Bone Sialoprotein mRNA Expression Research Articles

Brain-Bone Crosstalk in a Murine Polytrauma Model Promotes Bone Remodeling but Impairs Neuromotor Recovery and Anxiety-Related Behavior.

Traumatic brain injury (TBI) and long bone fractures are a common injury pattern in polytrauma patients and modulate each other's healing process. As only a limited number of studies have investigated both traumatic sites, we tested the hypothesis that brain-bone polytrauma mutually impacts neuro- and osteopathological outcomes. Adult female C57BL/6N mice were subjected to controlled cortical impact (CCI), and/or osteosynthetic stabilized femoral fracture (FF), or sham surgery. Neuromotor and behavioral impairments were assessed by neurological severity score, open field test, rotarod test, and elevated plus maze test. Brain and bone tissues were processed 42 days after trauma. CCI+FF polytrauma mice had increased bone formation as compared to FF mice and increased mRNA expression of bone sialoprotein (BSP). Bone fractures did not aggravate neuropathology or neuroinflammation assessed by cerebral lesion size, hippocampal integrity, astrocyte and microglia activation, and gene expression. Behavioral assessments demonstrated an overall impaired recovery of neuromotor function and persistent abnormalities in anxiety-related behavior in polytrauma mice. This study shows enhanced bone healing, impaired neuromotor recovery and anxiety-like behavior in a brain-bone polytrauma model. However, bone fractures did not aggravate TBI-evoked neuropathology, suggesting the existence of outcome-relevant mechanisms independent of the extent of brain structural damage and neuroinflammation.

Sphingosine-1-phosphate receptor 2 agonist induces bone formation in rat apicoectomy and alveolar bone defect model

Background/purposeSphingosine-1-phosphate (S1P) is a lipid mediator that exerts its physiological functions in vivo through receptors. In the bone, S1P induces osteoblast differentiation. Here, we investigated the effects of S1P receptor agonists on the expression of osteoblast differentiation markers locally in the bone. Then, a rat apicoectomy and alveolar bone defect model was established to extend S1P applications to endodontics, and the effect of local administration of S1P receptor agonist on postoperative bone formation was examined. Materials and methodsSphingosine-1-phosphate receptor (S1PR) 1/S1PR3 agonists, S1PR2 agonists, and their signal-related agents were intraperitoneally administered to mice. Using the mRNA collected from the tibial bone, the expression of osteoblast differentiation markers was evaluated by real-time reverse-transcriptase quantitative polymerase chain reaction. An apicoectomy and alveolar bone defect model was established on the mesial root of the rat mandibular first molar. Bone formation parameters were measured by micro-computed tomography analysis 3 weeks after the procedure. ResultsIntraperitoneal administration of S1PR2 agonist significantly increased the mRNA expression of osteoblast differentiation markers, including alkaline phosphatase (ALP), osteopontin (OPN), bone sialoprotein (BSP), and osteocalcin, in the local tibial bone of mice. The S1PR2/Rho-associated coiled-coil forming kinase (ROCK) signaling was thought to be involved in the upregulated mRNA expression of ALP, OPN, and BSP. In the rat apical defects, bone formation parameters significantly increased following local administration of S1PR2 agonist. ConclusionIn the rat apicoectomy and alveolar bone defect model, therapeutic agents targeting S1PR2 agonist are effective against osteogenesis.

The relationship between BSP mRNA expression and 25(OH)D/OPG in peripheral blood of newly diagnosed T2DM patients with different bone mass

