Osteoblast differentiation of Gli1⁺ cells via Wnt and BMP signaling pathways during orthodontic tooth movement.

OBJECTIVES: Factors that induce bone formation during orthodontic tooth movement (OTM) remain unclear. Gli1 was recently identified as a stem cell marker in the periodontal ligament (PDL). Therefore, we evaluated the mechanism of differentiation of Cre/LoxP-mediated Gli1/Tomato+ cells into osteoblasts during OTM. METHODS: After the final administration of tamoxifen to 8-week-old Gli1-CreERT2/ROSA26-loxP-stop-loxP-tdTomato mice for 2 days, nickel-titanium closed coil springs were attached between the upper anterior alveolar bone and the first molar. Immunohistochemical localizations of ß-catenin, Smad4, and Runx2 were observed in the PDL on 2, 5, and 10 days after OTM initiation. RESULTS: In the untreated tooth, few Gli1/Tomato+ cells were detected in the PDL. Two days after OTM initiation, the number of Gli1/Tomato+ cells increased in the PDL on the tension side. On this side, 49.3 ±â€¯7.0% of ß-catenin+ and 48.7 ±â€¯5.7% of Smad4+ cells were found in the PDL, and Runx2 expression was detected in some Gli1/Tomato+ cells apart from the alveolar bone. The number of positive cells in the PDL reached a maximum on day 5. In contrast, on the compression side, ß-catenin and Smad4 exhibited less immunoreactivity. On day 10, Gli1/Tomato+ cells were aligned on the alveolar bone on the tension side, with some expressing Runx2. CONCLUSIONS: Gli1+ cells in the PDL differentiated into osteoblasts during OTM. Wnt and bone morphogenetic proteins signaling pathways may be involved in this differentiation.

Granzyme B promotes matrix metalloproteinase-1 (MMP-1) release from gingival fibroblasts in a PAR1- and Erk1/2-dependent manner: A novel role in periodontal inflammation.

OBJECTIVE: To gain insights into how proteases signal to connective tissues cells in the periodontium. BACKGROUND: The connective tissue degradation observed in periodontitis is largely due to matrix metalloproteinase (MMP) release by gingival fibroblasts. Granzyme B (GzmB) is a serine protease whose role in periodontitis is undefined. METHODS: Human gingival crevicular fluid (GCF) samples were obtained from sites with periodontal disease and healthy control sites. GzmB was quantified in the GCF ([GzmB]GCF ) by ELISA. Gingival fibroblasts (GF) were cultured in the presence or absence of recombinant GzmB. Culture supernatants were analyzed by ELISA to quantify GzmB-induced release of interstitial collagenase (MMP-1). In some experiments, cells were pre-treated with the inhibitor PD98059 to block MEK/ERK signaling. The protease-activated receptor-1 (PAR-1) was blocked with ATAP-2 neutralizing antibody prior to GzmB stimulation. Systemic MMP-1 levels were measured in plasma from wild-type (WT) and granzyme-B-knockout (GzmB-/- ) mice. RESULTS: The [GzmB]GCF in human samples was ~4-5 fold higher at sites of periodontal disease (gingivitis/periodontitis) compared to healthy control sites, suggesting an association between GzmB and localized matrix degradation. GzmB induced a ~4-5-fold increase in MMP-1 secretion by cultured fibroblasts. GzmB induced phosphorylation of Erk1/2, which was abrogated by PD98059. GzmB-induced upregulation of MMP-1 secretion was also reduced by PD98059. Blockade of PAR-1 function by ATAP-2 abrogated the increase in MMP-1 secretion by GF. Circulating MMP-1 was similar in WT and GzmB-/- mice, suggesting that GzmB's effects on MMP-1 release are not reflected systemically. CONCLUSION: These data point to a novel GzmB-driven signaling pathway in fibroblasts in which MMP-1 secretion is upregulated in a PAR1- and Erk1/2-dependent manner.

Esthetics of Orthodontic Appliances: Objective Evaluation by Spectrophotometry vs Subjective Evaluation Using the Visual Analog Scale Method.

