Birgit Rath-Deschner

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.

Mechanical signals control SOX-9, VEGF, and c-Myc expression and cell proliferation during inflammation via integrin-linked kinase, B-Raf, and ERK1/2-dependent signaling in articular chondrocytes

IntroductionThe importance of mechanical signals in normal and inflamed cartilage is well established. Chondrocytes respond to changes in the levels of proinflammatory cytokines and mechanical signals during inflammation. Cytokines like interleukin (IL)-1β suppress homeostatic mechanisms and inhibit cartilage repair and cell proliferation. However, matrix synthesis and chondrocyte (AC) proliferation are upregulated by the physiological levels of mechanical forces. In this study, we investigated intracellular mechanisms underlying reparative actions of mechanical signals during inflammation.MethodsACs isolated from articular cartilage were exposed to low/physiologic levels of dynamic strain in the presence of IL-1β. The cell extracts were probed for differential activation/inhibition of the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling cascade. The regulation of gene transcription was examined by real-time polymerase chain reaction.ResultsMechanoactivation, but not IL-1β treatment, of ACs initiated integrin-linked kinase activation. Mechanical signals induced activation and subsequent C-Raf-mediated activation of MAP kinases (MEK1/2). However, IL-1β activated B-Raf kinase activity. Dynamic strain did not induce B-Raf activation but instead inhibited IL-1β-induced B-Raf activation. Both mechanical signals and IL-1β induced ERK1/2 phosphorylation but discrete gene expression. ERK1/2 activation by mechanical forces induced SRY-related protein-9 (SOX-9), vascular endothelial cell growth factor (VEGF), and c-Myc mRNA expression and AC proliferation. However, IL-1β did not induce SOX-9, VEGF, and c-Myc gene expression and inhibited AC cell proliferation. More importantly, SOX-9, VEGF, and Myc gene transcription and AC proliferation induced by mechanical signals were sustained in the presence of IL-1β.ConclusionsThe findings suggest that mechanical signals may sustain their effects in proinflammatory environments by regulating key molecules in the MAP kinase signaling cascade. Furthermore, the findings point to the potential of mechanosignaling in cartilage repair during inflammation.

The Role of Periodontal Ligament Cells in Delayed Tooth Eruption in Patients with Cleidocranial Dysostosis