The objective of the study was to detect the levels of osteoprotegerin (OPG) and 25-hydroxyvitamin D [25(OH)D], as well as the expression of bone sialoprotein (BSP) mRNA, in the peripheral blood of patients with newly diagnosed type 2 diabetes mellitus (T2DM) under different bone mass conditions, and to explore its role and significance in the development process of T2DM combined with osteoporosis (OP). A total of 225 patients hospitalised in the Endocrinology Department and General Department from May 2017 to May 2018 were enrolled and categorised into five groups: the pure T2DM group (group A, 45 patients), the bone mass reduction group (group B, 45 patients), the T2DM + bone mass reduction group (group C, 45 patients), the OP group (group D, 45 patients), and the T2DM + OP group (group E, 45 patients); meanwhile, age-matched healthy subjects undergoing physical examination in our hospital were collected as the normal control group (group NC, 45 cases). Logistic regression analysis was used to analyse the influencing factors of bone mass in patients with T2DM. Compared with group B, the expression levels of glycated haemoglobin (HbA1c), 25(OH)D, N-terminal propeptide of type I procollagen (PINP), fasting plasma glucose (FPG), fasting plasma insulin (FINS), high-density lipoprotein cholesterol (HDL-C), and BSP mRNA were significantly increased while OPG and b-collagen degradation products (b-CTX) were significantly decreased in group A. The expression of BSP mRNA and the decrease of 25(OH)D and OPG in peripheral blood may participate in the development of diabetes and osteoporosis.

Interleukin-8 promotes prostate cancer bone metastasis through upregulation of bone sialoprotein.

The aim of the present study was to investigate whether interleukin-8 (IL-8) enhances the ability of prostate cancer bone metastasis by influencing the coding level of bone sialoprotein (BSP). Cultured prostate cancer cell lines LNCaP (androgen dependent) and DU145 (androgen independent) were divided into three groups: IL-8 treatment group; IL-8 receptor inhibitor (SB225002) treatment group; and control group. Western blotting and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to detect BSP protein and mRNA expression levels. Matrigel and bone adhesion experiments were used to detect the invasiveness of cancer cells and bone adhesion changes. Compared with the control group, western blotting and RT-qPCR results indicated that BSP protein and mRNA levels in LNCaP and DU145 were significantly upregulated following IL-8 treatment. Matrigel experiments indicated that following IL-8 treatment, the invasiveness of LNCaP and DU145 cells was significantly increased. The results of bone adhesion experiments indicated that following IL-8 treatment, the number of DU145 cells adhered to the surface of the bone was increased, compared with the control group. Following treatment of both cell lines with SB225002, western blotting and RT-qPCR results indicated that the expression levels of BSP protein and mRNA were significantly downregulated. Matrigel experiments indicated that following SB225002 treatment, the invasiveness of LNCaP and DU145 cells was significantly reduced. The number of DU145 cells adhered to the surface of the bone was reduced, compared with the untreated group. Therefore, IL-8 may promote prostate cancer bone metastasis by enhancing BSP regulation.

Recombinant amelogenin regulates the bioactivity of mouse cementoblasts in vitro

Amelogenin (AMG) is a cell adhesion molecule that has an important role in the mineralization of enamel and regulates events during dental development and root formation. The purpose of the present study was to investigate the effects of recombinant human AMG (rhAMG) on mineralized tissue-associated genes in cementoblasts. Immortalized mouse cementoblasts (OCCM-30) were treated with different concentrations (0.1, 1, 10, 100, 1000, 10,000, 100,000 ng · mL-1) of recombinant human AMG (rhAMG) and analyzed for proliferation, mineralization and mRNA expression of bone sialoprotein (BSP), osteocalcin (OCN), collagen type I (COL I), osteopontin (OPN), runt-related transcription factor 2 (Runx2), cementum attachment protein (CAP), and alkaline phosphatase (ALP) genes using quantitative RT-PCR. The dose response of rhAMG was evaluated using a real-time cell analyzer. Total RNA was isolated on day 3, and cell mineralization was assessed using von Kossa staining on day 8. COL I, OPN and lysosomal-associated membrane protein-1 (LAMP-1), which is a cell surface binding site for amelogenin, were evaluated using immunocytochemistry. F-actin bundles were imaged using confocal microscopy. rhAMG at a concentration of 100,000 ng · mL-1 increased cell proliferation after 72 h compared to the other concentrations and the untreated control group. rhAMG (100,000 ng · mL-1) upregulated BSP and OCN mRNA expression levels eightfold and fivefold, respectively. rhAMG at a concentration of 100,000 ng · mL-1 remarkably enhanced LAMP-1 staining in cementoblasts. Increased numbers of mineralized nodules were observed at concentrations of 10,000 and 100,000 ng · mL-1 rhAMG. The present data suggest that rhAMG is a potent regulator of gene expression in cementoblasts and support the potential application of rhAMG in therapies aimed at fast regeneration of damaged periodontal tissue.

