Alkaline Phosphatase mRNA Research Articles

Cyclic stretch promotes the ossification of ligamentum flavum by modulating the Indian hedgehog signaling pathway.

The Indian hedgehog (IHH) signaling pathway is an important pathway for bone growth and development. The aim of the present study was to examine the role of the IHH signaling pathway in the development of the ossification of ligamentum flavum (OLF) at the cellular and tissue levels. The expression levels and localization of the osteogenic genes Runt-related transcription factor 2 (RUNX2), Osterix, alkaline phosphatase (ALP), osteocalcin (OCN) and IHH were evaluated in OLF tissues by reverse transcription-quantitative PCR (RT-qPCR) and immunohistochemistry. Non-ossified ligamentum flavum (LF) sections were used as control samples. The tissue explant method was used to obtain cultured LF cells. In addition, OLF cells were subjected to cyclic stretch application for 0, 6, 12 or 24 h. The expression levels of osteogenic genes, and the IHH signaling pathway genes IHH, Smoothened (SMO), GLI family zinc finger 1 (GLI1), GLI2 and GLI3 were evaluated with RT-qPCR and western blotting. Osteogenic differentiation was further evaluated by assessing ALP activity and staining. Moreover, the effect of cyclopamine (Cpn), an IHH signaling inhibitor, on osteogenic differentiation was examined. The RT-qPCR and immunohistochemical results indicated that the mRNA and protein expression levels of RUNX2, Osterix, ALP, OCN and IHH were significantly higher in the OLF group compared with the LF group. Furthermore, application of cyclic stretch to OLF cells resulted in greater ALP activity, and significant increases in mRNA and protein expression levels of RUNX2, Osterix, ALP and OCN in a time-d00ependent manner. Cyclic stretch application also led to significant increases in IHH signaling pathway genes, including IHH, SMO, GLI1 and GLI2, while no significant effect was found on GLI3 expression level. In addition, it was found that Cpn significantly reversed the effect of cyclic stretch on the ALP activity, and the expression levels of RUNX2, Osterix, ALP, OCN, GLI1 and GLI2. Collectively, the present results suggested that the IHH signaling pathway may mediate the effect of cyclic stretch on the OLF cells.

Delayed tooth movement in Runx2+/− mice associated with mTORC2 in stretch-induced bone formation

Runt-related transcription factor 2 (Runx2) is an essential transcription factor for osteoblast differentiation, and is activated by mechanical stress to promote osteoblast function. Cleidocranial dysplasia (CCD) is caused by mutations of RUNX2, and CCD patients exhibit malocclusion and often need orthodontic treatment. However, treatment is difficult because of impaired tooth movement, the reason of which has not been clarified. We examined the amount of experimental tooth movement in Runx2+/− mice, the animal model of CCD, and investigated bone formation on the tension side of experimental tooth movement in vivo. Continuous stretch was conducted to bone marrow stromal cells (BMSCs) as an in vitro model of the tension side of tooth movement. Compared to wild-type littermates the Runx2+/− mice exhibited delayed experimental tooth movement, and osteoid formation and osteocalcin (OSC) mRNA expression were impaired in osteoblasts on the tension side of tooth movement. Runx2 heterozygous deficiency delayed stretch-induced increase of DNA content in BMSCs, and also delayed and reduced stretch-induced alkaline phosphatase (ALP) activity, OSC mRNA expression, and calcium content of BMSCs in osteogenic medium. Furthermore Runx2+/− mice exhibited delayed and suppressed expression of mammalian target of rapamycin (mTOR) and rapamycin-insensitive companion of mTOR (Rictor), essential factors of mTORC2, which is regulated by Runx2 to phosphorylate Akt to regulate cell proliferation and differentiation, in osteoblasts on the tension side of tooth movement in vivo and in vitro. Loss of half Runx2 gene dosage inhibited stretch-induced PI3K dependent mTORC2/Akt activity to promote BMSCs proliferation. Furthermore, Runx2+/− BMSCs in osteogenic medium exhibited delayed and suppressed stretch-induced expression of mTOR and Rictor. mTORC2 regulated stretch-elevated Runx2 and ALP mRNA expression in BMSCs in osteogenic medium. We conclude that Runx2+/− mice present a useful model of CCD patients for elucidation of the molecular mechanisms in bone remodeling during tooth movement, and that Runx2 plays a role in stretch-induced proliferation and osteogenesis in BMSCs via mTORC2 activation.

Adhesion, intracellular signalling and osteogenic differentiation of mesenchymal progenitor cells and preosteoblasts on poly(epsilon)caprolactone films functionalized by peptides derived from fibronectin and/or BMP-9.

