-containing serum on H_2O_2-induced proliferation and osteogenic differentiation of MC3T3-E1 cells via BK channels / 中国中药杂志

This study aimed to investigate the effects of Erxian Decoction(EXD)-containing serum on the proliferation and osteogenic differentiation of MC3T3-E1 cells under oxidative stress through BK channels. The oxidative stress model was induced in MC3T3-E1 cells by H_2O_2, and 3 mmol·L~(-1) tetraethylammonium(TEA) chloride was used to block the BK channels in MC3T3-E1 cells. MC3T3-E1 cells were divided into a control group, a model group, an EXD group, a TEA group, and a TEA+EXD group. After MC3T3-E1 cells were treated with corresponding drugs for 2 days, 700 μmol·L~(-1) H_2O_2 was added for treatment for another 2 hours. CCK-8 assay was used to detect cell proliferation activity. The alkaline phosphatase(ALP) assay kit was used to detect the ALP activity of cells. Western blot and real-time fluorescence-based quantitative PCR(RT-qPCR) were used to detect protein and mRNA expression, respectively. Alizarin red staining was used to detect the mineralization area of osteoblasts. The results showed that compared with the control group, the model group showed significantly blunted cell proliferation activity and ALP activity, reduced expression of BK channel α subunit(BKα), collagen Ⅰ(COL1), bone morphogenetic protein 2(BMP2), osteoprotegerin(OPG), and phosphorylated Akt, decreased mRNA expression levels of Runt-related transcription factor 2(RUNX2), BMP2, and OPG, and declining area of calcium nodules. EXD-containing serum could significantly potentiate the cell proliferation activity and ALP activity, up-regulate the protein expression of BKα, COL1, BMP2, OPG, and phosphorylated Akt, and forkhead box protein O1(FoxO1), promote the mRNA expression of RUNX2, BMP2, and OPG, and enlarge the area of calcium nodules. However, BK channel blockage by TEA reversed the effects of EXD-containing serum in promoting the protein expression of BKα, COL1, BMP2, OPG, and phosphorylated Akt and FoxO1, increasing the mRNA expression of RUNX2, BMP2, and OPG, and enlarging the area of calcium nodules. EXD-containing serum could improve the proliferation activity, osteogenic differentiation, and mineralization ability of MC3T3-E1 cells under oxidative stress, which might be related to the regulation of BK channels and downstream Akt/FoxO1 signaling pathway.

Regulation of osteogenic differentiation under hypoxia by EphrinB2/EphB4 in MC3T3-E1 cells / 口腔疾病防治

Objective @# To investigate the effect of erythropoietin producing hepatocyte kinase receptor ligand B2-erythropoietin producing hepatocyte kinase receptor B4 (EphrinB2/EphB4) on the osteogenic differentiation of MC3T3-E1 cells in a hypoxic environment to provide experimental evidence for hypoxia regulation of osteoblast differentiation.@*Methods @# Control groups and cobalt chloride (CoCl2)-induced hypoxia groups were set up first. qRT-PCR was used to detect the mRNA expression of the osteogenic markers alkaline phosphatase (ALP), collogen1 (COL I), runt-related transcription factor 2 (RUNX2) and osteocalcin (OCN). ALP staining was used to detect the activity of cell alkaline phosphatase after osteogenic induction. The mRNA and protein expression levels of hypoxia inducible factor-1α (HIF-1α), EphrinB2 and EphB4 in the two groups were detected via qRT-PCR and Western blot. Then, the CoCl2 + inhibitor group was established. NVP-BHG712, an EphB4 phosphorylation inhibitor, was added to this group to prevent EphrinB2 from binding to EphB4 and producing signals. qRT-PCR and Western blot were used to detect the mRNA and protein expression of osteogenic markers, including ALP, RUNX2, COL I, and OCN. ALP staining and Alizarin red S staining were used to measure osteoblast differentiation and mineralization. @*Results @# Compared with the control group, the mRNA expression of the osteogenic differentiation markers ALP, RUNX2, COL-1, and OCN in MC3T3-E1 cells increased, and ALP activity and mineralization were enhanced under CoCl2-induced hypoxia in vitro (P<0.05). Additionally, the expression of HIF-1α, EphrinB2 and EphB4 was upregulated at the mRNA and protein levels under hypoxia (P<0.05). When NVP-BHG712 was used to block the connection between EphrinB2 and EphB4, the expression of osteogenic markers and ALP activity and mineralization were decreased (P<0.05).@*Conclusion@#EphrinB2/EphB4 can promote osteogenic differentiation of MC3T3-E1 cells and increase the expression of osteogenic markers and tissue mineralization in a hypoxic environment.

