Osteocalcin mRNA Levels Research Articles

Effects of i-PRF, A-PRF+, and EMD on Osteogenic Potential of Osteoblasts on Titanium.

The study evaluates three biologically active substances with known bone-inductive potential on previously decontaminated titanium (Ti) discs. Rough and smooth Ti surfaces were contaminated with a multispecies biofilm and cleaned with a chitosan brush. Discs were treated either with injectable-platelet-rich fibrin (i-PRF), advanced platelet-rich fibrin (A-PRF+), or enamel matrix derivatives (EMDs) before osteoblast seeding. Biocompatibility, adhesion, migration, and gene expression of runt-related transcription factor 2 (RUNX2), collagen Type I Alpha 2 (COL1a2), alkaline phosphatase (ALP), osteocalcin (OC), and osteonectin (ON) were performed. All the tested biologic agents similarly increased cell viability. Specifically, osteoblasts seeded over i-PRF and EMD-treated surfaces showed improvement in adhesion and migration and significantly increased ALP, OC, ON, RUNX-2, and COL1a2 mRNA levels up to 2.8 fold (p < 0.05) with no differences between Ti surfaces. i-PRF and EMD possess beneficial bioactive properties that enhance tissue healing and promote regeneration on thoroughly sterilized surfaces. Biologically active materials may hold the potential to influence the process of implant re-osseointegration, which warrants more research since sterilization of the affected surfaces under clinical conditions is still not reliably possible and remains one of the greatest challenges.

Relaxin-2 promotes osteoblastic differentiation mediated by epidermal growth factor and epidermal growth factor receptor signaling.

Loss of osteogenic differentiation potential of osteoblasts has been associated with the pathogenesis of osteoporosis. Thus, stimulation of osteoblastic differentiation is a therapeutic strategy for osteoporosis. Relaxin-2 is a peptide hormone with potent biological functions. However, the effects of Relaxin-2 in osteoblastic differentiation and osteoporosis have not been reported before. Here, we report a novel physiological role of Relaxin-2 in promoting osteoblastic differentiation and mineralization of MC3T3-E1 cells. Our results indicate that exposure to Relaxin-2 upregulated the expression, and elevated the activity of alkaline phosphatase (ALP) when MC3T3-E1 cells were cultured in osteogenic differentiation medium (OM). Additionally, Relaxin-2 upregulated the mRNA levels of osteocalcin (ocn), osteopontin (opn), and collagen type I alpha 1 (Col1a1). The alizarin red S staining assay revealed that Relaxin-2 promoted the mineralization of MC3T3-E1 cells. We also found that Relaxin-2 increased the expression of Runx-2 as well as the epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR). Importantly, silencing of EGF abolished the effects of Relaxin-2 in osteoblastic differentiation and related gene expression. These findings suggest that Relaxin-2 stimulates osteogenic differentiation through activating EGF/EGFR signaling.

Evaluation of the osteogenic potential of demineralized and decellularized bovine bone granules following implantation in rat calvaria critical-size defect model.

The aim of this study was to compare the ability of demineralized (DMB) and decellularized (DCC) bovine bone granules to support bone regeneration in rat calvaria critical-size defects. DMB and DCC were prepared using a previously published method. The granule size used ranged between 500 and 750 μm. A total of forty-eight Sprague-Dawley rats were divided into two groups (n = 24). A pair of 5 mm diameter defects were created on the calvaria of the rats in the right and left parietal bone in both groups. Group A animals received DMB granules and Group B received DCC granules in the right parietal defect side while the left parietal untreated defect acted as sham surgery for both groups. Four animals per group were euthanized in a CO2 chamber at day 7, 14 and 21 post-surgery and the calvaria implantation site biopsy harvested was subjected to osteogenic gene expression analysis. Another four animals per group were euthanized at days 15, 30 and 60 post surgery and the calvaria implantation site biopsy harvested was subjected to histological, immunohistochemistry, RAMAN spectroscopy and Micro-CT analysis at the mentioned time points. Statistical analysis was conducted using t-tests and ANOVA. Histomorphometry showed significantly higher new bone formation in the DCC sites (p<0.05) compared to DMB. Both DMB and DCC implantation sites showed distinct staining for osteocalcin and osteopontin proteins compared to their respective sham sites. By day 21 after implantation, DCC sites demonstrated significantly elevated mRNA levels of osteonectin (p<0.001), osteopontin (p<0.001), osteocalcin (p<0.0001), ALP (p<0.01), and BMP-2 (p<0.001) compared to DMB. However, VEGF expression showed no significant differences at this time point between the two groups. Micro-CT analysis also showed enhanced defect closure and higher bone density in DCC implanted sites while RAMAN spectra demonstrated increased abundance of collagen and bone minerals, especially, PO43- ions than DMB. In conclusion, both DMB and DCC granules demonstrated favorable osteogenic potential in critical-sized defects, with DCC exhibited superior osteoconductive, osteoinductive and osteogenesis properties.

