Sigbjørn Løes

Coordination of trigeminal axon navigation and patterning with tooth organ formation: epithelial-mesenchymal interactions, and epithelial Wnt4 and Tgfβ1 regulate semaphorin 3a expression in the dental mesenchyme

During development, trigeminal nerve fibers navigate and establish their axonal projections to the developing tooth in a highly spatiotemporally controlled manner. By analyzing Sema3a and its receptor Npn1 knockout mouse embryos, we found that Sema3a regulates dental trigeminal axon navigation and patterning, as well as the timing of the first mandibular molar innervation, and that the effects of Sema3a appear to be mediated by Npn1 present in the axons. By performing tissue recombinant experiments and analyzing the effects of signaling molecules, we found that early oral and dental epithelia, which instruct tooth formation, and epithelial Wnt4 induce Sema3a expression in the presumptive dental mesenchyme before the arrival of the first dental nerve fibers. Later, at the bud stage, epithelial Wnt4 and Tgfβ1 regulate Sema3a expression in the dental mesenchyme. In addition, Wnt4 stimulates mesenchymal expression of Msx1 transcription factor, which is essential for tooth formation, and Tgfβ1 proliferation of the dental mesenchymal cells. Thus, epithelial-mesenchymal interactions control Sema3a expression and may coordinate axon navigation and patterning with tooth formation. Moreover, our results suggest that the odontogenic epithelium possesses the instructive information to control the formation of tooth nerve supply.

European Maxillofacial Trauma (EURMAT) project: A multicentre and prospective study

The purpose of this study was to analyse the demographics, causes and characteristics of maxillofacial fractures managed at several European departments of oral and maxillofacial surgery over one year. The following data were recorded: gender, age, aetiology, site of facial fractures, facial injury severity score, timing of intervention, length of hospital stay. Data for a total of 3396 patients (2655 males and 741 females) with 4155 fractures were recorded. The mean age differed from country to country, ranging between 29.9 and 43.9 years. Overall, the most frequent cause of injury was assault, which accounted for the injuries of 1309 patients; assaults and falls alternated as the most important aetiological factor in the various centres. The most frequently observed fracture involved the mandible with 1743 fractures, followed by orbital-zygomatic-maxillary (OZM) fractures. Condylar fractures were the most commonly observed mandibular fracture. The results of the EURMAT collaboration confirm the changing trend in maxillofacial trauma epidemiology in Europe, with trauma cases caused by assaults and falls now outnumbering those due to road traffic accidents. The progressive ageing of the European population, in addition to strict road and work legislation may have been responsible for this change. Men are still the most frequent victims of maxillofacial injuries.

Expression of class 3 semaphorins and neuropilin receptors in the developing mouse tooth.

The semaphorins are a large family of secreted or cell-bound signals needed for the development of the nervous system. We compared mRNA expression of class 3 semaphorins (Sema3A, 3B, 3C and 3F) and their two receptors (Neuropilin-1 and -2) in the embryonic mouse first molar tooth germ (E10-18) by radioactive in situ hybridization. All genes showed distinct developmentally regulated expression patterns during tooth organogenesis. Interestingly, Sema3A and 3C were first detected in the early dental epithelium, and later both genes were present in the epithelial primary enamel knot, a putative signaling center of the embryonic tooth regulating tooth morphogenesis. Prior to birth, Sema3A was also observed in tooth-specific cells, preodontoblasts, which later differentiate into odontoblasts secreting dentin, and in the mesenchymal dental follicle cells surrounding the tooth germ. Sema3B appeared transiently in the dental mesenchyme in the bud and cap stage tooth while Sema3F was expressed in both epithelial and mesenchymal components of the tooth. Of note, Npn-1 expression pattern was largely complementary to that of Sema3A, and transcripts were restricted to the dental mesenchymal cells. Npn-1 expression was first seen in the developing dental follicle, and later transcripts also appeared in the dental papilla mesenchyme. In contrast, Npn-2 signal was seen in both epithelial and mesenchymal tissues such as in the primary enamel knot and preodontoblasts.

Identification of a novel putative signaling center, the tertiary enamel knot in the postnatal mouse molar tooth

The final shape of the molar tooth crown is thought to be regulated by the transient epithelial signaling centers in the cusp tips, the secondary enamel knots (SEKs), which are believed to disappear after initiation of the cusp growth. We investigated the developmental fate of the signaling center using the recently characterized Slit1 enamel knot marker as a lineage tracer during morphogenesis of the first molar and crown calcification in the mouse. In situ hybridization analysis showed that after Fgf4 downregulation in the SEK, Slit1 expression persisted in the deep compartment of the knot. After the histological disappearance of the SEK, Slit1 expression was evident in a novel epithelial cell cluster, which we call the tertiary enamel knot (TEK) next to the enamel-free area (EFA)-epithelium at the cusp tips. In embryonic tooth, Slit1 was also observed in the stratum intermedium (SI) and stellate reticulum cells between the parallel SEKs correlating to the area where the inner enamel epithelium cells do not proliferate. After birth, the expression of Slit1 persisted in the SI cells of the transverse connecting lophs of the parallel cusps above the EFA-cells. These results demonstrate the presence of a novel putative signaling center, the TEK, in the calcifying tooth. Moreover, our results suggest that Slit1 signaling may be involved in the regulation of molar tooth shape by regulating epithelial cell proliferation and formation of EFA of the crown.

