Effects of moderate intensity static magnetic fields on osteoclastic differentiation in mouse bone marrow cells.

Although we recently demonstrated that static magnetic fields (SMFs) of 3, 15, and 50 mT stimulate osteoblastic differentiation, the effects of SMFs on osteoclastogenesis are still poorly understood. This study focused on the suppressive effects of SMFs on receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis and bone resorption. Direct SMFs inhibit RANKL-induced multinucleated osteoclast formation, tartrate-resistant acid phosphatase activity, and bone resorption in mouse bone marrow-derived macrophage cells. The conditioned medium from osteoblasts treated with SMFs also resulted in the inhibition of osteoclast differentiation as well as resorption. The RANKL-induced expression of osteoclast-specific transcription factors, such as c-Fos and NFATc1, was remarkably downregulated by SMF at 15 mT. In addition, SMF inhibited RANKL-activated Akt, glycogen synthase kinase 3ß (GSK3ß), extracellular signal-regulated kinase, c-jun N-terminal protein kinase, mitogen-activated protein kinase (MAPK), and nuclear factor-κB (NF-κB) formation. These findings indicate that SMF-mediated attenuation of RANKL-induced Akt, GSK3ß, MAPK, and NF-κB pathways could contribute to the direct and indirect inhibition of osteoclast formation and bone resorption. Therefore, SMFs could be developed as a therapeutic agent against periprosthetic or peri-implant osteolysis. Additionally, these could be used against osteolytic diseases such as osteoporosis and rheumatoid arthritis. Bioelectromagnetics. 39:394-404, 2018. © 2018 Wiley Periodicals, Inc.

Bisphosphonates hinder osteoblastic/osteoclastic differentiation in the maxillary sinus mucosa-derived stem cells.

OBJECTIVES: Although bisphosphonates (BPs) are known to be associated with osteonecrosis of the maxilla, the precise effects of BPs on bone metabolism in human maxillary sinus mucosal cells (HMSMCs) are not yet known. The purposes of this study were to examine the effects of the BPs zoledronate (ZOL) and alendronate (ALN) on osteoblastic and osteoclastic differentiation in HMSMCs and to investigate the signaling pathways involved. MATERIALS AND METHODS: The effects of ZOL and ALN were assessed for osteoblast differentiation by alkaline phosphatase (ALP) activity, alizarin red staining, and RT-PCR for genes encoding Runx2 and osterix. Receptor activator of nuclear factor-κB ligand (RANKL)-mediated osteoclast differentiation in bone marrow macrophages (BMMs) was also examined. RESULTS: ZOL and ALN both suppressed osteoblastic differentiation, as evidenced by their effects on ALP activity, mineralization nodule formation, and the mRNA expression levels of osteoblastic transcript factors. The RANKL/osteoprotegerin ratio in HMSMCs was increased by ALN, whereas ZOL had the opposite effect. Conditioned medium obtained from ALN-treated HMSMCs stimulated osteoclast formation and upregulated NFATc1 expression, whereas conditioned medium from ZOL-treated cells did not. ALN was more cytotoxic and stimulated apoptosis more strongly than ZOL. BPs decreased the protein levels of the non-canonical Wnt signaling protein Wnt5a and calmodulin-dependent kinase II. Moreover, recombinant human Wnt5a reversed the effects of BPs on osteoblastic and osteoclastic differentiation. CONCLUSION: This study is the first demonstration that BPs exert negative effects on osteoblastic and osteoclastic processes via the non-canonical Wnt pathway in HMSMSCs. CLINICAL RELEVANCE: It suggests that patients taking BPs during the period of maxillary sinus lifting and amentation should be given special attention.

Effects of Melatonin and Its Underlying Mechanism on Ethanol-Stimulated Senescence and Osteoclastic Differentiation in Human Periodontal Ligament Cells and Cementoblasts.

