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BACKGROUND AND OBJECTIVE Previously, we demonstrated an inflammatory response of human PDL (hPDL) cells to mechanical loading. The cellular reaction was dampened by heat pre-treatment suggesting a protective role for heat shock proteins (HSP) during stress-induced ischemia. Here we explored if HSP70, which has already been documented in the pressure zone of tooth movement, might be regulatorily involved in the attenuation of the inflammatory response. MATERIALS AND METHODS Fifth passage hPDL cells were mechanically loaded in the presence of the HSP70 inhibitor VER155008. Cell morphology, HSP70 expression, viability, IL-6 and IL-8 expression were determined by means of microscopy, realtime-PCR and ELISA. The conditioned medium of mechanically loaded and pre-treated hPDL cells was used to culture monocytes to identify a potential impact on adhesion and osteoclastic differentiation capacity. RESULTS Mechanical cell stress resulted in a significant increase of pro-inflammatory parameters. HSP70 inhibition led to a further enhancement of cytokine expression. The conditioned medium of mechanically loaded hPDL cells significantly increased monocyte adhesion and differentiation along the osteoclastic pathway. VER155008 pronounced this effect significantly. CONCLUSION The results indicate a regulatory role for HSP70 in the control of the inflammatory hPDL cell response to mechanical loading and identify HSP70 as a target in the attempt to attenuate tissue damage during orthodontic tooth movement. Furthermore, the present findings point to the risk of increased periodontal destruction when medication targeting HSP70 is applied for severe medical conditions during orthodontic tooth movement.

Biomechanical orthodontics loading of the periodontium initiates a cascade of inflammatory signaling events that induce periodontal remodeling and finally facilitate orthodontic tooth movement. Pattern recognition receptors such as toll-like receptors (TLRs) have been well characterized for their ability to induce the activation of inflammatory, immunomodulatory cytokines. Here, we examined whether the cellular response of human periodontal ligament (hPDL) cells to mechanical stress involves TLR-4 signaling in vitro. Confluent hPDL cells were cultured in the presence of 5 μg/ml TLR-4 antibody (TLR-4ab) for 1 h prior to the induction of compressive forces by the use of round glass plates for 24 h. At harvest, interleukin-6 and interleukin-8 (IL-6, IL-8) mRNA and protein expression were analyzed by real-time PCR and ELISA. The immunomodulatory role of mechanical cell stress and TLR-4 signaling was addressed in co-culture experiments of hPDL and THP-1 cells targeting monocyte adhesion and by culturing osteoclastic precursors (RAW 264.7) in the presence of the conditioned medium of hPDL cells that had been mechanically loaded before. Basal expression of IL-6 and IL-8 was not affected by TLR-4ab, but increased significantly upon mechanical loading of hPDL cells. When cells were mechanically stressed in the presence of TLR-4ab, the effect seen for loading alone was markedly reduced. Likewise, monocyte adhesion and osteoclastic differentiation were enhanced significantly by mechanical stress of hPDL cells and this effect was partially inhibited by TLR-4ab. The results of the present study indicate a proinflammatory and immunomodulatory influence of mechanical loading on hPDL cells. Intracellular signaling involves a TLR-4-dependent pathway. These findings hold out the prospect of interfering with the cellular response to mechanical cell stress in order to minimize undesired side effects of orthodontic tooth movement.

Einleitung: In der kieferorthopadischen Lingualtherapie ist eine effektive Torquekontrolle der Schneidezahne aufgrund der biomechanischen Besonderheiten, die mit dem Kraftansatzpunkt und der engen Verknupfung von Abweichungen der dritten Ordnung mit der vertikalen Zahnposition verbunden sind, von besonderer Bedeutung. Studienziel: In der vorliegenden In-vitro-Studie sollte daher die Torquekapazitat einer vollstandig individuellen lingualen Apparatur (WIN) in Verbindung mit verschiedenen Drahtdimensionen, wie sie ublicherweise in der Finishingphase Verwendung finden, untersucht werden. Methode: An einem Oberkiefertypodonten mit WIN-Apparatur wurden slotfullende und untermasige β-Titaniumbogen einligiert und horizontale Krafte zwischen 0 und 100cN am zentralen Schneidezahn mittels Federwaagen appliziert. Die resultierenden angularen Abweichungen wurden aufgezeichnet und die korrespondierenden Drehmomente berechnet. Ergebnisse: Bei den slotfullenden Bogen (0,018×0,018″ und 0,018×0,025″ β-Titanium) musste ein initiales Torquespiel von 0–2° uberwunden werden, ehe sich ein effektives Drehmoment entwickelte. Anschliesend war eine lineare Korrelation zwischen Torquewinkel und Drehmoment fur beide Bogendimensionen mit steilerem Kurvenverlauf fur die hohermasigen Drahte zu verzeichnen. Ein fur eine effektive Torquekorrektur notwendiges Drehmoment von 2Nmm wurde nach mindestens 2–3° Verwindung der 0,018×0,018″ β-Titaniumbogen erreicht, verglichen mit 2–4° fur das 0,018×0,025″ β-Titanium Untersuchungsgut. Bei der Analyse untermasiger Bogen (0,0175×0,0175″ β-Titanium) betrug das gemessene Torquespiel 5–7°. Ab einem Torquewinkel von 8–12° wurde die notwendige Schwelle von 2Nmm erreicht. Auch hier ergab sich eine lineare Beziehung zwischen dem Ausmas der Drahtverwindung und resultierendem Drehmoment mit generell flacherem Kurvenverlauf im Vergleich zu den Berechnungen fur die slotfullenden Bogen. Schlussfolgerung: Aufgrund der hohen Prazision der Bracketslot-Bogen-Kombination kann mit der WIN-Apparatur klinisch eine effektive Torquekontrolle erreicht werden.

