Bone Morphogenetic Protein-2 Levels Research Articles

MiR-25 overexpression promotes fracture healing by activating the Wnt signaling pathway.

To study the mechanism of micro-ribonucleic acid (miR)-25 in regulating the fracture healing in rats. A total of 45 male Sprague-Dawley (SD) rats were selected and randomly divided into group A [Phosphate Buffered Saline (PBS), n=15], group B (mimics NC, n=15) and group C (miR-25 mimics, n=15). The fracture model in rats was established via operation in all groups. From 1 d after the successful modeling, 50 μL (2 nmoL) of PBS was intraperitoneally injected into rats in group A, an equal amount of mimics NC was injected into rats in group B, and an equal amount of miR-25 mimics was injected into rats in group C. The above agents were injected once a week for consecutive 6 weeks. Fracture healing in rats was evaluated via X-ray imaging. At the same time, miR-25 expression in the three groups was detected via Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Protein expressions of β-catenin, proliferating cell nuclear antigen (PCNA) and bone morphogenetic protein-2 (BMP-2) in the three groups were detected via Western blotting. The OCN-, PCNA- and BMP-2-positive osteoblasts in the three groups were detected via immunohistochemical staining and were further quantified. Moreover, the biomechanical properties of femoral fracture healing in the three groups were analyzed via the 4-point bending flexural test. The X-ray examination of the femoral fracture healing at postoperative 1 and 7 weeks revealed that the fracture line disappeared, and both callus formation and fracture healing were good in miR-25 mimics group. In PBS group and mimics NC group, a few fracture lines could be observed, and both callus formation and fracture healing were poor. RT-PCR data showed that the expression level of miR-25 significantly increased in the miR-25 mimics group compared with that in the other two groups, and the differences were statistically significant (p<0.01). Western blotting analyses showed upregulated levels of β-catenin, PCNA and BMP-2 in the miR-25 mimics group compared with those in the control group, and the differences were statistically significant (p<0.01). Immunohistochemical staining manifested that the numbers of OCN-, PCNA- and BMP-2-positive osteoblasts in miR-25 mimics group markedly increased compared with that in the other two groups (p<0.01), suggesting that osteoblast differentiation in miR-25 mimics group was affected. The above immunohistochemical results indicated that the osteoblast differentiation at the fracture end in miR-25 mimics group was markedly enhanced compared with that in control groups. The results of the biomechanical test of femur specimens at 7 weeks after operation showed that in miR-25 mimics group, the maximum load, fracture energy and stiffness increased by 188%, 333% and 90%, respectively, compared with those in the PBS group (p<0.01). It is indicated that miR-25 promoted the mechanical properties of fracture healing. The overexpression of miR-25 in the fracture in rats promotes fracture healing by activating the Wnt signaling pathway.

Endogenous BMP-4/ROS/COX-2 Mediated IPC and Resveratrol Alleviated Brain Damage.

The objective of the study was to examine the therapeutic role of combined ischemic preconditioning (IPC) and resveratrol (RES) on brain ischemia/reperfusion injury (BI/RI) by modulating endogenous bone morphogenetic protein-4 (BMP-4)/reactive oxygen species (ROS)/cyclooxygenase-2 (COX-2) in rats. Sprague Dawley (SD) rats were pretreated with 20 mg/kg RES (20 mg/kg RES was administered once a day via intraperitoneal injection 7 days prior to the I/R procedure) and IPC (equal volumes of saline were administered once a day by intraperitoneal injection over 7 days, and the bilateral common carotid arteries were separated for clamp 5 minutes followed by 5 minutes of reperfusion prior to the I/R procedure), and then subjected to 2 hours of ischemia and 22 hours of reperfusion. Blood and cerebral tissues were collected, cerebral pathological injuries and infarct sizes were investigated, serum interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) levels were measured, the activities of superoxide dismutase (SOD) and ROS were calculated, the contents of methane dicarboxylic aldehyde (MDA), IL-6, TNF-α and hemodynamic change were estimated, and expression levels of b-cell lymphoma-2 (Bcl-2), bcl-2-associated x (Bax), BMP-4 and COX-2 were assessed in cerebral tissues. IPC, RES and a combination of IPC and RES preconditioning ameliorated the pathological damage and infarct sizes, reduced cerebral oxidative stress damage, alleviated inflammatory damage, restrained apoptosis, and downregulated the expression levels of BMP-4 and COX-2 compared with those of the ischemia/reperfusion (I/R) group. This study suggested a combined strategy that could enhance protection against BI/RI in clinical brain disease.

