CD8+ T cells mediate the regenerative PTH effect in hPDL cells via Wnt10b signaling.

It was the aim of the present investigation to examine whether the stimulating effect of parathyroid hormone (PTH) on human periodontal ligament (hPDL) cell proliferation and differentiation would be enhanced by hPDL/T-cell interaction involving Wnt10b signaling as a mediating pathway. hPDL cells were cultured from healthy premolar tissues of three adolescent orthodontic patients and exposed to PTH(1-34) in monocultures or co-cultures with CD8+ T cells. At harvest, proliferation, alkaline phosphatase-specific activity (ALP), and osteocalcin production were determined by immunofluorescence cytochemistry, real-time PCR, biochemical assay, and ELISA. Wnt10b signaling was analyzed by the use of a specific WNT10b neutralizing antibody. PTH(1-34) stimulation of T cells significantly increased Wnt10b expression and production. Wnt10b exposure of hPDL cells enhanced proliferation and differentiation. PDL cells co-cultured with T cells showed a Wnt10b-dependent regulation of proliferation and differentiation parameters. The addition of a Wnt10b-neutralizing Ab to the co-culture medium resulted in a significant inhibition of the PTH(1-34) effect on proliferation, ALP-specific activity, and osteocalcin protein expression. Our findings provide novel insight into the mechanism of action of PTH on hPDL cells and establish the interplay of T cells and hPDL cells via the Wnt10b pathway as a modulating factor for the anabolic properties of the hormone in periodontal regeneration.

Short-term heat pre-treatment modulates the release of HMGB1 and pro-inflammatory cytokines in hPDL cells following mechanical loading and affects monocyte behavior.

OBJECTIVE: Heat shock proteins (HSP) act as cell-protective molecules that are upregulated upon thermal insult, hypoxia, and ischemia. Such ischemic conditions can be found during tissue remodeling associated with orthodontic tooth movement or trauma when compression forces lead to cell necrosis and subsequent clearance of cellular debris by immune competent cells. Host immune overreaction can result in undesired side effects such as tooth root resorption. Here, we analyzed whether heat pre-treatment would affect the initially catabolic host immune response induced by mechanical loading of human periodontal ligament (hPDL) cells, which represent major constituents of the tooth supporting apparatus involved in the regulation of periodontal remodeling. MATERIALS AND METHODS: Fifth passage hPDL cells were exposed to an elevated temperature of 43° for 1 h prior to mechanical loading. Cell morphology, high mobility group box protein 1 (HMGB1), interleukin (IL)-6, and IL-8 expression were analyzed microscopically and by ELISA. The physiological relevance for monocyte behavior was tested in monocyte adhesion and osteoclast differentiation assays. RESULTS: Short-term heat pre-treatment did not show any visible effect on hPDL cell morphology, but resulted in a significant downregulation of pro-inflammatory cytokines when being additionally loaded mechanically. Supernatants of heat-exposed hPDL cell cultures demonstrated a reduced impact on monocyte adhesion and osteoclastic differentiation. CONCLUSIONS: Heat pre-treatment of hPDL cells induces cell-protective mechanisms towards mechanical stress and favors the reduction of cell stress associated effects on monocyte/macrophage physiology. CLINICAL RELEVANCE: These data present the induction of heat shock proteins as a promising treatment option to limit undesired side effects of periodontal remodeling.

Reference genes for valid gene expression studies on rat dental, periodontal and alveolar bone tissue by means of RT-qPCR with a focus on orthodontic tooth movement and periodontitis.