Expression of ephrin-A ligands and EphA receptors in the developing mouse tooth and its supporting tissues

Ephrins are cell-membrane-bound ligands for Eph receptor tyrosine kinases and regulate a variety of developmental processes. In order to investigate the potential roles of the ephrin-Eph system in tooth formation, we studied the cellular mRNA expression of Ephrin-A1–A5 and EphA2, EphA3, EphA4, EphA7, and EphA8 receptors during embryonic histomorphogenesis of the mouse first molar (embryonic days 11.5–18.5). Ephrin-A1, ephrin-A5, EphA2, EphA3, EphA4, and EphA7 were expressed in the tooth germ at the epithelial thickening stage, and later, ephrin-A1, ephrin-A5, EphA2, EphA4, and EphA7 showed distinct expression patterns in the enamel organ undergoing epithelial folding morphogenesis. Prior to birth, ephrin-A1, ephrin-A5, EphA2, and EphA4 transcripts were present in the cuspal area of the dental papilla including the preodontoblasts. In addition, ephrin-A1 and ephrin-A5 were seen in the forming blood vessels and alveolar bone, respectively. In contrast, ephrin-A2, ephrin-A3, and ephrin-A4 showed ubiquitous expression during odontogenesis, whereas EphA8 transcripts were not observed. During dental trigeminal axon pathfinding (embryonic days 12.5–13.5), ephrin-A2, ephrin-A4, and ephrin-A5 were evenly distributed in the trigeminal ganglion, whereas EphA7 was expressed in a subset of ganglion cells. These results suggest regulatory roles for ephrin-A/EphA signaling in the formation of the tooth organ proper and its supporting tissues.

Slit1 is specifically expressed in the primary and secondary enamel knots during molar tooth cusp formation.

The shape and diversity of the mammalian molar teeth is suggested to be regulated by the primary and secondary enamel knots, which are putative epithelial signaling centers of the tooth. In search of novel molecules involved in tooth morphogenesis, we analyzed mRNA expression of Slit1, -2 and -3, earlier characterized as secreted signals needed for axonal pathfinding and their two receptors Robo1 and -2 (Roundabout1 and -2) in the developing mouse first molar. In situ hybridization analysis showed that Slit1 mRNAs were expressed in the primary enamel knot of the bud and cap stage tooth germ and later the expression continued in the secondary enamel knots of the late cap and bell stage tooth. In contrast, expression of Slit2 and -3 as well Robo1, and -2 was largely restricted to mesenchymal tissue components of the tooth until the bell stage. At the late bud stage, however, Robo1 transcripts were evident in the primary enamel knot, and at the cap stage a pronounced expression was noted in the middle of the tooth germ covering the primary enamel knot and dental papilla mesenchyme. During the bell stage, Robo1 and Slit2 expression became restricted to the dental epithelia, while Slit3 continued in the dental mesenchyme. Prior to birth, Robo1 and -2 were co-localized in the predontoblasts. These results indicate that Slits and Robos display distinct, developmentally regulated expression patterns during tooth morphogenesis. In addition, our results show that Slit1 is the second known gene specifically located in the primary and secondary enamel knots.

Mouse rudimentary diastema tooth primordia are devoid of peripheral nerve fibers

The tooth is a well-defined peripheral target organ for trigeminal nerve fibers. However, only limited information is available regarding pioneer axon guidance to the developing tooth target field. In rodents there is a toothless diastema region between incisors and molars that in the mouse maxilla contains three rudimentary tooth anlagen. Their development stop at the early bud stage when the primary nerve axons grow towards the developing first molar tooth germs. In order to provide background information for studies of regulatory mechanisms of pioneer axon guidance to the developing tooth germs, we investigated the distribution of nerve fibers in the mouse diastema tooth buds, and compared it to the axon growth to the maxillary and mandibular first molar tooth germs by immunohistochemical localization of peripherin and PGP9.5. Analysis of serial sections showed that trigeminal nerve fibers emerging from the trigeminal maxillary and mandibular nerve trunks started to grow towards the developing molar tooth germ at the early bud stage, and subsequently they diverged into buccal and lingual branches next to the condensed dental mesenchyme. During the cap stage, nerve fibers were observed around the tooth germ in the dental follicle region. In contrast, no nerve fibers were located in the vicinity of the diastema tooth primordia at any stage studied, nor did any nerve fibers appear to grow towards this region. Our results show that the development and subsequent disappearance of the diastema tooth primordia takes place without peripheral trigeminal innervation. The diastema tooth primordia may therefore be a useful model system for future studies on molecular regulatory mechanisms of pioneer axon guidance to the tooth germs, and possibly also for evolutionary studies of peripheral axon guidance mechanisms.