Biomaterials that can control the behaviour of stem cells play a major role in regenerative medicine and tissue engineering. We previously showed that poly(epsilon)caprolactone (PCL) films functionalized with adhesive peptides containing sequences of both cell binding domain (RGD) and synergistic site (PHSRN) of the fibronectin (pFibro) enhanced the osteoblastic commitment of C3H10T1/2 mesenchymal progenitor cells (C3H10T1/2 cells) induced by soluble BMP-9 or its derived peptide SpBMP-9. Here, the effect of PCL films functionalized with pFibro and/or SpBMP-9 or its negative peptide NSpBMP-9 on adhesion and intracellular signalling of C3H10T1/2 cells was determined. The differentiation of adherent C3H10T1/2 cells and MC3T3-E1 preosteoblasts into osteoblasts was also analysed with or without IGF-2, since this growth factor can favour the osteoblastic differentiation induced by BMP-9. We found that pFibro and SpBMP-9 co-functionalization on PCL films promoted the adhesion of C3H10T1/2 cells with well-organized focal adhesion points and FAK activation. In these mesenchymal progenitor cells, PCL-SpBMP-9 and PCL-pFibro/SpBMP-9 induced the activation and nuclear translocation of Smad 1/5 after 4h, and enhanced the protein expression of RUNX2 (3 d) and alkaline phosphatase (ALP) activity (7 d), two markers of osteoblast differentiation. No PPARγ, a marker of adipogenic differentiation, was detected. C3H10T1/2 cells attached to PCL-SpBMP-9 also contained more SOX9, a marker of chondroblastic lineage, compared with other experimental conditions. The use of inactive peptides NSpBMP-9 confirmed the specificity and effectiveness of SpBMP-9 on cell adhesion, intracellular signalling and osteoblastic differentiation. Adding IGF-2 only significantly improved the differentiation of MC3T3-E1 preosteoblasts into osteoblasts as shown by the increase in gene expression encoding Osterix (mRNA Sp7) and ALP (mRNA Alpl), probably because of the lack of serum in the medium. Therefore, material surface co-functionalized with pFibro and SpBMP-9 could be most useful for developing scaffolds with both osseointegrative and osteoinductive properties for bone application and tissue engineering strategy when combined with IGF-2 in serum free medium.

Microfluidic fabrication of alendronate-loaded chitosan nanoparticles for enhanced osteogenic differentiation of stem cells

AimsIn this study, we used a cross-junction microfluidic device for preparation of alendronate-loaded chitosan nanoparticles with desired characteristics to introduce a suitable element for bone tissue engineering scaffolds. Main methodsBy controlling the reaction condition in microfluidic device, six types of alendronate-loaded chitosan nanoparticles were fabricated which had different physical properties. Hydrodynamic diameter of synthetized particles was evaluated by dynamic light scattering (102 to 215 nm). Nanoparticle morphology was determined by SEM and AFM images. The osteogenic effects of prepared selected nanoparticles on human adipose stem cells (hA-MSCs) were evaluated by assessment of alkaline phosphatase (ALP) activity, calcium deposition, ALP and osteopontin gene expression. Key findingsThe highest loading efficiency percentage (%LE) was %32.42 ± 2.02. Based on MTT assessment, two samples which had no significant cytotoxicity were chosen for further studies (particle sizes and %LE were 142 ± 6.1 nm, 198 ± 16.56 nm, %16.76 ± 3.91 and %32.42 ± 2.02, respectively). In vitro release behavior of nanoparticles displayed pH responsive characteristics. Significant faster release was seen in acidic pH = 5.8 than neutral pH = 7.4. The selected nanoparticles demonstrated higher ALP activity at 14 days in comparison to selected blank sample and osteogenic differentiation media (ODM) and a downregulation at 21 days in comparison to 14 days. Calcium content assay at 21 days displayed significant differences between alendronate-loaded nanoparticles and ODM. ALP and osteopontin mRNA expression was significantly higher than the cells cultured in ODM at 14 and 21 days. SignificanceWe concluded that our prepared nanoparticles significantly enhanced osteogenic differentiation of hA-MSCs and can be a suitable compartment of bone tissue engineering scaffolds.

Effects of Novel Laser Dental Implant Microtopography on Human Osteoblast Proliferation and Bone Deposition

