Kohji Hasegawa

Sulfated Polysaccharides Enhance the Biological Activities of Bone Morphogenetic Proteins

Bone morphogenetic proteins (BMPs), which have been shown to be heparin-binding proteins, induce osteoblast differentiation in mesenchymal cells. In the present study, we examined the effects of heparin on the BMP activities in C2C12 myoblasts. Heparin dose dependently enhanced the osteoblast differentiation induced by not only homodimers of BMP-2 or BMP-4 but also heterodimers of BMP-2/6 or BMP-2/7. However, the osteoblast differentiation induced by the constitutively active BMPR-IA, a functional BMP type I receptor, was not affected by heparin. Heparan sulfate and dextran sulfate also enhanced the BMP-2 activity, although the chemically desulfated heparin-derivatives have lost this stimulatory capacity. Heparin dose-dependently suppressed the accumulation of BMP-2 from the culture media into the cell layer or BMPR-IA, and retained a large amount of BMP-2 in the culture media. The biological activity of BMP-2, which was evaluated using a BMP-responsive reporter gene expression, was prolonged in the presence of heparin. Taken together, these results suggest that sulfated polysaccharides enhance the biological activity of both homodimers and heterodimers of BMPs by continuously serving the ligands to their signaling receptors expressed on cell membranes.

Two distinct stem cell lineages in murine bone marrow.

Mesenchymal stem cells (MSC), a distinct type of adult stem cell, are easy to isolate, culture, and manipulate in ex vivo culture. These cells have great plasticity and potential for therapeutic application, but their properties are poorly understood because of their low frequency and the lack of knowledge on cell surface markers and their location of origin. The present study was designed to address the undefined lineage relationship of hematopoietic and mesenchymal stem cells. Genetically marked, highly purified hematopoietic stem cells (HSCs) were transplanted into wild‐type animals and, after bone marrow repopulation, the progeny were rigorously investigated for differentiation potential into mesenchymal tissues by analyzing in vitro differentiation into mesenchymal tissues. None/very little of the hematopoietic cells contributed to colony‐forming units fibroblast activity and mesenchymal cell differentiation; however, unfractionated bone marrow cells resulted in extensive replacement of not only hematopoietic cells but also mesenchymal cells, including MSCs. As a result, we concluded that purified HSCs have no significant potency to differentiate into mesenchymal lineage. The data strongly suggest that hematopoietic cells and mesenchymal lineage cells are derived from individual lineage‐specific stem cells. In addition, we succeeded in visualizing mesenchymal lineage cells using in vivo microimaging and immunohistochemistry. Flow cytometric analysis revealed CD140b (PDGFRβ) could be a specific marker for mesenchymal lineage cells. The results may reinforce the urgent need for a more comprehensive view of the mesenchymal stem cell identity and characteristics.

Mechanisms Involved in Apoptosis of Human Macrophages Induced by Lipopolysaccharide from Actinobacillus actinomycetemcomitans in the Presence of Cycloheximide

ABSTRACT Actinobacillus actinomycetemcomitans is a major periodontopathic bacterium with multiple virulence factors, including lipopolysaccharide (LPS). Previous reports have demonstrated that LPS induced apoptosis in a murine macrophage-like cell line, J744.1, as well as in peritoneal macrophages from C3H/HeN mice in the presence of cycloheximide (CHX). However, the detailed molecular mechanisms involved in the apoptosis of macrophages induced by LPS and CHX are not well known. To clarify the possible role of LPS in the induction of macrophage apoptosis, we investigated cell death induced by LPS from A. actinomycetemcomitans and CHX in human macrophage-like U937 cells, which were differentiated by 12-O-tetradecanoylphorbol 13-acetate (TPA), and also assessed the molecular mechanisms involved in the process. We found that TPA-differentiated U937 cells usually showed resistance to LPS-induced apoptosis. However, in the presence of CHX, LPS induced release of cytochrome c without modifying steady-state levels of Bcl-2, Bcl-xL, Bax, and Bak. Treatment with LPS in the presence of CHX also led to activation of caspase-3 and apoptosis via, in part, the CD14/toll-like receptor 4 (TLR4). The induction of cytochrome c release may have been due to dephosphorylation of Akt and Bad, which were cooperatively induced by CHX and LPS. However, endogenous tumor necrosis factor alpha- and Fas-induced signals, extracellular signal-regulated kinase kinase/mitogen-activated protein kinases and I-κBα/nuclear factor-κB (NF-κB) were not required for caspase-3-dependent apoptosis. These results emphasize the possible important role of the mitochondrial apoptotic pathway leading to caspase-3 activation in LPS-induced apoptosis of human macrophages in the presence of CHX.

