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Summary Background Orthodontic therapy aims to treat misaligned teeth and jaws to improve dental occlusion as well as the function and aesthetics of the masticatory system. Continuous data collection to check treatment quality is of great importance for the constant optimization of orthodontic care. Objective The aim of this retrospective multicentre cohort study was to systematically determine the outcome and quality of orthodontic treatment by applying the internationally established Index of Orthodontic Treatment Need (IOTN) and Peer Assessment Rating (PAR) index in multiple clinical settings for a representative number of patient cases. Materials and methods A total of 1509 consecutive orthodontic patient cases (treatment completion between January 2018 and December 2020) from three representative orthodontic centres (University clinic, city office, small town office) were analysed in a multicentre study. The pre- and post-treatment casts were scanned, digitally measured, and partially automatically evaluated using the IOTN and PAR indices. Results A statistically significant improvement in occlusion was observed for medically necessary treatment of IOTN grades 4 and 5 in 97.30 per cent of the analysed cases and for treatment-requiring grades 2 and 3 in 94.08 per cent of the analysed cases. The average percentage PAR improvement was 76.51 per cent. 72.50 per cent of cases showed improvement of more than 70 per cent. The mean PAR index score was reduced from 28.19 ± 9.49 to 6.22 ± 5.41 points. Conclusion The present data demonstrate that orthodontic treatment is efficient in inducing significant improvement of malocclusions in general and has a high success rate with severe dysgnathia.

Cells : open access journal 11(19), 2949 (2022). doi:10.3390/cells11192949 special issue: "Special Issue "The Role of Fibroblasts in Acute and Chronic Inflammatory Processes" / Special Issue Editor: Dr. Scott Levick, Guest Editor", Published by MDPI, Basel

Porphyromonas gingivalis lipopolysaccharide (PG-LPS) is an important virulence factor potentially contributing to periodontal tissue destruction. Toll-like receptor 4 (Tlr4) is a key mediator of NF-kB activation during pathogen recognition. Previous work using Tlr4-specific antibodies demonstrated a partial neutralization of PG-LPS effects on murine cementoblasts, which can affect cell function and regulate gene expression of osteoclastic markers. PG-LPS also potentially influence the inflammation process and the resorption of mineralized tissues. Yet, such inflammatory responses and cell signaling events remain to be characterized at the protein level. We thus investigated the effect of 1 and 10 µg/ml of PG-LPS, respectively, on cell morphology, cell viability, and selected key downstream molecules of the Tlr4 signaling cascade in cementoblasts. High concentrations of PG-LPS (10 µg/ml) significantly reduced cell viability after 48 h. Upon PG-LPS-stimulation, Tlr4 was significantly downregulated. Equally, IκBα, a downstream molecule, was downregulated in terms of phosphorylation and protein production. Furthermore, downstream signaling kinases, like serine/threonine kinase phospho-AKT and the mitogen-activated protein kinase (MAPK)-family, specifically phospho-ERK1/2, were significantly upregulated under high PG-LPS-concentrations. We provide new insights into PG-LPS-triggered intracellular signaling pathways in cementoblasts and thus deliver a basis for further research in PG-mediated periodontal inflammation.

International journal of molecular sciences 23(15), 8062 (2022). doi:10.3390/ijms23158062 special issue: "Special Issue "The Role of Toll-Like Receptors (TLR) in Infection and Inflammation" / Special Issue Editors: Ralf Kircheis, Guest Editor; Oliver Planz, Guest Editor", Published by Molecular Diversity Preservation International, Basel

