Bone Morphogenetic Protein-2 Protein Expression Research Articles

Therapeutic mechanism of basic fibroblast growth factor on spinal cord injury in rats based on the Notch/signal transducer and activator of transcription 3 signaling pathway

To explore the therapeutic effect of basic fibroblast growth factor (bFGF) on spinal cord injury (SCI) in rats and the influence of Notch/signal transducer and activator of transcription 3 (STAT3) signaling pathway. A total of 40 10-week-old male Sprague Dawley (SD) rats were selected to establish T 10-segment SCI model by a free falling object. Among them, 32 successful models were randomly divided into model group and bFGF group, with 16 in each group. Another 16 SD rats were selected as sham-operation group, with only T 10 processes, dura mater, and spinal cord exposed. After modeling, the rats in bFGF group were intraperitoneally injected with 100 μg/kg bFGF (once a day for 28 days), and the rats in model group and sham-operation group were injected with normal saline in the same way. The survival of rats in each group were observed after modeling. Basso-Beattie-Bresnahan (BBB) scores were performed before modeling and at immediate, 14 days, and 28 days after modeling to evaluate the functional recovery of hind limbs. Then, the spinal cord tissue at the site of injury was taken at 28 days and stained with HE, Nissl, and propidium iodide (PI) to observe the pathological changes, neuronal survival (number of Nissl bodies) and apoptosis (number of PI red stained cells) of the spinal cord tissue; immunohistochemical staining and ELISA were used to detect the levels of astrocyte activation markers [glial fibrillary acidic protein (GFAP)] and inflammatory factors [interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α), interferon γ (IFN-γ)] in tissues, respectively. Western blot was used to detect the expressions of Notch/STAT3 signaling pathway related proteins [Notch, STAT3, phosphoryl-STAT3 (p-STAT3), bone morphogenetic protein 2 (BMP-2)] in tissues. All rats survived until the experiment was completed. At immediate after modeling, the BBB scores in model group and bFGF group significantly decreased when compared to sham-operation group ( P<0.05). At 14 and 28 days after modeling, the BBB scores in model group significantly decreased when compared to sham-operation group ( P<0.05); the bFGF group showed an increase compared to model group ( P<0.05). Compared with before modeling, the BBB scores of model group and bFGF group decreased at immediate after modeling, and gradually increased at 14 and 28 days, the differences between different time points were significant ( P<0.05). The structure of spinal cord tissue in sham-operation group was normal; in model group, there were more necrotic lesions in the spinal cord tissue and fewer Nissl bodies with normal structures; the number of necrotic lesions in the spinal cord tissue of the bFGF group significantly reduced compared to the model group, and some normally structured Nissl bodies were visible. Compared with sham-operation group, the number of Nissl bodies in spinal cord tissue significantly decreased, the number of PI red stained cells, GFAP, IL-1β, TNF-α, IFN-γ, Notch, p-STAT3 /STAT3, BMP-2 protein expression levels significantly increased in model group ( P<0.05). The above indexes in bFGF group significantly improved when compared with model group ( P<0.05). bFGF can improve motor function and pathological injury repair of spinal cord tissue in SCI rats, improve neuronal survival, and inhibit neuronal apoptosis, excessive activation of astrocytes in spinal cord tissue and inflammatory response, the mechanism of which may be related to the decreased activity of Notch/STAT3 signaling pathway.

Mechanism of the Notch1 signaling pathway regulating osteogenic factor influences lumbar disc calcification

To explore the mechanism of the Notch1 signaling pathway in regulating osteogenic factors and influencing lumbar disc calcification. Primary annulus fibroblasts from SD rats were isolated and subcultured in vitro. The calcification-inducing factors bone morphogenetic protein-2 (BMP-2) and basic fibroblast growth factor (b-FGF) were added to separate groups to induce calcification, which were referred to as the BMP-2 group and the b-FGF group, respectively. A control group was also set up, which was cultured in normal medium. Subsequently, cell morphology and fluorescence identification, alizarin red staining, ELISA, and quantitative real-time polymerase chain reaction (QRT-PCR) were performed to determine the effect of calcification induction. Cell grouping was performed again, including the control group, the calcification group (adding the inducer BMP-2), the calcification + LPS group(adding the inducer BMP-2 and the Notch1 pathway activator LPS), and the calcification + DAPT group (adding the inducer BMP-2 and the Notch1 pathway inhibitor DAPT). Alizarin red staining and flow cytometry were used to detect cell apoptosis, ELISA was used to detect the content of osteogenic factors, and Western blot was used to detect the expression of BMP-2, b-FGF, and Notch1 proteins. The induction factor screening results showed that the number of mineralized nodules in fibroannulus cells in BMP-2 group and b-FGF group was significantly increased, and the increase was greater in the BMP-2 group Meanwhile, ELISA and Western blot results showed that BMP-2, b-FGF and mRNA expression levels of BMP-2, b-FGF and Notch1 in the induced group were significantly increased (P<0.01). The results of the mechanism of Notch1 signaling pathway affecting lumbar disc calcification showed that compared to calcified group, the number of fibroannulus cell mineralization nodules, apoptosis rate, BMP-2, b-FGF content, the expression levels of BMP-2, b-FGF, and Notch1 proteins were further increased significantly However, the number of mineralization nodules, apoptosis rate, BMP-2 and b-FGF levels, BMP-2, b-FGF and Notch1 protein expression levels were decreased in the calcified +DAPT group (P<0.05 or P<0.01). Notch1 signaling pathway promotes lumbar disc calcification through positive regulation of osteogenic factors.

