Disrupted odontoblast differentiation and dentin dysplasia in Epiprofin-deficient mice.

Tooth formation is a process tightly regulated by reciprocal interactions between epithelial and mesenchymal tissues. These epithelial-mesenchyme interactions regulate the expression of target genes via transcription factors. Among the regulatory elements governing this process, Epiprofin/Sp6 is a zinc finger transcription factor which is expressed in the embryonic dental epithelium and in differentiating pre-odontoblasts. Epiprofin knockout (Epfn-/-) mice present severe dental abnormalities, such as supernumerary teeth and enamel hypoplasia. Here, we describe dentin defects in molars and incisors of Epfn-/- mice. We observed that in the absence of Epfn, markers of early odontoblast differentiation, such as alkaline phosphatase activity, Dsp/Dpp expression, and Collagen Type I deposition, are downregulated. In addition, the expression of tight and gap junction proteins was severely impaired in the predontoblastic cell layer of developing Epfn-/- molars. Altogether, our data shows that Epfn is crucial for the proper differentiation of dental mesenchymal cells towards functional odontoblasts and subsequent dentin-matrix deposition.

Dentinogenesis imperfecta type II in Swedish children and adolescents.

BACKGROUND: Dentinogenesis imperfecta (DGI) is a heritable disorder of dentin. Genetic analyses have found two subgroups in this disorder: DGI type I, a syndromic form associated with osteogenesis imperfecta (OI), and DGI type II, a non-syndromic form. The differential diagnosis between types I and II is often challenging. Thus, the present cross-sectional study had two aims: to (i) investigate the prevalence and incidence of DGI type II among Swedish children and adolescents and (ii) search out undiagnosed cases of DGI type I by documenting the prevalence of clinical symptoms of OI in these individuals. We invited all public and private specialist pediatric dental clinics (n = 47) in 21 counties of Sweden to participate in the study. We then continuously followed up all reported cases during 2014-2017 in order to identify all children and adolescents presenting with DGI type II. Using a structured questionnaire and an examination protocol, pediatric dentists interviewed and examined patients regarding medical aspects such as bruising, prolonged bleeding, spraining, fractures, hearing impairment, and family history of osteoporosis and OI. Joint hypermobility and sclerae were assessed. The clinical oral examination, which included a radiographic examination when indicated, emphasized dental variables associated with OI. RESULTS: The prevalence of DGI type II was estimated to be 0.0022% (95% CI, 0.0016-0.0029%) or 1 in 45,455 individuals. Dental agenesis occurred in 9% of our group. Other findings included tooth retention (17%), pulpal obliteration (100%), and generalized joint hypermobility (30%). Clinical and radiographic findings raised a suspicion of undiagnosed OI in one individual, a 2-year-old boy; he was later diagnosed with OI type IV. CONCLUSIONS: These results show a significantly lower prevalence of DGI type II than previously reported and point to the importance of excluding OI in children with DGI.

The fine tuning role of microRNA-RNA interaction in odontoblast differentiation and disease.

Dentinogenesis imperfecta and dentin dysplasia are two common types of genetic oral diseases resulted from the aberrant differentiation of odontoblast. Understanding the mechanisms of odontoblast differentiation is crucial for finding the diagnosis candidate genes and treatment targets for such kinds of diseases. Previous work has identified a battery of transcription factors and growth factors regulating odontoblast differentiation; however, the post-transcriptional regulating mechanisms of them are poorly studied. MicroRNAs (miRNA) are a group of non-coding RNAs widely studied in organ development, inflammation, and tumorigenesis because of its inhibitory effects on the target mRNAs. Also, miRNAs along with their binding targets form a complex competing endogenous RNA (ceRNA) network where miRNAs serve as the fine tuning balancers between their targets. Recent reports demonstrated the essential role of the miRNA pathway in dentinogenesis and the regulatory role of several specific miRNAs in the in vitro model of odontoblast differentiation. Herein, we will discuss the general roles of miRNA in diseases, the function of miRNAs during odontoblast differentiation, and finally the potential pathological mechanisms through which miRNAs cause the odontoblast-related diseases.

Rough endoplasmic reticulum trafficking errors by different classes of mutant dentin sialophosphoprotein (DSPP) cause dominant negative effects in both dentinogenesis imperfecta and dentin dysplasia by entrapping normal DSPP.

