Inverse genetics tracing the differentiation pathway of human chondrocytes.

OBJECTIVE: Mammalian somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) via the forced expression of Yamanaka reprogramming factors. However, only a limited population of the cells that pass through a particular pathway can metamorphose into iPSCs, while the others do not. This study aimed to clarify the pathways that chondrocytes follow during the reprogramming process. DESIGN: The fate of human articular chondrocytes under reprogramming was investigated through a time-coursed single-cell transcriptomic analysis, which we termed an inverse genetic approach. The iPS interference technique was also employed to verify that chondrocytes inversely return to pluripotency following the proper differentiation pathway. RESULTS: We confirmed that human chondrocytes could be converted into cells with an iPSC phenotype. Moreover, it was clarified that a limited population that underwent the silencing of SOX9, a master gene for chondrogenesis, at a specific point during the proper transcriptome transition pathway, could eventually become iPSCs. Interestingly, the other cells, which failed to be reprogrammed, followed a distinct pathway toward cells with a surface zone chondrocyte phenotype. The critical involvement of cellular communication network factors (CCNs) in this process was indicated. The idea that chondrocytes, when reprogrammed into iPSCs, follow the differentiation pathway backward was supported by the successful iPS interference using SOX9. CONCLUSIONS: This inverse genetic strategy may be useful for seeking candidates for the master genes for the differentiation of various somatic cells. The utility of CCNs in articular cartilage regeneration is also supported.

Millimeter-wave interferometry and far-forward scattering for density fluctuation measurements on LTX-ß.

The λ ≈ 1 mm (f = 288 GHz) interferometer for the Lithium Tokamak Experiment-ß (LTX-ß) will use a chirped-frequency source and a centerstack-mounted retro-reflector mirror to provide electron line density measurements along a single radial chord at the midplane. The interferometer is unique in the use of a single source (narrow-band chirped-frequency interferometry) and a single beam splitter for separating and recombining the probe and reference beams. The current work provides a documentation of the interferometry hardware and evaluates the capabilities of the system as a far-forward collective scattering diagnostic. As such, the current optical setup is estimated to have a detection range of 0.4 ≲ k ⊥ ≲ 1.7 cm-1, while an improved layout will extend the upper k ⊥ limit to ∼3 cm-1. Measurements with the diagnostic on LTX are presented, showing interferometry results and scattered signal data. These diagnostics are expected to provide routine measurements on LTX-ß for high frequency coherent density oscillations (e.g., Alfvénic modes during neutral beam injection) as well as for broadband turbulence.

Observation of Flat Electron Temperature Profiles in the Lithium Tokamak Experiment.

It has been predicted for over a decade that low-recycling plasma-facing components in fusion devices would allow high edge temperatures and flat or nearly flat temperature profiles. In recent experiments with lithium wall coatings in the Lithium Tokamak Experiment (LTX), a hot edge (>200 eV) and flat electron temperature profiles have been measured following the termination of external fueling. Reduced recycling was demonstrated by retention of ∼60% of the injected hydrogen in the walls following the discharge. Electron energy confinement followed typical Ohmic confinement scaling during fueling, but did not decrease with density after fueling terminated, ultimately exceeding the scaling by ∼200%. Achievement of the low-recycling, hot edge regime has been an important goal of LTX and lithium plasma-facing component research in general, as it has potentially significant implications for the operation, design, and cost of fusion devices.

A frequency-modulated continuous-wave reflectometer for the Lithium Tokamak Experiment.

The frequency-modulated continuous-wave reflectometer on LTX (Lithium Tokamak Experiment) and the data analysis methods used for determining electron density profiles are described. The diagnostic uses a frequency range of 13.1-33.5 GHz, for covering a density range of 0.21-1.4×1013 cm-3 (in O-mode polarization) with a time resolution down to 8 µs. The design of the diagnostic incorporates the concept of an "optimized" source frequency sweep, which minimizes the large variation in the intermediate frequency signal due to a long dispersive transmission line. The quality of the raw data is dictated by the tuning characteristics of the microwave sources, as well as the group delay ripple in the transmission lines, which can generate higher-order nonlinearities in the frequency sweep. Both effects are evaluated for our diagnostic and best practices are presented for minimizing "artifacts" generated in the signals. The quality of the reconstructed profiles is also improved using two additional data analysis methods. First, the reflectometer data are processed as a radar image, where clutter due to echoes from the wall and backscattering from density fluctuations can be easily identified and removed. Second, a weighed least-squares lamination algorithm POLAN (POLynomial ANalysis) is used to reconstruct the electron density profile. Examples of density profiles in LTX are presented, along with comparisons to measurements from the Thomson scattering and the λ = 1 mm interferometer diagnostics.

