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Dentin sialophosphoprotein (Dspp) is mainly expressed in teeth by the odontoblasts and preameloblasts. The Dspp mRNA is translated into a single protein, Dspp, and cleaved into two peptides, dentin sialoprotein and dentin phosphoprotein, that are localized within the dentin matrix. Recently, mutations in this gene were identified in human dentinogenesis imperfecta II (Online Mendelian Inheritance in Man (OMIM) accession number 125490) and in dentin dysplasia II (OMIM accession number 125420) syndromes. Herein, we report the generation of Dspp-null mice that develop tooth defects similar to human dentinogenesis imperfecta III with enlarged pulp chambers, increased width of predentin zone, hypomineralization, and pulp exposure. Electron microscopy revealed an irregular mineralization front and a lack of calcospherites coalescence in the dentin. Interestingly, the levels of biglycan and decorin, small leucine-rich proteoglycans, were increased in the widened predentin zone and in void spaces among the calcospherites in the dentin of null teeth. These enhanced levels correlate well with the defective regions in mineralization and further indicate that these molecules may adversely affect the dentin mineralization process by interfering with coalescence of calcospherites. Overall, our results identify a crucial role for Dspp in orchestrating the events essential during dentin mineralization, including potential regulation of proteoglycan levels.

Studying the mechanisms of molecular interactions in developing tissues demands sensitive molecular biological in vivo and in vitro techniques. Laser capture microdissection (LCM) allows for the isolation of mRNA in histological sections even from single cells, thus enabling the identification of in vivo gene expression products in closely circumscribed tissue areas. The aims of this study were to assess the optimal fixation, processing, and staining conditions to retrieve RNA from microdissected odontoblasts. Fluorometric assays and RT-PCR analysis of &#102 1(I) collagen, dentin sialophosphoprotein (Dspp), and osteocalcin (OC) confirmed that the total RNA isolated from day 0 and day 3 captured odontoblasts was sufficient in quantity and quality. Our results indicate that individual odontoblasts obtained by LCM are morphologically intact and chemically unaltered, allowing accurate molecular and biochemical analyses.

Tooth eruption is a complex and tightly regulated process that involves cells of the tooth organ and the surrounding alveolus. Mononuclear cells (osteoclast precursors) must be recruited into the dental follicle prior to the onset of eruption. These cells, in turn, fuse to form osteoclasts that resorb alveolar bone, forming an eruption pathway for the tooth to exit its bony crypt. Some of the molecules possibly involved in the signaling cascades of eruption have been proposed in studies from null mice, osteopetrotic rodents, injections of putative eruption molecules, and cultured dental follicle cells. In particular, recruitment of the mononuclear cells to the follicle may require colony-stimulating factor-one (CSF-1) and/or monocyte chemotactic protein-1 (MCP-1). Osteoclastogenesis is needed for the bone resorption and may involve inhibition of osteoprotegerin transcription and synthesis in the follicle, as well as enhancement of receptor activator of NFκB ligand (RANKL), in the adjacent alveolar bone and/or in the follicle. Paracrine signaling by parathyroid-hormone-related protein and interleukin -1α, produced in the stellate reticulum adjacent to the follicle, may also play a role in regulating eruption. Osteoblasts might also influence the process of eruption, the most important physiologic role likely being at the eruptive site, in the formation of osteoclasts through signaling via the RANKL/OPG pathway. Evidence thus far supports a role for an osteoblast-specific transcription factor, Cbfa1 (Runx2), in molecular events that regulate tooth eruption. Cbfa1 is also expressed at high levels by the dental follicle cells. This review concludes with a discussion of the several human conditions that result in a failure of or delay in tooth eruption.

