Dentin Maturation Research Articles

Cell Surface Accumulation of Intracellular Leucine Proline-Enriched Proteoglycan 1 Enhances Odontogenic Potential of Human Dental Pulp Stem Cells.

Primary dental pulp cells can be differentiated into odontoblast-like cells, which are responsible for dentin formation and mineralization. Successful differentiation of primary dental pulp cells can be verified using a few markers. However, odontoblast-specific cell surface markers have not been fully studied yet. LEucine PRoline-Enriched Proteoglycan 1 (LEPRE1) is a basement membrane-associated proteoglycan. LEPRE1 protein levels are increased during odontoblastic differentiation of human dental pulp cells (hDPCs). Intracellular and cell surface accumulation of this protein completely disappeared during dentin maturation and mineralization. Cell surface binding of an anti-LEPRE1 monoclonal antibody that could recognize an extracellular region was gradually increased in the odontoblastic stage. Overexpression and knockdown experiments showed that accumulation of intracellular LEPRE1 could lead to inefficient odontoblastic differentiation and that the movement of LEPRE1 from intracellular region to the cell surface was required for odontoblastic differentiation. Indeed, when LEPRE1 already located on the cell surface was blocked by the anti-LEPRE1 monoclonal antibody, odontoblastic differentiation of hDPCs was inhibited. In this study, we looked at other aspects of LEPRE1 function as a cell surface molecule rather than its known intracellular hydroxylase activity. Our results indicate that this protein has potential as a specific cell surface marker in odontoblastic differentiation.

Rationale for the use of bioactive cements by in vitro simulation of accidental pulp opening

To study the features of reparative dentinogenesis when using biomaterials during accidental opening of the pulp in an experiment. In an experiment on 3 dwarf pigs of the Svetlogorsk breed a traumatic opening of the tooth cavity was carried out in 18 teeth (6 molars, 6 premolars, 6 incisors), then Biodentin (9 teeth) and Aurosil (9 teeth) were applied on the opened pulp horn. After 14, 30 and 60 days, dwarf pigs were euthanized, and jaw blocks taken. Histological examination of the teeth was carried out using the method of embedding in methyl methacrylate (OsteoBead, Sigma, USA). Thin sections of 35-50 microns were made from the samples and stained using the «Heavenly Trichrome» method. Documentation was carried out using a Leica DM100 microscope and an EC3 digital camera in reflected and transmitted light. Histological examination of the thin sections of teeth stained according to the «Heavenly Trichrome» method revealed that 2 weeks after the pulp opening and applying of Aurosil and Biodentin, comparable processes occurred in the tooth cavity. Thus, the proliferation of odontoblasts was noted on the lateral surfaces of the chamber. New dentin layers and odontoblasts spread over the lower surface of the material with the formation of dentinal bridges by 4 weeks of observation in both groups. By week 12, there was a decrease in the proliferative activity of odontoblasts and dentin maturation in all cases. At the initial stages of the experiment, some pulp edema and moderate plethora were noted, which was no longer observed at 4 weeks. By 12 weeks, because of the proliferation of odontoblasts, the volume of the tooth cavity was reduced by a third. By the 6th month in the experiment, no significant changes in the structure of dentin and pulp were revealed. According to morphological data the studied biomaterials (Aurosil and Biodentin) contribute to dentin regeneration equally.

A case report of dentinogenesis imperfecta inherited in a family of four generations

In dental anomaly, dentinogenesis imperfecta (DI) is one of the hereditary developmental disorders aff ecting both formation and maturation of dentin. This autosomal dominant disorder is characterized by discolored and translucent teeth ranging from gray to brownish-blue or amber. It can aff ect both primary and permanent teeth. The enamel may split readily from the dentin on occlusal stress. Radiographically, there is evidence of cervical constrictions, short root, and pulp chambers. The aff ected teeth may have smaller or completely obliterated root canals. This article reports a case of a family, which is aff ected by type I DI over four generations.

MicroRNA 665 Regulates Dentinogenesis through MicroRNA-Mediated Silencing and Epigenetic Mechanisms.