Low-intensity pulsed ultrasound (LIPUS) treatment of cultured chondrocytes stimulates production of CCN family protein 2 (CCN2), a protein involved in the regeneration of articular cartilage: mechanism underlying this stimulation.

OBJECTIVE: CCN family protein 2/connective tissue growth factor (CCN2/CTGF) promotes cartilage regeneration in experimental osteoarthritis (OA) models. However, CCN2 production is very low in articular cartilage. The aim of this study was to investigate whether or not CCN2 was promoted by cultured chondrocytes treated with low-intensity pulsed ultrasound (LIPUS) and to clarify its mechanism. METHODS: Human chondrocytic cell line (HCS)-2/8, rat primary epiphyseal and articular cartilage cells, and Ccn2-deficient chondrocytes that impaired chondrocyte differentiation, were treated with LIPUS for 20 min at 3.0 MHz frequency and 60 mW/cm2 power. Expressions of chondrocyte differentiation marker mRNAs were examined by real-time PCR (RT-PCR) analysis from HCS-2/8 cells and Ccn2-deficient chondrocytes at 30 min and 1 h after LIPUS treatment, respectively. CCN2 production was examined by Western blotting after 5 h of LIPUS treatment. Moreover, Ca2+ influx was measured by using a Fluo-4 probe. RESULTS: The gene expression of chondrocyte differentiation markers and CCN2 production were increased in cultured chondrocytes treated with LIPUS. In addition, Ca2+ influx and phosphorylation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK)1/2 were increased by LIPUS treatment, and the stability of TRPV4 and BKca channel mRNAs was decreased by siRNA against CCN2. Consistent with those findings, the LIPUS-induced the gene expressions of type II collagen (COL2a1) and Aggrecan (ACAN) observed in wild-type cells were not observed in the Ccn2-deficient chondrocytes. CONCLUSION: These data indicate that chondrocyte differentiation represented by CCN2 production was mediated via MAPK pathways activated by LIPUS-stimulated Ca2+ influx, which in turn was supported by the induced CCN2 molecules in articular chondrocytes.

Characterisation of circadian rhythms of various duckweeds.

The plant circadian clock controls various physiological phenomena that are important for adaptation to natural day-night cycles. Many components of the circadian clock have been identified in Arabidopsis thaliana, the model plant for molecular genetic studies. Recent studies revealed evolutionary conservation of clock components in green plants. Homologues of clock-related genes have been isolated from Lemna gibba and Lemna aequinoctialis, and it has been demonstrated that these homologues function in the clock system in a manner similar to their functioning in Arabidopsis. While clock components are widely conserved, circadian phenomena display diversity even within the Lemna genus. In order to survey the full extent of diversity in circadian rhythms among duckweed plants, we characterised the circadian rhythms of duckweed by employing a semi-transient bioluminescent reporter system. Using a particle bombardment method, circadian bioluminescent reporters were introduced into nine strains representing five duckweed species: Spirodela polyrhiza, Landoltia punctata, Lemna gibba, L. aequinoctialis and Wolffia columbiana. We then monitored luciferase (luc+) reporter activities driven by AtCCA1, ZmUBQ1 or CaMV35S promoters under entrainment and free-running conditions. Under entrainment, AtCCA1::luc+ showed similar diurnal rhythms in all strains. This suggests that the mechanism of biological timing under day-night cycles is conserved throughout the evolution of duckweeds. Under free-running conditions, we observed circadian rhythms of AtCCA1::luc+, ZmUBQ1::luc+ and CaMV35S::luc+. These circadian rhythms showed diversity in period length and sustainability, suggesting that circadian clock mechanisms are somewhat diversified among duckweeds.

Alternative optical concept for electron cyclotron emission imaging.

The implementation of advanced electron cyclotron emission imaging (ECEI) systems on tokamak experiments has revolutionized the diagnosis of magnetohydrodynamic (MHD) activities and improved our understanding of instabilities, which lead to disruptions. It is therefore desirable to have an ECEI system on the ITER tokamak. However, the large size of optical components in presently used ECEI systems have, up to now, precluded the implementation of an ECEI system on ITER. This paper describes a new optical ECEI concept that employs a single spherical mirror as the only optical component and exploits the astigmatism of such a mirror to produce an image with one-dimensional spatial resolution on the detector. Since this alternative approach would only require a thin slit as the viewing port to the plasma, it would make the implementation of an ECEI system on ITER feasible. The results obtained from proof-of-principle experiments with a 125 GHz microwave system are presented.

