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Current Protocols Jan 2022Enamel is the hardest tissue in mammalian organisms and is the layer covering the tooth. It consists of hydroxyapatite (HAP) crystallites, which mineralize on a protein...
Enamel is the hardest tissue in mammalian organisms and is the layer covering the tooth. It consists of hydroxyapatite (HAP) crystallites, which mineralize on a protein scaffold known as the enamel matrix. Enamel matrix assembly is a very complex process mediated by enamel matrix proteins (EMPs). Altered HAP deposition or disintegration of the protein scaffold can cause enamel defects. Various methods have been established for enamel phenotyping, including MicroCT scanning with various resolutions from 9 µm for in vivo imaging to 1.5 µm for ex vivo imaging. With increasing resolution, we can see not only the enamel layer itself but also a detailed map of mineralization. To study enamel microstructure, we combine the MicroCT analysis with scanning electron microscopy (SEM), which enables us to perform element analyses such as calcium-carbon ratio. However, the methods mentioned above only show the result-already formed enamel. Stimulated emission depletion (STED) microscopy provides extra information about protein structure in the form of EMP localization and position before enamel mineralization. A combination of all these methods allows analyzing the same sample on multiple levels-starting with the live animal being scanned harmlessly and quickly, followed by sacrifice and high-resolution MicroCT scans requiring no special sample preparation. The biggest advantage is that samples remain in perfect condition for SEM or STED microscopic analysis. © 2022 Wiley Periodicals LLC. Basic Protocol 1: In vivo MicroCT scanning of mouse Basic Protocol 2: Ex vivo HR-MicroCT of the teeth Basic Protocol 3: SEM for teeth microstructure Basic Protocol 4: Stimulated emission depletion (STED) microscopy.
Topics: Animals; Durapatite; Mice; Microscopy, Electron, Scanning; Tooth; Tooth Calcification; X-Ray Microtomography
PubMed: 35007410
DOI: 10.1002/cpz1.340 -
Journal of Bone and Mineral Research :... May 2015Bone is a strong and tough material composed of apatite mineral, organic matter, and water. Changes in composition and organization of these building blocks affect...
Bone is a strong and tough material composed of apatite mineral, organic matter, and water. Changes in composition and organization of these building blocks affect bone's mechanical integrity. Skeletal disorders often affect bone's mineral phase, either by variations in the collagen or directly altering mineralization. The aim of the current study was to explore the differences in the mineral of brittle and ductile cortical bone at the mineral (nm) and tissue (µm) levels using two mouse phenotypes. Osteogenesis imperfecta model, oim(-/-) , mice have a defect in the collagen, which leads to brittle bone; PHOSPHO1 mutants, Phospho1(-/-) , have ductile bone resulting from altered mineralization. Oim(-/-) and Phospho1(-/-) were compared with their respective wild-type controls. Femora were defatted and ground to powder to measure average mineral crystal size using X-ray diffraction (XRD) and to monitor the bulk mineral to matrix ratio via thermogravimetric analysis (TGA). XRD scans were run after TGA for phase identification to assess the fractions of hydroxyapatite and β-tricalcium phosphate. Tibiae were embedded to measure elastic properties with nanoindentation and the extent of mineralization with backscattered electron microscopy (BSE SEM). Results revealed that although both pathology models had extremely different whole-bone mechanics, they both had smaller apatite crystals, lower bulk mineral to matrix ratio, and showed more thermal conversion to β-tricalcium phosphate than their wild types, indicating deviations from stoichiometric hydroxyapatite in the original mineral. In contrast, the degree of mineralization of bone matrix was different for each strain: brittle oim(-/-) were hypermineralized, whereas ductile Phospho1(-/-) were hypomineralized. Despite differences in the mineralization, nanoscale alterations in the mineral were associated with reduced tissue elastic moduli in both pathologies. Results indicated that alterations from normal crystal size, composition, and structure are correlated with reduced mechanical integrity of bone.
