Regulation of the autophagy-marker Sequestosome 1 in periodontal cells and tissues by biomechanical loading.

PURPOSE: Orthodontic treatment is based on the principle of force application to teeth and subsequently to the surrounding tissues and periodontal cells. Sequestosome 1 (SQSTM1) is a well-known marker for autophagy, which is an important cellular mechanism of adaptation to stress. The aim of this study was to analyze whether biomechanical loading conditions regulate SQSTM1 in periodontal cells and tissues, thereby providing further information on the role of autophagy in orthodontic tooth movement. METHODS: Periodontal ligament (PDL) fibroblasts were exposed to cyclic tensile strain of low magnitude (3%, CTSL), and the regulation of autophagy-associated targets was determined with an array-based approach. SQSTM1 was selected for further biomechanical loading experiments with dynamic and static tensile strain and assessed via real-time polymerase chain reaction (RT-PCR) and immunoblotting. Signaling pathways involved in SQSTM1 activation were analyzed by using specific inhibitors, including an autophagy inhibitor. Finally, SQSTM1 expression was analyzed in gingival biopsies and histological sections of rats in presence and absence of orthodontic forces. RESULTS: Multiple autophagy-associated targets were regulated by CTSL in PDL fibroblasts. All biomechanical loading conditions tested increased the SQSTM1 expression significantly. Stimulatory effects of CTSL on SQSTM1 expression were diminished by inhibition of the c­Jun N­terminal kinase (JNK) pathway and of autophagy. Increased SQSTM1 levels after CTSL were confirmed by immunoblotting. Orthodontic force application also led to significantly elevated SQTSM1 levels in the gingiva and PDL of treated animals as compared to control. CONCLUSIONS: Our in vitro and in vivo findings provide evidence of a role of SQSTM1 and thereby autophagy in orthodontic tooth movement.

Damage-regulated autophagy modulator 1 in oral inflammation and infection.

OBJECTIVES: Damage-regulated autophagy modulator (DRAM) 1 is a p53 target gene with possible involvement in oral inflammation and infection. This study sought to examine the presence and regulation of DRAM1 in periodontal diseases. MATERIAL AND METHODS: In vitro, human periodontal ligament fibroblasts were exposed to interleukin (IL)-1ß and Fusobacterium nucleatum for up to 2 days. The DRAM1 synthesis and its regulation were analyzed by real-time PCR, immunocytochemistry, and ELISA. Expressions of other autophagy-associated genes were also studied by real-time PCR. In vivo, synthesis of DRAM1 in gingival biopsies from rats and patients with and without periodontal disease was examined by real-time PCR and immunohistochemistry. For statistics, ANOVA and post-hoc tests were applied (p < 0.05). RESULTS: In vitro, DRAM1 was significantly upregulated by IL-1ß and F. nucleatum over 2 days and a wide range of concentrations. Additionally, increased DRAM1 protein levels in response to both stimulants were observed. Autophagy-associated genes ATG3, BAK1, HDAC6, and IRGM were also upregulated under inflammatory or infectious conditions. In vivo, the DRAM1 gene expression was significantly enhanced in rat gingival biopsies with induced periodontitis as compared to control. Significantly increased DRAM1 levels were also detected in human gingival biopsies from sites of periodontitis as compared to healthy sites. CONCLUSION: Our data provide novel evidence that DRAM1 is increased under inflammatory and infectious conditions in periodontal cells and tissues, suggesting a pivotal role of DRAM1 in oral inflammation and infection. CLINICAL RELEVANCE: DRAM1 might be a promising target in future diagnostic and treatment strategies for periodontitis.

Role of Cathepsin S in Periodontal Inflammation and Infection.