The Role of Nephronectin on Proliferation and Differentiation in Human Dental Pulp Stem Cells.

Aim The purpose of the current study was to investigate the effects of nephronectin (Npnt) in human dental pulp stem cells (hDPSCs). Methodology Npnt was coated to nontissue culture-treated polystyrene (non-PS) plates. The presence of immobilized protein on the surface was detected by polyclonal rabbit primary anti-Npnt antibody. Then the cell number was counted and compared with PBS-, bovine serum albumin- (BSA-), fish scale type I collagen- (FCOL1-), and human fibronectin- (Fn-) coated wells. Cell proliferation was assessed using CCK-8 assay. Cell morphology was observed under light microscopy and fluorescence microscopy. Lastly, the mRNA expression profiles of integrins, dentin sialophosphoprotein (DSPP), bone sialoprotein (BSP), and mineralization capacity of hDPSCs were investigated by real time RT-PCR and alizarin red staining, respectively. Results Npnt mediates hDPSC adhesion and spreading partially via the Arg-Gly-Asp (RGD) motif. Npnt enhanced the mRNA expression of ITGA1, ITGA4, ITGA7, and ITGB1 on day five. Npnt downregulated DSPP but significantly upregulated BSP mRNA expression at day 28. Further, Npnt and FCOL1 accelerated the matrix mineralization in hDPSCs. Conclusions The current findings implicate that Npnt would be favorable to recruit hDPSCs and conducive to mineralization in hDPSCs. The combination of Npnt with hDPSCs may offer a promising approach for hard tissue regeneration.

Potential of l-thyroxine to differentiate osteoblast-like cells via Angiopoietin1

Angiogenesis is closely associated with osteoblast differentiation. Previously, we demonstrated that bone formation can be accelerated by treatment with COMP-Angiopoietin1, a known angiogenic factor. Angiopoietin1 (Ang1) is a specific growth factor that generates stable and mature vasculature through the Tie2 receptor. In this study, we aimed to identify a novel drug that can activate endogenous Ang1 expression as a pharmacological treatment for bone formation. Therefore, Ang1 expression was examined in U2OS osteoblast-like cells treated with 770 drugs from a library of Food and Drug Administration (FDA)-approved drugs by using ELISA for Ang1. l-thyroxine was selected as a novel drug candidate. l-Thyroxine is a synthetic form of the hormone thyroxine, which is used to treat patients with hypothyroidism. Enzyme-linked immunosorbent assays (ELISAs) were performed to test whether Ang1 is induced in a dose-dependent manner in human osteoblast-like cell lines, U2OS and MG63. The effects of l-thyroxine on osteoblast differentiation and mineralization were evaluated by alkaline phosphatase (ALP) activity and Alizarin red s staining. To determine the molecular mechanism, the expression of proteins related to bone formation and differentiation, such as type I collagen (COL1A1), osteocalcin (OC), bone sialoprotein (BSP), distal-less homeobox 5 (Dlx5), Runt-related transcription factor 2 (Runx2), osterix (OSX), and ALP, was tested by Western blotting analysis. Consequently, l-thyroxine induced Ang1 expression in a dose-dependent manner in both U2OS and M63 cells, which was confirmed by ELISA and Western blotting. Also, l-thyroxine activated ALP activity in U2OS and MG63 cells as well as ALP expression. Furthermore, l-thyroxine enhanced the expression of COL1A1, Runx2, OC, BSP, Dlx5, and OSX mRNA and proteins. Taken together, we demonstrated that l-thyroxine increased Ang1 expression and induces bone formation, differentiation, and mineralization in U2OS and MG63 cell lines, which suggests that l-thyroxine could be a potential bone production agent.

A Putative Association of a Single Nucleotide Polymorphism in GPR126 with Aggressive Periodontitis in a Japanese Population.