Studies of proteins involved in microRNA (miRNA) processing, maturation, and silencing have indicated the importance of miRNAs in skeletogenesis, but the specific miRNAs involved in this process are incompletely defined. Here, we identified miRNA 665 (miR-665) as a potential repressor of odontoblast maturation. Studies with cultured cell lines and primary embryonic cells showed that miR-665 represses the expression of early and late odontoblast marker genes and stage-specific proteases involved in dentin maturation. Notably, miR-665 directly targeted Dlx3 mRNA and decreased Dlx3 expression. Furthermore, RNA-induced silencing complex (RISC) immunoprecipitation and biotin-labeled miR-665 pulldown studies identified Kat6a as another potential target of miR-665. KAT6A interacted physically and functionally with RUNX2, activating tissue-specific promoter activity and prompting odontoblast differentiation. Overexpression of miR-665 reduced the recruitment of KAT6A to Dspp and Dmp1 promoters and prevented KAT6A-induced chromatin remodeling, repressing gene transcription. Taken together, our results provide novel molecular evidence that miR-665 functions in an miRNA-epigenetic regulatory network to control dentinogenesis.

Peritubular dentin, a highly mineralized, non-collagenous, component of dentin: isolation and capture by laser microdissection

We demonstrate the capability and technique to perform microdissection and isolation of select regions of untreated, mineralized dentin using laser capture. Dentin is a complex, non-homogeneous tissue comprised of a mineralized collagenous matrix (intertubular dentin [ITD]), odontoblastic processes (ODPs), a void space (tubules) that forms within the ITD left behind by the retraction of ODPs during dentin maturation, and a highly mineralized non-collagenous component that exists at the interface between the tubules and ITD known as peritubular dentin (PTD). PTD forms as the dentin matures. The ODPs retract toward the direction of the pulp; leaving very little PTD at either the DEJ or near the pulp. Statistical analysis of thin cross-sections of coronal bovine dentin imaged by light microscopy reveal that the area occupied by PTD >50%. To examine the nature of PTD and its relation to both the tubules and ITD, we devised a series of steps to carefully prepare sections of coronal bovine dentin so that areas of the dentin tissue could be cut and isolated for further analysis. We demonstrate that it is possible to selectively isolate targeted regions of dentin for analysis and that high resolution analysis of such sections can be performed using electron microscopy. Results show that the mineralized PTD has a different texture than mineralized ITD and that there is a distinct boundary between the PTD and the ITD. Selective isolation of mineralized tissue components for further analytical study opens the door for the investigation of similar enigmatic mineralized structures.

Vitamin D receptor deficiency affects dentin maturation in mice

Mutation of vitamin D receptors ( vdr) results in resistance to the vitamin's normal effects which may compromise dentin formation. The objective of this study was to investigate the effect of vdr deficiency on post-natal dentin maturation in mice. The dentin in mandibular incisors of 70.5-day-old vdr wild-type and vdr knockout mice was compared at different levels along the long axis. Expression of biglycan and decorin was detected by immunolocalisation. Scanning electron microscopy was used to observe the ultrastructure of the dentin, and micro-computerised tomography was used to determine the degree of dentin mineralisation density. In the vdr knockout mice, the pulp chamber was larger and the dentin wall was thinner compared with the wild-type mice. In addition, the pre-dentin layer was thickened with an irregular front line and diffuse expression of biglycan and decorin. Fewer tubules, lower mineralisation density and pore-like defects were observed in the dentin at the eruptive region and level with the first molar. In conclusion, vdr deficiency compromises dentin maturation.

Changes in matrix phosphorylation during bovine dentin development

Phosphorylation of the organic matrix proteins of dentin is important for the initiation of mineralization, but its relevance in later mineralization stages is controversial. The objective of this study was to analyze changes in the total matrix phosphate content during dentin development and to identify their origin. Amino acid and total matrix phosphate analyses of microdissected developing mantle and circumpulpal fetal bovine dentin specimens were performed. The amino acid composition showed few changes during mantle and circumpulpal dentin maturation. However, the total matrix phosphate content showed a significant, positive correlation with tissue maturation in both mantle and circumpulpal dentin, with a two- and a three-fold increase, respectively, being observed. The data indicate that changes occur in the pattern of phosphorylation of matrix proteins during dentin maturation, which we suggest may play a functional role in later stages of tooth mineralization.

Diagnosis of X-linked hypophosphatemic vitamin D resistant rickets.

Recent advances in the pathophysiology and diagnosis of X-linked hypophosphatemic rickets (XLH) are reviewed. The recent discovery of the gene that is responsible for XLH especially enables us to understand the mechanism of hypophosphatemia in XLH. Laboratory and radiological findings are important for the diagnosis. However, dental abnormalities such as spontaneous dental abscess and a defect of dentin maturation are also notable findings.