Cathepsin S is a cysteine protease and regulator of autophagy with possible involvement in periodontitis. The objective of this study was to investigate whether cathepsin S is involved in the pathogenesis of periodontal diseases. Human periodontal fibroblasts were cultured under inflammatory and infectious conditions elicited by interleukin-1ß and Fusobacterium nucleatum, respectively. An array-based approach was used to analyze differential expression of autophagy-associated genes. Cathepsin S was upregulated most strongly and thus further studied in vitro at gene and protein levels. In vivo, gingival tissue biopsies from rats with ligature-induced periodontitis and from periodontitis patients were also analyzed at transcriptional and protein levels. Multiple gene expression changes due to interleukin-1ß and F. nucleatum were observed in vitro. Both stimulants caused a significant cathepsin S upregulation. A significantly elevated cathepsin S expression in gingival biopsies from rats with experimental periodontitis was found in vivo, as compared to that from control. Gingival biopsies from periodontitis patients showed a significantly higher cathepsin S expression than those from healthy gingiva. Our findings provide original evidence that cathepsin S is increased in periodontal cells and tissues under inflammatory and infectious conditions, suggesting a critical role of this autophagy-associated molecule in the pathogenesis of periodontitis.

Regulation of p53 under hypoxic and inflammatory conditions in periodontium.

OBJECTIVES: Different studies suggest that inflammation as well as hypoxia leads to an increase of p53 protein levels. However, the implication of p53 during oral inflammatory processes is still unknown. The aim of this study was therefore to investigate the effect of hypoxia and inflammation on p53 regulation in human periodontium in vitro and in vivo. MATERIALS AND METHODS: Under hypoxic and normoxic conditions, human primary periodontal ligament (PDL) fibroblasts (n = 9) were stimulated with lipopolysaccharides (LPS) from Porphyromonas gingivalis (P.g.), a periodontal pathogenic bacterium. After different time points, cell viability was tested; p53 gene expression, protein synthesis, and activation were measured using quantitative RT-PCR, immunoblotting, and immunofluorescence. Moreover, healthy and inflamed periodontal tissues were obtained from 12 donors to analyze p53 protein in oral inflammatory diseases by immunohistochemistry. RESULTS: LPS-P.g. and hypoxia initially induced a significant upregulation of p53 mRNA expression and p53 protein levels. Nuclear translocation of p53 after inflammatory stimulation supported these findings. Hypoxia first enhanced p53 levels, but after 24 h of incubation, protein levels decreased, which was accompanied by an improvement of PDL cell viability. Immunohistochemistry revealed an elevation of p53 immunoreactivity in accordance to the progression of periodontal inflammation. CONCLUSIONS: Our data indicate that p53 plays a pivotal role in PDL cell homeostasis and seems to be upregulated in oral inflammatory diseases. CLINICAL RELEVANCE: Upregulation of p53 may promote the destruction of periodontal integrity. A possible relationship with carcinogenesis may be discussed.

Hypoxia and P. gingivalis synergistically induce HIF-1 and NF-κB activation in PDL cells and periodontal diseases.

Periodontitis is characterized by deep periodontal pockets favoring the proliferation of anaerobic bacteria like Porphyromonas gingivalis (P. gingivalis), a periodontal pathogen frequently observed in patients suffering from periodontal inflammation. Therefore, the aim of the present study was to investigate the signaling pathways activated by lipopolysaccharide (LPS) of P. gingivalis (LPS-PG) and hypoxia in periodontal ligament (PDL) cells. The relevant transcription factors nuclear factor-kappa B (NF-κB) and hypoxia inducible factor-1 (HIF-1) were determined. In addition, we analyzed the expression of interleukin- (IL-) 1ß, matrix metalloproteinase-1 (MMP-1), and vascular endothelial growth factor (VEGF) in PDL cells on mRNA and protein level. This was accomplished by immunohistochemistry of healthy and inflamed periodontal tissues. We detected time-dependent additive effects of LPS-PG and hypoxia on NF-κB and HIF-1α activation in PDL cells followed by an upregulation of IL-1ß, MMP-1, and VEGF expression. Immunohistochemistry performed on tissue samples of gingivitis and periodontitis displayed an increase of NF-κB, HIF-1, and VEGF immunoreactivity in accordance with disease progression validating the importance of the in vitro results. To conclude, the present study underlines the significance of NF-κB and HIF-1α and their target genes VEGF, IL-1ß, and MMP-1 in P. gingivalis and hypoxia induced periodontal inflammatory processes.