OBJECTIVES: To obtain valid results in relative gene/mRNA-expression analyses by RT-qPCR, a careful selection of stable reference genes is required for normalization. Currently there is little information on reference gene stability in dental, periodontal and alveolar bone tissues of the rat, especially regarding orthodontic tooth movement and periodontitis. We therefore aimed to identify the best selection and number of reference genes under these experimental as well as physiological conditions. MATERIALS AND METHODS: In 7 male Fischer344-rats the upper left first and second molars were moved orthodontically for 2 weeks and in 7 more animals additionally subjected to an experimental periodontitis, whereas 7 animals were left untreated. Tissue samples of defined size containing both molars (without crowns) as well as the adjacent periodontal and alveolar bone tissue were retrieved and RNA extracted for RT-qPCR analyses. Nine candidate reference genes were evaluated and ranked according to their expression stability by 4 different algorithms (geNorm, NormFinder, BestKeeper, comparative ΔCq). RESULTS: PPIB/YWHAZ were the most stabile reference genes for the combined dental, periodontal and alveolar bone tissue of the rat overall, in untreated animals and rats with additional periodontitis, whereas PPIB/B2M performed best in orthodontically treated rats with YWHAZ ranking third. Gene-stability ranking differed considerably between investigated groups. A combination of two reference genes was found to be sufficient for normalization in all cases. CONCLUSIONS: The substantial differences in expression stability emphasize the need for valid reference genes, when aiming for meaningful results in relative gene expression analyses. Our results should enable researchers to optimize gene expression analysis in future studies by choosing the most suitable reference genes for normalization.

Orthodontic forces add to nicotine-induced loss of periodontal bone : An in vivo and in vitro study.

OBJECTIVES: Nicotine is considered an etiologic factor for chronic inflammatory phenomena within the periodontal ligament that may result in loss of periodontal attachment. Considering that smokers account for 26% of adult and 12% of adolescent patients in orthodontic practice, we performed in vivo and in vitro studies as to whether orthodontic forces may add to the nicotine-induced loss of periodontal bone. METHODS: Fourteen male rats (Fischer 344 inbred) were used. Seven of these served as controls, while the other seven received daily subcutaneous injections of 1.89 mg L-nicotine per kg body weight. Both groups were exposed to orthodontic mesialization of the first two upper left molars using a NiTi closed-coil spring, the contralateral side serving as control. Periodontal bone loss was assessed by cone-beam computed tomography (CBCT). Human periodontal fibroblasts were stressed by compression (2 g/cm(2)) and/or nicotine (3/5/7.5 µmol), and the expression of cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), interleukin-6 (IL-6), osteoprotegerin (OPG), and receptor activator of nuclear factor κB ligand (RANKL) was determined at the transcriptional level by quantitative real-time polymerase chain reaction (qRT-PCR) and at the translational level by enzyme-linked immunosorbent assay (ELISA). In addition, differentiation of co-cultured murine RAW264.7 cells to osteoclast-like cells was quantified by tartrate-resistant acid phosphatase (TRAP) staining. RESULTS: Orthodontic force application in vivo led to a significant increase in nicotine-induced periodontal bone loss, and cell compression in vitro to increased COX-2, PGE2, IL-6, and RANKL expression, reduced OPG expression, and enhanced differentiation of RAW264.7 cells to osteoclast-like cells compared to nicotine alone. CONCLUSION: Additional loss of periodontal bone must be expected during orthodontic treatment of smokers. Clinicians should inform their patients of this increased risk and refrain from performing tooth movements before cessation of smoking.

Expression of glutathione peroxidase 1 in the spheno-occipital synchondrosis and its role in ROS-induced apoptosis.

BACKGROUND/OBJECTIVE: Chondrogenesis is an integral part of endochondral bone formation, by which the midline cranial base is developed. Reactive oxygen species (ROS) are required in chondrogenic differentiation and antioxidant enzymes regulate their levels. The aim of this study was to localize the antioxidant enzyme glutathione peroxidase 1 (Gpx1) at the spheno-occipital synchondrosis, as well as its effect on ROS challenge and its expression pattern in the course of differentiation. MATERIALS AND METHODS: Gpx1 was semiquantified in immunohistochemically stained sections of spheno-occipital synchondroses of rats. The effect of Gpx1 on ROS-induced apoptosis was investigated by manipulating the expression of Gpx1 in ATDC5 cells. The temporal pattern of Gpx1 expression was determined during chondrocyte differentiation for 21 days in vitro. RESULTS: Proliferating chondrocytes exhibited the greatest Gpx1 immunoreactivity and hypertrophic ones the lowest (P = 0.02). Cells transfected with Gpx1-siRNA had the highest apoptotic rate, while cells overexpressing Gpx1 the lowest one (P < 0.001). Gpx1 was significantly increased on days 10 (P = 0.02) and 14 (P = 0.01). CONCLUSIONS: Hypertrophic chondrocytes have the lowest Gpx1 activity in the spheno-occipital synchondrosis. Gpx1 is implicated in the ROS-induced apoptosis in chondrocytes. Its expression was not constitutive during chondrogenic differentiation.