Evaluation of tooth movement after retainer debonding in retainer-associated misalignment cases.This pilot study is based on a retrospective data analysis. Adult patients (age 25.5 ± 4.9 years) wearing fixed twistflex retainers and having visible retainer-associated misalignment were included and examined for tooth movement after retainer debonding. Orthodontic study models were taken at retainer debonding (t0) and 14 (±1) weeks later (t1). They were digitally superimposed using 2D/3D dental imaging software and tooth movement was analyzed in all three dimensions.A total of 23 teeth (12 upper teeth: 10 incisors, 2 canines; 11 lower teeth: 7 incisors, 4 canines) were analyzed. Mean overall tipping was 1.11 ± 0.82° in the mesial/distal direction (angulation, x‑axis), 2.02 ± 1.9° in the buccal/lingual direction (inclination, y‑axis) and 1.28 ± 0.99° around the tooth axis (z-axis). Mean overall bodily movement was 0.30 ± 0.31 mm in the mesial/distal direction (angulation, x‑axis), 0.10 ± 0.13 mm in the buccal/lingual direction (inclination, y‑axis), and mean in- or extrusion 0.22 ± 0.24 mm (z-axis). Mean tipping and bodily movement were more pronounced in the upper jaw.The present data shows that tooth movement after debonding of twistflex retainers can be expected in misalignment cases.ZIEL: Evaluation von Zahnbewegungen nach Retainerentfernung in Fällen von retainerassoziierter Zahnfehlstellungen.Diese Pilotstudie basiert auf einer retrospektiven Datenanalyse. Erwachsene Patienten (Alter 25,5 ± 4,9 Jahre) mit sichtbarer retainerassoziierter Zahnfehlstellung bei intakten Twistflex-Retainern wurden in die Studie einbezogen und hinsichtlich einer spontanen Zahnstellungskorrektur nach Retainerentfernung untersucht. Dazu wurden Gipsmodelle vom Zeitpunkt der Retainerentfernung (t0) sowie 14 (±1) Wochen danach (t1) in einer 2‑/3-D-Bildverarbeitungssoftware digital überlagert und die Zahnstellungsänderungen wurden in allen 3 Dimensionen analysiert.Insgesamt 23 Zähne (12 Oberkieferzähne: 10 Inzisiven, 2 Eckzähne; 11 Unterkieferzähne: 7 Inzisiven, 4 Eckzähne) wurden analysiert. Die durchschnittliche Kippbewegung in beiden Kiefern betrug1,11 ± 0,82° in mesiodistaler Richtung (Angulation, x‑Achse), 2,02 ± 1,9° in bukkolingualer Richtung (Inklination, y‑Achse) und 1,28 ± 0,99° um die eigene Achse (z-Achse). Die durchschnittliche körperliche Bewegung betrug 0,30 ± 0,31 mm in mesiodistaler (Angulation, x‑Achse) und 0,10 ± 0,13 mm in bukkolingualer Richtung (Inklination, y‑Achse), die durchschnittliche In- bzw. Extrusion 0,22 ± 0,24 mm (z-Achse). Das durchschnittliche Ausmaß der Zahnbewegung war deutlicher im Oberkiefer.Die vorgelegten Daten zeigen, dass nach dem Debonding von Twistflex-Retainern bei Zahnfehlstellungen eine Zahnbewegung erwartet werden kann.

Scientific reports 10(1), 10893 (2020). doi:10.1038/s41598-020-67449-w, Published by Macmillan Publishers Limited, part of Springer Nature, [London]

Journal of cellular and molecular medicine : JCMM 26(23), 5832-5845 (2022). doi:10.1111/jcmm.17604, Published by Wiley-Blackwell, Hoboken, NJ

Basic & clinical pharmacology & toxicology : BCPT 130(1), 132-140 (2022). doi:10.1111/bcpt.13681, Published by Wiley-Blackwell, Oxford

Scientific reports 12(1), 14970 (2022). doi:10.1038/s41598-022-19220-6, Published by Macmillan Publishers Limited, part of Springer Nature, [London]

Scientific reports 11(1), 4007 (2021). doi:10.1038/s41598-021-82274-5, Published by Macmillan Publishers Limited, part of Springer Nature, [London]

Advanced healthcare materials 10(1), 2001269 (2021). doi:10.1002/adhm.202001269, Published by Wiley-VCH, Weinheim

Biomolecules : open access journal 11(7), 932 (2021). doi:10.3390/biom11070932 special issue: "Special Issue "Oral Regenerative Medicine: Current and Future" / Special Issue Editors: Prof. Dr. Taneaki Nakagawa, Guest Editor, Dr. Takehito Ouchi, Guest Editor", Published by MDPI, Basel