Content of bone morphogenetic protein 2 in demineralized bone matrix prepared from different long bones and study of the osteogenic properties in vitro / 中国修复重建外科杂志

OBJECTIVE@#To measure the concentration of bone morphogenetic protein 2 (BMP-2) in demineralized bone matrix (DBM) prepared from different long bones and to evaluate the osteoinductivity of different DBM on MC3T3-E1 cells.@*METHODS@#Different bones from the same cadaver donor were used as the initial materials for making DBM, which were divided into ulna group (uDBM), humerus group (hDBM), tibia group (tDBM), and femur group (fDBM) according to the origins, and boiled DBM (cDBM) was taken as the control group. The proteins of DBM were extracted by guanidine hydrochloride, and the concentrations of BMP-2 were determined by ELISA assay. Then the DBM were co-cultured with MC3T3-E1 cells, the proliferation of MC3T3-E1 cells was observed by cell counting kit 8 (CCK-8) assay. The osteogenic differentiation ability of MC3T3-E1 cells was qualitatively observed by alizarin red, alkaline phosphatase (ALP), and Van Gieson staining, and the osteogenic differentiation ability of MC3T3-E1 cells was quantitatively analyzed by ALP content. Linear regression was used to analyze the effect of BMP-2 concentration in DBM on ALP synthesis.@*RESULTS@#There were significant differences in the concentration of BMP-2 among the DBM groups (P<0.05). The concentrations of BMP-2 in the lower limb long bone were higher than those in the upper limb long bone, and the concentration of BMP-2 in the fDBM group was about 35.5 times that in the uDBM group. CCK-8 assay showed that the cells in each group continued to proliferate within 5 days of co-culture, and the absorbance (A) values at different time points were in the order of cDBM group<uDBM group<hDBM group<tDBM group<fDBM group. After co-culture for 14 days, the expressions of ALP, calcified nodules, and collagen fibers in each group were consistent with the distribution of BMP-2 concentration in DBM. The order of ALP content from low to high was cDBM group<uDBM group<hDBM group<tDBM group<fDBM group, and the differences between the groups were significant (P<0.05). Linear regression analysis showed that y

Overview of the main biological mechanisms linked to changes in periodontal ligament stem cells and the inflammatory microenvironment / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Periodontitis is a complex chronic inflammatory disease. The invasion of pathogens induces the inflammatory microenvironment in periodontitis. Cell behavior changes in response to changes in the microenvironment, which in turn alters the local inflammatory microenvironment of the periodontium through factors secreted by cells. It has been confirmed that periodontal ligament stem cells (PDLSCs) are vital in the development of periodontal disease. Moreover, PDLSCs are the most effective cell type to be used for periodontium regeneration. This review focuses on changes in PDLSCs, their basic biological behavior, osteogenic differentiation, and drug effects caused by the inflammatory microenvironment, to provide a better understanding of the influence of these factors on periodontal tissue homeostasis. In addition, we discuss the underlying mechanism in detail behind the reciprocal responses of PDLSCs that affect the microenvironment.