Evaluation of implant-materials as cell carriers for dental stem cells under in vitro conditions.

BACKGROUND: Dental stem cells in combination with implant materials may become an alternative to autologous bone transplants. For tissue engineering different types of soft and rigid implant materials are available, but little is known about the viability and the osteogenic differentiation of dental stem cells on these different types of materials. According to previous studies we proposed that rigid bone substitute materials are superior to soft materials for dental tissue engineering. METHODS: We evaluated the proliferation, the induction of apoptosis and the osteogenic differentiation of dental stem/progenitor cells on a synthetic bone-like material and on an allograft product. The soft materials silicone and polyacrylamide (PA) were used for comparison. Precursor cells from the dental follicle (DFCs) and progenitor cells from the dental apical papilla of retained third molar tooth (dNC-PCs) were applied as dental stem cells in our study. RESULTS: Both dental cell types attached and grew on rigid bone substitute materials, but they did not grow on soft materials. Moreover, rigid bone substitute materials only sustained the osteogenic differentiation of dental stem cells, although the allograft product induced apoptosis in both dental cell types. Remarkably, PA, silicone and the synthetic bone substitute material did not induce the apoptosis in dental cells. CONCLUSIONS: Our work supports the hypothesis that bone substitute materials are suitable for dental stem cell tissue engineering. Furthermore, we also suggest that the induction of apoptosis by bone substitute materials may not impair the proliferation and the differentiation of dental stem cells.

Impact of radiation history, gender and age on bone quality in sites for orthodontic skeletal anchorage device placement.

AIMS: Stability of orthodontic miniscrew implants is prerequisite to their success and durability in orthodontic treatment. As investigations revealed a positive correlation of miniscrew stability to periimplant bone quality, it has been the aim of this study to analyze the bone structure of resection preparations of human mandibles histologically by investigating the samples according to age, gender and exposure to radiotherapy. METHODS: Inflammation- and tumor-free alveolar bone sections from human mandibles (n = 31) with previously diagnosed carcinoma, chronic osteomyelitis or cysts were analyzed histomorphologically and histomorphometrically as to the dimension of trabeculae in cancellous areas. Group A investigated the impact of a history of radiation therapy, group B of gender and group C contrasted biopsies from individuals aging under 60 or over 60 years. Statistics were performed using the Kruskal-Wallis-test. RESULTS: Radiation, gender and age did not significantly influence bone density. The mean bone density averaged 40.7 ± 15.0% of spongiosa for the total collective with a median age of 58.4 years ± 14.7 years. CONCLUSIONS: Our findings provide new information on bone quality, thus contributing to a more precise evaluation of the parameters affecting and those not affecting miniscrew implant stability. On the basis of these results, the formulation of clinical guidelines for risk assessment of therapeutic approaches in patients prior to insertion of orthodontic skeletal anchorage devices seems to be conceivable.

Effect of growth factors on antimicrobial peptides and pro-inflammatory mediators during wound healing.