Outcome in high-risk patients with refractory or relapsed germ cell tumours (GCT) remains poor. Novel strategies enhancing therapeutic efficacy whilst limiting therapeutic burden are warranted, yet immunotherapy approaches geared towards activating endogenous antitumor responses have not been successful thus far. Redirection of cytotoxic effector cells by bispecific antibodies represents a promising approach in this setting. We demonstrate that the Epithelial Cell Adhesion Molecule (EpCAM) is broadly expressed in GCT cell lines of different histologic origin including seminoma, choriocarcinoma (CHC), and embryonal carcinoma (EC). In these GCT lines of variable EpCAM surface expression, targeting T cells by the prototypic bispecific EpCAM/CD3-antibody (bAb) Catumaxomab together with natural killer (NK) cell engagement via the Fc domain promotes profound cytotoxicity across a broad range of antibody dilutions. In contrast, tumor cell lysis mediated by either immune cell subset alone is influenced by surface density of the target antigen. In the CHC line JAR, NK cell-dependent cytotoxicity dominates, which may be attributed to differential surface expression of immunomodulatory proteins such as MHC-I, CD24, and Fas receptors on CHC and EC. In view of redirecting T cell therapy mediated by bispecific antibodies, such differences in GCT immunophenotype potentially favoring immune escape are worth further investigation.

Introduction Bond failure during fixed orthodontic treatment is a frequently occurring problem. As bracket rebonding is associated with reduced shear bond strength, the aim of the present investigation is to analyse the effect of different innovative rebonding systems to identify optimised rebonding protocols for orthodontic patient care. Methods Metallic brackets were bonded to the frontal enamel surfaces of 240 bovine lower incisors embedded in resin bases. Teeth were randomly divided into two major experimental groups: in group 1 a hydrophilic primer (Assure™ PLUS) was compared to commonly used orthodontic adhesives (Transbond XT™, BrackFix®, Grengloo™) and a zero control. In group 2 different rebonding systems were analysed using a hydrophilic primer (Assure™ PLUS), a methyl methacrylate-consisting primer (Plastic Conditioner) and a conventional adhesive (Transbond XT™). All teeth were tested for shear bond strength according to the DIN-13990 standard, the Adhesive Remnant Index and enamel fracture rate. Results The hydrophilic primer enhanced shear bond strength at first bonding (Assure™ PLUS 20.29 ± 4.95 MPa vs. Transbond XT™ 18.45 ± 2.57 MPa; BrackFix® 17 ± 5.2 MPa; Grengloo™ 19.08 ± 3.19 MPa; Meron 8.7 ± 3.9 MPa) and second bonding (Assure™ PLUS 16.76 ± 3.71 MPa vs. Transbond XT™ 13.06 ± 3.19 MPa). Using Plastic Conditioner did not seem to improve shear bond strength at rebonding (13.57 ± 2.94). When enamel etching was left out, required shear bond strength could not be achieved (Plastic Conditioner + Assure™ PLUS 8.12 ± 3.34 MPa; Plastic Conditioner: 3.7 ± 1.95 MPa). Hydrophilic priming systems showed decreased ARI-scores (second bonding: 2.63) and increased enamel fracture rates (first bonding: 55%; second bonding 21,05%). Conclusions Based on the present study we found that rebonding strength could be compensated by the use of hydrophilic priming systems. The additional use of a methyl methacrylate-consisting primer does not seem to enhance shear bond strength. No etching approaches resulted in non-sufficient bond strength.

