Mapping and Identification of Native Proteins of Developing Teeth in Mouse Mandibles.

Mass spectrometry imaging is a powerful tool of increasing utility due to its ability to spatially resolve molecular biomarkers directly from sectioned tissues. One hindrance to its universality is that no single protocol is sufficient for every tissue type, fixation, and pretreatment. Mineralized tissues are uniquely challenging as extensive decalcification protocols are necessary to achieve thin sections. In this study, we optimized a method to image tryptic peptides by matrix-assisted laser desorption ionization mass spectrometry of decalcified, formalin-fixed paraffin-embedded mouse hemimandibles. Using a combination of on-tissue MS/MS and hydrogel extraction LC-MS/MS, peptides from the enamel, dentin, periodontal ligament, alveolar bone, pulp, and other regions are identified and mapped. This breakthrough method provides a comprehensive approach to biomarker discovery in dental and craniofacial tissues which is highly relevant given that diseases originating from this region of the body are the most prevalent across all populations.

Bmp2 deletion causes an amelogenesis imperfecta phenotype via regulating enamel gene expression.

Although Bmp2 is essential for tooth formation, the role of Bmp2 during enamel formation remains unknown in vivo. In this study, the role of Bmp2 in regulation of enamel formation was investigated by the Bmp2 conditional knock out (Bmp2 cKO) mice. Teeth of Bmp2 cKO mice displayed severe and profound phenotypes with asymmetric and misshaped incisors as well as abrasion of incisors and molars. Scanning electron microscopy analysis showed that the enamel layer was hypoplastic and enamel lacked a typical prismatic pattern. Teeth from null mice were much more brittle as tested by shear and compressive moduli. Expression of enamel matrix protein genes, amelogenin, enamelin, and enamel-processing proteases, Mmp-20 and Klk4 was reduced in the Bmp2 cKO teeth as reflected in a reduced enamel formation. Exogenous Bmp2 up-regulated those gene expressions in mouse enamel organ epithelial cells. This result for the first time indicates Bmp2 signaling is essential for proper enamel development and mineralization in vivo.

Overexpression of ameloblastin in secretory ameloblasts results in demarcated, hypomineralized opacities in enamel.

Introduction: Developmental defects of the enamel manifest before tooth eruption and include amelogenesis imperfecta, a rare disease of underlying gene mutations, and molar-incisor hypomineralization (MIH), a prevalent disease in children originating from environmental and epigenetic factors. MIH enamel presents as the abnormal enamel marked by loss of translucency, demarcation between the healthy and affected enamel, and reduced mineral content. The pathophysiology of opaque, demarcated enamel lesions is not understood; however, the retention of enamel proteins in the matrix has been suggested. Ameloblastin (Ambn) is an enamel protein of the secreted calcium-binding phosphoproteins (SCPPs) critical for enamel formation. When the Ambn gene is mutated or deleted, teeth are affected by hypoplastic amelogenesis imperfecta. Methods: In this study, enamel formation in mice was analyzed when transgenic Ambn was overexpressed from the amelogenin promoter encoding full-length Ambn. Ambn was under- and overexpressed at six increasing concentrations in separate mouse lines. Results: Mice overexpressing Ambn displayed opaque enamel at low concentrations and demarcated lesions at high concentrations. The severity of enamel lesions increased starting from the inner enamel close to the dentino-enamel junction (DEJ) to span the entire width of the enamel layer in demarcated areas. Associated with the opaque enamel were 17-kDa Ambn cleavage products, a prolonged secretory stage, and a thin basement membrane in the maturation stage. Ambn accumulations found in the innermost enamel close to the DEJ and the mineralization front correlated with reduced mineral content. Demarcated enamel lesions were associated with Ambn species of 17 kDa and higher, prolonged secretory and transition stages, a thin basement membrane, and shortened maturation stages. Hypomineralized opacities were delineated against the surrounding mineralized enamel and adjacent to ameloblasts detached from the enamel surface. Inefficient Ambn cleavage, loss of contact between ameloblasts, and the altered basement membrane curtailed the endocytic activity; thus, enamel proteins remained unresorbed in the matrix. Ameloblasts have the ability to distinguish between Ambn concentration and Ambn cleavage products through finely tuned feedback mechanisms. The under- or overexpression of Ambn in murine secretory ameloblasts results in either hypoplastic amelogenesis imperfecta or hypomineralization with opaque or sharply demarcated boundaries of lesions, similar to MIH.