Background Twist1 and Twist2 are highly homologous bHLH transcription factors that exhibit extensive highly overlapping expression profiles during development. While both proteins have been shown to inhibit osteogenesis, only Twist1 haploinsufficiency is associated with the premature synostosis of cranial sutures in mice and humans. On the other hand, biallelic Twist2 deficiency causes only a focal facial dermal dysplasia syndrome or additional cachexia and perinatal lethality in certain mouse strains. It is unclear how these proteins cooperate to synergistically regulate bone formation. Methods Twist1 floxed mice (Twist1f/f) were bred with Twist2-Cre knock-in mice (Twist2Cre/+) to generate Twist1 and Twist2 haploinsufficient mice (Twist1f/+; Twist2Cre/+). X-radiography, micro-CT scans, alcian blue/alizarin red staining, trap staining, BrdU labeling, immunohistochemistry, in situ hybridizations, real-time PCR and dual luciferase assay were employed to investigate the overall skeletal defects and the bone-associated molecular and cellular changes of Twist1f/+;Twist2Cre/+ mice. Results Twist1 and Twist2 haploinsufficient mice did not present with premature ossification and craniosynostosis; instead they displayed reduced bone formation, impaired proliferation and differentiation of osteoprogenitors. These mice exhibited decreased expressions of Fgf2 and Fgfr1–4 in bone, resulting in a down-regulation of FGF signaling. Furthermore, in vitro studies indicated that both Twist1 and Twist2 stimulated 4.9 kb Fgfr2 promoter activity in the presence of E12, a Twist binding partner. Conclusion These data demonstrated that Twist1- and Twist2-haploinsufficiency caused reduced bone formation due to compromised FGF signaling.

We recently identified a frame-shift mutation in the PAX9 gene as the underlying cause for hypodontia involving permanent molar teeth segregating in an autosomal dominant pattern in a single large family (Stockton et al. 2000). Here we report a small nuclear family in which a father and his daughter are affected with severe hypodontia, involving agenesis of all primary and permanent molars, evidently caused by deletion of the entire PAX9 gene. Hemizygosity at the PAX9 locus in the two affected individuals was initially discovered when an informative single nucleotide polymorphism, identified while sequencing the gene for mutations, appeared to demonstrate non-Mendelian inheritance. Fluorescence in situ hybridization (FISH) analysis with a cosmid containing the PAX9 gene yielded a signal on only one chromosome 14 homologue and confirmed the presence of a deletion encompassing the PAX9 locus. Analysis of microsatellite loci in the vicinity of PAX9 delineated one breakpoint of the deletion. These data, in concert with FISH analysis with cosmids encompassing a 199 kb region, indicated that the deletion is between approximately 44 kb and 100 kb. PAX9 is one of two genes, and the only odontogenic gene within the deletion interval, thus supporting the model of haploinsufficiency for PAX9 as the underlying basis for hypodontia.

Odontogenesis or tooth development is a highly regulated process that involves complex epithelial-mesenchymal signaling interactions that lead to cuspal morphogenesis, cell differentiation and the subsequent formation of the specialized matrices of enamel, dentin, cementum and bone. Although studies on tooth epithelial-mesenchymal signaling interactions have greatly increased our understanding of molecules that regulate tooth initiation and early morphogenesis (review: Jernvall and Thesleff, Mech. Dev. 92 (2000) 19), the precise nature of the molecular events controlling late morphogenesis and terminal cytodifferentiation is not known. We have recently reported a unique phenotype involving dentition in mice lacking a functional Runx2 gene (D'Souza et al., Development 126 (1999) 2911). The markedly hypoplastic tooth organs as well as defects in the maturation of ameloblasts and odontoblasts point to an important and non-redundant role for Runx2 in tooth morphogenesis and cytodifferentiation. In order to identify genes that are affected by the absence of Runx2, a cDNA library was generated from Runx2(−/−) and Runx2(+/+) first molar organs. Thus far, our analysis has revealed several tooth-specific downstream target genes of Runx2 that include extracellular matrix proteins, kinases, receptors, growth factors, mitochondrial proteins and transcription molecules. Sequence analysis of 61 differentially expressed genes revealed that 96.03% of the clones matched previously described genes in the GenBank/EBML database and 3.96% did not match any entries in the database. Our preliminary expression analysis of one of the differentially expressed clones which encodes for a zinc finger transcription factor termed Zfp reveals that the gene is temporally regulated during tooth development. In conclusion, we have successfully generated a library enriched in genes expressed in Runx2(+/+) molar tooth organs and performed preliminary studies to assess the role of Zfp in tooth development.

