Amelogenins regulate expression of genes associated with cementoblasts in vitro

Amelogenins are major proteins expressed by ameloblasts during development of the crown (enamel and dentin). These matrix proteins guide crystal habits of the mineral phase of developing enamel and are possible regulators of other genes/proteins during development and maturation of crown and root (dentin and cementum). This study focused on defining the effect that a specific proteolytic cleavage product of amelogenin, tyrosine-rich amelogenin peptide (TRAP), has on cementoblast behavior. Immortalized cementoblasts (OCCM-30) were exposed to TRAP in vitro . Cells treated with TRAP were evaluated for cell proliferation, gene expression for osteocalcin (OCN), osteopontin (OPN), and bone sialoprotein (BSP), and induction of mineral nodule formation. No significant difference in cell proliferation was found between vehicle-treated cells and those treated with TRAP for up to 9 d after treatment. Gene expression of OCN, OPN, and BSP in TRAP-treated cementoblasts showed down-regulation, up-regulation, and no significant change, respectively, relative to vehicle control. A marked decrease in mineral nodule formation was found in cells treated with TRAP compared with the vehicle control, in a dose-dependent manner. These data, along with our previous results demonstrating similar activity with full-length amelogenin and leucine-rich amelogenin peptide (LRAP), suggest that amelogenin-like molecules regulate mesenchymal cell behavior.     

Growth factors regulate expression of osteoblast-associated genes

BACKGROUND: The goal of periodontal regenerative therapies is to reconstruct periodontal tissues such as bone, cementum, and periodontal ligament cells (PDL). The need to establish predictable treatment modalities is important for reconstruction of these tissues. The aim of this study was to determine the effects of a low molecular extract of bovine bone protein (BP) containing bone morphogenetic proteins (BMPs) 2, 3, 4, 6, 7, 12, and 13, alone or in combination with platelet-derived growth factor (PDGF) and/or insulin-like growth factor (IGF) on osteoblast differentiation in vitro. METHODS: BP, mixed with a collagen matrix, was added to a poly (DL-lactide-co-glycolide) polymer (PLG) and placed at orthotopic sites in the skullcaps of Sprague-Dawleys rats. At day 28, rats were sacrificed for histological analysis. All sites treated with the polymer/BP produced bone while control sites (without BP) showed no bone formation. Having established the biological activity of BP, in vitro studies were initiated using MC3T3-E1 cells, a mouse osteoprogenitor cell line. The ability of BP and other growth factors to alter cell proliferation was determined by Coulter counter, and differentiation was determined by Northern analysis for specific genes. RESULTS: When compared with cells treated with 2% serum alone, PDGF enhanced cell numbers at 10 and 20 ng/ml; IGF produced no significant effect at these doses; and BP at 10 and 20 microg/ml decreased cell proliferation. Northern analysis revealed that PDGF blocked gene expression of osteopontin (OPN) and osteocalcin (OCN), while BP and IGF promoted gene expression of bone sialoprotein (BSP) and OPN. The combination of BP and IGF enhanced expression of OPN beyond that of either BP or IGF alone. PDGF was able to block the effects of IGF on gene expression, but not those of BP. CONCLUSIONS: These results indicate that BP, PDGF, and IGF influence cell activity differently, and thus raise the possibility that combining factors may enhance the biological activity of cells.     