Hyperglycemia and xerostomia are key determinants of tooth decay in type 1 diabetic mice.

Insulin-dependent type 1 diabetes mellitus (DM) and oral diseases are closely interrelated. Poor metabolic control in diabetics is associated with a high risk of gingivitis, periodontitis and tooth loss. Salivary flow declines in diabetics and patients suffer from xerostomia. Reduced saliva predisposes to enamel hypomineralization and caries formation; however, the mechanisms that initiate and lead to progression of tooth decay and periodontitis in type 1 DM have not been explored. To address this issue, we analyzed tooth morphology in Akita ⁻/⁻ mice that harbor a point mutation in the Ins2 insulin gene, which leads to progressive hyperglycemia. Mandibles from Akita ⁻/⁻ and wild-type littermates were analyzed by microCT, scanning EM and histology; teeth were examined for amelogenin (Amel) and ameloblastin (Ambn) expression. Mice were injected with pilocarpine to assess saliva production. As hyperglycemia may alter pulp repair, the effect of high glucose levels on the proliferation/differentiation of cultured MD10-F2 pulp cells was also analyzed. Results showed that Akita ⁻/⁻ mice at 6 weeks of age showed chalky white incisors that correlated with marked hyperglycemia and impaired saliva production. MicroCT of Akita ⁻/⁻ teeth revealed excessive enamel wearing and hypomineralization; immunostaining for Amel and Ambn was decreased. A striking feature was invasion of dentinal tubules with Streptococcus mitis and microabcesses that originated in the coronal pulp and progressed to pulp necrosis and periapical periodontitis. High levels of glucose also inhibited MD10-F2 cell proliferation and differentiation. Our findings provide the first evidence that hyperglycemia in combination with reduced saliva in a model of type1 DM leads to decreased enamel mineralization/matrix proteins and predisposes to excessive wearing and decay. Importantly, hyperglycemia adversely affects enamel matrix proteins and pulp repair. Early detection and treatment of hyperglycemia and hyposalivation may provide a useful strategy for preventing the dental complications of diabetes and promoting oral health in this population.

Angiogenic activity of an enamel matrix derivative (EMD) and EMD-derived proteins: an experimental study in mice.

OBJECTIVES: To determine whether all or only certain proteins in an enamel matrix derivative (EMD) are angiogenic. MATERIALS AND METHODS: The angiogenic effect was analysed using an in vivo angiogenesis assay. Silicon tubes were filled with or without potential and known angiogenic-modulating factors: (i) an EMD parent, (ii) nine pools of EMD proteins, (iii) fibroblast growth factor/vascular endothelial growth factor and (iv) amelogenin. Silicon tubes were implanted subcutaneously in mice. Dextran-fluorescein isothiocyanate (FITC) was injected via the tail vein, mice were euthanized and tubes were retrieved. Neovascularization was determined by measuring the amount of dextran-FITC within the tubes. RESULTS: The greatest angiogenic potential of the EMD parent was at a weight of 125 ng, resulting in a 4.3-fold increase compared with the negative control. Five pools of EMD proteins showed a stronger angiogenic activity than the EMD parent. Pool 5 showed the greatest angiogenic activity, when compared with the negative control (8.1-fold increase) and with 125 ng of the EMD parent (4.2-fold increase). Amelogenin demonstrated a significantly higher angiogenic activity than the negative control (increase up to 4.0-fold) and the EMD parent (increase up to 1.6-fold). CONCLUSIONS: EMD parent, recombinant porcine amelogenin and certain pools of EMD proteins induced significant angiogenesis compared with the controls using a standardized in vivo assay.