Anabolic properties of high mobility group box protein-1 in human periodontal ligament cells in vitro.

High mobility group box protein-1 (HMGB1) is mainly recognized as a chemoattractant for macrophages in the initial phase of host response to pathogenic stimuli. However, recent findings provide evidence for anabolic properties in terms of enhanced proliferation, migration, and support of wound healing capacity of mesenchymal cells suggesting a dual role of the cytokine in the regulation of immune response and subsequent regenerative processes. Here, we examined potential anabolic effects of HMGB1 on human periodontal ligament (PDL) cells in the regulation of periodontal remodelling, for example, during orthodontic tooth movement. Preconfluent human PDL cells (hPDL) were exposed to HMGB1 protein and the influence on proliferation, migration, osteogenic differentiation, and biomineralization was determined by MTS assay, real time PCR, immunofluorescence cytochemistry, ELISA, and von Kossa staining. HMGB1 protein increased hPDL cell proliferation, migration, osteoblastic marker gene expression, and protein production as well as mineralized nodule formation significantly. The present findings support the dual character of HMGB1 with anabolic therapeutic potential that might support the reestablishment of the structural and functional integrity of the periodontium following periodontal trauma such as orthodontic tooth movement.

Cellular response to orthodontically-induced short-term hypoxia in dental pulp cells.

Orthodontic force application is well known to induce sterile inflammation, which is initially caused by the compression of blood vessels in tooth-supporting apparatus. The reaction of periodontal ligament cells to mechanical loading has been thoroughly investigated, whereas knowledge on tissue reactions of the dental pulp is rather limited. The aim of the present trial is to analyze the effect of orthodontic treatment on the induction and cellular regulation of intra-pulpal hypoxia. To investigate the effect of orthodontic force on dental pulp cells, which results in circulatory disturbances within the dental pulp, we used a rat model for the immunohistochemical analysis of the accumulation of hypoxia-inducible factor-1α in the initial phase of orthodontic tooth movement. To further examine the regulatory role of circulatory disturbances and hypoxic conditions, we analyze isolated dental pulp cells from human teeth with regard to their specific reaction under hypoxic conditions by means of flow cytometry, immunoblot, ELISA and real-time PCR on markers (Hif-1α, VEGF, Cox-2, IL-6, IL-8, ROS, p65). In vivo experiments showed the induction of hypoxia in dental pulp after orthodontic tooth movement. The induction of oxidative stress in human dental pulp cells showed up-regulation of the pro-inflammatory and angiogenic genes Cox-2, VEGF, IL-6 and IL-8. The present data suggest that orthodontic tooth movement affects dental pulp circulation by hypoxia, which leads to an inflammatory response inside treated teeth. Therefore, pulp tissue may be expected to undergo a remodeling process after tooth movement.

Association of dentoskeletal morphology with incisor inclination in angle class II patients: a retrospective cephalometric study.

INTRODUCTION: The purpose of this study was to identify possible dentoskeletal parameters associated with variation of anterior tooth inclination in Angle Class II subdivisions. METHODS: Pre-treatment lateral radiographs of 144 Class II patients (68 males, 76 females) aged 9 to 17 years were classified for upper incisor inclination into three groups (proclined, normally inclined, retroclined) homogeneous for gender and skeletal jaw relationship. The effect of age on the 22 cephalometric variables was controlled by covariance analysis. RESULTS: Multivariate analysis of the cephalometric parameters indicated significant inter-group differences. Systematic associations with incisor inclination were revealed using rank correlation: Lower incisor proclination, Wits appraisal and gonial angle significantly decreased (0.04 ≥ p ≥ 0.002), while intercisal angle, mandibular total and corpus length and nasolabial angle increased (0.04 ≥ p ≥ 0.001) with decreasing incisor proclination. CONCLUSIONS: Clear-cut classification criteria and control of confounding effects may clarify conflicting previous findings on dentoskeletal differences between Class II subdivisions in the mixed dentition. Only minor dentoskeletal differences appear to be associated with incisor inclination. The increased interincisal and nasolabial angle in Class II division 2 subjects are due to reclination of both upper and lower incisors. Jaw positions and chin prominence are not significantly different between the subdivisions. However, Wits appraisal is decreased in Class II division 2. The increased mandibular length observed in Class II division 2 requires further scrutinization.