The present study evaluated the protective effects of melatonin in ethanol (EtOH)-induced senescence and osteoclastic differentiation in human periodontal ligament cells (HPDLCs) and cementoblasts and the underlying mechanism. EtOH increased senescence activity, levels of reactive oxygen species (ROS) and the expression of cell cycle regulators (p53, p21 and p16) and senescence-associated secretory phenotype (SASP) genes (interleukin [IL]-1ß, IL-6, IL-8 and tumor necrosis factor-α) in HPDLCs and cementoblasts. Melatonin inhibited EtOH-induced senescence and the production of ROS as well as the increased expression of cell cycle regulators and SASP genes. However, it recovered EtOH-suppressed osteoblastic/cementoblastic differentiation, as evidenced by alkaline phosphatase activity, alizarin staining and mRNA expression levels of Runt-related transcription factor 2 (Runx2) and osteoblastic and cementoblastic markers (glucose transporter 1 and cementum-derived protein-32) in HPDLCs and cementoblasts. Moreover, it inhibited EtOH-induced osteoclastic differentiation in mouse bone marrow⁻derived macrophages (BMMs). Inhibition of protein never in mitosis gene A interacting-1 (PIN1) by juglone or small interfering RNA reversed the effects of melatonin on EtOH-mediated senescence as well as osteoblastic and osteoclastic differentiation. Melatonin blocked EtOH-induced activation of mammalian target of rapamycin (mTOR), AMP-activated protein kinase (AMPK), mitogen-activated protein kinase (MAPK) and Nuclear factor of activated T-cells (NFAT) c-1 pathways, which was reversed by inhibition of PIN1. This is the first study to show the protective effects of melatonin on senescence-like phenotypes and osteoclastic differentiation induced by oxidative stress in HPDLCs and cementoblasts through the PIN1 pathway.

N-methyl-D-aspartate (NMDA) impairs myogenesis in C2C12 cells.

INTRODUCTION: N-methyl-d-aspartate (NMDA) is expressed in sensory neurons and plays important roles in peripheral pain mechanisms. The aim of this study was to examine the effects and molecular mechanisms of NMDA on C2C12 myoblast proliferation and differentiation. METHODS: Cytotoxicity and differentiation were examined by the MTT assay, reverse transcription-polymerase chain reaction, and immunofluorescence. RESULTS: NMDA had no cytotoxicity (10-500 µM) and inhibited myoblastic differentiation of C2C12 cells, as assessed by F-actin immunofluorescence and levels of mRNAs encoding myogenic markers such as myogenin and myosin heavy-chain 2. It inhibited phosphorylation of mammalian target of rapamycin (mTOR) by inactivating mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38. It induced reactive oxygen species production. Furthermore, NMDA-suppressed expression of F-actin was reversed by adding the antioxidant N-acetylcysteine. CONCLUSIONS: Collectively, these results indicate that NMDA impairs myogenesis or myogenic differentiation in C2C12 cells through the mTOR/MAPK signaling pathways and may lead to skeletal muscle degeneration. Muscle Nerve 56: 510-518, 2017.

Effects of static magnetic fields on bone regeneration of implants in the rabbit: micro-CT, histologic, microarray, and real-time PCR analyses.

OBJECTIVES: The aim of this study was to investigate the effects of static magnetic fields (SMFs) on bone regeneration around titanium implants by µCT, histologic analysis, microarrays, and quantitative real-time PCR (qRT-PCR). MATERIALS AND METHODS: Neodymium magnets provided the source of SMFs, the specimens were grade 5 titanium implants, and the animals were twenty-seven adult male New Zealand white rabbits. These implants were divided into six groups according to the presence of a magnet and predetermined healing period (1, 4, and 8 weeks). Each group comprised six specimens for µCT (n = 6) and histologic examination, and three specimens (n = 3) for microarrays and qRT-PCR, yielding a total of 54 specimens. RESULTS: The µCT data showed that SMFs increased bone volume fraction (bone volume/total volume, BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th). Histologic observation indicated that SMFs promoted new bone formation and direct bony contact with implants. Microarray analysis identified 293 genes upregulated (>twofold) in response to SMFs. The upregulated genes included extracellular matrix (ECM)-related genes (COL10A1, COL9A1, and COL12A1) and growth factor (GF)-related genes (CTGF and PDGFD), and the upregulation was confirmed by qRT-PCR. Gene Ontology (GO) and pathway analysis revealed the involvement of the mitogen-activated protein kinase (MAPK), Wnt, and PPAR-gamma signaling pathways in implant healing. CONCLUSIONS: µCT, histology, microarrays, and real-time PCR indicate that SMFs could be an effective approach to improving bone regeneration around dental implants.