Differences in bone microarchitecture between genetic and secondary iron-overload mouse models suggest a role for hepcidin deficiency in iron-related osteoporosis.

The aim of the study was to investigate the respective roles of iron excess and hepcidin, the systemic iron regulator, in the development of iron-related osteoporosis. We used mice models with genetic iron overload (GIO) related to hepcidin deficiency (Hfe-/- and Bmp6-/- ) and secondary iron overload (SIO) exhibiting a hepcidin increase secondary to iron excess. Iron concentration and transferrin saturation levels were evaluated in serum and hepatic, spleen, and bone iron concentrations were assessed by ICP-MS and Perl's staining. Gene expression was evaluated by quantitative RT-PCR. Bone micro-architecture was evaluated by micro-CT. The osteoblastic MC3T3 murine cells that are able to mineralize were exposed to iron and/or hepcidin. Despite an increase of bone iron concentration in all overloaded mice models, bone volume/total volume (BV/TV) and trabecular thickness (Tb.Th) only decreased significantly in GIO, at 12 months for Hfe-/- and from 6 months for Bmp6-/- . Alterations in bone microarchitecture in the Bmp6-/- model were positively correlated with hepcidin levels (BV/TV (ρ = +.481, p < .05) and Tb.Th (ρ = +.690, p < .05). Iron deposits were detected in the bone trabeculae of Hfe-/- and Bmp6-/- mice, while iron deposits were mainly visible in bone marrow macrophages in secondary iron overload. In cell cultures, ferric ammonium citrate exposure abolished the mineralization process for concentrations above 5 μM, with a parallel decrease in osteocalcin, collagen 1, and alkaline phosphatase mRNA levels. Hepcidin supplementation of cells had a rescue effect on the collagen 1 and alkaline phosphatase expression level decrease. Together, these data suggest that iron in excess alone is not sufficient to induce osteoporosis and that low hepcidin levels also contribute to the development of osteoporosis.

Zuo Gui Wan Promotes Osteogenesis via PI3K/AKT Signaling Pathway: Network Pharmacology Analysis and Experimental Validation.

Osteogenesis is vitally important for bone defect repair, and Zuo Gui Wan (ZGW) is a classic prescription in traditional Chinese medicine (TCM) for strengthening bones. However, the specific mechanism by which ZGW regulates osteogenesis is still unclear. The current study is based on a network pharmacology analysis to explore the potential mechanism of ZGW in promoting osteogenesis. A network pharmacology analysis followed by experimental validation was applied to explore the potential mechanisms of ZGW in promoting the osteogenesis of bone marrow mesenchymal stem cells (BMSCs). In total, 487 no-repeat targets corresponding to the bioactive components of ZGW were screened, and 175 target genes in the intersection of ZGW and osteogenesis were obtained. And 28 core target genes were then obtained from a PPI network analysis. A GO functional enrichment analysis showed that the relevant biological processes mainly involve the cellular response to chemical stress, metal ions, and lipopolysaccharide. Additionally, KEGG pathway enrichment analysis revealed that multiple signaling pathways, including the phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) signaling pathway, were associated with ZGW-promoted osteogensis. Further experimental validation showed that ZGW could increase alkaline phosphatase (ALP) activity as well as the mRNA and protein levels of ALP, osteocalcin (OCN), and runt related transcription factor 2 (Runx 2). What's more, Western blot analysis results showed that ZGW significantly increased the protein levels of p-PI3K and p-AKT, and the increases of these protein levels significantly receded after the addition of the PI3K inhibitor LY294002. Finally, the upregulated osteogenic-related indicators were also suppressed by the addition of LY294002. ZGW promotes the osteogenesis of BMSCs via PI3K/AKT signaling pathway.

Methoxyflavones isolated from the whole plant of Scutellaria rubropunctata Hayata var. rubropunctata promote osteoblast differentiation in MC3T3-E1 cells.