Heart donation in Japan before and after the revision of the Japanese Transplantation Act.

INTRODUCTION: After the revision of the Organ Transplant Act in July 2010, brain dead organ donation increased from 13 to 45 per year, and heart donation increased. The purpose of this study was to review 166 consecutive brain dead heart donors to evaluate our strategies to identify and manage organ donors. METHODS: This study reviewed 166 consecutive brain dead heart donors since the Act was issued. Whereas 69 heart donations were performed between October 1997 and July 2010 before the revision of the Act, 97 heart donations were performed for the 3 years after the revision. Since November 2002, special transplant management doctors were sent to donor hospitals to assess donor organ function and to identify which organs could be transplanted. They also intensively cared for the donors to stabilize hemodynamics and to improve cardiac function by giving intravenous antidiuretic hormones and by pulmonary toileting via bronchofiberscope. RESULTS: The mean heart donor age increased from 41.0 to 43.9 years after the revision. Notably, 11 hearts from donors more than 60 years old were transplanted successfully after the revision. Before the revision, the cause of death was 37 cerebrovascular disease (SAH 34, stroke 1, bleeding 2), 18 head trauma, 13 asphyxia, and 2 postresuscitation brain damage. After the revision, there were 49 cerebrovascular disease (SAH 37, stroke 2, bleeding 16, and other 4), 17 head trauma, 10 asphyxia, and 11 postresuscitation brain damage. A total of 58 donors had a history of cardiac arrest, 58 required a high dose of catecholamine drip infusion, and only 1 recipient died of primary graft dysfunction. Patient survival rate at 3 years after heart transplantation was not different before and after the revision of the Act (98.6% vs 92.2%). CONCLUSIONS: Although donor age was increased and donors who died of cerebral bleeding or postresuscitation after the revision of the Act increased, the outcome after heart transplantation was not changed.

Japanese strategies to maximize heart and lung availabilities: experience from 100 consecutive brain-dead donors.

OBJECTIVE: Because the donor shortage is extremely severe in Japan because of a strict organ transplantation law, special strategies have been established to maximize heart and lung transplantations (HTs and LTs, respectively). We reviewed 100 consecutive brain-dead donors to evaluate our strategies to identify and manage heart and lung donors. METHODS: We retrospectively reviewed all 100 consecutive brain-dead donors procured since the law was issued in 1997. There were 56 mens and the overall mean donor age was 43.5 years. The causes of death were cerebrovascular disease (n = 62), head trauma (n = 20), and asphyxia (n = 16): Since November 2002, special transplant management doctors were sent to donor hospitals to assess cardiac and lung functions, seeking to identify transplant opportunities. They stabilized donor hemodynamics and lung function by administering antidiuretic hormone intravenously and performing bronchofibroscopy for pulmonary toilet. RESULTS: Seventy-nine HTs, 1 heart-lung transplantations, and 78 LTs (46 single and 32 bilateral) were performed. By applying these strategies organs per donor were increased from 4.5 to 6.8. Among heart donors, 61/80 were marginal: high inotrope requirement (n = 29), cardiopulmonary resuscitation (n = 28), and/or >55 years old (n = 20). None of the 80 HT recipients died of primary graft failure (PGF). Patient survival rate at 10 years after HT was 95.4%. Among lung donors, 48/65 were marginal: pneumonia (n = 41), chest trauma (n = 4), and >55 years old (n = 9). Only 2/78 LT recipients died of PGF. Patient survival rate at 3 years after LT was 72.2%. After inducing frequent pulmonary toilet, lung procurement and patient survival rates increased significantly after LT. CONCLUSIONS: Although the number of cases was still small, the availability of organs has been greater and the outcomes of HT/LT acceptable.

Impaired glycolytic metabolism causes chondrocyte hypertrophy-like changes via promotion of phospho-Smad1/5/8 translocation into nucleus.