European Maxillofacial Trauma (EURMAT) in children: A multicenter and prospective study

OBJECTIVE The aim of this study is to present and discuss the results of a European multicentre prospective study about pediatric maxillofacial trauma epidemiology during a year. STUDY DESIGN The following data were recorded: gender, age, etiology, site of fracture, date of injury. Of the 3396 patients with maxillofacial fractures admitted within the study period, 114 (3.3%) were children aged 15 years and younger, with a male/female ratio of 2.6:1. Mean age was 10.9 years. Most patients (63%) were aged 11-15 years. RESULTS The most frequent cause of injury was fall (36 patients). Sport injuries and assaults were almost limited to the oldest group, whereas falls were more uniformly distributed in the 3 groups. The most frequently observed fracture involved the mandible with 47 fractures. In particular, 18 condylar fractures were recorded, followed by 12 body fractures. CONCLUSIONS Falls can be acknowledged as the most important cause of facial trauma during the first years of life. The high incidence of sport accidents after 10 years may be a reason to increase the use of mouthguards and other protective equipment. Finally, the mandible (and in particular the condyle) was confirmed as the most frequent fracture site.

Vascular endothelial growth factors signalling in normal human dental pulp: a study of gene and protein expression.

In the well-vascularized dental pulp vascular endothelial growth factor A (VEGF-A) is expressed. Vascular endothelial growth factor A is a member of the VEGF family, which includes VEGFs-B, -C, and -D. The latter three have not been investigated in the pulp. Vascular endothelial growth factors C and D are the only ligands for vascular endothelial growth factor receptor (VEGFR)-3, which is usually expressed in lymphatic endothelium. They can also activate VEGFR-2, the main angiogenic receptor. We aimed to study VEGFs signalling in human dental pulp at the gene level and to identify the cellular source for protein expression using immunolabelling. All VEGFs (-A, -B, -C, and -D) were expressed in the pulp and may exert both autocrine and paracrine effects in blood vessels and immune cells found to be equipped with VEGFRs-2 and -3. Lymphatic vessel endothelial hyaluronan receptor-positive macrophages, known to be involved in angiogenesis, were found in the pulp, whereas lymphatic vessels were not detected. Twenty-six of 84 VEGF signalling genes, including VEGFR-3, were expressed at a significantly higher level in the pulp than in the control periodontal ligament. In conclusion, the normal human pulp represents a tissue with relatively high VEGF signalling involving both immune responses and vascular activity.

Localization and Signaling Patterns of Vascular Endothelial Growth Factors and Receptors in Human Periapical Lesions

INTRODUCTION Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are key players in vasculogenesis and are also involved in pathologic conditions with bone destruction. Vasculogenesis is critical for disease progression, and bone resorption is a hallmark of apical periodontitis. However, the localization of VEGFs and VEGFRs and their gene signaling pathways in human apical periodontitis have not been thoroughly investigated. The aim of this study was to localize VEGFs and VEGFRs and analyze their gene expression as well as signaling pathways in human periapical lesions. METHODS Tissue was collected after endodontic surgery from patients diagnosed with chronic apical periodontitis. Periodontal ligament samples from extracted healthy wisdom teeth was also collected and used as control tissue. In lesion cryosections, VEGFs/VEGFRs were identified by immunohistochemistry/double immunofluorescence by using specific antibodies. A human VEGF signaling polymerase chain reaction array system was used for gene expression analysis comparing lesions with periodontal ligament samples. RESULTS The histologic evaluation revealed heterogeneous morphology of the periapical lesions with various degrees of inflammatory infiltrates. In the lesions, all investigated factors and receptors were identified in blood vessels and various immune cells. No lymphatic vessels were detected. Gene expression analysis revealed up-regulation of VEGF-A and VEGFR-3, although not significant. Phosphatidylinositol-3-kinases, protein kinase C, mitogen-activated protein kinases, and phospholipases, all known to be involved in VEGF-mediated angiogenic activity, were significantly up-regulated. CONCLUSIONS The cellular and vascular expressions of VEGFs and VEGFRs in chronic apical periodontitis, along with significant alterations of genes mediating VEGF-induced angiogenic responses, suggest ongoing vascular remodeling in established chronic periapical lesions.