AbstractObjective:The clinical appearance of patients with cleidocranial dysplasia (CCD), which is caused by mutations in the RUNX2 gene, is characterized by anomalies of the clavicles, thorax, spine, pelvis and extremities and by disturbances of the skull and tooth development. Of orthodontic relevance are multiple supernumerary teeth associated with delayed tooth eruption. The present investigation is based on the hypothesis that an altered phenotypic expression of periodontal ligament (PDL) cells from CCD patients and a reduced ability of those cells to support the differentiation of bone-resorbing osteoclasts might contribute to delayed tooth eruption.Materials and Methods:To test this hypothesis, PDL cells from healthy donors and from two patients with clinically and molecular biologically diagnosed CCD were characterized for the basal and induced mRNA expression of osteoblast marker genes. The physiological relevance of the findings for the differentiation of osteoclasts was examined in an osteoclast assay, as well as in a co-culture model of PDL cells and osteoclast precursors.Results:Both CCD patients displayed missense mutations of the RUNX2 gene. The in vitro experiments revealed an unaltered expression of RUNX2 mRNA, however especially in CCD patient 2 there was a reduced basal expression of mRNA for the key regulatory gene for bone remodeling RANKL. Furthermore, compared to the control cells from healthy donors, these factors were less inducible by stimulation of the cultures with 1α,25(OH)2D3. In the osteoclast assays as well as in the co-culture experiments, PDL cells from the CCD patients showed a reduced capacity to induce the differentiation of active osteoclasts.Conclusions:These data indicate that PDL cells from CCD patients express a less distinctive osteoblastic phenotype resulting in an impaired ability to support osteoclastogenesis which might, in part, account for the delayed tooth eruption that can be observed clinically.ZusammenfassungZielsetzung:Das klinische Bild der Cleidocranialen Dysplasie (CCD), das durch Mutationen im Bereich des Transkriptionsfaktors RUNX2 verursacht wird, ist durch Schlüsselbeinanomalien, Störungen der Schädel- bzw. Zahnentwicklung sowie Veränderungen im Bereich von Thorax, Wirbelsäule, Becken und Extremitäten geprägt. Besonders auffällig und von kieferorthopädischer Relevanz sind multiple überzählige Zähne sowie ein verzögerter Zahndurchbruch. Der vorliegenden Untersuchung liegt die Hypothese zugrunde, dass eine veränderte phänotypische Expression der Zellen der Parodontalligaments (PDL) von CCD-Patienten und daraus resultierend eine eingeschränkte Fähigkeit zur Unterstützung der Aktivität von knochenresorbierenden Osteoklasten für diesen erschwerten Zahndurchbruch mitverantwortlich sein könnten.Material und Methodik:Zur Überprüfung dieser Annahme wurden PDL-Zellen von gesunden Spendern und solchen mit klinisch und molekularbiologisch diagnostizierter CCD bezüglich des Auftretens und der Induzierbarkeit osteoblastärer Markergene auf mRNA-Ebene charakterisiert und die physiologische Relevanz dieser Befunde für die Differenzierung von Osteoklasten aus entsprechenden Vorläuferzellen in einem Osteoklastenassay sowie in einem Kokulturmodell vergleichend näher untersucht.Ergebnisse:Dabei konnte zunächst bei beiden CCD-Patienten eine Missense-Mutation im Bereich des RUNX2-Gens nachgewiesen werden. In den In-vitro-Versuchen zeigte sich für die PDLZellen eine unveränderte Expression von RUNX2 mRNA, jedoch insbesondere bei CCD-Patient 2 eine reduzierte basale Expression der mRNA für das osteoklastenregulatorische Molekül RANKL. Außerdem ließen sich diese Faktoren im Vergleich zur Kontrollgruppe der gesunden Donoren nur vermindert durch Stimulation der Kulturen mit 1α,25(OH)2D3 induzieren. In den durchgeführten Osteo klastenassays sowie in den Kokulturversuchen zeigten die PDL-Zellen der CCD-Patienten eine verringerte Potenz zur Induktion von aktiven Osteoklasten.Schlussfolgerungen:Die erhobenen Daten implizieren einen geringer gradig ausgeprägten osteoblastären Phänotyp der PDLZellen bei den untersuchten CCD-Patienten mit einer resultierenden eingeschränkten Fähigkeit zur Unterstützung der Osteoklastogenese, die letztlich für den klinisch zu beobachtenden erschwerten Zahndurchbruch mitverantwortlich sein könnte.

Regulatory effects of biomechanical strain on the insulin-like growth factor system in human periodontal cells

During mastication, dental trauma and functional dental habits the tissues that surround and support the teeth, i.e. the periodontium, are subject to complex biomechanical forces. The exact mechanisms mediating the anabolic and catabolic biomechanical effects on the periodontium are yet poorly understood. Therefore, the objective of this in-vitro study was to determine if continuous tensile strain (CTS) regulates the synthesis of components of the insulin-like growth factor (IGF) system in human periodontal ligament (PDL) cells. PDL cells from six donors were phenotyped, seeded on collagen type-I coated silicone membranes, and subjected to CTS of low (3%) or high (20%) magnitudes for 4 and 24 h. The gene expression of IGF1, IGF2, IGF1 receptor (IGF1R), insulin receptor substrate (IRS)1, and IGF-binding proteins (IGFBPs) was detected by real-time PCR. The protein synthesis was determined by immunoblotting. For statistical analysis, ANOVA and the Tukey test (p<0.05) were applied. When cells were subjected to low CTS for 4 h, the IGF1 expression was significantly increased, whereas high CTS or CTS applied for 24 h reduced the constitutive IGF1 synthesis. Although PDL cells also expressed IGF2, IGF1R, and IRS1, no significant differences for these molecules were found between stretched cells and controls. High CTS caused a significant upregulation of IGFBP1 and significant downregulation of IGFBP3 and IGFBP5 at 24 h. In conclusion, this in-vitro study suggests that biomechanical forces may regulate several components of the local IGF system in the human periodontium.

IGF-I, IGF-IR and IRS1 expression as an early reaction of PDL cells to experimental tooth movement in the rat.