Effects of Systemic Administration of Resveratrol on New Bone Formation in Critical-Sized Bone Defects in Rats

Antioxidant agents stimulate new bone formation during the process of repair of bone defects. This study aimed to examine the effects of systemic administration of resveratrol, an antioxidant agent, on new bone formation in a rat model of experimentally induced bone defects. The study consisted of four groups. Each group was divided into 2 subgroups on the basis of the date on which the animals were sacrificed. We formed a 5-mm bone defect in the right mandible of the animals in each group. Control group (C) received no further treatment, first experimental group (G) received a bovine bone graft, second experimental group (R) received systemic administration of resveratrol (10 mg/kg), third experimental group (GR) received systemic administration of resveratrol (10 mg/kg) with a bovine bone graft. The results of histopathological examination on the 14th and 28th day showed significant differences in the degree of ossification, fibrosis, angiogenesis, and the density of inflammatory cells between the groups (p &lt; 0.05). Results of biochemical analysis showed significant differences in the serum levels of bone morphogenetic protein-2 (BMP-2) and tumor necrosis factor α (TNF-α) between the groups (p &lt; 0.05). Thus, our results showed that systemic resveratrol administration with graft placement may have positive effects on bone healing in critical-sized bone defects.

Bone Morphogenetic Protein 2-Conjugated Silica Particles Enhanced Early Osteogenic Differentiation of Adipose Stem Cells on the Polycaprolactone Scaffold.

Silica particles (SPs) induce cell proliferation and osteogenic differentiation. We reported that SPs in the scaffold induced early stage osteogenic differentiation. A polycaprolactone (PCL) scaffold was fabricated with a 10wt% SPs. The surface of PCL scaffold was coated with a 10µg/mL collagen solution. Next, the scaffold was conjugated with 2μM SPs, 2μg/mL bone morphogenetic protein 2 (BMP2), or 2μM BMP2-conjugated SPs (BCSPs). Green fluorescent protein-coupled BMP2 was applied to fabricate the scaffold. The fluorescence intensity was analyzed by confocal microscopy. The mRNA levels of the early osteogenic differentiation marker, alkaline phosphatase (ALP), were analyzed by real-time quantitative polymerase chain reaction. Levels of BMP2, RUNX2, ERK1/2, and AKT were assessed by western blotting. ALP mRNA levels were significantly higher in the BCSP-conjugated scaffold than in the other scaffolds. In the early stage of osteogenic differentiation, the protein levels of BMP2, RUNX2, ERK1/2, and AKT in cells were significantly higher in the BCSP-conjugated scaffold than in other scaffolds. Thus, the BCSP composite scaffold induced rapid osteogenic differentiation. These results suggest that BCSP composite can be used to promote early stage osteogenic differentiation and show promise as a material for use in scaffolds for bone regeneration.

Estradiol-induced RORα expression positively regulates osteoblast differentiation

The retinoic acid receptor-related orphan receptor alpha (RORα) is a member of the nuclear hormone receptor superfamily. Several studies show that estradiol is related to RORα expression. However, the link between estradiol and RORα in osteoblast differentiation remains unknown. Here, we showed that estradiol induces RORα expression in C3H10T1/2 and MC3T3-E1 cells. RORα overexpression increased the expression of osteogenic genes including bone morphogenetic protein 2 (BMP2), distal-less homeobox 5, inhibitor of DNA binding, runt-related transcription factor 2 (Runx2), and osteocalcin. In addition, RORα increased phosphorylation of smad1/5/9. Furthermore, RORα knockdown suppressed estradiol-induced BMP2 and Runx2 protein level. Also, we confirmed that estradiol-induced ALP staining and matrix mineralization was attenuated in RORα knockdown. Summarily, these results suggest that estradiol-induced RORα promotes osteoblast differentiation.