The aim of this study was to compare how two innovative laser titanium surfaces and sandblasted and acid-etched surfaces influence human osteoblast behavior during osteogenesis and the initial phases of bone deposition. Human osteoblasts from human adipose stem cells were sorted by flow cytometric analysis and induced to differentiate. After 40 days, the osteogenic differentiation was detected by alizarin red staining, and the alkaline phosphatase (ALP) was evaluated with western blot (WB) and real-time reverse transcriptase-polymerase chain reaction (RT-PcR) analysis. After confluence, human osteoblasts were cultured onto two different innovative laser-obtained titanium surfaces (L1 and L2) and compared with one sandblasted and acid-etched (SBAE) surface as the control. At different times, human osteoblast behavior was evaluated with cell proliferation viability assay (MTT), scanning electron microscopy (SEM), energy-dispersive x-rays (EDAX), osteogenic markers with RT-PcR, and WB analysis of matrix extracellular phosphoglycoprotein (MEPE), ALP, and osteocalcin (OCN). After cell sorting, the human osteoblasts from human adipose stem cells showed increasing values of ALP mRNA and protein expression. The osteogenic differentiation was confirmed by quantitative alizarin red staining assay. Profilometric and SEM analysis showed relevant differences between SBAE, L1, and L2 specimens. After 20 days of culture onto titanium samples, SEM evaluation showed a small number of human osteoblasts and isolated sites of bone matrix deposition in SBAE specimens. At the same time, on L1 surfaces, only an osteoblast mono-layer with initial bone deposition was found, while on L2 specimens, there was a thick network with flattened large stellate cells, many cellular interconnections with strong titanium adhesion, and a large complex mineralized structure of crystal bone. After 20 days, for all titanium samples, human osteoblasts culturing EDAX analysis showed the absence of impurities and a higher bone matrix deposition in L2 specimens compared with L1 and SBAE samples. The innovative microtopography and nanotopography laser-induced surface showed high biocompatibility with primary human osteoblast cultures and the absence of impurities. The innovative laser texture was capable of influencing the osteogenic process, confirming the critical role of titanium surface characteristics in the cell adhesion and bone deposition during the early phases of osseointegration. The association of human adipose stem cells and titanium surfaces laser-induced with an innovative procedure could generate promising improvements and developments in orthopedics, maxillofacial, and dental implant surgery.

Reduction of inflammation in a chronic periodontitis model in rats by TNF-α gene silencing with a topically applied siRNA-loaded calcium phosphate paste

We developed a calcium phosphate-based paste containing siRNA against TNF-α and investigated its anti-inflammatory and bone-healing effects in vitro and in vivo in a rat periodontitis model. The bioactive spherical CaP/PEI/siRNA/SiO2 nanoparticles had a core diameter of 40–90 nm and a positive charge (+23 mV) that facilitated cellular uptake. The TNF- α gene silencing efficiency of the nanoparticles in J774.2 monocytes, gingival-derived cells, and bone marrow-derived cells was 12 ± 2%, 36 ± 8%, and 35 ± 22%, respectively. CaP/PEI/siRNA/SiO2 nanoparticles cancelled the suppression of alkaline phosphatase (ALP) activity in LPS-stimulated bone marrow-derived cells. In vivo, ALP mRNA was up-regulated, TNF-α mRNA was down-regulated, and the amount of released TNF-α was significantly reduced after topical application of the calcium phosphate-based paste containing siRNA-loaded nanoparticles. The number of TNF-α-positive cells in response to CaP/PEI/siRNA/SiO2 nanoparticle application was lower than that observed in the absence of siRNA. Elevated ALP activity and numerous TRAP-positive cells (osteoclasts) were observed in response to the application of all calcium phosphate pastes. These results demonstrate that local application of a paste consisting of siRNA-loaded calcium phosphate nanoparticles successfully induces TNF-α silencing in vitro and in vivo and removes the suppression of ALP activity stimulated by inflammation. Statement of significanceWe developed a calcium phosphate-based paste containing nanoparticles loaded with siRNA against TNF-α. The nanoparticles had a core diameter of 40–90 nm and positive charge (+23 mV). The anti-inflammatory and osteoinductive effects of the paste were investigated in vitro and in vivo in a rat periodontitis model. In vitro, the TNF-α gene silencing efficiency of the nanoparticles in J774.2 monocytes, gingival-derived cells, and bone marrow-derived cells was 12 ± 2%, 36 ± 8%, and 35 ± 22%, respectively. The ALP activity of bone marrow-derived cells was recovered. In vivo, TNF-α mRNA was down-regulated and the amount of released TNF-α was significantly reduced, whereas the ALP mRNA was up-regulated. Elevated ALP activity and TRAP-positive cells were observed by immunohistochemistry.

Lipopolysaccharide inhibits osteogenic differentiation of periodontal ligament stem cells partially through toll-like receptor 4-mediated ephrinB2 downregulation.

This study aimed to investigate the possible crosstalk between LPS/toll-like receptor 4 (TLR4) and ephrinB2 signaling in mediating osteogenic differentiation of PDLSCs. Human periodontal ligament stem cells (hPDLSCs) were harvested and treated with different concentrations of LPS under osteogenic induction. qPCR, alkaline phosphatase (ALP) staining, and Alizarin Red S staining were performed to assess osteogenic gene expression, ALP activity, and mineralized nodule formation. EphrinB2 mRNA and protein expressions after LPS treatment were also determined. To explore the role of ephrinB2 in LPS-impaired osteogenic differentiation of hPDLSCs, hPDLSCs were stimulated with ephrinB2-Fc or transfected with ephrinB2 lentivirus, and then, the osteogenic differentiation capacity was evaluated. LPS inhibited osteogenic differentiation of hPDLSCs and downregulated ephrinB2 expression in hPDLSCs during osteogenic differentiation. Blockage of TLR4 partially reversed LPS-induced decrease in ephrinB2 expression. EphrinB2-Fc promoted mineralized nodule formation and increased the expression of ALP, osteocalcin (OCN), and bone morphogenetic protein 2 (BMP2) in hPDLSCs. EphrinB2-overexpressing hPDLSCs treated with LPS expressed higher ALP and BMP2 mRNA and higher ALP activity and showed more mineralized nodule formation, when compared with wide-type hPDLSCs treated with LPS. Our data suggested that LPS decreased the osteogenic differentiation capacity of hPDLSCs partially through downregulation of ephrinB2 expression via LPS/TLR4 signaling. Upregulation of ephrinB2 partially reversed the impaired osteogenic potential of hPDLSCs induced by LPS. Our results provided a new insight of mechanism underling LPS-mediated osteogenic differentiation inhibition of PDLSCs and clarified a potential target for the management of periodontitis.