AIM AND OBJECTIVE: In this study, we quantified the color of brackets and archwire appliances for an objective evaluation and investigated its relationship with subjective esthetic evaluation. MATERIALS AND METHODS: Five types of commercially available brackets (ceramic brackets C1, C2, and C3; plastic brackets P1 and P2) and three types of archwires (coated nickel-titanium archwires W1, W2, and W3) were used. The reflectance (%) and color (lightness: L*, hue: a*, b*) of each sample were quantified using a spectrophotometer (n = 5). Fifteen combinations of brackets and archwires were used. The esthetic evaluation was performed using the visual analog scale (VAS) method, and responses were obtained from 30 laypersons and 15 orthodontists. The mean VAS score was calculated, and the relationship between the reflectance and color of the bracket and archwire was discussed. RESULTS: The reflectance and L* of the brackets showed significantly higher values for C3 and C1 than for the others and lower values for P1 and P2. The reflectance and L* of the archwire showed significant differences among all samples. There was a high positive correlation between the reflectance and L*. There were statistically significant positive correlations between the layperson and orthodontist groups, between the VAS score and reflectivity, and between VAS score and L*. CONCLUSION: Our results showed that as the lightness and reflectance of the brackets and archwires increased, the subjective evaluation concerning their esthetic value was higher. CLINICAL SIGNIFICANCE: It is extremely difficult to evaluate esthetics despite the fact that patients' demands for esthetics have been increasing in recent years. If a method for evaluating esthetics is established, it should help in the development and selection of esthetic devices. The results of this study will facilitate the development of future study designs.

Degradation and Biocompatibility of AZ31 Magnesium Alloy Implants In Vitro and In Vivo: A Micro-Computed Tomography Study in Rats.

In current orthodontic practice, miniscrew implants (MSIs) for anchorage and bone fixation plates (BFPs) for surgical orthodontic treatment are commonly used. MSIs and BFPs that are made of bioabsorbable material would avoid the need for removal surgery. We investigated the mechanical, degradation and osseointegration properties and the bone-implant interface strength of the AZ31 bioabsorbable magnesium alloy to assess its suitability for MSIs and BFPs. The mechanical properties of a Ti alloy (TiA), AZ31 Mg alloy (MgA), pure Mg and poly-L-lactic acid (PLA) were investigated using a nanoindentation test. Also, pH changes in the solution and degradation rates were determined using immersion tests. Three-dimensional, high-resolution, micro-computed tomography (CT) of implants in the rat femur was performed. Biomechanical push-out testing was conducted to calculate the maximum shear strength of the bone-implant interface. Scanning electron microscopy (SEM), histological analysis and an evaluation of systemic inflammation were performed. MgA has mechanical properties similar to those of bone, and is suitable for implants. The degradation rate of MgA was significantly lower than that of Mg. MgA achieved a significantly higher bone-implant bond strength than TiA. Micro-CT revealed no significant differences in bone density or bone-implant contact between TiA and MgA. In conclusion, the AZ31 Mg alloy is suitable for both MSIs and BFPs.

Cementocyte cell death occurs in rat cellular cementum during orthodontic tooth movement.

OBJECTIVE: To clarify the mechanism of root resorption during orthodontic treatment, we examined cementocyte cell death and root resorption in the cellular cementum on the pressure side during experimental tooth movement. MATERIALS AND METHODS: Using 8-week-old male Wistar rats, the right first molar was pushed mesiobuccally with a force of 40 g by a Ni-Ti alloy wire while the contralateral first molar was used as a control. Localization and number of cleaved caspase-3-positive and single-stranded DNA (ssDNA) - positive cells were evaluated using dual-label immunohistochemistry with anticleaved caspase-3 and anti-ssDNA antibodies. In addition, tartrate-resistant acid phosphatase (TRAP)-positive cells in the cellular cementum were evaluated using TRAP histochemical staining. RESULTS: Caspase-3- and ssDNA-positive cells appeared at 12 hours, but were restricted to the compressed periodontal ligament (PDL) and not the cellular cementum. Cleaved caspase-3-positive cementocytes were observed in the cellular cementum adjacent to the compressed PDL on day 1. From days 2 to 4, the number of caspase-3- and ssDNA-positive cementocytes increased. TRAP-positive cells appeared on the cellular cementum at the periphery of the hyalinized tissue on day 7, and resorption progressed into the broad surface of the cementum by day 14. CONCLUSION: Cementocytes adjacent to the hyalinized tissue underwent apoptotic cell death during orthodontic tooth movement, which might have been associated with subsequent root resorption.