OBJECTIVE: Hypertrophy-like changes are often observed in chondrocytes during the development of osteoarthritis (OA). These changes play a crucial part in the OA-associated cartilage degradation and osteophyte formation. However, the pathogenesis leading to such changes is still unknown. In this study, we investigated the mechanism by which these hypertrophy-like changes are induced from the viewpoint of impaired glycolytic metabolism. METHODS: The effect of sodium fluoride (NaF) on glycolytic metabolism of cultured chondrocytes was confirmed by measurement of intracellular adenosine triphosphate (ATP) production. Translocation of phosphorylated Smad1/5/8 to the nucleus was evaluated by subcellular fractionation and Western blotting. Chondrocyte hypertrophy-like changes were investigated by real-time RT-PCR and Western blot analysis of differentiation markers. RESULTS: ATP production was dose-dependently decreased by NaF in the human chondrocytic cell line HCS-2/8. In addition, both chondrocyte proliferation and differentiation were inhibited, whereas cell death was promoted by treatment with NaF. Interestingly, combinational treatment with NaF and lactate enhanced translocation of phospho-Smad1/5/8 to the nucleus, as well as gene expression of ALP, VEGF, COL10a1, and matrix metalloproteinase13 (MMP13), which were the markers of late mature and hypertrophic chondrocytes. Furthermore, the production of type X collagen and activation of MMP9 were also promoted under the same conditions. CONCLUSIONS: These findings suggest that decreased ATP production by NaF promotes hypertrophy-like changes via activation of phospho-Smad1/5/8 in the presence of lactate. Novel metabolic aspects of OA pathogenesis are indicated herein.

Design of a millimeter-wave polarimeter for NSTX-Upgrade and initial test on DIII-D.

Polarimetry is a powerful diagnostic technique to probe plasma equilibria and magnetic fluctuations in fusion plasmas. In a high beta plasma such as the National Spherical Torus eXperiment (NSTX), these measurements are important to understand plasma stability and anomalous transport. A 288 GHz polarimeter operating along a major radial chord in retroreflection geometry has been developed and is being tested on the DIII-D tokamak to prepare for future implementation on NSTX-Upgrade. The system launches a rotating linearly polarized beam and detects the phase shift directly related to the polarization change caused by the plasma. To accomplish this, a pair of orthogonal linearly polarized beams with a stable difference frequency is generated using a single sideband modulation technique, then combined and transformed to be counter-rotating circularly polarized. To improve phase resolution, quasi-optical isolation, using Faraday rotators and polarizers, is utilized to eliminate a multi-path feedback effect, which is found to be the primary source of phase error. The bench tests in the laboratory and DIII-D power supply test discharges indicate ≤1° phase resolution.

2D full wave modeling for a synthetic Doppler backscattering diagnostic.

Doppler backscattering (DBS) is a plasma diagnostic used in tokamaks and other magnetic confinement devices to measure the fluctuation level of intermediate wavenumber (k(θ)ρ(s) ~ 1) density fluctuations and the lab frame propagation velocity of turbulence. Here, a synthetic DBS diagnostic is described, which has been used for comparisons between measurements in the DIII-D tokamak and predictions from nonlinear gyrokinetic simulations. To estimate the wavenumber range to which a Gaussian beam would be sensitive, a ray tracing code and a 2D finite difference, time domain full wave code are used. Experimental density profiles and magnetic geometry are used along with the experimental antenna and beam characteristics. An example of the effect of the synthetic diagnostic on the output of a nonlinear gyrokinetic simulation is presented.

Estimation of the kinetic-optimized stimulus intensity envelope for drop foot gait rehabilitation.

The purpose of present study is to estimate the optimal stimulus intensity envelope for drop foot rehabilitation based on a kinetic perspective. The voluntary and electric-stimulated elicited dorsiflexion torque responses of 11 healthy subjects were measured. During dorsiflexion, we recorded the tibialis anterior (TA) electromyography (EMG) or the stimulation intensity at four angles of the ankle joint. From these measurements, we derived two approximate equations that estimate dorsiflexion produced by either voluntary contraction or by electrical stimulation using a sigmoid function and a stepwise-regression analysis. We then tested the predictive capability of the model using Pearson correlation. Both equations indicated high correlation coefficients. Finally, we derived a relation between the TA EMG amplitude and stimulation intensity. From the obtained equation, we determined the optimal stimulus amplitude. We assume that the derived stimulus intensity envelope, calculated from EMG amplitude and angle of ankle joint, satisfies kinetic demand.

Deletion of thyrotropin receptor residue Asp403 in a hyperfunctioning thyroid nodule provides insight into the role of the ectodomain in ligand-induced receptor activation.

