Immunohistochemical Localization Of Nell-1During Murine Molar Development And In Pulp-dentin Complex Of Human Teeth

Objective:Nel-like type I gene(nell-1) is a novel gene which was found overexpressed in pre-fused areas of craniosynostosis patients,because of its very similar structure with the nel gene specifically expressed in chicken nerve,so named nel-like type1gene. Nell-1gene plays an important role in regulating osteogenesis related gene expression, inducing osteoblast differentiation and promoting bone formation. Its coding protein Nell-1, with EGF(epidermal cell growth factor)-like coding structure, is a recently discovered secreted protein and is preferentially expressed in neural crest-derived tissues like craniofacial region. Animal studies and in vitro studies show that Nell-1can specifically induce osteoblast differentiation, promote mineralization and induce bone formation and regeneration.Rencent studies show that Nell-1is likely a downstream target of Runx2and is directly regulated by Runx2through its binding to osteoblast-specific binding element2(OSE2) sites in its promoter. Runx2is an important transcription factor that controls bone and tooth development by regulating osteoblast and odontoblast differentiation and contolling the expression of related target genes.Dental tissue and craniofacial bone are all neural crest-derived tissues. Odontoblasts and osteoblasts are all differentiated from mesenchymal cells, sharing several similarities in genetic structure.In addition, Nell-1is directly regulated by Runx2, an important transcription factor that controls bone and tooth development. So we hypothesized that Nell-1might be also involved in tooth development, odontoblast differentiation and dentin formation.Therefore, the aim of this study was to investigate the expression patterns of Nell-1during murine molar development and in pulp-dentin complex by immunohistochemistry.Methods, Results and Conclusions:Experiment1:Immunohistochemistry was used to investigate the expression patterns of Nell-1at different stages of murine molar development. Nell-1protein was expressed during molar development in embryonic and postnatal Kunming mice, but its expression levels and patterns at various developmental stages differed. At embryonic day13.5(E13.5) and E14.5, Nell-1was found in both the entire enamel organ and the underlying mesenchyme. At E16.5, it was detected in the inner and outer enamel epithelia, stratum intermedium, secondary enamel knot, and dental papilla. At E18.5, Nell-1was expressed in the differentiating ameloblasts, differentiating odontoblasts, and stratum intermedium. Positive staining was also found in the outer enamel epithelium. At postnatal day2.5(P2.5), P5, and P7, Nell-1appeared in the secretory and mature ameloblasts and odontoblasts (odontoblastic bodies and processes) as well as immature enamel. Hertwig’s epithelial root sheath also stained positively at P7. At P13.5, positive staining was restricted to the reduced dental epithelium and odontoblasts, whereas Nell-1disappeared in the mature enamel. During tooth eruption, Nell-1was observed only in the odontoblastic bodies, odontoblastic processes, and endothelial cells of blood vessels. The spatiotemporal expression patterns of Nell-1during murine tooth development suggest that it might play an important role in ameloblast and odontoblast differentiation, secretion and mineralization of the extracellular enamel matrix, molar crown morphogenesis, as well as root formation.Experiment2:The purpose of this study was to investigate the expression and localization of Nell-1in human pulp-dentin complex by immunohistochemistry. We found that Nell-1was expressed primarily in odontoblasts (odontoblastic bodies and processes), pulp fibroblasts and endothelial cells of the blood vessels both in normal and in carious teeth. Furthermore, we found Nell-1expression on the surface of the pulp stone,disseminated calcification particles and fibrosis of root pulp tissue in pulp-dentin complex with pulp calcification. Our data suggest that Nell-1might play a role in odontoblast differentiation, dentin matrix mineralization and dentin formation of human teeth.