Objective Heat pre-treatment of mechanically loaded human periodontal ligament cells (hPDL) dampens the inflammatory cellular response, as evidenced by a reduced expression of pro-inflammatory cytokines, inhibition of monocyte adhesion and osteoclastic differentiation. These findings imply heat shock proteins (HSP) as cell protective molecules acting in the PDL that are up-regulated upon ischemia caused by mechanical loading. HSP70 and its inhibition by VER155008 as the active agent in several pharmaceuticals are established targets and strategies, respectively, in the treatment of neoproliferative diseases. However, the effect of both players on periodontal remodeling in unknown. Therefore, we analyzed the role of HSP70 and its frequently used inhibitor VER155008 in the regulation of physiological hPDL cell functions and immune cell interaction. Materials and Methods Fifth passage hPDL cells were cultured in the presence of 25 μm HSP70 inactivating agent VER155008. At harvest, HSP70 expression, cell proliferation, and parameters of cell interaction, colony formation and wound healing were analyzed by means of real-time PCR, immunohistochemistry, Western blot, biochemical MTS assay, microscopy, and functional assays for monocyte adhesion and differentiation. Results Basal HSP70 expression and hPDL cell morphology were not affected by HSP70 inhibitor VER155008. In contrast, cell proliferation, tissue defect healing, and colony formation were reduced significantly following HSP70 inhibition, whereas apoptosis and necrosis, monocyte adhesion and osteoclastic differentiation were markedly increased. Conclusions The present data indicate a regulatory role for HSP70 protein in hPDL cell biology. Clinical relevance These findings identify HSP70 as a promising target in the attempt to modify periodontal remodeling and point to potential periodontal side effects of HSP70 pharmaceutical usage.

Recent studies have demonstrated the interplay of human periodontal ligament cells (hPDLs) with immune cells, such as macrophages, during tissue repair. High-mobility group box protein-1 (HMGB1) is released into the extracellular milieu by damaged cells and functions as an alarmin to mediate the inflammatory host response. The present study addressed the role of HMGB1 released by hPDLs in the regulation of macrophage differentiation, migration and activity. The aim was to examine the inflammatory potential of HMGB1 itself and in combination with other mediators. The induction of sterile necrosis by thermal insult of hPDLs resulted in HMGB1 translocation from the nucleus to the cytoplasm and on to the extracellular space, as determined by immunocytochemistry/ELISA. Exposure of human macrophages to the conditioned PDL cell medium increased the expression of macrophage differentiation/activation markers CD14, CD23, CD64 and CD163. Chemotactic migration and osteoclastic differentiation of macrophages were also enhanced. Supplementation of the conditioned medium with a saturating concentration of HMGB1-Ab reduced these effects. Challenge with recombinant HMGB1 protein induced less migration and osteoclast differentiation than thermal insult. These data point to the immune modulatory capacity of hPDLs by the release of mediators, including HMGB1, which modify macrophage differentiation, migration and activity during periodontal repair, and indicate an enhanced HMGB1 activity when acting in concert with other mediators.

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.