Strontium stimulates alkaline phosphatase and bone morphogenetic protein-4 expression in rat chondrocytes cultured in vitro

The trace element strontium has a significant impact on cartilage metabolism. However, the direct effects of strontium on alkaline phosphatase (ALP), a marker of bone growth, and bone morphogenetic protein-4 (BMP-4), which plays a key role in the regulation of bone and cartilage development, are not entirely clear. In order to understand the mechanisms involved in these processes, the chondrocytes were isolated from Wistar rat articular cartilage by enzymatic digestion and cultured under standard conditions. They were then treated with strontium at 0.5, 1.0, 2.0, 5.0, 20.0 and 100.0 μg/mL for 72 h. The mRNA abundance and protein expression levels of ALP and BMP-4 were measured using real-time polymerase chain reaction (real-time PCR) and Western blot analysis. The results showed that the levels of expression of ALP and BMP-4 in chondrocytes increased as the concentration of strontium increased relative to the control group, and the difference became significant at 1.0 μg/mL strontium (P<0.05). These results indicated that strontium could be involved in cartilage development via regulating ALP and BMP-4 expression.

Comparison of Transforming Growth Factor-Beta1 and Lovastatin on Differentiating Mesenchymal Stem Cells toward Nucleus Pulposus-like Phenotype: An In Vitro Cell Culture Study.

Study DesignIn Vitro cell culture study.PurposeThis study aims to investigate the impact of transforming growth factor-beta1 (TGF-β1) and lovastatin on differentiating human mesenchymal stem cells (MSCs) toward nucleus pulposus (NP)-like phenotype.Overview of LiteratureMSCs offer a cell source to the cell-based therapy for intervertebral disc degeneration. TGF-β1 is used to induce MSCs to differentiate into NP-like cells; however, an undesired expression of collagen type I has been reported. Statins reportedly stimulate expression of bone morphogenetic protein-2 (BMP-2) and promote the chondrogenic phenotype to NP cells. However, the effects of statins with or without TGF-β1 on the differentiation of MSCs into NP-like cells remain unclear.MethodsHuman MSCs were treated with TGF-β1 alone, lovastatin alone, and simultaneous or sequential treatment with TGF-β1 and lovastatin. After the proposed stimulation, the total RNA was extracted to assess the expression profile of NP cells-specific genes. Hematoxylin–eosin staining was used for examining the microscopic morphology. Furthermore, we detected the syntheses of S-100 protein, aggrecan, and collagen type II in the extracellular matrix using immunohistochemical staining.ResultsSimultaneous or sequential treatment of TGF-β1 and lovastatin could further augment the BMP-2 overexpression compared with lovastatin-alone treatment. However, the mRNA expression of aggrecan and collagen type II was not compatible with the expression level of BMP-2. Immunohistochemical studies revealed compatible production of aggrecan, collagen type II, and S-100 protein in all three groups treated with lovastatin. Cells in groups treated with lovastatin were less populated than that in the group treated with TGF-β1 alone.ConclusionsThis study demonstrates a promising role of lovastatin in inducing human MSCs into NP-like cells. However, further optimization of cell density before lovastatin treatment, treatment duration, and combination with TGF-β1 are warranted to attain better stimulatory effects.

Angiogenesis and bone regeneration by mesenchymal stem cell transplantation with danshen in a rabbit model of avascular necrotic femoral head

The present study aimed to explore the potential of combined treatment with mesenchymal stem cells (MSCs) and danshen for angiogenesis and bone regeneration in a rabbit model of avascular necrosis of femoral head (ANFH). A rabbit model of ANFH was established using the Shwartzman reaction with methylprednisolone and Escherichia coli endotoxin injection. Magnetic resonance imaging (MRI) and histopathological examination were used to evaluate the rabbit model of ANFH. The rabbits were randomly divided into the danshen group, the MSCs group, the danshen combined with MSCs group and the model group (treated with physiological saline). The expression level of monocyte chemoattractant protein-1 (MCP-1) and stromal cell-derived factor-1 (SDF-1) were determined by reverse transcription polymerase chain reaction (RT-PCR). The expression level of bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) were detected by immunofluorescence and the mRNA expression of BMP-2 and VEGF were detected by RT-PCR. Typical osteonecrosis occurred in the rabbit model of ANFH, which indicated that the model was successfully established. MCP-1 and SDF-1 were significantly increased in the model group compared with the normal group (P<0.05). Following the administration of MSCs and Salvia miltiorrhiza (danshen), MSCs labeled with 5-bromo-2-deoxyuridine were observed to be gathered in the necrotic area. The increased migration of MSCs to the necrotic area may be due to the upregulated expression of the chemokines MCP-1 and SDF-1. ANFH treated with danshen combined with MSCs may promote revascularization by increasing the expression of VEGF and BMP-2 in the femoral head, promoting re-ossification and revascularization. Danshen combined with the transplantation of MSCs may be regarded as a novel therapy for the treatment of ANFH in a clinical setting.

