OBJECTIVE: To investigate the effect of stretch on long non-coding RNA taurine upregulated gene 1 (TUG1)-mediated miR-545-3p/cannbinoida receptor 2 (CNR2) pathway regulating bone regeneration in the distraction area of rats during distraction osteogenesis. METHODS: Thirty-six 10-week-old male Sprague Dawley rats were randomly divided into 3 groups ( n=12 in each group): group A (femoral fracture+injection of interfering RNA), group B (distraction osteogenesis+injection of interfering RNA), and group C (distraction osteogenesis+injection of TUG1). Groups A and B were injected with 60 μg of interfering RNA at the beginning of incubation period (immediate after operation), the beginning of distraction phase (7 days after operation), and the end of distraction phase (21 days after operation), and group C was injected with 60 μg of synthetic TUG1 in vivo interfering sequence at the same time. The general situation of rats in each group was observed during the experiment. The mineralization of fracture space or distraction area was observed by X-ray films at 21, 35, and 49 days after operation. At 49 days after operation, the samples of the distraction area were taken for HE staining to observe the mineralization, and real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the expressions of osteoblast-related genes such as TUG1, miR-545-3p, CNR2, alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN). Blood samples were collected from the abdominal aorta of the rats, and the expressions of ALP and C terminal telopeptide of type Ⅰ (CTX-Ⅰ) protein were detected by ELISA assay. RESULTS: The results of X-ray film and HE staining observations showed that osteogenesis in group C was superior to groups A and B at the same time point. The results of qRT-PCR showed that the relative mRNA expressions of TUG1, CNR2, ALP, OCN, and OPN in group C were significantly higher than those in group A and group B, and the relative mRNA expression of miR-545-3p in group C was significantly lower than that in group A and group B ( P<0.05). The relative mRNA expressions of TUG1 and ALP in group B were significantly higher than those in group A, and the relative mRNA expression of miR-545-3p in group B was significantly lower than that in group A ( P<0.05). There was no significant difference in the relative mRNA expressions of CNR2, OCN, and OPN between group A and group B ( P>0.05). The results of ELISA showed that the expressions of ALP and CTX-Ⅰ protein were significantly higher in group C than in group A and group B, and in group B than in group A ( P<0.05). CONCLUSION Under the action of stretch, the expression of TUG1 in the femoral distraction area of rats increases, which promotes the expression of CNR2 by inhibiting the expression of miR-545-3P, which is helpful to the mineralization of the extension area and osteogenesis.
Animals ; MicroRNAs/genetics* ; Rats, Sprague-Dawley ; Male ; Osteogenesis, Distraction/methods* ; Rats ; RNA, Long Noncoding/metabolism* ; Osteopontin/genetics* ; Osteogenesis ; Bone Regeneration ; RNA, Small Interfering/genetics* ; Osteocalcin/genetics* ; Alkaline Phosphatase/metabolism* ; Osteoblasts/cytology* ; Signal Transduction ; Femoral Fractures/surgery*