The beagle dog with naturally occurring periodontal disease is one of the most widely used animal models in periodontal research for histological studies on disease pathogenesis and on the effect of potential therapeutic regimens. However, previous studies were restricted to morphological assessment of immunocompetent cells because of the lack of available cell-specific markers. In this study we systematically characterized the specificity and immunoreactivity of a panel of anti-human antibodies for identification (ABC method) of immunocompetent cells in formalin-fixed, EDTA-decalcified, paraffin-embedded inflamed periodontal tissues obtained from six beagle dogs. Canine lymph nodes and a panel of different human tissues served as positive controls. Polyclonal anti-CD3 immunolabeled canine T-lymphocytes specifically. Anti-CD79alpha (clone HM57) reacted with B-lymphocytes and plasma cells, and CD79alpha (clone JCP117) showed no staining in canine tissues. Neutrophils, monocytes, small macrophages, and keratinocytes reacted with an anti-myeloid/histiocyte antibody (clone MAC387). Anti-CD68 (clones PG-M1 and EBM11) immunolabeled large macrophages and plasma cells. Clone EBM11 also stained osteoclasts and cementoclasts. With the exception of JCB117, all antibodies revealed similarly favorable immunolabeling of canine and human immunocompetent cells. Long-term EDTA decalcification appeared to weaken immunostaining of plasma cells with HM57. MAC387 and CD68 can be used to distinguish macrophages in different differentiation stages in canine periodontal tissues. (J Histochem Cytochem 46:1443-1454, 1998)

Among the complex network of cytokines that influence odontoblast function during development and repair, TGF-beta1 is unique in its dual abilities to function as a potent immunosuppressant and as an inducer of extracellular matrix production. These properties underscore the importance of this molecule in maintaining the homeostasis of the dentin-pulp complex after injury. The purpose of this paper is to describe new findings of our phenotypic analysis of dentition in mice in which the TGF-beta1 gene has been disrupted. The major phenotype of TGF-beta1(-/-) offspring is one of diffuse immune system activation with progressive inflammation, wasting and death. Our studies of adult TGF-beta1(-/-) dentition show widespread pulpal and periapical inflammation and necroses. In addition, the coronal surfaces of occluding molars show marked attrition. To determine whether the phenotypic changes in TGF-beta1(-/-) dentition are directly linked to the loss of TGF-beta1 rather than the inflammatory process itself, we studied adult dentition in TGF-beta1(-/-) mice backcrossed into immunodeficient backgrounds. Results of our histopathologic and radiographic analyses show that teeth of TGF-beta1(-/-) immunodeficient mice retain vitality in pulpal and periapical regions but show excessive wear of occlusal surfaces.

In long bone development, a regulating role of OP-1 is suggested by the local correlated expression of both OP-1 ligand and OP-1 binding receptors in developing mouse hind limbs. OP-1 is expressed in the interdigital mesenchyme, whereas OP-1 binding receptors are found in the bordering perichondrium, and both OP-1 ligand and receptors are present in the zone of (pre)hypertrophic chondrocytes. We investigated the role of OP-1 in long bone development experimentally by treating organ cultures of embryonic mouse metatarsals with rhOP-1. The mRNA expression patterns of type I, II, X collagen, and matrix Gla protein (MGP) were studied using in situ hybridization and cell proliferation using [3H]thymidine and BrdU labeling. In the epiphyseal perichondrium, treatment with 40 ng/ml OP-1 enhanced cell proliferation after day 2, while 6-day treatment caused a shift in expression from type I collagen to type II collagen mRNA. This supports previous histochemical findings that OP-1 induced the transition of perichondrium into cartilage. In the center of the rudiment, OP-1 inhibited the expression of type X collagen mRNA, indicating inhibition of chondrocyte hypertrophy. An arrest of differentiation at the (pre)hypertrophic chondrocyte stage was also indicated by the large area of cells expressing MGP mRNA in the OP-1-treated rudiments. We conclude that OP-1 affected the expression of marker genes of chondrocyte differentiation by acting on two steps in endochondral ossification. First, cell proliferation was enhanced, particularly so in the perichondrium where cells started to express the chondrocyte phenotype. Second, the terminal differentiation of mature chondrocytes into hypertrophic chondrocytes was inhibited. These results, combined with the known pattern of OP-1 ligand and BMP receptor expression in the embryo, suggest that OP-1 plays a local role in the cascade of events during endochondral ossification.