Alendronate induces postnatal maxillary bone growth by stimulating intramembranous ossification and preventing premature cartilage mineralization in the midpalatal suture of newborn rats

Cleft palate is a common malformation of craniofacial development, and postnatal deficiencies in palate formation may occur. The aim of this study was to determine whether alendronate treatment could induce maxillary mineralization and thus reduce the need for surgical procedures. The effects of alendronate on maxillary bone development, the midpalatal suture, and the levels of transforming growth factor beta-1 (TGF-β1), bone morphogenetic protein 2 (BMP-2), collagen I and II, and V-ATPase were evaluated in newborn rats. Thirty newborn rats were placed in a control group and 30 in a group that received intraperitoneal alendronate (2.5 mg/kg/day). The animals were euthanized on day 7 or 12, and the heads were subjected to histological and immunohistochemical analyses. Specimens from rats that received alendronate presented larger bone matrix deposition in areas of intramembranous ossification of the maxillary bone when compared to controls. Furthermore, higher levels of TGF-β1, BMP-2, and collagen I were observed, whereas osteoclasts showed no V-ATPase. The alendronate group also showed higher levels of TGF-β1 and collagen II in the midpalatal suture, whereas BMP-2 levels were lower than in controls. These results coincided with an expansion of the chondroid. In conclusion, alendronate increased the intramembranous ossification in the maxillary bone in association with increased expression of TGF-β1, BMP-2, and collagen I and decreased V-ATPase. The drug induced an expansion of chondrocytes and a decrease in mineral bone deposition despite the high levels of TGF-β1 in this area. Alendronate may therefore be useful in the treatment of diseases affecting bone growth.

BONE MORPHOGENETIC PROTEIN-2 AND COLLAGEN TYPE 1 FROM DIFFERENT SOURCES OF DEMINERALIZED DENTINE MATRIX: RELEASE KINETIC AND CHEMOTAXIS POTENTIAL FOR OSTEOPROGENITOR CELLS

Objective: To investigate the release of bone morphogenetic protein-2 (BMP-2) and collagen type I proteins (COL1) from different sources ofdemineralized dentine matrix (DDM) and their chemotaxis to mouse osteoprogenitor cells.Methods: The release kinetic of BMP-2 and COL1 was measured from custom-made DDM (CMDDM) and commercially available DDM (CADDM).Using Urist physicochemical method, CMDDM was collected from the extracted teeth in a certified dental clinic. Levels of BMP-2 and COL1 releasedwere measured at days 1, 2, 3, 5, 7, 9, 11, and 13. Next, mouse osteoprogenitor cells, MC3T3-E1, were cultured with a variety of materials as follows:CMDDM, CADDM, Bio-Oss®, and blank control in transwell system. The number of cell migration was determined by crystal violet staining to explorechemotaxis of different DDMs to mouse osteoprogenitor cells.Results: BMP-2 was detected at 588.32 ± 14.53 pg/ml from 5 g of CMDDM. In the release kinetic assay, the concentration of BMP-2 in the CMDDMgroup increased rapidly and peaked at 113.9 pg/ml on day 5, almost four times higher than that of CADDM. The release of COL1 showed similarpattern in both CMDDM and CADDM; however, the amount was significantly higher in the CMDDM group. In cell culture experiment, the number ofmigrated MC3T3-E1 was ranked as the highest in CMDDM, followed by CADDM and Bio-Oss® (p&lt;0.05).Conclusion: CMDDM released BMP-2 and COL1 greater than CADDM, which can induce more osteoblast-like cell migration. These results demonstrateda release kinetic of proteins and osteoinductivity of CMDDM, which supports a benefit of using autogenous bone graft.

Heparin-Conjugated Decellularized Bone Particles Promote Enhanced Osteogenic Signaling of PDGF-BB to Adipose-Derived Stem Cells in Tissue Engineered Bone Grafts.