Recent studies have shown that periodontal ligament (PDL) cells interact with macrophages from the immune system during orthodontically induced repair of periodontal tissue. Hypothesizing that high-mobility group box 1 (HMGB1) protein is released by mechanically stressed PDL cells into the extracellular space and has a role in mediating the local immune response by acting as an “alarmin”, this study was performed to further elucidate these cellular interactions, with a special focus on the impact of proinflammatory mediators secreted by PDL cells on macrophage physiology. The study included an in vivo part in which orthodontic stress was induced in rats and their PDL analyzed for expression of HMGB1 by immunohistochemistry after 5 days of tooth movement. In the in vitro part, human PDL cells were subjected to compressive loading, followed by stimulating human macrophages with conditioned supernatants of these stressed PDL cells and analyzing how mediators that had been released by these cells into the medium would impact macrophage physiology. Assays for macrophage migration and osteoclast differentiation were used in addition to immunohistochemistry, enzyme-linked immunosorbent assays, and western blotting. Induction of mechanical stress was found to upregulate HMGB1 expression both in vivo and in vitro. At the same time, translocation HMGB1 from nuclei into cytoplasm was observed. Culturing macrophages in conditioned PDL cell medium was associated with enhanced chemotactic migration and osteoclast differentiation. Addition of anti-HMGB1 antibodies to inhibit HMGB1 in the conditioned medium was found to significantly attenuate these effects. A less marked increase of migration and osteoclast differentiation by macrophages was observed after isolated addition of HMGB1, at its observed pathological concentration, to nonconditioned medium. This study clearly indicates an immunomodulatory potential of human PDL cells via release of mediators, including HMGB1 protein. Our finding that these mediators modify the migration and differentiation of macrophages as a function of periodontal repair during orthodontic treatment broadens the theoretical basis toward developing interventional strategies to avoid orthodontically induced root resorption.

SUMMARy IntRODUCtIOn: the chromatin-binding protein high mobility group box protein 1 (HMGB1) can be released into the extracellular milieu by necrotic and damaged cells and functions as an alarmin that is recognized by the innate immune system to initiate and modulate tissue repair. However, little is known about the function of HMGB1 within orthodontic tooth movement. therefore, it was the aim of the present study to investigate HMGB1 expression by periodontal ligament (PDL) cells challenged by mechanical loading similar to force levels being applied in orthodontic treatment in vitro and to transfer these findings to an in vivo microenvironment in an animal model of tooth movement in rats. In addition, we addressed the question of whether the regulation of HMGB1 expression is modulated in a time-dependent manner in zones of compressive forces. MEtHODS AnD RESULtS: Protein analysis revealed a basal HMGB1 expression in PDL cell cultures and an increased expression when orthodontic forces were applied. In a rat model of tooth movement including 25 animals that received orthodontic tooth movement for 3, 6, 9, and 12 days, HMGB1 protein expression was demonstrated to be regulated in a time-dependent manner as determined by means of immunohis- tochemistry and histomorphometrical analyses. COnCLUSIOn: these data indicate a potential role for HMGB1 protein originating from PDL cells in the regu- lation of orthodontic tooth movement and the periodontal remodelling process by modifying the local microenvironment.

Intermittent parathyroid hormone (PTH) exerts anabolic effects on bone and has been approved for osteoporosis therapy. The dual actions of PTH are mediated primarily through the parathyroid hormone 1 receptor (PTH1R). Upon ligand binding, PTH1R activates diverse signaling pathways, including cAMP/protein kinase A (PKA)- and phospholipase C/protein kinase C (PLC/PKC)-dependent pathways. PTH1R has been abundantly studied in bone cells. Knowledge on PTH1R characteristics and physiology in periodontal ligament (PDL) cells is still in its infancy. We characterized PTH1R in PDL cells in terms of its cellular localization, binding affinity, and signal transduction and compared these characteristics to those of MG63 osteoblast-like cells. PTH1R mRNA/protein was identified in PDL and MG63 cells. PTH1R was mainly localized on the plasma membrane, in vesicular structures inside the cell, and, to some extent, in the nucleus of both cell types. Binding characteristics of PTH1R were cell type specific, with PDL cells demonstrating a lower binding affinity. The response of cAMP and active PKC production in MG63 cells was dose dependent with increasing PTH(1-34) concentration, whereas in PDL cells, it was regulated biphasically. However, we observed a cross talk between the cAMP/PKA and PLC/PKC signaling pathways, which were regulated diametrically opposed at a given concentration of PTH(1-34). These data indicate that, albeit the similarity in its subcellular distribution, PTH1R in PDL cells exhibits characteristics different from those in MG63 cells, pointing to the cell type specificity of this receptor. The findings further elucidate the characteristics of PTH action in dental tissues and widen the theoretical basis for the development of anabolic treatment strategies.