OBJECTIVE: To initially investigate the function of neuronal pentraxin 1 (NPTX1) gene on osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs). METHODS: hBMSCs were induced to undergo osteogenic differentiation, and then RNA was collected at different time points, namely 0, 3, 7, 10 and 14 d. The mRNA expression levels of key genes related with osteogenic differentiation, including runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osteocalcin (OCN), and NPTX1, were detected on the basis of quantitative real-time polymerase chain reaction (qPCR) technology. In order to establish a stable NPTX1-knockdown hBMSCs cell line, NPTX1 shRNA lentivirus was constructed and used to infect hBMSCs. ALP staining, alizarin red (AR) staining, and qPCR were employed to assess the impact of NPTX1-knockdown on the osteogenic differentiation ability of hBMSCs. RESULTS: The results showed that during the osteogenic differentiation of hBMSCs in vitro, the mRNA expression levels of osteogenic genes RUNX2, ALP and OCN significantly increased compared with 0 d, while NPTX1 expression decreased markedly (P < 0.01) as the osteogenic induction period exten-ded. At 72 h post-infection with lentivirus, the result of qPCR indicated that the knockdown efficiency of NPTX1 was over 60%. After knocking down NPTX1 in hBMSCs, RNA was extracted from both the NPTX1-knockdown group (sh NPTX1 group) and the control group (shNC group) cultured in regular proliferation medium. The results of qPCR showed that the expression levels of osteogenic-related genes RUNX2 and osterix (OSX) were significantly higher in the sh NPTX1 group compared with the shNC group (P < 0.01). ALP staining revealed a significantly deeper coloration in the sh NPTX1 group than in the shNC group at the end of 7 d of osteogenic induction. AR staining demonstrated a marked increase in mineralized nodules in the sh NPTX1 group compared with the shNC group at the end of 14 d of osteogenic induction. CONCLUSION NPTX1 exerts a modulatory role in the osteogenic differentiation of hBMSCs, and its knockdown has been found to enhance the osteogenic differentiation of hBMSCs. This finding implies that NPTX1 could potentially serve as a therapeutic target for the treatment of osteogenic abnormalities, including osteoporosis.
Humans ; Mesenchymal Stem Cells/cytology* ; Osteogenesis/genetics* ; Cell Differentiation/genetics* ; Nerve Tissue Proteins/genetics* ; Cells, Cultured ; C-Reactive Protein/genetics* ; RNA, Small Interfering/genetics* ; Core Binding Factor Alpha 1 Subunit/metabolism* ; Bone Marrow Cells/cytology* ; Gene Knockdown Techniques ; Osteocalcin/metabolism* ; Alkaline Phosphatase/metabolism* ; RNA, Messenger/metabolism*

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.
Animals ; Female ; Mice ; Cell Differentiation ; Cells, Cultured ; Core Binding Factor Alpha 1 Subunit/metabolism* ; Cyclic AMP Response Element-Binding Protein/metabolism* ; Mesenchymal Stem Cells ; Mice, Inbred C57BL ; MicroRNAs/metabolism* ; Osteocalcin/metabolism* ; Osteogenesis/genetics* ; RNA, Messenger/genetics*

Objective: To investigate the effect of resveratrol on peak bone mineral density and bone mass in growing rats. Methods: Thirty-six female healthy Wistar rats were randomly divided into control group, icariin group and resveratrol group with 12 rats in each group. Icariin (25 mg·kg^-1·d^-1), resveratrol (8.4 mg·kg^-1·d^-1) or equal volume of distilled water were given by gavage to icariin group, resveratrol group and control group, respectively. The rats were sacrificed after 12 weeks. The organ indexes were calculated and pathology sections were observed; the bone mineral density (BMD), bone biomechanics, serum bone metabolism index, and results of micro-CT scan were analyzed. Results: During the experiment, the body weight of rats showed an increasing trend and there was no significant difference among three groups (P0.05). There were no significant differences in organ index of vital organs and pathological changes among the groups (all P0.05). Compared with the control group, the whole body BMD, and the BMDs of femur and vertebrae in icariin and resveratrol groups were significantly increased after 12 weeks (all P<0.05). The maximum load values of femur and vertebrae, as well as elastic modulus of vertebrae in icariin and resveratrol groups were significantly higher than those in control group (P<0.05 or P<0.01). Micro-CT scan showed that the volumetric BMD, number of trabecular, trabecular thickness and bone volume/tissue volume of the cancellous bone in icariin and resveratrol groups were significantly higher and the trabecular separation was significantly lower than those in the control group (P<0.05 or P<0.01); while there was no significant difference in volumetric BMD of cortical bone for femur. The contents of osteocalcin in icariin and resveratrol groups were significantly higher than those in control group (all P<0.05), while the contents of tartarte-resistant acid phosphatase 5b (TRACP5b) were significantly lower than those in control group (all P<0.05).Conclusion: Resveratrol can inhibit bone resorption and enhance bone formation, so as to improve the peak bone mass and bone density, enhance bone strength and improve the microstructure of bone tissue in young rats.
Animals ; Bone Density ; drug effects ; Bone and Bones ; diagnostic imaging ; drug effects ; Female ; Femur ; drug effects ; Osteocalcin ; genetics ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Rats, Wistar ; Resveratrol ; pharmacology ; Tartrate-Resistant Acid Phosphatase ; genetics ; metabolism

OBJECTIVETo observe the expression profiles of osteoblast-related genes in human mesenchymal stem cells (MSCs) derived from bone marrow during osteogenic differentiation.