Panax quinquefolius saponin inhibits vascular smooth muscle cell calcification via activation of nuclear factor-erythroid 2-related factor 2

BackgroundPanax quinquefolius saponin (PQS) is the main active component of Panax quinquefolius. Emerging evidence suggests that PQS exerts beneficial effects against cardiovascular diseases. However, the role and mechanism of PQS in vascular calcification are not unclear. The present study investigated the effects of PQS on the calcification of vascular smooth muscle cell (VSMCs).MethodsThe present study used calcification medium containing 3 mM inorganic phosphate (Pi) to induce rat VSMCs calcification. We investigated the effects of PQS on VSMCs calcification using alizarin red staining and alkaline phosphatase (ALP) activity assays. The intracellular reactive oxygen species (ROS) levels and the transcriptional activity of nuclear factor-erythroid 2-related factor 2 (Nrf2) were determined. The mRNA and protein expression levels of Nrf2, the antioxidant gene heme oxygenase-1 (HO-1), osteogenic markers, including runt-related transcription factor 2 (Runx2) and bone morphogenetic protein 2 (BMP2), and Kelch-like ECH-associated protein 1 (Keap1) were also measured.ResultsTreatment with Pi significantly increased intracellular calcium deposition and ALP activity, which were suppressed by PQS in a concentration-dependent manner. During VSMCs calcification, PQS inhibited the mRNA and protein expression of Runx2 and BMP2. PQS treatment reduced intracellular ROS production and significantly upregulated Nrf2 transcriptional activity and the expression of Nrf2 and its target antioxidant gene HO-1. PQS suppressed the Pi-induced protein expression of Keap1, which is an endogenous inhibitor of Nrf2. Keap1 siRNA treatment induced Nrf2 expression and downregulated Runx2 expression in the presence of Pi and PQS.ConclusionTaken together, these findings suggest that PQS could effectively inhibit VSMCs calcification by ameliorating oxidative stress and regulating osteogenic genes via the promotion of Nrf2 expression.

Therapeutic Mechanism of Chinese Medicine on the Healing of Early and Middle Fractures in Rabbits Under the Expression Level of Bone Morphogenetic Protein-2 (BMP-2) in Bone Tissue

In order to explore the therapeutic mechanism of Chinese medicine on the healing of rabbits early and middle fractures, a rabbit fracture model was established in this study. The study was divided into several groups, i.e., treatment group (TG) (fed with Chinese medicine Capsule) and control group (CG) (fed with normal saline (NS)). The materials were collected at 1, 3, and 5 weeks after the start of the experiment for analysis. The experiment content included: callus Hematoxylin-Eosin staining (HE staining); Bone Morphogenetic protein-2 (BMP-2) protein level detection; Type I and type II bone collagen (BC) detection; and serum biochemical factors detection. The experimental results showed that the formation of callus in the TG was better than in the CG; the BMP-2 protein expression level in the TG was higher than in the CG, and there were statistically significant differences (SSDs); the type I and type II BC levels in the TG were higher than the CG, there were SSDs; the levels of serum calcium (SC), phosphorus ion (PI), and alkaline phosphatase (ALP) in the TG were also higher than in the CG, and there were SSDs.

Evaluation of Three Growth Factors with the Most Controversial Expression Pattern in Osteoarthritic and Non-osteoarthritic Cartilage

There is no clear consensus regarding the expression pattern of many growth factors in healthy and osteoarthritic cartilage. In this study, the expression pattern of three controversial cartilage growth factors was compared between osteoarthritic (n=27) ‎and non-osteoarthritic cartilage samples (n=14). The candidate growth factor included Insulin-like ‎growth factor 1 (IGF-1), Fibroblast Growth Factor 2 (FGF-2), and Bone Morphogenetic Protein 2 (BMP-2). The candidate markers' gene and protein expression were analyzed using relative quantification relative to real-time PCR and ELISA, respectively. The mean IGF-I ‎mRNA and protein expression were 11.1, and 2 folds greater in osteoarthritic cartilage, respectively (p&lt;0.001 for both). The mean FGF-2 ‎mRNA and protein expression were 6.7 and 1.3 folds greater in osteoarthritic cartilage, respectively (p&lt;0.001 and p=0.003). The mean BMP-2 ‎mRNA and protein expression were 3.48 and 1.51 folds greater in osteoarthritic cartilage, respectively (p=0.001 and p&lt;0.001). A significant positive correlation was found between the mRNA and protein expression of all candidate growth factors. In conclusion, the gene and protein expression of IGF-I, FGF-2, and BMP-2 were significantly higher in the ‎osteoarthritic cartilages when compared with non-osteoarthritic, suggesting them as‎‏ a therapeutic ‏target in OA treatment.

