Stefan Lossdörfer

IMMUNOHISTOCHEMICAL CHARACTERIZATION OF NANOCRYSTALLINE HYDROXYAPATITE SILICA GEL (NANOBONE(R)) OSTEOGENESIS: A STUDY ON BIOPSIES FROM HUMAN JAWS

OBJECTIVES Bone substitute biomaterials may be osteogenic, osteoconductive or osteoinductive. To test for these probable characteristics in a new nanoporous grafting material consisting of nanocrystalline hydroxyapatite embedded in a porous silica gel matrix (NanoBone(s)), applied in humans, we studied biopsies from 12 patients before dental implantation following various orofacial augmentation techniques with healing times of between 3.5 and 12 months. MATERIAL AND METHODS Sections from decalcified specimens were investigated using histology, histochemistry [periodic acid Schiff, alcian blue staining and tartrate-resistant acid phosphatase (TRAP)] and immunohistochemistry, with markers for osteogenesis, bone remodelling, resorption and vessel walls (alkaline phosphatase, bone morphogenetic protein-2, collagen type I, ED1, osteocalcin, osteopontin, runx2 and Von-Willebrand factor). RESULTS Histologically, four specific stages of graft transformation into lamellar bone could be characterized. During early stages of healing, bone matrix proteins were absorbed by NanoBone(s) granules, forming a proteinaceous matrix, which was invaded by small vessels and cells. We assume that the deposition of these molecules promotes early osteogenesis in and around NanoBone(s) and supports the concomitant degradation probably by osteoclast-like cells. TRAP-positive osteoclast-like cells were localized directly on the granular surfaces. Runx2-immunoreactive pre-osteoblasts, which are probably involved in direct osteogenesis forming woven bone that is later transformed into lamellar bone, were attracted. Graft resorption and bone apposition around the graft granules appear concomitantly. CONCLUSIONS We postulate that NanoBone(s) has osteoconductive and biomimetic properties and is integrated into the hosts physiological bone turnover at a very early stage.

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.

PTH(1-34) Affects Osteoprotegerin Production in Human PDL Cells in vitro

Since periodontal ligament (PDL) cells exhibit several osteoblastic traits, we hypothesized that human PDL cells will respond to hormonal stimulation in an osteoblast-like manner. Confluent and pre-confluent PDL cells from six patients were challenged with PTH(1-34). Cell number, ALP, osteocalcin, osteoprotegerin, and RANKL expression were determined. Intermittent PTH(1-34) treatment of confluent PDL cells caused a significant increase in proliferation, whereas differentiation and osteoprotegerin production decreased significantly. In pre-confluent PDL cells, this treatment regimen induced a biphasic decrease in proliferation, but a biphasic increase in differentiation and osteoprotegerin production. Continuous PTH(1-34) exposure enhanced proliferation but inhibited osteocalcin production in confluent cells and stimulated osteoprotegerin production in pre-confluent PDL cells. RANKL was hardly detectable and unaffected by PTH(1-34) treatment. These results indicate that human PDL cells respond to PTH(1-34) in an osteoblast-like manner, and that the PTH(1-34) effect depends on the maturation state of the cells and on the mode of administration.

PTH(1-34)-induced changes in RANKL and OPG expression by human PDL cells modify osteoclast biology in a co-culture model with RAW 264.7 cells

Parathyroid hormone (PTH) is widely accepted as an anabolic agent when administered intermittently. Here, we explored the influence of intermittent PTH(1-34) on the expression of local factors by human periodontal ligament (PDL) cells that modify osteoclast biology. This approach aimed at a further elucidation of the role of the hormone and of PDL cells in the regulation of periodontal tissue homeostasis and of repair processes. In a co-culture model of mature PDL cells and RAW 264.7 cells, intermittent PTH(1-34) induced an increased gene expression for tartrate-resistant acid phosphatase (+84%), cathepsin K (+56%), and vitronectin-receptor (+56%); and an enhanced resorptive activity of differentiated osteoclasts (+154%). These findings were correlated with a reduction of the osteoprotegerin (OPG)/receptor activator of nuclear factor kappaB ligand (RANKL) ratio in the presence of PTH(1-34; −44%). Similar results were obtained when RAW cells were cultured with the conditioned medium of PTH(1-34)-stimulated PDL cells. In contrast, when less mature PDL cells were co-cultured with RAW cells, PTH(1-34) induced an inhibition of osteoclastic differentiation (TRAP, −35%; cathepsin K, −28%; vitronectin-receptor, −35%), a reduction of the resorbed substrate area (−77%) and an increase of the OPG/RANKL ratio (+11%). The conditioned medium of PTH(1-34)-pretreated less mature PDL cells led to a down-regulation of the number and activity of multinucleated cells. These data indicate that intermittent PTH(1-34) modifies the expression of membrane-bound and secreted factors by PDL cells which then in turn alter osteoclast biology. The PDL cell response to PTH(1-34) is specific in terms of cell maturation and the mechanism involved.