Enamel formation genes are associated with high caries experience in Turkish children

There is evidence for a genetic component in caries susceptibility, and studies in humans have suggested that variation in enamel formation genes and their interaction with Streptococcus mutans levels may contribute to caries. For the present study, we used DNA samples collected from 173 unrelated children from Istanbul: 91 children with 4 or more affected tooth surfaces and 82 caries-free children. Six single-nucleotide polymorphism markers were genotyped in selected candidate genes (ameloblastin, amelogenin, enamelin, tuftelin 1 and tuftelin interacting protein 11) that influence enamel formation. Allele and genotype frequencies were compared between groups with distinct caries experience. Regression analysis was used for the evaluation of individual gene effects, environmental effects and gene-environment interactions. Overrepresentation of the C allele of the amelogenin marker was seen in cases with dmft scores higher than 8 (p = 0.01) when compared to controls. Also, overrepresentation of the T allele of the ameloblastin marker was seen in cases with dmfs scores higher than 10 (p = 0.05) when compared to controls. In addition, the CT genotype of the tuftelin rs3790506 marker was overrepresented in cases with dmft scores higher than 5 (p = 0.05) and dmfs scores higher than 6 (p = 0.05) compared to controls. The best-fitting model showed that dmfs is increased when the following factors are present: (1) females and both the anterior and posterior teeth are affected simultaneously, (2) when the T allele of the tuftelin rs3790506 is involved, and (3) the C allele of the amelogenin rs17878486 is involved. Our study provides support that genes involved in enamel formation modify caries susceptibility in humans.     

Dynamic expression of E-cadherin in ameloblasts and cementoblasts in mice

During embryonic development, E-cadherin mediates intercellular adhesion in a variety of epithelia in a spatio-temporal pattern. We have analyzed the distribution of this cell adhesion molecule in the mouse during odontogenesis, at both mRNA and protein levels, in the mandibular first molars and incisors. E-cadherin was strongly expressed at the bell stage by the cells of the dental organ, and by the pre-secretory ameloblasts and the cells of stratum intermedium at the early mineralization stage. At the onset of enamel secretion, E-cadherin disappeared from the apical pool of the ameloblasts and was later absent from the post-secretory ameloblasts. E-cadherin was also found in Hertwig's root sheath and later in the cells producing acellular cementum. These findings indicate that E-cadherin may be involved in the polarization of the ameloblasts and in the early stages of cementogenesis.     

Comparison of gene expression profile of cementoblasts with periodontal ligament cells in mouse mandible with laser capture microdissection

Cementum is an essential tissue to maintain tooth function and should be closely correlated to tooth root development and periodontal tissue regeneration. However, detailed features of the periodontium including cementum and specific markers for cementoblasts are unknown. Moreover, the molecular mechanism of periodontal tissue development, homeostasis and regeneration remains unknown. Previous studies have usually examined cementum or periodontalligament (PDL) tissue obtained by manual curettage, resulting in difficulties in isolating pure cementum or PDL. We employed laser capture microdissection (LCM) to isolate cementoblasts and PDL cells from undecalcified frozen sections of murine mandible and to obtain RNA of good quality for subsequent genetic analysis. Over 500 cementoblasts and PDL cells were separately laser captured under microscopy. A bioanalyzer detected peaks of 18S and 28S rRNA both in the laser-dissected cementoblasts and in PDL cells, suggesting that the RNA was of sufficient quality. The RNA samples were amplified due to their small amount and a comparative analysis of mRNA expression by GeneChip showed that about 2,000 genes were differentially expressed between cementoblasts and PDL cells. Both cementoblast-positive and PDL cell-negative genes were serially analyzed by quantitative RT-PCR using RNA samples obtained from mandibles and femurs. Several genes were expressed at higher levels in the mandible than in the femur, suggesting that some might be cementoblast-specific markers. We established a novel experimental system with which to isolate target tissues from single cells in undecalcified frozen sections and to obtain intact RNA. These methodologies could be useful for further investigation of mineralized tissues and to explore tissue-specific factors.     

