In vitro and in vivo evaluation of a novel polymer-free everolimus-eluting stent by nitrogen-doped titanium dioxide film deposition.

Inflammation and thrombosis are linked to the use of polymer-based drug-eluting stents (DES). The aim of this study was to develop a polymer-free everolimus (EVL)-eluting stent using nitrogen-doped titanium dioxide (N-TiO2) and verify its efficacy by in vitro and in vivo assessment in a porcine coronary model. Various analytical approaches such as scanning electron microscopy and atomic force microscopy, electron spectroscopy, Fourier transform infrared spectrometry and contact angle measurement were employed for the characterization. As a part of biocompatibility assessment, platelet adhesion and smooth muscle cell (SMC) proliferation were examined. Bare metal stent (BMS), N-TiO2 stent, everolimus-eluting N-TiO2 (N-TiO2-EVL) stent, and commercialized EVL-eluting stent (EES) were randomly placed in forty coronary arteries in twenty pigs. After four weeks of implantation, the stents were subjected to histological and quantitative analysis. The N-TiO2 film used in this study was well coated without any cracks or peeling. Surface hydrophilicity (88.8% of angle decrement) could be associated with the decrease in surface roughness post N-TiO2 deposition (37.0%). The platelet adhesion on the N-TiO2 surfaces was less than that on the BMS surface. The proliferation of SMC was suppressed in the N-TiO2-EVL group (30.2%) but not in the BMS group. In the animal study, the percent area restenosis was significantly decreased in the N-TiO2-EVL group compared to that in the BMS group. The results (BMS; 47.0 ±â€¯11.00%, N-TiO2-EVL; 31.7 ±â€¯10.50%, and EES; 29.1 ±â€¯11.21%, n = 10, p < 0.05) were almost at par with those of the commercialized EVL-eluting stent. The introduction of N-TiO2 deposition during fabrication of polymer-free DES may be an efficient accessorial process for preventing in-stent restenosis and thrombosis.

Tectorigenin Promotes Osteoblast Differentiation and in vivo Bone Healing, but Suppresses Osteoclast Differentiation and in vivo Bone Resorption.

Although tectorigenin (TG), a major compound in the rhizome of Belamcanda chinensis, is conventionally used for the treatment of inflammatory diseases, its effects on osteogenesis and osteoclastogenesis have not been reported. The objective of this study was to investigate the effects and possible underlying mechanism of TG on in vitro osteoblastic differentiation and in vivo bone formation, as well as in vitro osteoclast differentiation and in vivo bone resorption. TG promoted the osteogenic differentiation of primary osteoblasts and periodontal ligament cells. Moreover, TG upregulated the expression of the BMP2, BMP4, and Smad-4 genes, and enhanced the expression of Runx2 and Osterix. In vivo studies involving mouse calvarial bone defects with µCT and histologic analysis revealed that TG significantly increased new bone formation. Furthermore, TG treatment inhibited osteoclast differentiation and the mRNA levels of osteoclast markers. In vivo studies of mice demonstrated that TG caused the marked attenuation of bone resorption. These results collectively demonstrated that TG stimulated osteogenic differentiation in vitro, increased in vivo bone regeneration, inhibited osteoclast differentiation in vitro, and suppressed inflammatory bone loss in vivo. These novel findings suggest that TG may be useful for bone regeneration and treatment of bone diseases.

Bilirubin coating attenuates the inflammatory response to everolimus-coated stents.

The aim of this study was to evaluate the effects of bilirubin- and/or everolimus (EVL)-coated stents to prevent arterial neointimal hyperplasia and inflammation in vitro and in vivo. The stents were prepared by spray coating bare metal stents (BMS) with bilirubin and/or EVL. Study groups were divided into (1) BMS, (2) bilirubin-coated stents (BES), (3) commercialized stents (Synergy™; EES), and (4) bilirubin/EVL-coated stents (B-EES). The coating thickness and drug release rates were comparable to previous reports (i.e., <4 µm thickness and 50% drug release in 7 days). Smooth muscle cell migration was inhibited in both EVL-containing groups (20.5 ± 3.80% in EES and 18.4 ± 2.55% in B-EES) compared to the non-EVL-containing groups (78.0 ± 6.41% in BMS and 76.1 ± 4.88% in BES) (n = 10, p < 0.05). Stents were randomly implanted to 40 coronary arteries in 20 pigs and subjected to various analyses after 4 weeks of implantation. As results, the inflammation score was dramatically increased in the EES group (2.1 ± 0.42) compared to that of the other groups (1.5 ± 0.55, 1.3 ± 0.23, and 1.5 ± 0.27 for BMS, BES, and B-EES, respectively, n = 10, p < 0.05). Immunofluorescence analysis revealed that inflammation was prevented in the bilirubin-containing groups (BES and B-EES). However, the percent area of restenosis was decreased in the EVL-containing groups (20.5 ± 4.11% for EES and 18.4 ± 3.61% for B-EES) compared to the non-EVL-containing groups (32.3 ± 6.41% for BMS and 29.6 ± 5.95% for BES, n = 10, p < 0.05). The percent areas of restenosis determined by histopathology, optical coherence tomography, and micro-computed tomography were consistent. In addition, the stent was barely covered in the EES and B-EES groups at 4 weeks postimplantation. These dual drug-coated stents may be especially beneficial to patients who have an increased risk of inflammation. These stents have great potential for use in cardiovascular applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1486-1495, 2018.

