The prevailing model of osteology is that bones constantly undergo a remodeling process, and that the differentiation and functions of osteoblasts are partially regulated by leptin through different central hypothalamic pathways. The finding that bone remodeling is regulated by leptin suggested possible endocrinal effects of bones on energy metabolism. Recently, a reciprocal relationship between bones and energy metabolism was determined whereby leptin influences osteoblast functions and, in turn, the osteoblast-derived protein osteocalcin influences energy metabolism. The metabolic effects of bones are caused by the release of osteocalcin into the circulation in an uncarboxylated form due to incomplete gamma-carboxylation. In this regard, the Esp gene encoding osteotesticular protein tyrosine phosphatase is particularly interesting because it may regulate gamma-carboxylation of osteocalcin. Novel metabolic roles of osteocalcin have been identified, including increased insulin secretion and sensitivity, increased energy expenditure, fat mass reduction, and mitochondrial proliferation and functional enhancement. To date, only a positive correlation between osteocalcin and energy metabolism in humans has been detected, leaving causal effects unresolved. Further research topics include: identification of the osteocalcin receptor; the nature of osteocalcin regulation in other pathways regulating metabolism; crosstalk between nutrition, osteocalcin, and energy metabolism; and potential applications in the treatment of metabolic diseases.
Bone Remodeling/physiology ; Bone and Bones/*metabolism ; *Energy Metabolism ; Humans ; Leptin/metabolism ; Osteocalcin/genetics/*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

OBJECTIVE: To investigate the effect of adiponectin on the osteoblast differentiation and its signal transduction. METHODS: Adipopnectin receptor (AdipoR) was detected by immunoblot analysis. Alkaline phosphatase (ALP) activity was measured by enzyme-linked immunosorbent assay. Osteocalcin was measured by a specific radioimmunoassay kit, and the extent of mineralized matrix was determined. RNA interference was used to down-regulate the expression of AdipoR1 in human osteoblasts, and the effect of adiponectin on osteoblast differentiation was investigated. RESULTS: Only AdipoR1 protein was detected in human osteoblasts. Adiponectin could promote osteoblast differentiation, and result in a dose-dependent increase in ALP activity, osteocalcin secretion, and an increase in mineralized nodules. Suppression of AdipoR1 with siRNA could abolish the adiponectin induced ALP expression. Adiponectin could induce the activation of p38 and JNK, but not ERK1/2 in osteoblasts, and the pretreatment of osteoblasts with the p38 inhibitor (SB203580) could block the adiponectin-induced ALP activity. CONCLUSION Adiponectin can induce human osteoblast differentiation via AdipoR1/p38 pathway.
Adiponectin ; pharmacology ; Alkaline Phosphatase ; metabolism ; Cell Differentiation ; drug effects ; Cells, Cultured ; Humans ; Osteoblasts ; cytology ; metabolism ; Osteocalcin ; analysis ; RNA, Small Interfering ; genetics ; Receptors, Adiponectin ; biosynthesis ; Signal Transduction

OBJECTIVETo investigate the changes of mRNA and protein expression of CaN in the bone of rats with chronic fluorosis, and the mechanism of skeletal fluorosis.

METHODSThirty-six SD rats were divided into three groups (12 in each group, half male and half female selected according to body weight): control, low-dose and high-dose fluorosis groups. Controls were fed tap water (NaF < 0.5 mg/L), experimental animals in the low- or high-dose groups were fed water containing NaF of 5.0 and 50.0 mg/L, respectively. The rats were sacrificed after 6 months of treatment with fluoride. The serum was kept for testing bone metabolic marker bone gla protein (BGP) by enzyme-linked immunosorbent assay (ELISA), the protein and mRNA levels of CaN in distal femur of the rats with chronic flurosis were assessed by immunohistochemistry and in-situ hybridization.

RESULTSThe levels of BGP (1.99 ± 0.62, 2.38 ± 0.16)µg/L in the low- or high-dose fluorosis groups were higher than that in the control group (0.15 ± 0.03) µg/L; and the high fluorosis group showed higher level than the low fluorosis group (all P < 0.05). Compared to the control group (131.11 ± 1.95, 111.82 ± 2.39), the protein and mRNA levels of CaN were higher in the low- or high-dose fluorosis groups (142.69 ± 1.17, 157.54 ± 1.88 and 121.28 ± 3.27, 134.63 ± 3.19, respectively), and the high fluorosis group showed higher levels than the low fluorosis group (all P < 0.05).

CONCLUSIONSBGP content could be used as a bone metabolic index in endemic fluorosis disease. Fluoride might up-regulate the mRNA and protein expression of CaN, and the changes in CaN level may be involved in the increase of the bone turnover and could be one of the pathogenetic factors in fluorosis.

