Dominik Kraus

Interactions of Adiponectin and Lipopolysaccharide from Porphyromonas gingivalis on Human Oral Epithelial Cells

Background Periodontitis is an inflammatory disease caused by pathogenic microorganisms, such as Porphyromonas gingivalis, and characterized by the destruction of the periodontium. Obese individuals have an increased risk for periodontitis and show decreased serum levels of adiponectin. This in-vitro study was established to examine whether adiponectin modulates critical effects of lipopolysaccharide (LPS) from P. gingivalis on oral epithelial cells (OECs). Methodology/Principal Findings The presence of adiponectin and its receptors in human gingival tissue samples and OECs was analyzed by immunohistochemistry and PCR. Furthermore, OECs were treated with LPS and/or adiponectin for up to 72 h, and the gene expression and protein synthesis of pro- and anti-inflammatory mediators, matrix metalloproteinases (MMPs) and growth factors were analyzed by real-time PCR and ELISA. Additionally, cell proliferation, differentiation and in-vitro wound healing were studied. The nuclear translocation of NFκB was investigated by immunofluorescence. Gingival tissue sections showed a strong synthesis of adiponectin and its receptors in the epithelial layer. In cell cultures, LPS induced a significant up-regulation of interleukin (IL) 1β, IL6, IL8, MMP1 and MMP3. Adiponectin abrogated significantly the stimulatory effects of LPS on these molecules. Similarly, adiponectin inhibited significantly the LPS-induced decrease in cell viability and increase in cell proliferation and differentiation. Adiponectin led to a time-dependent induction of the anti-inflammatory mediators IL10 and heme oxygenase 1, and blocked the LPS-stimulated NFκB nuclear translocation. Conclusions/Significance Adiponectin may counteract critical actions of P. gingivalis on oral epithelial cells. Low levels of adiponectin, as observed in obese individuals, may increase the risk for periodontal inflammation and destruction.

Human β-defensins differently affect proliferation, differentiation, and mineralization of osteoblast-like MG63 cells.

Purpose of this study was to investigate whether human β‐defensins (hBDs) affect maturation and proliferation of osteoblast‐like MG63 cells in vitro. Osteoblast‐like MG63 cells were stimulated with hBD‐1, ‐2, and ‐3 under control conditions and with hBD‐2 during experimental inflammation (induced by interleukin‐1β, tumor necrosis factor‐α, toll‐like receptor‐2 and ‐4 agonists). Expression of different osteogenic markers and hBDs were analyzed by real‐time PCR, immunohistochemistry, and enzyme‐linked immunosorbent assay. In addition, alkaline phosphatase (ALP) enzyme activity and biomineralization as markers for differentiation were monitored. All tested hBDs were expressed on mRNA and protein level in MG63 cells. Only stimulation with hBD‐2 elevated the proliferation rate. hBD‐2 and hBD‐3 positively affected the differentiation of osteoblast‐like cells provided by increased transcript levels of osteogenic markers, up‐regulated ALP enzyme activity and enhanced mineralized nodule formation. All pro‐inflammatory stimuli enhanced interleukin‐6 and hBD‐2 expression and down‐regulated markers of osteoblastic differentiation. In accordance, inflammation increased transcript level of Notch‐1 (an inhibitor of osteoblastic differentiation). hBD‐2 was not able to revert effects of inflammation on differentiation. In bone cells human β‐defensins exhibit further functions than antimicrobial peptide activity. These include stimulation of proliferation and differentiation. Differentiation arrest due to inflammation could not be overcome by hBD‐2 alone. J. Cell. Physiol. 227: 994–1003, 2012.

