Abstract:

Objective To investigate the spatiotemporal expression characteristics and biological functions of CD44 during the development of the mandibular first molar crown in mice. Methods Immunohistochemical staining was performed to examine CD44 protein localization and expression in the mandibular first molar tooth germs at different developmental stages from E11.5 （embryonic day 11.5） to PN7 （postnatal day 7）. Real-time quantitative PCR was used to analyze the mRNA expression level of CD44. To further explore the function of CD44，mouse mandibular first molar dental germs at E16.5 were isolated for organ culture and transfected with small interfering RNA to knock down CD44 expression. The overall morphology of the dental germs，their width and height，as well as the differentiation status of ameloblasts and odontoblasts，collagen secretion，and the formation of predentin were evaluated using HE staining，Masson trichrome staining，and immunofluorescence staining techniques. Results At the initiation stage of tooth germ development （E11.5），CD44 was not significantly expressed in the dental plate epithelium and the underlying mesenchyme. In the bud stage （E13.5），CD44 showed weak expression in the dental epithelial cells. From the cap stage （E14.5） to the late bell stage （E18.5），CD44 was expressed in all types of cells in the enamel organ，and was highly expressed in the inner enamel epithelium and stratum intermedium. After birth （PN1 to PN7），CD44 continued to be highly expressed in the ameloblasts and stratum reticulum. From PN3 onwards，positive signals were also detected in the sub-odontoblastic layer near the odontoblasts at the tooth cusps. RT-qPCR results showed that the expression level of CD44 mRNA reached its peak at PN5. The culture experiment of dental germs showed that compared with the control group，the width and height of the dental germ in the siCD44 group were reduced； the differentiation of ameloblasts and odontoblasts was inhibited； collagen secretion was significantly decreased，and the formation of predentin was impaired. Conclusion CD44 exhibits dynamic spatiotemporal expression characteristics during the development of the mandibular first molar crown in mice. Inhibiting its expression can affect the morphology of the dental germ，key cell differentiation，and the formation of predentin，suggesting that CD44 plays an important role in this process.

Key words: CD44, dental crown development, enamel organ, immunohistochemical staining, organ culture