Capsaicin-sensitive Innervation Modulates the Development of Apical Periodontitis.

INTRODUCTION: Nociceptive neurons play a critical role in the detection of stimuli evoking actual or potential tissue injury. In addition, they are involved in neurogenic inflammation by the peripheral release of neuropeptides such as calcitonin gene-related peptide (CGRP). The dental pulp and periradicular tissues are innervated by capsaicin-sensitive neurons known to release CGRP. However, the role of these capsaicin-sensitive neurons in the development of apical periodontitis is largely unknown. The aim of this study was to evaluate the contribution of peptidergic neurons to the development of apical periodontitis. METHODS: Neonatal Sprague-Dawley rats were injected with vehicle (control group) or a single subcutaneous capsaicin dose to cause the selective ablation of peptidergic neurons (neonatal capsaicin group). Ablation of capsaicin-sensitive neurons was verified with confocal microscopy, capsaicin-induced eye-wipe nocifensive behavior test, and by measurement of immunoreactive CGRP levels in the dental pulp. Five weeks after ablation, standardized pulp exposures were made in the mandibular left first molars. Mandibles were harvested at 7, 14, 21, and 28 days after pulp exposure and imaged with micro-computed tomography (µCT) to quantify apical lesion volume. Data were analyzed by using 2-way ANOVA analysis with Bonferroni post hoc test. RESULTS: Rats in the control group displayed a robust capsaicin-induced nocifensive behavior, which was nearly abolished in the neonatal capsaicin group. In addition, the neonatal capsaicin group showed a significant depletion of susceptible neurons and CGRP in the dental pulp compared with control. Importantly, micro-computed tomography analysis showed larger periradicular lesions at 7 and 14 days after pulp exposure in the neonatal capsaicin group when compared with control. CONCLUSIONS: Results identify a protective role for capsaicin-sensitive neurons in the initial phase of apical periodontitis. Thus, interventions or disorders that alter activity of capsaicin-sensitive fibers are likely to alter the development of apical periodontitis.

Maturation stage enamel malformations in Amtn and Klk4 null mice.

Amelotin (AMTN) and kallikrein-4 (KLK4) are secreted proteins specialized for enamel biomineralization. We characterized enamel from wild-type, Amtn(-/-), Klk4(-/-), Amtn(+/-)Klk4(+/-) and Amtn(-/-)Klk4(-/-) mice to gain insights into AMTN and KLK4 functions during amelogenesis. All of the null mice were healthy and fertile. The mandibular incisors in Amtn(-/-), Klk4(-/-) and Amtn(-/-)Klk4(-/-) mice were chalky-white and chipped. No abnormalities except in enamel were observed, and no significant differences were detected in enamel thickness or volume, or in rod decussation. Micro-computed tomography (µCT) maximum intensity projections localized the onset of enamel maturation in wild-type incisors distal to the first molar, but mesial to this position in Amtn(-/-), Klk4(-/-) and Amtn(-/-)Klk4(-/-) mice, demonstrating a delay in enamel maturation in Amtn(-/-) incisors. Micro-CT detected significantly reduced enamel mineral density (2.5 and 2.4gHA/cm(3)) in the Klk4(-/-) and Amtn(-/-)Klk4(-/-) mice respectively, compared with wild-type enamel (3.1gHA/cm(3)). Backscatter scanning electron microscopy showed that mineral density progressively diminished with enamel depth in the Klk4(-/-) and Amtn(-/-)Klk4(-/-) mice. The Knoop hardness of the Amtn(-/-) outer enamel was significantly reduced relative to the wild-type and was not as hard as the middle or inner enamel. Klk4(-/-) enamel hardness was significantly reduced at all levels, but the outer enamel was significantly harder than the inner and middle enamel. Thus the hardness patterns of the Amtn(-/-) and Klk4(-/-) mice were distinctly different, while the Amtn(-/-)Klk4(-/-) outer enamel was not as hard as in the Amtn(-/-) and Klk4(-/-) mice. We conclude that AMTN and KLK4 function independently, but are both necessary for proper enamel maturation.

Correlation of ameloblastin with enamel mineral content.

