The sensory nerve regulates stem cell homeostasis through Wnt5a signaling

Description

Increasing evidence indicates that nerves play a significant role in regulating stem cell homeostasis and developmental processes. To explore the impact of nerves on epithelial stem cell homeostasis during tooth development, the regulation of the inferior alveolar nerve (IAN) and sensory nerves on stem cell homeostasis was investigated using a rat model of incisor development. Impaired mineralization, decreased enamel thickness, and fractured enamel rods of the incisor were observed after denervation. Transcriptome sequencing analysis and staining revealed that the IAN group had decreased expression of Wnt5a and enamel-related genes kallikrein-4, odontogenic ameloblast-associated protein, and amelotin. The decreased expression of Wnt5a in ameloblasts was coupled with the downregulation of calcium ion-related calmodulin kinase II. These results implicate that the sensory nerves, as the most critical component of the inferior alveolar nerve, are essential in stem cell homeostasis for enamel mineralization and development.

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Animal Administration Seven-week-old Sprague–Dawley rats purchased from Charles River Laboratory, were raised under specific pathogen-free conditions. Eighteen rats were randomly allocated into 3 groups (n = 6 per group). All experimental procedures and the feeding protocols were approved by the Animal Ethics Committee of Nanjing Agricultural University (No. PZW2022054) and followed the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guideline. Histology and immunohistochemical analysis of the incisor. After fixed and decalcified, the sections were stained with routine H&E and Masson's trichrome staining (MTS). Immunohistochemical staining was performed to detect the protein expression levels of β-catenin (Service- bio), CaMK-II (Abcam) and Ror2 (Abcam). Immunofluorescence analysis The expression of PGP9.5 (Abcam), CGRP (CST), Wnt5a (Proteintech), Amelx (Abcam) and KLK4 (LifeSpan Biosciences) were detected using immunofluorescence staining. The images were acquired using a confocal laser microscope (Nikon Instruments Co.). Micro-Computed Tomography Analysis All rat incisors were scanned using micro-CT with a voxel resolution of 18 μm (Bruker micro-CT). The data were imported into DataViewer software (Bruker) for analysis and reconstruction of the incisors. mRNA sequencing analysis mRNA sequencing was used to detect differences in incisor mRNA expression between the IAN and sham groups. The VAHTS Universal V5 RNA-seq Library Prep kit was used to construct a transcriptome library according to the instructions. Transcriptome sequencing and analyses were performed by OE Biotech Co., Ltd. Hard tissue sectioning assay After fixing the tissues were dehydrated and embedded in a light-curing monomer resin (Technovit 7200VLC). Tissue sections were prepared using an EKAKT E300 sectioning and grinding system (Ekakt). Examination and photography were performed using a transmitted light microscope (Zeiss Axioscope 2 plus). Scanning electron microscopy. The sagittal sections were etched with 37% phosphoric acid for 20 seconds. A 30 nm gold-palladium sputtering coating was applied, and the surface morphology of the sample was observed using a scanning electron microscope (S-3400N). SEM images were obtained to observe the surface morphology of the enamel. Vickers hardness testing. The prepared specimens were placed on the testing table of an HV-1000 microhardness tester (Yanrun). The surface hardness values of each specimen within the five regions were determined, and the average value was taken as the average Vickers hardness of the specimen.

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