BACKGROUND: Antimicrobial peptides (AMPs), such as human beta-defensin-2 (hBD-2) and the CC-chemokine ligand 20 (CCL20), exhibit direct microbicidal effects and mediator-like activity. It was hypothesized that wounding induces the expression of AMPs and pro-inflammatory mediators and that endogenous mediators, such as insulin-like growth factor-1 (IGF-1) and transforming growth factor-alpha (TGF-alpha), modulate this induced expression. MATERIAL AND METHODS: Monolayers of gingival epithelial cells (GECs) and gingival fibroblast (HGFs) from three different donors were wounded using the scratch assay (in vitro wounding) in the presence (test group) or absence (control group) of IGF-1 and TGF-alpha. In vitro wound closure was monitored over time (0, 6, 24, 48, 72 h), and wound areas were microscopically analyzed (Axio-Vision® Software, Zeiss). Gene expression analysis of the GAPDH, hBD-2, CCL20, interleukin-1 beta (IL-1 beta), and interleukin-8 (IL-8) was performed by qPCR. RESULTS: In comparison to control cells, IGF-1 and TGF-alpha significantly enhanced in vitro wound closure (P < 0.05). In GECs, IGF-1 induced the gene expression of IL-1 beta and IL-8 when compared to control cells (P < 0.05). In HGFs, wounding per se induced the messenger RNA of hBD-2, CCL20, and IL-1 beta, whereas IGF-1 and TGF-alpha reversed this effect (P < 0.05). CONCLUSION: In gingival cells, the gene expression of AMPs was altered by injury, and endogenous growth factors further influenced the expression profiles, but with high interindividual differences.

LPS from P. gingivalis and hypoxia increases oxidative stress in periodontal ligament fibroblasts and contributes to periodontitis.

Oxidative stress is characterized by an accumulation of reactive oxygen species (ROS) and plays a key role in the progression of inflammatory diseases. We hypothesize that hypoxic and inflammatory events induce oxidative stress in the periodontal ligament (PDL) by activating NOX4. Human primary PDL fibroblasts were stimulated with lipopolysaccharide from Porphyromonas gingivalis (LPS-PG), a periodontal pathogen bacterium under normoxic and hypoxic conditions. By quantitative PCR, immunoblot, immunostaining, and a specific ROS assay we determined the amount of NOX4, ROS, and several redox systems. Healthy and inflamed periodontal tissues were collected to evaluate NOX4 and redox systems by immunohistochemistry. We found significantly increased NOX4 levels after hypoxic or inflammatory stimulation in PDL cells (P < 0.001) which was even more pronounced after combination of the stimuli. This was accompanied by a significant upregulation of ROS and catalase (P < 0.001). However, prolonged incubation with both stimuli induced a reduction of catalase indicating a collapse of the protective machinery favoring ROS increase and the progression of inflammatory oral diseases. Analysis of inflamed tissues confirmed our hypothesis. In conclusion, we demonstrated that the interplay of NOX4 and redox systems is crucial for ROS formation which plays a pivotal role during oral diseases.

Bone substitute material composition and morphology differentially modulate calcium and phosphate release through osteoclast-like cells.

The aim of this study was to determine the material composition and cell-mediated remodelling of different calcium phosphate-based bone substitutes. Osteoclasts were cultivated on bone substitutes (Cerabone, Maxresorb, and NanoBone) for up to 5 days. Bafilomycin A1 addition served as the control. To determine cellular activity, the supernatant content of calcium and phosphate was measured by inductively coupled plasma optical emission spectrometry. Cells were visualized on the materials by scanning electron microscopy. Material composition and surface characteristics were assessed by energy-dispersive X-ray spectroscopy. Osteoclast-induced calcium and phosphate release was material-specific. Maxresorb exhibited the highest ion release to the medium (P = 0.034; calcium 40.25mg/l day 5, phosphate 102.08 mg/l day 5) and NanoBone the lowest (P = 0.021; calcium 8.43 mg/l day 5, phosphate 15.15 mg/l day 5); Cerabone was intermediate (P = 0.034; calcium 16.34 mg/l day 5, phosphate 30.6 mg/l day 5). All investigated materials showed unique resorption behaviours. The presented methodology provides a new perspective on the investigation of bone substitute biodegradation, maintaining the material-specific micro- and macrostructure.

Autoregulation of insulin-like growth factor 2 and insulin-like growth factor-binding protein 6 in periodontal ligament cells in vitro.