In vitro cytocompatibility and osteogenic potential of calcium silicate-based dental cements in a root canal-filling model.

ObjectiveTo investigate the in vitro cytocompatibility and osteogenic potential of an experimental calcium silicate-based cement and the inflammatory response in human periodontal ligament stem cells (hPDLSCs).MethodsCellular responses, osteogenic-related gene expression, and the production of inflammatory cytokines including interleukin (IL)-6 and IL-8 were studied in hPDLSCs exposed to the experimental root canal-filling material C-Root, the commercial tricalcium silicate-based material BioRoot RCS, and the epoxy resin-based material AH Plus. Differences were analyzed using one-way ANOVA with Bonferroni-adjusted pairwise comparison.ResultsExposure to BioRoot and C-Root caused time-dependent increases in cell proliferation. Significantly more mineralized nodules were formed in cells exposed to AH Plus and BioRoot compared with the negative control. Alkaline phosphatase (ALP) activity was significantly lower in AH Plus cells compared with negative control, BioRoot, and C-Root cells. ALP, osteocalcin (OCN), and runt-related transcription factor2 (RUNX2) mRNA expression levels were all significantly higher in C-Root compared with AH Plus cells at day 7. IL-6 and IL-8 levels differed significantly among the experimental groups, with the highest IL-8 levels in BioRoot cells at days 7 and 14.ConclusionThe experimental root canal-filling material C-Root has similar in vitro cytocompatibility to BioRoot and better osteogenic potential than AH Plus.

Interpenetrated nano- and submicro-fibrous biomimetic scaffolds towards enhanced mechanical and biological performances.

Developing fibrous scaffolds with hierarchical structures that closely mimic natural extracellular matrix (ECM) is highly desirable. However, fabricating scaffolds with true nanofibers (<100 nm) and submicrofibers (<1 μm) remains a big challenge. In this work, to mimic the fibrillar structure of natural ECM, bacterial cellulose (BC) nanofibers were hybridized with cellulose acetate (CA) submicrofibers for the first time. The interpenetrated nano-submicron fibrous BC/CA scaffold was fabricated using the combined electrospinning and modified in situ biosynthesis method. The BC/CA scaffold has an integrated symmetrical nanostructure in which BC nanofibers (42 nm in diameter) penetrate into the submicrofibrous CA (820 nm in diameter) scaffold. The BC/CA scaffold shows an interconnected porous structure with a high porosity of >90%. Additionally, the combination of CA submicrofibers with BC nanofibers leads to significantly improved mechanical properties over nanofibrous BC and submicrofibrous CA scaffolds and enlarged pores over nanofibrous BC scaffold. In addition, the biological behaviors of prepared BC/CA on MC3T3-E1 cells were investigated. Results suggested that BC/CA scaffold is beneficial for cell migration and proliferation. Moreover, the BC/CA scaffold shows higher alkaline phosphatase (ALP) activity, and calcium depositions. In addition, the hierarchical structures can effectively improve the expression of osteogenic gene (ALP mRNA and Runx2 mRNA) and protein (ALP). We believe that the methodology might provide biomimetic morphological microenvironments for enhanced tissue regeneration.

MiR-128 inhibits the osteogenic differentiation in osteoporosis by down-regulating SIRT6 expression.

Background: MicroRNAs (miRNAs) are involved in the regulation of osteogenic differentiation and chondrification in vivo. The purpose of the present study was to explore the potential mechanism of miR-128 in osteoporosis (OP).Methods: Quantitative real-time PCR (qRT-PCR) was used to determine the expression of miR-128 in femoral neck trabecular bones of OP patients (n=40) and non-OP patients (n=40). C2C12 cells were transfected with miR-128 mimic or inhibitor to determine the effect of miR-128 on osteoblastic differentiation of C2C12 cells. Bioinformatics and luciferase reporter genes were used to determine the molecular mechanism of miR-128 in osteoblastic differentiation of C2C12 cells.Results: The qRT-PCR results showed that the expression level of miR-128 in bone samples of OP patients was significantly higher than that of non-OP patients, while miR-128 was significantly down-regulated during the osteogenic differentiation of C2C12 cells. In addition, the results showed that overexpression of miR-128 significantly inhibited the mRNA and protein expression levels of osteocalcin (OC), alkaline phosphatase (ALP) and collagen I type-α1 (COL1A1) in C2C12 cells, while miR-128 inhibitor could reverse this effect. Bioinformatics analysis and dual-luciferase reporter assay found that silencing information regulatory protein 6 (SIRT6) was a direct target of miR-128. The qRT-PCR and Western Blot results found that miR-128 significantly down-regulated the mRNA and protein expressions of SIRT6. Furthermore, silencing SIRT6 significantly inhibited the promoting effect of the miR-128 inhibitor on the expression of osteoblast markers.Conclusion: The above results confirmed that miR-128 inhibited osteoblast differentiation in OP by down-regulating SIRT6 expression, thus accelerating the development of OP.