Recent studies indicate that high mobility group box protein 1 (HMGB1) can be released by necrotic and damaged cells and functions as an alarmin that is recognized by the innate immune system. Little is known about the role of HMGB1 within the periodontal ligament (PDL). Therefore, we examined HMGB1 expression by PDL cells in vitro and compared the findings to an in vivo model of orthodontically induced tooth root resorption. In addition, we addressed the question of whether a potentially anabolic intermittent administration of parathyroid hormone (iPTH) would modulate the expression of HMGB1. In confluent PDL cell cultures, HMGB1 messenger RNA (mRNA) expression was quantified by real-time polymerase chain reaction. In a rat model comprising 25 animals, mechanical loading for 5 days was followed by administration of either iPTH (1–34) systemically or sham injections for up to 56 days. HMGB1 expression was determined by means of immunohistochemistry and histomorphometry. The in vitro experiments revealed an inhibitory effect of iPTH on basal HMGB1 mRNA expression in confluent PDL cells. In vivo, the mechanical force-induced enhanced HMGB1 protein expression declined time dependently. Intermittent PTH further inhibited HMGB1 expression. The significantly higher basal HMGB1 protein expression in the former compression side was followed by a more pronounced time- and iPTH-dependent decline in the same area. These data indicate a major role for HMGB1 in the regulation of PDL wound healing following mechanical load-induced tissue injury. The findings point to the potential benefit of iPTH in the attempt to support these immune-associated reparative processes.

Periodontal ligament (PDL) cells have been associated with the regulation of periodontal repair processes by the differential expression of osteoprotegerin and RANKL in response to intermittent parathyroid hormone (PTH) resulting in a modified activity of bone-resorbing osteoclasts. Here, we examined the intracellular signaling pathways that PDL cells use to mediate the PTH(1-34) effect on osteoprotegerin production and hypothesized that those would be dependent on the cellular maturation stage. Two stages of confluence served as a model for cellular maturation of 5th passage human PDL cells from six donors. Intermittent PTH(1-34) (10(-12) M) and PTH(1-31), the latter lacking the protein kinase C (PKC) activating domain, induced a significant decrease of osteoprotegerin production in confluent cultures, whereas the signal-specific fragments PTH(3-34) and PTH(7-34), which both are unable to activate protein kinase A (PKA), had no effect. The addition of the PKA inhibitor H8 antagonized the PTH(1-34) effect, whereas the PKC inhibitor RO-32-0432 did not. In pre-confluent, less mature cultures, intermittent PTH(1-34) resulted in a significant increase of osteoprotegerin. Similar results were obtained when PTH(1-31) substituted for PTH(1-34) as opposed to a lack of an effect of PTH(3-34) and PTH(7-34). Likewise, in confluent cultures, H8 inhibited the PTH(1-34) effect in pre-confluent cultures contrasted by RO-32-0432 which had no effect. These findings indicate that PTH(1-34) signaling targeting osteoprotegerin production in PDL cells involves a PKA-dependent pathway. The PTH(1-34) effect is dependent on cell status, whereas intracellular signal transduction is not. Clinical trials will have to prove whether those in vitro data are of physiological relevance for interference strategies.

Lossdorfer S, Kraus D, Abuduwali N, Jager A. Intermittent administration of PTH(1–34) regulates the osteoblastic differentiation of human periodontal ligament cells via protein kinase C- and protein kinase A-dependent pathwaysin vitro. J Periodont Res 2011; 46: 318–326.© 2011 John Wiley & Sons A/S 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.

Lossdorfer S, Kraus D, Jager A. Aging affects the phenotypic characteristics of human periodontal ligament cells and the cellular response to hormonal stimulation in vitro. J Periodont Res 2010; 45: 764–771. © 2010 John Wiley & Sons A/S 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.

SUMMARY The aim of this study was to investigate the role of extracellular matrix components, such as collagen type I, fi bronectin, 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 fi rst 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 nonexperimental 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 fi rst signs of cementum repair were seen on day 10. The newly produced cementum was of the acellular extrinsic fi bre type (AEFC) and reattachment was achieved with the principal periodontal ligament (PDL) fi bres 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 fi bronectin 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 fi broblastic phenotype. Cells with a defi ned osteoblastic phenotype seemed to be of minor importance in this repair process.

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.