Annatto-Derived Tocotrienol Promotes Mineralization of MC3T3-E1 Cells by Enhancing BMP-2 Protein Expression via Inhibiting RhoA Activation and HMG-CoA Reductase Gene Expression.

PurposeAnnatto-derived tocotrienol (AnTT) has been shown to improve bone formation in animal models of osteoporosis and promote differentiation of pre-osteoblastic cells. However, the mechanism of action of AnTT in achieving these effects is unclear. This study aims to investigate the mechanism of action of AnTT on MC3T3-E1 pre-osteoblasts via the mevalonate pathway.MethodsMurine pre-osteoblastic cells, MC3T3-E1, were cultured with the density of 1 × 104 cells/mL and treated with 4 concentrations of AnTT (0.001–1 µg/mL). Expression of HMG-CoA reductase (HMGR) gene was carried out using qPCR after treatment with AnTT for 21 days. RhoA activation and bone morphogenetic protein-2 (BMP-2) were measured using immunoassay after 9 and 15 days of AnTT treatment. Lovastatin was used as the positive control. Mineralized nodules were detected using Von Kossa staining after 21 days of AnTT treatment.ResultsThe results showed that HMGR was up-regulated in the lovastatin group on day 9 and 21 compared to the control. Lovastatin also inhibited RhoA activation (day 9 and 15) and increased BMP-2 protein (day 15). On the other hand, AnTT at 0.001 μg/mL (day 3) and 0.1 μg/mL (day 21) significantly down-regulated HMGR gene expression compared to the control. On day 21, HMGR gene expression was significantly reduced in all groups compared to day 15. AnTT at 0.1 μg/mL significantly decreased RhoA activation on day 9 compared to the control. AnTT at 1 μg/mL significantly increased BMP-2 protein on day 15 compared to the control (P<0.05). Mineralized calcium nodules were more abundant in AnTT treated groups compared to the control on day 21.ConclusionAnTT suppresses the mevalonate pathway by downregulating HMGR gene expression and inhibiting RhoA activation, leading to increased BMP-2 protein in MC3T3-E1 cells. This explains the stimulating effects of AnTT on osteoblast mineralization.

Regulatory effect of miR-421 on humeral fracture and heterotopic ossification in elderly patients.

The present study aimed to investigate the role of miR-421 and bone morphogenetic protein-2 (BMP-2) in the bone tissues and blood of elderly patients with humeral fractures and heterotopic ossification. A total of 38 patients with humeral fractures, including 16 patients who received surgery within 1-7 days of fracture and 22 patients who received surgery within 8-14 days of fracture, were enrolled. An additional 18 patients who had heterotopic ossification and 26 patients who had humeral fracture and not heterotopic ossification were also included. Bone tissues and blood were collected. Reverse transcription-quantitative polymerase chain reaction was performed to determine the miR-421 and BMP-2 mRNA expression levels in the samples. Western blotting and ELISA were performed to detect BMP-2 protein levels in bone tissues and blood, respectively. Dual-luciferase reporter assays were performed to verify whether BMP-2 is the direct target gene of miR-421. Compared with the patients who received surgery 1-7 days after fracture, the patients who accepted the surgery 8-14 days after fracture had significantly increased levels of BMP-2 mRNA and protein in their bone tissues and blood (P<0.05). Contrastingly, the expression level of miR-421 decreased in the samples from patients who accepted the surgery 8-14 days after fracture compared with the level in those who received surgery 1-7 days after fracture (P<0.05). Compared with the patients without heterotopic ossification, the patients with heterotopic ossification had increased BMP-2 mRNA and protein expression levels in their bone tissues and blood, whereas the expression of miR-421 was significantly decreased (P<0.05). The dual-luciferase reporter assay demonstrated that BMP-2 was the direct target gene of miR-421. The upregulation of BMP-2 may be associated with the downregulation of miR-421. miR-421 may regulate the recovery of humeral fracture and heterotopic ossification through BMP-2. The results of the present study may provide a theoretical basis for the diagnosis and treatment of humeral fracture and heterotopic ossification.

Conditioned medium from bone marrow-derived mesenchymal stem cells inhibits vascular calcification through blockade of the BMP2\u2013Smad1/5/8 signaling pathway