Animals ; Bone and Bones ; metabolism ; Calcineurin ; genetics ; metabolism ; Female ; Fluoride Poisoning ; metabolism ; pathology ; Fluorides ; metabolism ; urine ; Fluorosis, Dental ; metabolism ; pathology ; Male ; Osteoblasts ; metabolism ; Osteocalcin ; blood ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley ; Sodium Fluoride ; poisoning

This study sought to elucidate the effect of mechanical strain on the differentiation of mesenchymal stem cells into osteoblasts. Under the conditons of inducing osteoblasts, Immunohistochemical methods and RT-PCR technology were applied in osteogenic supplements medium to detect: (1) the expression of Alkaline phosphatase (ALP), Type I collagen (COL I ), Osterx (Osx) and Osteocalcin (OCN) mRNA, with cyclic strain (3%, 0.5 Hz) applied for 15 min, 30 min, 1 h, 2 h, 4 h, 3 d, 7 d, 14 d; (2) the expression of Osx mRNA and OCN mRNA with 3% strain for 1 h. The results showed: (1) ALP mRNA expression was higher at 7 days; COL I mRNA expression was greater obviously at 7 days and 14 days than that at 3 days and that of the unstrained cells; (2) the expression of Osx mRNA was up-regulated after 15min by strain stimulation,which was significantly increased at 30 min and 1 h in the unstrained cells. The expression of OCN mRNA was not affected in the unstrained cells at 15 min, whereas strain could promote the expression of OCN mRNA at this period. The expression of OCN mRNA was more obviously upregulated in the strained cells at 30 min and 1 h when compared with that in the unstrained cells; (3) the strain (1% and 3%) significantly promoted the expression of Osx mRNA; 10% strain had a little effect on Osx mRNA expression. The expression of OCN mRNA was up-regulated by 3% strain, whereas it had little effect at 1% and 10% strain. In summary, mechanical strain can promote the differentiation of mesenchymal stem cells into osteoblasts.
Animals ; Bone Marrow Cells ; cytology ; Cell Differentiation ; Cells, Cultured ; Mechanoreceptors ; physiology ; Mechanotransduction, Cellular ; physiology ; Mesenchymal Stromal Cells ; cytology ; Mice ; Osteoblasts ; cytology ; Osteocalcin ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Sp7 Transcription Factor ; Stress, Mechanical ; Transcription Factors ; genetics ; 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*

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

This study is to compare the effects of kaempferide and anhydroicaritin on biomineralization of rat osteoblasts (ROB) in vitro. Calvarias were dissected aseptically from newborn SD rats, the osteoblasts were obtained by enzyme digestion and were cultured in MEM containing 10% FBS. The medium was changed every three days, and serial subculture was performed when cells covered with 90% of the dish. Kaempferide and anhydroicaritin were separately added with final concentrations of 1 x 10(-4), 1 x 10(-5), 1 x 10(-6) and 1 x 10(-7) mol x L(-1) under the conditions of osteogenic differentiation. The proliferation was measured by MTT, and the optimal concentration was detected by the ALP activity at the 9th day after osteogenic induction culture. The osteogenic indexes of kaempferide, anhydroicaritin and control group with the optimal concentration were compared. The result showed that the anhydroicaritin at concentration of 1 x 10(-5) mol x L(-1) had significantly promoted the activity of ALP, calcium content and osteocalcin content, increased the number of CFU-F(ALP) and mineralized nodules, enhanced the mRNA level of BMP-2, OSX and Runx-2, which are key genes of osteogenic differentiation, and raised the protein content of collagen-I. However, the kaempferide group had not significantly represented the ability that promoted osteogenic differentiation of ROB. The difference of osteogenic differentiation on ROB between kaempferide and anhydroicaritin was caused by the prenyl group on C-8 of icariin.
Alkaline Phosphatase ; metabolism ; Animals ; Benzopyrans ; pharmacology ; Bone Morphogenetic Protein 2 ; genetics ; metabolism ; Calcium ; metabolism ; Cell Proliferation ; drug effects ; Cells, Cultured ; Collagen Type I ; metabolism ; Core Binding Factor Alpha 1 Subunit ; genetics ; metabolism ; Kaempferols ; pharmacology ; Osteoblasts ; cytology ; metabolism ; Osteocalcin ; metabolism ; Osteogenesis ; drug effects ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley ; Transcription Factors ; genetics ; metabolism

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

PURPOSE: The objective of this study was to determine the phenotypic characterization of ligamentum flavum cells from patients with ossification of the ligamentum flavum (OLF). MATERIALS AND METHODS: Ligamentum flavum tissues were harvested from OLF and non-OLF patients during surgery. OLF and non-OLF cells were isolated from explant cultures. Cultured cells were analyzed using immunofluorescence staining and reverse transcription-polymerase chain reaction. RESULTS: OLF cells exhibited various appearances compared with the typical fibroblast-like morphology of non-OLF cells. Expressions of collagen type I and collagen type III were observed in OLF and non-OLF cells. OLF cells uniquely expressed osteocalcin, which is a marker for osteoblasts, and collagen type II which is a marker for chondrocytes, whereas they were negative in non-OLF cells. CONCLUSION: These findings indicate that OLF cells have phenotypic characterization of osteoblasts and chondrocytes which could play a role in the pathophysiology of OLF.
Adolescent ; Adult ; Aged ; Cells, Cultured ; Collagen Type I/genetics/metabolism ; Collagen Type II/genetics/metabolism ; Collagen Type VI/genetics/metabolism ; Female ; Humans ; Ligamentum Flavum/metabolism/*pathology ; Male ; Microscopy, Fluorescence ; Middle Aged ; Ossification, Heterotopic/metabolism/*pathology ; Osteocalcin/genetics/metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Young Adult