In VitroandIn VivoAssociation of Dentin Phosphophoryn with a1CB6 Peptide of Type I Collagen

A small number of molecules of phosphophoryns, dentin phosphoproteins, are associated to collagen tightly with the maturation of dentin. As an in vitro model of the process, we examined non-covalent association of phosphophoryns and collagen CNBr peptides. Cyanogen bromide peptides of type I collagen were separated with electrophoresis and transferred to a membrane, which was probed with labeled phosphophoryns. Phosphophoryns were bound preferentially to alpha1CB6 peptide. Another experiment using a cleavable crosslinking agent also demonstrated the affinity between phosphophoryns and the alpha1CB6 region of collagen. The matrix-bound fraction of phosphophoryns was solubilized by CNBr digestion of bovine dentin, and was partially purified. Compositional analysis revealed that the fraction was composed of association products of phosphophoryns and collagen at the ratio of 2:3. Considering the high molecular weight of the fraction, one phosphophoryn molecule should be associated with several collagen peptides. The fraction reacted with the antibody against alphalCB6 peptide. When the fraction was digested with lysyl endopeptidase, several peptides that coincided with peptides from alpha1CB6 were released. One of the peptides was sequenced and identified to be a peptide with Asp 975 of the alpha1(I) chain as an amino terminal residue. The alpha1CB6 peptide may be involved in the matrix-bound fraction, although involvement of other collagen peptides cannot be ruled out. Phosphophoryns may associate with collagen at the alpha1CB6 region at first, and then be immobilized on it, possibly by covalent crosslinking, with maturation of dentin. Binding of several alpha1CB6 peptides to a single phosphophoryn molecule is possible in current 3-dimensional models of collagen packing in mineralized tissue, which allows alignment of several hole zones in register.

The Distribution of Magnesium in Developing Rat Incisor Dentin

Previous studies have shown that rat incisor dentin contains a considerable amount of magnesium that is distributed heterogeneously. The cementum-related dentin, especially its incisal portion, is richest in magnesium. It was the purpose of the present study to investigate the changes that occur in the magnesium content during dentin maturation. Cross-sections were prepared from rat incisors at the apical, middle, and incisal levels. By means of an electron microprobe, tracings were made of the Ca-, Mg-, and P- signal frequencies. Comparison of corresponding dentin layers within and between the cross-sections showed that the Mg/P molar ratio was always higher in the cementum-related dentin (CRD) than in the enamel-related dentin (ERD) and increased from the apex toward the incisal edge. Especially in the incisal cross-section, an increase in Mg/P was found from the older (peripheral) toward the younger (central) dentin layers. As the Mg/P ratio varied from 0.07 to 0.33, the Ca/P ratio was found to fluctuate from 1.48 to 1.15. The two ratios appeared to be highly correlated (r = -0.97; p less than 0.001), suggesting that Mg replaces Ca and is bound to phosphate.

Crystal growth in matrix vesicles of permanent tooth germs in kittens.

Dentinogenesis of permanent tooth germs in kittens was examined in a transmission electron microscope. Crystals of various sizes (3-30 A in width) appeared in the matrix vesicles. Electron diffractional features revealed that crystal size in electron micrographs reflected various stages of crystal growth. Pairs of small crystals and dislocated crystals were observed in some crystal-containing matrix vesicles. However, these matrix vesicles were not observed in unmineralized matrix just adjacent to calcifying fronts and in mineralized dentin. Furthermore, long crystals were abundant in mature dentin. These findings suggest that crystal growth in matrix vesicles is due to fusion and that dentin maturation progresses through crystal growth along the long axis.

A pound of Vitamin-C

Mutation of vitamin D receptors (vdr) results in resistance to the vitamin's normal effects which may compromise dentin formation. The objective of this study was to investigate the effect of vdr deficiency on post-natal dentin maturation in mice. The dentin in mandibular incisors of 70.5-day-old vdr wild-type and vdr knockout mice was compared at different levels along the long axis. Expression of biglycan and decorin was detected by immunolocalisation. Scanning electron microscopy was used to observe the ultrastructure of the dentin, and micro-computerised tomography was used to determine the degree of dentin mineralisation density. In the vdr knockout mice, the pulp chamber was larger and the dentin wall was thinner compared with the wild-type mice. In addition, the pre-dentin layer was thickened with an irregular front line and diffuse expression of biglycan and decorin. Fewer tubules, lower mineralisation density and pore-like defects were observed in the dentin at the eruptive region and level with the first molar. In conclusion, vdr deficiency compromises dentin maturation.