BackgroundArterial calcification is associated with cardiovascular disease as a complication of advanced atherosclerosis and is a significant contributor to cardiovascular morbidity and mortality. Osteoblastic differentiation of vascular smooth muscle cells (VSMCs) plays an important role in arterial calcification and is characterized by cellular necrosis, inflammation, and lipoprotein and phospholipid complexes, especially in atherosclerotic calcification. The conditioned medium from bone marrow-derived mesenchymal stem cells (MSC-CM) is well known as a rich source of autologous cytokines and is universally used for tissue regeneration in current clinical medicine. Here, we demonstrate that MSC-CM inhibits beta-glycerophosphate (β-GP)-induced vascular calcification through blockade of the bone morphogenetic protein-2 (BMP2)–Smad1/5/8 signaling pathway.MethodsVSMC calcification was induced by β-GP followed by treatment with MSC-CM. Mineral deposition was assessed by Alizarin Red S staining. Intracellular calcium content was determined colorimetrically by the o-cresolphthalein complexone method and alkaline phosphatase (ALP) activity was measured by the para-nitrophenyl phosphate method. Expression of BMP2, BMPR1A, BMPR1B, BMPR2, msh homeobox 2 (Msx2), Runt-related transcription factor 2 (Runx2), and osteocalcin (OC), representative osteoblastic markers, was assessed using real-time polymerase chain reaction analysis while the protein expression of BMP2, Runx2, and phosphorylated Smad1/5/8 was detected by western blot analysis.ResultsOur data demonstrated that MSC-CM inhibits osteoblastic differentiation and mineralization of VSMCs as evidenced by decreased calcium content, ALP activity, and decreased expression of BMP-2, Runx2, Msx2, and OC. MSC-CM suppressed the expression of phosphorylated Smad1/5/8 and the β-GP-induced translocation from the cytoplasm to the nucleus. Further study demonstrated that human recombinant BMP-2 overcame the suppression of VSMC calcification by MSC-CM.ConclusionMSC-CM may act as a novel therapy for VSMC calcification by mediating the BMP2–Smad1/5/8 signaling pathway

Effects of water extracts of Davallia formosana on ovariectomized mice

ObjectiveThe Taiwanese native fern, Davallia formosana, is used to treat bone diseases in traditional Chinese medicine, but, very few animal tests to verify. To study how this fern and its active components alter bone metabolism. MethodsThe effects of low and high doses of D. formosana water extracts (DFW, 50 and 200 mg/(kg·d)) and (-)-epicatechin-3-O-D-allopyranoside isolates of DFW (ECAP, 0.06 and 2.5 mg/(kg·d)) on protein levels of important indicators of osteoclast and osteoblast activities were examined. Ovariectomized mice were used as a model of osteoporosis. Bone morphogenetic protein 2 (BMP-2), collagen 1 (COL-1), alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), and osteopontin (OPN) were analyzed using western blotting of femur tissue. Interleukin (IL)-1β and tumor necrosis factor (TNF)-α were measured in the serum, and hematoxylin and eosin staining were used to assess bone morphology. ResultsTreatment with DFW or ECAP significantly induced BMP-2, COL-1, ALP, RUNX2, OCN, and OPN protein expression, indicating stimulation of osteogenesis. The treatments also reduced IL-1β and TNF-α expression, indicating inhibition of osteoclastogenesis. In histological examinations, mice treated with DFW or ECAP had more bone trabeculae. The results demonstrate that DFW and ECAP inhibit osteoclast differentiation and promote osteoblast differentiation, and effectively ameliorate ovariectomy (OVX)-induced osteoporosis. ConclusionECAP is a major component of DFW, which may have therapeutic potential for the treatment of diseases associated with excessive osteoclastic and insufficient osteogenic activity. ECAP may not be the specific active molecule because it is unstable in the stomach. However, the major active molecule needs to be confirmed using a relevant pharmacological model.

Effects of Davallia formosana Hayata Water and Alcohol Extracts on Osteoblastic MC3T3-E1 Cells.

The Taiwanese native fern Davallia formosana Hayata (DFH) is used to treat bone diseases in classical Chinese medicine. We analyzed MC3T3E1 osteoblasts treated with different concentrations of water and ethanol extracts (10, 25, and 50 [both], and 100μg/mL [DFE only]) using cell viability, expression of osteoblast differentiation markers [bone morphogenetic protein 2 (BMP-2), collagen 1 (CoL-1), alkaline phosphatase (ALP), and Runt-related transcription factor 2 (Runx 2)], and mineralization. These were significantly increased by DFW or DFE after 24-h incubation compared with the untreated controls. Compared with other treatments, DFW 50 and DFE 100μg/mL significantly increased MC3T3E1 cell survival. DFW 25 and 50μg/mL increased bone BMP-2, CoL-1, ALP, and Runx2 protein expression, ALP activity, and mineralization more than DFE did. Repeated chromatographic separation of DFW yielded compound (-)-epicatechin-3-O-d-allipyranoside (ECAP), which was characterized using 1 H and 13 C nuclear magnetic resonance spectroscopy. (-)-Epicatechin-3-O-d-allipyranoside (0.01μg/mL) significantly increased cell survival (118.9%) and mineralization (218.7%) compared with that of the control treatment. We inferred that ECAP could mediate the main activity of DFW in bone formation, likely through BMP-2-induced Runx2 transcription, which increased bone cell differentiation factors ALP and CoL-1 and promoted mineralization. (-)-Epicatechin-3-O-d-allipyranoside could be an anti-osteoporotic agent. Copyright © 2017 John Wiley & Sons, Ltd.