The Effect of Adding Calcium Lactate to Xylitol Chewing Gum on Remineralization of Enamel Lesions

The purpose of the study was to determine whether adding calcium lactate to chewing gum containing xylitol enhances remineralization of enamel surfaces using an early caries lesion model. Enamel slabs were cut from human extracted sound teeth and artificial subsurface lesions created within each. Half the enamel slabs were used as controls and stored in a humidifier while half were mounted into oral appliances worn by 10 volunteers (22–27 years old, 2 males and 8 females) in a three-leg trial, during which they wore the appliance without chewing gum, chewed gum containing xylitol + calcium lactate or chewed gum containing only xylitol 4 times a day for 2 weeks. Calcium concentrations in the enamel surfaces of control and test slabs were measured by X-ray spectrometry and degrees of remineralization were calculated. The mean degree of remineralization was greater after chewing xylitol-Ca gum (0.46 ± 0.10) than after no gum (0.16 ± 0.14) or after chewing xylitol gum (0.33 ± 0.10) (p < 0.01). In conclusion, chewing gum containing xylitol + calcium lactate could enhance remineralization of enamel surface compared to chewing gum containing only xylitol or no gum chewing.

Intracellular Interleukin-1α Production in Human Gingival Fibroblasts is Differentially Regulated by Various Cytokines

Interleukin-1 (IL-1) may play a critical role in immune and inflammatory responses in inflamed gingiva, and it is synthesized by a wide variety of host cells. In this study, we examined the regulatory effects of various cytokines on bioactive membrane IL-1 and intracellular IL-1α production in cultured human gingival fibroblasts (HGF). Recombinant human (rh) IL-lp stimulated membrane IL-1 activity, which was mainly attributed to IL-la. rhIL-1β and rh tumor necrosis factor (TNF)-a stimulated HGF to produce intracellular IL-la, whereas rh interleukin-6 (IL-6), rh interleukin-4 (IL-4), and rh interferon (IFN)-γ did not do so. Intracellular IL-la production induced by rhIL-1β or rhTNF-a may be partially related to protein kinase C (PKC) activation, because rhIL-1β or rhTNF-a-induced intracellular IL-la production was stimulated by pre-treatment with 12-o-tetradecanoylphorbol 13-acetate (TPA), a PKC activator, but was suppressed by the pre-treatment with 1-(5-isoquinoline-sulfonyl) -2-methylpiperazine dihydrochloride (H-7), which is a PKC inhibitor. rhIL-4 inhibited rhIL-1β- or rhTNF-a-induced intracellular IL-la production, but rhIL-6 had no effect on this production. Pre-treatment with rh IFN-γ remarkably enhanced intracellular IL-la production induced by subsequent treatment with rhIL-1β or rhTNF-a. Simultaneous treatment with rhIFN-γ and rhIL-1β inhibited rhIL-1β-induced intracellular IL-la production, but co-treatment with rhIFN-γ and rhTNF-a enhanced rhTNF-a-induced intracellular IL-la production. These results suggest that in inflamed gingiva, pro-inflammatory cytokines such as IL-1β and TNF-a may induce bioactive intracellular IL-la production in human gingival fibroblasts and that this production can be differentially modulated by T-cell-derived cytokines such as IFN-γ or IL-4.

Actinobacillus actinomycetemcomitans lipopolysaccharide stimulates collagen phagocytosis by human gingival fibroblasts

INTRODUCTION Collagen phagocytosis by fibroblasts is involved in the intracellular pathway related to collagen breakdown in soft connective tissues. The possible role of lipopolysaccharide (LPS) in regulating this fibroblast function has not been elucidated so we investigated the effect of LPS from Actinobacillus actinomycetemcomitans, a periodontopathic bacterium, on collagen phagocytic activity in human gingival fibroblasts and associated regulatory mechanisms. METHODS LPS pretreatment stimulated binding of collagen-coated beads to cells and, subsequently, their internalization. RESULTS The LPS-activated collagen phagocytic process was enhanced in the presence of the soluble form of CD14 (sCD14) or LPS-binding protein (LBP), while the LPS/LBP treatment activated Akt and induced actin reorganization. Furthermore, these LPS/LBP-induced effects were partially suppressed by adding phosphatidyl-inositol-3 kinase (PI3K) inhibitors. CONCLUSION These results suggest that A. actinomycetemcomitans LPS disturbs the homeostasis of collagen metabolism within gingival tissue by facilitating collagen phagocytosis by gingival fibroblasts, and serum sCD14 and LBP positively regulate the action of LPS. In addition, the PI3K/Akt signaling is thought to partially mediate the LPS/LBP-stimulated collagen phagocytic pathway, which may be dependent on actin cytoskeletal rearrangement.

