Increased tooth mobility after fixed orthodontic appliance treatment can be selectively utilized for case refinement via positioner therapy - a pilot study.

BACKGROUND: Increased tooth mobility persists after fixed orthodontic appliance removal, which is therapeutically utilized for post-treatment finishing with positioners. As such a fine adjustment is only required for selected teeth, the aim of this pilot study was to investigate tooth mobility in vivo on corrected and uncorrected subgroups under positioner therapy. METHODS: Mobility was measured on upper teeth of 10 patients (mean age 16.8) by applying loadings for 0.1, 1.0 and 10.0 s with a novel device directly after multibracket appliance debonding as much as 2d, 1, 2 and 6 weeks later. Positioners were inserted at day 2. Specimens were divided into Group C (teeth corrected via positioner), Group N (uncorrected teeth adjacent to teeth from group C), and Group U (uncorrected teeth in an anchorage block). Untreated individuals served as controls (n = 10, mean age 22.4). Statistics were performed via Kolmogorov-Smirnov test and Welch's unequal variances t-test for comparisons between groups. P < 0.05 was considered statistically significant. RESULTS: After 1 week, tooth mobility in Group U almost resembled controls (13.0-15.7 N), and reached physiological values after 6 weeks (17.4 N vs. 17.3 N in controls). Group C (9.0-13.4 N) and Group N (9.2-14.7 N) maintained increased mobility after 6 weeks. Tooth mobility was generally higher by reason of long loading durations (10.0 s). CONCLUSIONS: Positioner therapy can selectively utilized increased tooth mobility upon orthodontic fixed appliance treatment for case refinements. Here, uncorrected teeth in anchorage blocks are not entailed by unwanted side effects and recover after 6 weeks post treatment. Corrected teeth and their neighbors exhibit enhanced mobility even after 6 weeks, which represents a necessity for the proper correction of tooth position, and concurrently arouses the requirement for an adequate retention protocol.

Human periodontal ligament cells facilitate leukocyte recruitment and are influenced in their immunomodulatory function by Th17 cytokine release.

The objective of this in vitro study was to examine the immunomodulatory impact of human periodontal ligament (PDL) cells on the nature and magnitude of the leukocyte infiltrate in periodontal inflammation, particularly with regard to Th17 cells. PDL cells were challenged with pro-inflammatory cytokines (IL-1ß, IL-17A, and IFN-γ) and analyzed for the expression of cytokines involved in periodontal immunoinflammatory processes (IL-6, MIP-3 alpha, IL-23A, TGFß1, IDO, and CD274). In order to further investigate a direct involvement of PDL cells in leukocyte function, co-culture experiments were conducted. The expression of the immunomodulatory cytokines studied was significantly increased under pro-inflammatory conditions in PDL cells. Although PDL cells did not stimulate leukocyte proliferation or Th17 differentiation, these cells induced the recruitment of leukocytes. The results of our study suggest that PDL cells might be involved in chronic inflammatory mechanisms in periodontal tissues and thus in the transition to an adaptive immune response in periodontitis.

Antigen-presenting cell marker expression and phagocytotic activity in periodontal ligament cells.