METHODSMSCs were induced to differentiate with MSC osteogenic differentiation medium for 7, 14, 21 and 28 days respectively. Alizarin Red staining was used to detect matrix mineralization. Expression of osteoblast-related genes, including osteocalcin, osteopontin, Runt-related transcription factor 2 (Runx2), alkaline phosphatase and collagen type 1, was assessed with quantitative reverse transcription-polymerase chain reaction.

RESULTSOn day 14 after induction of differentiation, cells were stained positively with Alizarin Red. The expression levels of these genes exhibited an upward trend as induction time was prolonged. Exposure to osteogenic differentiation medium less than 21 days did not significantly induce osteocalcin expression; osteocalcin expression levels in the differentiated cells induced for 21 and 28 days were 1.63 and 2.46 times as high as the undifferentiated cells respectively (all P<0.05). Stimulation with MSC osteogenic differentiation medium over 14 days significantly enhanced bone marrow-derived MSCs to express osteopontin and Runx2 genes (all P<0.05). Osteogenic differentiation medium could significantly induce the expressions of alkaline phosphatase and collagen type1 genes (all P<0.05). Their expressions reached the peak levels on day 21, which were increased more than 4- and 3-fold respectively.

CONCLUSIONHuman bone marrow-derived MSCs could exhibit the sequential expression pattern of osteoblast marker genes during osteogenic differentiation in vitro.

Alkaline Phosphatase ; genetics ; Cell Differentiation ; Cells, Cultured ; Collagen Type I ; genetics ; Core Binding Factor Alpha 1 Subunit ; genetics ; Genetic Markers ; Humans ; Mesenchymal Stromal Cells ; metabolism ; Osteocalcin ; genetics ; Osteogenesis ; Transcriptome

PURPOSE: This study is intended to investigate the effects of plants or plant-derived antioxidants on prevention of osteoporosis through the maintenance of reactive oxygen species (ROS) at a favorable level. MATERIALS AND METHODS: In this study, a novel antioxidant, namely 3,4,5-Trihydroxy-N-[4-(5-hydroxy-6-methoxy-pyrimidin-4-ylsulfamoyl)-phenyl]-benzamide (ZXHA-TC) was synthesized from gallic acid and sulfadimoxine. Its effect on osteoblast metabolism was investigated via the detection of cell proliferation, cell viability, production of ROS, and expression of osteogenic-specific genes including runt-related transcription factor 2 (RUNX2), bone sialoprotein (BSP), osteocalcin (OCN), alpha-1 type I collagen (COL1A1), and osteogenic-related proteins after treatment for 2, 4, and 6 days respectively. RESULTS: The results showed that ZXHA-TC has a stimulating effect on the proliferation and osteogenic differentiation of primary osteoblasts by promoting cell proliferation, cell viability, and the expression of genes BSP and OCN. Productions of bone matrix and mineralization were also increased by ZXHA-TC treatment as a result of up-regulation of COL1A1 and alkaline phosphatase (ALP) at the early stage and down-regulation of both genes subsequently. A range of 6.25x10(-3) microg/mL to 6.25x10(-1) microg/mL is the recommended dose for ZXHA-TC, within which 6.25x10(-2) microg/mL showed the best performance. CONCLUSION: This study may hold promise for the development of a novel agent for the treatment of osteoporosis.
Alkaline Phosphatase/metabolism ; Bone Morphogenetic Proteins/pharmacology ; Cell Differentiation/*drug effects ; Cell Proliferation/*drug effects ; Collagen Type I/genetics ; Core Binding Factor Alpha 1 Subunit ; Down-Regulation ; Gallic Acid ; Osteoblasts/*drug effects ; Osteocalcin/metabolism ; Osteogenesis/drug effects ; Osteoporosis/*prevention & control ; Reactive Oxygen Species ; Up-Regulation