Human Beta-Defensin-1, -2, and -3 Exhibit Opposite Effects on Oral Squamous Cell Carcinoma Cell Proliferation

The objective of this study was to investigate the impact of human beta-defensins (hBDs) on oral squamous cell carcinoma (OSCC) proliferation and hBD expression in vitro. BHY-OSCC cell lines were stimulated with hBD-1, -2, and -3. Proliferation of BHY cells was ascertained and hBD-mRNA expression was evaluated by real-time PCR. Proliferation of BHY cells decreased by 25% in response to hBD-1 stimulation but increased after stimulation with hBD-2 and -3. HBD-1 stimulation enhanced hBD-3 expression, whereas HBD-2 stimulation decreased early hBD-3 expression. HBD-3 stimulation enhanced hBD-1 expression. HBDs profoundly impact on OSCC proliferation and hBD expression in vitro. Therefore, hBD-1 might function as a tumor suppressor gene in OSCCs, while hBD-2 and -3 might be protooncogenes.

Aging affects the phenotypic characteristics of human periodontal ligament cells and the cellular response to hormonal stimulation in vitro.

BACKGROUND AND OBJECTIVE Aging modulates the proliferative activity and organic matrix production of cells in vivo and in vitro. Here, we explore how aging affects the phenotypic characteristics of human periodontal ligament cells and their response to hormonal stimulation. MATERIAL AND METHODS Fifth passage periodontal ligament cells from subjects aged 12-14 (group 1), 41-55 (group 2) and 61-70 years (group 3) were characterized for the expression of mesenchymal marker genes and proteins by real-time PCR and flow cytometry. Confluent cultures were exposed to 10(-12) m parathyroid hormone(1-34) [PTH(1-34)] intermittently for three cycles. At harvest, cell number, alkaline phosphatase activity and osteocalcin production were determined by cell count, biochemical assay and ELISA. RESULTS The characterization of the cells revealed a decreased expression of osteoblast-specific marker genes along with a lower percentage of cells presenting the respective proteins with age. An intermittent exposure of the cultures to 10(-12) m PTH(1-34) induced an increase of the cell number as opposed to a significant decrease of alkaline phosphatase activity and osteocalcin production. The cellular response to PTH(1-34) was strongest in group 1. Basal osteoprotegerin levels were highest in the cultures from the oldest donors and inhibited by intermittent PTH(1-34) in all groups. CONCLUSION Our data indicate that periodontal ligament cells from older subjects display a less differentiated phenotype and a reduced response to intermittent PTH, suggesting a compromised ability to maintain tissue homeostasis and a limited possibility to support periodontal repair processes with age. The high basal osteoprotegerin expression in older subjects might serve as a compensatory mechanism.

Ghrelin promotes oral tumor cell proliferation by modifying GLUT1 expression

In our study, ghrelin was investigated with respect to its capacity on proliferative effects and molecular correlations on oral tumor cells. The presence of all molecular components of the ghrelin system, i.e., ghrelin and its receptors, was analyzed and could be detected using real-time PCR and immunohistochemistry. To examine cellular effects caused by ghrelin and to clarify downstream-regulatory mechanisms, two different oral tumor cell lines (BHY and HN) were used in cell culture experiments. Stimulation of either cell line with ghrelin led to a significantly increased proliferation. Signal transduction occurred through phosphorylation of GSK-3β and nuclear translocation of β-catenin. This effect could be inhibited by blocking protein kinase A. Glucose transporter1 (GLUT1), as an important factor for delivering sufficient amounts of glucose to tumor cells having high requirements for this carbohydrate (Warburg effect) was up-regulated by exogenous and endogenous ghrelin. Silencing intracellular ghrelin concentrations using siRNA led to a significant decreased expression of GLUT1 and proliferation. In conclusion, our study describes the role for the appetite-stimulating peptide hormone ghrelin in oral cancer proliferation under the particular aspect of glucose uptake: (1) tumor cells are a source of ghrelin. (2) Ghrelin affects tumor cell proliferation through autocrine and/or paracrine activity. (3) Ghrelin modulates GLUT1 expression and thus indirectly enhances tumor cell proliferation. These findings are of major relevance, because glucose uptake is assumed to be a promising target for cancer treatment.

Human α-defensin (DEFA) gene expression helps to characterise benign and malignant salivary gland tumours.