Expression of matrix Gla protein and bone morphogenetic protein 2 in renal papillary tissues in patients with calcium oxalate kidney stones

To compare expression levels of matrix Gla protein (MGP) and bone morphogenetic protein 2 (BMP-2) in Randall's plaque of renal papillary tissues in patients with calcium oxalate kidney stones and the underlying mechanism for stone formation. Methods: A total of 30 samples of Randall's plaque in renal papillary tissues from patients with calcium oxalate kidney stones were collected from the Department of Urology of Xiangya Hospital of Central South University from April, 2015 to December, 2015 and served as an experimental group. Ten samples of renal papillary tissues in patients undergone renal tumor nephrectomy were collected from the same hospital and served as a control group. The expressions of MGP and BMP-2 mRNA and protein were detected by quantitative real-time PCR and Western blot.Meanwhile, immunohistochemical technique was used to observe the expressions of MGP and BMP-2 in different parts of renal papillary tissues in the 2 groups. Results: 1) The mRNA expression levels of MGP in the experimental group and the control group were 0.760±0.804 and 1.365±0.348, respectively, with significant difference between them (P<0.05). Them RNA levels of BMP-2 in the experimental group and the control group were 2.500±0.725 and 1.485±0.870, respectively, with significant difference between them (P<0.05). The expression levels of MGP protein in the experimental group and the control group were 0.130±0.424 and 0.202±0.704, respectively, with no significant difference between them (P>0.05). The expression levels of BMP-2 protein in the experimental group and the control group were 0.885±0.220 and 0.682±0.272, respectively, with significant difference between them (P<0.05). The immunohistochemistry showed that the protein expression of MGP in the experimental group was lower than that in the control group, while the protein expression of BMP-2 in the experimental group was higher than that in the control group (both P<0.05). Conclusion: The BMP-2 expression is increased while MGP expression is decreased in renal papillary tissues in patients with calcium oxalate kidney stones, and the formation of calcium oxalate kidney stone might be a kind of osteogenetic reaction or ectopic calcification.

Osteoblast differentiation of rabbit adipose-derived stem cells by polyethylenimine-mediated BMP-2 gene transfection in vitro

The aim of this study was to examine the efficiency of polyethylenimine-mediated transfection of the human bone morphogenetic protein-2 (BMP-2) gene into rabbit adipose-derived stem cells (ADSCs), and its effect on osteoblast differentiation. Adipose tissue was isolated from the necks of adult Japanese white rabbits and cultured in vitro to obtain ADSCs. Gene delivery of BMP-2 was mediated by polyethylenimine and stable transformants were selected by G-418. The expression of BMP-2 mRNA was confirmed by reverse transcription-polymerase chain reaction, and of the BMP-2 protein by ELISA. Osteocalcin and collagen type I were detected by western blot and by an alkaline phosphatase kit. Alizarin red S stain was also utilized to examine osteogenesis. The non-transfected group was considered as a control. In this study, we successfully derived ADSCs from rabbit adipose tissue. Through passages 3-6, the expression of CD29 and CD44 gradually increased, whereas the expression of CD34 and CD45 gradually decreased. Both mRNA and protein expression of BMP-2 were confirmed following polyethylenimine-mediated BMP-2 gene delivery. In addition, the expression of alkaline phosphatase, osteocalcin, and collagen type I was found to be upregulated and alizarin red S staining was positive in transfected ADSCs, indicating BMP-2-induced osteogenesis. Therefore, this study determined that polyethylenimine was able to mediate BMP-2 gene delivery and induce osteogenic differentiation of ADSCs.

Substance P promotes expression of bone morphogenetic protein 2 in bone marrow stromal cells and its relationship with Wnt pathway

Objective To explore how substance P(SP) regulates expression of bone morphogenetic protein 2 (BMP-2) in bone marrow stromal cells (BMSCs) in the respects of time and concentration effects and relationship with Wnt signaling pathway. Methods The third passage of BMSCs from SD rats were divided into 4 groups (n = 3) which were added respectively with: phosphate buffered saline (PBS) (control), SP (10-8 mol/L), SP (10-10 mol/L), and SP (10-12 mol/L). At 7, 14 and 21 days after culture, cellular morphology was observed by light microscopy, expression of alkaline phosphatase (ALP) and BMP-2 was detected using Western blot. After the best concentration of SP was determined, the third passage BMSCs from SD rats were then divided into 3 groups (n = 3) which were added respectively with: PBS (control), SP, and SP + DKK1 (dickkopf 1). Protein expression of BMP-2 and β-catenin was detected using western blot. Results At 14 days, ALP protein was significantly more in SP (10-8 mol/L) group (1.428±0.166) and SP (10-10 mol/L) group (1.742±0.221) than in control group (P < 0.05). At 14 days, BMP-2 was significantly more in SP(10-8 mol/L) group(1.801±0.239), SP (10-10 mol/L) group (4.070±0.297) and SP (10-12 mol/L) group (1.943±0.196) than in control group(P < 0.05). At 21 days, BMP-2 was significantly more in SP (10-10 mol/L) group (2.960±0.324) and SP (10-12 mol/L) group (2.652±0.265) than in control group(P < 0.05). The best concentration of SP was 10-10 mol/L. BMP-2 at 7, 14 and 21 days (4.300±0.297, 2.290±0.175 and 3.160±0.234) was significantly more in SP group than in control group and SP + DKK1 group(P < 0.05). β-catenin at 14 and 21 days in SP group (1.553±0.210 and 1.471±0.170) was significantly more than in SP + DKK1 group and control group (P < 0.05). Conclusions SP can promote expression of BMP-2 in BMSCs. The regulation of BMP-2 expression in BMSCs by SP has time and concentration effects and may be associated with the Wnt signaling pathway. Key words: Substance P; Bone marrow cells; Mesenchymal stem cells; Bone morphogenetic proteins; Signaling transduction