Dual Effect of Curcumin/BMP-2 Loaded in HA/PLL Hydrogels on Osteogenesis In Vitro and In Vivo.

In the present study, we evaluated the potential of poly-l-lysine/hyaluronic acid (HA/PLL) hydrogels containing curcumin (CUR) and bone morphogenetic protein-2 (BMP-2) as bone tissue regeneration scaffolds. Hydrogels HP-1˜2 were formed by amide bonds via the condensation reactions between 0.02 µmol HA and 0.06­0.12 µmol poly-l-lysine · hydrobromide (PLL · HBr). Physical, chemical, and thermal analyses revealed that the amount of PLL · HBr significantly influenced hydrogel properties. Based on an In Vitro MG-63 cell proliferation test, HP-1˜2 were cytocompatible, and all hydrogels containing different amounts of CUR and BMP-2, except for HA0.02/PLL0.06/CUR20/BMP-2100 (HPCB-4), resulted in cell proliferation above 80%. An In Vitro release test showed that CUR and BMP-2 were consistently released from HA0.02/PLL0.06/CUR15 (HPC), HA0.02/PLL0.06/BMP-2100 (HPB), HA0.02/PLL0.06/CUR15/BMP-210 , 50 , or 100 (HPCB-1˜3), and HA0.02/PLL0.06/CUR10 or 20/BMP-2100 (HPCB-4˜5) for 7 and 28 days, respectively. In Vitro ALP activity and calcium deposition and In Vivo micro-computed tomography (micro-CT) tests demonstrated the potential application of HPCB-3 as bone tissue regeneration scaffolds, suggesting that bone tissue regeneration can be optimized by controlling the amounts of CUR and BMP-2.

Supramolecular Hydrogels Based on MPEG-Grafted Hyaluronic Acid and α-CD Containing HP-ß-CD/Simvastatin Enhance Osteogenesis In Vivo.

Simvastatin (SIM) accelerates new bone formation both in vitro and In Vivo by enhancing the expression of recombinant human bone morphogenetic protein-2 (rhBMP-2). In this study, we evaluated the effect of water-solubility of SIM on new bone formation by preparing two types of supramolecular hydrogels: pseudopolyrotaxanes (PPRXs) based on metoxy polyethyleneglycol-grafted hyaluronic acid (MPEG-g-HA) and α-cyclodextrin (α-CD) containing water-soluble hydroxypropyl ß-cyclodextrin/simvastatin inclusion complex (HP-ß-CD-ic-SIM; MPEG-g-HA/α-CD/HP-ß-CD-ic-SIM) or only SIM (MPEG-g-HA/α-CD/SIM). As compared to MPEG-g- HA/α-CD/SIM, SIM was more rapidly released from MPEG-g-HA/α-CD/HP-ß-CD-ic-SIM in a sustained manner owing to increased water-solubility. New bone actively formed at the calvarial defect site in a rabbit model 4 weeks after implantation, as examined by micro computed tomography (micro CT), hematoxylin and eosin (H&E) staining, and Goldner's trichrome staining. The results showed that the water-solubility of SIM plays a significant role in enhancing new bone formation in vivo.

In Vitro Osteogenic Differentiation Enhanced by Zirconia Coated with Bone Morphogenetic Protein-2.

In this study we report on the effectiveness of click chemistry-enhanced zirconium dioxide (ZrO2-3) for the immobilization of biomolecules, and the enhancement of osteoblastic differentiation of MC3T3-E1 cells by bone morphogenetic protein-2 (BMP-2) immobilized on ZrO2-6. The surfaces of ZrO2-1 through 6 were characterized by scanning electron microscopy (SEM), static contact angles, and X-ray photoelectron spectroscopy (XPS) measurements. The results from these tests indicated that ZrO2-1 was successfully surface-modified via click chemistry (ZrO2-3). Through quantitative analysis of heparin immobilized on ZrO2-5, we found that ZrO2-3 was a useful tool for immobilizing biomolecules such as heparin. Release tests of BMP-2 from ZrO2-6 showed well-controlled release kinetics over a period of 28 days. MC3T3-E1 cell proliferation tests indicated that ZrO2-6 was highly biocompatible with these cells. Through In Vitro tests such as alkaline phosphatase (ALP) activity, calcium deposition, and real-time polymerase chain reaction (real-time PCR), we found that ZrO2-6 was a useful tool for enhancing osteoblastic differentiation of MC3T3-E1 cells.