Assessment of Proliferation, Clonogenic Assay, and Osteogenic Differentiation of Human Periodontal Ligament Stem Cells Following Application of Orthodontic Forces

Context: The proliferation and differentiation of human periodontal ligament stem cells (hPDLSC) into other cell types are also mediated by mechanical stresses; they might offer therapeutic benefits in tissue regeneration and angiogenesis. Objectives: The study was planned to assess the proliferation, clonogenic potential, and osteogenic differentiation of human periodontal ligament stem cells (PDLSC) following the application of light and heavy orthodontic forces. Materials and Methods: A couple forces of 50 gm (light force) were applied on the 1st premolar on the one side and 250 gm (heavy force) on the contralateral side in the upper arch of patients requiring orthodontic treatment with extraction of all 1st premolars. After 30 days, periodontal tissues were scrapped from extracted teeth for the establishment of PDLSC in vitro. PDLC from the lower premolar teeth where no orthodontic force was applied acted as the control group. Morphology, viability, proliferating rate and population doubling time, clonogenicity, and alkaline phosphatase activity were analysed. Result: The osteogenic potential was confirmed by Alizarin red staining and the expression of the osteogenic markers by qRT?PCR. The morphology, growth kinetics, potency, and osteogenic lineage characteristics inferred the application of high force reduced the proliferative ability and osteogenesis of PDLSC, though the difference was not significant. Conclusion: The established PDLSCs demonstrated their MSC?like properties based on morphology, growth kinetics, colony forming ability, and AP activity. The culture?expanded PDLSCs showed their differentiation potential into osteocytes. The application of high force reduced the proliferative ability and osteogenesis of PDLSCs, variations were not significant.differentiation

CCDC134 regulates the osteogenic differentiation of human dental pulp stem cells / 口腔疾病防治

Objective @#To study the regulatory effect of coiled-coil domain containing 134 (CCDC134) on the osteogenic differentiation of human dental pulp stem cells (hDPSCs).@*Methods @# HDPSCs were isolated and cultured from dental pulp tissue and transfected with NC-CCDC134, shCCDC134 and CCDC134 lentiviruses. They were divided into the control group, negative control group, CCDC134 downregulation (shCCDC134) group and CCDC134 overexpression (CCDC134) group. Surface markers of hDPSCs (Stro-1, CD105, CD34, CD45) were detected by flow cytometry; colony formation was analyzed by toluidine blue staining; ALP expression was estimated by ALP staining; mineralized nodule formation was evaluated by alizarin red staining; lipid droplet formation was examined by oil red staining; and gene and protein expression of CCDC134, Runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), bone morphogenetic protein-2 (BMP-2), and mothers against decapentaplegic homolog 1 (SMAD1) was detected by qPCR and western blot, respectively. Further, a BMP-2 activator (BMP-2) and inhibitor (Dorsomorphin) were used to down-regulate and up-regulate CCDC134, respectively (shCCDC134, shCCDC134+BMP-2, CCDC134, CCDC134+Dorsomorphin), in hDPSCs. The hDPSC aggregates were subcutaneously transplanted into nude mice for 2 months, and new bone formation was detected by H&E staining. The BMP-2/SMAD1 signaling in each group was detected by qPCR.@*Results@#hDPSCs showed high expression of mesenchymal markers and low expression of hematopoietic markers. Compared with the control group, the expression of CCDC134 was increased in the osteogenic-induced hDPSCs (P < 0.05). Compared with the negative control group, the expression of CCDC134 was decreased in the shCCDC134 group, whereas it was increased in the CCDC134 group (P < 0.05). The mineralized nodules, osteogenic genes and proteins in the shCCDC134 group were decreased (P < 0.05), while they were increased in the CCDC134 group (P < 0.05). The expression of BMP-2/SMAD1 signaling decreased in the shCCDC134 group, while it increased in the CCDC134 group (P < 0.05). Compared to the shCCDC134 group, osteogenic genes and proteins increased in the shCCDC134+BMP-2 group, and subcutaneous new bone formation increased in nude mice (P < 0.05). The indexes of the CCDC134+Dorsomorphin group decreased compared with the CCDC134 group (P < 0.05).@*Conclusion@#CCDC134 promotes the osteogenic differentiation of hDPSCs by regulating the BMP-2/SMAD1 signaling pathway.

