Petros Papagerakis

The Circadian Clock in Oral Health and Diseases

Most physiological processes in mammals display circadian rhythms that are driven by the endogenous circadian clock. This clock is comprised of a central component located in the hypothalamic suprachiasmatic nucleus and subordinate clocks in peripheral tissues. Circadian rhythms sustain 24-hour oscillations of a large number of master genes controlling the correct timing and synchronization of diverse physiological and metabolic processes within our bodies. This complex regulatory network provides an important communication link between our brain and several peripheral organs and tissues. At the molecular level, circadian oscillations of gene expression are regulated by a family of transcription factors called “clock genes”. Dysregulation of clock gene expression results in diverse human pathological conditions, including autoimmune diseases and cancer. There is increasing evidence that the circadian clock affects tooth development, salivary gland and oral epithelium homeostasis, and saliva production. This review summarizes current knowledge of the roles of clock genes in the formation and maintenance of oral tissues, and discusses potential links between “oral clocks” and diseases such as head and neck cancer and Sjögren’s syndrome.

Functions of KLK4 and MMP-20 in dental enamel formation.

Abstract Two proteases are secreted into the enamel matrix of developing teeth. The early protease is enamelysin (MMP-20). The late protease is kallikrein 4 (KLK4). Mutations in MMP20 and KLK4 both cause autosomal recessive amelogenesis imperfecta, a condition featuring soft, porous enamel containing residual protein. MMP-20 is secreted along with enamel proteins by secretory-stage ameloblasts. Enamel protein-cleavage products accumulate in the space between the crystal ribbons, helping to support them. MMP-20 steadily cleaves accumulated enamel proteins, so their concentration decreases with depth. KLK4 is secreted by transition- and maturation-stage ameloblasts. KLK4 aggressively degrades the retained organic matrix following the termination of enamel protein secretion. The principle functions of MMP-20 and KLK4 in dental enamel formation are to facilitate the orderly replacement of organic matrix with mineral, generating an enamel layer that is harder, less porous, and unstained by retained enamel proteins.

Investigation of osteocalcin, osteonectin, and dentin sialophosphoprotein in developing human teeth

Biochemical investigations in rodents have shown that numerous mineralized matrix proteins share expression in bone, dentin, and cementum. Little information is available regarding the expression pattern of these proteins in human tissues, particularly during tooth formation. The aim of this study was to identify the expression pattern of the two major noncollagenous proteins of bone and dentin, osteocalcin (OC) and osteonectin (ON), in comparison to the dentin-specific protein, dentin sialophosphoprotein (DSPP). Mandibles from fetuses (5-26 weeks), neonate autopsies, forming teeth from 10-12-year-old patients, third molars extracted for orthodontic reasons, and bone tumors were collected with approval from the National Ethics Committee. Human OC, ON, and DSPP mRNAs were detected by reverse transcription-polymerase chain reaction (RT-PCR) in fetal mandibles (5-11 weeks) and in primary cell cultures of dental pulp. In addition, OC, ON, and DSPP proteins were localized in forming human mineralized tissues using immunohistochemistry. In vivo, DSPP expression was associated with tooth terminal epithelial-mesenchymal interaction events, amelogenesis and dentinogenesis. Transient DSPP expression was seen in the presecretory ameloblasts with continuous expression in the odontoblasts. In contrast, both osteoblasts and odontoblasts showed a temporal gap between OC and ON expression in early development. ON was expressed in the initial stages of cytodifferentiation, whereas OC was expressed only during the later stages, especially in the teeth. At the maturation stage of enamel formation, both proteins were detected in odontoblasts and their processes within the extracellular matrix. In contrast to bone, OC was not localized extracellularly within the collagen-rich dentin matrix (predentin or intertubular dentin), but was found in the mature enamel. ON was present mostly in the nonmineralized predentin. These results demonstrate for the first time that both OC and ON are produced by human odontoblasts and determine the expression pattern of DSPP in human teeth, and suggest that OC and ON move inside the canalicule via odontoblast cell processes becoming localized to specific extracellular compartments during dentin and enamel formation. These distinct extracellular patterns may be related to the nature of DSPP, OC, and ON interactions with other matrix-specific macromolecules (i.e., amelogenin, dentin matrix protein-1) and/or to the polarized organization of odontoblast secretion as compared with osteoblasts.