Enamel Matrix Derivative Promotes Human Periodontal Ligament Cell Differentiation and Osteoprotegerin Production in vitro

Enamel matrix derivative (EMD) has been used successfully to aid periodontal repair. We sought to elucidate the mechanism of action of EMD and hypothesized that combined exposure to EMD and parathyroid hormone (PTH), which acts anabolicly when administered intermittently, would enhance periodontal ligament cell proliferation, differentiation, and local factor production. Confluent human periodontal ligament cells were exposed to EMD continuously or to PTH(1-34) intermittently, or a combination of both. Cell number, alkaline phosphatase activity, osteocalcin, and osteoprotegerin production were determined. Continuous challenge with EMD resulted in an increase of the differentiation parameters and osteoprotegerin production, while simultaneously inhibiting proliferation. Intermittent PTH(1-34) administration exerted opposite effects. Combined administration of EMD and PTH(1-34) weakened or even nullified the effects seen for the agents alone. These results suggest that EMD promotes periodontal ligament cell differentiation and osteoprotegerin production, potentially resulting in a microenvironment supporting periodontal repair, whereas combining EMD and PTH(1-34) failed to prove beneficial in this respect.

Cellular and extracellular factors in early root resorption repair in the rat

The aim of this study was to investigate the role of extracellular matrix components, such as collagen type I, fibronectin, and osteopontin (OPN) during cementum repair following experimentally induced tooth movement, and to characterize the cells taking part in the regenerative process. The upper right first molars were moved mesially in 21 three-month-old male Wistar rats using a coil spring with a force of 0.5 N. After 9 days, the appliance was removed and the animals were killed in groups of three immediately after withdrawal of the force and 5, 7, 10, 12, 14, and 17 days later. Three rats served as non-experimental control animals. The maxillae were prepared and processed for histological analysis. Together with the disappearance of the multinucleated odontoclasts from the resorption lacunae, signs of repair were visible 5 days after the release of the orthodontic force. The first signs of cementum repair were seen on day 10. The newly produced cementum was of the acellular extrinsic fibre type (AEFC) and reattachment was achieved with the principal periodontal ligament (PDL) fibres orientated almost perpendicular to the root surface. The initial interface formed between the old and new cementum, as well as the new AEFC, was characterized by a strong immunoreaction with OPN and collagen I antibody, but only a weak immunoreaction with the fibronectin antibody. Only a small number of mononuclear cells, which were involved in the repair process, showed a positive immunoreaction with the osteoblastic lineage markers runt-related transcription factor 2 and osteocalcin. These same cells stained sparsely with muscle segment homeobox homologue 2, but not with the E11 antibody. Thus, most of the cells associated with this reparative activity on the surface of the lacunae were differentiated PDL cells of the fibroblastic phenotype. Cells with a defined osteoblastic phenotype seemed to be of minor importance in this repair process.

Analysis of the torque capacity of a completely customized lingual appliance of the next generation

IntroductionIn lingual orthodontic therapy, effective torque control of the incisors is crucial due to the biomechanical particularities associated with the point of force application and the tight link between third order deviations and vertical tooth position.AimThe aim of the present in vitro investigation was to analyze the torque capacity of a completely customized lingual appliance of the next generation (WIN) in combination with different finishing archwire dimensions.MethodsUsing a typodont of the upper arch carrying the WIN appliance, slot filling and undersized individualized β-titanium archwires were engaged. Horizontal forces ranging from 0 to 100 cN were applied at the central incisor by means of spring gauges. The resulting angular deviations were recorded and the corresponding torque moments were calculated.ResultsFor fullsize archwires (0.018”×0.018” β-titanium and 0.018”×0.025” β-titanium), an initial torque play of 0-2° had to be overcome prior to the development of an effective torque moment. Thereafter, a linear correlation between torque angle and torque moment developed for both archwire dimensions with steeper slopes calculated for the specimens with the larger dimension. A torque moment of 2 Nmm required for effective torque correction was noted after a minimum of 2-3° of twist for the 0.018”×0.018” β-titanium wires as compared to 2-4° for the 0.018”×0.025” β-titanium study sample. When undersized archwires were analyzed (0.0175”×0.0175” β-titanium), the measured torque play ranged from 5-7°. After 8-12° of torque angle, the threshold of 2 Nmm was reached. A linear relationship between twist angle and torque moment in which the steepness of the slopes was generally flatter than the ones calculated for the slot filling archwires was noted.ConclusionsGiven the high precision of the bracket slot-archwire-combination provided with the WIN appliance, an effective torque control can be clinically realized.

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.

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

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

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

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