Local application of Usag-1 siRNA can promote tooth regeneration in Runx2-deficient mice.

Runt-related transcription factor 2 (Runx2)-deficient mice can be used to model congenital tooth agenesis in humans. Conversely, uterine sensitization-associated gene-1 (Usag-1)-deficient mice exhibit supernumerary tooth formation. Arrested tooth formation can be restored by crossing both knockout-mouse strains; however, it remains unclear whether topical inhibition of Usag-1 expression can enable the recovery of tooth formation in Runx2-deficient mice. Here, we tested whether inhibiting the topical expression of Usag-1 can reverse arrested tooth formation after Runx2 abrogation. The results showed that local application of Usag-1 Stealth small interfering RNA (siRNA) promoted tooth development following Runx2 siRNA-induced agenesis. Additionally, renal capsule transplantation of siRNA-loaded cationized, gelatin-treated mouse mandibles confirmed that cationized gelatin can serve as an effective drug-delivery system. We then performed renal capsule transplantation of wild-type and Runx2-knockout (KO) mouse mandibles, treated with Usag-1 siRNA, revealing that hindered tooth formation was rescued by Usag-1 knockdown. Furthermore, topically applied Usag-1 siRNA partially rescued arrested tooth development in Runx2-KO mice, demonstrating its potential for regenerating teeth in Runx2-deficient mice. Our findings have implications for developing topical treatments for congenital tooth agenesis.

Direct evidence for the age-dependent demise of GNAS-mutated cells in oral fibrous dysplasia.

OBJECTIVE: Fibrous dysplasia (FD) is a benign bone disease characterized by fibro-osseous lesions. FD is caused by somatic mutations in the gene, guanine nucleotide-binding protein, alpha stimulating activity polypeptide 1 (GNAS), which encodes the G protein subunit, Gsα. FD manifests early in life, but the growth of lesions usually ceases in adulthood. FD lesions often exhibit somatic mutation mosaicism. In this study, the relationship between lesion growth and mutation prevalence within a lesion was investigated. DESIGN: Lesions from five FD patients were characterized by radiographical, histological and immunohistochemical methods. To accurately calculate the prevalence of mutations within lesions, GNAS codon 201 in genomic DNA isolated from fresh surgical FD specimens was sequenced. RESULTS: Uniquely, a lesion in one 46-year-old patient was still growing, enabling simultaneous analysis of both stable-old and active-new FD lesions in the same patient. Immunohistochemical analysis indicated that a newer, proximal lesion was growing while an older, distal lesion was not. The mutation prevalence differed between these lesions; it was low in the old and high in the new lesion. Thus, the frequency of mutated cells had decreased in the older lesion. CONCLUSIONS: This is the first direct evidence for the age-dependent demise of mutated cells in FD, helping to explain why FD lesion growth generally ceases in adulthood.

Gender differences in morphological and functional outcomes after mandibular setback surgery.

PURPOSE: The aim of this study was to examine and compare morphological and functional outcomes after either isolated mandibular setback or bimaxillary surgery in males and females. MATERIALS AND METHODS: A retrospective study was done on 52 patients, in whom surgical correction for mandibular prognathism was performed either by isolated mandibular setback (30 cases) or by bimaxillary surgery (22 cases). Morphological changes were studied using cephalograms and functional changes studied using impulse oscillometry (IOS) taken before surgery (T0), 3 months (T1) and 1 year after surgery (T2). Also 3% oxygen desaturation index (ODI) was measured at T0 and T2. RESULT: Posterior airway space decreased significantly in both groups and both sexes but more so in males after mandibular setback surgery and in females after bimaxillary surgery. Changes in supine R20 (central airway resistance at 20 Hz) and supine R5 (total airway resistance at 5 Hz) in IOS statistically significantly increased in the period T0-T1 in males compared with females after mandibular setback surgery (p < 0.05). CONCLUSION: Gender dimorphism is present according to morphological and functional outcomes, with males at a higher risk for obstructive sleep apnea (OSA) after mandibular setback surgery and females after bimaxillary surgery; however, compensatory changes act as a barrier against this.

Loss of Stemness, EMT, and Supernumerary Tooth Formation in Cebpb-/-Runx2+/- Murine Incisors.

