Yong Hee P Chun

Enamel Formation and Amelogenesis Imperfecta

Dental enamel is the epithelial-derived hard tissue covering the crowns of teeth. It is the most highly mineralized and hardest tissue in the body. Dental enamel is acellular and has no physiological means of repair outside of the protective and remineralization potential provided by saliva. Enamel is comprised of highly organized hydroxyapatite crystals that form in a defined extracellular space, the contents of which are supplied and regulated by ameloblasts. The entire process is under genetic instruction. The genetic control of amelogenesis is poorly understood, but requires the activities of multiple components that are uniquely important for dental enamel formation. Amelogenesis imperfecta (AI) is a collective designation for the variety of inherited conditions displaying isolated enamel malformations, but the designation is also used to indicate the presence of an enamel phenotype in syndromes. Recently, genetic studies have demonstrated the importance of genes encoding enamel matrix proteins in the etiology of isolated AI. Here we review the essential elements of dental enamel formation and the results of genetic analyses that have identified disease-causing mutations in genes encoding enamel matrix proteins. In addition, we provide a fresh perspective on the roles matrix proteins play in catalyzing the biomineralization of dental enamel.

Comparative Clinical Study of the Effectiveness of Three Different Bleaching Methods

OBJECTIVE The current study assessed the efficacy of three current bleaching methods. METHODS Seventy-five healthy subjects (45 female; 30 male) with anterior teeth, having a Vita Shade score of A2 or darker, participated in the study. The subjects were randomly assigned to one of three treatment groups: Group A: home-bleaching (illumine Home, 10% carbamide peroxide, trays, overnight, for two weeks), Group B: in-office bleaching (Illumine Office, 15% hydrogen peroxide, trays for 45 minutes, three times over three weeks), Group C: Whitestrips (strips, twice a day, 30 minutes each for two weeks). Following the screening visit, three weeks prior to the baseline examination, all subjects received a dental prophylaxis. The color of the teeth was determined using a colorimeter (ShadeEye NCC) and a custom-made stent at baseline (E0), immediately after completion of the bleaching (E3) and three months after treatment (E4). All subjects received oral hygiene instructions and a toothbrush and toothpaste for oral home care during the study period. The change of tooth color was determined for each treatment regimen between baseline and E3 and baseline and E4 and was statistically analyzed performing the Kruskal Wallis test and the Mann-Whitney-U test. The significance level was set atp < 0.01. RESULTS The dropout rate was 0%. Mean (SD) deltaE* (overall color change) from baseline to immediately after treatment was 6.57 (2.13) for Group A, 5.77 (1.72) for Group B and 3.58 (1.57) for Group C. The mean (SD) tooth color change from baseline to three months after treatment deltaE* was: 4.98 (1.34) for Group A, 4.59 (1.42) for Group B and 2.99 (1.39) for Group C. Significant differences were found between home bleaching and Whitestrips, as well as between in-office bleaching and Whitestrips, but not between home-bleaching and in-office bleaching during the same time. CONCLUSION Using an objective color measurement device, home bleaching and in-office bleaching were found to be superior to Whitestrips. Home bleaching and in-office bleaching were equally efficient for bleaching teeth and maintaining the results for up to three months.

Cleavage Site Specificity of MMP-20 for Secretory-stage Ameloblastin

Ameloblastin is processed by protease(s) during enamel formation. We tested the hypothesis that MMP-20 (enamelysin) catalyzes the cleavages that generate secretory-stage ameloblastin cleavage products. We isolated a 23-kDa ameloblastin cleavage product from developing enamel and determined its N-terminus sequence. Ameloblastin was stably expressed and secreted from HEK293-H cells, purified, and digested with MMP-20 or Klk4 (kallikrein 4). The digests were analyzed by SDS-PAGE and Western blotting, and cleavage products were characterized by N-terminal sequencing. Six fluorescent peptides were digested with MMP-20 and Klk4 and analyzed by RP-HPLC and by mass spectrometry. MMP-20 cleaved each peptide exactly at the sites corresponding to ameloblastin cleavages catalyzed in vivo. Klk4 cleaved ameloblastin and the fluorescent peptides at sites not observed in vivo, and cleaved at only a single correct site: before Leu171. We conclude that MMP-20 is the enzyme that processes ameloblastin during the secretory stage of amelogenesis, and we present a hypothesis about the sequence of ameloblastin cleavages.

Role of Bcl2 in osteoclastogenesis and PTH anabolic actions in bone.

Introduction: B‐cell leukemia/lymphoma 2 (Bcl2) is a proto‐oncogene best known for its ability to suppress cell death. However, the role of Bcl2 in the skeletal system is unknown. Bcl2 has been hypothesized to play an important anti‐apoptotic role in osteoblasts during anabolic actions of PTH. Although rational, this has not been validated in vivo; hence, the impact of Bcl2 in bone remains unknown.

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.

Restoring strength of incisors with veneers and full ceramic crowns.

PURPOSE The aim was to determine the in vitro fracture resistance of incisors restored with veneers and full ceramic crowns compared to unrestored teeth. MATERIALS AND METHODS Seventy intact, extracted human maxillary central incisors were randomized and assigned to 7 groups (n = 10). The teeth in group 1 remained intact (control). The teeth in groups 2 to 6 were prepared and IPS Empress restorations were conditioned and bonded using an adhesive luting cement, Variolink II/Syntac (group 2: labial veneer with incisal overlap, group 3: 3/4 veneer with margin in enamel, group 4: 3/4 veneer with margin in dentin, group 5: crown with margin in enamel, group 6: crown with margin in dentin group 7: veneer on worn tooth. After finishing and polishing, specimens were stored in water and thermocycled for 2000 cycles between 5 degrees C and 55 degrees C. The maximal fracture load of the specimens (40-degree inclination) was determined using the universal testing machine (Zwick) at a constant crosshead speed (0.5 mm/min). The statistical analysis was performed using the Kruskal-Wallis test with Bonferroni correction (p < 0.05). Fracture surfaces were qualitatively analyzed by SEM. RESULTS All restored teeth with cervical preparation margins in enamel showed a fracture load not significantly different from the intact teeth (control). Restored teeth with cervical preparation margins in dentin showed a significantly lower fracture load. All restorations showed a fracture load far above 400 N, serving as functional reference for anterior teeth. The failures were predominantly cohesive. CONCLUSION For the restoration of tooth strength, defining the finishing lines of veneers and crowns in enamel is recommended. Restorations with finishing lines in dentin resulted in significant loss of strength. Three-quarter veneers with finishing lines in enamel are functionally equal to crowns with the advantage of conserving tooth structure.

Transgenic Rescue of Enamel Phenotype in Ambn Null Mice

Ameloblastin null mice fail to make an enamel layer, but the defects could be due to an absence of functional ameloblastin or to the secretion of a potentially toxic mutant ameloblastin. We hypothesized that the enamel phenotype could be rescued by the transgenic expression of normal ameloblastin in Ambn mutant mice. We established and analyzed 5 transgenic lines that expressed ameloblastin from the amelogenin (AmelX) promoter and identified transgenic lines that express virtually no transgene, slightly less than normal (Tg+), somewhat higher than normal (Tg++), and much higher than normal (Tg+++) levels of ameloblastin. All lines expressing detectable levels of ameloblastin at least partially recovered the enamel phenotype. When ameloblastin expression was only somewhat higher than normal, the enamel covering the molars and incisors was of normal thickness, had clearly defined rod and interrod enamel, and held up well in function. We conclude that ameloblastin is essential for dental enamel formation.

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.

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.

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. J. Cell. Physiol. 230: 1871–1882, 2015.