Effect of silencing HDAC9 on the proliferation and differentiation of periodontal ligament stem cells / 口腔疾病防治

Objective @#To investigate the effect of silencing histone deacetylase 9 (HDAC9) expression on the proliferation and osteogenic differentiation of periodontal ligament stem cells (PDLSCs).@*Methods@# PDLSCs were isolated, cultured and identified in vitro. An siRNA construct specific for HDAC9 was transfected into PDLSCs (siHDAC9 group), and a nontargeting siRNA was used as a control (siNC group). The interference effect was determined by qRT-PCR. The cell cycle progression of PDLSCs was detected using flow cytometry. The proliferation activity of PDLSCs was detected via CCK-8 assay. Western blotting was used to detect the protein expression of proliferating cell nuclear antigen (PCNA). The mRNA expression of runt-related transcription factor 2 (RUNX2) and alkaline phosphatase (ALP) was investigated by qRT-PCR. The protein expression of RUNX2 was detected by western blotting. In addition, the formation of mineralized nodules was assessed by alizarin red staining. @*Results@#Compared with that in the siNC group, the mRNA expression of HDAC9 in the siHDAC9 group was lower (P < 0.01). Moreover, compared with those in the siNC group, the proliferation index (P<0.01), proliferation activity (P<0.05) and protein expression of PCNA (P<0.01) in the siHDAC9 group were all increased. Compared with the siNC group, the siHDAC9 group exhibited higher mRNA expression of RUNX2 and ALP (P < 0.05), and the protein expression of RUNX2 showed the same results (P < 0.01). The results of alizarin red staining showed that compared to the siNC group, the siHDAC9 group formed more mineralized nodules.@* Conclusion@#Silencing HDAC9 expression can promote the proliferation and osteogenic differentiation of PDLSCs.

Effects of fluoride exposure on proliferation, apoptosis and osteogenic differentiation of mouse bone marrow mesenchymal stem cells / 中华地方病学杂志