The insulin-like growth factor (IGF) system plays an important role in tissue development and presumably also governs pathophysiology of the periodontal ligament (PDL). It has been the aim of this study to elucidate the specific expression pattern of IGF2 and IGFBP6 in PDL cells and to determine whether PDL cells feature autoregulatory mechanisms upon exposure to these IGF components. Human PDL cells (n=6) were exposed to IGF2 (100 ng/ml), IGFBP6 (450 ng/ml, 675 ng/ml, 1125 ng/ml) or a combination of 100 ng/ml IGF2 and 675 ng/ml IGFBP6 for 1, 3 or 5d. qRT-PCR was run for IGF2, IGFBP6, Ki67, ALP, osteocalcin. Immunocytochemical quantification was performed for IGF2 and IGFBP6. Results showed a time-dependent increase in IGF2 and IGFBP6 gene expression, as opposed to a general decrease at the protein level. At the transcriptional and protein level, challenge with IGF2 and IGFBP6 dampened the expression of both molecules at all time points investigated. Only in the case of IGF2 did combined treatment with IGF2 and IGFBP6 contrarily increased protein expression in both nuclear and cytoplasmatic structures compared to the vehicle treated controls. Analyses of PDL cell proliferation and differentiation revealed Ki67 downregulation by IGF2 and IGFBP6 alone or in combination. Beyond this, the osteogenic differentiation potential of PDL cells was suppressed as ALP and osteocalcin expression was reduced. Our results indicate that IGF2 and IGFBP6 appear to govern various regulatory feedback mechanisms in PDL cells. Thus, the functional properties of these molecules in oral structures are presumably self-controlled under impact of different biological processes such as expression levels of these IGF components, cell proliferation and differentiation.

In vivo differentiation of human periodontal ligament cells leads to formation of dental hard tissue.

OBJECTIVE: Following trauma, periodontal disease, or orthodontic tooth movement, residual periodontal ligament (PDL) cells at the defect site are considered mandatory for successful regeneration of the injured structures. Recent developments in tissue engineering focus, as one pillar, on the transplantation of PDL cells to support periodontal regeneration processes. Here, we examined the ability of osteogenically predifferentiated PDL cells to undergo further osteoblastic or cementoblastic differentiation and to mineralize their extracellular matrix when transplanted in an in vivo microenvironment. MATERIALS AND METHODS: Using collagen sponges as carriers, osteogenically predifferentiated human PDL cells were transplanted subcutaneously into six immunocompromised CD-1® nude mice. Following explantation after 28 days, osteogenic and cementogenic marker protein expression was visualized immunohistochemically. RESULTS: After 28 days, transplanted PDL cells revealed both cellular, cytoplasmatic and extracellular immunoreactivity for the chosen markers alkaline phosphatase, osteopontin, PTH-receptor 1, and osteocalcin. Specific osteogenic and cementoblastic differentiation was demonstrated by RUNX2 and CEMP1 immunoreactivity. Early stages of mineralization were demonstrated by calcium and phosphate staining. CONCLUSION: Our results reinforce the previously published reports of PDL cell mineralization in vivo and further demonstrate the successful induction of specific osteogenic and cementogenic differentiation of transplanted human PDL cells in vivo. These findings reveal promising possibilities for supporting periodontal remodeling and regeneration processes with PDL cells being potential target cells with which to influence the process of orthodontically induced root resorption.

Continuous PTH modulates alkaline phosphatase activity in human PDL cells via protein kinase C dependent pathways in vitro.