Effects of oxidized low density lipoprotein on transformation of valvular myofibroblasts to osteoblast-like phenotype.

In order to investigate the roles of Wnt signal pathway in transformation of cardiac valvular myofibroblasts to the osteoblast-like phenotype, the primary cultured porcine aortic valve myofibroblasts were incubated with oxidized low density lipoprotein (ox-LDL, 50 mg/L), and divided into four groups according to the ox-LDL treatment time: control group, ox-LDL 24-h group, ox-LDL 48-h group, and ox-LDL 72-h group. Wnt signal pathway blocker Dickkopf-1 (DDK-1, 100 μg/L) was added in ox-LDL 72-h group. The expression of a-smooth muscle actin (α-SMA), bone morphogenetic protein 2 (BMP2), alkaline phosphatase (ALP), and osteogenic transcription factor Cbfa-1 was detected by Western blotting, and that of β-catenin, a key mediator of Wnt signal pathway by immunocytochemical staining method. The Wnt/β-catenin was observed and the transformation of myofibroblasts to the osteoblast-like phenotype was examined. The expression of α-SMA, BMP2, ALP and Cbfa-1 proteins in the control group was weaker than in the ox-LDL-treated groups. In ox-LDL-treated groups, the protein expression of a-SMA, BMP2, ALP, and Cbfa-1 was significantly increased in a time-dependent manner as compared with the control group, and there was significant difference among the three ox-LDL-treated groups (P<0.05 for all); β-catenin protein was also up-regulated in the ox-LDL-treated groups in a time-dependent manner as compared with the control group (P<0.05), and its transfer from cytoplasm to nucleus and accumulation in the nucleus were increased in the same fashion (P<0.05). After addition of DKK-1, the expression of α-SMA, bone-related proteins and β-catenin protein was significantly reduced as compared with ox-LDL 72-h group (P<0.05). The Wnt/ β-catenin signaling pathway may play an important role in transformation of valvular myofibroblasts to the osteoblast-like phenotype.

A comparative study of mechanical strain, icariin and combination stimulations on improving osteoinductive potential via NF-kappaB activation in osteoblast-like cells

BackgroundThe combination of drugs and exercise was the effective treatment in bone injure and rebuilding in clinic. As mechanical strain has potential in inducing the differentiation of osteoblasts in our previous study, the further research to investigate the combination of mechanical strain and icariin stimulation on inducing osteoblast proliferation, differentiation and the possible mechanism in MC3T3-E1 cell line.MethodsA whole cell enzyme-linked immunosorbent assay that detects the bromodeoxyuridine incorporation during DNA synthesis was applied to evaluate the proliferation. The mRNA expression of alkaline phosphatase (ALP), osteocalcin (OCN), type I collagen (Col I), bone morphogenetic protein-2 (BMP-2) and BMP-4 was detected by real-time reverse-transcription polymerase chain reaction. The activity of ALP was analyzed by ELISA and the protein expression of OCN, Col I and BMP-2 was assessed by western blot. Moreover, the activity of nuclear transcription factor kappa-B (NF-κB) signaling pathway was investigated with the expression of inhibitor of κB (IκB) α, phosphorylation of IκB-α (P-IκB-α), p65, P-p65 by western blot.ResultsWe observed that compared to single mechanical strain or icariin stimulation, the mRNA and protein expressions of ALP (P < 0.05 or P < 0.01), OCN (P < 0.01) and Col I (P < 0.05 or P < 0.01) were increased significantly by the combination of mechanical strain and icariin stimulation. Moreover, the combination of mechanical strain and icariin stimulation could up-regulate the expression of BMP-2 (P < 0.01) and BMP-4 compared to single mechanical strain or icariin stimulation. The combination of mechanical strain and icariin stimulation could activate NF-κB signaling pathway by increasing the expression of IκB α, P-IκB-α, p65, P-p65 (P < 0.01).ConclusionThe combination of mechanical strain and icariin stimulation could activate the NF-κB pathway to improve the proliferation, differentiation of osteoblast-like cells.