Correlation of condylar kinematics in children with gender, facial type and weight.

OBJECTIVE: The aim of the study has been to describe the normal range of mandibular movements and condylar kinematics in children as well as to test the null hypothesis that these variables are not associated with gender, facial type and weight. MATERIALS AND METHODS: The sample was comprised of 92 healthy children (7.2-10.6 years old) and 40 adult controls (18-34.7 years old). Examinations included the maximal mouth opening capacity and laterotrusion to the right and to the left. The condylar path inclination angle was calculated at 3mm and 5mm protrusion of the mandible. Kinematic variables were registered using the ultrasonic JMA system. RESULTS: Maximal mouth opening capacity averaged 46.73 mm for the children and 53.53 mm for the adults. The mean values of the lateral movements were 9.36 mm to the right and 9.62 mm to the left for the boys, and 9.91 mm and 9.68 mm for the girls, respectively. Mean condylar path inclination in the children was 36.5° (right) and 36.2° (left) at 3mm of protrusive movement, and 34.3° (right) and 34.0° (left) at 5mm of protrusive movement. Associations of the kinematic variables with gender, weight, or facial type were insignificant. CONCLUSION: Younger school children have not yet reached the maximum mouth-opening capacity. Correlation analysis suggests some weak, but insignificant associations of gender, facial type and weight with mouth opening, laterotrusion and the condylar path inclination angle. The null hypothesis was not rejected.

Endotoxins potentiate COX-2 and RANKL expression in compressed PDL cells.

OBJECTIVE: This study aims to demonstrate in vitro the synergistic effect of orthodontic forces and periodontal pathogens on cyclooxygenase-2 regulation and the subsequent receptor activator of nuclear factor kappa-B ligand (RANKL) production from periodontal ligament (PDL) cells. MATERIALS AND METHODS: In comparison to a control group, three experimental groups were formed from human primary PDL cells stressed with compressive forces, bacterial endotoxins, or a combination of both. Gene expression of cyclooxygenase-2 and RANKL was analysed with RT real-time PCR. The prostaglandin E2 production was determined with ELISA. A co-culture of PDL cells and an osteoclast-progenitor cell line was used in order to demonstrate the osteoclast formation effect caused by the simultaneous combined stress. RESULTS: The simultaneous combined stress resulted in a 56-fold up-regulation of cyclooxygenase-2 gene expression with a subsequent noticeable rise in the prostaglandin E2 in the culture medium. The RANKL/osteoprotegerin gene expression ratio was 50-fold up-regulated and the osteoclast formation assay revealed 153.5 ± 15.7 tartrate-resistant acid phosphatase (TRAP)-positive cells per well compared with 42.3 ± 3.8 TRAP-positive cells per well of the control group. CONCLUSION: The synergistic action of periodontal pathogens and orthodontic forces leads to an increased expression of cyclooxygenase-2 from PDL cells that intensify the RANKL production which in turn induces osteoclast differentiation and subsequent osteoclastogenesis. CLINICAL RELEVANCE: The present study puts an emphasis on the detrimental effect of orthodontic forces on patients with an active periodontal disease by underlining the significance of cyclooxygenase-2 activity and RANKL binding on the osteoclastogenesis process.

Effect of excessive methionine on the development of the cranial growth plate in newborn rats.