BACKGROUND: Periodontal ligament (PDL) cells are the main cellular constituents of the periodontium, maintain the integrity of the connective tissue, and impact pathology in periodontitis. The aim of this study was to analyze whether PDL cells recognize foreign particles and participate in the immune response to periodontal pathogens. METHODS: Expression of surface proteins characteristic of antigen-presenting cells (APCs) (major histocompatibility complex [MHC] class II, CD40, CD80, CD86) was analyzed in PDL cells after challenge with the cytokines interleukin (IL)-1ß, IL-17A, and interferon-gamma (IFN-γ) or with heat-killed Aggregatibacter actinomycetemcomitans using real-time PCR and flow cytometry. Confocal laser scanning microscopy, transmitted light microscopy, flow cytometry, and time-lapse microscopy were applied to analyze their phagocytotic capacity of collagen (carboxylate-modified microspheres), non-periodontal (Escherichia coli) and periodontal (Aggregatibacter actinomycetemcomitans) pathogens. Furthermore, it was examined whether cytokine activation of PDL cells affects the phagocytosis of collagen or bacteria. RESULTS: PDL cells upregulated MHC class II after cytokine stimulation on transcriptional level, whereas co-stimulatory molecules characteristic of professional APCs were not induced. Analyses on protein level revealed that MHC class II was not constitutively expressed in all PDL cell lines used. PDL cells phagocytosed both collagen and bacteria via acidic vesicles, suggesting the formation of phagosomes. Phagocytosis could be partially inhibited by inhibitors of phagocytosis, i.e., dynasore and wortmannin. Pre-incubation with cytokines did not further enhance the phagocytosis rate of collagen or bacteria. CONCLUSIONS: These results suggest that PDL cells do not only represent bystanders in periodontal infections, but display non-professional APC characteristics, suggesting possible participation in immune reactions of the oral cavity.

A novel serum-free medium for the isolation, expansion and maintenance of stemness and tissue-specific markers of primary human periodontal ligament cells.

PURPOSE: Periodontal ligament (PDL) cell cultures are classically maintained in serum-containing media. However, unwanted side-effects of these conditions on cellular and molecular characteristics demand a serum-free alternative. Even though these limitations are well known and efforts for the development of adequate serum-free alternatives have been made, these approaches for replacement remained unsuccessful so far. This study aimed at developing a well-defined, serum-free formulation supporting both isolation from tissue samples and efficient expansion of PDL cells. Here, of particular focus was the perpetuation of tissue-characteristic markers detectable in primary tissues and of stemness features. BASIC PROCEDURES: Primary PDL cell cultures from generally healthy human donors (n = 3) were maintained in basal media N2B27 and E6 together with different concentrations of growth and attachment factors. Cell proliferation was recorded via microscopy and WST assay. Gene expression of RUNX2, Periostin, ALP, CD73, CD90, CD105, CD45, SOX10 and SOX2 was compared to primary PDL explants via qRT-PCR. Immunocytochemistry was performed for anti-CD105, SSEA-3, CD271, HNK1. Serum-containing sDMEM medium served as control. MAIN FINDINGS: N2B27 medium substituted with 25 ng/mL EGF, 25 ng/mL IGF1, 0.5 mg/mL Fetuin plus gelatine coating (designated N2B27-PDLsf) emerged as potent serum-free formulation ensuring adequate culture isolation and expansion. Here, PDL primary tissue signature markers RUNX2 and Periostin remained stable in N2B27-PDLsf compared to controls (229.0-fold ±101.0 and 83.2-fold ±9.6 increase). Additionally, stemness markers ALP and CD105 were significantly upregulated on transcriptional, and CD105 and SOX2 on protein level. PRINCIPAL CONCLUSIONS: This investigation identified a novel serum-free medium for the isolation, and expansion of primary human PDL cells with constantly high proliferation rates. Here, purity and stemness properties are maintained. Thus, N2B27-PDLsf represents a valid replacement for serum-containing media in PDL cultures.

The stability of different housekeeping genes in human periodontal ligament cells under inflammatory conditions.