251. Additive manufactured Ti-6Al-4V/polyetheretherketone composite porous cage for interbody fusion: bone growth and biocompatibility evaluation in a porcine model

BACKGROUND CONTEXT Several materials have been used to manufacture the cages, the most common being polyetheretherketone (PEEK) and titanium (Ti) alloy (Ti–6Al–4V). Each material has its advantages and disadvantages. PURPOSE The purpose is to utilize the advantages of each material in combination with additive manufacturing technology to develop a new porous Ti alloy/PEEK composite interbody cage. STUDY DESIGN/SETTING In vitro mechanical tests and cell biocompatibility study; in vivo posterior instrumented interbody fusion porcine model. PATIENT SAMPLE In vitro cell culture analysis for Porous Ti alloy/PEEK composite cages. In vivo animal study used 20 female pigs through an anterior intervertebral lumbar fusion and posterior pedicle screws augmentation. Each level was randomly implanted with one of five testing cages. OUTCOME MEASURES In vitro mechanical tests for shear strength of the interface layer. In vitro cell culture analysis for alkaline phosphatase (ALP) activity, ALP and osteocalcin mRNA expression for in vitro study. Micro–computed tomography (CT), back-scattered-electrons SEM (BSE-SEM) and histological analyses for in vivo study. METHODS In vitro and in vivo study compared 5 groups of cages. The first was a commercialized pure PEEK cage (group 1). The second was a Ti alloy/PEEK composite cage with nonporous Ti alloy end plates (group 2). The third, fourth, and fifth groups were composite cages with porosity of 40%, 60%, and 80% (groups 3, 4, and 5), respectively. Mechanical tests, alkaline phosphatase activity and mRNA expression, osteocalcin mRNA expression, SEM were performed for in vitro study. Micro–computed tomography (CT), back-scattered-electrons SEM (BSE-SEM) and histological analyses were performed for in vivo study. RESULTS The shear strength reached 33.4 MPa at the Ti alloy/PEEK interface. ALP activity and ALP mRNA expression were positively correlated with the porosity rate and peaked at 60% porosity. Micro-CT and BSE-SEM demonstrated that structures with high porosities, especially 60% and 80%, facilitated more bone formation inside the implant but not outside the implants. Histological analysis also showed that bone formation was better in Ti alloy groups than in the PEEK group. CONCLUSIONS In vitro cell culture showed higher porous structure exhibited superior bone growth. Micro-CT and histological analyses showed improved bone growth in high-porosity composite cage groups. Composite cage implant takes the biological advantages of Ti alloy porous end plates and the mechanical and radiographic advantages of PEEK central core to incorporate as a single implant suitable for intervertebral fusion. FDA DEVICE/DRUG STATUS Unavailable from authors at time of publication.

High Expression of Acid-Sensing Ion Channel 2 (ASIC2) in Bone Cells in Osteoporotic Vertebral Fractures.

Little is known about the function of acid-sensing ion channels (ASICs) in bone cells or osteoporotic vertebral fractures (OVF). This study delineated ASICs expression in adult human bone marrow-mesenchymal stem cells- (BM-MSC-) derived osteoblasts and in OVF bone cells. Adult BM-MSC-derived osteoblasts were isolated and cultured in different pH values. Osteogenic markers as alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OC) mRNA were assessed. Western blots method was applied to analyze ASICs protein expression in different pH values. Amiloride was added into the osteogenic media to analyze the Na+/K+ ATPase change. We harvested the vertebral cancellous bone through a bone biopsy needle in 26 OVF patients when performing percutaneous vertebroplasty. Six vertebral bone specimens obtained from 4 patients with high-energy vertebral fractures were used as the control. The reverse transcription polymerase chain reaction was performed to analyze the quantitative mRNA expression of ASICs. Osteogenic markers as ALP, OPN, and OC mRNA were higher expressed in increasing pH values throughout osteoblastogenesis. ASIC proteins were higher expressed in lower pH media, especially ASIC3, and ASIC4. The highest protein expression at days 7, 14, and 21 was ASIC2, ASIC4, and ASIC3, respectively. Expression of Na+/K+ ATPase was significantly decreased in cultured osteoblasts by addition of amiloride into the pH 6.9 osteogenic media. ASIC2 mRNA was most highly expressed with a 65.93-fold increase in the biopsied vertebral bone cells in OVF compared with the control. In conclusion, we found osteoblastogenesis was reduced in an acidic environment, and ASIC2, ASIC3, and ASIC4 were most highly expressed in turn during osteoblastogenesis within acidic media. ASIC2 was the most abundantly expressed gene in human bone cells in OVF compared with the control. ASIC2 could be crucial in the pathogenesis of osteoporosis and could serve as a therapeutic target for antiosteoporotic therapies.