OBJECTIVE To characterize in vivo the role of IGF-I and its signalling, as an early reaction in the mechanotransduction process and to analyse changes of the local expression related to the magnitude of the applied force. MATERIALS AND METHODS Forces of 0.1N, 0.25 N and 0.5 N were applied to move the right upper first molars of 12 anaesthetized rats mesially. These forces were kept constant for 4h. The untreated contralateral side served as a control. Paraffin-embedded sections of the resected jaws were prepared for immunohistochemistry to localize insulin-like growth factor-I (IGF-I), its receptor (IGF-IR), and insulin receptor substrate 1 (IRS1). Histomorphometric analysis was performed to count the percentage of immunoreactive cells in different parts of the periodontal ligament. RESULTS IGF-I, IGF-IR and IRS1 positive cells were observed in the periodontal tissues of the control and loaded teeth. In the experimental group, the number of IGF-I-, IGF-IR- and IRS1-positive cells increased significantly on the tension side and decreased on the compression side. CONCLUSIONS These data indicate a close relationship between mechanical loading of the PDL and the autocrine/paracrine expression of IGF components as an early step in the mechanotransduction process leading in the long term to an organized remodelling of the alveolar bone.

Insulin-like growth factor 1 (IGF1) affects proliferation and differentiation and wound healing processes in an inflammatory environment with p38 controlling early osteoblast differentiation in periodontal ligament cells

OBJECTIVE The objective of this study was to investigate effects of insulin-like growth factor 1 (IGF1) on proliferation, wound healing and differentiation processes of human periodontal ligament (PDL) cells under inflammatory conditions and whether the protective, anabolic effects of IGF1 can attenuate unfavorable effects of interleukin-1β (IL-1β). DESIGN Inflammation was mimicked through cell stimulation with IL-1β. PDL cells were characterized in respect to the presence of components of the IGF system and the responsive potential on IL-1β incubation. Gene expression levels were analyzed by quantitative real-time PCR. Cellular localization of target proteins was visualized using fluorescent-based immunohistochemistry. Effects on cell division were investigated by proliferation assays. Wound healing was analyzed using light microscopic techniques. Differentiation was quantified by measuring biomineralization and osteoblast-specific alkaline phosphatase enzyme activity. RESULTS PDL cell proliferation and wound healing were positively affected by IGF1 and the combination of IGF1 with IL-1β, while only IL-1β showed negative effects. Biomineralization was enhanced by IGF1, IL-1β, and the combination of both stimulants. Osteoblast differentiation was increased by IL-1β and the combination of IL-1β with IGF1, whereas only IGF1 negatively affected ALP activity. Phosphorylation of p38 was regulated by IL-1β and IGF1. CONCLUSIONS The data presented in this work showed a potential of IGF1 to improve wound healing and proliferation processes and to sustain cell differentiation under inflammatory stimuli in PDL cells.