Icariin enhances differentiation and maturation of rat calvarial osteoblasts in collagen hydrogel three-dimensional culture

To investigate the effects of icariin on the differentiation and maturation of rat calvarial osteoblasts(ROB) in collagen hydrogel three-dimensional culture. ROB were obtained by enzyme digestion from the segregated neonatal SD rats skull and were embedded in 2 mg/mL rat tail collagen for three-dimensional culture. The growth state of ROB was observed by FDA/PI staining, HE staining and scanning electron microscopy. ROB were treated with icariin at the concentration of 1 × 10⁻⁴, 1 × 10⁻⁵, 1 × 10⁻⁶ and 1 × 10⁻⁷ mol/L respectively. The activity of alkaline phosphatase(ALP) was detected after 3, 6, 9 d of icariin treatment. Three-dimensional cultured ROB were treated with optimal concentration icariin for 12, 24, 36, 48 h and total RNA was extracted and the mRNA expressions of bone morphogenetic protein-2 (BMP-2), Runt-related transcription factor 2 (RUNX-2) and Osterix were detected by real time RT-PCR. The protein expression of BMP-2, RUNX-2 and Osterix were examined by Western-blotting. ROB were cultured in collagen hydrogel successfully. FDA/PI staining, HE staining, and scanning electron microscopy showed that ROB adhered with collagen tightly and distributed homogeneously. Icariin at final concentration of 1 × 10⁻⁵, 1 × 10⁻⁶ and 1×10⁻⁷ mol/L all enhanced the activity of ALP of collagen hydrogel three-dimensional cultured ROB, and 1 × 10⁻⁶ mol/L was the optimal concentration. Besides, icariin (1 × 10⁻⁶ mol/L) increased mRNA and protein expression of BMP-2、RUNX-2 and Osterix compared to control group. Icariin can enhance the expression of osteogenic markers of ROB in collagen hydrogel three-dimensional culture significantly.

7-Dehydrocholesterol reductase regulated the palatal development by the sonic hedgehog-bone morphogenetic protein 2 signal pathway

To investigate the effect of 7- dehydrocholesterol reductas (Dhcr-7) gene silencing on the palatal development by sonic hedgehog (Shh)-bone morphogenetic protein2(BMP-2) signal pathway in vitro. A total of 60 pairs of palatal shelves fromgestation day (GD) 13.5 mouse embryos were divided into three groups (A, B, C) of 20 randomly. In group A (control), palatal shelves were cultured with medium containing no cholesterol.In group B (Dhcr-7-siRNA), palatal shelves were cultured without cholesterol medium but containing Dhcr-7 siRNA adenovirus. After 48h, the culture medium of groups A and B were changed with medium without cholesterol. In group C (cholesterol), palatal shelves were cultured without cholesterol medium but containing Dhcr-7 siRNA adenovirus. After 48h, the culture medium of group C was changed with medium containing 600 mg/L cholesterol. After 72h again, tissues dyeing and scanning electron microscope (SEM) technique were used to observe morphological changes of palates. Both RT-PCR and Western blottingtechniques were used to measure mRNA and protein expressions for Dhcr-7, Shh, and BMP-2, respectively. The tissues dyeing and SEM showedthat the palates fusedin groups A and Candthe palates did not fuse in group B eventually. The expression of both mRNA and proteins for Shh and BMP-2 in group B wasdecreased with the Dhcr-7 reduction. In group B, the mRNA and protein expression of Shh was separately 0.063±0.018 and 0.092±0.065;the mRNA and protein expression quantity of BMP- 2 was separately 0.054±0.018 and 0.049±0.021. In group A, the mRNA and protein expression of Shh was separately 0.667±0.093 and 0.639±0.078;the mRNA and protein expression of BMP-2 was separately 0.591 ± 0.043 and 0.569 ± 0.081. The difference of Shh and BMP- 2 mRNA and protein expression between A and B group were statistically significant separately (P < 0.05). The expression of both mRNA and protein for Dhcr-7 (0.074±0.034 and 0.075±0.028) did not changebasicallyin group C, compared with the Dhcr- 7expression of mRNA and protein (0.083±0.045; 0.067±0.065) in group B, the difference wasnot statistically significant(P > 0.05). In group C, the mRNA and protein expressionof Shh (0.649±0.085 and 0.608±0.092) and BMP-2 (0.578±0.062 and 0.548±0.065) were significantly increased. The difference of Shh and BMP-2 mRNA and protein expression between B and C group were statistically significant separately (P < 0.05). Dhcr-7 could influence the expression of Shh and BMP-2. Dhcr-7 reductase regulated the palatal development by the Shh-BMP-2 signal pathway.