Objective:To investigate the effects of fluoride exposure on proliferation, apoptosis and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in mice.Methods:BMSCs were isolated and cultured from femur bone marrow of C57BL/6 mice (6 - 8 weeks). The cells in passage 3 were used to detect the surface markers of stem cells by flow cytometry. The cells were cultured in media with a final fluoride concentration of 0.0, 0.1, 1.0, 5.0, 10.0, 15.0, 20.0 and 40.0 mg/L, respectively. The effects of different fluoride concentrations on BMSCs cell proliferation (CCK8 method), apoptosis (flow cytometry analysis), osteogenic differentiation ability [alizarin red and alkaline phosphatase (ALP) staining] were detected. Western blot was applied to detect the levels of apoptosis-related proteins [poly ADP-ribose polymerase (PARP)], mitogen-activated protein kinase (MAPK) pathway member proteins [extracellular regulated protein kinase 1/2 (ERK1/2), c-Jun amino-terminal kinase (JNK), p38 and phosphorylated ERK, JNK, p38 (p-ERK, p-JNK, p-p38)], osteogenic differentiation-related protein [Runt-related transcription factor 2 (Runx2), ALP] and Wnt/β-catenin pathway member proteins [glycogen synthase kinase-3β (GSK3β), phosphorylated GSK3β (p-GSK3β) and β-catenin]. Immunocytofluorescense staining was applied to evaluate the expression levels of p-GSK3β and β-catenin. The two pathways (MAPK and Wnt/β-catenin) were blocked by SP600125 and DKK-1, respectively, to testify their involvement in mechanisms of apoptosis and osteogenic differentiation.Results:The mouse BMSCs were successfully isolated and cultured. Flow cytometry analysis showed that the mesenchymal stem cell surface biomarkers (CD73, CD90 and CD105) were positively expressed. The comparison of cell proliferation at three time points (24, 48 and 72 h) in each concentration group was statistically significant ( F = 65.36, 160.04 and 365.32, P < 0.001), and the comparison of early apoptosis (24 h) in each concentration group was statistically significant ( F = 214.04, P < 0.001); compared with the 0.0 mg/L group, the cell proliferation in 15.0, 20.0 and 40.0 mg/L groups decreased, and the early apoptosis rate in 10.0, 15.0 and 20.0 mg/L groups increased ( P < 0.05). When cells were treated with 15.0 mg/L fluoride for 0 - 24 h, the p-JNK/JNK ratio was higher at 2, 4, 8, 12, 18 and 24 h compared with that at 0 min ( P < 0.05); compared with the fluoride group (15.0 mg/L), the early apoptosis rate of cells after SP600125 block decreased ( P < 0.05), and the protein expression levels of PARP and p-JNK decreased ( P < 0.05). After osteogenic induction, compared with the 0.0 mg/L group, in 0.1 and 1.0 mg/L groups ALP staining was enhanced and the number of calcified nodules increased, and the protein expression levels of Runx2 and ALP in the 0.1 and 1.0 mg/L groups were higher ( P < 0.05). After osteogenic induction, compared with the 0.0 mg/L group, the p-GSK3β/GSK3β ratio and β-catenin protein level were significantly higher in the 0.1 and 1.0 mg/L groups ( P < 0.05); and compared with the fluoride group (1.0 mg/L), addition of DKK-1 significantly decreased the protein expression levels of p-GSK3β and β-catenin and reduced the nuclear entry of β-catenin, and ALP staining decreased and the number of calcified nodules decreased. Conclusions:High concentration of fluoride (> 10.0 mg/L) inhibits the proliferation and promotes apoptosis of BMSCs, while low concentration of fluoride (0.1, 1.0 mg/L) promotes osteogenic differentiation. The MAPK/JNK pathway and the classical Wnt pathway are involved in the above cellular processes, respectively.

Preparation and osteogenic properties of hydrogel scaffolds with different concentrations of laponite / 中华创伤骨科杂志

Objective:To prepare the hydrogel scaffolds with different concentrations of laponite and compare their osteogenic properties.Methods:The scaffolds of gelatin/sodium alginate hydrogel into which laponite was added according to the mass ratios of 0%, 1%, 2%, and 3% were assigned into groups T0, T1, T2, and T3. In each group, the compressive modulus was measured and the leaching solution for 24 h extracted to measure the ion release. Bone marrow mesenchymal stem cells (BMSCs) were cultured in the extract medium from each group and common medium (blank group) ( n=3) in the in vitro experiments to determine the expression of osteogenic genes Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP) and type I collagen after 7 days of culture. In the in vivo experiments, the scaffolds were implanted into the femoral condyle defects in rats, and a blank group with no scaffolds was set. The bone repair in each group was evaluated by hematoxylin-eosin(HE) staining and immunohistochemical staining. Results:The compressive modulus in group T2 [(139.05±6.43) kPa] was significantly higher than that in groups T0, T1 and T3 [(68.83±3.76) kPa, (101.18±3.68) kPa and (125.40±3.28) kPa] ( P<0.05). The ion contents of lithium, magnesium and silicon released from the 24 h leaching solution in group T2 were (0.031±0.005) μg/mL, (3.047±0.551) μg/mL and (5.243±0.785) μg/mL, insignificantly different from those in group T3 ( P> 0.05) but significantly larger than those in group T1 ( P>0.05). The in vitro experiments showed that the expression levels of Runx2, ALP and type I collagen in group T2 were 1.59±0.11, 2.02±0.08 and 1.06±0.17, significantly higher than those in the other groups ( P<0.05). HE staining showed that the implanted hydrogel was tightly bound to the bone tissue. Immunohistochemical staining showed that the numbers of Runx2 and osteocalcin positive cells in group T2 were significantly higher than those in the other groups. Conclusions:With ideal biocompatibility, hydrogel scaffolds with different concentrations of laponite can slowly release the decomposed ions of lithium, magnesium and silicon to promote the osteogenic differentiation of BMSCs and the repair of bone defects in vivo. A 2% concentration of laponite in the hydrogel scaffolds may result in the best results.

