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Notes: Nitric oxide is known to be an important inflammatory mediator, and is implicated in the pathophysiology of a range of inflammatory disorders. The aim of this study was to determine the localization and distribution of endothelial NOS (NOSII) in human gingival tissue, and to ascertain if human gingival fibroblasts express NOS-II when stimulated with interferon gamma (IFN-γ) and bacterial lipopolysaccharide (LPS). The distribution of NOS-II in inflamed and non-inflamed specimens of human gingivae was studied using a monoclonal antibody against nitric oxide synthase II. Cultures of fibroblasts derived from healthy human gingivae were used for the cell culture experiments. The results from immunohistochemical staining of the tissues indicated an upregulation of NOS-II expression in inflamed compared to non-inflamed gingival tissue. Fibroblasts and inflammatory cells within the inflamed connective tissue were positively stained for NOS-II. In addition, basal keratinocytes also stained strongly for NOS-II, in both healthy and inflamed tissue sections. When cultured human gingival fibroblasts were stimulated by INF-γ and Porphyromonas gingivalis LPS, NOS-II was more strongly expressed than when the cells were exposed to LPS or IFN-γ alone. These data suggest that, as for other inflammatory diseases, NO plays a role in the pathophysiology of periodontitis.

Notes: Objective:  The aim of this study was to evaluate the influence of Emdogain® (EMD) on cultured gingival fibroblasts, periodontal ligament fibroblasts and dermal fibroblasts, using an in vitro model of wound healing.Background:  Enamel matrix derivative has been demonstrated to promote periodontal regeneration. However, the precise mechanisms by which this agent acts are still unclear.Methods:  The effect of EMD on proliferation of the cells was studied using subconfluent cultures of gingival fibroblasts and periodontal ligament fibroblasts. The cells were made quiescent overnight and then stimulated with various concentrations of EMD (10, 50, 100 and 150 μg/ml) for 24 h. Negative and positive controls were cells cultured in media containing 0.2% and 10% fetal calf serum (FCS). The DNA synthesis was measured by the cellular uptake of [3H]thymidine. For in vitro wounding the cells were cultured, wounded and stimulated with 0.2% FCS, 10% FCS and EMD at a concentration of 20 μg/ml. The percentage of wound fill after treatment was measured after d 1, 4, 6, 12 and 16. The proliferation of cells was also calculated by the extent of incorporation of crystal violet.Results:  The results demonstrated that cells in vitro fill an empty space by a combination of proliferation and cell migration. The most rapid closure of a wound area occurred where both proliferation and migration can occur as was seen when wounded cultures were maintained in 10% FCS or at a concentration of 20 μg/ml EMD which promoted proliferation.Conclusions:  Therefore, EMD appears to exert an influence on cells that is compatible with improved wound healing.

Notes: The expression of hard tissue associated proteins may be used to identify periodontal fibroblasts with the capability to facilitate periodontal regeneration. The aim of this study was to describe, by immunohistochemistry, the distribution of osteocalcin, osteopontin, bone sialoprotein and bone morphogenic proteins-2 and -4 (BMP-2 and BMP-4) within the human periodontium. Furthermore, the expression of mRNA for the above proteins and alkaline phosphatase by gingival and periodontal ligament fibroblasts in vitro was also assessed by reverse transcriptase polymerase chain reaction (RT-PCR). Localization of osteopontin, osteocalcin, BMP-2 and BMP-4 within sections of human periodontal structures was stronger in the periodontal ligament compared to the gingiva. Bone sialoprotein was not detected in either of the soft tissues but, along with osteopontin and osteocalcin, it was localized in the cementum and bone. In vitro, both the gingival and periodontal ligament fibroblasts expressed mRNA for alkaline phosphatase, BMP-2, BMP-4 and osteopontin. Although there were no differences in the expression of alkaline phosphatase and BMP-4 mRNA between the two cell types, we noted significantly higher mRNA levels of osteopontin in the periodontal ligament and BMP-2 in the gingival fibroblasts. Osteocalcin and bone sialoprotein mRNA expression was only noted in the cultured periodontal ligament fibroblasts. From these results, it can be concluded that distinct differences exist between the two fibroblast populations in terms of the localization and mRNA expression of the majority of the hard tissue associated proteins. Furthermore, the elevated in vitro mRNA expression for osteocalcin, osteopontin and bone sialoprotein may be used to identify cells with the potential to facilitate hard tissue formation and hence periodontal regeneration.

Notes: Background:  Growth hormone (GH) is a potent regulator of bone formation. The proposed mechanism of GH action is through the stimulation of osteogenic precursor cell proliferation and, following clonal expansion of these cells, promotion of differentiation along the osteogenic lineage.Objectives:  We tested this hypothesis by studying the effects of GH on primary cell populations of human periodontal ligament cells (PLC) and alveolar bone cells (ABC), which contain a spectrum of osteogenic precursors.Methods:  The cell populations were assessed for mineralization potential after long-term culture in media containing β-glycerophosphate and ascorbic acid, by the demonstration of mineral deposition by Von Kossa staining. The proliferative response of the cells to GH was determined over a 48-h period using a crystal violet dye-binding assay. The profile of the cells in terms of osteogenic marker expression was established using quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for alkaline phosphatase (ALP), osteopontin, osteocalcin, bone sialoprotein (BSP), as well as the bone morphogenetic proteins BMP-2, BMP-4 and BMP-7.Results:  As expected, a variety of responses were observed ranging from no mineralization in the PLC populations to dense mineralized deposition observed in one GH-treated ABC population. Over a 48-h period GH was found to be non-mitogenic for all cell populations. Quantitative reverse transcriptase polymerase chain reaction (RT-PCR) BSP mRNA expression correlated well with mineralizing potential of the cells. The change in the mRNA expression of the osteogenic markers was determined following GH treatment of the cells over a 48-h period. GH caused an increase in ALP in most cell populations, and also in BMP expression in some cell populations. However a decrease in BSP, osteocalcin and osteopontin expression in the more highly differentiated cell populations was observed in response to GH.Conclusion:  The response of the cells indicates that while long-term treatment with GH may promote mineralization, short-term treatment does not promote proliferation of osteoblast precursors nor induce expression of late osteogenic markers.

Notes: Cell-surface proteoglycans participate in several biological functions including interactions with a variety of growth factors and cytokines. Regulation of syndecan-1 and -2 gene expression was investigated in human periodontal ligament fibroblasts (PDLF), osteoblasts (OB) and gingival fibroblasts (GF), in response to platelet-derived growth factor (PDGF-BB), transforming growth factor (TGF-β1), and interleukin (IL-1β) by Northern blot analyses. We also compared the effect of PDGF-BB and TGF-β1, separately and in combination, in the prolonged presence of IL-1β on the expression of both syndecan genes. The results demonstrated that the three cell lines regulated the expression of syndecan-1 and -2 in response to growth factors and cytokines in different manners. These cell lines increased syndecan-1 mRNA levels in response to either PDGF-BB or TGF-β1 and decreased levels in response to IL-1β. The effect of IL-1β on syndecan-1 mRNA synthesis was partially reversed after adding PDGF-BB and TGF-β1, separately or in combination, in the presence of IL-1β. In contrast, syndecan-2 mRNA level was markedly upregulated in response to either TGF-β1 or IL-1β in OB when compared with the other two cell lines. However, the stimulatory effect of TGF-β1 on syndecan-2 mRNA production in OB was abolished in the prolonged presence of IL-1β. These findings lend support to the notion that syndecan-1 and syndecan-2 have distinct functions which correlate with their source and functions within the periodontium.