Expression and possible immune-regulatory function of ghrelin in oral epithelium.

Originally found in stomach mucosa, ghrelin is a peptide appetite hormone that has been implicated as an immuno-modulatory factor. Ghrelin has also been found in salivary glands and saliva; however, its expression patterns and biological properties in the oral cavity remain unclear. Therefore, we investigated the expression patterns of ghrelin in saliva, gingival crevicular fluid (GCF), and gingival tissue, as well as its in vitro effects on IL-8 production by TNF-α or LPS-stimulated oral epithelial cells. In the clinical samples obtained from 12 healthy volunteers, the concentration of ghrelin in GCF remarkably exceeded that detected in saliva. The expression of ghrelin mRNAs and growth hormone secretagogue (GHS) receptors could be detected in human oral epithelial cells. Immunohistochemical analysis revealed the expression of ghrelin in gingival epithelium, as well as in fibroblasts in the lamina propria. Ghrelin increased intracellular calcium mobilization and cAMP levels in oral epithelial cells, suggesting that ghrelin acts on epithelial cells to induce cell signaling. Furthermore, synthetic ghrelin inhibited the production of IL-8 from TNF-α or LPS-stimulated oral epithelial cells. These results indicate that ghrelin produced in the oral cavity appears to play a regulatory role in innate immune responses to inflammatory infection.

Differences in clinical grading associated with instructor status.

PURPOSE: The purpose of this study was to determine whether the clinical evaluation of pre-doctoral students is associated with instructor status. The hypothesis was that there would be no association between instructor status and clinical evaluation grades. MATERIALS AND METHODS: Retrospective analysis of pre-doctoral clinical evaluations for class II amalgams, class III composites, and periodontal scaling and root planing was performed. The grade averages were based on a rank scale. Descriptive statistics were computed to summarise the predictor and outcome variables. Bivariate statistics were computed to evaluate any associations between the predictors and outcomes. Multiple linear regression models were computed to evaluate the simultaneous effects of multiple predictors on clinical evaluations. RESULTS: The study sample consisted of 238 class II amalgams, 246 class III composites, and 675 scaling and root planings which occurred between August 2003 and June 2005. The procedure averages for these procedures were 1.8 +/- 0.67, 1.8 +/- 0.66 and 2.1 +/- 0.56 respectively. The management averages were 2.0 +/- 0.63, 1.5 +/- 0.58 and 1.4 +/- 0.54 respectively. In bivariate analyses, faculty status was associated with treatment averages for all three procedures. CONCLUSIONS: Faculty status was associated with treatment score for all three procedures evaluated. Full-time faculty gave the best grades for restorative procedures. For periodontal procedures, part-time faculty gave the best grades. More studies are warranted to elucidate the nature behind these differences.

Diminished forkhead box P3/CD25 double-positive T regulatory cells are associated with the increased nuclear factor-kappaB ligand (RANKL+) T cells in bone resorption lesion of periodontal disease.

Periodontal disease involves multi-bacterial infections accompanied by inflammatory bone resorption lesions. The abundant T and B lymphocyte infiltrates are the major sources of the osteoclast differentiation factor, receptor activator for nuclear factor-kappaB ligand (RANKL) which, in turn, contributes to the development of bone resorption in periodontal disease. In the present study, we found that the concentrations of RANKL and regulatory T cell (T(reg))-associated cytokine, interleukin (IL)-10, in the periodontal tissue homogenates were correlated negatively, whereas RANKL and proinflammatory cytokine, IL-1beta, showed positive correlation. Also, according to the fluorescent-immunohistochemistry, the frequency of forkhead box P3 (FoxP3)/CD25 double-positive cells was diminished strikingly in the bone resorption lesion of periodontal disease compared to healthy gingival tissue, while CD25 or FoxP3 single positive cells were still observed in lesions where abundant RANKL+ lymphocytes were present. Very importantly, few or no expressions of FoxP3 by the RANKL+ lymphocytes were observed in the diseased periodontal tissues. Finally, IL-10 suppressed both soluble RANKL (sRANKL) and membrane RANKL (mRANKL) expression by peripheral blood mononuclear cells (PBMC) activated in vitro in a bacterial antigen-specific manner. Taken together, these results suggested that FoxP3/CD25 double-positive T(reg) cells may play a role in the down-regulation of RANKL expression by activated lymphocytes in periodontal diseased tissues. This leads to the conclusion that the phenomenon of diminished CD25+FoxP3+ T(reg) cells appears to be associated with the increased RANKL+ T cells in the bone resorption lesion of periodontal disease.

Collagen XII mutation disrupts matrix structure of periodontal ligament and skin.