In this study, we isolated two new methoxyflavones (1 and 2) and eight known methoxyflavones (3-10) from the whole plant of Scutellaria rubropunctata Hayata var. rubropunctata (SR). Based on spectroscopic analyses, the methoxyflavones were identified as 5,8,2',6'-tetramethoxy-6,7-methylenedioxyflavone (1) and 5,2',6'-trimethoxy-6,7-methylenedioxyflavone (2). We reported SR might have effects on promoting osteoblast differentiation and stimulating estrogen receptor (ER) in the previous study. Then, the effects of 1-10 on pre-osteoblast MC3T3-E1 cells were investigated, and 1, 2, and 9 were observed to promote alkaline phosphatase activity. To evaluate their effect on osteogenesis-related genes, we performed gene expression analysis using quantitative real-time PCR after treatment of MC3T3-E1 cells with these compounds. Although 2 was only effective at lower concentrations, 1 and 9 upregulated the mRNA levels of Runx2, Osterix, Osteopontin, Osteocalcin, Smad1, and Smad4. These results indicate that 1 and 9 may induce osteoblast differentiation by activating Runx2 via the BMP/Smad pathway and may play a central role in the promotion of osteoblast differentiation by SR. The ER agonist activity of 1-10 were tested using a luciferase reporter assay in HEK293 cells. However, none of the compounds exhibited remarkable activity. Thus, SR may contain other compounds that contribute to its ER agonist activity.

Tumor Necrosis Factor-α Blunts the Osteogenic Effects of Muscle Cell-Derived Extracellular Vesicles by Affecting Muscle Cells.

Extracellular vesicles (EVs) play crucial roles in physiological and pathophysiological processes. Although studies have described muscle-bone interactions via humoral factors, we reported that EVs from C2C12 muscle cells (Myo-EVs) suppress osteoclast formation. Current clinical evidence suggests that inflammation induces both sarcopenia and osteoporosis. Although tumor necrosis factor-α (TNF-α) is a critical proinflammatory factor, the influences of TNF-α on muscle-bone interactions and Myo-EVs are still unclear. In the present study, we investigated the effects of TNF-α stimulation of C2C12 cells on osteoclast formation and osteoblastic differentiation modulated by Myo-EVs in mouse cells. TNF-α significantly decreased the protein amount in Myo-EVs, but did not affect the Myo-EV size distribution. TNF-α treatment of C2C12 myoblasts significantly decreased the suppression of osteoclast formation induced by Myo-EVs from C2C12 myoblasts in mouse bone marrow cells. Moreover, TNF-α treatment of C2C12 myoblasts in mouse preosteoclastic Raw 264.7 cells significantly limited the Myo-EV-induced suppression of osteoclast formation and decreased the Myo-EV-induced increase in mRNA levels of osteoclast formation-related genes. On the other hand, TNF-α treatment of C2C12 muscle cells significantly decreased the degree of Myo-EV-promoted mRNA levels of Osterix and osteocalcin, as well as ALP activity in mouse mesenchymal ST-2 cells. TNF-α also significantly decreased miR196-5p level in Myo-EVs from C2C12 myoblasts in quantitative real-time PCR. In conclusion, TNF-α stimulation of C2C12 muscle cells blunts both the osteoclast formation suppression and the osteoblastic differentiation promotion that occurs due to Myo-EVs in mouse cells. Thus, TNF-α may disrupt the muscle-bone interactions by direct Myo-EV modulation.

Human Recombinant Lactoferrin Promotes Differentiation and Calcification on MC3T3-E1 Cells

Lactoferrin (LF), known to be present in mammalian milk, has been reported to promote the proliferation of osteoblasts and suppress bone resorption by affecting osteoclasts. However, the mechanisms underlying the effects of human sources LF on osteoblast differentiation have not yet been elucidated, and almost studies have used LF from bovine sources. The presented study aimed to characterize the molecular mechanisms of bovine lactoferrin (IF-I) and human recombinant lactoferrin (LF-II) on MC3T3-E1 pre-osteoblast cells. MC3T3-E1 cells were treated with LF, ascorbic acid, and β-glycerophosphate (β-GP). Cell proliferation was analyzed using the MTT assay. Alkaline phosphatase activation and osteopontin expression levels were evaluated via cell staining and immunocytochemistry. The differentiation markers were examined using quantitative real-time PCR. The cell viability assay showed the treatment of 100 μg/mL LF significantly increased; however, it was suppressed by the simultaneous treatment of ascorbic acid and β-GP. Alizarin red staining showed that the 100 μg/mL treatment of LF enhanced calcification. Quantitative real-time PCR showed a significant increase in osterix expression. The results suggest that treatment with both LFs enhanced MC3T3-E1 cell differentiation and promoted calcification. The mechanisms of calcification suggest that LFs are affected by an increase in osterix and osteocalcin mRNA levels.