Should dental school faculty be measured and compensated using academic productivity models? Two viewpoints.

Operationalizing faculty contributions in ways that align with organizational mission can be difficult, particularly when monetizing effort. Conventional compensation methods may result in faculty effort going undefined, resulting in more subjectivity in recognition and compensation. Inequities lead to faculty marginalization, fragmentation, decreased motivation, and attrition. Dental faculty retirements are expected to increase, as 81% of men and 19% of women faculty aged 60 years and older in 2015-2016. We present opposing perspectives on the use of educational value units (EVUs) in academic dentistry. The first viewpoint articulates that such models improve recruitment and retention by objectifying (a) faculty performance measurement, (b) academic productivity improvements, and (c) compensation determination. The counterpoint suggests EVUs are deterrents to faculty retention due to challenges with objectively quantifying performance measures, a potential inherent bias linked to gender, and the undervaluing of teaching quality or collaborative practices.

Impact of Er:YAG laser on wound healing following nonsurgical therapy: A pilot study.

The purpose is to examine early wound healing through histological analysis by characterizing connective tissue distribution and organization in the treated periodontium following nonsurgical therapy. Periodontal disease is a multifactorial pathological process that leads to the loss of the surrounding periodontium. Traditional periodontal therapies have proven beneficial in halting the progression of disease. The aim of this study is to investigate early wound healing in periodontal patients following hand/ultrasonic instrumentation alone, erbium-doped yttrium aluminum garnet laser instrumentation alone, or a combination of hand/ultrasonic instrumentation and Er:YAG laser instrumentation for the nonsurgical treatment of periodontitis by histologic evaluation. Twenty-one patients were randomized to receive nonsurgical therapy for the treatment of chronic periodontitis with three modalities prior to surgical therapy. Baseline clinical measurements were obtained prior to treatment. Wound healing was assessed by obtaining an otherwise discarded tissue sample following nonsurgical therapy of the selected study site. Samples were obtained at 2 or 6 weeks following initial therapy with a step-back incision and fixated for histological and immunohistochemical analysis. There were minimal between-group differences in the amount of collagen distribution when analyzing the Mallory-Heidenhain Azan trichrome, Picrosirus Red stain, and proliferating cell nuclear antigen at both time points. Descriptive analysis of baseline measurements showed no differences in probing depth change, bleeding on probing, and clinical attachment level following initial therapy between the three treatment groups at 2 or 6 weeks. Each treatment modality was effective in treating moderate to severe chronic periodontitis; however, the results of this study are inconclusive regarding superiority of any one treatment approach from a histologic and immunohistochemical perspective. Based on this assessment, there was increased fibroblast proliferation and collagen maturation between the 2- and 6-week time point after treatment in all treatment groups, with few apparent differences between treatment groups. This pilot study qualitatively evaluated early wound healing in periodontal patients following non surgical therapy with various treatment modalities. When comparing descriptive outcomes of Er:YAG laser therapy and hand/ultrasonic instrumentation there were minimal differences in collagen distribution and density between groups. The evaluated modalities were each effective treating periodontal patients with non surgical therapy.

Toothbrush abrasivity in a long-term simulation on human dentin depends on brushing mode and bristle arrangement.

