Enamel Defects Associated With Dentin Sialophosphoprotein Mutation in Mice

Dentin sialophosphoprotein (DSPP) is an extracellular matrix protein that is highly expressed in odontoblasts, but only transiently expressed in presecretory ameloblasts during tooth development. We previously generated a knockin mouse model expressing a mouse equivalent (DSPP, p.P19L) of human mutant DSPP (p.P17L; referred to as “DsppP19L/+”), and reported that DsppP19L/+ and DsppP19L/P19L mice manifested a dentin phenotype resembling human dentinogenesis imperfecta (DGI). In this study, we analyzed pathogenic effects of mutant P19L-DSPP on enamel development in DsppP19L/+ and DsppP19L/P19L mice. Micro-Computed Tomography (μCT) analyses of 7-week-old mouse mandibular incisors showed that DsppP19L/P19L mice had significantly decreased enamel volume and/or enamel density at different stages of amelogenesis examined. Acid-etched scanning electron microscopy (SEM) analyses of mouse incisors demonstrated that, at the mid-late maturation stage of amelogenesis, the enamel of wild-type mice already had apparent decussating pattern of enamel rods, whereas only minute particulates were found in DsppP19L/+ mice, and no discernible structures in DsppP19L/P19L mouse enamel. However, by the time that incisor enamel was about to erupt into oral cavity, distinct decussating enamel rods were evident in DsppP19L/+ mice, but only poorly-defined enamel rods were revealed in DsppP19L/P19L mice. Moreover, μCT analyses of the mandibular first molars showed that DsppP19L/+ and DsppP19L/P19L mice had a significant reduction in enamel volume and enamel density at the ages of 2, 3, and 24weeks after birth. Backscattered and acid-etched SEM analyses revealed that while 3-week-old DsppP19L/+ mice had similar pattern of enamel rods in the mandibular first molars as age-matched wild-type mice, no distinct enamel rods were observed in DsppP19L/P19L mice. Yet neither DsppP19L/+ nor DsppP19L/P19L mice showed well-defined enamel rods in the mandibular first molars by the age of 24weeks, as judged by backscattered and acid-etched SEM. In situ hybridization showed that DSPP mRNA level was markedly reduced in the presecretory ameloblasts, but immunohistochemistry revealed that DSP/DSPP immunostaining signals were much stronger within the presecretory ameloblasts in Dspp mutant mice than in wild-type mice. These results suggest that mutant P19L-DSPP protein caused developmental enamel defects in mice, which may be associated with intracellular retention of mutant DSPP in the presecretory ameloblasts.

Effects of DSPP Gene Mutations on Periodontal Tissues

Dentin sialophosphoprotein (DSPP) gene mutations cause autosomal dominantly inherited diseases. DSPP gene mutations lead to abnormal expression of DSPP, resulting in a series of histological, morphological, and clinical abnormalities. A large number of previous studies demonstrated that DSPP is a dentinal-specific protein, and DSPP gene mutations lead to dentin dysplasia and dentinogenesis imperfecta. Recent studies have found that DSPP is also expressed in bone, periodontal tissues, and salivary glands. DSPP is involved in the formation of the periodontium as well as tooth structures. DSPP deficient mice present furcation involvement, cementum, and alveolar bone defect. We speculate that similar periodontal damage may occur in patients with DSPP mutations. This article reviews the effects of DSPP gene mutations on periodontal status. However, almost all of the research is about animal study, there is no evidence that DSPP mutations cause periodontium defects in patients yet. We need to conduct systematic clinical studies on DSPP mutation families in the future to elucidate the effect of DSPP gene on human periodontium.

Dentin Sialophosphoprotein Deletion Leads to Femoral Head Cartilage Attenuation and Subchondral Bone Ill-mineralization

Dentin sialophosphoprotein (DSPP), which expresses and synthesizes in odontoblasts of dental pulp, is a critical protein for normal teeth mineralization. Originally, DSPP was identified as a dentin-specific protein. In 2010, DSPP was also found in femoral head cartilage, and it is still unclear what roles DSPP play in femoral head cartilage formation, growth, and maintenance. To reveal biological functions of DSPP in the femoral head cartilage, we examined Dspp null mice compared with wild-type (WT) mice to observe DSPP expression as well as localization in WT mice and to uncover differences of femoral head cartilage, bone morphology, and structure between these two kinds of mice. Expression data demonstrated that DSPP had heterogeneous fragments, expressed in each layer of femoral head cartilage and subchondral bone of WT mice. Dspp null mice exhibited a significant reduction in the thickness of femoral head cartilage, with decreases in the amount of proliferating cartilage cells and increases in apoptotic cells. In addition, the subchondral bone mineralization decreased, and the expressions of vessel markers (vascular endothelial growth factor [VEGF] and CD31), osteoblast markers (Osterix and dentin matrix protein 1 [DMP1]), osteocyte marker (sclerostin [SOST]), and osteoclast marker (tartrate-resistant acid phosphatase [TRAP]) were remarkably altered. These indicate that DSPP deletion can affect the proliferation of cartilage cells in the femoral head cartilage and endochondral ossification in subchondral bone. Our data clearly demonstrate that DSPP plays essential roles in the femoral head cartilage growth and maintenance and subchondral biomineralization.