Effects of cosmetics on the skin microbiome of facial cheeks with different hydration levels.

Basic cosmetics was used by volunteers belonging to high (HHG) and low (LHG) hydration groups for 4 weeks, and bacterial communities and biophysical parameters in facial skin were analyzed. Hydration level increases and transepidermal water loss and roughness decreases were observed in both groups after cosmetic use. Bacterial diversity was greater in LHG than HHG, and increased after cosmetic use in both groups. Bray-Curtis dissimilarities that were higher in LHG than HHG increased in HHG after cosmetic use, whereas they decreased in LHG. The phyla Actinobacteria, Proteobacteria, Firmicutes, and Bacteroidetes and the genera Propionibacterium, Ralstonia, Burkholderia, Staphylococcus, Corynebacterium, Cupriavidus, and Pelomonas were identified as common groups and they were not significantly different between LHG and HHG except for Propionibacterium that was more abundant in HHG. After cosmetic use, Propionibacterium, Staphylococcus, and Corynebacterium decreased, whereas Ralstonia, not a core genus, increased, as did KEGG categories of lipid metabolism and xenobiotics biodegradation and metabolism, suggesting that Ralstonia in skin may have the ability to metabolize cosmetics components. Bacterial communities after cosmetic use were different from those in both LHG and HHG before the cosmetic use, indicating that bacterial communities in LHG were not shifted to resemble those in HHG by cosmetics use.

Hydrophilic surface modification of coronary stent using an atmospheric pressure plasma jet for endothelialization.

The first two authors contributed equally to this study. Bioactivity and cell adhesion properties are major factors for fabricating medical devices such as coronary stents. The aim of this study was to evaluate the advantages of atmospheric-pressure plasma jet in enhancing the biocompatibility and endothelial cell-favorites. The experimental objects were divided into before and after atmospheric-pressure plasma jet treatment with the ratio of nitrogen:argon = 3:1, which is similar to air. The treated surfaces were basically characterized by means of a contact angle analyzer for the activation property on their surfaces. The effect of atmospheric-pressure plasma jet on cellular response was examined by endothelial cell adhesion and XTT analysis. It was difficult to detect any changeable morphology after atmospheric-pressure plasma jet treatment on the surface. The roughness was increased after atmospheric-pressure plasma jet treatment compared to nonatmospheric-pressure plasma jet treatment (86.781 and 7.964 nm, respectively). The X-ray photoelectron spectroscopy results showed that the surface concentration of the C-O groups increased slightly from 6% to 8% after plasma activation. The contact angle dramatically decreased in the atmospheric-pressure plasma jet treated group (22.6 ± 15.26°) compared to the nonatmospheric-pressure plasma jet treated group (72.4 ± 15.26°) ( n = 10, p < 0.05). The effect of the increment in hydrophilicity due to the atmospheric-pressure plasma jet on endothelial cell migration and proliferation was 85.2% ± 12.01% and 34.2% ± 2.68%, respectively, at 7 days, compared to the nonatmospheric-pressure plasma jet treated group (58.2% ± 11.44% in migration, n = 10, p < 0.05). Taken together, the stent surface could easily obtain a hydrophilic property by the atmospheric-pressure plasma jet method. Moreover, the atmospheric-pressure plasma jet might affect re-endothelialization after stenting.

Sirolimus coating on heparinized stents prevents restenosis and thrombosis.

