| Background and Objective: Dentin-pulp complex is an important structure of teeth,which is of great significance in the development and repair of teeth.The odontoblast cells are the most representative functional cells which can secrete and form dentin.Mastication is one of the crucial functions of teeth,and the bite force produced by its process can affect the development of periodontal ligament,root,jaw and tooth eruption.However,the effect of bite force on the development of dentin-pulp complex and its mechanism are still unclear.It was found that with the increase of age,the rate of dentin formation decreased significantly,the pulp cavity becomes smaller,and the time node coincided with the establishment of occlusal relationship.In this study,a mouse model of reduced bite force was established to preliminarily explore the influence of bite force on the development of dentin-pulp complex,providing a new theoretical basis for the formation of dentin-pulp complex.Methods:The adult male and female BALB/c mice were caged together.After conception,the pregnant mice were reared separately until the newborn mice were born.Newborn rats were marked as P0.5(Postnatal 0.5),and they were weaned on P21.5 days.Then they were randomly divided into hard food group and soft food group,with 5 mice in each group.A weakened bite force model of BALB/c mice was established by diet method.According to literature reports,the mandibular bones of neonatal mice were collected at P0.5(late bell-shaped tooth embryo),P7.5(late embryonic tooth development),P15.5(early tooth eruption),P21.5(early occlusal contact),P27.5(middle occlusal contact),P33.5(late occlusal contact),and P60.5(completed construction).The collected samples were stained with HE and aniline blue,to observe the changes of the number of odontoblasts and the amount of dentin formation in the dentin-pulp complex of BALB/c mice from lactation period to occlusal contact.PCNA and Caspase-3 Immunohistochemistry(IHC)staining were used to observe the effect of reduced bite force on the proliferation and apoptosis of odontoblast cells.Then,the dentine deposition rate was calculated by the dentine thickness obtained by aniline blue staining.Next,the in vivo double-labeling method was used to observe the difference in dentin formation between 3 days and 9 days after P21.5 days of soft and hard food groups,to evaluate the effect of bite force on the rate of dentin formation in the dentin-pulp complexResults:1.The mice in the soft and hard food group were weighed,and results showed that there was no significant difference in the weight of mice between the two groups(p>0.05).2.From P0.5 day to P60.5 day after birth,the odontoblasts of mice gradually changed from a high columnar monolayer to flat pseudostratified arrangement at the dentin-pulp interface.And from P0.5 to P60.5,The number of odontoblasts per unit area increased significantly(p<0.05).There was no significant difference in the morphology and number of odontoblasts between the hard and soft food group(p>0.05).3.The results of aniline blue staining showed that the thickness of dentin in both the hard food group and the soft food group increased with age.However,the thickness of the soft food group at P27.5,P33.5,P60.5 was greater than that of the hard food group(p<0.05),and the difference of dentin thickness between the soft-food group and the hard-food group showed a decreasing trend with the increase of age.4.The results of PCNA IHC staining showed that the expression of odontoblast cells in both the hard food group and the soft food group were positive,and there was no significant difference between the two groups.And the expression of Caspase-3 was negative in both the hard food group and the soft food group.5.According to the dentine thickness of the soft and hard food group measured by aniline blue,the dentine deposition rate in each period was calculated.During P21.5 to P27.5 days,the dentin formation rate in the soft food group was significantly higher than that in the hard food group(p<0.0001).However,the rate of dentin formation in the hard-eating group was significantly higher than that in the soft-eating group during P27.5 to P33.5(p<0.0001).There was no significant difference in the dentin formation rate between the hard and soft food groups from P33.5 to P60.5.6.In vivo fluorescence double labeling method was used to detect the amount and rate of dentin deposition in the soft food group and the hard food group,respectively.The measurement of the distance between the two fluorescence lines showed that the increase of dentin in the soft food group was 13.10±0.90 μm,and that in the hard food group was 7.70±1.00 μm,the dentine growth of the soft food group was significantly higher than that of the hard food group(p<0.0001);In the 9-day interval group,there was no significant difference in the dentin growth between the hard and soft food groups(p>0.05).7.By measuring and analyzing the periodontal ligament thickness in apical area of the soft food group and the hard food group at P27.5,P33.5 and P60.5 of HE staining,the results showed that the periodontal ligament thickness in the hard food group was greater than that in the soft food group during each period,and the difference was statistically significant(p<0.05).Conclusion: In this study,a mouse model of reduced bite force was successfully established by dietary method.The morphology of odontoblast cells was observed by HE staining and the odontoblast cells were counted per unit area.PCNA and Caspase-3 IHC staining was used to observe the proliferation and apoptosis of odontoblast cells.Dentin deposition rate was measured by aniline blue and in vivo staining.The results showed that the weakened bite force did not affect the morphology,number,proliferation and apoptosis of odontoblasts.However,the weakened occlusal force can promote the formation of dentin.The increase of dentin is not caused by the increase in the number of odontoblasts,but by changes in the secretion rate of odontoblasts.The mechanism is still needed to be further studied. |
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