ANALYSIS OF LYSATE PLATELET-RICH FIBRIN EFFECTS ON HUMAN DENTAL PULP STEM CELL DIFFERENTIATION THROUGH DENTIN SIALOPHOSPHOPROTEIN EXPRESSION

Objective: In vitro, the culture media in which human dental pulp stem cells (hDPSCs) are grown are supplemented with specific growth factors thatinduce cell cycle entry and differentiation. Lysate platelet-rich fibrin (L-PRF) is a unique and stable growth factor supplement produced from plateletslysed by freezing-thawing. In this study, we aimed to analyze the potential effects of L-PRF on hDPSC differentiation.Methods: We divided hDPSCs isolated from human third molars at the second passage into five culture media groups treated with 1%, 5%, 10%,and 25% L-PRF or 10% fetal bovine serum (control). After 7 days, we evaluated hDPSC differentiation using an enzyme-linked immunosorbent assayspecific for dentin sialophosphoprotein and Alizarin-Red staining.Results: None of our analyses revealed any significant differences between the L-PRF- and control-treated cells.Conclusion: L-PRF could potentially induce the differentiation of hDPSCs in vitro.

Shikonin induces odontoblastic differentiation of dental pulp stem cells via AKT–mTOR signaling in the presence of CD44

In our previous study, we demonstrated that hyaluronan induces odontoblastic differentiation of dental pulp stem cells via interactions with CD44. However, it remains unclear whether CD44 expression by dental pulp stem cells is required for odontoblastic differentiation. Therefore, we searched for a compound that induces odontoblastic differentiation of dental pulp stem cells, regardless of the chemical structure and function of hyaluronan, and examined whether CD44 is involved in the induction of odontoblastic differentiation by the compound. Because vitamin K analogues can promote bone formation and tissue calcification, we focused on derivatives of naphthoquinone, the skeleton of vitamin K; we verified whether those compounds could induce odontoblastic differentiation of dental pulp stem cells. We found that dentin sialophosphoprotein, a marker of odontoblasts, was expressed in dental pulp stem cells after treatment with shikonin. The shikonin-induced expression of dentin sialophosphoprotein was inhibited by PI3K, AKT, and mTOR inhibitors. In addition, in dental pulp stem cells transfected with siRNA against CD44, the shikonin-induced expression of dentin sialophosphoprotein was inhibited. Thus, shikonin can stimulate dental pulp stem cells to undergo odontoblastic differentiation through a mechanism involving the AKT–mTOR signaling pathway and CD44. Hyaluronan stimulated dental pulp stem cells to undergo CD44-mediated odontoblastic differentiation in our previous study; the present study indicated that CD44 is necessary for dental pulp stem cells to undergo odontoblastic differentiation. Although expression of CD44 is important for inducing odontoblastic differentiation of dental pulp stem cells, the relationship between the AKT–mTOR signaling pathway and CD44 expression, in the context of shikonin stimulation, has not yet been elucidated. This study suggested that shikonin may be useful for inducing odontoblastic differentiation of dental pulp stem cells, and that it may have clinical applications, including protection of dental pulp.

Mutant Dentin Sialophosphoprotein Causes Dentinogenesis Imperfecta

Dentin sialophosphoprotein (DSPP) is an extracellular matrix protein highly expressed by odontoblasts in teeth. DSPP mutations in humans may cause dentinogenesis imperfecta (DGI), an autosomal dominant dentin disorder. We recently generated a mouse model (named “ DsppP19L/+ mice”) that expressed a mutant DSPP in which the proline residue at position 19 was replaced by a leucine residue. We found that the DsppP19L/+ and DsppP19L/P19L mice at a younger age displayed a tooth phenotype resembling human DGI type III characterized by enlarged dental pulp chambers, while the teeth of older DsppP19L/+ and DsppP19L/P19L mice had smaller dental pulp chambers mimicking DGI type II. The teeth of DsppP19L/+ and DsppP19L/P19L mice had a narrower pulp chamber roof predentin layer, thinner pulp chamber roof dentin, and thicker pulp chamber floor dentin. In addition, these mice also had increased enamel attrition, accompanied by excessive deposition of peritubular dentin. Immunohistochemistry, in situ hybridization, and real-time polymerase chain reaction analyses showed that the odontoblasts in both DsppP19L/+ and DsppP19L/P19L mice had reduced DSPP expression, compared to the wild-type mice. We also observed that the levels of DSPP expression were much higher in the roof-forming odontoblasts than in the floor-forming odontoblasts in the wild-type mice and mutant mice. Moreover, immunohistochemistry showed that while the immunostaining signals of dentin sialoprotein (N-terminal fragment of DSPP) were decreased in the dentin matrix, they were remarkably increased in the odontoblasts of the DsppP19L/+ and DsppP19L/P19L mice. Consistently, our in vitro studies showed that the secretion of the mutant DSPP was impaired and accumulated within endoplasmic reticulum. These findings suggest that the dental phenotypes of the mutant mice were associated with the intracellular retention of the mutant DSPP in the odontoblasts of the DSPP-mutant mice.