Collagen XII has been postulated to organize the extracellular matrix (ECM) architecture of dense connective tissues such as the periodontal ligament (PDL) and skin. The objective of this study was to test this hypothesis in transgenic mice carrying a dominant interference mutation of collagen XII. The truncated alpha1(XII) collagen minigene construct MXIINC3(-), driven by the mouse alpha2(I) collagen promoter, was prepared and used to generate transgenic mouse lines. The PDL matrix fibers of molar teeth lost the ordered architecture characteristic of ligament tissue without noticeable inflammation. Cellular cement appeared to be disrupted at the PDL insertion. By confocal laser scanning microscopy, the PDL of transgenic mice demonstrated swollen and irregularly arranged collagen fibers associated with internal porosity. The skin of transgenic mice revealed the lack of matrix fiber structure in the papillary dermis. These results indicated that the dominant interference mutation of collagen XII disorganized the ECM architecture of PDL and skin.

The effect of insulin therapy on osseointegration in a diabetic rat model.

As patients become edentulous, dental implants have been one treatment alternative. Although studies indicate that dental implants inserted in healthy patients have been successful, their placement in the diabetic patient remains controversial. The purpose of this study utilizing histometric parameters compares the course of osseous healing around endosseous implants in normal non-diabetic and insulin controlled diabetic rats. Diabetes was induced by a single intraperitoneal injection of streptozotocin. Blood glucose was monitored by the glucose-oxidase method and controlled with daily insulin injections. Sterile custom fabricated commercially pure solid cylinder titanium implants, with a titanium plasma-sprayed surface were placed in the femora of each animal. The results indicate that insulin therapy was able to upregulate the formation of bone around implants inserted in the streptozotocin-induced diabetic rat model. However, histometric parameters utilized indicated that although the total quantity of bone formation was greater in the insulin controlled group, there was significantly less bone-to-implant contact in the insulin controlled diabetic group as compared to normal non-diabetic controls.

Structural variation of type XII collagen at its carboxyl-terminal NC1 domain generated by tissue-specific alternative splicing.

This paper reports the identification of two structural variations in the NC1 domain of rat and mouse type XII collagen. The long NC1 domain encoding 74 amino acids showed homology to chicken type XII and XIV collagens. The short NC1 domain was composed of 19 amino acids. Through genomic DNA analyses, two alternative exons were identified, each of which contained the variable NC1 sequence. With the amino-terminal NC3 splicing alternatives, we propose here a new descriptive nomenclature: types XIIA-1 and XIIB-1 which include a long NC1 sequence encoded by exon 1 (from the 3'-end), and types XIIA-2 and XIIB-2 which include a short NC1 sequence encoded by exon 2. Types XIIA-1 and XIIB-1, the predominant transcripts in 15-day old mouse embryos, showed decreased expression in 17-day old embryos when type XIIB-2 expression was sustained at constant levels. In adult mice, type XIIB-1 associates with ligament and tendon, whereas type XIIB-2 is expressed in various other tissues. The long NC1 domain contains an extended acidic region (pI = 3.4) followed by a terminal basic region (pI = 13.8). Because the short NC1 domain lacks these features, structural variations in the type XII collagen NC1 domain suggests different functional roles in a tissue-specific fashion.

Wound healing around endosseous implants in experimental diabetes.

Wound healing has been shown to be altered in diabetes mellitus. The aim of this study was to identify the effects of streptozotocin-induced diabetes on osseointegration. Diabetes was induced in 40-day-old rats by intraperitoneal injection of 70 mg per kg streptozotocin. At 14 days postinjection, implants were placed in the femora of 10 diabetic and 10 age-matched normal rats. Animals were sacrificed at 28 and 56 days following implantation. Histometric results indicated that the quantity of bone formation was similar for diabetic and control animals (P > .05). However, less bone-implant contact was observed for diabetic compared to control animals at both 28 and 56 days (P < .0001). This study demonstrates that the process of osseointegration is affected by streptozotocin-induced diabetes.

The expression of collagen I and XII mRNAs in Porphyromonas gingivalis-induced periodontitis in rats: the effect of doxycycline and chemically modified tetracycline.

Tissue remodeling is a dynamic state in which a balance is achieved between the proteolytic breakdown and synthesis of the extracellular matrix. Type I collagen is a major component of the gingival connective tissue (GCT) and the periodontal ligament (PDL) throughout development, while type XII collagen has been found in the mature forms of these tissues. The purpose of this study was to investigate the effects of periodontitis on the expression of type I and XII collagen and subsequently to investigate the effects of doxycycline (DOXY) and chemically modified non-antimicrobial tetracycline (CMT-1) on the expression of these molecules in this model. Adult barrier-raised male Sprague-Dawley rats were inoculated with Porphyromonas gingivalis obtained from humans to create the experimental periodontitis. The animals with the P. gingivalis-induced periodontitis were then split into the following groups: Group A served as infected untreated controls (PGI group); group B was treated with doxycycline (DOXY group); and group C was treated with chemically modified tetracycline-1 (CMT-1 group). Group D contained uninfected animals that served as uninfected controls (NIC group). The expression of type I and XII collagen mRNAs was examined by in situ hybridization in each group, with the co-expression of these molecules representing mature and functional gingival connective tissue. In the NIC group, cells hybridized with digoxygenine-labeled cDNA probes encoding rat alpha2(I) or alpha1(XII) collagens were found distributed uniformly throughout the periodontal connective tissue. The PGI group showed little hybridization in the areas of infection, while both the DOXY and CMT-1 groups showed co-expression of the alpha2(I) and alpha1(XII) probes in the GCT and coronal part of the PDL. This study demonstrates that doxycycline and CMT-1 moderate or reduce the inhibitory effects of periodontal infection on the expression of type I and type XII collagen mRNAs. These results suggest that doxycycline and a form of non-antimicrobial tetracycline, chemically modified tetracycline-1, can reduce periodontal destruction by reversing the inhibitory effect of periodontal infection on collagen synthesis.