OBJECTIVE: Methionine is an essential amino acid and pivotal for normal growth and development. However, previous animal studies have shown that excessive maternal intake of methionine causes growth restrictions, organ damages, and abnormal growth of the mandible in newborn animals. However, the effect of excessive methionine on the development of the cranial growth plate is unknown. This study investigated histological alterations of the cranial growth plate induced by high methionine administration in newborn rats. DESIGN: Twenty pregnant dams were divided into a control and an experimental group. The controls received a diet for rats and the experimental group was fed from the 18th gestational day with a special manufactured high methionine diet for rats. The high methionine diet was maintained until the end of the lactation phase (day 20). The offspring of both groups were killed at day 10 or 20 postnatally and their spheno-occipital synchondroses were collected for histological analysis. RESULTS: The weight of the high-dose methionine treated experimental group was considerably reduced in comparison to the control group at day 10 and 20 postnatally. The cartilaginous area of the growth plate and the height of the proliferative zone were markedly reduced at postnatal day 10 in the experimental group. CONCLUSIONS: In summary, the diet-induced hypermethioninemia in rat dams resulted in growth retardations and histomorphological changes of the spheno-occipital synchondrosis, an important craniofacial growth centre in newborns. This finding may elucidate facial dysmorphoses reported in patients suffering from hypermethioninemia.

Strontium promotes cell proliferation and suppresses IL-6 expression in human PDL cells.

This study was designed to investigate the effect of strontium on human PDL cells in vitro. Strontium is used to treat osteoporosis because of its bone formation promoting effect on osteoblast cells. This investigation presents evidence that strontium promotes PDL cell proliferation. Simultaneously, strontium suppresses the expression of the inflammation-promoting cytokine IL-6. The observed effect of strontium on PDL cells supports its use it in guided dental tissue regeneration.

Electron-beam irradiation of polymer bracket materials.

INTRODUCTION: Electron-beam irradiation can be used to influence the properties of polymers. Electron beams cause cross-linking that enhances the molecular mass of the polymer; this leads to branched chains until, ultimately, a 3-dimensional network is formed. The aim of this study was to evaluate the effect of electron-beam irradiation on polymer bracket materials. METHODS: Since polymers are commonly used materials for brackets, polyoxymethylene, polycarbonate, and polyurethane were chosen for this study. The acceleration voltage of the electron-beam device was 10 MeV, and the energy dose was 100 kGy with an electron accelerator (BGS beta gamma service, Rhodotron, Bruchsal, Germany). Three-medium wear, fracture toughness, and Vickers hardness tests were performed. The irradiated samples were compared with untreated control groups. RESULTS: Polycarbonate and polyurethane bracket materials have enhanced fracture toughness and Vickers hardness after electron-beam irradiation of 100 kGy and 10 MeV. Polyoxymethylene bracket materials showed significantly lower fracture toughness values after irradiation compared with the untreated control group. Polyoxymethylene had the best mechanical properties, followed by polycarbonate and polyurethane. Almost the same effects could be measured during the 3-medium wear test. CONCLUSIONS: Electron-beam postcuring improved Vickers hardness and fracture toughness of polymers with lower mechanical properties (polycarbonate and polyurethane). Polyoxymethylene, with sufficient hardness and fracture toughness, could not be improved with electron-beam postcuring.

Effect of strontium on human Runx2+/- osteoblasts from a patient with cleidocranial dysplasia.

Strontium represents a new generation of anti-osteoporotic agents that exert anti-catabolic and anabolic effects on bone cells at once. We used strontium in vitro in order to examine its potential to stimulate bone marker transcription and hydroxyapatite formation on isolated Runx2(+/-) osteoblasts from a patient with cleidocranial dysplasia. This disease is evoked by heterozygous mutations of Runx2, an important transcription factor for osteoblast maturing and transcription of osteogenic genes, which results in insufficient gene dosage of Runx2. This genetic defect is responsible, for example, for patent fontanels, sometimes throughout the life, supernumerary teeth, and aplasia or hypoplasia of clavicles and mimics symptoms of hypophosphatasia. In this trial, we investigated the effect of strontium on gene expression of bone marker proteins, the formation of hydroxyapatite and the cell proliferation of strontium-treated Runx2(+/-)-osteoblasts. Unlike normal osteoblasts, gene expression of bone marker proteins was not affected in strontium-treated Runx2(+/-) osteoblasts, while improved hydroxyapatite formation was noted in the extracellular matrix. A WST-1 cell proliferation assay with strontium-treated Runx2(+/-)-osteoblasts showed that strontium induces cell proliferation and growth. This effect might be responsible for the improved mineralisation of the extracellular matrix of strontium-treated Runx2(+/-)-osteoblasts observed.