Adipose-derived stem cells (ASCs) are a promising cell source for regenerating critical-sized craniofacial bone defects, but their clinical use is limited due to the supraphysiological levels of bone morphogenetic protein-2 required to induce bone formation in large grafts. It has been recently reported that platelet-derived growth factor-BB (PDGF) directly enhances the osteogenesis of ASCs when applied at physiological concentrations. In this study, a biomimetic delivery system that tethers PDGF to decellularized bone matrix (DCB) is developed to enhance osteogenic signaling in bone grafts by colocalizing PDGF-extracellular matrix cues. Heparin is conjugated to DCB particles (HC-DCB) to promote sustained binding of PDGF via electrostatic interactions. HC-DCB particles bind to PDGF with >99% efficiency and release significantly less PDGF over 21 days compared to nonconjugated DCB particles (1.1% vs 22.8%). HC-DCB-PDGF signaling in polycaprolactone (PCL)-fibrin grafts promotes >40 µg Ca2+ µg-1 DNA deposition by ASCs during in vitro osteogenic culture compared to grafts without HC-DCB or PDGF. Furthermore, more bone formation is observed in grafts with HC-DCB-PDGF at 12 weeks following implantation of grafts into murine critical-sized calvarial defects. Collectively, these results demonstrate that HC-DCB enhances the osteogenic signaling of PDGF to ASCs and may be applied to promote ASC-mediated bone regeneration in critical-sized defects.

Influences of hucMSC-exosomes on VEGF and BMP-2 expression in SNFH rats.

To investigate the influences of human umbilical cord mesenchymal stem cell-derived exosomes (hucMSC-exosome) on steroid-induced necrosis of the femoral head (SNFH) and the expressions of vascular endothelial growth factor (VEGF) and bone morphogenetic protein-2 (BMP-2) in rats. A total of 20 male Sprague-Dawley rats were randomly divided into SNFH group and SNFH + hucMSC-exosome group using a random number table. Prednisolone acetate (24.5 mg/kg) was injected twice a week to establish the rat model of SNFH, and hucMSC-exosome in a certain dose was additionally injected into the marrow cavity in SNFH + hucMSC-exosome group. After 3 weeks, the influences of hucMSC-exosome on the pathological changes and apoptosis of the femoral head in SNFH rats were detected via hematoxylin-eosin (H&E) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. In addition, the expressions of cluster of differentiation 31 (CD31), VEGF, and BMP-2 in bone tissues in both groups were detected via immunohistochemical staining, and the messenger ribonucleic acid (mRNA) and protein expression levels of VEGF and BMP-2 in necrotic bone tissues in both groups were detected via Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Western blotting. The results of H&E staining revealed that the fibrous callus formation was good, the new trabecular structure was more obvious, the number of vacuum cleft declined, and there were fewer enlarged adipocytes in SNFH + hucMSC-exosome group compared with SNFH group. The results of TUNEL staining showed that the number of apoptotic cells in femoral head tissues was smaller in SNFH + hucMSC-exosome group (p<0.05). According to the results of immunohistochemistry, hucMSC-exosome could increase the expression of vascular endothelial marker CD31 in SNFH rats (p<0.05). Besides, the results of RT-PCR, immunostaining and Western blotting manifested that both the mRNA and protein levels of BMP-2 and VEGF in femoral head tissues were significantly increased in SNFH + hucMSC-exosome group (p<0.05). HucMSC-exosome can improve SNFH in rats, whose mechanism may be related to the up-regulation of VEGF and BMP-2 by exosomes.

Serum Sclerostin and Bone Morphogenetic Protein-2 Levels in Patients with Ankylosing Spondylitis: A Meta-Analysis.

Various studies have investigated the serum sclerostin and bone morphogenetic protein-2 (BMP-2) levels in patients with ankylosing spondylitis (AS), but the results were inconsistent. The aim of this meta-analysis was to synthetically assess the associations of serum levels of sclerostin and BMP-2 with AS. Multiple electronic databases were searched to locate relevant articles published before November 2018. Pooled standard mean difference (SMD) with 95% confidence interval (CI) was calculated by the random-effect model. Totally, 21 studies were included. Meta-analysis results showed no significant difference between AS group and control group in serum sclerostin levels (SMD = 0.098, 95% CI - 0.395 to 0.591, p = 0.697). Nevertheless, serum BMP-2 levels in AS patients were higher than that in controls (SMD = 1.184, 95% CI 0.209 to 2.159, p = 0.017). Subgroup analysis demonstrated that European and South American AS patients had lower serum levels of sclerostin than controls. AS patients with age ≥ 40 years, erythrocyte sedimentation rate (ESR) ≤ 20mm/h and Bath Ankylosing Spondylitis Functional Index (BASFI) < 4 had statistically significant lower serum sclerostin concentrations compared to controls. Chinese and Korean AS patients as well as patients with lower CRP had higher serum BMP-2 levels than controls, and country may be a source of heterogeneity across the studies. No publication bias existed and sensitivity analysis confirmed the stability of results. Serum BMP-2, but not sclerostin levels may be closely related to the development of AS.