Effects of sodium tri- and hexameta-phosphate in vitro osteoblastic differentiation in Periodontal Ligament and Osteoblasts, and in vivo bone regeneration.

The present study was designed to assess the effects and underlying mechanism of two poly(P) compounds, sodium triphosphate (STP, Na5P3O10) and sodium hexametaphosphate (SHMP, Na15P13O40~Na20P18O40) on osteoblastic differentiation of human periodontal ligament cells (PDLCs) and osteoblasts in vitro, and bone formation in vivo. Differentiation was assessed by alkaline phosphatase (ALP) activity, mineralization, and mRNA expression for marker genes. To examine the osteogenic potential to regenerate bone, the critical-sized mouse calvarial defect model was utilized. Incubation of PDLCs and osteoblasts with STP and SHMP resulted in a dose- and time-dependent increase in growth, alkaline phosphatase (ALP) activity, mineralization and mRNA expression for marker genes. STP and SHMP increased phosphorylation of adenosine monophosphate-activated protein kinase (AMPK), Akt, and mammalian target of rapamycin (mTOR), and mitogen-activated protein kinases (MAPK). Treatment with the mTOR inhibitor, rapamycin, attenuatted STP- and SHMP-induced osteoblastic differentiation. Micro-CT and histologic analysis showed that STP significantly increased new bone formation in calvarial defects, compared with SHMP and control group. Collectively, this is the first study to demonstrate that STP and SHMP promotes the osteoblastic differentiation in vitro, whereas STP only stimulated bone repair in vivo. Therefore, STP may be useful therapeutic approach for the regeneration of bone or periodontal tissue.

Sonic Hedgehog Promotes Cementoblastic Differentiation via Activating the BMP Pathways.

Although sonic hedgehog (SHH), an essential molecule in embryogenesis and organogenesis, stimulates proliferation of human periodontal ligament (PDL) stem cells, the effects of recombinant human SHH (rh-SHH) on osteoblastic differentiation are unclear. To reveal the role of SHH in periodontal regeneration, expression of SHH in mouse periodontal tissues and its effects on the osteoblastic/cementoblastic differentiation in human cementoblasts were investigated. SHH is immunolocalized to differentiating cementoblasts, PDL cells, and osteoblasts of the developing mouse periodontium. Addition of rh-SHH increased cell growth, ALP activity, and mineralization nodule formation, and upregulated mRNA expression of osteoblastic and cementoblastic markers. The osteoblastic/cementoblastic differentiation of rh-SHH was abolished by the SHH inhibitor cyclopamine (Cy) and the BMP antagonist noggin. rh-SHH increased the expression of BMP-2 and -4 mRNA, as well as levels of phosphorylated Akt, ERK, p38, and JNK, and of MAPK and NF-κB activation, which were reversed by noggin, Cy, and BMP-2 siRNA. Collectively, this study is the first to demonstrate that SHH can promote cell growth and cell osteoblastic/cementoblastic differentiation via BMP pathway. Thus, SHH plays important roles in the development of periodontal tissue, and might represent a new therapeutic target for periodontitis and periodontal regeneration.

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.

In Vitro Osteogenic Differentiation Enhanced by Zirconia Coated with Nano-Layered Growth and Differentiation Factor-5.

Zirconia (Zr) is also known as a biocompatible material with favorable mechanical properties as well as low plaque adhesion. In this study, we examined the efficacy of Zr coated with growth and differentiation factor-5 (GDF-5) bonded via click reaction as a substrate to support osteogenic differentiation of MC3T3-E1 cells. Pristine and surface-modified Zr surfaces were characterized by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS), resulting that GDF-5 was successfully coated to the pristine Zr surface. GDF-5 coated to Zr surfaces was released for 28 days in a sustained manner. New bone formation onto GDF-5 coated Zr (Zr/GDF-5) surface was confirmed by in vitro test including cell proliferation, alkaline phosphatase activity and calcium deposition assays, and in vivo test including real-time polymerase chain reaction (qPCR) assay including osterix (OSX), runt-related transcription factor 2 (Runx 2), COL 1 (type I collagen) and osteocalcin (OC). Cell proliferation, alkaline phosphatase activity, and calcium deposition of MC3T3- E1 cells were significantly enhanced when the cells were cultured on Zr/GDF-5. Additionally, the results of qPCR revealed that genes related with osteogenic differentiation were up regulated when the cells were cultured on Zr/GDF-5. Our findings demonstrate that Zr/GDF-5 could be used as a material for enhancing the efficacy of osteogenic differentiation.

