Primary Observation Of The Role Of Posttranslational Modification Of Dentin Sialophosphoprotein On Postnatal Development Of Mandibular Condyle In Mice

Background:The mandibular condyle plays a key role in the function of the mandible.In recent years,people have become more and more interested in the molecular mechanism of mandibular condyle development,its unique structure and functional characteristics.Dentin sialophosphoprotein（DSPP）is a precursor protein,plays an important role in dentinogenesis and periodontium.through posttranslational modification,it is cleaved into the NH₂-terminal fragment（dentin sialoprotein,DSP and/or proteoglycan form of dentin sialoprotein,DSP-PG）,and COOH-terminal fragment（dentin phosphoprotein,DPP）.The proteolytic processing of DSPP is much important in dentinogenesis and periodontium.And DSPP mainly exist in the form of full-length in mandibular condylar cartilage,the results showed that the post-translational modification of DSPP in condylar cartilage was significantly different from dentin,suggesting its unique function in condylar cartilage was different from that of mineralized tissue.Previously,we showed that condylar thickness of Dspp KO mice decreased due to reduced proliferation of cartilage cells,which indicates that Dspp is a crucial factor for postnatal development and maintaining of condylar cartilage in mice.In addition,we hypothesized that a complete and full-length form DSPP rather than the conversion of DSPP to its fragments by proteolytic processing might control the biological role of the condylar cartilage.Objectives:To observe the full-length of DSPP on postnatal development of mandibular condyle in mice and explore the specific development mechanism of the NH₂-terminal fragments of DSPP on the mandibular condyle,while will provide new ideas and new targets for clinical treatment of mandibular condyle related diseases.Methods:1.To explore the function of full-length DSPP,four groups of mice were employed as experimental subjects:（1）wild type（WT）mice;（2）Dspp knockout（Dspp KO）mice;（3）mice expressing the normal DSPP transgene in the Dspp KO background（Dspp KO/normal Tg）;（4）mice expressing the uncleavable full-length DSPP in the Dspp KO background（Dspp KO/D452A Tg）.The four groups of the mandibular condyles were collected at 3 and 6 months postnatal,respectively.After fixation,plain X-ray Radiography and micro-computed Tomography were used to observe the condylar morphology changes;Hematoxylin&eosin（HE）and toluidine blue staining were applied to uncover the histological changes of mandibular condylar cartilage.2.To explore the function of the NH₂-terminal fragments（i.e.DSP/DSP-PG）,three groups of mice were employed as experimental subjects:（1）WT mice;（2）Dspp KO mice;（3）mice expressing the NH₂-terminal fragments of DSPP in the Dspp-null background（Dspp KO/DSP Tg）.The three groups of the mandibular condyles were collected at 3 and 6 months postnatal,respectively.The former strategies were utilized to examine the differences of condylar morphology and histological structures changes within three groups of mice.Results:1.Full-length DSPP partially reversed the mandibular condylar defects in Dspp KO mice.X-ray and mirco-CT showed the bone defects of condyles from Dspp KO mice were very marked in comparison with that of WT mice,either at the age of 3months or 6 months.There was no differences in the volume of subcjondral bone in between the Dspp KO/normal-Tg mice and WT mice.The mandibular condylar shapes of Dspp KO/D452A Tg mice also were closer to those of WT mice.HE staining and toluidine blue staining showed that,in comparison with WT mice,the mandibular condylar cartilage of Dspp KO mice was thinner,and the amount of mandibular condylar cartilage of cell numbers were less than that of WT mice.The mandibular condylar thickness and the amount of cell numbers of Dspp KO/normal Tg mice were almost consistent with that of WT mice.The thickness and cell numbers of the mandibular condylar cartilage of the D452A-Dspp mice were less than that of WT mice,but greater than that of Dspp KO mice.In addition,the surface of condylar cartilage of Dspp KO/normal Tg mice was smooth and intact.But the Dspp KO/D452A Tg mice also displayed some phenotypic changes,such as small cartilage fissures,sporadic cell clusters and the cell free areas.Transgenic full-length DSPP partially reversed mandibular condylar morphology and mandibular condylar cartilage thickness of Dspp KO mice.2.NH₂-terminal fragments of DSPP aggravates the mandibular condylar defects in Dspp KO mice.Analysis of X-ray and mirco-CT revealed that the defects of mandibular condyle of Dspp KO/DSP Tg mice were evident,manifested as smaller mandibular condyle and narrowed condylar neck,in comparison with that of WT mice and Dspp KO mice.HE staining and toluidine blue staining showed a decrease in the MCC thickness in Dspp KO/DSP Tg mice compared with Dspp KO mice at the age of3 months,but the thickness of mandibular condylar cartilage in Dspp KO/DSP Tg mice was greater than that of Dspp KO mice at the age of 6 months.The chondrocyte numbers in the mandibular condylar cartilage of 3-month-old Dspp KO/DSP Tg mice was less than that of Dspp KO mice,however,there was no differences in the amount of chondrocyte numbers in between the Dspp KO mice and Dspp KO/DSP Tg mice at the ages of 6 months.In addition,the surface of the mandibular condylar cartilage of Dspp KO/DSP Tg mice was flat,noticeable acellular regions and irregular cell arrangement.Transgenic DSP failed to reverse mandibular condylar morphology and mandibular condylar cartilage thickness of Dspp KO mice,but led to smaller mandibular condyle and disordered cartilage structure.Conclusions:DSPP posttranslational modification has a crucial role in postnatal development of mandibular condyle.The products of posttranslational processing,i.e.full-length DSPP and cleavage fragments all play a part in it.