Proteomic Analyses Discern the Developmental Inclusion of Albumin in Pig Enamel: A New Model for Human Enamel Hypomineralization

Excess albumin in enamel is a characteristic of the prevalent developmental dental defect known as chalky teeth or molar hypomineralization (MH). This study uses proteomic analyses of pig teeth to discern between developmental origin and post-eruptive contamination and to assess the similarity to hypomineralized human enamel. Here, the objective is to address the urgent need for an animal model to uncover the etiology of MH and to improve treatment. Porcine enamel is chalky and soft at eruption; yet, it hardens quickly to form a hard surface and then resembles human teeth with demarcated enamel opacities. Proteomic analyses of enamel from erupted teeth, serum, and saliva from pigs aged 4 (n = 3) and 8 weeks (n = 2) and human (n = 4) molars with demarcated enamel opacities show alpha-fetoprotein (AFP). AFP expression is limited to pre- and perinatal development and its presence in enamel indicates pre- or perinatal inclusion. In contrast, albumin is expressed after birth, indicating postnatal inclusion into enamel. Peptides were extracted from enamel and analyzed by nano-liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) after tryptic digestion. The mean total protein number was 337 in the enamel of all teeth with 13 different unique tryptic peptides of porcine AFP in all enamel samples but none in saliva samples. Similarities in the composition, micro-hardness, and microstructure underscore the usefulness of the porcine model to uncover the MH etiology, cellular mechanisms of albumin inclusion, and treatment for demarcated opacities

Osteopontin regulates type I collagen fibril formation in bone tissue

Osteopontin (OPN) is a non-collagenous protein involved in biomineralization of bone tissue. Beyond its role in biomineralization, we show that osteopontin is essential to the quality of collagen fibrils in bone. Transmission electron microscopy revealed that, in Opn−/− tissue, the organization of the collagen fibrils was highly heterogeneous, more disorganized than WT bone and comprised of regions of both organized and disorganized matrix with a reduced density. The Opn−/− bone tissue also exhibited regions in which the collagen had lost its characteristic fibrillar structure, and the crystals were disorganized. Using nanobeam electron diffraction, we show that damage to structural integrity of collagen fibrils in Opn−/- bone tissue and their organization causes mineral disorganization, which could ultimately affect its mechanical integrity

Mechanical and physicochemical multiscale analysis of cortical bone

36th Congress of the Society-of-Biomechanics, Besancon, FRANCE, AUG 31-SEP 02, 201

Finite element dependence of stress evaluationfor human trabecular bone

International audienceNumerical simulation using finite element models (FEM) has become more and moresuitable to estimate the mechanical properties of trabecular bone. The size and kind ofelements involved in the models, however, may influence the results. The purpose of thisstudy is to analyze the influence of hexahedral elements formulation on the evaluation ofmechanical stress applied to trabeculae bone during a compression test simulation.Trabecular bone cores were extracted from 18 L2 vertebrae (12 women and 6 men, meanage: 76711, BV/TV¼7.571.9%). Samples were micro-CT scanned at 20 mm isotropic voxelsize. Micro-CT images have been sub-sampled (20, 40 and 80 mm) to create 5.6mm cubicFEM. For each sample, a compression test FEM has been created, using either 8-nodeslinear hexahedral elements with full or reduced integration or 20-nodes quadratichexahedral elements fully integrated, resulting in nine models per samples. Bonemechanical properties have been assumed isotropic, homogenous and to follow a linearelastic behavior law (Young modulus: 8 GPa, Poisson ratio: 0.3).Despite micro-architecture modifications (loss of connectivity, trabeculae thickening)due to voxel size increase, apparent mechanical properties calculated with low resolutionmodels are significantly correlated with high resolution results, no matter the elementformulation. However, stress distributions are more sensitive to both resolution andelement formulation modifications. With linear elements, increasing voxel size leads toan alteration of stress concentration areas due to stiffening errors. On the opposite, the useof reduced integration induces severe smoothing and underestimation of stress fieldsresulting in stress raisers loss. Notwithstanding their high computational cost, quadraticelements are most appropriate for stress prediction in low resolution trabecular bone FEM.These observations are dependent on trabecular bone micro-architecture, and are moresignificant for low density sample displaying low trabecular thickness.In conclusion, we found that element formulation is almost important as element sizewhen evaluating trabecular bone mechanical behavior at trabeculae scale. Therefore,element type should be chosen carefully when evaluating trabecular bone behaviorusing FEM

Rapid post-eruptive maturation of porcine enamel

The teeth of humans and pigs are similar in size, shape, and enamel thickness. While the formation of human primary incisor crowns takes about 8 months, domestic pigs form their teeth within a much shorter time. Piglets are born after 115 days of gestation with some of their teeth erupted that must after weaning meet the mechanical demands of their omnivorous diet without failure. We asked whether this short mineralization time before tooth eruption is combined with a post-eruptive mineralization process, how fast this process occurs, and how much the enamel hardens after eruption. To address this question, we investigated the properties of porcine teeth at two, four, and sixteen weeks after birth (N = 3 animals per time point) through analyses of composition, microstructure, and microhardness. We collected data at three standardized horizontal planes across the tooth crown to determine the change of properties throughout the enamel thickness and in relation to soft tissue eruption. Our findings indicate that porcine teeth erupt hypomineralized compared to healthy human enamel and reach a hardness that is similar to healthy human enamel within less than 4 weeks.</jats:p

Table1_Rapid post-eruptive maturation of porcine enamel.xlsx

The teeth of humans and pigs are similar in size, shape, and enamel thickness. While the formation of human primary incisor crowns takes about 8 months, domestic pigs form their teeth within a much shorter time. Piglets are born after 115 days of gestation with some of their teeth erupted that must after weaning meet the mechanical demands of their omnivorous diet without failure. We asked whether this short mineralization time before tooth eruption is combined with a post-eruptive mineralization process, how fast this process occurs, and how much the enamel hardens after eruption. To address this question, we investigated the properties of porcine teeth at two, four, and sixteen weeks after birth (N = 3 animals per time point) through analyses of composition, microstructure, and microhardness. We collected data at three standardized horizontal planes across the tooth crown to determine the change of properties throughout the enamel thickness and in relation to soft tissue eruption. Our findings indicate that porcine teeth erupt hypomineralized compared to healthy human enamel and reach a hardness that is similar to healthy human enamel within less than 4 weeks.</p

Immature and Mature Collagen Crosslinks Quantification Using High-Performance Liquid Chromatography and High-Resolution Mass Spectrometry in Orbitrap

Different methodologies for collagen quantification have been described in the past. Introduction of mass spectrometry combined with high-performance liquid chromatography (HPLC) is a high-resolution tool, which has generated novel applications in biomedical research. In this study, HPLC coupled to electrospray ionization (ESI) tandem mass spectrometry (HPLC-ESI-MS/MS) was used to characterize tissue samples from AVFs done in rats. These findings helped create a protocol for identifying and quantifying components of immature and mature collagen crosslink moieties. Two different internal standards were used: epinephrine and pyridoxine. Quantification curves were drawn by means of these standards. The goal of the experiment was to achieve accurate quantification with the minimum amount of sample. Time and cost of experiment were considerably minimized. Up to date, this method has not been tested for crosslinking quantification