Background and Objective: Periodontal ligament (PDL) cells show traits that are typical of osteoblasts, such as osteoblastic marker gene expression and the ability to respond to parathyroid hormone (PTH) stimulation in an osteoblast-like manner with respect to differentiation and local factor production. In the present study, we hypothesized that human PDL cells might respond to PTH stimulation with changes in proliferation and cell survival and thereby provide another mechanism by which PTH might affect the reparative potential of PDL cells. We speculated that the maturation state of the cells and the mode of PTH(1-34) administration would have an impact on the cellular response. Material and Methods: PDL cells were challenged with PTH(1-34) intermittently or continuously at different maturation states. Cell number, 5-bromo-2-deoxyuridine (BrdU) incorporation, DNA fragmentation, nitric oxide production and the duration of the PTH(1-34) effect were determined. Results: Intermittent PTH(1-34) treatment of preconfluent cells caused a significant increase in proliferation and DNA fragmentation, whereas in more mature cells, proliferation was less enhanced while apoptosis was more pronounced than in immature cells. Continuous PTH(1-34) exposure did not alter proliferation in any maturation state but increased DNA fragmentation in preconfluent cells. PTH(1-34) prevented etoposide-induced apoptosis after 6 h but no longer after 24 h. Nitric oxide production was unaffected. Conclusion: These results indicate that human PDL cells respond to PTH(1-34) with changes in proliferative and apoptotic signaling in a maturation-state-dependent manner. Besides changes in local factor production, these findings provide a further possible mechanism to support the idea that PDL cells possess the potential to be involved in the regulation of dental hard tissue repair.

There is evidence that growth factors, such as the insulin-like growth factors (IGFs), are involved in biological and pathological processes in oro-dento-facial tissues. To investigate their roles in tooth movement, root resorption, and repair, the occurrence of components of the IGF system, including the ligands IGF-I and -II, the IGF receptor 1 (IGF1R) and six IGF-binding proteins (IGFBP-1 to -6), was investigated by immunohistochemistry on sections from rat maxillae where the first molar had been moved mesially by means of an orthodontic appliance for 9 d to induce root resorption. After force deactivation on day 0, early repair was studied after a further 5, 7, 10, 12, 14, and 17 d. The immunostaining pattern in the periodontal ligament, cementum, and bone of control animals showed similarities known from studies in human teeth. Increased immunostaining for nearly all components in pressure sides and resorption lacunae indicated an involvement in resorption processes and clastic activities. During early stages of repair, the occurrence of several components (e.g. IGF-II, IGFBP-5 or -6) within lacunae and in cementoblasts showed an involvement in the resorption-repair sequence, which is considered to be a coupling process as known from bone.

There is growing evidence that the insulin-like growth factor (IGF) system plays an important role in the biology of oro-dento-facial tissues and organs, including the development, homeostasis and regeneration of the periodontium. To obtain basic data on the occurrence and distribution of IGF components in human permanent teeth we immunohistochemically investigated 25 extracted, decalcified and paraffin-embedded teeth using mono and polyclonal antibodies against the ligands IGF-I and -II, the IGF1 receptor (IGF1R) and all six IGF binding proteins (IGFBP-1 to -6). In the extracellular matrix (ECM) of the adhering periodontal ligament (PDL), immunoreactivity for IGF-I, -II and IGFBP-1 and -6 was observed. PDL fibroblasts showed immunostaining for the IGF1R. For the cementum, in the acellular cementum only IGF-II could be detected, while outer cementum layers with inserting Sharpey's fibers reacted with all antibodies applied except for IGFBP-4 and -6. In the pulp, mainly fibrotic areas and areas around denticles were immunoreactive for IGF-I, IGFBP-1, -3, -5 and -6. Predentin and odontoblastic processes were stained for IGF-I and IGFBP-3. The spatially oriented occurrence of components of the IGF system in human permanent teeth indicates that specific functions of the IGFs may be localized in particular tissue compartments. In the cementum, several IGF components were found indicating roles in tissue homeostasis or attachment. The PDL may function as a reservoir for IGFs probably bound to ECM components. PDL fibroblasts could then respond in a paracrine manner. In the pulp, the IGF system may be involved in odontoblast biology, fibrosis and denticle formation.

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.

So-called epithelial rests of Malassez are derived from the Hertwig's root sheath and are located in the periodontal ligament, with still unknown functions. Different pathological conditions may lead to proliferation of these otherwise non-proliferative cell clusters. The insulin-like growth factor (IGF) system is an important growth factor system controlling proliferation and differentiation. In our study on Malassez cells from extracted human deciduous teeth, we investigated their structure by means of light and electron microscopy. Although they appeared as cellular clusters with a uniform epithelial phenotype, immunohistochemical analyses of components of the IGF system revealed an unique pattern: weak immunoreactivity could be seen for IGF-II while among all IGF binding proteins (IGFBPs) only IGFBP-6 and weakly IGFBP-4 were detectable in epithelial cells of Malassez. Since IGFBP-6 has a very high affinity for IGF-II and can inhibit its functions, we discuss that, in the normal periodontal ligament, autocrine IGFBP-6 may function as an antiproliferative molecule suppressing mitogenic effects of IGFs on Malassez cells.