The aim of this study was to evaluate the inhibitory effect of sirolimus coating on the occurrence of restenosis and thrombosis with heparinized stents. Heparin and dopamine were conjugated by chemical bonding and anchored on the stent surface by a mussel-inspired adhesion mechanism. Subsequently, sirolimus was coated with poly lactic-glycolic acid on the heparinized stent surface. The heparin was well attached to the surface, and the surface was smooth after sirolimus coating. The smoothness of the surface was maintained after expansion of the stent. The amount of sirolimus released from the stent was 67.3% ± 4.55% within 7 days, followed by continual release up to day 28. The proliferation of smooth muscle cells was successfully arrested (51.3% ± 2.25% at 7 days of culture) by sirolimus released from the stent. Platelet adhesion was clearly prevented in the heparin-coated group (78.0 ± 8.00/1.8 cm2) compared to that in the heparin noncoated group (5.0 ± 1.00/1.8 cm2). Animal studies showed that the heparin and sirolimus-coated stent group had no obvious inflammatory response and no change in the fibrin score compared to those in the other groups. However, restenosis clearly decreased in the heparin and sirolimus-coated group (12.3% ± 3.54%) compared to the bare-metal stent group (27.5% ± 8.52%) and the heparin-coated group (25.3% ± 11.79%). These results suggest that heparinized surface-based sirolimus coating may be a useful approach for the prevention of restenosis and stent thrombosis.

Multi-marker linkage disequilibrium mapping of quantitative trait loci.

Single nucleotide polymorphisms (SNPs), the most common genetic markers in genome-wide association studies, are usually in linkage disequilibrium (LD) with each other within a small genomic region. Both single- and two-marker-based LD mapping methods have been developed by taking advantage of the LD structures. In this study, a more general LD mapping framework with an arbitrary number of markers has been developed to further improve LD mapping and its detection power. This method is referred as multi-marker linkage disequilibrium mapping (mmLD). For the parameter estimation, we implemented a two-phase estimation procedure: first, haplotype frequencies were estimated for known markers; then, haplotype frequencies were updated to include the unknown quantitative trait loci based on estimates from the first step. For the hypothesis testing, we proposed a novel sequential likelihood ratio test procedure, which iteratively removed haplotypes with zero frequency and subsequently determined the proper degree of freedom. To compare the proposed mmLD method with other existing mapping methods, e.g. the adjusted single-marker LD mapping and the SKAT_C, we performed extensive simulations under various scenarios. The simulation results demonstrated that the mmLD has the same or higher power than the existing methods, while maintaining the correct type I errors. We further applied the mmLD to a public data set, 'GAW17', to investigate its applicability. The result showed the good performance of mmLD. We concluded that this improved mmLD method will be useful for future genome-wide association studies and genetic association analyses.

Comparison of dextran-based sirolimus-eluting stents and PLA-based sirolimus-eluting stents in vitro and in vivo.

The aim of this study was to compare dextran and Poly(l-lactide) (PLLA) polymer stent coatings as mediators for sirolimus (SRL) drug elution in a porcine coronary model. The bare metal stent (BMS) surface was first coated with a layer of SRL and then either dextran (DSS, a natural polymer) or PLA (PSS, a synthetic polymer). The release velocity of SRL was slightly faster in DSS than PSS over the first 7 days (78.5% and 62.3%, respectively, n = 10, p < 0.05) and continued to 28 days in both groups. The contact angle was dramatically decreased in DSS (38.7° ± 1.24) compared to BMS and PSS groups (72.7° ± 5.32 and 81.1º ± 1.70, respectively, n = 10, p < 0.05). Smooth muscle cell migration was arrested in both the DSS and PSS-treated groups compared to that in the nontreated group (4.2% ± 0.31, 5.8% ± 0.60, 80.0% ± 4.4, respectively, n = 10, p < 0.05). In the animal study, there were no significant differences in the injury score, the internal elastic lamina, and the lumen area among the groups. However, percent area stenosis was significantly decreased in the SRL-containing group (27.5% ± 2.52 in DSS and 27.9% ± 3.30 in PSS) compared to BMS (35.9% ± 3.51, p < 0.05). The fibrin score was higher in the PSS (2.9 ± 0.31) than BMS (2.1 ± 0.12) and DSS (2.5 ± 0.66). The inflammation score in the DSS (0.7 ± 0.21) was similar to that in the BMS (0.7 ± 0.12), which was dramatically lower than that PSS (1.5 ± 0.18, p < 0.005). Immunofluorescence analysis revealed that endothelialization was increased and inflammation prevented in the DSS. These results suggest that dextran may be useful for the fabrication of drug eluting stent as an alternative existing synthetic polymer. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 301-310, 2017.

Stimulation of Odontogenesis and Angiogenesis via Bioactive Nanocomposite Calcium Phosphate Cements Through Integrin and VEGF Signaling Pathways.