Periodontitis is an inflammatory disease causing loss of tooth-supporting periodontal tissue. Disease susceptibility to the rapidly progressive form of periodontitis, aggressive periodontitis (AgP), appears to be influenced by genetic risk factors. To identify these in a Japanese population, we performed whole exome sequencing of 41 unrelated generalized or localized AgP patients. We found that AgP is putatively associated with single nucleotide polymorphism (SNP) rs536714306 in the G-protein coupled receptor 126 gene, GPR126 [c.3086 G>A (p.Arg1029Gln)]. Since GPR126 activates the cAMP/PKA signaling pathway, we performed cAMP ELISA analysis of cAMP concentrations, and found that rs536714306 impaired the signal transactivation of GPR126. Moreover, transfection of human periodontal ligament (HPDL) cells with wild-type or mutant GPR126 containing rs536714306 showed that wild-type GPR126 significantly increased the mRNA expression of bone sialoprotein, osteopontin, and Runx2 genes, while mutant GPR126 had no effect on the expression of these calcification-related genes. The increase in expression of these genes was through the GPR126-induced increase of bone morphogenic protein-2, inhibitor of DNA binding (ID) 2, and ID4 expression. These data indicate that GPR126 might be important in maintaining the homeostasis of periodontal ligament tissues through regulating the cytodifferentiation of HPDL cells. The GPR126 SNP rs536714306 negatively influences this homeostasis, leading to the development of AgP, suggesting that it is a candidate genetic risk factor for AgP in the Japanese population.

In Vitro Effects of Pioglitazone on the Expression of Components of Wnt Signaling Pathway and Markers of Bone Mineralization.

Pioglitazone is an insulin-sensitizing thiazolidinedione (TZD) whose use is associated with bone loss. We examined the effects of pioglitazone on components of the Wnt signaling pathway (Wnt1, β-catenin) and markers of bone mineralization [osteoprotegerin (OPG), bone sialoprotein (BSP), fibroblast growth factor (FGF)23] as well as mineral content in human osteoblast hFOB 1.19 cells. hFOB 1.19 cells were cultured in K12/DMD medium with or without pioglitazone. PPARγ Wnt1, OPG, BSP, or FGF23 mRNA expression was measured using qRT-PCR; β-catenin, OPG, BSP, or FGF23 using ELISA; and calcium or phosphate content using colorimetry. Treatment with pioglitazone resulted in increased expression of PPARγ mRNA in hFOB 1.19 osteoblasts. Pioglitazone decreased Wnt1 mRNA levels and suppressed components of Wnt signaling pathway as evidenced by a decrease in β-catenin gene expression and secretion as well as β-catenin specific activity. The expression and the activity of OPG, BSP, and FGF23 were also reduced by pioglitazone together with total (but not specific) calcium and phosphate content. Pioglitazone affects Wnt1 signaling pathway and mineral matrix regulation components in human osteoblasts.

Potential Osteoinductive Effects of Calcitriol on the m-RNA of Mesenchymal Stem Cells Derived from Human Alveolar Periosteum.

This study characterized alveolar periosteum-derived mesenchymal stem cells (P-MSCs) and examined the hypothesis that 1,25-(OH)2D3 (calcitriol) exerts osteoinductive effects on P-MSCs. The mRNA expressions of alkaline phosphatase (ALP), bone sialoprotein (BSP), core-binding factor alpha-1 (CBFA1), collagen-1 (Col-1), osteocalcin (OCN), and vitamin D3 receptor (VDR) were assessed after incubation with calcitriol for 2 weeks. Vitamin C as positive control (Vit. C-p) increased ALP and CBFA1 mRNA expression at both 1 and 2 weeks and increased BSP and Col-1 mRNA expression only at the first week. A concentration of 10−8 M calcitriol enhanced ALP, CBFA1, Col-1, and OCN mRNA expression at both weeks and BSP mRNA expression at the first week. Furthermore, 10−7 M calcitriol increased the mRNA expressions of all compounds at both weeks, except that of CBFA1 at the first week. 10−8 M calcitriol and Vit. C-p enhanced ALP activity at the second and third weeks. The results revealed that 10−9, 10−8, and 10−7 M calcitriol induced osteoinduction in alveolar P-MSCs by increasing ALP, CBFA1, Col-1, and OCN mRNA expression. A 10−7 M calcitriol yielded a higher mRNA expression than Vit. Cp on VDR and OCN mRNA expression at both weeks and on Col-1 mRNA at the second week.