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. 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. 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. 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. These data present the induction of heat shock proteins as a promising treatment option to limit undesired side effects of periodontal remodeling.

Osteoblasts and osteoclasts are involved in bone formation and resorption. RANK and RANKL [receptor activator of nuclear factor kappaB (ligand)], two cytokine-like proteins of the tumor necrosis factor superfamily, are localized on these bone cells and are crucial for the regulation of osteoclastic cell differentiation from hematopoietic precursors and also for the upregulation of mature osteoclasts mediated by cell-to-cell contact and a subsequent cascade of diverse intracellular signaling processes in the osteoclasts. It was the aim of this study to examine the sites of expression of RANKL and RANK in the corresponding cells of human dental hard and periodontal tissues using immunohistochemical light microscopical methods on tissue sections of 15 paraffin-embedded human deciduous teeth undergoing root resorption. We detected granular cytoplasmic RANKL-immunoreactivity in odontoblasts, pulp fibroblasts, periodontal ligament fibroblasts, and in single odontoclasts, the latter finding suggesting an autocrine/paracrine role. RANK-positive cells were identified as multinucleated odontoclasts localized near the dentine surface in resorption lacunae or as mononucleated precursors. These findings indicate time that human dental cells express key mediators of hard tissue resorption and, though the RANK/RANKL-system may not be the sole regulator of tooth root resorption, these factors could at least contribute to this complex process under both physiological and pathological conditions.

Peri-implantitis (PI) is characterized by inflammation and bone resorption eventually leading to implant failure, but the characteristic pathologic determinants are undefined to date. This study aims to elucidate the parameters involved in PI pathogenesis, including intraoral implant retention time, extent of bone loss, smoking history, and identification of osteoimmunological markers for inflammation and bone loss. Peri-implant tissues (n = 21) displaying clinically diagnosed PI from patients with vertical bone loss ranging from 0–12 mm and implant function period between 1 and 60 months were evaluated by histochemistry and immunohistochemistry for TRAP, CD3, RANK, RANKL, OPG, and TNF-α. Statistical analyses were performed with the Welch test and correlation coefficients were calculated. Most bone resorption occurred during the first 12 months of implant function and correlated with the extent of inflammation, although histological signs of inflammation strongly varied between samples from minimal appearance of inflammatory cells to extended infiltrates. Implant function period and smoking history did not significantly affect the degree of inflammation. Higher RANK levels emerged in the first 12 months of implant function compared to longer retention times and were negatively correlated to the occurrence of RANKL. Additionally, histological signs of inflammation were about two-fold higher in specimens with bone resorption up from 5 mm compared to under 5 mm. CD3+ cells were more prevalent in extensive inflammatory infiltrates and samples derived from smokers. Our analyses proved that PI-induced bone loss is differentially influenced by the parameters evaluated in this study, but a distinct interconnection between disease severity and implant retention time can be established.

Item does not contain fulltext 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 (100ng/ml), IGFBP6 (450ng/ml, 675ng/ml, 1125ng/ml) or a combination of 100ng/ml IGF2 and 675ng/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.

Bracket slots and orthodontic archwires offering high dimensional precision are needed for fully customized lingual appliances. We aimed to investigate whether high-precision appliances of this type enable dentoalveolar compensation of class III malocclusion so that lower incisor inclination at the end of treatment will closely match the anticipated situation as defined in a pretreatment setup.This retrospective study included a total of 34 consecutive patients who had worn a fully customized lingual appliance to achieve dentoalveolar compensation for class III malocclusion by intermaxillary elastics, or proximal enamel reduction, or extraction of teeth in one or both jaws. Casts fabricated at different points in time were three-dimensionally scanned to analyze how precisely the lower incisor inclinations envisioned in the setup were implemented in clinical practice.Aside from minor deviations of ±3.75°, the lower incisor inclinations were clinically implemented as planned even in patients with major sagittal discrepancies.Treatment goals predefined in a setup of dentoalveolar compensation for class III malocclusion can be very precisely achieved via a customized lingual appliance. Correct planning can prevent undesirable lingual tipping of the lower incisors. This finding should not encourage a more liberal use of dentoalveolar compensation, but it should heighten clinicians' awareness of how essential it is to sufficiently consider the individual anatomy of the dentoalveolar complex during treatment planning.

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. 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. 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. 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.

