Search

Phosphophoryn (PP) and dentin sialoprotein (DSP) are the most dominant non-collagenous proteins in dentin. PP is an extremely acidic protein that can function as a mineral nucleator for dentin mineralization. DSP was first identified in 1981, yet its functional significance is still controversial. Historically, these two proteins were considered to be independently synthesized and secreted by dental pulp cells into the developing dentin matrix. However, with the identification of the DSP coding sequence in 1994, followed 2 years later by the finding that the PP coding sequence was located immediately downstream from the DSP sequence, it became immediately clear that DSP and PP proteins were derived from a single DSP-PP (i.e., dentin sialophosphoprotein, DSPP) transcript. Since DSPP cDNA became available, tremendous progress has been made in studying DSP-PP mRNA distribution and DSP generation from the DSP-PP precursor protein at specific cleavage sites by protease tolloid-related-1 (TLR1) or bone morphogenetic protein 1 (BMP1). The functions of DSP-PP and DSP were investigated via DSP-PP knockout (KO) and DSP knockin in DSP-PP KO mice. In addition, a number of in vitro studies aimed to elucidate DSPP and DSP function in dental pulp cells.

Objective: To compare the expression patterns of two multiple transcripts derived from DSP-PP gene during tooth development. One is DSP-only transcript (i.e. does not encode PP) and the other is DSP-PP 523 transcript, a main DSP-PP transcript., Design: Unique antisense and sense riboprobes were generated from DSP-only and DSPPP 523 cDNAs for in situ studies to examine DSP-only and DSP-PP 523 transcript expression in developing molars. Paraffin-embedded sections (5-7μ m) from embryonic 20day, postnatal 2, 3 and 6days were deparaffined and hydrated. Tissues were prehybridized, then hybridized with DSP-only and DSP-PP 523 anti-sense (AS) or sense (S) Digoxigenin labeled-riboprobes overnight, and washed. Anti-Digoxigenin antibodies conjugated to alkaline phosphatase were used to detect the presence of bound riboprobes by color reaction with NBT/BCIP. Stro-1 antibody was used for immunohistochemical analysis of Stro-1 protein expression in rat molars., Results: We found that unlike the DSP-PP 523 transcript, the DSP-only transcript does not express in the entire polarized mature odontoblasts but is expressed in the areas subjacent to the mature odontoblast layer. In addition, DSP-only transcript is expressed in the dental pulp. Interestingly, Stro-1 protein, a stem cell marker, was also identified in the areas subjacentto odontoblasts and in dental pulp., Conclusion: Differential expression of DSP-only and DSP-PP 523 transcripts suggest that these two kinds of transcripts may play different roles during dentinogenesis. DSP-PP 523 transcript is expressed in mature odontoblasts, which actively participates in dentin formation. DSP-only transcript might have a different function., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The craniofacial skeleton is derived from both neural crest cells and mesodermal cells; however, the majority of the bone, cartilage, and connective tissue is derived from the neural crest. Dentin sialophosphoprotein (DSPP) is a precursor protein that is expressed by the connective tissues of the craniofacial skeleton, namely, bone and dentin with high expression levels in the dentin matrix. Gene ablation studies have shown severe dental defects in DSPP-null mutant mice. Therefore, to elucidate the role of DSPP on the developing dental-craniofacial complex, we evaluated phenotypic changes in the structure of intramembranous bone and dentin mineralization using 3 different age groups of DSPP-null and wild-type mice. Results from micro-computed tomographic, radiographic, and optical microscopic analyses showed defective dentin, alveolar and calvarial bones, and sutures during development. The impaired mineralization of the cranial bone correlated well with low expression levels of Runx2, Col1, and OPN identified using calvarial cells from DSPP-null and wild-type mice in an in vitro culture system. However, the upregulation of MMP9, MMP2, FN, and BSP was observed. Interestingly, the null mice also displayed low serum phosphate levels, while calcium levels remained unchanged. Alizarin red and von Kossa staining confirmed the dysfunction in the terminal differentiation of osteoblasts obtained from the developing calvaria of DSPP-null mice. Immunohistochemical analysis of the developing molars showed changes in Runx2, Gli1, Numb, and Notch expression in the dental pulp cells and odontoblasts of DSPP-null mice when compared with wild-type mice. Overall, these observations provide insight into the role of DSPP in the normal development of the calvaria, alveolar bone, and dentin-pulp complex., (© International & American Associations for Dental Research 2015.)

