The effect of periodontal therapy on glycaemic control in a Hispanic population with type 2 diabetes: a randomized controlled trial.

OBJECTIVE: In the Mexican-American population, the prevalence of Type 2 diabetes mellitus (T2DM) is as high as 50% of the population. This randomized controlled clinical trial was designed to elucidate how treatment of periodontal disease affects HbA1c values in this population. MATERIALS AND METHODS: One hundred and fifty-four T2DM patients with periodontal disease were enrolled in the study. The test group was treated with scaling and root planing (SRP); the control group received oral hygiene instructions. At baseline and 4-6 weeks after therapy, a complete periodontal examination was performed. Blood was collected at baseline and 4 months later for HbA1c levels. RESULTS: One hundred and twenty-six individuals completed the study. Baseline mean ± SD HbA1c for the test and control groups were 9.0 ± 2.3% and 8.4 ± 2.0% respectively. Non-significant difference in HbA1c reductions (0.6 ± 2.1% and 0.3 ± 1.7%) was found between test and control groups at 4 months. Comparisons of the periodontal clinical parameters between the test and control groups found significant differences with improved results in the test subjects. CONCLUSIONS: No statistically significant differences were found in the changes of HbA1c levels between test and control groups. Non-surgical periodontal therapy improved the magnitude of change in periodontal parameters as compared to the control subjects. ClinicalTrials.gov Identifier: NCT01128374.

Molecular studies on the roles of Runx2 and Twist1 in regulating FGF signaling.

BACKGROUND: Supernumerary teeth are often observed in patients suffering from cleidocranial dysplasia due to a mutation in Runx2 that results in haploinsufficiency. However, the underlying molecular mechanisms are poorly defined. In this study, we assessed the roles of Runx2 and its functional antagonist Twist1 in regulating fibroblast growth factor (FGF) signaling using in vitro biochemical approaches. RESULTS: We showed that Twist1 stimulated Fgfr2 and Fgf10 expression in a mesenchymal cell line and that it formed heterodimers with ubiquitously expressed E12 (together with E47 encoded by E2A gene) and upregulated Fgfr2 and Fgf10 promoter activities in a dental mesenchyme-derived cell line. We further demonstrated that the bHLH domain of Twist1 was essential for its synergistic activation of Fgfr2 promoter with E12 and that the binding of E12 stabilized Twist1 by preventing it from undergoing lysosomal degradation. Although Runx2 had no apparent effects on Fgfr2 and Fgf10 promoter activities, it inhibited the stimulatory activity of Twist1 on Fgfr2 promoter. CONCLUSIONS: These findings suggest that Runx2 haploinsufficiency might result in excessive unbound Twist1 that can freely bind to E12 and enhance FGF signaling, thereby promoting the formation of extra teeth.

A customized self-assembling peptide hydrogel for dental pulp tissue engineering.

Root canal therapy is common practice in dentistry. During this procedure, the inflamed or necrotic dental pulp is removed and replaced with a synthetic material. However, recent research provides evidence that engineering of dental pulp and dentin is possible by using biologically driven approaches. As tissue engineering strategies hold the promise to soon supersede conventional root canal treatment, there is a need for customized scaffolds for stem cell delivery or recruitment. We hypothesize that the incorporation of dental pulp-derived stem cells with bioactive factors into such a scaffold can promote cell proliferation, differentiation, and angiogenesis. In this study, we used a cell adhesive, enzyme-cleavable hydrogel made from self-assembling peptide nanofibers to encapsulate dental pulp stem cells. The growth factors (GFs) fibroblast growth factor basic, transforming growth factor ß1, and vascular endothelial growth factor were incorporated into the hydrogel via heparin binding. Release profiles were established, and the influence of GFs on cell morphology and proliferation was assessed to confirm their bioactivity after binding and subsequent release. Cell morphology and spreading in three-dimensional cultures were visualized by using cell tracker and histologic stains. Subcutaneous transplantation of the hydrogel within dentin cylinders into immunocompromised mice led to the formation of a vascularized soft connective tissue similar to dental pulp. These data support the use of this novel biomaterial as a highly promising candidate for future treatment concepts in regenerative endodontics.

Dentin conditioning codetermines cell fate in regenerative endodontics.

