Evidence for changing nerve growth factor signalling mechanisms during development, maturation and ageing in the rat molar pulp.

AIM: To examine rat molar pulp innervation and identify complex cellular signalling systems involving nerve growth factor (NGF) and its p75 receptors (NGFR) at different stages of development, maturation and ageing. METHODOLOGY: Decalcified mandibular first molar mesial cusps from Wistar rats of ages 0 day; 1, 2, 3, 4, 6, 9, 12 and 24 weeks (n = 5 per group) were sectioned (10 µm) and incubated with antibodies for NGF, NGFR, calcitonin gene-related peptide (CGRP) and neurofilament. Nerve densities in worn and intact regions of 3- to 24-week-old rats were compared by anova, Bonferroni and t-tests. RESULTS: During odontogenesis, differences in NGF and NGFR expression were observed, with no evidence of nerve fibres, suggesting a signalling mechanism controlling cellular differentiation and dentine formation. Tooth wear in 4-week rats was associated with reduced NGF expression and significantly decreased CGRP axons within affected odontoblast regions. The underlying subodontoblasts started expressing NGF which continued until 9 weeks. This may promote a significant increase in CGRP nerve density in affected regions. Nerve density in intact odontoblast regions increased gradually and reached significant levels in 12-week rats. Reduction in nerve densities within worn and intact regions of cusps was observed at 24 weeks. CONCLUSIONS: Age-related changes and responses to tooth wear may be controlled by the NGF signalling mechanism, with roles in odontoblast/subodontoblast communication and control of sensory innervation at different stages of tooth development, maturation and ageing. Greater understanding of cellular and nerve regulation in the injured pulp may promote therapeutic strategies for pulp survival.

Evidence for programmed odontoblast process retraction after dentine exposure in the rat incisor.

OBJECTIVE: To re-examine the morphology and potential functions of odontoblasts in intact rat incisors and after cavity preparation into dentine. DESIGN: Intact incisors were fixed, decalcified, snap frozen and sectioned (10µm), before staining with rhodamine phalloidin or antibodies for cyto-skeletal proteins: vimentin and actin, ion transporter: NaK-ATPase, and dendritic cell marker: OX6. Samples with cavity were processed similarly and stained for actin and vimentin before comparing the lengths of odontoblast processes (OP) at baseline, 3h and 24h (n=5 for each group). RESULTS: Actin was expressed through the full length of OP, while vimentin immunoreactivity was not uniform, with 4 distinct regions. OP showed morphological complexity with fine branches emanating within different regions of dentine. Novel actin-positive tree-like OP were identified within predentine which reduced in intensity and length toward the incisal portion of the tooth. Specimens with cavities showed time-dependant pulpal retraction of OP. CONCLUSIONS: Differences in structural antibody expression suggest functional variations in OP within different regions of dentine. The role of actin positive OP in predentine is not known, but could be related to dentine deposition, cellular stability or sensing mechanisms. Cavity preparation into dentine was followed by programmed retraction of OP which could be controlled either mechanically by the spatial limitation of the OP within dentinal tubules or structurally by the presence of vimentin, in addition to actin, in the mid-dentine.

Complex cellular responses to tooth wear in rodent molar.

The arrangement and roles of the odontoblast and its process in sensing and responding to injuries such as tooth wear are incompletely understood. Evidence is presented that dentine exposure by tooth wear triggers structural and functional changes that aim to maintain tooth integrity. Mandibular first molars from freshly culled 8 week Wistar rats were prepared for light microscopy ground-sections (n=6), or fixed in 4% paraformaldehyde, decalcified in 17% EDTA, sectioned and stained with antibodies to cyto-skeletal proteins (vimentin (vim), α-tubulin (tub) and α-actin), cellular homeostatic elements (sodium potassium ATPase (NaK-ATPase) and sodium hydrogen exchanger (NHE-1)), and sensory nerve fibres (CGRP) (n=10) for fluorescence microscopy of worn and unworn regions of the mesial cusp. Immunoreactivity (IR) to vim, actin, NaK-ATPase and CGRP was confined to the pulpal third of odontoblast processes (OPs). IR to tub and nhe-1 was expressed by OPs in full dentine thickness. In areas associated with dentine exposure, the tubules contained no OPs. In regions with intact dentine, odontoblasts were arranged in a single cell layer and easily distinguished from the sub-odontoblast cells. In regions with open tubules, the odontoblasts were in stratified or pseudo-stratified in arrangement. Differences in structural antibody expression suggest a previously unreported heterogeneity of the odontoblast population and variations in different regions of the OP. This combined with differences in OPs extension and pulp cellular arrangement in worn and unworn regions suggests active and dynamic cellular responses to the opening of dentinal tubules by tooth wear.