Temporal and spatial expressions of type XII collagen in the remodeling periodontal ligament during experimental tooth movement.

This study tested the hypothesis that the remodeling processes of adult periodontal ligament (PDL) reiterate the cellular and molecular events that occur sequentially during development. Type XII collagen has been implicated in the three-dimensional organization of the PDL extracellular matrix, and its expression has been restricted to the terminally differentiated stages. This study focused on the examination of the temporal and spatial expression of type XII collagen during experimental PDL remodeling in the rat. The temporal expressions of types I and XII collagen mRNAs were examined by RNA transfer blot and RNase protection assays, respectively, and were found to be relatively stable in the control group throughout the experimental period. In the tooth movement group, the expression of type I collagen increased at 72 hours and sustained the high level of expression at one week, while an increase in the expression of type XII collagen was first noted at the one-week period. The temporal activation of types I and XII collagen expression in the remodeling occurred in a pattern similar to that found during the development of the PDL. The spatial expression of type XII collagen mRNA was examined by in situ hybridization in the one-week-tooth-movement specimens. Labeled cells, which were more evident in the tension side, typically exhibited a spindle shape and were surrounded by the mature PDL matrix. Our data suggest that the type XII collagen expression may be closely associated with the functional regeneration of the PDL.

A new animal model for molecular biological analysis of the implant-tissue interface: spatial expression of type XII collagen mRNA around a titanium oral implant.

The objective of this study was to develop an animal model to investigate the molecular biological healing events at the tissue-implant surface occurring in the alveolar bone. Newly designed mini-titanium implants (2mm in length and 1 mm in diameter) were placed in the maxilla of retired-breeder male Sprague-Dawley rats. The implants were placed in freshly drilled holes in the maxillary bone, or in an area close to the roots of the maxillary first molar. The healing phase in each group was studied histologically at 28 days and at 56 days by means of non-decalcified polymethylmethacrylate-embedded sections and decalcified paraffin-embedded sections. Initial osseointegration was observed at 28 days, with mature osseointegration seen at 56 days. Specimens with implants placed immediately adjacent to the root showed fibrous healing at the implant-tissue surface. As a pilot study, the expression of type XII collagen, a molecular marker specific to the mature periodontal ligament (PDL), was studied by in situ hybridization. There was an absence of type XII expression close to the implant surface, whereas there was a zone of type XII collagen expression closest to the bony wall. Our preliminary results indicated a significant molecular variation in the fibrous-implant interface. This model will be useful in studies of the wound-healing patterns of the extracellular matrix around oral implants specifically relevant to alveolar bone osseointegration and potential formation of PDL.

Site-specific expression of collagen I and XII mRNAs in the rat periodontal ligament at two developmental stages.

In mammals, the periodontal ligament (PDL) is a highly specialized tissue which facilitates tooth eruption and lends mechanical support to the tooth once in occlusion. The PDL extracellular matrix fibers play a major role in such functions. During its development, the spatial arrangement of the PDL extracellular matrix undergoes rapid changes. So that it could be determined whether the structural alteration in the PDL is associated with changes in the expression of collagenous proteins with different functional properties, the transcriptional patterns of collagens I and XII were examined. The maxillary dento-alveolar segments, each containing three molars, from 25-day-old and 40-day-old Sprague-Dawley rats were selected as being representative of developing and matured tissues, respectively. Rat alpha 2(I) collagen cDNA and rat alpha 1(XII) collagen cDNA were used as molecular probes for identification of the corresponding mRNAs by RNA transfer blot analysis, RNase protection assay, and in situ hybridization. The results showed that alpha 2(I) collagen mRNA was expressed in both developing and matured tissues. However, the level of expression decreased with maturity. In contrast, the expression of alpha 1(XII) collagen was increased in the matured tissue as compared with the developing tissue. In situ hybridization in these tissues indicated that the expression of alpha 1(XII) collagen mRNA was limited to the mature stage of PDL development. It is suggested that collagen fibril arrangement during PDL development may be related to the expression of collagen XII.