Immunohistochemical localization of very late activation integrins in healthy and diseased human gingiva

The β1-integrins (VLA family) are cellular adhesion molecules (CAM) that play a major role in cell-cell and cell-matrix interactions. The expression pattern of CAM was studied in 5 clinically normal volunteers with healthy gingiva and in 18 patients with clinically different stages of periodontitis. In healthy human gingiva α2. α3 and α6 integrin chains were found in a characteristic distribution, showing a broad continuous expression on the junctional and sulcular epithelium sites. The expression of these integrins was demonstrated primarily on the basal cell layers and in some cells of the stratum spinosum. Inflammatory stages of periodontitis revealed further upregulation of α2, α3 and α6 integrins into the junctional and sulcular epithelial cells, which correlated with the stage of the periodontitis and the extent of the cellular infiltration. α4 and α6 were found to be the predominant β1 integrin chains on inflammatory cells. The amount of δ4 and ş6 positive infiltrative cells increased with the number of inflammatory cells. VCAM-1. the corresponding cell-cell ligand of VLA-4 (α4) was present on the majority of subepithelial vessels in all stages of gingivitis and periodontitis. The α5 subunit was expressed on both endothelium and gingival connective tissue cells. Samples from advanced periodontitis cases showed a higher number of a5 positive mononuclear cells. In comparison to normal epidermis, a human gingival epithelial cells express higher levels of integrins. This expression is further upregulated in advanced stages of periodontitis, indicating changes of the β1 integrin organization.     

SHED repair critical-size calvarial defects in mice

Objective:  Stem cells from human exfoliated deciduous teeth (SHED) are a population of highly proliferative postnatal stem cells capable of differentiating into odontoblasts, adipocytes, neural cells, and osteo-inductive cells. To examine whether SHED-mediated bone regeneration can be utilized for therapeutic purposes, we used SHED to repair critical-size calvarial defects in immunocompromised mice.
Materials and methods:  We generated calvarial defects and transplanted SHED with hydroxyapatite/tricalcium phosphate as a carrier into the defect areas.
Results:  SHED were able to repair the defects with substantial bone formation. Interestingly, SHED-mediated osteogenesis failed to recruit hematopoietic marrow elements that are commonly seen in bone marrow mesenchymal stem cell-generated bone. Furthermore, SHED were found to co-express mesenchymal stem cell marker, CC9/MUC18/CD146, with an array of growth factor receptors such as transforming growth factor β receptor I and II, fibroblast growth factor receptor I and III, and vascular endothelial growth factor receptor I, implying their comprehensive differentiation potential.
Conclusions:  Our data indicate that SHED, derived from neural crest cells, may select unique mechanisms to exert osteogenesis. SHED might be a suitable resource for orofacial bone regeneration.     

The up-regulation of heme oxygenase-1 expression in human gingival fibroblasts stimulated with nicotine

BACKGROUND: Cigarette smoking is a major risk factor in the development and further progression of periodontal diseases. Heme oxygenase-1 (HO-1) is known as a stress-inducible protein and functions as an antioxidant enzyme. There is limited information on the expression of HO-1 in smoking-associated periodontal disease. OBJECTIVES: The aim of the present study was to investigate the effects of nicotine on the expression of HO-1 protein in cultured human gingival fibroblasts in vitro and further to compare HO-1 expression in gingival tissues obtained from cigarette smokers and non-smokers in vivo. METHODS: Western blot assay was used to investigate the effects on human gingival fibroblasts exposed to nicotine. In addition, antioxidants catalase, superoxide dismutase (SOD), and N-acetyl-l-cysteine (NAC) were added to test how they modulated the effects on nicotine-induced HO-1 expression. Gingival biopsies taken from the flap surgery of 20 male patients with periodontal disease (10 cigarette smokers and 10 non-smokers) were examined by immunohistochemistry. RESULTS: The exposure of quiescent human gingival fibroblasts to 10 mm nicotine resulted in the induction of HO-1 protein expression in a time-dependent manner (p < 0.05). The addition of glutathione (GSH) precursor NAC inhibited the nicotine-induced HO-1 protein expression (p < 0.05). However, SOD and catalase did not decrease the nicotine-induced HO-1 protein expression (p > 0.05). The results from immunohistochemistry demonstrated that HO-1 expression was significantly higher in cigarette smokers (p < 0.05). HO-1 was noted in the basal layers of epithelium, inflammatory cells, and fibroblasts in specimens from cigarette smoking. CONCLUSIONS: Taken together, these results suggest that HO-1 expression is significantly up-regulated in gingival tissues from cigarette smokers, and nicotine may, among other constituents, be responsible for the enhanced HO-1 expression in vivo. The regulation of HO-1 expression induced by nicotine is critically dependent on the intracellular GSH concentration.     