Regulation of Dental Enamel Shape and Hardness

Epithelial-mesenchymal interactions guide tooth development through its early stages and establish the morphology of the dentin surface upon which enamel will be deposited. Starting with the onset of amelogenesis beneath the future cusp tips, the shape of the enamel layer covering the crown is determined by five growth parameters: the (1) appositional growth rate, (2) duration of appositional growth (at the cusp tip), (3) ameloblast extension rate, (4) duration of ameloblast extension, and (5) spreading rate of appositional termination. Appositional growth occurs at a mineralization front along the ameloblast distal membrane in which amorphous calcium phosphate (ACP) ribbons form and lengthen. The ACP ribbons convert into hydroxyapatite crystallites as the ribbons elongate. Appositional growth involves a secretory cycle that is reflected in a series of incremental lines. A potentially important function of enamel proteins is to ensure alignment of successive mineral increments on the tips of enamel ribbons deposited in the previous cycle, causing the crystallites to lengthen with each cycle. Enamel hardens in a maturation process that involves mineral deposition onto the sides of existing crystallites until they interlock with adjacent crystallites. Neutralization of acidity generated by hydroxyapatite formation is a key part of the mechanism. Here we review the growth parameters that determine the shape of the enamel crown as well as the mechanisms of enamel appositional growth and maturation.

Enamel defects and ameloblast-specific expression in Enam knock-out/lacz knock-in mice.

Enamelin is critical for proper dental enamel formation, and defects in the human enamelin gene cause autosomal dominant amelogenesis imperfecta. We used gene targeting to generate a knock-in mouse carrying a null allele of enamelin (Enam) that has a lacZ reporter gene replacing the Enam translation initiation site and gene sequences through exon 7. Correct targeting of the transgene was confirmed by Southern blotting and PCR analyses. No enamelin protein could be detected by Western blotting in the Enam-null mice. Histochemical 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal) staining demonstrated ameloblast-specific expression of enamelin. The enamel of the Enam+/- mice was nearly normal in the maxillary incisors, but the mandibular incisors were discolored and tended to wear rapidly where they contacted the maxillary incisors. The Enam-/- mice showed no true enamel. Radiography, microcomputed tomography, and light and scanning electron microscopy were used to document changes in the enamel of Enam-/- mice but did not discern any perturbations of bone, dentin, or any other tissue besides the enamel layer. Although a thick layer of enamel proteins covered normal-appearing dentin of unerupted teeth, von Kossa staining revealed almost a complete absence of mineral formation in this protein layer. However, a thin, highly irregular, mineralized crust covered the dentin on erupted teeth, apparently arising from the formation and fusion of small mineralization foci (calcospherites) in the deeper part of the accumulated enamel protein layer. These results demonstrate ameloblast-specific expression of enamelin and reveal that enamelin is essential for proper enamel matrix organization and mineralization.

Endogenous Msx1 antisense transcript: in vivo and in vitro evidences, structure, and potential involvement in skeleton development in mammals.

Msx1 is a key factor for the development of tooth and craniofacial skeleton and has been proposed to play a pivotal role in terminal cell differentiation. In this paper, we demonstrated the presence of an endogenous Msx1 antisense RNA (Msx1-AS RNA) in mice, rats, and humans. In situ analysis revealed that this RNA is expressed only in differentiated dental and bone cells with an inverse correlation with Msx1 protein. These in vivo data and overexpression of Msx1 sense and AS RNA in an odontoblastic cell line (MO6-G3) showed that the balance between the levels of the two Msx1 RNAs is related to the expression of Msx1 protein. To analyze the impact of this balance in the Msx-Dlx homeoprotein pathway, we analyzed the effect of Msx1, Msx2, and Dlx5 overexpression on proteins involved in skeletal differentiation. We showed that the Msx1-AS RNA is involved in crosstalk between the Msx-Dlx pathways because its expression was abolished by Dlx5. Msx1 was shown to down-regulate a master gene of skeletal cells differentiation, Cbfa1. All these data strongly suggest that the ratio between Msx1 sense and antisense RNAs is a very important factor in the control of skeletal terminal differentiation. Finally, the initiation site for Msx1-AS RNA transcription was located by primer extension in both mouse and human in an identical region, including a consensus TATA box, suggesting an evolutionary conservation of the AS RNA-mediated regulation of Msx1 gene expression.

Expression of amelogenin in odontoblasts

Amelogenin is the major enamel protein produced by ameloblasts. Its expression has been shown to be down-regulated in ameloblasts of vitamin-D-deficient (-D) rats. The potential expression and localization of amelogenin in odontoblasts and its regulation by vitamin D were investigated in this study. RT-PCR and semi-quantitative Northern blot analyses were performed using the odontoblast cell line MO6-G3 and microdissected dental pulp mesenchyme. Both in vitro and in vivo odontoblasts expressed various alternatively spliced amelogenin transcripts. In situ hybridization studies showed that amelogenin expression was restricted to young odontoblasts during mantle dentin deposition. Electron microscopy studies localized the amelogenin protein in the odontoblast cell process cytoplasm and mantle dentin. Amelogenin immunolabeling was stronger in -D rats, suggesting an inverse regulation by vitamin D in odontoblasts. Furthermore, amelogenin mRNA steady-state levels were significantly increased in -D dental pulp mesenchyme. In addition, a temporal-spatial lengthening of the mantle dentin stage was observed in -D animals, suggesting that developmental perturbations occur in relation to the vitamin D status and/or amelogenin expression. These data show that amelogenin is expressed by odontoblasts selectively during mantle dentin deposition. This developmental regulated expression pattern is enhanced under vitamin-D-deficiency status and in a broader context may play an important role during ameloblast and odontoblast differentiation and function.