Aberrant receptor kinase signalling and tumour neovascularization are hallmarks of medulloblastoma development and are both considered valuable therapeutic targets. In addition to VEGFR1/2, expression of PDGFR α/β in particular has been documented as characteristic of metastatic disease correlating with poor prognosis. Therefore, we have been suggested that the clinically approved multi-kinase angiogenesis inhibitor Axitinib, which specifically targets these kinases, might constitute a promising option for medulloblastoma treatment. Indeed, our results delineate anti-neoplastic activity of Axitinib in medulloblastoma cell lines modelling the most aggressive c-myc-amplified Non-WNT/Non-SHH and SHH-TP53-mutated tumours. Exposure of medulloblastoma cell lines to Axitinib results in marked inhibition of proliferation and profound induction of cell death. The differential efficacy of Axitinib is in line with target expression of medulloblastoma cells identifying VEGFR 1/2, PDGFR α/β and c-kit as potential markers for drug application. The high specificity of Axitinib and the consequential low impact on the haematopoietic and immune system render this drug ideal multi-modal treatment approaches. In this context, we demonstrate that the clinically available PI3K inhibitor GDC-0941 enhances the anti-neoplastic efficacy of Axitinib against c-myc-amplified medulloblastoma. Our findings provide a rational to further evaluate Axitinib alone and in combination with other therapeutic agents for the treatment of most aggressive medulloblastoma subtypes.

Prognosis in patients suffering from high‐risk, refractory and relapsed germ cell tumours (GCT) often comprising of CD30‐positive embryonal carcinoma (EC) components remains poor. Thus, novel treatment strategies are warranted. The antibody‐drug conjugate (ADC) brentuximab vedotin delivers the potent antimitotic drug monomethyl auristatin E (MMAE) to CD30‐expressing tumour cells. After CD30 binding, internalization and intracellular linker cleavage cytotoxic MMAE can efflux and eradicate neighbouring CD30‐negative cells. To analyse cytotoxicity and a potential bystander effect of brentuximab vedotin in GCT, we established an in vitro coculture model mimicking GCT of heterogeneous CD30 positivity and measured cell viability, proliferation and apoptosis after exposure to brentuximab vedotin and unbound MMAE by MTS‐ and flow cytometry‐based CFSE/Hoechst assay. CD30 expression being assessed by quantitative RT‐PCR and immunohistochemistry was apparent in all EC cell lines with different intensity. Brentuximab vedotin abrogates cell viability of CD30‐positive GCT27 EC line exerting marked time‐dependent antiproliferative and pro‐apoptotic activity. CD30‐negative JAR cultured alone barely responds to brentuximab vedotin, while in coculture with GCT27 brentuximab vedotin induces clear dose‐dependent cytotoxicity. Cellular proliferation and cell death are significantly enhanced in CD30‐negative JAR cocultured with CD30‐positive GCT27 compared to JAR cultured alone in proof of substantial bystander activity of brentuximab vedotin in CD30‐negative GCT. We present first evidence that in an in vitro model mimicking GCT of heterogeneous histology, brentuximab vedotin exerts potent antiproliferative and pro‐apoptotic activity against both CD30‐positive as well as CD30‐negative GCT subsets. Our results strongly support translational efforts to evaluate clinical efficacy of brentuximab vedotin in high‐risk GCT of heterogeneous CD30 positivity.

Cementoblasts, located on the tooth root surface covered with cementum, are considered to have tooth protecting abilities. They prevent tissue damage and secure teeth anchorage inside the periodontal ligament during mechanical stress. However, the involvement of cementoblasts in mechanical compression induced periodontal remodeling needs to be identified and better understood. Here, we investigated the effect of static compressive stimulation, simulating the compression side of orthodontic force and cell confluence on a murine cementoblast cell line (OC/CM). The influence of cell confluence in cementoblast cells was analyzed by MTS assay and immunostaining. Furthermore, mRNA and protein expression were investigated by real-time RT-PCR and western blotting at different confluence grades and after mechanical stimulation. We observed that cementoblast cell proliferation increases with increasing confluence grades, while cell viability decreases in parallel. Gene expression of remodeling markers is regulated by compressive force. In addition, cementoblast confluence plays a crucial role in this regulation. Confluent cementoblasts show a significantly higher basal expression of Bsp, Osterix, Alpl, Vegfa, Mmp9, Tlr2 and Tlr4 compared to sub-confluent cells. After compressive force of 48 h at 60% confluence, an upregulation of Bsp, Osterix, Alpl, Vegf and Mmp9 is observed. In contrast, at high confluence, all analyzed genes were downregulated through mechanical stress. We also proved a regulation of ERK, phospho-ERK and phospho-AKT dependent on compressive force. In summary, our findings provide evidence that cementoblast physiology and metabolism is highly regulated in a cell confluence-dependent manner and by mechanical stimulation.