Effect of recombinant human platelet-derived growth factor-BB and bone morphogenetic protein-2 application to demineralized dentin on early periodontal ligament cell response

The purpose of this study is to investigate the early responses of human periodontal ligament cells attached to recombinant human platelet-derived growth factor-BB and bone morphogenetic protein-2 applied EDTA-demineralized dentin. One hundred and seventy-four root-planed flat dentin blocks were prepared from the mid-third of periodontally diseased human tooth roots. After demineralization with 24% EDTA (pH 7.02) 120 dentin blocks were treated with 0.5 and 1 microgram/ml rhPDGF-BB, 1 and 3 micrograms/ml rhBMP-2 and only MEM as control (24/group). Human periodontal ligament cells (HPLC) were seeded on these dentin surfaces and incubated. The alkaline phosphatase (ALP) activity and protein concentration of the attached cell were assessed at d 2, 4 and 7. Fifty-four dentin blocks were seeded with HPLC after application of 1 microgram/ml rhPDGF-BB, 3 micrograms/ml rhBMP-2 and MEM (18/group) and then incubated. At d 2, 4 and 7, the attached cells were stained and counted under light microscope. The results showed a significant increase of protein concentration and cell number in PDGF-BB treated groups than control (p < 0.05, p < 0.01) but not the ALP activity, and a significant increase of ALP activity was observed in BMP-2 treated groups than control (p < 0.05) but protein concentration and cell number remained almost the same over time. Thus, rhPDGF-BB and rhBMP-2 application to EDTA demineralized dentin surfaces promote the early human periodontal ligament cell responses by increasing cell proliferation and differentiation, respectively, which would ultimately enhance periodontal regeneration.     

Reduction of connexin 43 expression in aged human dental pulp

AIM: To investigate the expression of connexin 43 (CX43) mRNA in young and old human dental pulp tissues to determine the characteristics of CX43 expression. METHODOLOGY: Samples were obtained from human dental pulp of healthy young (17-23 years) and aged (>50 years) subjects. CX43 expression was determined by RT-PCR and by quantitative real-time RT-PCR (QRT-PCR). The threshold cycle (Ct) value, which reflects the amount of PCR, was calculated and the difference between value in the young pulp and that in the aged pulp was statistically analysed. RESULTS: RT-PCR analysis of human dental pulp tissue detected CX43 mRNA in all the samples. CX43 was abundantly expressed in young adult dental pulp, but expression of CX43 mRNA was dramatically decreased in aged human dental pulp. QRT-PCR analysis also showed the reduced expression of CX43 in aged pulp, and expression of CX43 in young pulp was significantly higher (about 10-fold, P < 0.01, Mann-Whitney U-test). CONCLUSION: Reduction of CX43 expression may be associated with the loss of viability in human dental pulp, and is considered as one characteristic of aged pulp.     

The effects of a triclosan/copolymer dentifrice on oral bacteria including those producing hydrogen sulfide