Although the precise mechanisms of the conversion of predentin to dentin are not well understood, several lines of evidence implicate the noncollagenous proteins (NCPs) as important regulators of dentin biomineralization. Here we compared the in vivo temporospatial expression patterns of two dentin NCP genes, dentin matrix protein 1 (Dmp1), and dentin sialophosphoprotein (DSPP) in developing molars. Reverse transcription-polymerase chain reaction was performed on embryonic day 13 to 1-day-old first molars using Dmp1- and DSPP-specific primer sets. Dmp1 transcripts appeared at the late bud stage, while DSPP mRNA was seen at the cap stage. Expression of both genes was sustained throughout odontogenesis. In situ hybridization analysis revealed interesting differences in the expression patterns of these genes. While Dmp1 and DSPP showed coexpression in young odontoblasts before the start of mineralization, the expression of these genes was notably distinct at later stages. Dmp1 expression decreased in secretory odontoblasts after the appearance of mineral, while high levels of DSPP were sustained in odontoblasts. In early secretory ameloblasts, DSPP expression was transient and down-regulated with the appearance of dentin matrix. Interestingly, Dmp1 expression became evident in ameloblasts during the maturative phase of amelogenesis. In contrast to Dspp expression that was tooth-specific, Dmp1 was expressed by osteoblasts throughout ossification in the skeleton. Probes directed to the “DSP” and “DPP” regions of the DSPP gene showed identical patterns of mRNA expression. These data show that the developmental expression patterns of Dmp1 and DSPP are distinct, implying that these molecules serve different biological functions in vivo.

D’Souza RN, Dunnvald M, Frazier-Bowers S, Polverini PJ, Wright JT, de Rouen T, Vieira AR (2013). Translational genetics: Advancing fronts for craniofacial health. J Dent Res 92:1058-1064. (Original DOI: 10.1177/0022034513507954 )

The TUNEL (transferase-mediated, dUTP-biotin nick end labeling) method for in situ labeling of DNA strands was utilized to localize DNA fragmentation in cells involved in tooth formation in the neonatal mouse and hamster. Positive reactions for the presence of DNA fragments were obtained in some epithelial cells of the cervical loop region of incisors, late secretory, transitional and early maturation stage ameloblasts, stratum intermedium cells and in shortened ameloblasts just before eruption. Also, cells of the periodontal ligament of the continuously erupting incisors stained positive shortly before eruption. Odontoblasts were negative but became strongly positive during the formation of physiological osteodentin at the tip of developing incisors. Osteodentin matrix and the surfaces of unerupted enamel and cementum just prior to eruption stained for DNA fragments as well. DNA fragmentation could be elicited in odontoblasts and underlying pulpal tissues of mature erupted molars after mechanical injury to the odontoblast processes during cavity preparation. We conclude that, in rodents, DNA fragmentation and cell death are biological processes which take place in a variety of cells involved in formation of teeth. The TUNEL staining technique is a simple but powerful tool to examine the fate of cells and tissues undergoing either programmed cell death (apoptosis) or fragmentation of nuclear DNA induced by external factors leading to pathological changes.

The extracellular proteins types I and III collagen are abundantly expressed during development. Here, the patterns of the pro alpha 1(I), pro alpha 2(I), and pro alpha 1(III) collagen mRNAs are systematically examined from 7.5 to 17.5 days of development (E7.5 to E17.5) in the mouse using in situ hybridization with specific riboprobes. Coordinated expression of pro alpha 1(I) and pro alpha 2(I) collagen mRNA was found throughout development in all regions examined. Widespread type I collagen expression starting at E8.5 occurred in embryonic mesoderm, sclerotomes, dermatomes, and in the forming connective tissues. After E14.5, regions of ossification showed highest levels of type I collagen expression. Pro alpha 1(III) collagen expression was specific to and coordinated with patterns of type I collagen expression in many fibroblast-containing tissues. No expression of type III collagen occurred in osteoblasts. This comprehensive study of the transcripts of abundantly expressed structural proteins should provide a basis for comparison of other key extracellular matrix molecules and serve as a reference for studies on the patterns of activities of various promoter/enhancer-reporter gene constructions of type I and III collagen genes in transgenic mice.