Bax inhibitor 1 inhibits vascular calcification in mice by activating optic atrophy 1 expression / 南方医科大学学报

OBJECTIVE@#To investigate the effects of Bax inhibitor 1 (BI- 1) and optic atrophy protein 1 (OPA1) on vascular calcification (VC).@*METHODS@#Mouse models of VC were established in ApoE-deficient (ApoE-/-) diabetic mice by high-fat diet feeding for 12 weeks followed by intraperitoneal injections with Nε-carboxymethyl-lysine for 16 weeks. ApoE-/- mice (control group), ApoE-/- diabetic mice (VC group), ApoE-/- diabetic mice with BI-1 overexpression (VC + BI-1TG group), and ApoE-/- diabetic mice with BI-1 overexpression and OPA1 knockout (VC+BI-1TG+OPA1-/- group) were obtained for examination of the degree of aortic calcification using von Kossa staining. The changes in calcium content in the aorta were analyzed using ELISA. The expressions of Runt-related transcription factor 2 (RUNX2) and bone morphogenetic protein 2 (BMP-2) were detected using immunohistochemistry, and the expression of cleaved caspase-3 was determined using Western blotting. Cultured mouse aortic smooth muscle cells were treated with 10 mmol/L β-glycerophosphate for 14 days to induce calcification, and the changes in BI-1 and OPA1 protein expressions were examined using Western blotting and cell apoptosis was detected using TUNEL staining.@*RESULTS@#ApoE-/- mice with VC showed significantly decreased expressions of BI-1 and OPA1 proteins in the aorta (P=0.0044) with obviously increased calcium deposition and expressions of RUNX2, BMP-2 and cleaved caspase-3 (P= 0.0041). Overexpression of BI-1 significantly promoted OPA1 protein expression and reduced calcium deposition and expressions of RUNX2, BMP-2 and cleaved caspase-3 (P=0.0006). OPA1 knockdown significantly increased calcium deposition and expressions of RUNX2, BMP-2 and cleaved caspase-3 in the aorta (P=0.0007).@*CONCLUSION@#BI-1 inhibits VC possibly by promoting the expression of OPA1, reducing calcium deposition and inhibiting osteogenic differentiation and apoptosis of the vascular smooth muscle cells.

Research progress on metformin in the prevention and treatment for oral diseases / 口腔疾病防治

@#Metformin is currently the first-line drug for the treatment of diabetes. In addition to its hypoglycemic effect, it has also been found to have other potential effects, such as anti-inflammatory, odontogenic differentiation-promoting, osteogenic differentiation-promoting, and antitumor effects. Previous studies have shown that metformin can promote the healing of periapical lesions, and its mechanism may be related to the promotion of osteogenic differentiation and the induction of dental pulp cell differentiation by activation of adenylate-activated protein kinase by dimethyldiphosphate. Clinical indexes, such as the probing depth, attachment loss level and probing bleeding index, were significantly improved in patients with periodontitis treated with metformin, which may play a role in the prevention and treatment of periodontal disease by promoting the proliferation, migration and osteogenic differentiation of periodontal ligament stem cells. Metformin has been proven to inhibit the growth and proliferation of tumor cells and plays an important role in the prevention and treatment of oral tumors such as oral squamous cell carcinoma. At present, research remains in the in vitro and animal experimental stage, and the related mechanism needs to be further explored. Clinical trials remain in the evaluation of clinical indicators, so large-scale, long-term, multicenter, randomized controlled clinical trials need to be further developed