BMP9 directly induces rapid GSK3-β phosphorylation in a Wnt-independent manner through class I PI3K-Akt axis in osteoblasts.

Bone morphogenetic protein (BMP)9 has been reported to be the most potent BMP to induce bone formation. However, the details of BMP9-transduced intracellular signaling remain ambiguous. Here, we have investigated signal transduction mechanisms of BMP9 in comparison to BMP2, another potent inducer of bone formation, in osteoblasts. In a mouse osteoblast cell line, BMP9 induced higher mRNA levels of alkaline phosphatase (ALP) and runt-related transcription factor 2 (Runx2) than BMP2 within 2 h. Unlike BMP2, BMP9 induced rapid phosphorylation of glycogen synthase kinase 3-β (GSK3-β) and protein kinase B (Akt) and increased the cellular protein content of β-catenin. BMP9 moderately increased mRNA levels of several canonical Wingless-related integration site to lower degrees than BMP2. Furthermore, BMP9-induced GSK3-β phosphorylation was not inhibited by pretreatment with actinomycin D, cycloheximide, or Brefeldin A, indicating it is independent of Wnt protein secretion. BMP9-induced GSK3-β phosphorylation was abrogated by Akt or class I PI3K-specific inhibitors. Moreover, inactivation of GSK3-β by LiCl did not further promote ALP and Runx2 mRNA induction by BMP9 as significantly as that by BMP2. Notably, BMP9-induced GSK3-β phosphorylation was inhibited by small interfering RNA against endoglin and GIPC PDZ domain-containing family, member 1. Taken together, our present findings have indicated that BMP9 directly activates GSK3β-β-catenin signaling pathway through class I PI3K-Akt Axis in osteoblasts, which may be essential for the potent osteoinductive activity of BMP9.-Eiraku, N., Chiba, N., Nakamura, T., Amir, M. S., Seong, C.-H., Ohnishi, T., Kusuyama, J., Noguchi, K., Matsuguchi, T. BMP9 directly induces rapid GSK3-β phosphorylation in a Wnt-independent manner through class I PI3K-Akt axis in osteoblasts.

Role of P2X7 receptor in the osteogenic differentiation of bone marrow stromal cells induced by electromagnetic fields

Objective The aim of this study was to elucidate the role of P2X7 receptors in the osteogenic differentiation of bone marrow stromal cells (BMSCs) induced by electromagnetic fields (EMF), and the underlying mechanism. Methods BMSCs of 4-week-old male SD rat were isolated and cultured in vitro. The cells at passage 3 were used in this study. To select the most effective frequency EMF, mRNA and protein levels of P2X7 receptoe in BMSCs, which were induced by 1 mT, 7.5-75.0 Hz positive metaphysical EMF for 1 week, were investigated by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and Western blotting. BMSCs cultured in osteogenic medium with or without A740003 (a specific P2X7 antagonist, 5 μmol/L) were stimulated by selected frequency EMF for 1 week, and then expression of runt related transcription factor-2 (Runx2), alkaline phosphatase (ALP), osteopontin (OPN) and activation of protein kinase B (Akt)/glycogen synthase kinase 3β (GSK3β)/β-catenin in BMSCs were investigated by Western blotting or RT-qPCR.Measurement data were expressed as (Mean ± SD) and ANOVA analysis of variance was used. Results 1 mT, 15 Hz EMF exhibited the optimal effect on expression of P2X7 receptor, and the relative expression of P2X7 mRNA and protein was 2.0 times and 1.9 times as much as that in control group, respectively. The relative expression of RUNX2, ALP and OPN mRNA in 1 mT, 15 Hz EMF group were 2.1, 5.3 and 3.7 times of that in control group, and the relative expression of RUNX2, ALP and OPN protein was 1.3, 1.6 and 1.5 times of that in control group, respectively. When BMSCs induced by A740003 medium were stimulated by 1 mT, 15 Hz EMF, the relative expression of RUNX2, ALP and OPN mRNA decreased by 36%, 42% and 41% than that in control group, and the relative expression of RUNX2, ALP and OPN protein decreased by 28%, 30% and 26% than that in control group, respectively. Use of A740003 or phosphoinositol 3 kinase (PI3K)/Akt pathway blocker LY294002 (5 μmol/L) alone decreased the levels of phosphorylated Akt and GSK3β induced by EMF, and also decreased the expression of intracellular β-catenin. Conclusion EMF increase the expression of P2X7 of rat BMSCs, which activates Akt/GSK3β/β-catenin axis and finally promotes the osteogenesis. Key words: Electromagnetic fields; Purinergic receptor P2X7; Bone mesenchymal stem cells; Osteogenic differentiation