While transforming growth factor-beta 1 (TGF-beta 1) and its related mammalian isoforms TGF-beta 2 and TGF-beta 3 are coexpressed in developing tooth organs, the specific biological role of each isoform is unknown. To delineate the role of TGF-beta 1 in odontogenesis, we have studied tissues from mice that lack a functional TGF-beta 1 gene. Histologic analyses revealed that in TGF-beta 1 (-/-) mice, tooth morphogenesis, cytodifferentiation and histogenesis were unaffected. Using in situ hybridization we studied the patterns of distribution of TGFs-beta 1, beta 2 and beta 3 in the TGF-beta 1 (+/+, +/- and -/-) genotypes. Our results indicate no detectable TGF-beta 1 mRNA in null tissues while TGFs-beta 2 and beta 3 showed normal temporal-spatial patterns of distribution. Using antibodies against TGF-beta 1, we observed immunoreactive TGF-beta 1 in tissues from null mice suggesting that maternally-derived TGF-beta 1 may be involved in the rescue of several developmental events in TGF-beta 1 knockout mice.

To begin to assess the transcriptional mechanisms that regulate type I collagen gene expression in differentiating osteoblasts, we have sought to determine the minimal promoter sequences that confer osteoblast-specific expression to the alpha 2(I) collagen gene during murine development. Transgenic mice were generated harboring DNA constructs in which the -2000, -500, and -350 to +54 regions located upstream of the start of transcription were linked to the Escherichia coli beta-galactosidase reporter gene (LacZ). Histochemical staining using X-gal indicated that the -2000 lacZ transgene was strongly expressed in newly differentiated and fully functional osteoblasts at intramembranous and endochondral sites of ossification. The promoter was also active in osteocytes in regions of bone remodeling within alveolar bone. The temporal and spatial activity of this region of the promoter closely resembled the developmental patterns of expression of the endogenous alpha 2(I) collagen gene as determined by in situ hybridization. The cis-acting elements within the 500 and 350 bp segments of the alpha 2(I) collagen promoter also drove reporter gene expression in forming osteoblasts, although levels of transgene expression were not as marked as that seen with the 2000 bp promoter. Furthermore, the synthesis and secretion of TGF-beta 1 in osteogenic zones coincided with areas where the alpha 2(I) collagen promoter constructs were transcriptionally active. Since a nuclear factor 1 binding site present at -300 has been shown to mediate the effects of TGF-beta 1 on the alpha 2(I) collagen promoter, these data support a role for TGF-beta 1 in the control of this gene during development.

A non-collagenous protein, extracted from rat incisor dentin, is a dentin sialoprotein (DSP). We examined immunohistochemically the developmental appearance and tissue distribution of DSP in 1 to 3-day-old rat molar and incisor tooth germs. The earliest staining for DSP was observed in newly differentiated odontoblasts. In more advanced stages, immunostaining for DSP gradually increased in pre-dentin, odontoblasts and dentin, and appeared in many cells of the dental papilla. In early stages of development before the breakdown of the dental basement membrane, pre-ameloblasts were also positive for DSP. This staining disappeared from the ameloblast cell body soon after deposition of the first layer of mineralized dentin. Radiolabelling of tooth matrix proteins with 14C-serine in vitro followed by immunoprecipitation and fluorography confirmed that DSP was synthesized by tooth-forming cells. The immunolocalization for DSP was different from that of either collagen type-I, osteocalcin or the amelogenins. Whereas collagen type-I and osteocalcin were restricted to the mesenchymal dental tissues, the amelogenins were detectable in both epithelial and mesenchymal dental cells and tissues at the epithelio-mesenchymal interface at early stages of development, prior to the onset of dentin mineralization. We conclude that DSP is expressed in and secreted by odontoblasts and some dental papilla cells from early stages of dentinogenesis onwards, i.e. later than type-I collagen, but before deposition of the first layer of mineralized dentin. In pre-mineralizing stages, some of the matrix proteins may be endocytosed from the pre-dentin by both cell types involved in the epithelio-mesenchymal interaction.