The extracellular matrix of human bone marrow adipocytes and glucose concentration differentially alter mineralization quality without impairing osteoblastogenesis

Bone marrow adipocytes (BMAds) accrue in various states of osteoporosis and interfere with bone remodeling through the secretion of various factors. However, involvement of the extracellular matrix (ECM) produced by BMAds in the impairment of bone marrow mesenchymal stromal cell (BM-MSC) osteoblastogenesis has received little attention. In type 2 diabetes (T2D), skeletal fragility is associated with several changes in bone quality that are incompletely understood, and BMAd quantity increases in relationship to poor glycemic control. Considering their altered phenotype in this pathophysiological context, we aimed to determine the contribution of the ECM of mature BMAds to osteoblastogenesis and mineralization quality in the context of chronic hyperglycemia.Human BM-MSCs were differentiated for 21 days in adipogenic medium containing either a normoglycemic (LG, 5.5 mM) or a high glucose concentration (HG, 25 mM). The ECM laid down by BMAds were devitalized through cell removal to examine their impact on the proliferation and differentiation of BM-MSCs toward osteoblastogenesis in LG and HG conditions. Compared to control plates, both adipocyte ECMs promoted cell adhesion and proliferation. As shown by the unmodified RUNX2 and osteocalcin mRNA levels, BM-MSC commitment in osteoblastogenesis was hampered by neither the hyperglycemic condition nor the adipocyte matrices. However, adipocyte ECMs or HG condition altered the mineralization phase with perturbed expression levels of type 1 collagen, MGP and osteopontin. Despite higher ALP activity, mineralization levels per cell were decreased for osteoblasts grown on adipocyte ECMs compared to controls. Raman spectrometry revealed that culturing on adipocyte matrices specifically prevents type-B carbonate substitution and favors collagen crosslinking, in contrast to exposure to HG concentration alone. Moreover, the mineral to organic ratio was disrupted according to the presence of adipocyte ECM and the glucose concentration used for adipocyte or osteoblast culture. HG concentration and adipocyte ECM lead to different defects in mineralization quality, recapitulating contradictory changes reported in T2D osteoporosis.Our study shows that ECMs from BMAds do not impair osteoblastogenesis but alter both the quantity and quality of mineralization partly in a glucose concentration-dependent manner. This finding sheds light on the involvement of BMAds, which should be considered in the compromised bone quality of T2D and osteoporosis patients more generally.

Effects of a Whole Plant Extract of Scutellaria rubropunctata var. rubropunctata on Bone Metabolism with Estrogen Receptor Activation

We screened natural resources for estrogen receptor (ER)-activating and bone metabolism-promoting activities with the aim of finding potential treatments for osteoporosis. A screen of 1531 extracts from Ryukyu Arc plants using a luciferase reporter assay identified an 80% MeOH extract of Scutellaria rubropunctata var. rubropunctata (SRE) with dose-dependent ER transcription-promoting activity. Importantly, SRE had no proliferative effect on human breast cancer cells. SRE enhanced the ALP activity of pre-osteoblast MC3T3-E1 cells after 72 h in culture and slightly enhanced mineralization at 14 and 21 d. SRE did not significantly affect the TRAP activity of RAW264.7 cells. Gene expression analysis in MC3T3-E1 cells by quantitative real-time PCR revealed that SRE upregulated the mRNA levels of Runx2, Osterix (Osx), Osteopontin (Opn), Osteocalcin (Ocn), Smad1, Smad4, and Smad5 at 72 h, and those of Runx2, Osx, Smad1, Smad4, and Smad5 at 21 d of osteogenic induction. Analysis of the expression levels of osteogenic markers suggested that SRE may promote osteogenic differentiation by acting at the early stage of differentiation rather than at the late stage of differentiation. These results indicate that SRE activates ER and induces osteoblast differentiation by activating Runx2 and Osx through the BMP/Smad pathway, suggesting that SRE may be useful for the prevention and treatment of postmenopausal osteoporosis.

Inhibition of Sphingosine-1-Phosphate Receptor 2 by JTE013 Promoted Osteogenesis by Increasing Vesicle Trafficking, Wnt/Ca2+, and BMP/Smad Signaling.