BackgroundBecause of the infrequence of salivary gland tumours and their complex histopathological diagnosis it is still difficult to exactly predict their clinical course by means of recurrence, malignant progression and metastasis. In order to define new proliferation associated genes, purpose of this study was to investigate the expression of human α-defensins (DEFA) 1/3 and 4 in different tumour entities of the salivary glands with respect to malignancy.MethodsTissue of salivary glands (n=10), pleomorphic adenomas (n=10), cystadenolymphomas (n=10), adenocarcinomas (n=10), adenoidcystic carcinomas (n=10), and mucoepidermoid carcinomas (n=10) was obtained during routine surgical procedures. RNA was extracted according to standard protocols. Transcript levels of DEFA 1/3 and 4 were analyzed by quantitative realtime PCR and compared with healthy salivary gland tissue. Additionally, the proteins encoded by DEFA 1/3 and DEFA 4 were visualized in paraffin-embedded tissue sections by immunohistochemical staining.ResultsHuman α-defensins are traceable in healthy as well as in pathological altered salivary gland tissue. In comparison with healthy tissue, the gene expression of DEFA 1/3 and 4 was significantly (p<0.05) increased in all tumours – except for a significant decrease of DEFA 4 gene expression in pleomorphic adenomas and a similar transcript level for DEFA 1/3 compared to healthy salivary glands.ConclusionsA decreased gene expression of DEFA 1/3 and 4 might protect pleomorphic adenomas from malignant transformation into adenocarcinomas. A similar expression pattern of DEFA-1/3 and -4 in cystadenolymphomas and inflamed salivary glands underlines a potential importance of immunological reactions during the formation of Warthin’s tumour.

Influence of the lubricant and the alloy on the wear behaviour of attachments

OBJECTIVES Wear of attachments leads to a loss of retention and reduces the function of overdentures. This study evaluated the retention force changes of an attachment system for overdentures. The influence of the lubricant and the alloy on wear constancy was examined. METHODS Cylindrical anchors of the Dalbo(®) -Z system were tested (Cendres+Métaux SA). Three groups of alloy-lubricant combinations were generated 1.Elitor(®) /NaCl-solution (EN) 2.Elitor(®) /Glandosane(®) aquadest. (EG) and 3.Valor(®) /Glandosane(®) /aquadest. (VG). Ten samples of each group were subjected to 10 000 insertion-separation cycles. RESULTS For the EN-group, this led to a large increase in retention force. The EG- and VG-group showed a constant decrease after an initial increase in retention force at the beginning of the wear simulation. The change of the alloy caused no statistically significant differences. The use of a more viscous lubricant reduced the retention force increase significantly. CONCLUSIONS The use of a lubricant which simulates clinical conditions is an absolute need for wear simulation because the retention force changes are influenced enormously. The change of the alloy at the Dalbo(®) -Z system did not influence the wear behaviour. As a slight decrease in retention force was recorded, it is useful for an attachment system to allow compensation with an adjustable matrix.

Conical crowns with electroplated gold copings: retention force changes caused by wear and combined off‐axial load

OBJECTIVES The aim of this study was to examine the wear behavior of conical crowns of gold alloy and zirconium dioxide ceramics facing electroplated gold copings. METHODS The conical crowns were milled in Group A of a cast gold alloy (Degunorm(®), DeguDent(®)) and B zirconium dioxide (Cercon(®), DeguDent(®)). Fifteen specimens were milled per group with a conicity of 2°. The electroplated coping was established by direct electroforming with the Solaris system (DeguDent(®)) and glued into a framework. An apparatus accomplishing 5000 wear cycles performed the wear test. After each of the cycles, extra axial forces (80 N) were loaded 1 million times on an extension of the framework. The retentive forces and the correlating distance during insertion and separation were measured. The wear test was separated in a start phase, an initial wear phase and the long-term wear period. The retention force value and the force-distance integral of the first 0.3 mm of each cycle were calculated. RESULTS The changes of retention force (ΔF) and integral (ΔF(d)) differed significantly between both groups in the start phase (ΔF: A -1.85 N, B +1.39 N, P=0.0028; ΔF(d): A -0.155 N mm, B +0.2685 N mm, P=0.0378) and the initial wear phase. The long-term wear period showed no differences in force and integral development. As a statistical test for the comparison of the median values of the two groups, the Mann-Whitney test was used. The median force never fell below 4 N or exceeded 12 N. CONCLUSIONS Within the limitations of this study, the conical crowns tested showed clinically acceptable retentive properties. The values of about 4-6 N mentioned as sufficient in the recent literature were always attained. As the most retentive elements for dentures, both groups showed the main changes in retentive forces during the first 2000 cycles. The retention force development of the zirconia group appears to be less excursive over the whole wear test.