Development dependent collagen gene expression in the rat cranial base growth plate.

The spheno-occipital synchondrosis is part of the cranial base growth plate and is of crucial importance in craniofacial development. In this investigation, we studied changes in collagen gene expression in the spheno-occipital synchondrosis in order to identify the developmental stages most important for extracellular matrix production and ossification of the rat cranial base growth plate. Gene transcripts of type II and X collagen were most abundant at day 10 postnatally in the spheno-occipital synchondrosis. This observation is assumed to be due to intrinsic genetic factors and local environmental factors.

The molecular mechanism behind bone remodelling: a review.

Bone is a connective tissue and guarantees protection and support of organ function. Contrary to the common view, bone is a dynamic tissue that constantly undergoes turnover in order to maintain stability and integrity. In this process called bone turnover or bone remodelling, two effector cell types are involved. Osteoclasts, specialised for bone resorption, and osteoblasts, responsible for bone formation, are key players in bone turnover. In the past decade, a lot of information about signal pathways, osteoblast-osteoclast communication and osteoclast activation concerning bone remodelling has arisen. In this publication, we aim to review molecular and biochemical insights with respect to the bone remodelling process. The bone remodelling process is of fundamental importance for craniofacial growth, orthodontic tooth movement and regenerative dentistry.

Putative spermine synthases from Thalassiosira pseudonana and Arabidopsis thaliana synthesize thermospermine rather than spermine.

Polyamines are involved in many fundamental cellular processes. Common polyamines are putrescine, spermidine and spermine. Spermine is synthesized by transfer of an aminopropyl residue derived from decarboxylated S-adenosylmethionine to spermidine. Thermospermine is an isomer of spermine and assumed to be synthesized by an analogous mechanism. However, none of the recently described spermine synthases was investigated for their possible activity as thermospermine synthases. In this work, putative spermine synthases from the diatom Thalassiosira pseudonana and from Arabidopsis thaliana could be identified as thermospermine synthases. These findings may explain the previous result that two putative spermine synthase genes in Arabidopsis produce completely different phenotypes in knock-out experiments. Likely, part of putative spermine synthases identifiable by sequence comparisons represents in fact thermospermine synthases.

A functional Ni-Ni-[4Fe-4S] cluster in the monomeric acetyl-CoA synthase from Carboxydothermus hydrogenoformans.

In anaerobic microorganisms employing the acetyl-CoA pathway, acetyl-CoA synthase (ACS) and CO dehydrogenase (CODH) form a complex (ACS/CODH) that catalyzes the synthesis of acetyl-CoA from CO, a methyl group, and CoA. Previously, a [4Fe-4S] cubane bridged to a copper-nickel binuclear site (active site cluster A of the ACS component) was identified in the ACS(Mt)/CODH(Mt) from Moorella thermoacetica whereas another study revealed a nickel-nickel site in the open form of ACS(Mt), and a zink-nickel site in the closed form. The ACS(Ch) of the hydrogenogenic bacterium Carboxydothermus hydrogenoformans was found to exist as an 82.2-kDa monomer as well as in a 1:1 molar complex with the 73.3-kDa CODHIII(Ch). Homogeneous ACS(Ch) and ACS(Ch)/CODHIII(Ch) catalyzed the exchange between [1-(14)C]acetyl-CoA and (12)CO with specific activities of 2.4 or 5.9 micromol of CO per min per mg, respectively, at 70 degrees C and pH 6.0. They also catalyzed the synthesis of acetyl-CoA from CO, methylcobalamin, corrinoid iron-sulfur protein, and CoA with specific activities of 0.14 or 0.91 micromol of acetyl-CoA formed per min per mg, respectively, at 70 degrees C and pH 7.3. The functional cluster A of ACS(Ch) contains a Ni-Ni-[4Fe-4S] site, in which the positions proximal and distal to the cubane are occupied by Ni ions. This result is apparent from a positive correlation of the Ni contents and negative correlations of the Cu or Zn contents with the acetyl-CoA/CO exchange activities of different preparations of monomeric ACS(Ch), a 2.2-A crystal structure of the dithionite-reduced monomer in an open conformation, and x-ray absorption spectroscopy.