A clinical procedure was developed to examine the effects of short-term and extended use of a triclosan/copolymer dentifrice and a commercial fluoride dentifrice on oral bacteria, including those producing hydrogen sulfide. Healthy adults volunteered for this double-blind, crossover design clinical study and provided saliva samples for culturing on enriched and indicator media to enumerate all salivary bacteria and those producing hydrogen sulfide (odorigenic), respectively. Subjects brushed with an assigned dentifrice for 7 d and were sampled on day 8 to assess the long-term effects on bacteria. Extended use of the triclosan/copolymer dentifrice resulted in a 49% and 66% reduction of salivary and odorigenic bacteria, respectively, compared with the fluoride dentifrice. To examine short-term effects, subjects subsequently brushed with their assigned dentifrice and were sampled at 2 h and 4 h post-brushing. At 2 h and 4 h post-brushing, the triclosan/copolymer dentifrice resulted in a 62% and 52% decrease for salivary bacteria and 79% and 72% decrease for odorigenic bacteria, respectively, vs. the fluoride dentifrice. The results indicate a significant decrease of all salivary bacteria and hydrogen sulfide-producing odorigenic bacteria following use of the triclosan/copolymer dentifrice and explain previous results on the efficacy of this dentifrice on oral malodor.     

Gamma-interferon enhances expression of CD14/MyD88 and subsequent responsiveness to lipopolysaccharide from Actinobacillus actinomycetemcomitans in human gingival fibroblasts

OBJECTIVES: CD14, toll-like receptor 4 (TLR4) and MyD88 have been shown to mediate responsiveness in host cells to lipopolysaccharide. We investigated here the regulatory effects of inflammatory cytokines on the expression of membrane CD14 (mCD14), TLR4 and MyD88, and on subsequent responsiveness to lipopolysaccharide from Actinobacillus actinomycetemcomitans in human gingival fibroblasts. MATERIALS AND METHODS: Following treatment with either interleukin-1beta, tumor necrosis factor-alpha (TNF-alpha) or gamma-interferon (IFN-gamma), expression of mCD14/TLR4 and MyD88 was determined by flow cytometry and western blotting, respectively. After pretreatment with IFN-gamma, cells were pre-incubated with either anti-CD14 antibody MY4 or anti-TLR4 antibody HTA125 and subsequently treated with A. actinomycetemcomitans lipopolysaccharide. Then, phosphorylation of mitogen-activated protein (MAP) kinases and IkappaBalpha was examined by western blotting, and production of interleukin-6 and interleukin-8 was measured by their respective enzyme-linked immunosorbent assay (ELISA) kits. RESULTS: IFN-gamma stimulated expression of mCD14, whereas -1beta and TNF-alpha did not. Expression of MyD88 but not TLR4 was also enhanced by IFN-gamma. The lipopolysaccharide activated MAP kinases, such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38, and IkappaBalpha and stimulated production of interleukin-6 and interleukin-8. The lipopolysaccharide-stimulated interleukin-6 and interleukin-8 production was markedly inhibited by MY4 or HTA125. Pretreatment with IFN-gamma augmented the following activation of MAP kinases and IkappaBalpha and production of interleukin-6 and interleukin-8 in response to the lipopolysaccharide. CONCLUSIONS: These results suggest that the augmentation by IFN-gamma of the responsiveness to A. actinomycetemcomitans lipopolysaccharide, such as activation of MAP kinases and IkappaBalpha and terminal cytokine production in human gingival fibroblasts, may be partially mediated by up-regulation of CD14 and MyD88 expression.     

Improving esthetically objectionable human enamel fluorosis with a simple microabrasion technique

Mild-to-moderately severe enamel fluorosis (EF) is an unsightly maturation-phase dental disorder. Despite extensive epidemiological studies on EF, little is known about individual treatment options. This study was carried out to determine whether a simple microabrasion technique is effective in improving the esthetics of EF . Patients with a variety of severities were treated using a water-cooled fine diamond polishing bur at high speed to remove the surface enamel layers. Photographs of the affected teeth before and after treatment were shown by computer to a panel of three judges (two lay and one experienced), who rated the appearance of the teeth using a newly developed visual analog scale. The severity of EF was rated randomly and blind for 52 individual teeth (26 before and 26 after treatment). Reteated-measures analysis of variance was used to analyze the results. The lay judges rated the appearance of the teeth with EF as significantly more objectionable before treatment. All judges found a significant improvement in the severity of EF after treatment. Using a newly developed visual analog scale, our study indicates that EF of an objectionable nature can be significantly improved with a simple microabrasion technique, thus conserving tooth structure and minimizing the cost of treating EF.