Dentin Sialophosphoprotein (DSPP) is the major non-collagenous protein of dentin and plays a significant role in dentin mineralization. Recently, animal models lacking DSPP have been developed and the DSPP KO phenotype has been characterized at the histological level. Little is known, however, about the DSPP KO dentin at nano- and meso-scale. Dentin is a hierarchical material spanning from nano- to macroscale, hence information on the effects of DSPP deficiency at the submicron scale is essential for understanding of its role in dentin biomineralization. To bridge this gap, we have conducted ultrastructural studies of dentin from DSPP KO animals. Transmission electron microscopy (TEM) studies of DSPP KO dentin revealed that although the overall ultrastructural organization was similar to the WT, the mineral particles were less organized. Scanning electron microscopy in the back-scattered mode (BS-SEM) of the DSPP KO dentin revealed that circumpulpal dentin comprises large areas of non-mineralized matrix, with numerous spherulitic mineralized inclusions, while the mantle dentin appeared largely unaffected. Analysis of the mineral distribution in the circumpulpal dentin of the DSPP KO mice suggests a reduction in the number of mineral nucleation sites and an increase in the nucleation barrier in DSPP KO dentin. These preliminary results indicate that in addition to the reduction of mineralized and total dentin volume in DSPP KO animals significant changes in the ultrastructural organization exist. These changes are likely related to the role of DSPP in the regulation of mineral formation and organization in dentin.

Dentin sialophosphoprotein (DSPP) is a large precursor protein that is proteolytically processed into a NH2 -terminal fragment [composed of dentin sialoprotein (DSP) and a proteoglycan form (DSP-PG)] and a COOH-terminal fragment [dentin phosphoprotein (DPP)]. In vitro studies indicate that DPP is a strong initiator and regulator of hydroxyapatite crystal formation and growth, but the role(s) of the NH2 -terminal fragment of DSPP (i.e., DSP and DSP-PG) in dentinogenesis remain unclear. This study focuses on the function of the NH2 -terminal fragment of DSPP in dentinogenesis. Here, transgenic (Tg) mouse lines expressing the NH2 -terminal fragment of DSPP driven by a 3.6-kb type I collagen promoter (Col 1a1) were generated and cross-bred with Dspp null mice to obtain mice that express the transgene but lack the endogenous Dspp (Dspp KO/DSP Tg). We found that dentin from the Dspp KO/DSP Tg mice was much thinner, more poorly mineralized, and remarkably disorganized compared with dentin from the Dspp KO mice. The fact that Dspp KO/DSP Tg mice exhibited more severe dentin defects than did the Dspp null mice indicates that the NH2 -terminal fragment of DSPP may inhibit dentin mineralization or may serve as an antagonist against the accelerating action of DPP and serve to prevent predentin from being mineralized too rapidly during dentinogenesis., (© 2013 Eur J Oral Sci.)

Dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP-1) are highly phosphorylated proteins that belong to the family of small integrin-binding ligand N-linked glycoproteins (SIBLINGs), and are essential for proper development of hard tissues such as teeth and bones. In order to understand how they contribute to tissue organization, DSPP and DMP-1 have been analyzed for over a decade using both in vivo and in vitro techniques. Among the five SIBLINGs, the DSPP and DMP-1 genes are located next to each other and their gene and protein structures are most similar. In this review we examine the phenotypes of the genetically engineered mouse models of DSPP and DMP-1 and also introduce complementary in vitro studies into the molecular mechanisms underlying these phenotypes. DSPP affects the mineralization of dentin more profoundly than DMP-1. In contrast, DMP-1 significantly affects bone mineralization and importantly controls serum phosphate levels by regulating serum FGF-23 levels, whereas DSPP does not show any systemic effects. DMP-1 activates integrin signalling and is endocytosed into the cytoplasm whereupon it is translocated to the nucleus. In contrast, DSPP only activates integrin-dependent signalling. Thus it is now clear that both DSPP and DMP-1 contribute to hard tissue mineralization and the tissues affected by each are different presumably as a result of their different expression levels. In fact, in comparison with DMP-1, the functional analysis of cell signalling by DSPP remains relatively unexplored., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

DSPP, which plays a crucial role in dentin formation, is processed into the NH(2)-terminal and COOH-terminal fragments. We believe that the proteolytic processing of DSPP is an essential activation step for its biological function in biomineralization. We tested this hypothesis by analyzing transgenic mice expressing the mutant D452A-DSPP in the Dspp-knock-out (Dspp-KO) background (referred to as "Dspp-KO/D452A-Tg" mice). We employed multipronged approaches to characterize the dentin of the Dspp-KO/D452A-Tg mice, in comparison with Dspp-KO mice and mice expressing the normal DSPP transgene in the Dspp-KO background (named Dspp-KO/normal-Tg mice). Our analyses showed that 90% of the D452A-DSPP in the dentin of Dspp-KO/D452A-Tg mice was not cleaved, indicating that D452A substitution effectively blocked the proteolytic processing of DSPP in vivo. While the expression of the normal DSPP fully rescued the dentin defects of the Dspp-KO mice, expressing the D452A-DSPP failed to do so. These results indicate that the proteolytic processing of DSPP is an activation step essential to its biological function in dentinogenesis.

Unlabelled: Matrix extracellular phosphoglycoprotein (MEPE) is predominantly expressed in osteoblasts, osteocytes, and odontoblasts and plays key biological roles in bone and dentin metabolism. Post-translational modifications are essential for its activation. This study tested the hypothesis that MEPE is activated through proteolytic processing by furin in dental pulp. MEPE was present in three sizes, 1 full-length and 2 cleaved fragments; the cleavage site was 146R↓147. The proprotein convertase family, particularly furin, was a candidate enzyme. Introducing a substitution at the cleavage site inhibited hydrolysis, but there was no cleavage of MEPE expressed in furin-deficient LoVo cells. Therefore, furin is a strong candidate for the proteolytic cleavage of MEPE. The C-terminal cleavage product promoted cell adhesion via its RGD motif. These results indicate that proteolytic processing by furin may activate MEPE during its secretion from odontoblasts and may play important roles in dentinogenesis and pulpal homeostasis., Abbreviations: MEPE, matrix extracellular phosphoglycoprotein; PTM, post-translational modifications; OLC, odontoblast-lineage cells.

Previously, non-collagenous matrix proteins, such as DMP1, were viewed with little biological interest. The last decade of research has increased our understanding of DMP1, as it is now widely recognized that this protein is expressed in non-mineralized tissues, as well as in cancerous lesions. Protein chemistry studies have shown that the full length of DMP1, as a precursor, is cleaved into two distinct forms: the C-terminal and N-terminal fragments. Functional studies have demonstrated that DMP1 is essential in the maturation of odontoblasts and osteoblasts, as well as in mineralization via local and systemic mechanisms. The identification of DMP1 mutations in humans has led to the discovery of a novel disease: autosomal-recessive hypophosphatemic rickets. Furthermore, the regulation of phosphate homeostasis by DMP1 through FGF23, a newly identified hormone that is released from bone and targeted in the kidneys, sets a new direction for research that associates biomineralization with phosphate regulation.