Somatic mutations of the TSH receptor (TSHR) gene are the main cause of autonomously functioning thyroid nodules. Except for mutations in ectodomain residue S281, all of the numerous reported activating mutations are in the TSHR membrane-spanning region. Here, we describe a patient with a toxic adenoma with a novel heterozygous somatic mutation caused by deletion of ectodomain residue Asp403 (Del-D403). Subsequent in vitro functional studies of the Del-D403 TSHR mutation demonstrated greatly increased ligand-independent constitutive activity, 8-fold above that of the wild-type TSHR. TSH stimulation had little further effect, indicating that the mutation produced near maximal activation of the receptor. In summary, we report only the second TSHR ectodomain activating mutation (and the first ectodomain deletion mutation) responsible for development of a thyroid toxic adenoma. Because Del-D403 causes near maximal activation, our finding provides novel insight into TSHR structure and function; residue D403 is more likely to be involved in the ligand-mediated activating pathway than in the ectodomain inverse agonist property.

Whole-mount bone and cartilage staining of chick embryos with minimal decalcification.

Whole-mount staining with Alcian blue for cartilage and alizarin red for bone has been widely used for visualizing the skeletal patterns of embryos and small adult vertebrates. The possibility of decalcification by the acidic Alcian blue solution is known, but standard staining protocols do not always avoid this issue. We investigated the effects of acidity on the stainability of developing bones in stage 36 chick embryos and developed an optimal procedure for obtaining reliable results with minimal decalcification. The diaphyses of long bone rudiments and the maxillofacial membranous bones progressively lost their stainability with alizarin red when the chick embryos were soaked for long periods in the preceding acidic Alcian blue staining solution for cartilage. Unless the acidity was neutralized with an alkaline solution, the remaining acidity in the specimens rendered the pH sufficiently low to prevent the subsequent alizarin red staining of the bones. These findings indicate that the mineralizing bones at the early stages of development are labile to acidity and become decalcified more substantially during the staining process than previously appreciated. The following points are important for visualizing such labile mineralizing bones in chick embryos: 1) fixing with formaldehyde followed by soaking in 70% ethanol, 2) minimizing the time that the specimens are exposed to the acidic Alcian blue solution, and 3) neutralizing and dehydrating the specimens by an alkaline-alcohol solution immediately after the cartilage staining. When the exact onset and/or an early phase of ossification are of interest, the current double-staining procedure should be accompanied by a control single-staining procedure directed only toward bone.

Interaction between Faraday rotation and Cotton-Mouton effects in polarimetry modeling for NSTX.

The evolution of electromagnetic wave polarization is modeled for propagation in the major radial direction in the National Spherical Torus Experiment with retroreflection from the center stack of the vacuum vessel. This modeling illustrates that the Cotton-Mouton effect-elliptization due to the magnetic field perpendicular to the propagation direction-is shown to be strongly weighted to the high-field region of the plasma. An interaction between the Faraday rotation and Cotton-Mouton effects is also clearly identified. Elliptization occurs when the wave polarization direction is neither parallel nor perpendicular to the local transverse magnetic field. Since Faraday rotation modifies the polarization direction during propagation, it must also affect the resultant elliptization. The Cotton-Mouton effect also intrinsically results in rotation of the polarization direction, but this effect is less significant in the plasma conditions modeled. The interaction increases at longer wavelength and complicates interpretation of polarimetry measurements.

A Ka-band tunable direct-conversion correlation reflectometer for NSTX.

The recent availability of broadband microwave quadrature mixers in the Ka-band (28-40 GHz) of frequencies has allowed the fabrication of low-cost direct-conversion detection circuits for use in the variable-frequency correlation reflectometer on the National Spherical Torus eXperiment (NSTX). The quadrature receiver in this case can be implemented as a simple homodyne circuit, without the complication of a single-sideband modulator or a feedforward tracking circuit present in more typical designs. A pair of direct-conversion receivers is coupled with broadband microwave voltage-controlled oscillators to construct a flexible dual-channel radar system with a fast frequency settling time of ∼160 µs. A detailed description of the design and a full characterization of the hardware are provided. Examples of turbulence measurements from radial and poloidal correlation reflectometry on NSTX using a poloidal array of antennas (oriented normal to the magnetic flux surfaces for conventional reflectometry) are presented.

Neurodegenerative influence of oxidative stress in the retina of a murine model of diabetes.