Periodontal ligament (PDL) cells, a major component of the tooth supporting apparatus, share osteoblastic characteristics including their responsiveness to parathyroid hormone (PTH). Clinical studies have already pointed to the benefit of PTH in supporting regenerative processes in the craniofacial region. However, those reports did not analyze which cells mediated the PTH effect on the alveolar bone. The aim of the present study has been to further elucidate the mechanism of action of continuous PTH application on human PDL-cells mimicking a local bolus application and to analyze its intracellular signalling pathways to widen the theoretical basis for future development of reliable local PTH delivery protocols. Analyses of PDL of extracted human teeth as well as cultured human PDL-cells demonstrated strong expression of PTH-receptor-1 by immune fluorescencecytochemistry/histochemistry. To examine the effect of short time continuous PTH treatment on PDL-cell osteogenic differentiation, PDL-cells were stimulated for 48h. Analyses for mRNA and protein expression of the early osteogenic marker alkaline-phosphatase revealed an enhanced expression. Pathways analyses mediating the PTH effect resulted in a similar effect when PDL-cells were stimulated with either the signal specific fragments lacking the PKA-activating domain PTH(3-34), PTH(7-34), second-messenger-analogues PKC (PMA) or inhibitors for PKA (H8). Inhibition of the PKC-dependent pathway by stimulation with PTH(1-31), PKA second-messenger-analogue (forskolin) or PKA-inhibitor (RO-32-0432) abolished the PTH effect These data indicate abundant expression of PTH1R within the PDL and a stimulatory effect of short time continuous PTH on PDL cell differentiation towards an osteogenic phenotype and suggested local PTH application protocols as a possible treatment option in periodontal therapy.

Gene analysis of signal transduction factors and transcription factors in periodontal ligament cells following application of dynamic strain.

OBJECTIVE: Orthodontic treatment is usually associated with the application of forces to teeth and periodontium. Instrumental in transmitting these forces are the cells of the periodontal ligament (PDL). In the present study, we used an established strain model to investigate the potential role of biophysical stimulation in modulating the gene expression pattern of these PDL cells. MATERIALS AND METHODS: PDL cells derived from non-carious and periodontally healthy teeth of six patients were grown on culture plates coated with collagen type I. Upon completion of culture, dynamic strain was applied to the cells for 24 h, using 3% of tensile force and a frequency of 0.05 Hz. This loading protocol for biomechanical stimulation was followed by extracting the RNA from the cells and using a RT(2) PCR array(®) for analysis. RESULTS: Compared to non-stimulated control cells, this analysis revealed the induction of several factors (e.g., RELA, IRF1, MAX, MYC, CDKN1B, BCL2, BCL2A1) known to influence tissue homeostasis by contributing essentially to cell proliferation, cell differentiation, and the inhibition of apoptosis. CONCLUSION: This study demonstrates that the biomechanical stimulation of PDL cells is an important factor in periodontal tissue homeostasis.

Osteoimmunological mechanisms involved in orthodontically and bacterially induced periodontal stress.

BACKGROUND AND OBJECTIVE: Orthodontic tooth movement is known to cause sterile inflammation of the periodontal ligament (PDL). It may also be accompanied by pathological effects of external apical root resorption, with interindividual differences in the incidence and extent of resorption. An involvement of autoimmunological mechanisms is currently under discussion. This study aimed to improve our understanding of similarities between the inflammatory mechanisms underlying the pathophysiology of periodontitis and root resorption. MATERIALS AND METHODS: Human PDL cells were stimulated with interleukin (IL)-1ß/IL-17A/IFN-γ, or left non-stimulated. Their potential for phagocytosis was then evaluated by incubation with dextran or E. coli or S. aureus particles, followed by flow cytometric and immunohistochemical analysis. Real-time polymerase chain reaction (PCR) was used to analyze receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) expression in PDL cells. Verification was obtained in vivo by studying IL-17A, RANKL, and OPG expression in biopsies of inflamed periodontal tissues and in biopsies of rat maxillae with mechanically induced root resorption. Statistical analysis included Wilcoxon's rank sum test to analyze gene expression data and one-way ANOVA in conjunction with Tukey's post hoc test to analyze flow cytometric data. RESULTS: PDL cells phagocytosed foreign particles under both inflammatory and non-inflammatory conditions. Furthermore, IL-17A significantly downregulated RANKL expression while significantly upregulating OPG expression in PDL cells. These immunomodulatory cytokines were also demonstrable in both inflammatorily altered periodontal tissues and root resorption lacunae, while the incidence of IL-7A was strikingly variable in resorption areas. CONCLUSION: PDL cells were demonstrated to effect phagocytosis and to express immunomodulatory molecules, which proves their capability of participating in periodontal osteoimmunological processes. The development of root resorption and periodontitis appears to be governed by similar pathophysiological mechanisms.

