Ultrastructure of Dental Enamel afflicted with Hypoplasia: An Atomic Force Microscopic Study

The ultrastructure of the human tooth enamel from a patient diagnosed with hypoplasia (HYP) was investigated using atomic force microscopy (AFM) and compared with the surface of normal human tooth enamel. Hypoplasia is a hereditary defect of dental enamel in which the enamel is deficient in either quality or quantity. AFM results presented for the HYP tooth enamel clearly demonstrate that the apatite crystal morphology in hypoplasia tooth enamel is perturbed in the diseased state which could result from a defective synthesis of the extracellular matrix proteins, e.g., amelogenin, by the ameloblasts. HYP enamel consisting of loosely packed, very small grains does not present a tendency for association, as in the case of the normal healthy tooth. Indeed, the enamel surface affected by HYP is porous and is made of much smaller grains. In some samples, the HYP part of enamel surface appeared in the form of a point-defect, which we believe may be associated with the early stages of the HYP deformation.     

Anti-TGF-beta antibody blocks enamel matrix derivative-induced upregulation of p21WAF1/cip1 and prevents its inhibition of human oral epithelial cell proliferation

We have previously demonstrated that porcine enamel matrix derivative (EMD) contains TGF-beta 1 (or a TGF-beta-like substance), and that EMD rapidly translocates smad2, which is an effector of the TGF-beta signaling pathway, into the nucleus and modulates the proliferation of both human gingival fibroblastic and oral epithelial cells in a cell type-specific manner. To investigate the involvement of TGF-beta in the growth modulatory action of EMD, two approaches have been used in the present study: i) a neutralizing anti-TGF-beta antibody to block EMD action, and ii) authentic porcine TGF-beta 1 to compare with EMD. Both in epithelial and fibroblastic cells, TGF-beta 1 closely mimicked EMD in nuclear accumulation of smad2, phosphorylation of MAP kinase family members, and consequent cell type-specific growth modulation. Anti-TGF-beta antibody, at levels which completely blocked TGF-beta 1-induced smad2 translocation, strongly blocked EMD-induced smad2 translocation. This antibody also blocked other actions of EMD in epithelial cells, i.e. p38-MAP kinase (p38-K) phosphorylation, p21WAF1/cip1 expression, and inhibition of DNA synthesis. In support of our previous proposal, these data suggest that TGF-beta 1 (or a TGF-beta-like substance), which is delivered as a principal bioactive factor in EMD, inhibits epithelial cell proliferation probably by a smad2-mediated, p21WAF1/cip1-dependent mechanism. However, the same neutralizing antibody failed to convincingly block EMD-induced fibroblastic proliferation, which suggests that EMD may contain additional unidentified mitogenic factor(s), which act in combination with TGF-beta to fully stimulate fibroblastic proliferation.     

Expression of integrins by human periodontal ligament and gingival fibroblasts and their involvement in fibroblast adhesion to enamel matrix-derived proteins

We showed recently that human periodontal ligament (PDL) and gingival fibroblasts adhere and spread on enamel matrix protein (EMP) coatings. In the present study, we investigated whether this interaction can be attributed to integrin expression. Human PDL and gingival fibroblasts were cultured for periods up to 24 h on EMP coatings, in the presence of synthetic RGD-containing peptide or an antibody against the beta1 integrin subunit. The cells were first cultured for 24 h under serum-free conditions and then cultured on EMP coatings for 48 h. Integrin expression levels were assessed by flow cytometry analysis. It was found that attachment and spreading on EMP was inhibited by the synthetic RGD-containing peptide, but not by a synthetic RGE-peptide. Both PDL and gingival fibroblasts showed expression of the integrin subunits, alpha2, alpha5, beta1, and the integrin, alphavbeta3. Incubation with an antibody against the beta1 subunit significantly inhibited the attachment and spreading of PDL and gingival fibroblasts on EMP coatings. We conclude that integrins are involved in the interaction of PDL and gingival fibroblasts with EMP.     

Porcine kallikrein-4 activation,glycosylation, activity,and expression in prokaryotic and eukaryotic hosts

Kallikrein-4 (KLK4) is a serine proteinase believed to be important in the normal development of dental enamel. We isolated native KLK4 from developing pig enamel and expressed four recombinant forms. Pig KLK4 was expressed in bacteria with and without the propeptide, and in two eukaryotic systems. Recombinant pig KLK4 was secreted as a zymogen by '293' cells and purified. The proKLK4 was activated in vitro by thermolysin and recombinant pig enamelysin, but not by native KLK4. These results were confirmed using a fluorescent peptide analog of the KLK4 propeptide-enzyme junction. Native KLK4 appears as a doublet at 37 kDa and 34 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Removal of N-linked oligosaccharides by digestion with deglycosidase-F reduced the doublet to a single band at approximately 28 kDa, demonstrating that the active enzyme is glycosylated, and that the 37 kDa and 34 kDa forms differ only in their number of glycosylations. Deglycosylation was also associated with a loss of proteolytic activity. We digested recombinant pig amelogenin with native KLK4 and characterized the cleavage products by N-terminal sequencing and mass spectrometry. Eleven cleavage sites in the amelogenin protein were identified, demonstrating that KLK4 degrades amelogenin and is likely to participate in the degradation of enamel proteins in vivo.     