Effect of mechanical strain-induced PGE2 production on bone nodule formation by rat calvarial progenitor cells.

High-magnitude mechanical strain inhibits bone nodule formation by reducing expression of bone morphogenetic protein-2 (BMP-2), Runt-related transcription factor 2 (Runx2), and muscle segment homeobox 2 (Msx2). Mechanical strain also induces production of proinflammatory factor prostaglandin E2 (PGE2) by osteoblasts. We measured the effect of mechanical strain-induced PGE2 production on bone nodule formation and expression levels of bone formation-related factors. Osteoblast-like cells isolated from fetal rat calvariae were loaded with 18% cyclic tension force (TF) for 48 h in the presence or absence of NS-398, a selective inhibitor of cyclooxygenase-2. To investigate the effect of TF-induced PGE2 on bone formation, bone nodule area on day 21 was measured by von Kossa staining. BMP-2, Runx2, and Msx2 expression levels were examined at 1 day after TF loading. Bone nodule formation was significantly inhibited by TF but was restored to control level by PGE2 inhibition. Furthermore, TF loading-induced reductions in expressions of these factors were restored to control level by PGE2 suppression. These results indicate that PGE2 production induced by high-magnitude mechanical strain inhibits bone nodule formation by reducing expression levels of bone formation-related factors.

MiR-214-3p delays fracture healing in rats with osteoporotic fracture through inhibiting BMP/Smad signaling pathway.

The purpose of this study was to explore the mechanism of micro-ribonucleic acid (miR)-214-3p in regulating fracture healing in rats with osteoporosis. A total of 30 female Sprague-Dawley rats were selected and randomly divided into 3 groups, including group A [phosphate-buffered saline (PBS), n=10], group B (AntagomiR-NC, n=10), and group C (AntagomiR-214-3p, n=10). All rats underwent ovariectomy, and the osteoporosis rat model was verified by dual-energy X-ray absorptiometry 8 weeks after the operation. Then the osteoporotic fracture was established in rats via a second operation. From the successful modeling until the 6th week, 50 μL PBS (2 nmoL) was intraperitoneally injected in group A, an equal amount of AntagomiR-NC was injected in group B, and an equal amount of AntagomiR-214-3p was injected in group C once a week. At the 6th week, fracture healing of osteoporosis rats was evaluated. At the same time, the expression of miR-214-3p in the three groups was detected via reverse Transcription-Polymerase Chain Reaction (RT-PCR). Furthermore, the protein expressions of bone morphogenetic protein 2 (BMP2) and Smad4 in the three groups were detected via Western blotting (WB). After ovariectomy, the bone mineral density in each group was significantly lower than that before ovariectomy, and the differences were statistically significant (p<0.05). Imaging evaluation demonstrated that compared with group A and B, there were significantly more callus tissues in group C. Meanwhile, the fracture line healing was better and blurred, and the internal fixation had no displacement and loosening. RT-PCR results indicated that the expression level of miR-214-3p in group C was significantly lower than that of the other two groups (p<0.05). WB results showed that the protein expression levels of BMP2 and Smad4 in group C were significantly higher than those of group A and group B (p<0.05). MiR-214-3p delays fracture healing in rats with osteoporotic fracture by inhibiting the BMP/Smad signaling pathway.