Expression of thymosin beta-4 in human periodontal ligament cells and mouse periodontal tissue and its role in osteoblastic/cementoblastic differentiation.

A recent report showed that thymosin beta-4 (Tß4) is expressed during the development of tooth germ, but its effect on osteoblastic/cementoblastic differentiation is a controversial topic. Furthermore, the precise expression and function of Tß4 in periodontal tissue remains unclear. Therefore, the purpose of this study was to investigate the immunolocalization of Tß4 in the developing periodontium of mouse, the function of Tß4 in osteoblastic/cementoblastic differentiation, and the underlying mechanism regulating periodontal regeneration in human periodontal ligament cells (hPDLCs), cementoblasts, and osteoblasts. Tß4 expression was observed in differentiating hPDLCs, osteoblasts of the periodontium during development, as well as in mature tissue. Higher Tß4 expression was observed in hPDLCs than in cementoblasts and osteoblasts in the developing periodontium. The expression of Tß4 mRNA and protein gradually increased during PDL cell differentiation. The downregulation of Tß4 expression by Tß4 siRNA transfection inhibited osteoblastic differentiation by decreasing calcium nodule formation, alkaline phosphatase (ALP) activity, and mRNA expression of differentiation markers in hPDLCs, cementoblasts, and osteoblasts. In contrast, Tß4 activation using a Tß4 peptide, promoted these processes by activation of Akt, p38, ERK MAPKs, and the NF-κB pathway. The expression of nuclear NFATc1 was upregulated by Tß4 peptide in hPDLCs. Inhibition of the calcineurin/NFATc1 pathway by cyclosporin A and FK506, attenuated Tß4-induced osteoblastic differentiation and activation of Wnt-related genes, as well as nuclear ß-catenin in hPDLCs. In conclusion, this study demonstrates, for the first time, that Tß4 is expressed in developing periodontal tissue and that its expression is associated with osteoblastic/cementoblastic differentiation. These results suggests that Tß4 is a potential therapeutic target for periodontal regeneration or bone disease.

Three-dimensional evaluation of the pharyngeal airway using cone-beam computed tomography following bimaxillary orthognathic surgery in skeletal class III patients.

OBJECTIVE: The study aims to evaluate the pharyngeal airway space (PAS) following bimaxillary surgery in skeletal class III patients and to compare the changes in PAS between genders using cone-beam computed tomography (CBCT). MATERIALS AND METHODS: In all, 38 patients (16 male and 22 female) with skeletal class III malocclusion underwent bimaxillary surgery. CBCT scans were acquired approximately 1 month before surgery, 3 months after surgery, and 6 months after surgery. The oropharyngeal volume and the minimum cross-sectional area (CSA) were characterized using the InVivoDental imaging software package at each time point. RESULTS: The volume and minimum CSA decreased significantly postoperatively, which was maintained until 6 months postoperatively (p < 0.01). The location of the minimum CSA tended to move into the retropalatal and retroglossal areas postoperatively. A strong correlation between volume and minimum CSA was found. The amount of mandibular setback was not correlated with the change in the airway. By gender, significant decreases in both the volume and minimum CSA were found in females (p < 0.05) but not in males. CONCLUSION: Bimaxillary surgery significantly affects PAS. Gender differences should also be considered when considering changes in PAS. CLINICAL RELEVANCE: An awareness of the effects of bimaxillary setback surgery on the airway should be considered when implementing an orthognathic treatment plan.

Combined effects of mineral trioxide aggregate and human placental extract on rat pulp tissue and growth, differentiation and angiogenesis in human dental pulp cells.