Formulating self-setting calcium phosphate cements (CPCs) with secondary phases particularly in the nanoscale order holds great promise to improve biological properties. Here, we focus on the effect that bioactive glass nanoparticles (BGN) incorporated in CPC compositions can have on the proliferation, odontogenic differentiation, and angiogenic stimulation of stem cells derived from human dental pulp (HDPSCs). These odontogenic and angiogenic events are of special importance in the dentin-pulp regeneration processes. In comparison to pure CPCs, nanocomposite cements exhibit a significantly improved proliferation of HDPSCs, and the improvement is more significant as the BGN content increases. The nanocomposite cements substantially enhance the adhesion of cells, and significantly up-regulate odontogenic differentiation, including alkaline phosphatase (ALP) activity and the expressions of odontogenic genes (sialophosphoprotein, dentin matrix protein I, ALP, osteopontin and osteocalcin). Furthermore, the use of nanocomposite cements result in stimulation of angiogenic gene expression (VEGF, FGF-2, VEGFRs, PECAM-1, and VE-cadherin) and protein production (VEGF, VEGFR-1). The angiogenic stimulation by the HDPSCs significantly affects the endothelial cell behaviors, that is, the endothelial cell migration and the tubular network formation are substantially improved when treated with HDPSC-conditioned medium, particularly with the help of nanocomposite cements. The integrin and VEGF signaling pathways are reasoned for the stimulation of the odontogenesis and angiogenesis of cells, where the nanocomposite cements up-regulate the integrin subsets α1, α2, α3, and ß1, and activate the integrin downstream signal pathways, such as p-FAK, p-Akt, p-paxillin, JNK, EK, and NF-κB, as well as other nuclear transcriptional factors, including CREB, STAT-3, and ELK-1. The current results indicate that the new formulation of the nanocomposite self-setting cements might provide some beneficial microenvironments for the regenerative processes of dentin-pulp complex tissues.

Prednisolone- and sirolimus-eluting stent: Anti-inflammatory approach for inhibiting in-stent restenosis.

Glucocorticoids are powerful anti-inflammatory, immunosuppressive, and anti-proliferative agents. The aim of this study was to evaluate the effectiveness of a prednisolone- (PDScs) and sirolimus-coated stent (SRLcs) in preventing artery vessel neointimal hyperplasia and inflammatory reactions in vitro and in vivo. PDS, a synthetic glucocorticoid, is a derivative of cortisol, which is used to treat a variety of inflammatory and autoimmune conditions. The stents were fabricated with PDS, SRL, or both agents using a layer-by-layer coating system (designated as PDScs, SRLcs, and PDSRLcs, respectively). The surface morphology of the PDScs showed an evenly dispersed and roughened shape, which was smoothened by the SRL coating. Half of the total drug amounts were released within seven days, followed by an additional release, which continued for up to 28 days. The proliferation of smooth muscle cells was inhibited in the SRLcs group (31.5 ± 4.08%), and this effect was enhanced by PDS addition (PDSRLcs, 46.8 ± 8.11%). Consistently, in the animal study, the restenosis rate was inhibited by the SRLcs and PDSRLcs (18.5 ± 6.23% and 14.5 ± 3.55%, respectively). Especially, fibrin expression and inflammation were suppressed in the PDS-containing group (PDScs, 0.6 ± 0.12 and 1.4 ± 0.33; PDSRLcs, 0.7 ± 0.48 and 1.7 ± 0.12, respectively) compared to PDS non-containing groups (BMS, 1.1 ± 0.12, and 1.8 ± 0.55; SRLcs, 1.6 ± 0.32 and 2.0 ± 0.62, respectively). Moreover, re-endothelialization was enhanced in the PDScs group as determined using immunohistochemistry with a cluster of differentiation (CD)-31 antibodies. These results suggest that the inhibitory effect of SRLcs on anti-restenosis can be accelerated by additional coating with PDS, which has promising properties as a bioactive compound with useful anti-inflammatory effects.

Expression of Caveolin-1 in Periodontal Tissue and Its Role in Osteoblastic and Cementoblastic Differentiation In Vitro.