N-CoR modulates osteogenic differentiation of rat mesenchymal stem cells through the PI3K/Akt-cell signaling pathway

The nuclear receptor corepressor (N-CoR) is involved in the regulation of diverse transcription factors. We previously found that N-CoR could regulate adipogenic differentiation of rat mesenchymal stem cells (MSCs),but whether it modulated osteogenic differentiation of this type of cells was uncertain. Therefore, in the present study, we investigated the effect and mechanism of N-CoRon osteogenic differentiation of rat MSCs. The results showed that MSCs cultured in osteogenic medium successfully differentiated into osteogenic cells. Overexpression of N-CoR decreased cell proliferation, alkaline phosphatase (ALP)activity, calcium accumulation, mRNA expression of genes including bone sialoprotein (BSP), osteocalcin (OCN), osteopontin (OPN), Osterix and Runx2, and protein expression of phosphor-Akt(pAkt). Conversely, knocking down cellular N-CoR by small interfering RNA (siRNA) promoted pAkt activity and cell differentiation. Overexpression or knockdown of N-CoRhad no significant influences on the protein expression of pyruvate dehydrogenase kinase isozyme 1 (PDK1), phosphatidylinositol 3-kinase (PI3K) and total Akt, indicating that N-CoR regulated the changes in the PI3K/Akt signaling pathway by targeting pAkt. To further prove the function of the PI3K/Akt signaling in N-CoR-regulated osteogenic differentiation, we used the PI3K inhibitor (LY294002) to block the activation of the PI3K/Akt pathway and found that overexpression of N-CoR showed no effects on ALP activity, calcium level and mRNA expression of BSP, osteocalcin OCN, OPN, Osterix and Runx2 in rat MSCs following the inhibition of the PI3K/Akt pathway. These findings suggest that N-CoR regulates osteogenic differentiation of rat MSCs through suppression of the PI3K/Akt signaling pathway.

Smoking Modulates Gene Expression of Type I Collagen, Bone Sialoprotein, and Osteocalcin in Human Alveolar Bone

Previous animal studies have shown the negative impact of smoking on bone-to-implant contact, and in humans, a decrease in bone density and implant survival over time. However, the effect of smoking on the human alveolar bone regarding the expression of bone-related markers is unknown. Therefore, the aim of this study was to evaluate the influence of smoking on the gene expression of molecules of bone metabolism in alveolar bone tissue from sites designed to receive dental implants. Biopsy specimens of alveolar bone were collected from smokers (n= 19) and nonsmokers (n= 19) from areas planned to receive dental implants. Gene expression of tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β, osteoprotegerin (OPG), type I collagen (COL-I), bone sialoprotein (BSP), and osteocalcin (OCN) was quantified by quantitative real-time polymerase chain reaction using glyceraldehyde-3-phosphate dehydrogenase as a reference gene. The results were assessed using multiple regression analysis, with a significance level of 5%. Multiple regression analysis indicated that smoking negatively affected mRNA expression of BSP and OCN and positively altered the expression of COL-I (P < .05) despite age, gender, and arch. Moreover, regression analysis did not show a significant correlation between smoking habit and mRNA levels of TNF-α, TGF-β, and OPG (P > .05). These results support the hypothesis that some bone markers in alveolar tissue are modulated by smoking, which could explain the negative impact of smoking on bone healing.

Lipopolysaccharide-regulated production of bone sialoprotein and interleukin-8 in human periodontal ligament fibroblasts: the role of toll-like receptors 2 and 4 and the MAPK pathway.