Sphingosine-1-phosphate receptor 2 (S1PR2) is a G protein-coupled receptor that regulates various immune responses. Herein, we determine the effects of a S1PR2 antagonist (JTE013) or a S1PR2 shRNA on osteogenesis by culturing murine bone marrow stromal cells (BMSCs) in osteogenic media with JTE013, dimethylsulfoxide (DMSO), a S1PR2 shRNA, or a control shRNA. Treatment with JTE013 or the S1PR2 shRNA increased alkaline phosphatase and alizarin red s staining, and enhanced alkaline phosphatase, RUNX2, osteocalcin, and osterix mRNA levels in BMSCs compared with the controls. Protein analysis revealed that a high dose of JTE013 (4 or 8 μM) increased vesicle trafficking-associated proteins (F-actin, clathrin, Early Endosome Antigen 1 (EEA1), and syntaxin 6) and Wnt/Ca2+ signaling. On the other hand, a low dose of JTE013 (1 to 2 μM) increased BMP/Smad signaling. In contrast, the S1PR2 shRNA reduced vesicle trafficking-associated proteins and attenuated Wnts and BMP/Smad signaling, but enhanced p-CaMKII compared with the control, suggesting that the S1PR2 shRNA influenced osteogenesis via different signaling pathways. Moreover, inhibiting protein trafficking by brefeldin A in BMSCs suppressed Wnts and BMPRs expressions. These data supported that enhanced osteogenesis in JTE013-treated BMSCs is associated with increased vesicle trafficking, which promotes the synthesis and transport of osteogenic protein and matrix vesicles and enhances matrix mineralization.

The Osteogenic Differentiation of Human Dental Pulp Stem Cells through G0/G1 Arrest and the p-ERK/Runx-2 Pathway by Sonic Vibration.

Mechanical/physical stimulations modulate tissue metabolism, and this process involves multiple cellular mechanisms, including the secretion of growth factors and the activation of mechano-physically sensitive kinases. Cells and tissue can be modulated through specific vibration-induced changes in cell activity, which depend on the vibration frequency and occur via differential gene expression. However, there are few reports about the effects of medium-magnitude (1.12 g) sonic vibration on the osteogenic differentiation of human dental pulp stem cells (HDPSCs). In this study, we investigated whether medium-magnitude (1.12 g) sonic vibration with a frequency of 30, 45, or 100 Hz could affect the osteogenic differentiation of HDPSCs. Their cell morphology changed to a cuboidal shape at 45 Hz and 100 Hz, but the cells in the other groups were elongated. FACS analysis showed decreased CD 73, CD 90, and CD 105 expression at 45 Hz and 100 Hz. Additionally, the proportions of cells in the G0/G1 phase in the control, 30 Hz, 45 Hz, and 100 Hz groups after vibration were 60.7%, 65.9%, 68.3%, and 66.7%, respectively. The mRNA levels of osteogenic-specific markers, including osteonectin, osteocalcin, BMP-2, ALP, and Runx-2, increased at 45 and 100 Hz, and the ALP and calcium content was elevated in the vibration groups compared with those in the control. Additionally, the western blotting results showed that p-ERK, BSP, osteoprotegerin, and osteonectin proteins were upregulated at 45 Hz compared with the other groups. The vibration groups showed higher ALP and calcium content than the control. Vibration, especially at 100 Hz, increased the number of calcified nodes relative to the control group, as evidenced by von Kossa staining. Immunohistochemical staining demonstrated that type I and III collagen, osteonectin, and osteopontin were upregulated at 45 Hz and 100 Hz. These results suggest that medium magnitude vibration at 45 Hz induces the G0/G1 arrest of HDPSCs through the p-ERK/Runx-2 pathway and can serve as a potent stimulator of differentiation and extracellular matrix production.