Research of simulated microgravity regulate MC3T3-E1 cells differentiation through the nuclear factor-kappa B signaling pathway

In this study, we aim to investigat the effect of microgravity on osteoblast differentiation in osteoblast-like cells (MC3T3-E1). In addition, we explored the response mechanism of nuclear factor-kappa B (NF-κB) signaling pathway to "zero- g" in MC3T3-E1 cells under the simulated microgravity conditions. MC3T3-E1 were cultured in conventional (CON) and simulated microgravity (SMG), respectively. Then, the expression of the related osteoblastic genes and the specific molecules in NF-κB signaling pathway were measured. The results showed that the mRNA and protein levels of alkaline phosphatase (ALP), osteocalcin (OCN) and type Ⅰ collagen (CoL-Ⅰ) were dramatically decreased under the simulated microgravity. Meanwhile, the NF-κB inhibitor α (IκB-α) protein level was decreased and the expressions of phosphorylation of IκB-α (p-IκB-α), p65 and phosphorylation of p65 (p-p65) were significantly up-regulated in SMG group. In addition, the IL-6 content in SMG group was increased compared to CON. These results indicated that simulated microgravity could activate the NF-κB pathway to regulate MC3T3-E1 cells differentiation.

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.

Forms of selenium in vitamin-mineral mixes differentially affect serum alkaline phosphatase activity, and serum albumin and blood urea nitrogen concentrations, of steers grazing endophyte-infected tall fescue.

The goal of this study was to test the hypothesis that sodium selenite (ISe), SEL-PLEX (OSe), vs. a 1:1 blend (MIX) of ISe and OSe in a basal vitamin-mineral mix would differentially affect serological and hepatic parameters of growing steers grazing toxic endophyte-infected tall fescue-mixed forage pasture. Predominately Angus steers (BW = 183 ± 34 kg) were randomly selected from herds of fall-calving cows grazing endophyte-infected tall fescue-mixed pasture and consuming vitamin-mineral mixes that contained 35 ppm Se as ISe, OSe, and MIX forms. Steers were weaned, depleted of Se for 98 d, and subjected to summer-long common grazing of an endophyte-infected tall fescue-mixed pasture (0.51 ppm total ergovaline + ergovalinine; 10.1 ha). Steers were assigned (n = 8 per treatment) to the same Se form treatments upon which they were raised. Se treatments were administered by daily top-dressing 85 g of vitamin-mineral mix onto 0.23 kg soyhulls, using in-pasture Calan gates. The PROC MIXED procedure of SAS was used to assess the effect of Se form treatments on serum parameters at day 0, 22, 43, 64, and 86. After slaughter, the effect of Se treatment on hepatic alkaline phosphatase (tissue nonspecific isoform, TNALP) mRNA, protein, and albumin protein content was assessed using the PROC GLM procedure of SAS. Fisher's protected LSD procedure was used to separate treatment means. Partial correlation analysis was used to evaluate the relationship among whole blood Se concentration and serum parameters, accounting for the effect of time. Across periods, MIX steers had more (P ≤ 0.04) serum albumin than OSe and ISe steers, respectively. However, the relative hepatic bovine serum albumin protein content was not affected (P = 0.28) by Se treatments. Serum alkaline phosphatase activity was greater (P ≤ 0.01) in MIX and OSe steers. Similarly, hepatic TNALP protein content in MIX steers was greater (P = 0.01) than ISe steers. Partial correlation analysis revealed that serum albumin, blood urea nitrogen, and alkaline phosphatase activity were correlated (r ≥ 0.23, P ≤ 0.02) with whole blood Se concentration. In summary, consumption of 3 mg Se/d as OSe or MIX forms of Se in vitamin-mineral mixes increased serum albumin concentration and alkaline phosphatase activity, the reduction of which is associated with fescue toxicosis. We conclude that the organic forms of Se ameliorated the depression of 2 of known serological biomarkers of fescue toxicosis.

Effect of cell culture density on dental pulp-derived mesenchymal stem cells with reference to osteogenic differentiation

Dental pulp stem cells (DPSCs) are a good source for tissue regeneration, however, the number of DPSCs in the pulp tissue is limited. Cell propagation is essential for tissue engineering using DPSCs and the cell culture conditions may affect the properties of DPSCs. The purpose of this study was to analyze the effect of cell culture condition, especially dense culture condition, on the property and differentiation pathway of DPSCs. We cultured DPSCs under sparse (sDPSCs; 5 × 103 cells/cm2) or dense (dDPSCs; 1 × 105 cells/cm2) conditions for 4 days and compared their properties. The populations of CD73+ and CD105+ cells were significantly decreased in dDPSCs. Both groups showed multi-differentiation potential, but mineralized nodule formation was enhanced in dDPSCs. The phosphorylation of focal adhesion kinase (FAK) and phosphoinositide 3-kinase (PI3K) proteins was promoted in dDPSCs, and alkaline phosphatase (ALP) mRNA expression in dDPSCs was abolished in the presence of pan-PI3K and FAK inhibitors. dDPSCs implanted into mouse bone cavities induced more mineralized tissue formation than sDPSCs and control. These findings indicate that dense culture conditions modified the properties of DPSCs and gave rise to osteogenic-lineage commitment via integrin signaling and suggest that dense culture conditions favor the propagation of DPSCs to be used for mineralized tissue regeneration.