Small integrin-binding ligand, N-linked glycoproteins (SIBLING) are believed to play key roles in the process of biomineralization. Reactionary dentin (RD), formed by odontoblasts in response to external stimuli, differs morphologically from primary dentin (PD). To test our hypothesis that the microscopic changes reflect variations in molecular mechanisms involved in formation of the two forms of dentin, and to characterize RD further, we compared the distributions of four SIBLING proteins [bone sialoprotein (BSP), osteopontin (OPN), dentin matrix protein 1 (DMP-1) and dentin sialophosphoprotein (DSPP)] in naturally occurring RD with those in PD. Molars of rats aged 12, 18, 24 and 36 wk were analyzed using immunohistochemistry with antibodies against BSP, OPN, DMP-1, and dentin sialoprotein (a fragment of DSPP). Differences in the distribution of the four SIBLING proteins were evident. Bone sialoprotein, not seen in PD, was consistently observed in RD. Osteopontin, almost absent from PD, was clearly observed in RD. The expression levels of DMP-1 and DSP in RD were lower than in PD. Elevated expression of BSP and OPN, along with a marked decrease of dentin sialoprotein and DMP-1 in RD, suggests a difference in the mechanism of formation of the two forms of dentin.

Unlabelled: Previous studies on dental pulp cell culture have described heterogenous mixtures of cells that differentiate into odontoblasts and form mineralized dentin., Objective: The aim of this study was to characterize the matrix extracellular phosphoglycoprotein (MEPE) expression by dental pulp stem cells (DPSC), related to cell differentiation., Design: DPSC differentiation to form mineralized nodules was characterized by Alizarin red staining and micro-Raman spectroscopy. Osteogenesis SuperArray analysis was used to broadly screen for osteogenesis-related genes altered by DPSC differentiation. Relative levels of expression of MEPE and DSP were determined by semiquantitative RT-PCR and Western blot., Results: Mineral analysis showed that as DPSC differentiated, they formed a carbonated hydroxyapatite mineral. Differentiation was initially marked by upregulation by Runx2, TGFbeta-related genes, EGFR and genes involved in collagen metabolism. ALP activity first increased, as DPSCs reached confluence but later decreased when cells further differentiated three weeks after confluence. MEPE was the only marker that was downregulated as DPSCs differentiated., Conclusion: DPSC differentiation can be characterized by downregulation of MEPE as other markers of DPSC differentiation, such as DSP, are upregulated. Expression of MEPE related to DSP and can be used to monitor DPSC as they are used for studies of odontoblast differentiation, tissue engineering or vital pulp therapy. The downregulation of MEPE as DPSC differentiate, suggests that MEPE is an inhibitor of mineralization.

Objective: To investigate the stage-specific and tissue expression of bone sialoprotein (BSP) and osteopontin (OPN) during reparative dentinogenesis in vivo., Methods: Direct pulp-capping with Ca(OH)2 developed a model used for the investigation of reparative dentinogenesis in the exposed dental pulp. In this model, standardized class V cavities were prepared close to the gingival margin on the buccal surface of each tooth. Animals were sacrificed 3, 7, and 10 days post-operation. Immunohistochemical staining determined the tissue-specific expression of BSP and OPN during the process., Results: Odontoblast-like cells and reparative dentin reacted positively with BSP and OPN. The expression of BSP reached a peak at day 7, then gradually decreased, while the expression of OPN showed no significant difference between day 7 and day 10., Conclusion: BSP and OPN may play different roles in reparative dentinogenesis. BSP may serve as a nucleator of hydroxyapatite crystal formation.