Premature Stop Codon in MMP20 Causing Amelogenesis Imperfecta

Proteolytic enzymes are necessary for the mineralization of dental enamel during development, and mutations in the kallikrein 4 (KLK4) and enamelysin (MMP20) genes cause autosomal-recessive amelogenesis imperfecta (ARAI). So far, only one KLK4 and two MMP20 mutations have been reported. We have identified an ARAI-causing point mutation (c.102G>A, g.102G>A, and p.W34X) in exon 1 of MMP20 in a proband with autosomal-recessive hypoplastic-hypomaturation amelogenesis imperfecta. The G to A transition changes the tryptophan (W) codon (TGG) at amino acid position 34 into a translation termination (X) codon (TGA). No disease-causing sequence variations were detected in KLK4. The affected enamel is thin, with mild spacing in the anterior dentition. The enamel layer is hypomineralized, does not contrast with dentin on radiographs, and tends to chip away from the underlying dentin. An intrinsic yellowish pigmentation is evident, even during eruption. The phenotype supports current ideas concerning the function of enamelysin.

Fabrication and characterization of bioactive and antibacterial composites for dental applications

There is an increasing clinical need to design novel dental materials that combine regenerative and antibacterial properties. In this work the characterization of a recently developed sol-gel-derived bioactive glass ceramic containing silver ions (Ag-BG) is presented. The microstructural characteristics, ion release profile, zeta potential value and changes in weight loss and pH value as a function of the immersion time of Ag-BG in Tris buffer are evaluated. Ag-BG is also incorporated into natural extracellular matrix (ECM) hydrogel to further enhance its regenerative properties. Then, the micro and macro architectures of these new composites (ECM/Ag-BG) are characterized. In addition, the antibacterial properties of these new composites are tested against Escherichia coli and Enterococcus faecalis, a bacterium commonly implicated in the pathogenesis of dental pulp infections. Cell-material interaction is also monitored in a primary culture of dental pulp cells. Our study highlights the benefits of the successful incorporation of Ag in the bioactive glass, resulting in a stable antibacterial material with long-lasting bactericidal activity. Furthermore, this work presents for the first time the fabrication of new Ag-doped composite materials, with inductive pulp-cell proliferation and antibacterial properties (ECM/Ag-BG). This advanced composite made of Ag-BG incorporated into natural ECM possesses improved properties that may facilitate potential applications in tooth regeneration approaches.

Altered desmoplakin expression at transcriptional and protein levels provides prognostic information in human oropharyngeal cancer

Desmoplakin, a desmosomal component, is a key protein involved in cell-cell adhesion. Down-regulation of desmosomal proteins is associated with the invasive and metastatic ability of tumor cells. We examined 37 cases of human primary oropharyngeal squamous cell carcinomas lacking overt distant metastases to gain further insights on the potential role of desmoplakin in oral cancer. Desmoplakin expression was evaluated using reverse transcriptase-polymerase chain reaction and immunohistochemistry on frozen unfixed sections. Western blotting was performed to characterize the relative expression levels for each of the 2 desmoplakin protein isoforms, I and II. Desmoplakin expression was compared with histopathological grade, clinical stage, and patient outcome. Desmoplakin expression was prominent in highly differentiated tumors and reduced or absent in poorly differentiated tumors that developed distant metastases within the 3 years of follow-up period. Desmoplakin mRNA levels tracked with protein levels, suggesting that lack of desmoplakin protein expression is due to down-regulation of mRNA expression at the transcription level. Western blot analysis demonstrated that the 2 desmoplakin isoforms displayed different patterns of subcellular distribution in tumors, with the desmoplakin II detected only in patients in which desmoplakin immunoreactivity displayed an abnormal cytoplasmic localization. Our findings suggest that down-regulation of desmoplakin expression may represent a useful marker for evaluating the risk of distant metastasis formation in oropharyngeal squamous cell carcinomas. Interestingly, desmoplakin II was detected only in tumors associated with a poor clinical outcome, suggesting a potential specific function for this isoform in oral carcinogenesis. Characterizing DSP expression may improve evaluation risk of distant metastasis formation in oral cancer patients.