Residual periodontal ligament (PDL) cells in the damaged tissue are considered a prerequisite for a successful regeneration of the periodontal architecture with all its components, including gingiva, PDL, cementum, and bone. Among other approaches, current concepts in tissue engineering aim at a hormonal support of the regenerative capacity of PDL cells as well as at a supplementation of lost cells for regeneration. Here, we investigated how far an anabolic, intermittent parathyroid hormone (iPTH) administration would enhance the osteoblastic differentiation of PDL cells and the cellular ability to mineralize the extracellular matrix in an in vivo transplantation model. PDL cells were predifferentiated in a standard osteogenic medium for 3 weeks before subcutaneous transplantation into CD-1 nude mice using gelatin sponges as carrier. Daily injections of 40 μg/kg body weight PTH(1-34) or an equivalent dose of vehicle for 4 weeks were followed by explantation of the specimens and an immunohistochemical analysis of the osteoblastic marker proteins alkaline phosphatase (ALP), osteopontin, and osteocalcin. Signs of biomineralization were visualized by means of alizarin red staining. For verification of the systemic effect of iPTH application, blood serum levels of osteocalcin were determined. The osteogenic medium stimulated the expression of ALP and PTH1-receptor mRNA in the cultures. After transplantation, iPTH resulted in an increased cytoplasmic and extracellular immunoreactivity for all markers investigated. In contrast to only sporadic areas of mineralization under control conditions, several foci of mineralization were observed in the iPTH group. Blood serum levels of osteocalcin were elevated significantly with iPTH. These data indicate that the osteoblastic differentiation of human PDL cells and their ability for biomineralization can be positively influenced by iPTH in vivo. These findings hold out a promising prospect for the support of periodontal regeneration.

Components of the insulin-like growth factor (IGF) system and intermittent parathyroid hormone (PTH) were demonstrated to exert anabolic effects on the periodontal ligament (PDL) and, thereby, contribute to the regeneration of structures that were lost because of inflammatory periodontal disease.In the present study, a PDL cell culture is created to examine whether a combination of IGFs and intermittent PTH would enhance the cellular response elicited by each factor alone and addressed the question of whether a pretreatment of the cells with bone morphogenetic protein (BMP)-7 alters the PDL cell phenotype and behavior on IGF-PTH stimulation.IGF-I and IGF-II enhanced proliferation and differentiation in preconfluent and confluent cultures. A BMP-7 pretreatment inhibited those effects in preconfluent cells but not in confluent cultures. Intermittent PTH (1-34) inhibited the proliferation but stimulated the differentiation of preconfluent cells, whereas the opposite effect was observed in confluent cultures. BMP-7 prestimulation made preconfluent cells respond like confluent cultures on intermittent PTH (1-34) challenge. A combined administration of PTH (1-34) and IGF-I or IGF-II mostly mirrored the effects seen for one of the individual agents alone, but synergistic effects were not observed.The present data indicate that both PTH (1-34) and IGF-I or IGF-II exert a maturation stage-dependent effect on PDL cell proliferation and differentiation. Combining PTH (1-34) and IGF does not seem to potentiate the effects seen for the agents alone. Furthermore, our data suggest that BMP-7 induces preconfluent cells to acquire a more differentiated phenotype and to respond to intermittent PTH (1-34) accordingly. Confluent cultures seem to be less susceptible to BMP-7.

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.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.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 1alpha,25(OH)(2)D(3). 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.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.

Jager A, Gotz W, Lossdorfer S, Rath-Deschner B. Localization of SOST/sclerostin in cementocytes in vivo and in mineralizing periodontal ligament cells in vitro. J Periodont Res 2009; doi: 10.1111/j.1600-0765.2009.01227.x. © 2009 John Wiley & Sons A/S 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 β-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.

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.

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 host's physiological bone turnover at a very early stage.

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-κB 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.

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.

There is evidence that growth factors, such as the insulin-like growth factors (IGFs), are involved in biological and pathological processes in oro-dento-facial tissues. To investigate their roles in tooth movement, root resorption, and repair, the occurrence of components of the IGF system, including the ligands IGF-I and -II, the IGF receptor 1 (IGF1R) and six IGF-binding proteins (IGFBP-1 to -6), was investigated by immunohistochemistry on sections from rat maxillae where the first molar had been moved mesially by means of an orthodontic appliance for 9 d to induce root resorption. After force deactivation on day 0, early repair was studied after a further 5, 7, 10, 12, 14, and 17 d. The immunostaining pattern in the periodontal ligament, cementum, and bone of control animals showed similarities known from studies in human teeth. Increased immunostaining for nearly all components in pressure sides and resorption lacunae indicated an involvement in resorption processes and clastic activities. During early stages of repair, the occurrence of several components (e.g. IGF-II, IGFBP-5 or -6) within lacunae and in cementoblasts showed an involvement in the resorption-repair sequence, which is considered to be a coupling process as known from bone.