IGF-1 deficiency in combination with a low basic hBD-2 and hBD-3 gene expression might counteract malignant transformation in pleomorphic adenomas in vitro.

This study investigated the IGF-1-influence on oncological relevant genes in pleomorphic adenomas. Therefore A64-tumor cells were stimulated by recombinant IGF-1. After RNA-extraction, transcript levels of hBD-1, hBD-2, hBD-3, DEFA1/3, DEFA4, S100A4, Psoriasin, DOC-1, EGF, EGFR, and IGFR were analyzed by qRT-PCR at t = 0, 4, 8, 24, 48, and 72 hr. The gene-products were visualized by immunostaining. A64-tumor-cells were deficient for hBD-1 and IGF-1. IGF-1 downregulates hBD-2 and hBD-3 without influencing hBD-1-expression. IGF-1 only slightly affects DEFA1/3-, DEFA4-, S100A4-, Psoriasin-, DOC-1-, EGF-, EGFR-, and IGFR-gene-expression. IGF-1-deficiency combined with low basic hBD-2-gene-expression and hBD-3-gene-expression might counteract, whereas hBD-1-deficiency promotes malignant transformation in pleomorphic adenomas.

Localization of SOST/sclerostin in cementocytes in vivo and in mineralizing periodontal ligament cells in vitro.

BACKGROUND AND OBJECTIVE: Cementum and bone are rather similar hard tissues, and osteocytes and cementocytes, together with their canalicular network, share many morphological and cell biological characteristics. However, there is no clear evidence that cementocytes have a function in tissue homeostasis of cementum comparable to that of osteocytes in bone. Recent studies have established an important role for the secreted glycoprotein sclerostin, the product of the SOST gene, as an osteocyte-derived signal to control bone remodelling. In this study, we investigated the expression of sclerostin in cementocytes in vivo as well as the expression of SOST and sclerostin in periodontal ligament cell cultures following induction of mineralization. MATERIAL AND METHOD: Immunolocalization of sclerostin was performed in decalcified histological sections of mouse and human teeth and alveolar bone. Additionally, periodontal ligament cells from human donors were cultured in osteogenic conditions, namely in the presence of dexamethasone, ascorbic acid and beta-glycerophosphate, for up to 3 wk. The induction of calcified nodules was visualized by von Kossa stain. SOST mRNA was detected by real-time PCR, and the presence of sclerostin was verified using immunohistochemistry and western blots. RESULTS: Expression of sclerostin was demonstrated in osteocytes of mouse and human alveolar bone. Distinct immunolocalization in the cementocytes was shown. In periodontal ligament cultures, following mineralization treatment, increasing levels of SOST mRNA as well as of sclerostin protein could be verified. CONCLUSION: The identification of SOST/sclerostin in cementocytes and mineralizing periodontal ligament cells adds to our understanding of the biology of the periodontium, but the functional meaning of these findings can only be unravelled after additional in vitro and in vivo studies.

Anabolic effect of intermittent PTH(1-34) on the local microenvironment during the late phase of periodontal repair in a rat model of tooth root resorption.

This study examined the histological changes and possible effects of intermittent parathyroid hormone (PTH) (1-34) treatment during the early and late phase of periodontal repair in a rat model of tooth root resorption. In a total of 70 animals, which either received intermittent PTH(1-34) systemically or sham injections for up to 70 days after discontinuation of an orthodontic force, histological characteristics were correlated to time-dependent distinct expression patterns of osteoprotegerin and receptor activator of nuclear factor kappaB ligand by PDL cells in the former compression and tension side of tooth movement by means of immunohistochemistry and histomorphometrical analysis. The balance of these key regulators of bone remodeling was demonstrated to be shifted in favor of hard tissue repair by intermittent PTH administration, which was demonstrated to exert anabolic effects in several cell culture and animal experiments as well as in humans, in the late phase of repair. These data indicate a role for PDL cells as potent regulators of periodontal repair by modifying the local microenvironment and point to the anabolic potential of an intermittent PTH administration to support these reparative processes.