Isoquercitrin, a flavonoid glucoside, exerts a positive effect on osteogenesis in vitro and in vivo

Isoquercitrin (quercetin-3-O-β-d-glucopyranoside) possess various pharmacological effect as a biologically active compound. The aim of the present study was to investigate its potential effects on the proliferation and osteoblastic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) in vitro and bone formation in the mid-palatal suture during rapid maxillary expansion (RME) in vivo. Cell proliferation of rat BMSCs was detected by cell-counting kit- 8 (CCK-8) assay. Alkaline phosphatase (ALP) activity assay and alizarin red staining were used to evaluate osteogenic differentiation of BMSCs. Furthermore, the expression levels of osteogenesis-related genes including runt-related transcription factor 2 (Runx2), bone sialoprotein (BSP) and activating transcription factor 6 (ATF6) were measured by real-time RT-PCR. In vivo, thirty-six male 6-week-old Wistar rats with or without suture expansion receive systemic administration of isoquercitrin or saline solution. Micro-CT, HE and Masson staining were used to compare the morphological changes between the groups. Bone morphogenetic protein 2 (BMP2) expression in the suture was detected using immunohistochemical staining. Our results showed that isoquercitrin significantly promoted cell proliferation, ALP activity and mineral deposition in the range from 0.01 to 1 μM. Moreover, the expression levels of Runx2, BSP and ATF6 were also upregulated. The measurement of micro-CT imaging and histological examinations demonstrated that daily oral administration of isoquercitrin (10 mg/kg) increased bone formation compared to the other groups. Furthermore, the expression level of BMP2 was also augmented in the presence of isoquercitrin. Consequently, those findings showed that isoquercitrin exerts stimulatory effects on osteogenesis in vitro and in vivo, suggesting that isoquercitrin could be a potential candidate for preventing relapse following RME within palatal sutures.

Role of TGF-β1 Signaling in Heart Valve Calcification Induced by Abnormal Mechanical Stimulation in a Tissue Engineering Model.

A tissue engineering model of heart valve calcification induced in a bioreactor was established to evaluate the calcification induced by abnormal mechanical stimulation and explore the underlying molecular mechanisms. Polyethylene glycol (PEG)-modified decellularized porcine aortic leaflets seeded with human valve interstitial cells (huVICs) were mounted on a Ti-Ni alloy frame to fabricate two-leaflet and threeleaflet tissue engineered valves. The two-leaflet model valves were exposed to abnormal pulsatile flow stimulation with null (group A), low (1000 mL/min, group B), medium (2000 mL/min, group C), and high velocity (3000 mL/min, group D) for 14 days. Morphology and calcification were assessed by von Kossa staining, alkaline phosphatase (ALP) content, and Runx2 immunostaining. Leaflet calcification and mRNA and protein expression of transforming growth factor (TGF)-β1, bone morphogenetic protein 2 (BMP2), Smad1, and MSX2 were measured at different time points. ALP content was examined in two-leaflet valves seeded with BMP2 shRNA plasmid-infected huVICs and exposed to the same stimulation conditions. The results showed that during 14 days of flow stimulation, huVICs on the leaflet surface proliferated to generate normal monolayer coverage in groups A, B, and C. Under mechanical stimulation, huVICs showed a parallel growth pattern in the direction of the fluid flow, but huVICs exhibited disordered growth in the high-velocity flow environment. von Kossa staining, ALP measurement, and immunohistochemical staining for Runx2 confirmed the lack of obvious calcification in group A and significant calcification in group D. Expression levels of TGF-β1, BMP2, and MSX2 mRNA and protein were increased under fluid stimulation. ALP production by BMP2 shRNA plasmid-infected huVICs on model leaflets was significantly reduced. In conclusion, abnormal mechanical stimulation in a bioreactor induced calcification in the tissue engineering valve model. The extent of calcification correlated positively with the flow velocity, as did the mRNA and protein levels of TGF-β1, BMP2, and MSX2. These findings indicate that TGF-β1/BMP2 signaling is involved in valve calcification induced by abnormal mechanical stimulation.

The effect of emphysema induced by tobacco smoke exposure and different rearing ways on the atherosclerosis formation