Families with nonsyndromic dentinogenesis imperfecta (DGI) and the milder, dentin dysplasia (DD), have mutations in one allele of the dentin sialophosphoprotein (DSPP) gene. Because loss of a single Dspp allele in mice (and likely, humans) causes no dental phenotype, the mechanism(s) underling the dominant negative effects were investigated. DSPP mutations occur in three classes. (The first class, the mid-leader missense mutation, Y6D, was not investigated in this report.) All other 5' mutations of DSPP result in changes/loss in the first three amino acids (isoleucine-proline-valine [IPV]) of mature DSPP or, for the A15V missense mutation, some retention of the hydrophobic leader sequence. All of this second class of mutations caused mutant DSPP to be retained in the rough endoplasmic reticulum (rER) of transfected HEK293 cells. Trafficking out of the rER by coexpressed normal DSPP was reduced in a dose-responsive manner, probably due to formation of Ca2+-dependent complexes with the retained mutant DSPP. IPV-like sequences begin many secreted Ca2+-binding proteins, and changing the third amino acid to the charged aspartate (D) in three other acidic proteins also caused increased rER accumulation. Both the leader-retaining A15V and the long string of hydrophobic amino acids resulting from all known frameshift mutations within the 3'-encoded Ca2+-binding repeat domain (third class of mutations) caused retention by association of the mutant proteins with rER membranes. More 5' frameshift mutations result in longer mutant hydrophobic domains, but the milder phenotype, DD, probably due to lower effectiveness of the remaining, shorter Ca2+-binding domain in capturing normal DSPP protein within the rER. This study presents evidence of a shared underlying mechanism of capturing of normal DSPP by two different classes of DSPP mutations and offers an explanation for the mild (DD-II) versus severe (DGI-II and III) nonsyndromic dentin phenotypes. Evidence is also presented that many acidic, Ca2+-binding proteins may use the same IPV-like receptor/pathway for exiting the rER.

OPG knockout mouse teeth display reduced alveolar bone mass and hypermineralization in enamel and dentin.

Recent studies showed that local injection or upregulation of OPG gene would result in early temporal retardation of tooth development. It was assumed that this retardation might cause defective tooth mineralization and pulp formation as the long-term effects. However, since those OPG treatments were transient, any possible long-term effects of OPG addition could not be assessed previously. In the present study, a high-resolution microCT was used to evaluate the long-term effect of OPG gene deprivation on the mineralization and morphology of mouse tooth. Our results showed that the mineralization of alveolar bone in OPG(-/-) mouse tooth was decreased while those of enamel and dentin were increased, compared with the wild-type (WT) group. The labial and lingual dentin thicknesses of OPG(-/-) group were significantly higher and with larger area in enamel and dentin than those of WT group. The size of pulp chamber was also substantially decreased in OPG(-/-) mouse incisor. Different responses in mineralization and morphogenesis to OPG gene deprivation were found between bone and tooth. These effects may be independent of the early odontogenesis, and further studies are warranted to investigate the molecular mechanism of the effect of OPG gene expression on bone formation and later tooth development.

Molecular basis of human dentin diseases.

In recent years, substantial progress has been made regarding the molecular etiology of human structural tooth diseases that alter dentin matrix formation. These diseases have been classified into two major groups with subtypes: dentin dysplasia (DD) types I and II and dentinogenesis imperfecta (DGI) types I-III. Genetic linkage studies have identified the critical loci for DD-II, DGI-II, and DGI-II to human chromosome 4q21. Located within the common disease loci for these diseases is cluster of dentin/bone genes that includes osteopontin (OPN), bone sialoprotein (BSP), matrix extracellular phosphoglycoprotein (MEPE), dentin matrix protein 1 (DMP1), and dentin sialophosphoprotein (DSPP). To date, only mutations within dentin sialophosphoprotein have been associated with the pathogenesis of dentin diseases including DGI types-II and -III and DD-II. In this article, we overview the recent literature related to these dentin genetic diseases, their clinical features, and molecular pathogenesis.

Mandibular phenotype of p20C/EBPbeta transgenic mice: Reduced alveolar bone mass and site-specific dentin dysplasia.