// Rogerio B. Craveiro 1, * , Michael Ehrhardt 1, * , Julia Velz 1 , Martin Olschewski 1 , Barbara Goetz 1 , Torsten Pietsch 2 and Dagmar Dilloo 1 1 Department of Pediatric Hematology and Oncology, Center for Pediatrics, University of Bonn Medical Center, D-53113 Bonn, Germany 2 Department of Neuropathology, University of Bonn, D-53105 Bonn, Germany * These authors have contributed equally to this work Correspondence to: Rogerio B. Craveiro, email: Rogerio.craveiro@ukb.uni-bonn.de Keywords: medulloblastoma, Vandetanib, GDC-0941, targeted therapy, multi-kinase inhibitor (MKI) Received: July 27, 2016 Accepted: December 26, 2016 Published: January 31, 2017 ABSTRACT Medulloblastoma is comprised of at least four molecular subgroups with distinct clinical outcome (WHO classification 2016). SHH-TP53-mutated as well as MYC-amplified Non-WNT/Non-SHH medulloblastoma show the worst prognosis. Here we present evidence that single application of the multi-kinase inhibitor Vandetanib displays anti-neoplastic efficacy against cell lines derived from high-risk SHH-TP53-mutated and MYC-amplified Non-WNT/Non-SHH medulloblastoma. The narrow target spectrum of Vandetanib along with a favourable toxicity profile renders this drug ideal for multimodal treatment approaches. In this context our investigation documents that Vandetanib in combination with the clinically available PI3K inhibitor GDC-0941 leads to enhanced cytotoxicity against MYC-amplified and SHH-TP53-mutated medulloblastoma. In line with these findings we show for MYC-amplified medulloblastoma a profound reduction in activity of the oncogenes STAT3 and AKT. Furthermore, we document that Vandetanib and the standard chemotherapeutic Etoposide display additive anti-neoplastic efficacy in the investigated medulloblastoma cell lines that could be further enhanced by PI3K inhibition. Of note, the combination of Vandetanib, GDC-0941 and Etoposide results in MYC-amplified and SHH-TP53-mutated cell lines in complete loss of cell viability. Our findings therefore provide a rational to further evaluate Vandetanib in combination with PI3K inhibitors as well as standard chemotherapeutics in vivo for the treatment of most aggressive medulloblastoma variants.

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.

Aberrant receptor kinase signalling and tumour neovascularization are hallmarks of medulloblastoma development and are both considered valuable therapeutic targets. In addition to VEGFR1/2, expression of PDGFR α/β in particular has been documented as characteristic of metastatic disease correlating with poor prognosis. Therefore, we have been suggested that the clinically approved multi‐kinase angiogenesis inhibitor Axitinib, which specifically targets these kinases, might constitute a promising option for medulloblastoma treatment. Indeed, our results delineate anti‐neoplastic activity of Axitinib in medulloblastoma cell lines modelling the most aggressive c‐myc‐amplified Non‐WNT/Non‐SHH and SHH‐TP53‐mutated tumours. Exposure of medulloblastoma cell lines to Axitinib results in marked inhibition of proliferation and profound induction of cell death. The differential efficacy of Axitinib is in line with target expression of medulloblastoma cells identifying VEGFR 1/2, PDGFR α/β and c‐kit as potential markers for drug application. The high specificity of Axitinib and the consequential low impact on the haematopoietic and immune system render this drug ideal multi‐modal treatment approaches. In this context, we demonstrate that the clinically available PI3K inhibitor GDC‐0941 enhances the anti‐neoplastic efficacy of Axitinib against c‐myc‐amplified medulloblastoma. Our findings provide a rational to further evaluate Axitinib alone and in combination with other therapeutic agents for the treatment of most aggressive medulloblastoma subtypes.