Adrenomedullin attenuates vascular calcification in fructose‐induced insulin resistance rats

To determine the therapeutic effects of adrenomedullin (ADM) on vascular calcification and related molecular mechanism in fructose-induced insulin resistance rats. Rats received ordinary drinking water or 10% fructose in drinking water for 12 weeks and subcutaneous injection of normal saline or ADM (3.6 μg kg(-1) ) twice a day for the last 4 weeks. Levels of ADM, calcitonin receptor-like receptors (CRLR), receptor activity-modifying proteins (RAMP) as well as calcium content, alkaline phosphatase (ALP) activity, osteoblastic and contractile smooth muscle markers in aortic media were measured. The levels of ADM, CRLR, RAMP2 and RAMP3 in aortic media were increased in fructose-fed rats. ADM treatment attenuated the fructose-induced insulin resistance, increased blood pressure, fasting glucose, insulin, triglycerides and cholesterol levels. It improved VSMCs proliferation and disordered arrangement and hyperplasia of elastic fibres in fructose-fed rats. Calcium deposits, calcium content and ALP activity in the aortic media were increased in fructose-fed rats, which were attenuated by ADM treatment. The osteoblastic markers such as osteopontin (OPN), bone morphogenetic protein 2 (BMP2) proteins and core binding factor alpha-1 (Cbfα-1) protein and mRNA expressions were increased in fructose-fed rats. ADM treatment increased the OPN protein expression, but reduced the BMP2 protein, Cbfα-1 protein and mRNA expression. Contractile smooth muscle markers such as α-actin and smooth muscle 22α (SM-22α) were downregulated in fructose-fed rats, which were recovered by ADM treatment. Administration of ADM attenuates insulin resistance, calcium deposition and osteogenic transdifferentiation in aortic media in fructose-fed rats.

Quantitative determination of matrix Gla protein (MGP) and BMP-2 during the osteogenic differentiation of human periodontal ligament cells

Background and objectiveMatrix Gla protein (MGP) has been recognized as a potent calcification inhibitor and a regulator for bone morphogenetic protein-2 (BMP-2). The periodontal ligament (PDL) is a non-mineralized connective tissue located between two mineralized tissues, the cementum and the alveolar bone. However, the mechanism by which PDL prevents mineralization has yet to be defined. This study aims to examine the expression pattern of MGP and BMP-2 during human periodontal ligament cells (hPDLCs) osteogenic differentiation in vitro, preliminarily exploring their roles in this process. Materials and methodshPDLCs were obtained and cultured in mineralizing medium. The expression of MGP and BMP-2 was confirmed by RT-PCR and immunofluorescence staining. In the process of osteogenic induction, alkaline phosphatase (ALP) activity, extracelluar calcium deposition, and mineralized nodules were measured. Quantitative real-time RT-PCR was performed to evaluate mRNA expression of MGP, BMP-2 and osteogenic marker genes, including ALP, bone sialoprotein (BSP), type I collagen (COLI), osteocalcin (OCN), and runt-related transcription factor 2 (Runx2). The protein expression of MGP and BMP-2 was analyzed by western blotting. ResultsCo-localization of MGP and BMP-2 was visualized in hPDLCs. After osteogenic induction, ALP activity, calcium deposition, mineralized nodules, and osteogenic marker genes were significantly up-regulated. mRNA expression of MGP and BMP-2 generally decreased, although MGP mRNA increased on day 14 and 21 compared with the control, and protein expression of MGP and BMP-2 was down-regulated. ConclusionOur results indicate that MGP might regulate hPDLCs osteogenic differentiation which might keep a potential relationship with BMP-2 in this process.

Short-Term Exposure to Tumor Necrosis Factor-Alpha Enables Human Osteoblasts to Direct Adipose Tissue-Derived Mesenchymal Stem Cells into Osteogenic Differentiation

Tumor necrosis factor-alpha (TNF-α) is one major inflammatory factor peaking at 24 h after bone fracture in response to injury; its role in bone healing is controversial. The aims of this study were to investigate whether the duration of exposure to TNF-α is crucial for the initiation of bone regeneration and to determine its underlying mechanism(s). We demonstrated that 24 h of TNF-α treatment significantly abrogated osteocalcin gene expression by human primary osteoblasts (HOBs). However, when TNF-α was withdrawn after 24 h, bone sialoprotein and osteocalcin gene expression levels in HOBs at day 7 were significantly up-regulated compared with the HOBs without TNF-α treatment. In contrast, continuous TNF-α treatment down-regulated bone sialoprotein and osteocalcin gene expression. In addition, in an indirect co-culture system, HOBs pretreated with TNF-α for 24 h induced significantly greater osteogenic differentiation of adipose tissue-derived mesenchymal stem cells (ASCs) than the HOBs without TNF-α treatment. TNF-α treatment also promoted endogenous bone morphogenetic protein 2 (BMP-2) production in HOBs, while blocking the BMP-2 signaling pathway with Noggin inhibited osteogenic differentiation of ASCs in the co-culture system. Furthermore, activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway after TNF-α treatment occurred earlier than BMP-2 protein expression. BMP-2 production by HOBs and osteogenic differentiation of ASCs in the co-culture system with HOBs was significantly decreased when HOBs were pretreated with TNF-α in combination with the p38 MAPK-specific inhibitor (SB203580). Taken together, we provide evidence that exposure duration is a critical element in determining TNF-α's effects on bone regeneration. We also demonstrate that the p38 MAPK signaling pathway regulates the expression of BMP-2 in osteoblasts, which then acts through a paracrine loop, to direct the osteoblast lineage commitment of mesenchymal stem cells.