Periodontal soft tissue management with a high pulse rate Er:YAG laser

Abstract The purpose of this study was to evaluate the effectiveness and safety of periodontal minor soft tissue surgery with the Er/YAG laser at a high pulse rate of 30 Hz and the subsequent clinical wound healing. Sixty-one patients were subjected to gingivectomy, removal of gingival melanin pigmentation, or frenectomy using the Er/YAG laser. Clinical parameters were evaluated before, immediately after, at 1 week and 4 weeks after surgery. The Er/YAG laser was capable of cutting or ablating gingival soft tissue easily and effectively with less or no bleeding as well as less pain. The healing process was uneventful without any complications or side effects. The results of this study suggest that the Er/YAG laser at 30 Hz could be used safely and effectively for minor periodontal soft tissue management without causing major thermal damage as well as the delay of wound healing.

The Incidence of Furca Involvements of Maxillary First Premolar and its Roentgenographic Diagnostic Technique

The diagnosis and the treatment of the furca involvements are clinical entity which require special attention of the periodontists because of its reported poor prognosis. However, the periodontal breakdown involving the furca of maxillary first premolar has been often over looked in daily practice because of the scarcity of information. This study was intended to enhance some practical knowlegde to this field.On 386 periodontal patients, the incidence of furcated maxillary first premolar and the incidence of furca involvements were investigated on the two dental X-ray films one of which having been taken ortho-radially and the other mesio-eccentrically to the maxillary first premolar.Two hundred and twenty-four out of 772 first premolar (29.0%) were interpreted as multirooted. Furca involvement were observed in 26 out of 224 multirooted premolar (11.6%). The incidence of furca involvements of the maxillary first premolar was found 3.3% (26/772×100).Some roentgenographic technique were applied to the furca of maxillary premolar to aid the more reliable roentgenographic interpretation. Computerized tomography was most helpful to determine the configulation of the furca area. Panagram together with the 25°mesio-eccentrically taken dental X-ray film was also helpful.

Interferon-γ inhibits collagen phagocytosis in human fibroblasts by inducing subcortical actin assembly and reducing ability of β1 integrin to bind to collagen

Abstract.Objective:We investigated the possible roles of interferon-γ (IFN-γ) in modulation of extracellular and intracellular routes of collagen digestion by human fibroblasts.Methods:Human gingival fibroblasts were treated with IFN-γ, after which matrix metalloproteinase-1 (MMP-1) activation was determined. Following the IFN-γ treatment, cells were further incubated with either activating antibody for β1 integrin or actin monomer-sequestering agent latrunculin B before incubation with collagen-coated fluorescent beads. Thereafter, the binding and internalization of the beads were assessed.Results:IFN-γ had no significant effect on MMP-1 activation, however, it reduced the binding of collagen-coated beads in the minimum affinity range and, subsequently, internalization of the beads. The inhibitory effects of IFN-γ were partially reversed by adding either the β1 integrin activating antibody or latrunculin B.Conclusions:Although IFN-γ does not appreciably moderate the extracellular route of collagen digestion by human fibroblasts, the reduced level of collagen phagocytosis by IFN-γ in the cells may contribute to fibrosis in inflamed connective tissues. Further, IFN-γ may decrease the binding of collagen and following phagocytosis in cells by inducing a subcortical actin assembly and reducing the ability of β1 integrin to bind to collagen.

Removal effects of subgingival calculus on root surface after Er:YAG laser irradiation at a high pulse rate

Abstract This aim of this study was to evaluate the clinical effectiveness of Er:YAG laser at a high pulse rate (ER-D1: HOYA continuum, Japan) for scaling of subgingival calculus and root planning. Sixty-one patients with calculus deposition were treated with the Er:YAG laser (panel setting 30–108 mJ/pulse, 30 Hz) using contact tips and the results were then subjected to clinical analysis. Clinical assessments of calculus index, pain, redness, swelling, discomfort, roughness of root surface, efficiency of scaling, PlI, PD, BOP and mobility were made prior to and at 1, 4 weeks and 3 months after treatment. Fifteen of the sixty-one patients were treated during the flap operation. The Er:YAG laser high-pulse-rate irradiation was capable of ablating the calculus on the root surface precisely and effectively. The roughness of root surface after debridement was evaluated to be adequately plane for 41 cases and still rough for 20 cases. BOP and PD were significantly improved throughout the 3-month examination of this study. No negative side effects or influences were observed on the healing of periodontal tissue following Er:YAG laser irradiation during this clinical study. In conclusion, the Er:YAG laser high-pulse-rate irradiation would be a safe and effective, alternative method for root surface treatment in both nonsurgical and surgical periodontal therapy.