Enamel malformations associated with a defined dentin sialophosphoprotein mutation in two families

Dentin sialophosphoprotein (DSPP) mutations cause dentin dysplasia type II (DD-II) and dentinogenesis imperfecta types II and III (DGI-II and DGI-III, respectively). We identified two kindreds with DGI-II who exhibited vertical bands of hypoplastic enamel. Both families had a previously reported DSPP mutation that segregated with the disease phenotype. Oral photographs and dental radiographs of four affected and one unaffected participant in one family and of the proband in the second family were used to document the dental phenotypes. We aligned the 33 unique allelic DSPP sequences showing variable patterns of insertions and deletions (indels), generated a merged dentin phosphoprotein (DPP) sequence that includes sequences from all DSPP length haplotypes, and mapped the known DSPP mutations in this context. Analyses of the DSPP sequence changes and their probable effects on protein expression, as well as published findings of the dental phenotype in Dspp null mice, support the hypothesis that all DSPP mutations cause pathology through dominant-negative effects. Noting that Dspp is transiently expressed by mouse pre-ameloblasts during formation of the dentino-enamel junction, we hypothesize that DSPP dominant-negative effects potentially cause cellular pathology in pre-ameloblasts that, in turn, causes enamel defects. We conclude that enamel defects can be part of the dental phenotype caused by DSPP mutations, although DSPP is not critical for dental enamel formation.     

Enamel matrix proteins bind to wound matrix proteins and regulate their cell-adhesive properties

Enamel matrix proteins (EMP) induce periodontal regeneration and accelerate dermal wound healing, but the cellular mechanisms of these processes are unclear. We investigated the binding of EMP to the wound matrix proteins fibronectin, laminin-1, collagen type I, and collagen type IV and analyzed the interaction of epithelial cells and periodontal ligament fibroblasts (PDLF) with EMP and composite matrices of EMP + fibronectin or EMP + collagen. The adhesion of PDLF to EMP was concentration- and integrin-dependent and did not require de novo protein synthesis. EMP supported PDLF migration. In contrast, keratinocytes did not adhere to EMP if their protein synthesis was blocked. EMP showed concentration-dependent binding of fibronectin, peaking at 100 microg ml(-1) (before the precipitation point) of EMP. Type I collagen binding to EMP peaked at a low (1 microg ml(-1)) and narrow concentration range. Neither laminin-1 nor type IV collagen bound to EMP. Collagen and fibronectin, bound to EMP, showed significantly reduced (> 50%) binding of both epithelial cells and PDLF compared with the equivalent concentration of these proteins alone. PDLF, but not epithelial cell, adhesion was rescued by increasing the EMP concentration. These findings show that EMP binds to wound extracellular matrix proteins and regulates their adhesive properties. Such interactions may favor fibroblast adhesion over epithelial cells, potentially promoting connective tissue regeneration.     

分泌型天冬氨酸蛋白酶基因表达差异及其影响因素

分泌型天冬氨酸蛋白酶(SAP)是白色假丝酵母菌最重要的毒力因子之一,由sap基因家族编码.在体外培养和体内外白色假丝酵母菌感染过程中,sap基因家族各成员间存在着表达和调控上的差异.本文就白色假丝酵母菌sap基因家族分子及其结构特点、sap基因表达差异及其影响因素作一综述.     