Objective To explore the relationship and its mechanism of emphysema induced by different ways of rearing and tobacco smoke exposure with the atherosclerosis formation. Methods Forty-eight SD rats were randomly divided into 6 groups (n=8, each): a normal diet + tobacco smoke exposure (TSE) group, a fat-rich diet + TSE group, a limit intake + TSE group, a normal diet group, a fat-rich diet group, and a limit intake group for 24 weeks.Lee's index, serum tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), adiponectin (APN) levels, mean linear intercept (MLI), mean alveoli number (MAN), the aortic pathology change, vascular endothelial growth factor (VEGF) of aorta, and bone morphogenetic protein-2 (BMP-2) level were collected. Results Tobacco smoke exposure, fat-rich diet, and limit intake increased the severity of emphysema.The microscopic features of aorta showed an early atherosclerosis in the tobacco smoke exposure groups, but no atherosclerosis in the non-smoking groups.Interactions between tobacco smoke exposure and different ways of rearing on serum APN level (F=10.68, P<0.05) were found.In the tobacco smoke exposure groups, Pearson correlation analysis showed positive correlations of aortic VEGF and BMP-2 levels with MLI, serum TNF-α and IL-6 levels (r=0.431, 0.471, 0, 448, 0.449, 0.428, 0.447, all P<0.05), and an negative correlation with MAN (r=-0.411, -0.442, P<0.05). Conclusions Both tobacco smoke exposure and different ways of rearing can influence the formation of emphysema.There is a positive correlation between the severities of emphysema and atherosclerosis, and systemic inflammation may be involved. Key words: Tobacco smoke pollution; Pulmonary emphysema; Atherosclerosis; Feeding methods

Hair follicle development and related gene and protein expression of skins in Rex rabbits during the first 8 weeks of life.

ObjectiveWe aimed to observe hair follicle (HF) development in the dorsal skin and elucidate the expression patterns of genes and proteins related to skin and HF development in Rex rabbits from birth to 8 weeks of age.MethodsWhole-skin samples were obtained from the backs of Rex rabbits at 0, 2, 4, 6, and 8 weeks of age, the morphological development of primary and secondary HFs was observed, and the gene transcript levels of insulin-like growth factor-I (IGF-I), epidermal growth factor (EGF), bone morphogenetic protein 2 (BMP2), transforming growth factor β-1, 2, and 3 (TGFβ-1, TGFβ-2, and TGFβ-3) were examined using quantitative real-time polymerase chain reaction (PCR). Additionally, Wnt family member 10b (Wnt10b) and β-Catenin gene and protein expression were examined by quantitative real-time PCR and western blot, respectively.ResultsThe results showed significant changes in the differentiation of primary and secondary HFs in Rex rabbits during their first 8 weeks of life. The IGF-I, EGF, TGFβ-2, and TGFβ-3 transcript levels in the rabbits were significantly lower at 2 weeks of age than at birth and gradually increased thereafter, while the BMP2 and TGFβ-1 transcript levels at 2 weeks of age were significantly higher than those at birth and gradually decreased thereafter. β-Catenin gene expression was also significantly affected by age, while the Wnt10b transcript level was not. However, the Wnt10b and β-catenin protein expression levels were the lowest at 2 and 4 weeks of age.ConclusionOur data showed that a series of changes in HFs in dorsal skin occurred during the first 8 weeks. Many genes, such as IGF-I, EGF, BMP2, TGFβ-1, TGFβ-2, TGFβ-3, and β-Catenin, participated in this process, and the related proteins Wnt10b and β-Catenin in skin were also affected by age.

Platelet-rich plasma decreases fibroblastic activity and woven bone formation with no significant immunohistochemical effect on long-bone healing: An experimental animal study with radiological outcomes.

This study aimed to analyze the immunohistochemical effect of platelet-rich plasma (PRP) on healing of long-bone fractures in terms of bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF), the Ki-67 proliferation index, and radiological and histological analyses. Sixteen adult rabbits, whose right femoral diaphysis was fractured and fixed with Kirschner wires, were randomly divided into two groups, control and PRP (groups A and B, respectively). PRP was given to group B at 1 week postoperatively, and all animals were euthanized after 12 weeks. Radiographic evaluations were performed periodically. Cortical callus formation, chondroid and woven bone area percentages, osteoblastic and fibroblastic activities, and mature bone formation were examined. The depths of BMP-2 and VEGF staining were measured. The Ki-67 proliferation index was also calculated. The mean radiological union score of group B was significantly higher than that of group A. There were also statistically significant differences between groups A and B in terms of cortical callus formation, woven bone area percentage, fibroblast proliferation, and mature bone formation. Group B had significantly more cortical callus and mature bone formation with less woven bone and fibroblast proliferation. Immunohistochemical analysis revealed no statistically significant difference between the groups in terms of BMP-2 and VEGF staining and the Ki-67 index. PRP had no effect on BMP-2 or VEGF levels with no increase in the Ki-67 proliferation index, although its application had a positive effect on bone healing by increasing callus and mature bone formation with decreased woven bone and fibroblast proliferation.