Adult Cebpb KO mice incisors present amelogenin-positive epithelium pearls, enamel and dentin allopathic hyperplasia, fewer Sox2-positive cells in labial cervical loop epitheliums, and reduced Sox2 expression in enamel epithelial stem cells. Thus, Cebpb acts upstream of Sox2 to regulate stemness. In this study, Cebpb KO mice demonstrated cementum-like hard tissue in dental pulp, loss of polarity by ameloblasts, enamel matrix in ameloblastic layer, and increased expression of epithelial-mesenchymal transition (EMT) markers in a Cebpb knockdown mouse enamel epithelial stem cell line. Runx2 knockdown in the cell line presented a similar expression pattern. Therefore, the EMT enabled disengaged odontogenic epithelial stem cells to develop supernumerary teeth. Cebpb and Runx2 knockdown in the cell line revealed higher Biglycan and Decorin expression, and Decorin-positive staining in the periapical region, indicating their involvement in supernumerary tooth formation. Cebpb and Runx2 acted synergistically and played an important role in the formation of supernumerary teeth in adult incisors.

The Relationship Between Cephalogram Analysis and Oxygen Desaturation Index During Sleep in Patients Submitted for Mandibular Setback Surgery.

OBJECTIVES: The aim of this study was to examine the relationship between morphologic factors of mandibular protrusion patients and clinical indices of obstructive sleep apnea (OSA). METHODS: Fifty-two Japanese patients divided into 2 groups: 1 jaw surgery group (30 patients) and 2 jaw surgery group (22 patients). Morphologic changes were studied using cephalograms taken before surgery and 1 year after surgery. Functional changes studied using impulse oscillometry and pulse oximetry during sleep, both of which are clinically useful measures in assessing OSA, taken before surgery and 1 year after surgery. RESULT: Lower face cage area significantly decreased in 1 jaw group than in 2 jaw group patients. Positive significant correlation was found between changes in 3% oxygen desaturation index (ODI) and changes of tongue area and vertical position of the hyoid bone in 1 jaw surgery group. Multiple regression analysis indicates that tongue area and airway area were independently significant predictors of 3% ODI in 1 jaw group patients. CONCLUSION: In 2 jaw surgery, maxillary surgery compensated for the effect of mandibular setback surgery. Mandibular setback surgery to mandibular protrusion patients was performed within the range of adequate movement distance, but precautions for risk of postoperative obstructive sleep apnea syndrome should be considered.

Antagonistic Functions of USAG-1 and RUNX2 during Tooth Development.

Supernumerary teeth and tooth agenesis are common morphological anomalies in humans. We previously obtained evidence that supernumerary maxillary incisors form as a result of the successive development of the rudimentary maxillary incisor tooth germ in Usag-1 null mice. The development of tooth germs is arrested in Runx2 null mice, and such mice also exhibit lingual epithelial buds associated with the upper molars and incisors. The aim of this study is to investigate the potential crosstalk between Usag-1 and Runx2 during tooth development. In the present study, three interesting phenomena were observed in double null Usag-1-/-/Runx2-/- mice: the prevalence of supernumerary teeth was lower than in Usag-1 null mice; tooth development progressed further compared than in Runx2 null mice; and the frequency of molar lingual buds was lower than in Runx2 null mice. Therefore, we suggest that RUNX2 and USAG-1 act in an antagonistic manner. The lingual bud was completely filled with odontogenic epithelial Sox2-positive cells in the Usag-1+/+/Runx2-/- mice, whereas almost no odontogenic epithelial Sox2-positive cells contributed to supernumerary tooth formation in the rudimentary maxillary incisors of the Usag-1-/-/Runx2+/+ mice. Our findings suggest that RUNX2 directly or indirectly prevents the differentiation and/or proliferation of odontogenic epithelial Sox2-positive cells. We hypothesize that RUNX2 inhibits the bone morphogenetic protein (BMP) and/or Wnt signaling pathways regulated by USAG-1, whereas RUNX2 expression is induced by BMP signaling independently of USAG-1.

ANKH Polymorphisms and Clicking of the Temporomandibular Joint in Dental Residents.