CCAAT enhancer binding proteins (C/EBP) comprise a family of basic-leucine zipper transcription factors that regulate cellular differentiation and function. To determine the role of C/EBP transcription factors in osteoblasts and odontoblasts, we generated a transgenic (TG) mouse model with Co1a1 (pOBCol3.6) promoter-targeted expression of a FLAG-tagged dominant negative C/EBP isoform, p20C/EBPbeta (previously LIP). Two of the four transgenic lines presented with abnormalities in the developing incisors, including breakage, overgrowth, and malocclusion. Histological examination revealed that the amount of alveolar bone was reduced in TG compared to wild-type (WT) mice. By microcomputed tomography (microCT), the bone volume fraction of the mandible was reduced at the level of the first and third molars, demonstrating a severe mandibular osteopenia. The lingual dentin morphology of TG incisors differed dramatically from WT. Labial dentin (enamel side) showed normal thickness and tubular dentin structure, whereas the lingual dentin was thinner (25-30% of WT at the alveolar crest) with an amorphous globular structure characteristic of dentin dysplasia. FLAG immunostaining was seen in both lingual and labial odontoblasts, indicating that the site-specific defect was not due to a lack of labial transgene expression. Northern blot analysis demonstrated reduced osteocalcin expression in TG mandibles, while bone sialoprotein was increased, consistent with prior results in calvariae and long bones. Dental sialophosphoprotein, a marker of the odontoblast lineage whose absence causes dentin dysplasia, was modestly reduced in TG mice by Northern blot and in situ hybridization analysis. By fluorescence microscopy, pOBCol2.3-GFP, a marker of the odontoblast lineage, was expressed in both labial and lingual odontoblasts, although GFP-marked lingual odontoblasts were more flattened than WT cells. Moreover, GFP-positive processes in the lingual dentin tubules were truncated and less organized than those in WT dentin. MicroCT analysis showed reduced tissue density in the lingual dentin. These data suggest that C/EBP transcription factors may be involved in the regulation of odontoblast polarization and dentin matrix production.

Dental structural diseases mapping to human chromosome 4q21.

Genetic diseases affecting tooth structure have been classified by the tissue affected enamel versus dentin, and their pattern of inheritance autosomal dominant, autosomal recessive, or X-linked. Advances in molecular genetics and the Human Genome Project have provided substantial progress regarding the identification of genes involved in the pathogenesis of human diseases. These include dental diseases affecting enamel and dentin formation: amelogenesis imperfecta (AI), dentinogenesis imperfecta (DGI) types II and III, and dentin dysplasia (DD) type II. Linkage studies using large informative families have provided insight identifying two proximal gene clusters on human chromosome 4q21 that contain the critical loci for five dental structural diseases. Studies related to the autosomal dominant forms of AI, representing approximately 85% of all cases, have established linkage to 4q21 for two forms: local hypoplastic and smooth hypoplastic AI. Two enamel matrix proteins, ameloblastin and enamelin, have been mapped within the critical regions for these diseases. Located more toward the telomere is another cluster containing loci for three dentin diseases: DGI type II, type III, and DD type II. Located within an overlapping segment of these diseases is a dentin/bone gene cluster that contains osteopontin, bone sialoprotein, matrix extracellular phosphoglycoprotein also known as osteoblast/osteocyte factor 45 or osteoregulin, dentin matrix protein 1, and dentin sialophosphoprotein. Continuing molecular genetic studies will facilitate the identification of novel tooth matrix proteins within these two tooth matrix gene clusters as well as the identification of additional autosomal dominant AI loci.

Ameloblastic odontosarcoma (ameloblastic fibro-odontosarcoma) in the mandible.

A case of ameloblastic odontosarcoma (ameloblastic fibro-odontosarcoma) originating in the mandible is reported. The patient was a 23-year-old Japanese male with diffuse swelling of the mandible from the left premolar to the ramus region. A biopsy specimen showed the histopathological features of ameloblastic fibrosarcoma. Histologic examination of the surgical specimen revealed various amounts of dysplastic dentin and dentinal matrix which were closely associated with both mesenchymal and epithelial components throughout the lesion. In addition, a small amount of abortive enamel matrix, which was in close contact with ameloblastoid cells of epithelial nests, was found by extensive sampling of the resected specimen. Therefore the present case was diagnosed as ameloblastic odontosarcoma. After 2 years, the patient died of uncontrollable local recurrence and extension to the cranial base.

Dentin dysplasia type II: absence of type III collagen in dentin.

A three-generation family with dentin dysplasia (DD) Type II is presented. Affected family members share common radiologic features with clinically varied expression of tooth discoloration and occlusal wear. Both the primary and the permanent dentition appear to be affected. No generalized connective tissue involvement is found. The mode of inheritance is autosomal dominant. Histologically, the findings are consistent with DD Type II. In indirect immunofluorescence, the irregular radicular dentin of an affected permanent tooth failed to stain with specific antibodies against Type III collagen and the N-terminal propeptide of Type III procollagen.