It has been previously reported that caveolin-1 (Cav-1) knockout mice exhibit increased bone size and stiffness. However, the expression and role of Cav-1 on periodontal tissue is poorly understood. The aim of this study was to investigate the immunohistochemical expression of Cav-1 in the mouse periodontium and explore the role of Cav-1 on osteoblastic and cementoblastic differentiation in human periodontal ligament cells (hPDLCs), cementoblasts, and osteoblasts. To reveal the molecular mechanisms of Cav-1 activity, associated signaling pathways were also examined. Immunolocalization of Cav-1 was studied in mice periodontal tissue. Differentiation was evaluated by ALP activity, alizarin red S staining, and RT-PCR for marker genes. Signal transduction was analyzed using Western blotting and confocal microscopy. Cav-1 expression was observed in hPDLCs, cementoblasts, and osteoblasts of the periodontium both in vivo and in vitro. Inhibition of Cav-1 expression by methyl-ß-cyclodextrin (MßCD) and knockdown of Cav-1 by siRNA promoted osteoblastic and cementoblastic differentiation by increasing ALP activity, calcium nodule formation, and mRNA expression of differentiation markers in hPDLCs, cementoblasts, and osteoblasts. Osteogenic medium-induced BMP-2 and BMP-7 expression, and phosphorylation of Smad1/5/8 were enhanced by MßCD and siRNA knockdown of Cav-1, which was reversed by BMP inhibitor noggin. MßCD and Cav-1 siRNA knockdown increased OM-induced AMPK, Akt, GSK3ß, and CREB phosphorylation, which were reversed by Ara-A, a specific AMPK inhibitor. Moreover, OM-induced activation of p38, ERK, JNK, and NF-κB was enhanced by Cav-1 inhibition. This study demonstrates, for the first time, that Cav-1 is expressed in developing periodontal tissue and in vitro in periodontal-related cells. Cav-1 inhibition positively regulates osteoblastic differentiation in hPDLCs, cementoblasts, and osteoblasts via BMP, AMPK, MAPK, and NF-κB pathway. Thus, Cav-1 inhibition may be a novel molecular target for therapeutic approaches in periodontitis or osteolytic disease.

Effect of a novel peptide, WKYMVm- and sirolimus-coated stent on re-endothelialization and anti-restenosis.

The drug-eluting stent still has limitations such as thrombosis and inflammation. These limitations often occur in the absence of endothelialization. This study investigated the effects of WKYMVm- and sirolimus-coated stents on re-endothelialization and anti-restenosis. The WKYMVm peptide, specially synthesized for homing endothelial colony-forming cells, was coated onto a bare-metal stent with hyaluronic acid through a simple dip-coating method (designated HA-Pep). Thereafter, sirolimus was consecutively coated to onto the HA-Pep (designated Pep/SRL). The cellular response to stents by human umbilical-vein endothelial cells and vascular smooth-muscle cells was examined by XTT assay. Stents were implanted into rabbit iliac arteries, isolated 6 weeks post-implantation, and then subjected to histological analysis. The peptide was well attached to the surface of the stents and the sirolimus coating made the surface smooth. The release pattern for sirolimus was similar to that of commercial sirolimus-coated stents (57.2% within 7 days, with further release for up to 28 days). Endothelial-cell proliferation was enhanced in the HA-Pep group after 7 days of culture (38.2 ± 7.62%, compared with controls). On the other hand, the proliferation of smooth-muscle cells was inhibited in the Pep/SRL group after 7 days of culture (40.7 ± 6.71%, compared with controls). In an animal study, the restenosis rates for the Pep/SRL group (13.5 ± 4.50%) and commercial drug-eluting stents (Xience Prime™; 9.2 ± 7.20%) were lower than those for bare-metal stents (25.2 ± 4.52%) and HA-Pep stents (26.9 ± 3.88%). CD31 staining was incomplete for the bare-metal and Xience Prime™ groups. On the other hand, CD31 staining showed a consecutive linear pattern in the HA-Pep and Pep/SRL groups, suggesting that WKYMVm promotes endothelialization. These results indicate that the WKYMVm coating could promote endothelial healing, and consecutive coatings of WKYMVm and sirolimus onto bare-metal stents have a potential role in re-endothelialization and neointimal suppression.

Expression of Phospholipase D in Periodontitis and Its Role in the Inflammatory and Osteoclastic Response by Nicotine- and Lipopolysaccharide-Stimulated Human Periodontal Ligament Cells.