Engineering oral tissues as a multidisciplinary approach to build complex structures such as bone, teeth or soft dental tissues remains a challenging endeavor which will also require significant additional development of materials chemistry before it will be successful. We will highlight areas of recent success and describe major challenges which the materials chemistry community, in collaboration with clinicians, must still overcome. The isolation of stem cell populations from various sources in the oral cavity and advances in utilizing their differentiation potential has been driving the field forward. So far, bioinert materials have mainly been used as carriers and delivery vehicles, relying on the intrinsic cellular competence to form tissues. As this may not suffice to induce regeneration, there is a need for novel biomimetic scaffolds capable of providing chemical and mechanical cues to promote multiple specific interactions between cells and matrix. These signals can orchestrate processes such as cell adhesion, migration, differentiation, matrix synthesis, mineralization, and/or vasculogenesis. In this review, we give a brief description of oral anatomy and pathology, state-of-the-art treatment methods and their shortcomings. We provide an overview of current strategies to fabricate bioactive matrices, with an emphasis on nanostructured materials, and we suggest design principles for scaffolding systems specifically tailored towards dental tissue regeneration. In this review, we envision future approaches based on these emerging areas that rely on recent developments in tissue engineering and stem cell research. At the interface between material science and biology, cellular response can be controlled by materials chemistry, and potential applications for regenerative strategies are evolving.

Rat dentin contains a major sialic acid-rich glycoprotein, DSP, with an overall composition similar to that of bone sialoproteins but whose biological role in dentinogenesis is unknown. Using polyclonal affinity-purified antibodies to rat DSP and four immunohistochemical methods of detection, we studied the cell and tissue localization of DSP and the time course of its appearance during odontoblast differentiation. DSP first appeared within young odontoblasts concomitant with early secretion of pre-dentin matrix and before the onset of mineralization but was absent in pre-odontoblasts. DSP immunostaining also localized within secretory odontoblasts and was intense in odontoblastic processes. Early pre-dentin stained positive for DSP, in contrast to more mature pre-dentin, where immunoreactivity was less intense and more restricted to odontoblastic processes. In the zone of mineralized dentin matrix, a moderate and uniform staining pattern was evident. Intense immunostaining was also seen within the cells and matrix of dental pulp during dentinogenesis. Other cells and tissues within the tooth organ and those surrounding it were non-reactive. These findings suggest that DSP is developmentally expressed in cells of the odontoblastic lineage and may be a biochemical marker of odontoblastic activity.

We identified a frameshift mutation in the paired domain of PAX9 following genome-wide analysis of a family segregating autosomal dominant oligodontia. Affected members have normal primary dentition but lacked most permanent molars.

Regulatory peptides of the TGF-beta family affect various aspects of embryonic development. Recent immunolocalization and in situ hybridization studies have demonstrated a specific time- and tissue-dependent expression of TGF-beta 1 in the developing mouse embryo. The purpose of this study was to evaluate the distribution of TGF-beta 1 within rat molars at different stages of development, using a well-characterized antibody, highly specific for TGF-beta 1, and immunohistochemical methods of detection. TGF-beta 1 was immunolocalized intensely within the ectodermally derived stellate reticulum and the mesenchyme of the dental papilla at the bell stage of development. Marked immunostaining was also evident in the papillary layer and the reduced dental organ subjacent to ameloblasts in the differentiation and secretory phases of amelogenesis. During the formation of coronal tissues and in the pre-eruptive phase, immunoreactive TGF-beta 1 was localized conspicuously within the dental follicle overlying the tooth germ. This temporospatial pattern of expression of TGF-beta 1 appears to correlate with specific events in morphogenesis, histogenesis and cytodifferentiation during tooth development.