INTRODUCTION: Recent successes in dental pulp engineering indicate that regenerative treatment strategies in endodontics are feasible. Clinically, revascularization procedures render completion of root formation in immature teeth. The generation of a pulp-like tissue after seeding of dental pulp stem cells into dentin discs or cylinders and transplantation in vivo is possible. In this experimental setup, which mimics the situation in the root canal, the pretreatment of dentin might influence cellular behavior at the cell-dentin interface. Thus, the objective of this study was to investigate whether dentin conditioning can determine cell fate. METHODS: Dental pulp stem cells (DPSCs) were seeded into a growth factor-laden peptide hydrogel, transferred into dentin cylinders, and transplanted subcutaneously into immunocompromised mice. Before cell seeding, dentin cylinders were either pretreated with sodium hypochloride (NaOCl) or conditioned with EDTA. The constructs were explanted after 6 weeks and subjected to histological and immunohistochemical analysis. RESULTS: In dentin treated with NaOCl, resorption lacunae were found at the cell-dentin interface created by multinucleated cells with clastic activity. After conditioning with EDTA, DPSCs adjacent to the dentin formed an intimate association with the surface, differentiated into odontoblasts-like cells that expressed dentin sialoprotein, and extended cellular processes into the dentinal tubules. A vascularized soft connective tissue similar to dental pulp was observed inside the dentin cylinder. CONCLUSIONS: Dentin conditioning considerably influences DPSC fate when seeded in close proximity to dentin. This information might be critical for optimized strategic planning for future regenerative endodontic treatment.

Bioengineering of dental stem cells in a PEGylated fibrin gel.

AIM: Postnatal stem cells can generate tooth-specific structures after transplantation in vivo, which makes them a valuable tool for dental tissue engineering. Scaffold materials that are compatible with dental stem cells, injectable and tunable for targeted regeneration are needed. A candidate material is fibrin, a biopolymer critical to hemostasis and wound healing. Rapid degradation of fibrin can be decelerated by modification with polyethylene glycol (PEG), thus creating a hybrid material for cell delivery. The aim of this study was to evaluate the suitability of PEGylated fibrin as a scaffold for dental stem cells. METHODS: A PEGylated fibrin hydrogel was combined with stem cells derived from dental pulp or periodontal ligament. Cell proliferation was assessed over a 4-week period, and alkaline phosphatase activity and expression levels of mineralization-associated genes after osteogenic induction were analyzed. Cell morphology, matrix degradation, collagen production and mineral deposition were evaluated by histology. Constructs of PEGylated fibrin with dental pulp stem cells in dentin disks were transplanted in immunocompromised mice for 5 weeks and examined for new tissue formation. RESULTS: All cell types proliferated in PEGylated fibrin. After osteogenic induction, alkaline phosphatase activity was higher and osteoblast-specific genes were upregulated. Dentin-specific markers increased in pulp-derived stem cells. Histologic analysis revealed degradation of fibrin, production of a collagenous matrix and mineral deposition. In vivo transplantation rendered a vascularized soft connective tissue similar to dental pulp. CONCLUSION: Fibrin allows for the growth and differentiation of dental stem cells, can be inserted into small defects and thus appears to be a promising biomaterial for tissue regeneration in the oral cavity.

Novel expression and transcriptional regulation of FoxJ1 during oro-facial morphogenesis.