AIM: This study aimed to carry out a case-control research study to assess occurrence of clicking of the temporomandibular joint (TMJ) in order to establish the relationship between TMJ clicking and the genotype of "ANKH inorganic pyrophosphate transport regulator" (ANKH) polymorphisms. MATERIALS AND METHOD: A sample of 41 first-year dental residents was selected. Each was examined using standard clinical procedures and genotyping techniques. RESULTS: The participation rate was 91.8 %. The prevalence of TMJ clicking was 51.2 % (95 % CI: 35.7-66.7 %). Occurrence of TMJ clicking was not related to age, gender and genotypes of ANKH-OR as well as ANKH-TR polymorphisms (p ≥ 0.165). CONCLUSION: A similar distribution of ANKH genotypes in TMJ clicking and asymptomatic individuals has been demonstrated by this study. A high percentage of TMJ clicking has been confirmed. Future investigations are indicated.

Prospective signs of cleidocranial dysplasia in Cebpb deficiency.

BACKGROUND: Although runt-related transcription factor 2 (RUNX2) has been considered a determinant of cleidocranial dysplasia (CCD), some CCD patients were free of RUNX2 mutations. CCAAT/enhancer-binding protein beta (Cebpb) is a key factor of Runx2 expression and our previous study has reported two CCD signs including hyperdontia and elongated coronoid process of the mandible in Cebpb deficient mice. Following that, this work aimed to conduct a case-control study of thoracic, zygomatic and masticatory muscular morphology to propose an association between musculoskeletal phenotypes and deficiency of Cebpb, using a sample of Cebpb-/-, Cebpb+/- and Cebpb+/+ adult mice. Somatic skeletons and skulls of mice were inspected with soft x-rays and micro-computed tomography (µCT), respectively. Zygomatic inclination was assessed using methods of coordinate geometry and trigonometric function on anatomic landmarks identified with µCT. Masseter and temporal muscles were collected and weighed. Expression of Cebpb was examined with a reverse transcriptase polymerase chain reaction (RT-PCR) technique. RESULTS: Cebpb-/- mice displayed hypoplastic clavicles, a narrow thoracic cage, and a downward tilted zygomatic arch (p < 0.001). Although Cebpb+/- mice did not show the phenotypes above (p = 0.357), a larger mass percentage of temporal muscles over masseter muscles was seen in Cebpb+/- littermates (p = 0.012). The mRNA expression of Cebpb was detected in the clavicle, the zygoma, the temporal muscle and the masseter muscle, respectively. CONCLUSIONS: Prospective signs of CCD were identified in mice with Cebpb deficiency. These could provide an additional aetiological factor of CCD. Succeeding investigation into interactions among Cebpb, Runx2 and musculoskeletal development is indicated.

Interactions between BMP-7 and USAG-1 (uterine sensitization-associated gene-1) regulate supernumerary organ formations.

Bone morphogenetic proteins (BMPs) are highly conserved signaling molecules that are part of the transforming growth factor (TGF)-beta superfamily, and function in the patterning and morphogenesis of many organs including development of the dentition. The functions of the BMPs are controlled by certain classes of molecules that are recognized as BMP antagonists that inhibit BMP binding to their cognate receptors. In this study we tested the hypothesis that USAG-1 (uterine sensitization-associated gene-1) suppresses deciduous incisors by inhibition of BMP-7 function. We learned that USAG-1 and BMP-7 were expressed within odontogenic epithelium as well as mesenchyme during the late bud and early cap stages of tooth development. USAG-1 is a BMP antagonist, and also modulates Wnt signaling. USAG-1 abrogation rescued apoptotic elimination of odontogenic mesenchymal cells. BMP signaling in the rudimentary maxillary incisor, assessed by expressions of Msx1 and Dlx2 and the phosphorylation of Smad protein, was significantly enhanced. Using explant culture and subsequent subrenal capsule transplantation of E15 USAG-1 mutant maxillary incisor tooth primordia supplemented with BMP-7 demonstrated in USAG-1+/- as well as USAG-1-/- rescue and supernumerary tooth development. Based upon these results, we conclude that USAG-1 functions as an antagonist of BMP-7 in this model system. These results further suggest that the phenotypes of USAG-1 and BMP-7 mutant mice reported provide opportunities for regenerative medicine and dentistry.