BACKGROUND: The aim of the present study is to investigate the expression of phospholipase D (PLD) 1 and PLD2 in periodontal patients and in human periodontal ligament cells (HPDLCs) exposed to nicotine plus lipopolysaccharide (LPS) from Porphyromonas gingivalis (Toll-like receptor 2 ligand). Furthermore, the effects of PLD isoform inhibition on the inflammatory response and osteoclast differentiation and its mechanisms were determined. METHODS: Proinflammatory mediators were examined by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. To silence the gene expression of the PLD isoforms, cells were transfected with small interfering RNA (siRNA) targeting PLD1 or PLD2. Mouse bone marrow-derived macrophages (BMMs) were used as osteoclast precursor cells for in vitro osteoclastogenesis. Western blot analysis and immunofluorescence were used to assess signaling pathways. RESULTS: Chronic smokers with periodontitis exhibited significantly higher PLD1 and PLD2 messenger RNA (mRNA) expression than non-smokers with periodontitis and healthy controls. Nicotine and LPS upregulated PLD1 and PLD2 mRNA expression in a dose-dependent manner in HPDLCs. Pharmacologic and siRNA-mediated inhibition of PLD1 and PLD2 attenuated the nicotine- and LPS-induced upregulation of inducible nitric oxide (NO) synthase and cyclooxygenase-2, production of NO, and prostaglandin E2, and mRNA expression and secretion of tumor necrosis factor-α, interleukin (IL)-1ß, and IL-8. The conditioned media from HPDLCs treated with PLD isoform inhibitors or siRNA against PLD inhibited receptor activator of nuclear factor-κB (NF-κB) ligand-mediated osteoclast differentiation, as well as protein expression of nuclear factor of activated T cells c1 and c-Fos, in BMMs. In addition, PLD isoform inhibitors and siRNA inhibited the nicotine- and LPS-induced activation of phosphoinositide 3-kinase, protein kinase C, p38, extracellular signal-regulated kinase, c-Jun N-terminal protein kinase, mitogen-activated protein kinase, and NF-κB. CONCLUSION: To the best of the authors' knowledge, this study is the first to demonstrate that PLD isoform inhibition has anti-inflammatory and antiosteoclastogenic effects and thus may be a therapeutic target for the treatment of periodontitis.

Odontogenic stimulation of human dental pulp cells with bioactive nanocomposite fiber.

The aim of the present study was to investigate the effects of a composite nanofibrous matrix made of biopolymer blend polycaprolactone-gelatin (BP) and mesoporous bioactive glass nanoparticles (BGNs) on the odontogenic differentiation of human dental pulp cells (HDPCs). BGN-BP nanomatrices, with BGN content of up to 20 wt%, were produced via electrospinning. The differentiation of the HDPCs was evaluated by using an ALP activity assay, calcified nodule formation, and mRNA expression for markers. Integrin and its underlying signal pathways were assessed via reverse transcriptase-polymerase chain reaction and Western blot analysis. Although cell growth and attachment on the BGN-BP nanomatrix was similar to that on BP, ALP activity, mineralized nodule formation, and mRNA, expressions involving ALP, osteocalcin, osteopontin, dentin sialophosphoprotein, and dentin matrix protein-1 were greater on BGN-BP. BGN-BP upregulated the key adhesion receptors (integrin components α1, α2, α5, and ß1) and activated integrin downstream pathways, such as phosphorylated-focal adhesion kinase (p-FAK), and p-paxillin. In addition, BGN-BP activated BMP receptors, BMP-2 mRNA, and p-Smad 1/5/8, and such activation was blocked by the BMP antagonist, noggin. Furthermore, BGN-BP induced phosphorylation of extracellular signal-regulated kinase, protein kinase 38, and c-Jun-N-terminal kinase mitogen-activated protein kinases and activated expression of the transcription factors Runx2 and Osterix in HDPCs. Collectively, the results indicated for the first time that a BGN-BP composite nanomatrix promoted odontogenic differentiation of HDPCs through the integrin, BMP, and mitogen-activated protein kinases signaling pathway. Moreover, the nanomatrix is considered to be promising scaffolds for the culture of HDPCs and dental tissue engineering.

Melatonin promotes hepatic differentiation of human dental pulp stem cells: clinical implications for the prevention of liver fibrosis.

Melatonin's effect on hepatic differentiation of stem cells remains unclear. The aim of this study was to investigate the action of melatonin on hepatic differentiation as well as its related signaling pathways of human dental pulp stem cells (hDPSCs) and to examine the therapeutic effects of a combination of melatonin and hDPSC transplantation on carbon tetrachloride (CCl4 )-induced liver fibrosis in mice. In vitro hepatic differentiation was assessed by periodic acid-Schiff (PAS) staining and mRNA expression for hepatocyte markers. Liver fibrosis model was established by injecting 0.5 mL/kg CCl4 followed by treatment with melatonin (5 mg/kg, twice a week) and hDPSCs. In vivo therapeutic effects were evaluated by histopathology and by means of liver function tests including measurement of alanine transaminase (ALT), aspartate transaminase (AST), and ammonia levels. Melatonin promoted hepatic differentiation based on mRNA expression of differentiation markers and PAS-stained glycogen-laden cells. In addition, melatonin increased bone morphogenic protein (BMP)-2 expression and Smad1/5/8 phosphorylation, which was blocked by the BMP antagonist noggin. Furthermore, melatonin activated p38, extracellular signal-regulated kinase (ERK), and nuclear factor-κB (NF-κB) in hDPSCs. Melatonin-induced hepatic differentiation was attenuated by inhibitors of BMP, p38, ERK, and NF-κB. Compared to treatment of CCl4 -injured mice with either melatonin or hDPSC transplantation alone, the combination of melatonin and hDPSC significantly suppressed liver fibrosis and restored ALT, AST, and ammonia levels. For the first time, this study demonstrates that melatonin promotes hepatic differentiation of hDPSCs by modulating the BMP, p38, ERK, and NF-κB pathway. Combined treatment of grafted hDPSCs and melatonin could be a viable approach for the treatment of liver cirrhosis.