Axenfeld-Rieger syndrome (ARS) patients with PITX2 point mutations exhibit a wide range of clinical features including mild craniofacial dysmorphism and dental anomalies. Identifying new PITX2 targets and transcriptional mechanisms are important to understand the molecular basis of these anomalies. Chromatin immunoprecipitation assays demonstrate PITX2 binding to the FoxJ1 promoter and PITX2C transgenic mouse fibroblasts and PITX2-transfected cells have increased endogenous FoxJ1 expression. FoxJ1 is expressed at embryonic day 14.5 (E14.5) in early tooth germs, then down-regulated from E15.5-E17.5 and re-expressed in the inner enamel epithelium, oral epithelium, tongue epithelium, sub-mandibular salivary gland and hair follicles during E18.5 and neonate day 1. FoxJ1 and Pitx2 exhibit overlapping expression patterns in the dental and oral epithelium. PITX2 activates the FoxJ1 promoter and, Lef-1 and beta-catenin interact with PITX2 to synergistically regulate the FoxJ1 promoter. FoxJ1 physically interacts with the PITX2 homeodomain to synergistically regulate FoxJ1, providing a positive feedback mechanism for FoxJ1 expression. Furthermore, FoxJ1, PITX2, Lef-1 and beta-catenin act in concert to activate the FoxJ1 promoter. The PITX2 T68P ARS mutant protein physically interacts with FoxJ1; however, it cannot activate the FoxJ1 promoter. These data indicate a mechanism for the activity of the ARS mutant proteins in specific cell types and provides a basis for craniofacial/ tooth anomalies observed in these patients. These data reveal novel transcriptional mechanisms of FoxJ1 and demonstrate a new role of FoxJ1 in oro-facial morphogenesis.

Expression of Runx2/Cbfa1/Pebp2alphaA during angiogenesis in postnatal rodent and fetal human orofacial tissues.

UNLABELLED: Transient expression of Runx2 is reported in endothelial cells and vascular smooth muscle cells during vessel formation in skin, stroma of forming bones and developing periodontal ligament, developing skeletal muscle cells, and fat tissue. The data suggest that Runx2 is expressed in a multipotential mesenchymal cell population that gives rise to various osseous and nonosseous cell lineages. INTRODUCTION: Runx2/Cbfa1 is a transcription factor essential for cells of osteogenic and dentinogenic lineages. Here we examined expression of Runx2/Cbfa1 (all isotypes) in several nonskeletal cell types present in developing orofacial tissues of neonatal rodents and human fetuses with special emphasis on vessel formation. MATERIALS AND METHODS: Sections obtained from heads or jaws of postnatal mice, hamster, and human fetuses were immunostained with monoclonal anti-Pebp2aA antibody. Mouse and human tissues were also examined by in situ hybridization. Sections of Runx2 null mutant mice with a LacZ reporter construct inserted in the Runx2 locus were stained for Runx2 promoter activity with anti-galactosidase. RESULTS: We found transient mRNA and protein expression in endothelial cells and in vascular smooth muscle cells of forming vessels in skin, alveoli of forming bone, and forming periodontal ligament. We also noticed weak and variable expression in some fibroblasts of embryonic skin, early differentiating cross-striated muscle cells, and differentiating fat cells. CONCLUSION: Runx2 is not an exclusive marker for chondrogenic, osteogenic, and dentinogenic tissues, but is much more widely present in an early multipotential mesenchymal cell population that gives rise to several other lineages.

Functional analysis of a mutation in PAX9 associated with familial tooth agenesis in humans.

Pax9 is a paired domain-containing transcription factor that plays an essential role in the patterning of murine dentition. In humans, mutations in PAX9 are associated with unique phenotypes of familial tooth agenesis that mainly involve posterior teeth. Among these, a frameshift mutation (219InsG) within the paired domain of PAX9 produces a protein product associated with a severe form of molar agenesis in a single family. The objectives of this study were to gain new insights into the molecular pathogenesis of the 219InsG mutation and its role in tooth agenesis. Here we describe functional defects in DNA binding and transactivation of mutant 219InsGPax9. Although wild type Pax9 binds to the high affinity paired domain recognition sequences, e5 and CD19-2(A-ins), the 219InsGPax9 mutant protein was unable to bind to these cognate DNA-binding sites. In co-transfection assays, wild type Pax9 activated reporter gene transcription although the mutant was transcriptionally inactive. Immunolocalization data show that Pax9 and 219InsGPax9 proteins are synthesized in mammalian cells but that the nuclear localization of the mutant Pax9 protein is altered. Furthermore, transactivation by the full-length Pax9 protein from paired domain binding sites was not impaired by the 219InsGPax9 mutant. The latter did not alter the DNA binding activities of wild type Pax9 in gel mobility shift assays. The combined defects in DNA binding activities and transactivation function of mutant 219InsGPAX9 likely alter the selective activation and/or repression of PAX9 effector genes during odontogenesis. This loss-of-function of PAX9 most likely results in its haploinsufficiency during the patterning of dentition and the subsequent loss of posterior teeth.