Human cementoblasts express enamel‐associated molecules in vitro and in vivo

Nuñez J, Sanz M, Hoz‐Rodríguez L, Zeichner‐David M, Arzate H. Human cementoblasts express enamel‐associated molecules in vitro and in vivo. J Periodont Res 2010; 45: 809–814. © 2010 John Wiley & Sons A/S Background and Objective: Cementum is a mineralized tissue that facilitates the attachment of periodontal ligament to the root and surrounding alveolar bone and plays a key role in the regeneration of periodontal tissues. The molecular mechanisms that regulate the proliferation and differentiation of cementoblasts, however, have not been elucidated to date. Enamel molecules are believed to regulate cementoblast differentiation and to initiate the formation of acellular extrinsic fiber cementum. The purpose of this study was therefore to isolate and culture human root‐derived cells (HRDC) in order to determine whether they are able to express both cementum and specific enamel proteins and subsequently to confirm these findings in vivo. Material and Methods: Human root‐derived cells were isolated and expanded in vitro. Cells were characterized using RT‐PCR, immunostaining, western blotting and by examination of total mRNA to determine the expression of cementum and enamel markers. Human periodontal tissues were also examined for the expression of enamel‐related proteins by immunostaining. Results: We showed that HRDC express mRNA corresponding to ameloblastin (AMBN), amelogenin (AMEL), enamelin (ENAM), tuftelin (TUFT) and cementum‐associated molecules such as cementum protein 1 (CEMP1) and cementum attachment protein (CAP). Western blotting revealed that HRDC express both AMEL and AMBN gene products, as well as the cementum markers CEMP1 and CAP. In vivo, we have showed that AMBN and AMEL are expressed by cementoblasts lining cementum, paravascular cells and periodontal ligament cells. Conclusion: These results suggest that enamel‐associated and cementum‐associated proteins could act synergistically in regulating cementoblast differentiation and cementum deposition and offer new approaches on how the cementogenesis process is regulated.     

Enamel defects associated with coeliac disease: putative role of antibodies against gliadin in pathogenesis

Enamel defects in the permanent teeth of patients with coeliac disease (CD) are often reported as an atypical manifestation, sometimes being suggestive of an undiagnosed atypical disease. We proposed to explore the pathogenesis of these oral defects, which are poorly studied. Sequence analyses of proteins from gluten (gliadins) and of proline-rich enamel proteins (amelogenin and ameloblastin) suggested the presence of common antigenic motifs. Therefore, we analyzed, by ELISA and western blotting, the reactivity of sera from patients with CD against gliadin and enamel-derived peptides. Correlation analyses between the levels of specific antibodies against gliadin and enamel derived peptides and inhibition experiments confirmed the presence of cross-reactive antibodies. Immunoblot analysis revealed that the most prominent component in enamel matrix derivative (of approximately 18.6 kDa), identified by an amelogenin-specific antibody, is recognized by sera from patients with CD; in addition, several fractions of pure gliadin were recognized by amelogenin-specific antibody. In agreement, sera from mice immunized with enamel matrix-derived proteins generated antibodies that recognized a peptide (of approximately 21.2 kDa) derived from gliadin. In conclusion, antibodies against gliadin generated in patients with CD can react in vitro with a major enamel protein. The involvement of anti-gliadin serum in the pathogenesis of enamel defects in children with untreated CD can be hypothesized on the basis of these novel results.     

Psychological factors associated with orofacial pains

This article develops the case for why trigeminal pain is a unique and challenging problem for clinicians and patients alike, and provides the reader with insights for effective trigeminal pain management based on an understanding of the interplay between psychologic and physiologic systems. There is no greater sensory experience for the brain to manage than unremitting pain in trigeminally mediated areas. Such pain overwhelms conscious experience and focuses the suffering individual like few other sensory events. Trigeminal pain often motivates a search for relief that can drain financial and emotional resources. In some instances, the search is rewarded by a treatment that immediately addresses an identifiable source of pain; in other cases, it can stimulate never-ending pilgrimages from one health provider to another.     

Dental anomalies associated with genetic factors

The article reviews the association between dental anomalies and genetic diseases. Knowledge of these hereditary diseases will enable the dentist to make a more extensive analysis of the families in which such diseases occur, and above all to evaluate more precisely the dental aspects that such conditions favour. This will lead to better management of diseases linked to malocclusion, congenital absence of teeth and anomalous development of dental tissues. Hereditary diseases are caused either by a single mutant gene, inherited from 1 parent and acting as dominant gene, or from a pair of mutant genes, inherited from both parents and acting recessively. Autosomal-dominant and autosomal-recessive inherited diseases are described, together with hereditary sex-linked conditions and those of multi-factorial origin. The purpose is to review such diseases so that the dentist can make an early diagnosis in clinical practice, check the predisposition toward such diseases and put in place preventive measures.