Effect of a Bone Morphogenetic Protein-2-derived peptide on the expression of tumor marker ZNF217 in osteoblasts and MCF-7 cells

Zinc Finger Protein 217 (ZNF217), a transcription factor and oncogene product, has been found to dysregulate Bone Morphogenetic Protein (BMP) signaling and induce invasion in breast tumors. In this study, the effect of BMP-2 or an active BMP-2 peptide, AISMLYLDEN, on the expression of ZNF217, BMP4 and CDK-inhibitor p21 gene, CDKN1A, was investigated in MCF-7 breast cancer cells. In parallel, the entire protein (BMP-2) as well as the aforementioned peptide were investigated in hDPSCs during osteogenic differentiation. The treatment of MCF-7 cancer cells with different concentrations of peptide AISMLYLDEN showed that the addition of 22.6 ng/ml was more effective in comparison to the other used concentrations. In particular, 48 h after treatment, CDKN1A and BMP4 mRNA levels were substantially increased in contrast to ZNF217 mRNA levels which were decreased. These results are strongly supported by BrdU assay that clearly indicated inhibition of cancer cell proliferation. Taken together, these results open ways for a concurrent use, at appropriate concentrations, of the peptide AISMLYLDEN during conventional therapeutic treatment in breast tumors with a metastatic tendency to the bones. Regarding the effect of the entire protein as well as its peptide on hDPSCs differentiation into osteocytes, the mRNA levels of osteocalcin, an osteogenic marker, showed that the peptide enhanced osteogenesis at a higher degree in comparison to the entire BMP-2 without however altering ZNF217, CDKN1A and BMP4 expression levels, which remained as expected of non-cancer cells.

Potential Privilege of Maltodextrin-α-Tocopherol Nano-Micelles in Seizing Tacrolimus Renal Toxicity, Managing Rheumatoid Arthritis and Accelerating Bone Regeneration.

BackgroundTacrolimus (TAC) is a powerful immunosuppressive agent whose therapeutic applicability is confined owing to its systemic side effects.ObjectiveHerein, we harnessed a natural polymer based bioconjugate composed of maltodextrin and α-tocopherol (MD-α-TOC) to encapsulate TAC as an attempt to overcome its biological limitations while enhancing its therapeutic anti-rheumatic efficacy.MethodsThe designed TAC loaded maltodextrin-α-tocopherol nano-micelles (TAC@MD-α-TOC) were assessed for their physical properties, safety, toxicological behavior, their ability to combat arthritis and assist bone/cartilage formation.ResultsIn vitro cell viability assay revealed enhanced safety profile of optimized TAC@MD-α-TOC with 1.6- to 2-fold increase in Vero cells viability compared with free TAC. Subacute toxicity study demonstrated a diminished nephro- and hepato-toxicity accompanied with optimized TAC@MD-α-TOC. TAC@MD-α-TOC also showed significantly enhanced anti-arthritic activity compared with free TAC, as reflected by improved clinical scores and decreased IL-6 and TNF-α levels in serum and synovial fluids. Unique bone formation criteria were proved with TAC@MD-α-TOC by elevated serum and synovial fluid levels of osteocalcin and osteopontin mRNA and proteins expression. Chondrogenic differentiation abilities of TAC@MD-α-TOC were proved by increased serum and synovial fluid levels of SOX9 mRNA and protein expression.ConclusionOverall, our designed bioconjugate micelles offered an excellent approach for improved TAC safety profile with enhanced anti-arthritic activity and unique bone formation characteristics.

Serpinb1a suppresses osteoclast formation

Serpinb1a, a serine protease inhibitor family protein, has been implicated in immunoregulation and several metabolic disorders, such as diabetes and obesity; however, its roles in bone remain unknown. Therefore, we herein investigated the physiological functions of Serpinb1a in osteoclastic and osteoblastic differentiation using mouse cell lines. Serpinb1a overexpression markedly reduced the number of tartrate-resistant acid phosphatase (TRAP)- and calcitonin receptor-positive multinucleated cells increased by receptor activator nuclear factor κB ligand (RANKL) in mouse preosteoclastic RAW 264.7 cells. Moreover, it significantly decreased the mRNA levels of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), TRAP and cathepsin K in these cells. Regarding osteoblasts, Serpinb1a overexpression significantly reduced the mRNA levels of alkaline phosphatase (ALP) and osteocalcin as well as ALP activity induced by bone morphogenetic protein-2 (BMP-2) in mouse mesenchymal ST2 cells, although it did not alter osteoblast differentiation in mouse osteoblastic MC3T3-E1 cells. Concerning the pathophysiological relevance of Serpinb1a, Serpinb1a mRNA levels were decreased in the soleus and gastrocnemius muscles of mice 4 weeks after bilateral sciatic nerve resection. In conclusion, we herein revealed for the first time that Serpinb1a inhibited osteoclast formation induced by RANKL in RAW 264.7 cells and suppressed BMP-2-induced ALP activity in ST2 cells.