AIMS/HYPOTHESIS: Diabetic retinopathy is a progressive neurodegenerative disease, but the underlying mechanism is still obscure. Here, we focused on oxidative stress in the retina, and analysed its influence on retinal neurodegeneration, using an antioxidant, lutein. METHODS: C57BL/6 mice with streptozotocin-induced diabetes were constantly fed either a lutein-supplemented diet or a control diet from the onset of diabetes, and their metabolic data were recorded. In 1-month-diabetic mice, reactive oxygen species (ROS) in the retina were measured using dihydroethidium and visual function was evaluated by electroretinograms. Levels of activated extracellular signal-regulated kinase (ERK), synaptophysin and brain-derived neurotrophic factor (BDNF) were also measured by immunoblotting in the retina of 1-month-diabetic mice. In the retinal sections of 4-month-diabetic mice, histological changes, cleaved caspase-3 and TUNEL staining were analysed. RESULTS: Lutein did not affect the metabolic status of the diabetic mice, but it prevented ROS generation in the retina and the visual impairment induced by diabetes. ERK activation, the subsequent synaptophysin reduction, and the BDNF depletion in the diabetic retina were all prevented by lutein. Later, in 4-month-diabetic mice, a decrease in the thickness of the inner plexiform and nuclear layers, and ganglion cell number, together with increase in cleaved caspase-3- and TUNEL-positive cells, were avoided in the retina of lutein-fed mice. CONCLUSIONS/INTERPRETATION: The results indicated that local oxidative stress that has a neurodegenerative influence in the diabetic retina is prevented by constant intake of a lutein-supplemented diet. The antioxidant, lutein may be a potential therapeutic approach to protect visual function in diabetes.

Permanent Genetic Resources added to Molecular Ecology Resources Database 1 August 2009-30 September 2009.

This article documents the addition of 238 microsatellite marker loci and 72 pairs of Single Nucleotide Polymorphism (SNP) sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Adelges tsugae, Artemisia tridentata, Astroides calycularis, Azorella selago, Botryllus schlosseri, Botrylloides violaceus, Cardiocrinum cordatum var. glehnii, Campylopterus curvipennis, Colocasia esculenta, Cynomys ludovicianus, Cynomys leucurus, Cynomys gunnisoni, Epinephelus coioides, Eunicella singularis, Gammarus pulex, Homoeosoma nebulella, Hyla squirella, Lateolabrax japonicus, Mastomys erythroleucus, Pararge aegeria, Pardosa sierra, Phoenicopterus ruber ruber and Silene latifolia. These loci were cross-tested on the following species: Adelges abietis, Adelges cooleyi, Adelges piceae, Pineus pini, Pineus strobi, Tubastrea micrantha, three other Tubastrea species, Botrylloides fuscus, Botrylloides simodensis, Campylopterus hemileucurus, Campylopterus rufus, Campylopterus largipennis, Campylopterus villaviscensio, Phaethornis longuemareus, Florisuga mellivora, Lampornis amethystinus, Amazilia cyanocephala, Archilochus colubris, Epinephelus lanceolatus, Epinephelus fuscoguttatus, Symbiodinium temperate-A clade, Gammarus fossarum, Gammarus roeselii, Dikerogammarus villosus and Limnomysis benedeni. This article also documents the addition of 72 sequencing primer pairs and 52 allele specific primers for Neophocaena phocaenoides.

Nicotine-induced CCN2: from smoking to periodontal fibrosis.

Since fibrosis is observed in smokers' gingiva, it was hypothesized that fibrosis was caused by nicotine in the periodontium. Therefore, in this study, we investigated the effects of nicotine on the induction of a profibrotic molecule, connective tissue growth factor (CCN2/CTGF), in human gingival fibroblasts (HGFs) and periodontal ligament (PDL) cells. With 1 microg/mL nicotine, vacuolization and attenuated proliferation were observed. Interestingly, 1 microg/mL nicotine increased the production of CCN2/CTGF protein in both cells without increasing mRNA expression. Furthermore, type I collagen mRNA and protein were also increased and were significantly blocked by a CCN2/CTGF neutralizing antibody. This is the first report to describe a relationship between nicotine and CCN2/CTGF in periodontal tissue cells. Analysis of our data also indicated that nicotine was cytotoxic, while it increased CCN2/CTGF and, eventually, type I collagen production. These findings suggest that periodontal fibrosis can be promoted by nicotine from smoking via effects on CCN2/CTGF.