PURPOSE: Stability of housekeeping genes as internal reference for RT-qPCR analyses is mandatory for a correct interpretation of results. As no normalization benchmark exists and reference gene validation is highly specific for individual experiments, it was the purpose of this study to identify stable candidates for investigations on periodontal inflammation. BASIC PROCEDURES: Human PDL cells from one cell line (Lonza) and three primary donors were challenged with IL-1ß (5 ng/ml) or centrifugation (170 × g) for 6 h under serum-free conditions. Unstimulated cells represented controls. qRT-PCR was performed with a TaqMan® array of 32 housekeeping genes (n = 3). Transcriptional stability was analyzed for (i) mean absolute CT values and (ii) relative fold changes. Finally, stability of mean CT values across specimens was evaluated for most stable candidates. Statistics were performed with one-way ANOVA and Bonferroni correction and one sample t-test, with 95% confidence level. Values represent mean ± SEM. MAIN FINDINGS: 18S was constant in experimental groups and specimens for mean absolute CT values and relative fold changes, and MT-APT6 for mean absolute CT values. Both genes exhibited low CT thresholds ranging from 20.2 ± 0.1 to 25.9 ± 0.2 for 18S, and from 18.9 ± 0.0-23.7 ± 0.1 for MT-APT6. Likewise stable YWHAZ ranged between 32.6 ± 0.2 and 37.2 ± 0.2 cycles. However, candidates were unstable across specimes. PRINCIPAL CONCLUSIONS: Reference validation is mandatory for RT-qPCR analyses in new experimental designs. Here, only three genes out of 32 turned out to be appropriate candidates. Due to low CT values and stability, 18S and MT-APT6 are most valid genes for data normalization in experiments with PDL cells under inflammatory conditions and are recommended as standards under these premises.

Impact of radiation history, gender and age on bone quality in sites for orthodontic skeletal anchorage device placement.

AIMS: Stability of orthodontic miniscrew implants is prerequisite to their success and durability in orthodontic treatment. As investigations revealed a positive correlation of miniscrew stability to periimplant bone quality, it has been the aim of this study to analyze the bone structure of resection preparations of human mandibles histologically by investigating the samples according to age, gender and exposure to radiotherapy. METHODS: Inflammation- and tumor-free alveolar bone sections from human mandibles (n = 31) with previously diagnosed carcinoma, chronic osteomyelitis or cysts were analyzed histomorphologically and histomorphometrically as to the dimension of trabeculae in cancellous areas. Group A investigated the impact of a history of radiation therapy, group B of gender and group C contrasted biopsies from individuals aging under 60 or over 60 years. Statistics were performed using the Kruskal-Wallis-test. RESULTS: Radiation, gender and age did not significantly influence bone density. The mean bone density averaged 40.7 ± 15.0% of spongiosa for the total collective with a median age of 58.4 years ± 14.7 years. CONCLUSIONS: Our findings provide new information on bone quality, thus contributing to a more precise evaluation of the parameters affecting and those not affecting miniscrew implant stability. On the basis of these results, the formulation of clinical guidelines for risk assessment of therapeutic approaches in patients prior to insertion of orthodontic skeletal anchorage devices seems to be conceivable.

Effect of archwire qualities and bracket designs on the force systems during leveling of malaligned teeth.

OBJECTIVES: The force systems during multiband treatment are influenced by the selection of the bracket-archwire combinations. Resin models replicated from casts reflecting the pretreatment intraoral situation of a patient's mandible were used to explore how different bracket systems and archwire qualities would affect the force systems developing during simulated orthodontic leveling of several malaligned teeth. MATERIALS AND METHODS: Leveling movements of the malaligned teeth 32, 33, and 34 were simulated using the orthodontic measurement and simulation system (OMSS). Two bracket types and three archwire qualities were compared, the former featuring a slot width of 0.022" (0.56 mm) and including one conventional (Freedom MIM Roth by ODS) and one passive self-ligating (Carriere MBT by ODS) design. Both were combined with three NiTi round 0.014" (0.36 mm) archwire products, two of them standard products (CuNiTi by Ormco; EuroArch by ODS) and one being a low-cost (NiTi Superelastic by Modern Arch) product. Measured parameters included force, torque, translation, and rotation. RESULTS: Archwire qualities are critical to the force systems developing in the leveling stage. On the other hand, the finding that lower force/torque values result in less tooth movement is not primarily due to wire selection. Our most striking result was that the ODS EuroArch wire resulted in very low force and torque values both with the conventional and with the self-ligating brackets. Almost identical patterns with these two bracket designs were found, and none of the measured parameters revealed a significant advantage for any of the bracket-archwire combinations over the others. CONCLUSION: In our experimental simulations of tooth leveling, wire-quality selection was found to be a key modifier of force, torque, translation, and rotation. Clearly, however, neither the wire qualities nor the bracket designs made a decisive difference to the amounts of leveling movement induced to malaligned teeth; other factors like tooth class or nature of the malalignment seem to be more important in this regard. A therapeutic benefit of self-ligating over conventional brackets was not demonstrable.