Background and Methods: The expressions of TGF-beta 1 and Type I collagen mRNA were studied by in situ hybridization and immunohistochemistry then the secretory pathway of dentin phosphoprotein was investigated electron microscopic radioautography in rat incisors., Results and Conclusions: Expression of TGF-beta 1 mRNA was observed in dental papilla cells before dentin formation. The signals were most intense in pre- and postodontoblasts and during dentinogenesis, but became weaker in the secretory region during the dentin formation. Type I collagen mRNA was expressed in essentially the same as that of TGF-beta 1. These results suggest that TGF-beta 1 plays an important role in the differentiation of, and collagen synthesis by odontoblasts. Radioautography showed radioactivity in the rough endoplasmic reticulum 5 min after injection of 3H-serine. Silver grains were observed over the cylindrical portions of the cis-face of the Golgi apparatus at 10 min and over the cylindrical portions of the transface at 20 min. The secretory granules showed the strongest reaction between 20 min and 1 h after injection. At 45 min, a significant labeled band appeared at the mineralization front. The pathway of 3H-proline was essentially the same as that of 3H-serine, but 3H-proline moved more slowly. Secretory granules were heavily labeled from 30 min; no labeling was found at the mineralization front at 45 min. The labeling pattern with 3H-serine appears to be closely related to the localization of phosphoproteins. Dentin phosphoproteins are related to secretory granules and are secreted by odontoblasts as the mineralization front, being involved in the process of dentin mineralization.

At neutral pH, poly-L-lysine-gold complexes labelled the predentine extensively, whereas in dentine the number of gold complexes was reduced by half. Hyaluronidase pretreatment of the section at pH 6.8, prior to labelling, suppressed most of the staining in predentine and did not affect dentine. In contrast, alkaline phosphatase pretreatment at pH 9 enhanced the gold complex labelling in predentine and removed most of the labelling in dentine. This proves that at pH 7.2, the polyanions which are stained include a heterogeneous population of glycosaminoglycans, located in predentine, and phosphoproteins, visualized in dentine. At acidic pH levels (2.9 and 1.1), the number of scored gold complexes decreased, but the ratio between predentine and dentine labelling remained constant. Hyaluronidase pretreatment removed or firmly reduced the gold complex labelling both in predentine and dentine, whereas alkaline phosphatase pretreatment of the sections at pH 9 prior to labelling did not induce any change. This argues in favour of an increased specificity of polylysine-gold complex staining for glycosaminoglycans, stained at low pH in both predentine and dentine. Differential staining of glycosaminoglycans and phosphoproteins according to the pH provides a useful tool for studying the role played respectively by the two matrix components in dentine mineralization.

Two fractions of isolated noncollagenous extracellular matrix proteins from dentine have been implanted into the base of exposed cavities in ferret canine teeth. After sealing the cavities, reparative dentinogenesis was assessed histologically after 14 and 28 days as compared to control cavities. Reparative dentinogenesis was enhanced in the presence of these matrix proteins with evidence of tubular dentine along the walls of the exposure and on the cavity floor demonstrating the in vivo morphogenetic properties of the dentine matrix proteins.

Using the rat incisor as a model, evidence is presented that enamel-related (ERD) and cementum-related (CRD) dentins exhibit differences in composition. Whereas the ERD is rich in highly phosphorylated phosphoproteins, the CRD is rich in lowly phosphorylated phosphoproteins. These differences are accompanied by and possibly related to differences in composition and physical properties of the mineral phase.

Matrix proteins (EDTA-soluble and collagenase-released) from rabbit dentine have been demonstrated to show in vivo morphogenetic activity in exposed pulps of ferret canine teeth. In young adult ferrets, the reparative dentine resembled normal primary tubular dentine with odontoblast-like cells lining its formative surface. The reparative response in older animals was seen as osteo-dentine deposition. In both cases, the reparative response was characterized by its intensity within a comparatively short time period and also by the low grade or complete absence of any localized inflammatory response. Dentine matrix proteins implanted in exposed pulps of teeth provide a good experimental model for analysing the cell-matrix interactions and events taking place during odontoblast differentiation and dentinogenesis.