Lipopolysaccharide (LPS) on the cell wall of periodontal pathogens is a major mediator of the inflammatory response and can enhance alveolar bone resorption in periodontitis. Bone sialoprotein is an early marker of osteoblast differentiation. The proinflammatory cytokine, interleukin-8 (IL-8), induces osteoclast differentiation, maturation and maintenance of bone resorption activity. However, the effects of LPS from periodontal pathogens on the expression of bone sialoprotein and IL-8 in human osteoblasts and the mechanism of periodontal bone metabolism regulation are rather unclear. The objectives of this study were to determine the effects of Porphyromonas gingivalis LPS on the production of bone sialoprotein and IL-8 in human periodontal ligament fibroblasts (hPDLFs), and to investigate whether toll-like receptor (TLR) 2, TLR4 and MAPKs pathways are involved in the regulation of production of bone sialoprotein and IL-8 by P.gingivalis LPS. The third-generation of hPDLFs were cultured with mineralization-inducing culture medium. After hPDLFs were treated with P.gingivalis LPS, bone sialoprotein and IL-8 mRNA expression were detected using Real time PCR. Then hPDLFs were transiently transfected with siTLR2 or siTLR4 (20nm) or inhibited by MAPK signaling pathways inhibitors, and then bone sialoprotein and IL-8 mRNA and protein expression were also detected using Real time PCR and western blotting. Treatments with 0.01 and 0.1mg/L of P.gingivalis LPS for 8h up-regulated bone sialoprotein mRNA expression, whereas 10 and 100mg/L of P.gingivalis LPS induced a significant decrease in the expression of bone sialoprotein mRNA. In contrast, IL8 mRNA levels were increased significantly by 10mg/L of P.gingivalis LPS. Interestingly, small interfering RNA (siRNA) knock down of the TLR2 and ERK1/2 inhibitor, PD98059, abolished the effects of P.gingivalis LPS on the bone sialoprotein mRNA level, whereas siRNA knock down of the TLR2 and p38 MAPK inhibitor, SB203580, blocked the effect of P.gingivalis LPS on IL-8 in hPDLFs. This study suggests that in hPDLFs, P.gingivalis LPS suppresses bone sialoprotein and enhances IL-8 gene and protein expression via TLR2 and ERK1/2 or the p38 MAPK signaling pathway, respectively.

Effect of epithelial rests of Malassez’ cells on RANKL mRNA expression and ALP activity by periodontal ligament fibroblasts stimulated with sonicated Porphyromonas gingivalis in vitro

ObjectivesThe purpose of this study was to investigate the effects of epithelial rests of Malassez’ (ERM) cells on RANKL expression and alkaline phosphatase (ALP) activity by periodontal ligament (PDL) fibroblasts in the presence of lipopolysaccharides (LPS) in vitro using a co-culture technique. Materials and methodsPorcine PDL fibroblasts and ERM cells were used. PDL fibroblasts were seeded in 6-well dishes. ERM cells plated in a chamber with a 0.4μm pore membrane were placed in the wells for 5 days, and then were stimulated with sonicated Porphyromonas gingivalis (P.g.) for 6h. PDL cells were then evaluated for mRNA and protein expression of RANKL and mRNA expression of bone sialoprotein (BSP) and ALP activity was also analyzed. ResultsRANKL mRNA expression by PDL cells co-cultured with ERM cells and sonicated P.g. was higher than when co-cultured with ERM cells without P.g. and when cultured without ERM cells. RANKL protein levels in the cytoplasm of PDL fibroblasts co-cultured with ERM cells were stronger than in PDL fibroblasts cultured without ERM cells. BSP mRNA expression and ALP activity of PDL fibroblasts co-cultured with ERM cells was significantly lower than in PDL fibroblasts cultured without ERM cells. ConclusionsIn the presence of sonicated P.g., ERM cells increase RANKL expression by PDL cells, but decrease BSP mRNA and ALP activity by PDL cells. Therefore, ERM cells secreted more RANKL to stimulate more osteoclasts differentiation to resorb the alveolar bone under the condition of co-culture in the environment of the presence of sonicated P.g.

Effects of CoCl2 on Osteogenic Differentiation of Human Mesenchymal Stem Cells

Objective. To investigate the effects of the hypoxia inducible factor-1 (HIF-1) activation–mimicking agent cobalt chloride (CoCl2) on the osteogenic differentiation of human mesenchy-mal stem cells (hMSCs) and elucidate the underlying mole-cular mechanisms. Study design. The dose and exposure periods for CoCl2 in hMSCs were optimized by cell viability assays. After confirmation of CoCl2-induced HIF-1α and vas-cular endothelial growth factor expression in these cells by RT-PCR, the effects of temporary preconditioning with CoCl2 on hMSC osteogenic differentiation were evaluated by RT- PCR analysis of osteogenic gene expression, an alkaline phos-phatase (ALP) activity assay and by alizarin red S staining. Results. Variable CoCl2 dosages (up to 500 µM) and exposure times (up to 7 days) on hMSC had little effect on hMSC survival. After CoCl2 treatment of hMSCs at 100 µM for 24 or 48 hours, followed by culture in osteogenic differentiating media, several osteogenic markers such as Runx-2, osteocal-cin and osteopontin, bone sialoprotein mRNA expression level were found to be up-regulated. Moreover, ALP acti-vity was increased in these treated cells in which an accele-rated osteogenic capacity was also verified by alizarin red S staining. Conclusions. The osteogenic differentiation poten-tial of hMSCs could be preserved and even enhanced by CoCl2 treatment.