OBJECTIVE: The aim of this study was to evaluate the susceptibility of dentin to brushing abrasion using four different toothbrushes (rotating-oscillating, sonic and two types of manual toothbrushes) with the same brushing forces. METHODS: Dentin samples (n = 72) were selected from 72 impacted third molars. Half of the surface of dentin samples was covered with an adhesive tape, creating a protected and a freely exposed area in the same specimen. Brushing was performed with either a: sonic (Sonicare PowerUp, Philips GmbH, Hamburg, Germany), b: oscillating-rotating (Oral B Vitality Precisions Clean, Procter & Gamble, Schwalbach am Taunus, Germany) or two different manual toothbrushes c: flat trim brush head toothbrush (Dr. Best: Original, Glaxo-Smith-Kline, Bühl, Germany) and d: rippled-shaped brush head toothbrush (Blend-a-Dent, Complete V-Interdental, Blend-a-med, Schwalbach, Germany) in a custom made automatic brushing machine. The brushing force was set to 2 N and a whitening toothpaste (RDA = 150) was used. The simulation period was performed over a calculated period to mimic a brushing behavior of two times a day brushing for eight years and six months. Dentin loss was quantitatively determined by profilometry and statistically analyzed by Wilcoxon and Mann-Whitney-U Test (p < 0.05). RESULTS: The mean (standard deviation) surface loss was 21.03 (±1.26) µm for the sonic toothbrush, 15.71 (±0.85) µm for the oscillating-rotating toothbrush, 6.13 (±1.24) µm for the manual toothbrush with flat trim brush head and 2.50 (±0.43) µm for the manual toothbrush with rippled-shaped brush head. Differences between all groups were statistically significant at p<0.05. CONCLUSION: Using the same brushing force and a highly abrasive toothpaste, manual toothbrushes are significantly less abrasive compared to power toothbrushes for an 8.5-year simulation.

Does Gingival Recession Require Surgical Treatment?

Gingival recession represents a clinical condition in adults frequently encountered in the general dental practice. Clinicians often face dilemmas of whether or not to treat such a condition surgically. An initial condensed literature search was performed using a combination of gingival recession and surgery controlled terms and keywords. An analysis of the search results highlights the limited understanding of the factors that guide the treatment of gingival recession. Understanding the cause, prognosis, and treatment of gingival recession continues to offer many unanswered questions and challenges in periodontics as we strive to provide the best care possible for our patients.

Emerging regenerative approaches for periodontal reconstruction: a consensus report from the AAP Regeneration Workshop.

BACKGROUND: Historically, periodontal regeneration has focused predominantly on bone substitutes and/or barrier membrane application to provide for defect fill and/or selected cell repopulation of the lesion. More recently, a number of technologies have evolved that can be viewed as emerging therapeutic approaches for periodontal regeneration, and these technologies were considered in the review paper and by the consensus group. The goal of this consensus report on emerging regenerative approaches for periodontal hard and soft tissue reconstruction was to develop a consensus document based on the accompanying review paper and on additional materials submitted before and at the consensus group session. METHODS: The review paper was sent to all the consensus group participants in advance of the consensus conference. In addition and also before the conference, individual consensus group members submitted additional material for consideration by the group. At the conference, each consensus group participant introduced themselves and provided disclosure of any potential conflicts of interest. The review paper was briefly presented by two of the authors and discussed by the consensus group. A discussion of each of the following topics then occurred based on the content of the review: a general summary of the topic, implications for patient-reported outcomes, and suggested research priorities for the future. As each topic was discussed based on the review article, supplemental information was then added that the consensus group agreed on. Last, an updated reference list was created. RESULTS: The application of protein and peptide therapy, cell-based therapy, genetic therapy, application of scaffolds, bone anabolics, and lasers were found to be emerging technologies for periodontal regeneration. Other approaches included the following: 1) therapies directed at the resolution of inflammation; 2) therapies that took into account the influence of the microbiome; 3) therapies involving the local regulation of phosphate and pyrophosphate metabolism; and 4) approaches directed at harnessing current therapies used for other purposes. The results indicate that, with most emerging technologies, the specific mechanisms of action are not well understood nor are the specific target cells identified. Patient-related outcomes were typically not addressed in the literature. Numerous recommendations can be made for future research priorities for both basic science and clinical application of emerging therapies. The need to emphasize the importance of regeneration of a functional periodontal organ system was noted. The predictability and efficacy of outcomes, as well as safety concerns and the cost-to-benefit ratio were also identified as key factors for emerging technologies. CONCLUSIONS: A number of technologies appear viable as emerging regenerative approaches for periodontal hard and soft tissue regeneration and are expanding the potential of reconstructing the entire periodontal organ system. The cost-to-benefit ratio and safety issues are important considerations for any new emerging therapies. Clinical Recommendation: At this time, there is insufficient evidence on emerging periodontal regenerative technologies to warrant definitive clinical recommendations.