Tooth agenesis is a clinically heterogeneous disorder affecting various teeth at different rates, and is strongly influenced by ethnicity.1,2 Evidence confirms the role of genetic heterogeneity in this common dental anomaly, and alterations in two genes have been identified as causing this condition namely, MSX1 and PAX9 . Interestingly, our understanding of the genetic aetiology of human tooth agenesis is still largely based on the selective agenesis of predominantly posterior teeth. Specifically, mutations in the transcription factor PAX9 were shown to be responsible for “molar oligodontia” in three independent families,3–7 and a large deletion of PAX9 in one nuclear family led to the absence of all posterior teeth.8 Moreover, point mutations in the transcription factor MSX1 have been identified in families affected with selective posterior tooth agenesis predominantly involving premolars and occasionally molars.9–12 Although recent studies of human tooth agenesis have greatly advanced our knowledge of the genetic basis of this disorder, very few studies have specifically considered the genetic aetiology of anterior tooth agenesis.13–15 In one study, a mutation in the SHH gene was identified in association with a solitary maxillary incisor14; however, this pattern of tooth agenesis presents as a clinical variation of the holoprosencephaly syndrome. Interestingly, in certain Asian populations (including Japanese, Chinese, and Malaysian people), the incidence of non-syndromic anterior tooth agenesis has been reported to be 2%,16,17 which contrasts with the 0.08%–0.23% occurrence rate in a white population.18 Despite the high prevalence of incisor hypodontia, there is little known about the genetic aetiology in this pattern of tooth agenesis. Previous studies1–7 have increased our current understanding of the biology of tooth development, specifically concerning the roles of PAX9 and MSX1 . Moreover, human genetic studies have provided interesting data …

Pulp tissue was obtained from clinically intact third molars and premolars, and from teeth affected by caries and periodontal disease. After incubation and homogenization, supernatants were centrifuged, sampled and assayed for lymphocyte activating factor-like activity using the thymidine-incorporated D10.G4.1 cell-line assay. Significantly higher levels of mitogenic activity were found in the pulp supernatants of symptomatic carious teeth, and in those of third molars with attendant pericoronitis, than in pulps from symptomatic and asymptomatic periodontally affected teeth and asymptomatic third molars and premolars. As both interleukin-1 (IL-1) and interleukin-2 (IL-2) may be involved in the proliferation of D10.G4.1 cells, supernatants from carious pulps were further tested for IL-2 activity using murine natural killer (NK) cells. Those that had elicited a proliferative response of D10.G4.1 cells did not enhance NK proliferation, indicating that the factor responsible for clonal replication of D10.G4.1 cells was IL-1. To confirm the presence of IL-1, immunohistochemistry using a monoclonal antibody to IL-1 was performed on frozen and paraffin-embedded sections of pulps from each group. IL-1 was immunolocalized within cells in pulps from the caries symptomatic group. The cell type producing IL-1 was further characterized as an alpha-naphthyl-acetate-esterase-positive macrophage within the connective tissue stroma of pulps from the caries symptomatic group. This novel demonstration of IL-1 and IL-1 producing cells in human dental pulp indicates the involvement of this mediator of inflammation in dental disease.

Periapical inflammatory responses occur as a consequence of bacterial infection of the dental pulp, as a result of caries, trauma, or iatrogenic insult. Periapical inflammation stimulates the formation of granulomas and cysts, with the destruction of bone. These inflammatory responses are complex and consist of diverse elements. Immediate-type responses-including vasodilatation, increased vascular permeability, and leukocyte extravasation-are mediated by endogenous mediators, including prostanoids, kinins, and neuropeptides. Non-specific immune responses-including polymorphonuclear leukocyte and monocyte migration and activation, and cytokine production-are elicited in response to bacteria and their products. Interleukin-1 and prostaglandins in particular have been implicated as central mediators of periapical bone resorption. Chronic periapical inflammation further involves specific T- and B-cell-mediated anti-bacterial responses, and activates a network of regulatory cytokines which are produced by Th 1- and Th2-type T-lymphocytes. Various naturally occurring and genetically engineered models of immunodeficiency are beginning to help elucidate those components of the immune system which protect the pulpal/periapical complex. Both specific and non-specific responses interface with and are regulated by the neural system. The modulation of these responses by immune response modifiers, cytokine antagonists, and other novel therapeutic agents is discussed. As an experimental model, periapical inflammation has many advantages which permit it to be used in studies of microbial ecology and pathogenesis, host response, neuroimmunology, and bone resorption and regeneration.