Wear of double crown systems: electroplated vs. casted female part

Objectives The wear of telescopic crowns is a common problem often reducing the patients satisfaction with the denture and resulting in a renewal of the denture. The study aims to compare the wear behavior of conical crowns using electroplated copings (group E) with standard telescopic crowns with cast female parts (group C). Material and Methods 10 conical crowns were milled for each group of a cast gold alloy. The specimen of group E had a conicity of 2º. The cast secondary crowns of group C had a 0º design. The electroplated coping was established by direct electroforming. An apparatus accomplishing 10,000 wear cycles performed the wear test. The retentive forces and the correlating distance during insertion and separation were measured. The wear test was separated in a start phase, an initial wear phase and the long term wear period. The retention force value and the force-distance integral of the first 0.33 mm of each cycle were calculated. Results The retentive forces were significantly higher for group E and the integrals were significantly lower for this group except the integral at cycle 10,000. The changes of retention force and integral did not differ significantly between both groups in all phases. The change of the integrals as well as the integral at the particular cycles showed higher interquartile distances for group C. Conclusions Within the limitations of this study the tested conical crowns showed clinically acceptable retentive properties. The values reached a range comparable to retentive elements tested in recent literature. The values of group C showed higher ranges. The force measured for group E was significantly higher than for group C but the integrals showed an opposite tendency. The results indicate that an exclusive analysis of the force is not sufficient as the integral is not equivalent to the force although it describes the retentive property of the system in a better way than the force over a distance is described. Both systems seem to be suitable for clinical practice.

Intermittent administration of PTH(1-34) regulates the osteoblastic differentiation of human periodontal ligament cells via protein kinase C- and protein kinase A-dependent pathways in vitro.

BACKGROUND AND OBJECTIVE Intermittent parathyroid hormone (PTH) is recognized as an anabolic agent in regenerative treatment strategies for bony tissues. Periodontal ligament (PDL) cells share features that are typical of osteoblasts, including an osteoblast-like response to stimulation with PTH, which implies a role for these cells in the regulation of repair processes following inflammatory periodontal disease. In the present study we explored the effect of intermittent administration of a PTH fragment [PTH(1-34)] on the osteoblastic differentiation of human PDL cells in vitro, and we investigated the signaling pathways used by the cells to mediate this effect. MATERIAL AND METHODS PDL cells at two stages of confluence were characterized and used as a model for the role of cell maturation in the cellular response. RESULTS In preconfluent, less mature cultures, intermittent administration of PTH(1-34) and PTH(1-31) fragments increased alkaline phosphatase (ALP) activity and osteocalcin production, whereas intermittent administration of PTH(3-34) and PTH(7-34) had no effect. RO-32-0432, a specific protein kinase C inhibitor, did not inhibit the PTH(1-34) effect, whereas the protein kinase A inhibitor, H8, antagonized the PTH(1-34)-induced increase in ALP activity and osteocalcin. In contrast, in confluent, more mature cultures, intermittent administration of PTH(1-34), PTH(3-34) and PTH(7-34) fragments, but not of the PTH(1-31) fragment, decreased ALP activity, and osteocalcin and RO-32-0432, but not H8, inhibited the effect. CONCLUSIONS This study showed that the PTH(1-34) effect on ALP activity and osteocalcin production in human PDL cells is maturation state-dependent and specific in terms of the pathways involved. Whereas in less mature cells the PTH effect is associated with cyclic AMP/protein kinase A-dependent signaling, more mature cells seem to mediate the PTH signal primarily via protein kinase C-dependent pathways.