Periodontal ligament cell behavior on different titanium surfaces

Aim. The purpose of this study was to investigate proliferation, morphology, mineralization and mRNA expressions of mineralized tissue associated proteins of PDL cells on smooth (S), sandblasted small-grit (SSG), sandblasted large-grit (SLG) and sodium titanate (NaTi) coated titanium alloys, in vitro. Methods and materials: PDL cells were cultured with DMEM media containing 10% FBS on the S, SSG, SLG and NaTi titanium surfaces. PDL cell proliferation, mineralization and immunohistochemistry experiments for Bone Sialoprotein (BSP) were performed. The morphology of the PDL cells was examined using confocal and scanning electron microscopy (SEM). Gene expression profiles of cells were evaluated using a quantitative-polymerase chain reaction (Q-PCR) for type I collagen (COL I), Osteocalcin (OCN), osteopontin (OPN) and Runt-related transcription factor-2 (Runx2) on days 7 and 14. Results. Proliferation results on days 6 and 10 were similar in groups, while those of day 13 revealed a decrease in the NaTi group when compared to the S group. NaTi surface induced BSP mRNA expression which was correlated with mineralization tests and BSP immunostaining results. Increased Runx2 mRNA expression was also noted in the NaTi surface when compared to other surfaces. Conclusions. This study considers the NaTi surface as a potential alternative to SSG and SLG surfaces. This surface might provide a promising environment for PDL ligament-anchored implants.

The effects of epithelial rests of Malassez cells on periodontal ligament fibroblasts against centrifugal forces in vitro

ObjectivesThe purpose of this study was to investigate the effects of epithelial rests of Malassez (ERM) cells on PDL fibroblasts in vitro against centrifugal forces using a co-culture system. Materials and methodsPDL fibroblasts and ERM cells derived from porcine were used. PDL fibroblasts were seeded in 6-well dishes. ERM cells plated in a chamber with a 0.4μm pore membrane were placed into the wells for 5days, and then were centrifuged as a mechanical stress. Vascular endothelial growth factor (VEGF), heat shock protein (HSP) 27, osteocalcin (OCN), bone sialoprotein (BSP), receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) mRNA levels were analyzed, and RANKL expression was analyzed at the protein level. ResultsVEGF mRNA expression by PDL fibroblasts co-cultured with ERM cells with or without force was higher than those cultured without ERM cells. HSP27 mRNA expression by PDL fibroblasts cultured without ERM cells with force was higher than those co-cultured with ERM cells. OCN and BSP mRNA expression by PDL fibroblasts co-cultured with ERM cells was significantly lower than without ERM cells with or without force. The expressions of RANKL by PDL fibroblasts co-cultured with ERM cells with force were higher than those without ERM cells. OPG mRNA expression by PDL fibroblasts co-cultured with ERM cells without force were lower than those without ERM cells. ConclusionThese results demonstrate that ERM cells play an important role in preventing PDL cells from being susceptible to dento-alveolar ankylosis during compression of the PDL.

Synthesis and Discovery of Novel Pyrazole Carboxamide Derivatives as Potential Osteogenesis Inducers

A series of novel N-aryl-3-aryl-1-arylmethyl-1H-pyrazole-5-carboxamide derivatives 4a-l was synthesized by the reaction of 3-aryl-1-arylmethyl-1H-pyrazole-5-carbonyl chloride with substituted aniline in good to excellent yields. Structures of the compounds were determined by IR, (1) H NMR, and HR-MS spectroscopy. The molecular structure was confirmed by the X-ray crystal analysis of one compound (4j) that was prone to crystallization. These compounds were used to induce mouse osteoblast precursors MC3T3-E1 into osteoblasts and the induction was assessed by alkaline phosphatase (ALP) activity and the gene expression of bone sialoprotein (BSP). The results showed that the compounds 4a-d, 4g, 4h, and 4k could increase the ALP activity in comparison with the negative control group and compound 4h was the most effective one which could induce osteogenesis. Furthermore, mRNA expression of BSP which is a marker of osteogenesis was up-regulated by the compound 4h.