Nuclear hBD-1 accumulation in malignant salivary gland tumours.

BACKGROUND: Whereas the antimicrobial peptides hBD-2 and -3 are related to inflammation, the constitutively expressed hBD-1 might function as 8p tumour suppressor gene and thus play a key role in control of transcription and induction of apoptosis in malignant epithelial tumours. Therefore this study was conducted to characterise proteins involved in cell cycle control and host defence in different benign and malignant salivary gland tumours in comparison with healthy salivary gland tissue. METHODS: 21 paraffin-embedded tissue samples of benign (n = 7), and malignant (n = 7) salivary gland tumours as well as healthy (n = 7) salivary glands were examined immunohistochemically for the expression of p53, bcl-2, and hBD-1, -2, -3. RESULTS: HBD-1 was distributed in the cytoplasm of healthy salivary glands and benign salivary gland tumours but seems to migrate into the nucleus of malignant salivary gland tumours. Pleomorphic adenomas showed cytoplasmic as well as weak nuclear hBD-1 staining. CONCLUSION: HBD-1, 2 and 3 are traceable in healthy salivary gland tissue as well as in benign and malignant salivary gland tumours. As hBD-1 is shifted from the cytoplasm to the nucleus in malignant salivary gland tumours, we hypothesize that it might play a role in the oncogenesis of these tumours. In pleomorphic adenomas hBD-1 might be connected to their biologic behaviour of recurrence and malignant transformation.

Subculture affects the phenotypic expression of human periodontal ligament cells and their response to fibroblast growth factor-2 and bone morphogenetic protein-7 in vitro.

BACKGROUND AND OBJECTIVE: Although periodontal ligament cells display several osteoblastic traits, their phenotypic expression is still not well established. It remains a matter of debate whether they resemble a terminally differentiated cell type or an intermediate maturation state that potentially can be directed towards a fibroblastic or an osteoblastic phenotype. MATERIAL AND METHODS: To explore the characteristics of periodontal ligament cells in greater detail, fourth-passage, sixth-passage and eighth-passage human periodontal ligament cells were cultured for up to 3 wk. Ki-67, alkaline phosphatase, osteocalcin, osteoprotegerin and receptor activator of nuclear factor-kappaB ligand (RANKL) mRNA expression was quantified by real-time polymerase chain reaction. Furthermore, the cellular response to fibroblast growth factor-2 and bone morphogenetic protein-7 was examined in first-passage and fourth-passage cells. Dermal fibroblasts (1BR.3.G) and osteoblast-like cells (MG63) served as reference cell lines. RESULTS: Proliferation decreased over time and was highest in fourth-passage cells. The expression of differentiation parameters, osteoprotegerin and RANKL increased with culture time and was higher in fourth-passage cells than in cells of later passages. The RANKL/osteoprotegerin ratio increased steadily until day 21. Administration of fibroblast growth factor-2 enhanced cell numbers in both passages, whereas alkaline phosphatase and osteocalcin production remained unchanged. By contrast, exposure of periodontal ligament cells to bone morphogenetic protein-7 resulted in a reduction of cell number in the first and fourth passages, whereas the production of alkaline phosphatase and osteocalcin was enhanced. In dermal fibroblasts, differentiation parameters did not respond to both stimuli. MG63 cells behaved similarly to periodontal ligament cells. CONCLUSION: These results indicate that subculture affects the phenotypic expression of human periodontal ligament cells with respect to the characteristics that these cells share with osteoblasts. Furthermore, the periodontal ligament cell phenotype can be altered by fibroblastic and osteoblastic growth factors.