Bone substitute material composition and morphology differentially modulate calcium and phosphate release through osteoclast-like cells.

The aim of this study was to determine the material composition and cell-mediated remodelling of different calcium phosphate-based bone substitutes. Osteoclasts were cultivated on bone substitutes (Cerabone, Maxresorb, and NanoBone) for up to 5 days. Bafilomycin A1 addition served as the control. To determine cellular activity, the supernatant content of calcium and phosphate was measured by inductively coupled plasma optical emission spectrometry. Cells were visualized on the materials by scanning electron microscopy. Material composition and surface characteristics were assessed by energy-dispersive X-ray spectroscopy. Osteoclast-induced calcium and phosphate release was material-specific. Maxresorb exhibited the highest ion release to the medium (P = 0.034; calcium 40.25mg/l day 5, phosphate 102.08 mg/l day 5) and NanoBone the lowest (P = 0.021; calcium 8.43 mg/l day 5, phosphate 15.15 mg/l day 5); Cerabone was intermediate (P = 0.034; calcium 16.34 mg/l day 5, phosphate 30.6 mg/l day 5). All investigated materials showed unique resorption behaviours. The presented methodology provides a new perspective on the investigation of bone substitute biodegradation, maintaining the material-specific micro- and macrostructure.

Autoregulation of insulin-like growth factor 2 and insulin-like growth factor-binding protein 6 in periodontal ligament cells in vitro.

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 (100 ng/ml), IGFBP6 (450 ng/ml, 675 ng/ml, 1125 ng/ml) or a combination of 100 ng/ml IGF2 and 675 ng/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.

Osteoimmunological mechanisms involved in orthodontically and bacterially induced periodontal stress.

BACKGROUND AND OBJECTIVE: Orthodontic tooth movement is known to cause sterile inflammation of the periodontal ligament (PDL). It may also be accompanied by pathological effects of external apical root resorption, with interindividual differences in the incidence and extent of resorption. An involvement of autoimmunological mechanisms is currently under discussion. This study aimed to improve our understanding of similarities between the inflammatory mechanisms underlying the pathophysiology of periodontitis and root resorption. MATERIALS AND METHODS: Human PDL cells were stimulated with interleukin (IL)-1ß/IL-17A/IFN-γ, or left non-stimulated. Their potential for phagocytosis was then evaluated by incubation with dextran or E. coli or S. aureus particles, followed by flow cytometric and immunohistochemical analysis. Real-time polymerase chain reaction (PCR) was used to analyze receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) expression in PDL cells. Verification was obtained in vivo by studying IL-17A, RANKL, and OPG expression in biopsies of inflamed periodontal tissues and in biopsies of rat maxillae with mechanically induced root resorption. Statistical analysis included Wilcoxon's rank sum test to analyze gene expression data and one-way ANOVA in conjunction with Tukey's post hoc test to analyze flow cytometric data. RESULTS: PDL cells phagocytosed foreign particles under both inflammatory and non-inflammatory conditions. Furthermore, IL-17A significantly downregulated RANKL expression while significantly upregulating OPG expression in PDL cells. These immunomodulatory cytokines were also demonstrable in both inflammatorily altered periodontal tissues and root resorption lacunae, while the incidence of IL-7A was strikingly variable in resorption areas. CONCLUSION: PDL cells were demonstrated to effect phagocytosis and to express immunomodulatory molecules, which proves their capability of participating in periodontal osteoimmunological processes. The development of root resorption and periodontitis appears to be governed by similar pathophysiological mechanisms.