中药五倍子、蜂房提取物对早期釉质龋再矿化作用的实验研究

目的通过pH循环实验研究中药五倍子、蜂房提取物对人工早期釉质龋的再矿化作用。方法应用离体牛切牙制备人工釉质龋标本40例,随机分为4组:(a)去离子水组(阴性对照)、(b)五倍子组、(c)蜂房组、(d)NaF组(阳性对照),进行体外pH循环实验后,采用显微硬度计测定标本脱矿前后和pH循环后的硬度值,观察釉质表面显微硬度的变化。通过偏光显微镜观察早期釉质龋损再矿化后的病理学改变。结果NaF组(阳性对照)和五倍子组能明显的提高早期釉质龋的表面显微硬度(P<0.05),蜂房组虽然也能提高釉质龋的表面显微硬度,但与去离子水组(阴性对照)比较无统计学意义(P>0.05)。偏光显微镜下,pH循环后NaF组(阳性对照)和五倍子组表层明显增厚,密度增加,病损体部出现呈负性双折射的再矿化条带,病损深度也明显变浅。结论五倍子能有效促进早期釉质龋再矿化,因而具有防龋功效。     

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本研究通过对比釉质磨除的氟斑牙与正常牙之间托槽抗剪切粘接破坏力的差异,探讨釉质局部磨除在提高氟斑牙患者托槽粘接的效果,为临床氟斑牙托槽粘接提供有效的方法。     

红碎茶对大鼠氟斑牙釉质形成及12种元素含量变化的影响

目的了解在接近人类饮茶习惯状态下,低超常氟含量红碎茶对牙齿釉质形态结构、化学元素含量的影响。方法对饮用氟浓度为8.2mg/L和16.4mg/L红碎茶水360天后的144只大鼠牙齿的牙釉质,使用偏光显微镜、扫描电子显微镜、X线荧光光谱仪观察牙釉质形态结构,检测釉质中的化学元素含量。结果两种氟含量的红碎茶水均导致动物氟斑牙釉质出现过度钙化和钙化不全,呈现出剂量-效应关系。检测到动物氟斑牙釉质中含有钙、磷、镁、铝等12种化学元素。两个实验组牙齿釉质的钙、磷、镁含量显著下降,铝、氯含量显著上升。饮氟含量8.2mg/L红碎茶水组硅、硫、铁含量低于对照组,而在氟含量16.4mg/L红碎茶水组,这3种元素含量显著高于8.2mg/L组和对照组。结论长期饮用低超常量氟含量红碎茶水可导致机体慢性氟中毒,低超常氟含量红碎茶对釉质形态结构的损害主要表现为过度钙化和钙化不全同时存在。釉质中钙、磷含量下降,铝、氯含量增加,以及其他几种化学元素含量的改变提示,这些化学元素可能参与了影响釉质的发育、矿化过程。     

大黄提取液对脱矿釉质中钙溶出量的影响

目的 观察大黄提取液对脱矿釉质中钙溶出量的影响.方法 用不同浓度的大黄提取液和去离子水处理牙面后进行脱矿,ASCA生化分析仪对釉质脱矿后的脱矿液进行钙离子浓度的检测.结果 2 mg/ml和4 mg/ml大黄提取液处理后釉质钙溶出量<去离了水(P<0.01)、<1 mg/ml大黄提取液组(P<0.05),>2%氟化钠组但差异无统计学意义(P>0.05).1 mg/ml大黄提取液处理后釉质钙溶出量>2%氟化钠组(P<0.01),<去离子水组但差异无统计学意义(P>0.05).结论 2mg/ ml和4 mg/ml大黄提取液具有抑制脱矿釉质中钙溶出作用:2mg/ml大黄提取液为抑制钙溶出的起始有效浓度.     

Neonatal Serum Phosphorus Levels and Enamel Defects in Very Low Birth Weight Infants

Background: Very low birth weight (VLBW) infants miss out on the period of greatest mineral accretion that occurs during the last trimester of pregnancy and are at higher risk of enamel defects. No studies have well described the relationship between neonatal nutrition and dental outcomes in preterm, VLBW infants. The objective of this study was to assess the differences in nutrition biomarkers, feeding intake, and comorbidities among VLBW infants with and without enamel defects. Methods: A retrospective chart review of VLBW infants recruited for an ongoing longitudinal dental study between 2007 and 2010 was done. Participants were classified as cases and controls according to the presence/absence of developmental defects of enamel at 8 and/or 18–20 and/or 36 months. Demographics and medical and nutrition data were abstracted from 76 subjects' medical charts. Results: Of the 76 VLBW subjects, 62% had enamel defects (hypoplasia and/or opacity). The only significant variable in the logistic regression analysis was that infants with a 1‐mg/dL increase in serum phosphorus levels had a 68% reduction in the odds of having enamel hypoplasia (odds ratio, 0.322; P = .024). Conclusion: Neonatal lower serum phosphorus levels are significantly associated with enamel hypoplasia in VLBW infants younger than 3 years.     

氟化泡沫与氟保护漆对离体乳牙釉质保护效果的比较

目的 比较局部使用氟化泡沫和氟保护漆对乳牙釉质脱矿的保护作用。方法 60颗牙冠完整的下颌乳切牙,在牙冠唇侧选定釉质处理区域,按照不同试剂处理分为A、B、C三组,A:氟化泡沫组,B:氟保护漆组,C:去离子水组。处理后的离体牙在脱矿液中浸泡72 h后用扫描电镜观察釉质表面形态结构差异;利用原子吸收分光光度计检测各组脱矿液中钙离子的溶出量。结果 A、B两组析出的Ca²⁺浓度均显著低于C组(P<0.05);B组析出的Ca²⁺浓度显著低于A组(P<0.05)。扫描电镜观察釉质表面显示C组较A、B两组脱矿明显,A、B两组的釉质表面可见再矿化物。结论 离体下颌乳切牙应用氟化泡沫及氟保护漆都可以增强其釉质的抗酸能力并促进再矿化,氟保护漆较氟化泡沫具有更强的保护作用。