OBJECTIVE: The aim of this study was to evaluate the combined effects of mineral trioxide aggregate (MTA) and human placental extract (HPE) on cell growth, differentiation and in vitro angiogenesis of human dental pulp cells (HDPCs) and to identify underlying signal transduction mechanisms. In vivo dental pulp responses in rats for a pulp-capping agent were examined. MATERIALS AND METHODS: MTS assay. ALP activity test, alizarin red S staining and RT-PCR for marker genes were carried out to evaluate cell growth and differentiation. HUVEC migration, mRNA expression and capillary tube formation were measured to evaluate angiogenesis. Signal transduction was analysed using Western blotting and confocal microscopy. The pulps of rat maxillary first molars were exposed and capped with either MTA or MTA plus HPE. Histologic observation and scoring were performed. RESULTS: Compared to treatment of HDPCs with either HPE or MTA alone, the combination of HPE and MTA increased cell growth, ALP activity, mineralized nodules and expression of marker mRNAs. Combination HPE and MTA increased migration, capillary tube formation and angiogenic gene expression compared with MTA alone. Activation of Akt, mammalian target of rapamycin (mTOR), p38, JNK and ERK MAPK, Akt, and NF-κB were significantly increased by combining HPE and MTA compared with MTA alone. Pulp capping with MTA plus HPE in rats showed superior dentin bridge formation, odontoblastic layers and dentinal tubules and lower inflammatory cell response, compared to the MTA alone group. CONCLUSIONS: This study demonstrates for the first time that the use of MTA with HPE promotes cell growth, differentiation and angiogenesis in HDPCs, which were associated with mTOR, MAPK and NF-κB pathways. Direct pulp capping with HPE plus MTA showed superior results when compared with MTA alone. Thus, the combination of MTA and HPE may be useful for regenerative endodontics.

Expression of inflammatory cytokines and MMPs on replanted teeth at different extra-alveolar time: an ex vivo and in vivo study.

BACKGROUND: Immediately after the avulsed tooth is replanted, a complex inflammatory response ensues. As part of the periodontium healing process, the extracellular matrix macromolecules are essential to create the cellular environment required during healing and morphogenesis. AIM: This study was designed to evaluate the correlation between different extra-alveolar dry times and inflammatory cytokines and matrix metalloproteinases (MMPs) as part of the periodontal ligament (PDL) gene expression. DESIGN: The first phase of the study aimed testing human PDL cells ex vivo. Extracted teeth were dried for 15 and 30 min. The PDL cells were extracted and analyzed by qRT-PCR. The second phase was performed in vivo, and 36 Sprague Dawley rat first maxillary molars were extracted and replanted after 15, 30, and 60 min extra-alveolar time. We tested the levels of inflammatory cytokines and MMPS in periodontal tissue at 3, 7, and 28 days after tooth replantation. The replanted area was dissected, grounded, and analyzed by RT-PCR. RESULTS: Expressions of IL-1ß, IL-6, TNF-α, and MMP-3 and MMP-9 were significantly higher in the replanted teeth. Extended dry time had a direct correlation with induction of pro-inflammatory cytokine and MMPs in PDL cells. CONCLUSION: Our study showed that pro-inflammatory cytokines were more significantly expressed in the tissues surrounding the replanted teeth. Future research must be undertaken to additionally confirm the release of these cytokines and be focused on the inhibition of these cytokines to reduce inflammation of replanted teeth.

HIF-2 Inhibition Supresses Inflammatory Responses and Osteoclastic Differentiation in Human Periodontal Ligament Cells.

Recent reports suggest that hypoxia inducible factor-2α (HIF-2α) is a key regulator of osteoarthritis cartilage destruction. However, the precise role of HIF-2α in the inflammatory response and osteoclast differentiation remains unclear. The purpose of this study was to investigate the effect of HIF-2α on inflammatory cytokines, extracellular matrix (ECM) destruction enzymes, and osteoclastic differentiation in nicotine and lipopolysaccharide (LPS)-stimulated human periodontal ligament cells (PDLCs). HIF-2α was upregulated in chronically inflamed PDLCs of periodontitis patients, and in nicotine- and LPS-exposed PDLC in dose- and time-dependent manners. HIF-2α inhibitor and HIF-2α siRNA attenuated the nicotine- and LPS- induced production of NO and PGE2 , upregulation of iNOS, COX-2, pro-inflammatory cytokines (IL-1ß, TNF-α, IL-1ß, IL-6, IL-8, IL-10, IL-11, and IL-17), and matrix metalloproteinases (MMPs; MMP-1, -8, -13, -2 and -9), and reversed the effect on TIMPs (TIMP-1 and -2) in PDLCs. The conditioned medium produced by nicotine and LPS-treated PDLCs increased the number of TRAP-stained osteoclasts, TRAP activity and osteoclast-specific genes, which has been blocked by HIF-2α inhibition and silencing. HIF-2α inhibitor and HIF-2α siRNA inhibited the effects of nicotine and LPS on the activation of Akt, JAK2 and STAT3, ERK and JNK MAPK, nuclear factor-κB, c-Jun, and c-Fos. Taken together, this study is the first to demonstrate that HIF-2α inhibition exhibits anti-inflammatory activity through the inhibition of inflammatory cytokines and impairment of ECM destruction, as well as blocking of osteoclastic differentiation in a nicotine- and periodontopathogen-stimulated PDLCs model. Thus, HIF-2α inhibition may be a novel molecular target for therapeutic approaches in periodontitis.