In vitro biocompatibility, inflammatory response, and osteogenic potential of 4 root canal sealers: Sealapex, Sankin apatite root sealer, MTA Fillapex, and iRoot SP root canal sealer.

INTRODUCTION: The objective of this study was to compare the cytotoxicity, inflammatory response, osteogenic effect, and the signaling mechanism of these biologic activities of 4 calcium compound-based root canal sealers (ie, Sealapex [Sybron Kerr, WA], apatite root sealer [ARS; Dentsply Sankin, Tokyo, Japan], MTA Fillapex [Angelus Indústria de Produtos Odontológicos S/A, Londrina, PR, Brazil], and iRoot SP [Innovative BioCreamix Inc, Vancouver, Canada]) in human periodontal ligament cells. METHODS: Cytotoxicity was assessed using the 3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide assay. Levels of inflammatory mediators were measured by enzyme-linked immunosorbent assay, reverse-transcription polymerase chain reaction, and Western blot analysis. Osteogenic potential was evaluated by alkaline phosphatase activity, alizarin red staining, and marker genes by reverse-transcription polymerase chain reaction. The signal transduction pathways were examined by Western blotting. RESULTS: None of the sealers were cytotoxic. ARS, MTA Fillapex, and iRoot SP induced a lower expression of proinflammatory mediators than Sealapex. All sealers increased ALP activity and the formation of mineralized nodules and up-regulated the expression of osteoblastic marker messenger RNA. ARS, MTA Fillapex, and iRoot SP showed superior osteogenic potential compared with Sealapex. The expression and/or activation of integrin receptors and downstream signaling molecules, including focal adhesion kinase, paxillin, Akt, mitogen-activated protein kinase, and nuclear factor κB, was induced by ARS, MTA Fillapex, and iRoot SP treatment but not by Sealapex treatment. CONCLUSIONS: We show for the first time that ARS, MTA Fillapex, and iRoot SP induce a lower expression of inflammatory mediators and enhance osteoblastic differentiation of PDLCs via the integrin-mediated signaling pathway compared with Sealapex.

Effects of glutamine on proliferation, migration, and differentiation of human dental pulp cells.

INTRODUCTION: Although glutamine (Gln) is mitogenic in various cell types, little is known about its role in human dental pulp cells (HDPCs). This study investigated the effects of Gln on proliferation, migration, and odontoblastic differentiation of HDPCs and the underlying signal pathway mechanisms. METHODS: Growth and migration were assessed by cell counting and colorimetric cell migration kits. Differentiation was measured as alkaline phosphatase activity, calcified nodule formation by alizarin red staining, and marker mRNA expression by reverse transcriptase-polymerase chain reaction (RT-PCR). Chemokine expression was also evaluated by RT-PCR. Signal transduction pathways were examined by RT-PCR and Western blot analysis. RESULTS: Gln dose-dependently increased proliferation, migration, alkaline phosphatase activity, mineralized nodule formation, and odontoblast-marker mRNA of HDPCs. Gln also up-regulated expression of interleukin-6, interleukin-8, MCP-1, MIP-3α, CCL2, CCL20, and CXCL1. Gln increased BMP-2 and BMP-4 mRNA, phosphorylation of Smad 1/5/8, ß-catenin, and key proteins of the Wnt signaling pathway. Furthermore, Gln resulted in up-regulation of extracellular signal-regulated kinase, p38, and c-Jun N-terminal kinase. In addition, noggin, DKK1, inhibitors of p38, ERK, and JNK significantly attenuatted Gln-induced growth, migration, and odontoblastic differentiation. CONCLUSIONS: Collectively, this study demonstrated that Gln promoted growth, migration, and differentiation in HDPCs through the BMP-2, Wnt, and MAPK pathways, leading to improved pulp repair and regeneration.

Effects of calcium silicate endodontic cements on biocompatibility and mineralization-inducing potentials in human dental pulp cells.