Deregulation of the Phosphoinositide 3-kinase (PI3K)/AKT signalling network is a hallmark of oncogenesis. Also medulloblastoma, the most common malignant brain tumor in children, is characterized by high levels of AKT phosphorylation and activated PI3K signalling in medulloblastoma is associated with enhanced cellular motility, survival and chemoresistency underscoring its role of as a potential therapeutic target. Here we demonstrate that GDC-0941, a highly specific PI3K inhibitor with good clinical tolerability and promising anti-neoplastic activity in adult cancer, also displays anti-proliferative and pro-apoptotic effects in pediatric human medulloblastoma cell lines. Loss in cell viability is accompanied by reduced phosphorylation of AKT, a downstream target of PI3K. Furthermore, we show that GDC-0941 attenuates the migratory capacity of medulloblastoma cells and targets subpopulations expressing the stem cell marker CD133. GDC-0941 also synergizes with the standard medulloblastoma chemotherapeutic etoposide. In an orthotopic xenograft model of the most aggressive human medulloblastoma variant we document that oral adminstration of GDC-0941 impairs tumor growth and significantly prolongs survival. These findings provide a rational to further investigate GDC-0941 alone and in combination with standard chemotherapeutics for medulloblastoma treatment.

Regardless of the recent advances in cytotoxic therapies, 30% of children diagnosed with medulloblastoma. succumb to the disease. Therefore, novel therapeutic approaches are warranted. Here we demonstrate that Pazopanib a clinically approved multi-kinase angiogenesis inhibitor (MKI) inhibits proliferation and apoptosis in medulloblastoma cell lines. Moreover, Pazopanib profoundly attenuates medulloblastoma cell migration, a prerequisite for tumor invasion and metastasis. In keeping with the observed anti-neoplastic activity of Pazopanib, we also delineate reduced phosphorylation of the STAT3 protein, a key regulator of medulloblastoma proliferation and cell survival. Finally, we document profound in vivo activity of Pazopanib in an orthotopic mouse model of the most aggressive c-myc amplified human medulloblastoma variant. Pazopanib reduced the growth rate of intracranial growing medulloblastoma and significantly prolonged the survival. Furthermore, to put these results into a broader perspective we analysed Pazopanib side by side with the MKI Sorafenib. Both compounds share a similar target profile but display different pharmacodynamics and pharmacokinetics with distinct cytotoxic activity in different tumor entities. Thus, we identified Pazopanib as a new promising candidate for a rational clinical assessment for targeted paediatric medulloblastoma therapy.

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.

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.

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.

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

Chicken acidic leucine-rich EGF-like domain-containing brain protein (CALEB), also known as chondroitin sulfate proteoglycan (CSPG)5 or neuroglycan C, is a neural chondroitin sulfate-containing and epidermal growth factor (EGF)-domain-containing transmembrane protein that is implicated in synaptic maturation. Here, we studied the role of CALEB within the developing cerebellum. Adult CALEB-deficient mice displayed impaired motor coordination in Rota-Rod experiments. Analysis of the neuronal connectivity of Purkinje cells by patch-clamp recordings demonstrated impairments of presynaptic maturation of inhibitory synapses. GABAergic synapses on Purkinje cells revealed decreased evoked amplitudes, altered paired-pulse facilitation and reduced depression after repetitive stimulation at early postnatal but not at mature stages. Furthermore, the elimination of supernumerary climbing fiber synapses on Purkinje cells was found to occur at earlier developmental stages in the absence of CALEB. For example, at postnatal day 8 in wild-type mice, 54% of Purkinje cells had three or more climbing fiber synapses in contrast to mutants where this number was decreased to less than 25%. The basic properties of the climbing fiber Purkinje cell synapse remained unaffected. Using Sholl analysis of dye-injected Purkinje cells we revealed that the branching pattern of the dendritic tree of Purkinje cells was not impaired in CALEB-deficient mice. The alterations observed by patch-clamp recordings correlated with a specific pattern and timing of expression of CALEB in Purkinje cells, i.e. it is dynamically regulated during development from a high chondroitin sulfate-containing form to a non-chondroitin sulfate-containing form. Thus, our results demonstrated an involvement of CALEB in the presynaptic differentiation of cerebellar GABAergic synapses and revealed a new role for CALEB in synapse elimination in Purkinje cells.