Study On The Function Of Dentin Sialophosphoprotein During The Tooth Development,mineralization And Dental Pulp Reparation After Injury

Dentin sialophosphoprotein (DSPP) is one of dentin non-collagenous proteins and mainly expressed by odontoblasts .Jt抯 regarded as a dentin specific protein. The gene structure and the expression pattern of DSPP at transcript level are well known in recent years, however, the expression pattern of DSPP protein, and it抯 possible role during tooth development and dental pulp reparation after injury are not clear yet.Trus, this study was aimed to get a better understanding on these questions. 1. Expression of DSPP during mouse tooth and cartilage development Expression of DSPP during tooth development (after bud stage) was examined by both immunohistochemistry and in situ hybridization. These two stainings were showed similar results ,which indicated that the expression patterns of DSPP had temporospacial character. DSPP was first detected in odontoblasts at middle bell stage and continued at the later stages, while detected transiently in mature ameloblasts as well as in preameloblasts. In addition, we found that DSPP was also expressed in nasal condrocytes. We cloned a 719bp fragment ,which was 99.7% homologous to the published DSPP eDNA sequence, by reverse transcriptase-polymerase chain reaction (RT-PCR) from mouse nasal cartilage. Further investigation showed that the expression in cartilage could be detected in postnatal ~ d - 6th d mice but not in postnatal 8th d mice. These data suggested that DSPP may not be regarded as the tooth special marker due to the expression in cartilage 5 tissue. 2. Effects of DSPP antisense oligodeoxynucleotide on development and mineralization of mouse molars in vitro In order to better understand the function role played by DSPP during tooth develo lpment, antisense o ligodeoxynueleotide(ODN) strategy was carried out in vitro. In the control experiment, explants of tooth germs from embryonic 1 7th d mice were cultured in agarose semi-solid medium under serum-free and chemically defined conditions for I 6ds and 1 Ods respectively, examined by HE, von Kossa staining and transmisson electron microscope. Other explants were cultured with l5bp antisense or sense ODN targeted to DSPP mRNA. Both the control and the sense-treated explants grew well, while antisense-treated explants were smaller in size. Von Kossa staining showed a dark band between enamel and predentin in control germs but not in the AS-ODN germs. Ultrastructure analyses showed that large cisternae of the rough endoplasmic reticulum(RER) in the odontoblasts at the tip of the cusp of antisense-treated explants. However, the average thickness of dentin matrix was thinner compared with the control ones. Further more, the collagen fibers in extracellular matrix were disorgnized. These findings provided, for the first time, a direct experimental evidence ,indicating that arrested DSPP translation in cultured tooth germs might result in delay in the tooth development,as well as inhibition of dentin mineralization, suggested that DSPP play an important role in dentin mineralization and may also in keeping tooth normal development by maintaining odontoblasts?secreting ability and controling fiber structure and orientation. 3. Immunohistochemical staining and in situ hybridization of DSPP on 6 dental pulp after injury...