The Significance of RUNX2 in Postnatal Development of the Mandibular Condyle

Objective:RUNX2, in the Runt gene family, is one of the most important transcription factors in the development of the skeletal system. Research in recent decades has shown that this factor plays a major role in the development, growth and maturation of bone and cartilage. It is also important in tooth development, mechanotransduction and angiogenesis, and plays a significant role in various pathological processes, i.e. tumor metastasization. Mutations in the RUNX2 gene correlate with the cleidocranial dysplasia (CCD) syndrome, important to dentistry, particularly orthodontics because of its dental and orofacial symptoms. Current research on experimentally-induced mouse mutants enables us to study the etiology and pathogenesis of these malformations at the cellular and molecular biological level. This study’s aim is to provide an overview of the RUNX2 gene’s function especially in skeletal development, and to summarize our research efforts to date, which has focused on investigating the influence of RUNX2 on mandibular growth, which is slightly or not at all altered in many CCD patients.Materials and Methods:Immunohistochemical analyses were conducted to reveal RUNX2 in the condylar cartilage of normal mice and of heterozygous RUNX2 knockout mice in early and late growth phases; we also performed radiographic and cephalometric analyses.Results:We observed that RUNX2 is involved in normal condylar growth in the mouse and probably plays a significant role in osteogenesis and angiogenesis. The RUNX2 also has a biomechanical correlation in relation to cartilage compartmentalization. At the protein level, we noted no differences in the occurrence and distribution of RUNX2 in the condyle, except for a short phase during the 4th and 6th postnatal weeks, so that one allele might suffice for largely normal growth; other biological factors may have compensatory effects. However, we did observe small changes in a few cephalometric parameters concerning the mandibles of heterozygous knockout animals. We discuss potential correlations to our findings by relating them to the most current knowledge about the RUNX2 biology.ZusammenfassungZiel:Zu den wichtigsten Transkriptionsfaktoren für die Entwicklung des Skelettsystems gehört RUNX2 aus der Familie der Runt- Gene. Die Forschungen der letzten Jahrzehnte zeigten, dass dieser Faktor eine große Rolle in der Entwicklung, dem Wachstum und der Reifung von Knochen und Knorpel spielt. Darüber hinaus ist er für Zahnentwicklung, Mechanotransduktion und Angiogenese wichtig und hat eine Bedeutung bei verschiedenen pathologischen Prozessen wie z.B. der Tumormetastasierung. Mutationen im RUNX2-Gen sind mit dem Syndrom der Dysplasia cleidocranialis (CCD) korreliert, das aufgrund seiner dentalen und orofazialen Symptomatik für die Zahnmedizin und insbesondere die Kieferorthopädie von Wichtigkeit ist. Die Untersuchung experimentell induzierter Mausmutanten erlaubt heutzutage Forschungen zur Ätiologie und Pathogenese dieser Fehlbildungen auf zell- und molekularbiologischer Ebene. Die vorliegende Arbeit soll eine Übersicht über die Funktion des RUNX2-Gens vor allem in der Skelettentwicklung und eine Zusammenfassung bisheriger eigener Forschungsarbeiten geben. Dabei ging es vor allem um die Frage des Einflusses von RUNX2 auf das Wachstum der Mandibula, die bei vielen CCD-Patienten nicht oder nur gering verändert ist.Material und Methodik:Immunhistochemische Untersuchungen wurden zum Nachweis von RUNX2 am Kondylenknorpel normaler Mäuse sowie von heterozygoten RUNX2-Knockout- Mäusen in frühen und späten Wachstumsphasen durchgeführt sowie röntgenologisch-kephalometrische Untersuchungen.Ergebnisse:RUNX2 war am normalen Kondylenwachstum der Maus beteiligt und wahrscheinlich auch in der Osteo- und Angiogenese sowie im Zusammenhang mit einer möglicherweise biomechanisch korrelierten Kompartimentierung des Knorpels von Bedeutung. Auf Proteinebene konnten bis auf eine kurze Phase während der 4. bis 6. postnatalen Woche keine Unterschiede im Auftreten und in der Verteilung von RUNX2 im Kondylus festgestellt werden, so dass womöglich ein Allel für ein weitgehend regelrechtes Wachstum ausreicht bzw. Kompensationen durch andere biologische Faktoren vermutet werden können. Dennoch konnten für einige kephalometrische Parameter an den Mandibulae heterozygoter Knockout-Tiere Veränderungen im Sinne einer reduzierten Dimension festgestellt werden. Mögliche Zusammenhänge zwischen diesen Befunden werden auf den Grundlagen neuer Erkenntnisse zur Biologie von RUNX2 diskutiert.

Development of an experimental device for the application of static and dynamic tensile strain on cells

The aim of this work was the development of new devices for the application of tensile strain on cells. A basic system was intended to stretch cells with physiological static forces. However, as biological systems are dynamic, a second device was built as an automatic dynamic apparatus. To establish a standardized process, commercial BioFlex® plates (Flexcell International, NC, USA) were integrated as cell adhesion membranes. In preliminary studies the mechanical behavior of these cell plate membranes was analyzed. In both systems the membranes were pulled over cylindrical stamps to get evenly distributed strains. The construction of the static system predominantly consisted of polyoxymethylene (POM), the dynamic system was reinforced with aluminum. The dynamic setup was equipped with a high-resolution linear translation stage and was computer-controlled with a program written in the LabView™ software. Various cell types were stressed with diverse strains over different time periods and first results have been published. Measurements of the 3% strained cell plate membrane and of there adnated cells resulted in cell elongations of 3.7%. The results demonstrated a high repeatability and the systems were regarded as being easy to use.