The impact of the size of bone substitute granules on macrophage and osteoblast behaviors in vitro

ObjectiveBone substitute (BS) size might influence the clinical outcomes of guided bone regeneration (GBR) procedures. The aim of the present study was to investigate the influence of BS size on macrophage (Mφ) and osteoblast behaviors in vitro.Materials and methodsTwo different granule sizes (S and M/L) were assessed for four different commercial BSs: deproteinized bovine bone mineral (DBBM), biphasic calcium phosphate type 1 (BCP1), BCP type 2 (BCP2), and carbonate apatite (CO3Ap). The BSs were compared for their impacts on the cell viability and differentiation potential of THP-1-derived Mφs and human osteoblast-like Saos-2 cells.ResultsThe smaller granules showed higher material volumes and surface areas than the larger granules. Significantly higher viability of Mφs and Saos-2 cells was observed with the DBBM_L-size granules than with the DBBM_S-size granules. Gene expression experiments in Mφs revealed few differences between the two sizes of each BS, although higher CD206 mRNA levels were observed in the BCP1_L group and the CO3Ap_M group than in the respective S-size groups on day 1. Only DBBM showed significantly higher mRNA levels of osteogenic markers, including Runx2 and osteocalcin, in Saos-2 cells in the S-size group than in the L-size group.ConclusionsThe S-size and L-size DBBM granules exhibited clear differences in cell outcomes: cells cultured on the S-size granules exhibited lower cell viability, higher osteopromotive ability, and no noticeable Mφ polarization changes.Clinical relevanceA smaller granule size might be advantageous due to greater bone regeneration potential in the use of DBBM granules to treat defects.

Physicochemical properties of anodized-hydrothermally treated titanium with a nanotopographic surface structure promote osteogenic differentiation in dental pulp stem cells

Purpose Implants made of anodized-hydrothermally treated commercially pure titanium with a nanotopographic surface structure (SA-treated c.p.Ti) may advantageously promote contact osteogenesis during the early stages of healing. We hypothesized that utilizing SA-treated c.p.Ti with dental pulp stem cells (DPSCs) might improve osteoconduction during the process of osseointegration. This in vitro study investigated the effect of initial adhesion of DPSCs to SA-treated c.p.Ti compared with conventional c.p.Ti and anodic oxide (AO) c.p.Ti.Methods DPSCs were obtained from the mandibular incisors of Sprague-Dawley rats and cultured without osteogenic induction medium on c.p.Ti, AO c.p.Ti, and SA-treated c.p.Ti disks for up to 14 days. The morphology, proliferation, and differentiation of DPSCs were assessed by scanning electron microscopy, an MTT assay, and Alizarin Red S staining, respectively. A real-time quantitative polymerase chain reaction was used to quantify the mRNA expression of osteocalcin, osteopontin, and bone sialoprotein.Results On all disks, the DPSCs appeared flattened with the formation of extensions over time. The filopodium-like extensions were closely bound to the SA-treated c.p.Ti surface. The proliferation of DPSCs was not significantly different among the c.p.Ti treatments. However, DPSCs on SA-treated c.p.Ti showed the greatest mRNA levels of osteopontin, osteocalcin, and bone sialoprotein, as well as increased Alizarin Red S staining.Conclusions The results of the present in vitro study demonstrate that the surface properties of SA-treated c.p.Ti disks enhance osteogenic differentiation of DPSCs and may facilitate mineralized matrix formation on SA-treated c.p.Ti implant surfaces, which can enhance early bone regeneration.

Effects of PIN on Osteoblast Differentiation and Matrix Mineralization through Runt-Related Transcription Factor.

Styrax Japonica Sieb. et Zucc. has been used as traditional medicine in inflammatory diseases, and isolated compounds have shown pharmacological activities. Pinoresinol glucoside (PIN) belonging to lignins was isolated from the stem bark of S. Japonica. This study aimed to investigate the biological function and mechanisms of PIN on cell migration, osteoblast differentiation, and matrix mineralization. Herein, we investigated the effects of PIN in MC3T3-E1 pre-osteoblasts, which are widely used for studying osteoblast behavior in in vitro cell systems. At concentrations ranging from 0.1 to 100 μM, PIN had no cell toxicity in pre-osteoblasts. Pre-osteoblasts induced osteoblast differentiation, and the treatment of PIN (10 and 30 μM) promoted the cell migration rate in a dose-dependent manner. At concentrations of 10 and 30 μM, PIN elevated early osteoblast differentiation in a dose-dependent manner, as indicated by increases in alkaline phosphatase (ALP) staining and activity. Subsequently, PIN also increased the formation of mineralized nodules in a dose-dependent manner, as indicated by alizarin red S (ARS) staining, demonstrating positive effects of PIN on late osteoblast differentiation. In addition, PIN induced the mRNA level of BMP2, ALP, and osteocalcin (OCN). PIN also upregulated the protein level of BMP2 and increased canonical BMP2 signaling molecules, the phosphorylation of Smad1/5/8, and the protein level of Runt-related transcription factor 2 (RUNX2). Furthermore, PIN activated non-canonical BMP2 signaling molecules, activated MAP kinases, and increased β-catenin signaling. The findings of this study indicate that PIN has biological roles in osteoblast differentiation and matrix mineralization, and suggest that PIN might have anabolic effects in bone diseases such as osteoporosis and periodontitis.