PURPOSE: To investigate the molecular responses of various genes and proteins related to disc degeneration upon treatment with cytokines that affect disc-cell proliferation and phenotype in living human intervertebral discs (IVDs). Responsiveness to these cytokines according to the degree of disc degeneration was also evaluated. MATERIALS AND METHODS: The disc specimens were classified into two groups: group 1 (6 patients) showed mild degeneration of IVDs and group 2 (6 patients) exhibited severe degeneration of IVDs. Gene expression was analyzed after treatment with four cytokines: recombinant human bone morphogenic protein (rhBMP-2), transforming growth factor-beta (TGF-beta), interleukin-1beta (IL-1beta), and tumor necrosis factor-alpha (TNF-alpha). Molecular responses were assessed after exposure of cells from the IVD specimens to these cytokines via real-time polymerase chain reaction and immunofluorescence staining. RESULTS: mRNA gene expression was significantly greater for aggrecan, type I collagen, type II collagen, alkaline phosphatase, osteocalcin, and Sox9 in group 1 than mRNA gene expression in group 2, when the samples were not treated with cytokines. Analysis of mRNA levels for these molecules after morphogen treatment revealed significant increases in both groups, which were much higher in group 1 than in group 2. The average number of IVD cells that were immunofluorescence stained positive for alkaline phosphatase increased after treatment with rhBMP-2 and TGF-beta in group 1. CONCLUSION: The biologic responsiveness to treatment of rhBMP-2, TGF-beta, TNF-alpha, and IL-1beta in the degenerative living human IVD can be different according to the degree of degeneration of the IVD.
Adult ; Aggrecans/genetics/metabolism ; Alkaline Phosphatase/genetics/metabolism ; Biological Products/pharmacology/*therapeutic use ; Bone Morphogenetic Protein 2/pharmacology/therapeutic use ; Collagen Type I/genetics/metabolism ; Collagen Type II/genetics/metabolism ; Cytokines/*pharmacology/*therapeutic use ; Female ; Fluorescent Antibody Technique ; Gene Expression Regulation/drug effects ; Humans ; Interleukin-1/pharmacology/therapeutic use ; Intervertebral Disc/*drug effects/*pathology ; Intervertebral Disc Degeneration/*drug therapy/genetics/*pathology ; Male ; Middle Aged ; Osteocalcin/genetics/metabolism ; RNA, Messenger/genetics/metabolism ; Recombinant Proteins/pharmacology/therapeutic use ; SOX9 Transcription Factor/genetics/metabolism ; Transforming Growth Factor beta/pharmacology/therapeutic use ; Tumor Necrosis Factor-alpha/pharmacology

Retaining or improving periodontal ligament (PDL) function is crucial for restoring periodontal defects. The aim of this study was to evaluate the physiological effects of low-power laser irradiation (LPLI) on the proliferation and osteogenic differentiation of human PDL (hPDL) cells. Cultured hPDL cells were irradiated (660 nm) daily with doses of 0, 1, 2 or 4 J⋅cm(-2). Cell proliferation was evaluated by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, and the effect of LPLI on osteogenic differentiation was assessed by Alizarin Red S staining and alkaline phosphatase (ALP) activity. Additionally, osteogenic marker gene expression was confirmed by real-time reverse transcription-polymerase chain reaction (RT-PCR). Our data showed that LPLI at a dose of 2 J⋅cm(-2) significantly promoted hPDL cell proliferation at days 3 and 5. In addition, LPLI at energy doses of 2 and 4 J⋅cm(-2) showed potential osteogenic capacity, as it stimulated ALP activity, calcium deposition, and osteogenic gene expression. We also showed that cyclic adenosine monophosphate (cAMP) is a critical regulator of the LPLI-mediated effects on hPDL cells. This study shows that LPLI can promote the proliferation and osteogenic differentiation of hPDL cells. These results suggest the potential use of LPLI in clinical applications for periodontal tissue regeneration.
Adenine ; analogs & derivatives ; pharmacology ; Adenylyl Cyclase Inhibitors ; Alkaline Phosphatase ; analysis ; genetics ; radiation effects ; Anthraquinones ; Bone Morphogenetic Protein 2 ; genetics ; Calcium ; metabolism ; radiation effects ; Cell Culture Techniques ; Cell Differentiation ; radiation effects ; Cell Line ; Cell Proliferation ; radiation effects ; Coloring Agents ; Core Binding Factor Alpha 1 Subunit ; genetics ; Cyclic AMP ; antagonists & inhibitors ; radiation effects ; Gene Expression ; radiation effects ; Humans ; L-Lactate Dehydrogenase ; analysis ; Lasers, Semiconductor ; Low-Level Light Therapy ; instrumentation ; Osteocalcin ; genetics ; Osteogenesis ; genetics ; radiation effects ; Periodontal Ligament ; cytology ; radiation effects ; Radiation Dosage ; Real-Time Polymerase Chain Reaction ; Reverse Transcriptase Polymerase Chain Reaction ; Tetrazolium Salts ; Thiazoles

OBJECTIVETo investigate the effects of naringin on the proliferation, differention and maturaion of rat calvarial osteoblasts (ROB).