Periodontal ligament (PDL) cells exhibit several osteoblastic traits and are parathyroid hormone (PTH)-responsive providing evidence for a role of these cells in dental hard-tissue repair. To examine the hypothesis that PDL cells respond to PTH stimulation with changes in proliferation and apoptotic signaling through independent but convergent signaling pathways, PDL cells were cultured from human bicuspids obtained from six patients. PDL cells at different states of maturation were challenged with PTH(1-34) intermittently for 0, 1, or 24 h/cycle or exposed continuously. Specific inhibitors to protein kinases A and C (PKA, PKC) and the mitogen-activated protein kinase cascade (MAPK) were employed. At harvest, the cell number, BrdU incorporation, and DNA fragmentation were determined by means of cell counting and immunoassays. Intermittent PTH(1-34) caused a significant increase in cell number in confluent cells as opposed to a reduction in pre-confluent cells. In confluent cells, the effect resulted from a significant increase in proliferation, whereas DNA fragmentation was reduced when PTH(1-34) was administered for 1 h/cycle but increased after PTH(1-34) for 24 h/cycle. Inhibition of PKC inhibited PTH(1-34)-induced proliferation but enhanced apoptosis. Inhibition of PKA enhanced proliferation and DNA fragmentation. Similar results were obtained in less mature cells, although, in the presence of the PKA inhibitor, the PTH(1-34)-induced changes were more pronounced than in confluent cells. In the presence of the MAPK inhibitor, all of the parameters examined were reduced significantly in both maturation states. Thus, PTH(1-34) mediates proliferative and apoptotic signaling in human PDL cells in a maturation-state-dependent manner via PKC-dependent and PKA-dependent pathways.

Background and objectives: Parathyroid hormone (parathyroid hormone) has been shown to be capable of exerting anabolic effects on bone when administered intermittently. We hypothesized that parathyroid hormone will modulate the response of periodontal ligament cells in terms of anabolic effects with respect to proliferation, differentiation and the production of key regulatory factors of bone remodeling such as osteoprotegerin and receptor activator of nuclear factor kappaB ligand (RANKL) in a maturation-state dependent manner. Methods: Periodontal ligament cells were cultured from human bicuspids obtained from six patients. Following characterization, confluent and preconfluent periodontal ligament cells were challenged with parathyroid hormone (1–34) for 0, 1, 3, 6 or 24 h within three incubation cycles of 48 h each. At harvest, the cell number, alkaline phosphatase specific activity and osteocalcin, osteoprotegerin and RANKL production were determined by means of semiquantitative polymerase chain reaction (PCR) and immunoassays. Dermal fibroblasts and MG63 osteoblast-like cells served as a reference. Results: Intermittent parathyroid hormone treatment of confluent periodontal ligament cells caused a significant increase in proliferation (+32% maximum) whereas alkaline phosphatase activity, osteocalcin and osteoprotegerin decreased at the transcriptional and translational level (−59.7% maximum). In preconfluent periodontal ligament cells, parathyroid hormone induced a decrease in proliferation (−66.3% maximum) but an increase in differentiation and osteoprotegerin production (+49.2% maximum). RANKL was hardly detectable and unaffected by parathyroid hormone treatment. Similar results were obtained in MG63 cells, whereas parathyroid hormone stimulation did not alter any of the parameters examined in dermal fibroblasts. Conclusion: These results indicate that human periodontal ligament cells respond to an intermittent parathyroid hormone exposure with changes in proliferation, differentiation and osteoprotegerin production in a maturation-state dependent manner and therefore might be regulatorily involved in periodontal regeneration.

So-called epithelial rests of Malassez are derived from the Hertwig's root sheath and are located in the periodontal ligament, with still unknown functions. Different pathological conditions may lead to proliferation of these otherwise non-proliferative cell clusters. The insulin-like growth factor (IGF) system is an important growth factor system controlling proliferation and differentiation. In our study on Malassez cells from extracted human deciduous teeth, we investigated their structure by means of light and electron microscopy. Although they appeared as cellular clusters with a uniform epithelial phenotype, immunohistochemical analyses of components of the IGF system revealed an unique pattern: weak immunoreactivity could be seen for IGF-II while among all IGF binding proteins (IGFBPs) only IGFBP-6 and weakly IGFBP-4 were detectable in epithelial cells of Malassez. Since IGFBP-6 has a very high affinity for IGF-II and can inhibit its functions, we discuss that, in the normal periodontal ligament, autocrine IGFBP-6 may function as an antiproliferative molecule suppressing mitogenic effects of IGFs on Malassez cells.

Introduction In 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. Aim The 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. Methods Using 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. Results For 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. Conclusions Given the high precision of the bracket slot-archwire-combination provided with the WIN appliance, an effective torque control can be clinically realized.