Epigenetic silencing of KLF2 by long non-coding RNA SNHG1 inhibits periodontal ligament stem cell osteogenesis differentiation

BackgroundExploring the effects of lncRNA SNHG1 in the process of osteogenic differentiation of periodontal ligament stem cells (PDLSCs) would provide novel therapeutic strategies for tissue regeneration.MethodsLoss-of-function and gain-of-function assays were induced by lentivirus. The osteogenic differentiation of PDLSCs were assessed by ALP staining and Alizarin Red staining as well as the mRNA and protein levels of osteogenic marker genes osterix, osteocalcin, and alkaline phosphatase through qRT-PCR and western blot. RNA immunoprecipitation assay and chromatin immunoprecipitation assays were performed to uncover the interaction between SNHG1 and EZH2.ResultsOur analysis revealed that SNHG1 was downregulated and KLF2 was upregulated during the osteogenic induction differentiation of PDLSCs. SNHG1 inhibited while KLF2 promoted osteogenic differentiation of PDLSCs. SNHG1 directly interact with the histone methyltransferase enhancer of the zeste homolog 2 (EZH2) and modulate the histone methylation of promoter of Kruppel-like factor 2 (KLF2) and altered the progress osteogenic differentiation of PDLSCs.ConclusionsTaken together, SNHG1 inhibited the osteogenic differentiation of PDLSCs through EZH2-mediated H3K27me3 methylation of KLF2 promotor and provided a novel class of therapeutic targets for regenerate dental tissues.

Effects of the amide alkaloid piperyline on apoptosis, autophagy, and differentiation of pre-osteoblasts

BackgroundAmide alkaloidsare typical constituents in plants of the Piperaceae family. Most of the pharmacological properties of Piper nigrum L. are attributed to the major amide alkaloid, piperine. Piperyline (PIPE) is a further amide alkaloid that has been isolated from P. nigrum. Hypothesis/PurposeThis study was performed to examine the biological effects of PIPE on pre-osteoblasts and elucidate the underlying mechanisms. Study DesignWe investigated the effects of PIPE in MC3T3E-1 cells, which are widely used for studying osteoblast behavior in in vitro cell systems. MethodsWe evaluated cell viability based on the MTT assay, apoptosis by TUNEL staining, adhesion and migration by cell adhesion and migration assays, and osteoblast differentiation by alkaline phosphatase activity and staining. Western blot and immunocytochemical analyses were used to investigate cell signaling pathways. ResultsWe found that at concentrations ranging from 1 to 30 μM, PIPE inhibited cell growth and induced apoptosis in pre-osteoblasts, which was accompanied by the upregulation of apoptotic proteins but downregulation of anti-apoptotic proteins. In contrast, PIPE had no appreciable effect on the autophagy pathway. Nevertheless, PIPE reduced cell adhesion and migration via the inactivation of non-receptor tyrosine kinase (Src)/focal adhesion kinase (FAK) and mitogen-activated protein kinases, and also promoted the downregulation of matrix metalloproteinase 2 and 9 levels. Furthermore, at concentrations of 10 and 30 μM, PIPE suppressed osteoblast differentiation, as indicated by reductions in alkaline phosphatase staining and activity. In addition, PIPE reduced the protein levels of phospho-Smad1/5/8 and runt-related transcription factor 2, and the mRNA levels of osteopontin, alkaline phosphatase, and osteocalcin. ConclusionThe findings of this study indicate that PIPE has biological effects associated with cell adhesion, migration, proliferation, and osteoblast differentiation, and suggest a potential role for this alkaloid in the treatment of bone diseases.