METHODSegregated neonatal SD rat skull, enzyme digestion to obtain ROB. The culture medium was replaced every three days. Serial subcultivation proceeded when cells covered with 80% culture dish. Naringin supplemented into the culture at 1 x 10(-4), 1 x 10(-5), 1 x 10(-6), 1 x 10(-7) mol x L(-1) respectively. MTT method was adopted in proliferation analysis and the activity of ALP was examined after induced 9 days. Search the best concentration and supplemented into the medium, then the osteogenic differentiation markers including the secretion amount of osteocalcin, osteopontin and bone morphogenetic protein-2 were compared between the naringin-supplemented group and the control. Total RNA was isolated and the mRNA level of bFGF, IGF-1, Runx-2, Osterix, ERa and ERbeta was investigated by Real time RT-PCR. Total protein also was isolated and the expression ERa, ERbeta and collagen I was examined by Western blot. After the addition of ICI 182.780, an inhibitor of the estrogen signal pathway, these index also was examined and the changes were compared.

RESULTThe ROB proliferation was motivated by naringin dose-dependently. And it evidently leads to osteogenic process and maturation. 1 x 10(-5) mol x L(-1) is the best concentration. Naringin improved the secretion of osteocalcin, osteopontin, bone morphogenetic protein-2 and collagen I significantly. Besides, it can also enhanced the mRNA level of bFGF, IGF-1, Runx-2, Osterix, ERalpha and ERbeta. While all these effects can be restrained by ICI 182.780.

CONCLUSIONThe naringin with final concentration of 1 x 10(-5) mol x L(-1) enhances the osteogenic differentiation and maturation of ROB significantly, while the promoting effects vanished after the addition of ICI 182.780. These results suggesting that naringin is one of the phytoestrogens and have the activity of bone formation may via estrogen signal pathway, it can be developed into a new drug for osteoporosis therapy.

Alkaline Phosphatase ; genetics ; metabolism ; Animals ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Flavanones ; pharmacology ; Insulin-Like Growth Factor I ; genetics ; metabolism ; Osteoblasts ; cytology ; drug effects ; metabolism ; Osteocalcin ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Skull ; cytology ; drug effects ; metabolism

BACKGROUNDBone morphogenetic protein 9 (BMP9) and Wnt/β-catenin signaling pathways are able to induce osteogenic differentiation of mesenchymal stem cells (MSCs), but the role of Wnt/β-catenin signaling pathway in BMP9-induced osteogenic differentiation is not well understood. Thus, our experiment was undertaken to investigate the interaction between BMP9 and Wnt/β-catenin pathway in inducing osteogenic differentiation of MSCs.

METHODSC3H10T1/2 cells were infected with recombinant adenovirus expressing BMP9, Wnt3a, and BMP9+Wnt3a. ALP, the early osteogenic marker, was detected by quantitative and staining assay. Later osteogenic marker, mineral calcium deposition, was determined by Alizarin Red S staining. The expression of osteopotin (OPN), osteocalcin (OC), and Runx2 was analyzed by Real time PCR and Western blotting. In vivo animal experiment was carried out to further confirm the role of Wnt3a in ectopic bone formation induced by BMP9.

RESULTSThe results showed that Wnt3a enhanced the ALP activity induced by BMP9 and increased the expressions of OC and OPN, with increase of mineral calcium deposition in vitro and ectopic bone formation in vivo. Furthermore, we also found that Wnt3a increased the level of Runx2, an important nuclear transcription factor of BMP9.

CONCLUSIONCanonical Wnt/β-catenin signal pathway may play an important role in BMP9-induced osteogenic differentiation of MSCs, and Runx2 may be a linkage between the two signal pathways.

Blotting, Western ; Cell Differentiation ; genetics ; physiology ; Core Binding Factor Alpha 1 Subunit ; genetics ; metabolism ; Growth Differentiation Factor 2 ; genetics ; metabolism ; Humans ; Osteocalcin ; genetics ; metabolism ; Osteogenesis ; genetics ; physiology ; Wnt3A Protein ; genetics ; metabolism