Inhibition of Insulin-like Growth Factor-1 (IGF-1) Expression by Prolonged Transforming Growth Factor-β1 (TGF-β1) Administration Suppresses Osteoblast Differentiation

TGF-β1 can regulate osteoblast differentiation not only positively but also negatively. However, the mechanisms of negative regulation are not well understood. We previously established the reproducible model for studying the suppression of osteoblast differentiation by repeated or high dose treatment with TGF-β1, although single low dose TGF-β1 strongly induced osteoblast differentiation. The mRNA expression and protein level of insulin-like growth factor-1 (IGF-1) were remarkably decreased by repeated TGF-β1 administration in human periodontal ligament cells, human mesenchymal stem cells, and murine preosteoblast MC3T3-E1 cells. Repeated TGF-β1 administration subsequently decreased alkaline phosphatase (ALP) activity and mRNA expression of osteoblast differentiation marker genes, such as RUNX2, ALP, and bone sialoprotein (BSP). Additionally, repeated administration significantly reduced the downstream signaling pathway of IGF-1, such as Akt phosphorylation in these cells. Surprisingly, exogenous and overexpressed IGF-1 recovered ALP activity and mRNA expression of osteoblast differentiation marker genes even with repeated TGF-β1 administration. These facts indicate that the key mechanism of inhibition of osteoblast differentiation induced by repeated TGF-β1 treatment is simply due to the down-regulation of IGF-1 expression. Inhibition of IGF-1 signaling using small interfering RNA (siRNA) against insulin receptor substrate-1 (IRS-1) suppressed mRNA expression of RUNX2, ALP, BSP, and IGF-1 even with single TGF-β1 administration. This study showed that persistence of TGF-β1 inhibited osteoblast differentiation via suppression of IGF-1 expression and subsequent down-regulation of the PI3K/Akt pathway. We think this fact could open the way to use IGF-1 as a treatment tool for bone regeneration in prolonged inflammatory disease.

An Ectopic Study of Apatite-Coated Silk Fibroin Scaffolds Seeded with AdBMP-2-Modified Canine bMSCs

The present study was undertaken to evaluate ectopic new bone formation effects of apatite-coated silk fibroin scaffolds (mSS) seeded with adenovirus-mediated bone morphogenic protein-2 gene (AdBMP-2) transduced canine bone marrow stromal cells (bMSCs) in nude mice. In this study, bMSCs derived from canine were cultured and transduced with AdBMP-2 adenovirus-mediated enhanced green fluorescent protein gene (AdEGFP) in vitro. Osteogenic differentiation of bMSCs was determined by alkaline phosphatase (ALP) activity analysis, and the transcript levels for BMP-2, osteopontin (OPN), osteocalcin (OCN) and bone sialoprotein (BSP) genes via real-time quantitative PCR (RT-qPCR) analysis. The ectopic bone formation effects of mSS seeded with AdBMP-2-modified bMSCs were evaluated through histological and histomorphological analysis 4, 8 and 12 weeks post-operation in nude mice. ALP activity was statistically increased in the AdBMP-2 group, when compared with control groups. The mRNA expression of BMP-2, OPN, OCN and BSP was also statistically up-regulated 6 and 9 days after AdBMP-2 transduction. Significantly higher bone volume was achieved in AdBMP-2-transduced bMSCs/mSS constructs than that of AdEGFP-transduced bMSCs/mSS or bMSCs/mSS groups at 4, 8 and 12 weeks (P < 0.01). These results demonstrated that mSS seeded with AdBMP-2-transduced canine bMSCs can promote ectopic new bone formation and maturation in nude mice, suggesting the potential of this silk-scaffold-based tissue-engineered bone for further bone regeneration studies in canine models.