Correlation of spontaneous adipocyte generation with osteogenic differentiation of porcine skin-derived stem cells.

The objective of this study was to examine effects of spontaneous adipocyte generation on osteogenic differentiation of porcine skin-derived stem cells (pSSCs). Correlation between osteogenic differentiation and adipocyte differentiation induced by osteocyte induction culture was determined using different cell lines. Osteogenic differentiation efficiency of pSSCs was then analyzed by controlling the expression of adipocyte-specific transcription factors during osteogenic induction culture. Among four cell lines, pSSCs-II had the lowest lipid droplet level but the highest calcium content (p < 0.05). It also expressed significantly low levels of peroxisome proliferator-activated receptor gamma 2 (PPARγ2) and adipocyte protein 2 (aP2) mRNAs but very high levels of runt-related transcription factor 2 (Runx2) and alkaline phosphatase (ALP) mRNAs as osteogenic makers (p < 0.05). Oil red O extraction was increased by 0.1 µM troglitazone (TGZ) treatment but decreased by 50 µM bisphenol A diglycidyl ether (BADGE) (p < 0.05). Calcium content was drastically increased after BADGE treatment compared to that in osteogenic induction control and TGZ-treated pSSCs (p < 0.05). Relative expression levels of PPARγ2 and aP2 mRNAs were increased by TGZ but decreased by BADGE. Expression levels of Rucx2 and ALP mRNAs, osteoblast-specific marker genes, were significantly increased by BADGE treatment (p < 0.05). The expression level of BCL2 like 1 was significantly higher in BADGE-treated pSSCs than that in TGZ-treated ones (p < 0.05). The results demonstrate that spontaneous adipocyte generation does not adversely affect osteogenic differentiation. However, reducing spontaneous adipocyte generation by inhibiting PPARγ2 mRNA expression can enhance in vitro osteogenic differentiation of pSSCs.

MicroRNA-29a-3p enhances dental implant osseointegration of hyperlipidemic rats via suppressing dishevelled 2 and frizzled 4

BackgroundFine osseointegration is the basis of long-term survival of implant. In our previous study, we observed a strong correlation between hyperlipidemia and compromised osseointegration. MicroRNA-29a-3p (miR-29a-3p) has been discovered to participate in bone marrow mesenchymal stem cells (BMSCs) differentiation. However, the role and the underlying mechanisms of hyperlipidemia and miR-29a-3p in osseointegration still remain obscure.ResultsIn peri-implant bone tissues of hyperlipidemia rats, bone mass, mineralization and bone trabecula formation were weakened. Alkaline phosphatase (ALP) and runt-related transcription factor 2 (Runx2), and miR-29a-3p expression were reduced. While in normal rats, implant-bone interfaces were filled with dense new bone and ALP, Runx2 and miR-29a-3p were up-regulated. Overexpressed miR-29a-3p can reverse the adverse effect of hyperlipidemia on osseointegration. Implants were tightly integrated with the surrounding dense new bone tissues, and ALP as well as Runx2 mRNAs were enhanced in miR-29a-3p overexpressed and hyperlipidemia rats, while little peri-implant bone tissue existed, ALP and Runx2 deregulated on miR-29a-3p inhibited rats. Dishevelled 2 (Dvl2) mRNA was declined in peri-implant bone tissue of high-fat (HF) group than normal group, while frizzled 4 (Fzd4) mRNA declined on day 5 and increased from day 10 to day 20 after implantation in hyperlipidemia rats than in normal rats. Next, BMSCs were cultured under HF or normal medium in vitro. In the HF group, ALP activity and mineralization, ALP and Runx2 mRNAs and proteins expression, and miR-29a-3p expression were suppressed, while adipogenesis was increased, as a result, cytoskeletons were sparse and disordered compared to control group. However, when miR-29a-3p was overexpressed in BMSCs, ALP activity, ALP, Runx2, Dvl2 and Fzd4 mRNAs and proteins expressions were up-regulated. As miR-29a-3p was inhibited in BMSCs, the reverse results were obtained. In addition, promoter assay revealed that miR-29a-3p can directly suppress Wnt/β-catenin pathway related Dvl2 and Fzd4 through binding to their 3′-UTR.ConclusionsMiR-29a-3p facilitated implant osseointegration via targeting Wnt/β-catenin pathway-related Dvl2 and Fzd4. MiR-29a-3p/Dvl2/Fzd4 may serve as a promising therapeutic target for hyperlipidemia osseointegration.