In enamel formation, the deposition of minerals as crystallites starts when the mineralization front first forms at the start of the secretory stage. During maturation, the enamel layer accumulates significant amounts of new mineral as the crystallites grow in volume. Inversely related to mineral gain is loss of protein and water from the forming enamel. Both ameloblastin (Ambn) and enamelin are essential components for formation of a functional enamel layer. The aim of this study was to quantify the proportion of mineral and non-mineral material present in developing enamel relative to Ambn concentration using Ambn mutant mice mated with others overexpressing full-length Ambn from the mouse amelogenin promoter at lower (+), similar (++) or higher (+++) concentration than normal. Mandibular incisors (age: 7 weeks, n = 8) were imaged by micro-computed tomography and the enamel was analyzed from the apical region to the incisal edge in sequential 1.0 mm volumes of interest. Mineral density was determined using a series of hydroxyapatite (HA) phantoms to calibrate enamel density measurements. At the site where the mandibular incisor emerged into the oral cavity, the enamel volume, mineral weight, and mineral density were reduced when Tg Ambn was expressed at lower or higher levels than normal. While in wild-type the % mineral was >95%, it was negligible in Ambn-/-, 22.3% in Ambn-/-, Tg(+), 75.4% in Ambn-/-, Tg(++), and 45.2% in Ambn-/-, Tg(+++). These results document that the deposition of mineral and removal of non-mineral components are both very sensitive to expressed Ambn concentrations.

Lumbar vertebral body bone microstructural scaling in small to medium-sized strepsirhines.

Bone mass, architecture, and tissue mineral density contribute to bone strength. As body mass (BM) increases any one or combination of these properties could change to maintain structural integrity. To better understand the structural origins of vertebral fragility and gain insight into the mechanisms that govern bone adaptation, we conducted an integrative analysis of bone mass and microarchitecture in the last lumbar vertebral body from nine strepsirhine species, ranging in size from 42 g (Microcebus rufus) to 2,440 g (Eulemur macaco). Bone mass and architecture were assessed via µCT for the whole body and spherical volumes of interest (VOI). Allometric equations were estimated and compared with predictions for geometric scaling, assuming axial compression as the dominant loading regime. Bone mass, microarchitectural, and vertebral body geometric variables predominantly scaled isometrically. Among structural variables, the degree of anisotropy (Tb.DA) was the only parameter independent of BM and other trabecular architectural variables. Tb.DA was related to positional behavior. Orthograde primates had higher average Tb.DA (1.60) and more craniocaudally oriented trabeculae while lorisines had the lowest Tb.DA (1.25), as well as variably oriented trabeculae. Finally, lorisines had the highest ratio of trabecular bone volume to cortical shell volume (∼3x) and while there appears to be flexibility in this ratio, the total bone volume (trabecular + cortical) scales isometrically (BM(1.23) , r(2) = 0.93) and appears tightly constrained. The common pattern of isometry in our measurements leaves open the question of how vertebral bodies in strepsirhine species compensate for increased BM.

Specimen size and porosity can introduce error into microCT-based tissue mineral density measurements.

The accurate measurement of tissue mineral density, rho(m), in specimens of unequal size or quantities of bone mineral using polychromatic microCT systems is important, since studies often compare samples with a range of sizes and bone densities. We assessed the influence of object size on microCT measurements of rho(m) using (1) hydroxyapatite rods (HA), (2) precision-manufactured aluminum foams (AL) simulating trabecular bone structure, and (3) bovine cortical bone cubes (BCt). Two beam-hardening correction (BHC) algorithms, determined using a 200 and 1200 mg/cm(3) HA wedge phantom, were used to calculate rho(m) of the HA and BCt. The 200 mg/cm(3) and an aluminum BHC algorithm were used to calculate the linear attenuation coefficients of the AL foams. Equivalent rho(m) measurements of 500, 1000, and 1500 mg HA/cm(3) rods decreased (r(2)>0.96, p<0.05 for all) as HA rod diameter increased in the 200 mg/cm(3) BHC data. Errors averaged 8.2% across these samples and reached as high as 29.5%. Regression analyses suggested no size effects in the 1200 mg/cm(3) BHC data but differences between successive sizes still reached as high as 13%. The linear attenuation coefficients of the AL foams increased up to approximately 6% with increasing volume fractions (r(2)>0.81, p<0.05 for all) but the strength of the size-related error was also BHC dependent. Equivalent rho(m) values were inversely correlated with BCt cube size (r(2)>0.92, p<0.05). Use of the 1200 mg/cm(3) BHC ameliorated the size-related artifact compared to the 200 mg/cm(3) BHC but errors with this BHC were still significant and ranged between 5% and 12%. These results demonstrate that object size, structure, and BHC algorithm can influence microCT measurements of rho(m). Measurements of rho(m) of specimens of unequal size or quantities of bone mineral must be interpreted with caution unless appropriate steps are taken to minimize these potential artifacts.