Comparison of the effects of human dental pulp stem cells and human bone marrow-derived mesenchymal stem cells on ischemic human astrocytes in vitro.

This study assesses the cytoprotective effects of human dental pulp stem cells (hDPSCs) and conditioned medium from hDPSCs (CM-hDPSCs) on ischemic human astrocytes (hAs) in vitro compared with human bone marrow-derived mesenchymal stem cells (hMSCs). Ischemia of hAs was induced by oxygen-glucose deprivation (OGD). CM-hDPSCs and hMSCs were collected after 48 hr of culture. Cell death was determined by 3-[4,5-dimethylthialzol-2-yl]-2,5-diphenyltetrazolium bromide and cellular ATP assays. The expression of glial fibrillary acidic protein (GFAP) and musashi-1 as markers of reactive astrogliosis was examined with immunochemical staining. mRNA expression and reactive oxygen species (ROS) were analyzed by RT-PCR and flow cytometry, respectively. OGD increased cytotoxicity in a time-dependent manner and decreased cellular ATP content concomitantly in hAs. Pretreatment and posttreatment with hDPSCs were associated with greater recovery from OGD-induced cytotoxicity in hAs compared with hMSCs. Similarly, CM-hDPSCs had a greater effect on OGD-induced cytotoxicity in a dose-dependent manner. Pre- and posttreatment with CM-hDPSCs or CM-hMSCs attenuated OGD-induced GFAP, nestin, and musashi-1 expression in hAs. Furthermore, treatment of cells with CM-hDPSCs and hMSCs blocked OGD-induced ROS production and interleukin-1ß upregulation. This study demonstrates for the first time that hDPSCs and CM-hDPSCs confer superior cytoprotection against cell death in an in vitro OGD model compared with hMSCs as shown by cell viability assay. Reactive gliosis, ROS production, and inflammatory mediators might contribute to this protective effect. Therefore, hDPSCs could represent an alternative source of cell therapy for ischemic stroke.

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.

Effects of moderate intensity static magnetic fields on human bone marrow-derived mesenchymal stem cells.

This study aimed to explore effects of static magnetic fields (SMFs) of moderate intensity (3-50 mT) as biophysical stimulators of proliferation and osteoblastic differentiation of human bone marrow-derived mesenchymal stem cells (MSCs). MSCs were exposed to SMFs of three intensities: 3, 15, and 50 mT. Proliferation was assessed by cell counting and bromodeoxyuridine incorporation, and differentiation by measuring alkaline phosphatase (ALP) activity, calcium content, mineralized nodule formation, and transcripts of osteogenic markers. Moderate intensity SMFs increased cell proliferation, ALP activity, calcium release, and mineralized nodule formation in a dose- and time-dependent manner, which peaked at 15 mT. In the same manner, they upregulated expression of osteogenic marker genes such as ALP, bone sialoprotein 2 (BSP2), collagen1a1 (COL1a1), osteocalcin (OCN), osteonectin (ON), osteopontin (OPN), osterix (OSX), and runt-related transcription factor 2 (RUNX2) with peak at 15 mT after 14 or 21 days of exposure. Results demonstrate that moderate intensity SMFs promote proliferation and osteoblastic differentiation of MSCs. This effect could help to improve MSC responses during osseointegration between a dental implant and surrounding bone.