Piero Römer

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.

The Role of Hypoxia in Orthodontic Tooth Movement

Orthodontic forces are known to have various effects on the alveolar process, such as cell deformation, inflammation, and circulatory disturbances. Each of these conditions affecting cell differentiation, cell repair, and cell migration, is driven by numerous molecular and inflammatory mediators. As a result, bone remodeling is induced, facilitating orthodontic tooth movement. However, orthodontic forces not only have cellular effects but also induce vascular changes. Orthodontic forces are known to occlude periodontal ligament vessels on the pressure side of the dental root, decreasing the blood perfusion of the tissue. This condition is accompanied by hypoxia, which is known to either affect cell proliferation or induce apoptosis, depending on the oxygen gradient. Because upregulated tissue proliferation rates are often accompanied by angiogenesis, hypoxia may be assumed to fundamentally contribute to bone remodeling processes during orthodontic treatment.

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

ObjectiveThis 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 methodsIn 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.ResultsThe 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.ConclusionThe 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 relevanceThe 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.

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.

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.

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.

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.

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.

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.

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.