INTRODUCTION: The objective of this study was to evaluate the biocompatibility, inflammatory response, and odontoblastic potential of Biodentine (Septodont, Saint Maur des Fosses, France), Ortho-MTA (OMTA; BioMTA, Seoul, Korea), Angelus-MTA (AMTA; Angelus, Londrina, Brazil), and IRM (Dentsply Tulsa Dental, Tulsa, OK) in human dental pulp cells. The underlying signaling mechanisms were also investigated. METHODS: Biocompatibilities were examined by the 3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide assay. Differentiation was assessed by alkaline phosphatase activity, alizarin red S staining, and reverse-transcription polymerase chain reaction for marker genes. The levels of inflammatory mediators and cytokines were measured by reverse-transcription polymerase chain reaction and enzyme-linked immunosorbent assay. Signal transduction analysis was performed by Western blotting. RESULTS: Biodentine, OMTA, and AMTA showed favorable cell proliferation, alkaline phosphatase activity, formation of mineralized nodules, and expression of odontoblastic marker genes that were similar to those of IRM. The levels of proinflammatory mediators including nitric oxide, prostaglandin E2, inducible nitric oxide synthase, and cyclooxygenase-2 were lower for Biodentine, OMTA, and AMTA compared with the IRM group. All test materials induced reactive oxygen species production and the expression of hemeoxygenase-1, nuclear factor-E2-related factor-2, and mitogen-activated protein kinases. CONCLUSIONS: These data indicate for the first time that the biocompatibility, inflammatory response, and odontoblastic differentiation of Biodentine were similar to that of OMTA and AMTA in HDPCs, which suggests that Biodentine could be good alternative pulp capping agent.

Combined effects of dentin sialoprotein and bone morphogenetic protein-2 on differentiation in human cementoblasts.

The aim of this study is to determine the effects of the combination of recombinant human BMP-2 (rh-BMP-2) and dentin sialoprotein (rh-DSP) on growth and differentiation in human cementoblasts and determine the underlying signal transduction mechanism. Compared to treatment of cementoblasts with either rh-BMP-2 or rh-DSP alone, the combination of rh-BMP-2 and rh-DSP synergistically increased cell growth, ALP activity, nodule formation and expression of differentiation markers. The differentiation-promoting effect was also observed in periodontal ligament cells and an osteoblastic cell line. Likewise, combination of rh-DSP and rh-BMP-2 increased BMP-2 mRNA expression and Smad1/5/8 phosphorylation, which was blocked by the BMP antagonist noggin. The expression levels of α2ß1 integrin and RhoA, as well as the phosphorylation status of FAK and Akt, were increased by the combination of rh-BMP-2 and rh-DSP in a time-dependent manner. In addition, rh-BMP-2 and rh-DSP enhanced expression of Wnt ligands, ß-catenin activation and GSK-3ß phosphorylation, all of which were inhibited by the Wnt receptor antagonist DKK1. Furthermore, treatment with rh-DSP plus rh-BMP-2 resulted in phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 and also induced the nuclear translocation of the NF-κB p65 subunit, which was blocked by noggin. This study demonstrates for the first time that rh-DSP and rh-BMP-2 act synergistically, enhancing each other's ability to stimulate cementoblastic cell growth and differentiation in vitro via autocrine BMP, integrin, Wnt/ß-catenin, MAP kinase and NF-κB pathways. These results support the therapeutic potential of a combination strategy for aiding periodontal regeneration.

Effects of ProRoot MTA, Bioaggregate, and Micromega MTA on odontoblastic differentiation in human dental pulp cells.

INTRODUCTION: The aim of this study was to compare the biocompatibility and odontogenic potential of newly developed Bioaggregate (BA) and Micromega MTA (MMTA) with ProRoot MTA (PMTA) and intermediate restorative material (IRM) by using human dental pulp cells. METHODS: Biocompatibility was assessed by an 3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide assay and scanning electron microscopy. Differentiation was evaluated by alkaline phosphatase (ALP) activity, alizarin red staining, and reverse transcriptase-polymerase chain reaction for the maker genes. The levels of inflammatory mediators and cytokines were measured by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS: PMTA, BA, and MMTA exhibited equally good biocompatibility, whereas IRM showed cytotoxicity compared with these materials. PMTA, BA, and MMTA increased the ALP activity, promoted mineralization nodule formation, and enhanced the mRNA expression level of the osteogenic/odontogenic markers (ALP, osteopontin, osteocalcin, dentin sialophosphoprotein, and dentin matrix protein-1) compared with IRM. The levels of proinflammatory mediators and proinflammatory cytokines were lower in PMTA, BA, and MMTA compared with the IRM group. CONCLUSIONS: Collectively, the biocompatibility, odontogenic potentials, and inflammatory response of BA and MMTA are equal to those of PMTA and superior to those of IRM.