Bone morphogenetic proteins (BMPs) form a family of growth factors originally isolated from extracellular bone matrix that are capable of inducing bone formation ectopically. We studied the expression, tissue localization, and function of BMP-7 (OP-1) during tooth development in rodents. Patterns of BMP-7 gene expression and peptide distribution indicated that BMP-7 was present in dental epithelium during the dental lamina, bud, and cap stages. During the bell stage, BMP-7 mRNA expression and protein distribution shifted from dental epithelium toward the dental mesenchyme. With advancing differentiation of odontoblasts, BMP-7 protein staining in the dental papilla became restricted to the layer of fully functional odontoblasts in the process of depositing (pre)dentin. Secretory-stage ameloblasts exhibited weak immunostaining for BMP-7. A restricted pattern of staining in ameloblasts became apparent in post-secretory stages of amelogenesis. Also, cells of the forming periodontal ligament were immunopositive. Histological analysis of tooth development in neonatal BMP-7-deficient mice did not reveal obvious changes compared with wild-type mice. We conclude that, in developing dental tissues, BMP-7 has distribution and expression patterns similar to those of other BMP members but is not an essential growth factor for tooth development, possibly because of functional redundancy with other BMP members or related growth factors.

During primary dentin formation, differentiating primary odontoblasts secrete an organic matrix, consisting principally of type I collagen and non-collagenous proteins, that is capable of mineralizing at its distal front. In contrast to ameloblasts that form enamel and undergo programed cell death, primary odontoblasts remain metabolically active in a functional tooth. When dentin is exposed to caries or by operative procedures, and when exposed dentinal tubules are treated with therapeutic dental materials, the original population of odontoblasts is often injured and destroyed. The characteristics of the replacement pool of cells that form reparative dentin and the biologic mechanisms that modulate the formation of this matrix are poorly understood. Based on the hypothesis that events governing primary dentinogenesis are reiterated during dentin repair, the present study was designed to test whether cells that form reparative dentin are odontoblast-like. Cervical cavities were prepared in rat first molars to generate reparative dentin, and animals were killed at various time intervals. In situ hybridization with gene-specific riboprobes for collagen types I and III was used to study de novo synthesis by cells at the injured dentin-pulp interface. Polyclonal antibodies raised against dentin sialoprotein (DSP), a dentin-specific protein that marks the odontoblast phenotype, were used in immunohistochemical experiments. Data from our temporal and spatial analyses indicated that cells forming reparative dentin synthesize type I but not type III collagen and are immunopositive for DSP. Our results suggest that cells that form reparative dentin are odontoblast-like.

Osteoblasts and odontoblasts, cells that are responsible for the formation of bone and dentin matrices respectively, share several molecular characteristics. Recently, Cbfa1 was shown to be a critical transcriptional regulator of osteoblast differentiation. Mutations in this gene cause cleidocranial dysplasia (CCD), an autosomal dominant disorder in humans and mice characterized by defective bone formation. CCD also results in dental defects that include supernumerary teeth and delayed eruption of permanent dentition. The dental abnormalities in CCD suggest an important role for this molecule in the formation of dentition. Here we describe results of studies aimed at understanding the functions of Cbfa1 in tooth formation. RT-PCR and in situ hybridization analyses show that Cbfa1 has a unique expression pattern in dental mesenchyme from the bud to early bell stages during active epithelial morphogenesis. Unlike that observed in osteoblast differentiation, Cbfa1 is downregulated in fully differentiated odontoblasts and is surprisingly expressed in ectodermally derived ameloblasts during the maturation phase of enamel formation. The role of Cbfa1 in tooth morphogenesis is further illustrated by the misshapen and severely hypoplastic tooth organs in Cbfa1−/− mice. These tooth organs lacked overt odontoblast and ameloblast differentiation and normal dentin and enamel matrices. Epithelial-mesenchymal recombinants demonstrate that dental epithelium regulates mesenchymal Cbfa1 expression during the bud and cap stages and that these effects are mimicked by the FGFs but not by the BMPs as shown by our bead implantation assays. We propose that Cbfa1 regulates the expression of molecules in mesenchyme that act reciprocally on dental epithelium to control its growth and differentiation. Taken together, our data indicate a non-redundant role for Cbfa1 in tooth development that may be distinct from that in bone formation. In odontogenesis, Cbfa1 is not involved in the early signaling networks regulating tooth initiation and early morphogenesis but regulates key epithelial-mesenchymal interactions that control advancing morphogenesis and histodifferentiation of the epithelial enamel organ.