Topics: Animals; Biomechanical Phenomena; Bone Matrix; Calcification, Physiologic; Calcium Phosphates; Crystallization; Elasticity; Mice; Minerals; Powders; Thermogravimetry; Tibia; X-Ray Diffraction
PubMed: 25418329
DOI: 10.1002/jbmr.2414 -
Bone Feb 2018The periodontal complex is essential for tooth attachment and function and includes the mineralized tissues, cementum and alveolar bone, separated by the unmineralized...
The periodontal complex is essential for tooth attachment and function and includes the mineralized tissues, cementum and alveolar bone, separated by the unmineralized periodontal ligament (PDL). To gain insights into factors regulating cementum-PDL and bone-PDL borders and protecting against ectopic calcification within the PDL, we employed a proteomic approach to analyze PDL tissue from progressive ankylosis knock-out (Ank) mice, featuring reduced PP, rapid cementogenesis, and excessive acellular cementum. Using this approach, we identified the matrix protein osteopontin (Spp1/OPN) as an elevated factor of interest in Ank mouse molar PDL. We studied the role of OPN in dental and periodontal development and function. During tooth development in wild-type (WT) mice, Spp1 mRNA was transiently expressed by cementoblasts and strongly by alveolar bone osteoblasts. Developmental analysis from 14 to 240days postnatal (dpn) indicated normal histological structures in Spp1 comparable to WT control mice. Microcomputed tomography (micro-CT) analysis at 30 and 90dpn revealed significantly increased volumes and tissue mineral densities of Spp1 mouse dentin and alveolar bone, while pulp and PDL volumes were decreased and tissue densities were increased. However, acellular cementum growth was unaltered in Spp1 mice. Quantitative PCR of periodontal-derived mRNA failed to identify potential local compensators influencing cementum in Spp1 vs. WT mice at 26dpn. We genetically deleted Spp1 on the Ank mouse background to determine whether increased Spp1/OPN was regulating periodontal tissues when the PDL space is challenged by hypercementosis in Ank mice. Ank; Spp1 double deficient mice did not exhibit greater hypercementosis than that in Ank mice. Based on these data, we conclude that OPN has a non-redundant role regulating formation and mineralization of dentin and bone, influences tissue properties of PDL and pulp, but does not control acellular cementum apposition. These findings may inform therapies targeted at controlling soft tissue calcification.
Topics: Alveolar Process; Animals; Calcification, Physiologic; Cementogenesis; Dentin; Female; Male; Mice; Mice, Knockout; Osteogenesis; Osteopontin; Periodontal Ligament
PubMed: 29313816
DOI: 10.1016/j.bone.2017.12.004 -
Oral and Maxillofacial Surgery Jun 2017The current systematic review investigated the results of application of some of the most commonly used scaffolds in conjugation with stem cells and growth factors in... (Review)
Review
PURPOSE
The current systematic review investigated the results of application of some of the most commonly used scaffolds in conjugation with stem cells and growth factors in animal and clinical studies.
METHODS
A comprehensive electronic search was conducted according to the PRISMA guidelines in NCBI PMC and PubMed from January 1970 to December 2015 limited to English language publications with available full texts. In vivo studies in relation to "bone healing," "bone regeneration," and at least one of the following items were investigated: allograft, β-tricalcium phosphate, deproteinized bovine bone mineral, hydroxyapetite/tricalcium phosphate, nanohydroxyapatite, and composite scaffolds.
RESULTS
A total of 1252 articles were reviewed, and 46 articles completely fulfilled the inclusion criteria of this study. The highest bone regeneration has been achieved when combination of all three elements, given scaffolds, mesenchymal stem cells, and growth factors, were used. Among studies being reported in this review, bone marrow mesenchymal stem cells are the most studied mesenchymal stem cells, β-tricalcium phosphate is the most frequently used scaffold, and platelet-rich plasma is the most commonly used growth factor.
CONCLUSION
The current review aimed to inform reconstructive surgeons of how combinations of various mesenchymal stem cells, scaffolds, and growth factors enhance bone regeneration. The highest bone regeneration has been achieved when combination of all three elements, given scaffolds, mesenchymal stem cells, and growth factors, were used.
Topics: Allografts; Animals; Biocompatible Materials; Bone Regeneration; Calcification, Physiologic; Calcium Phosphates; Humans; Hydroxyapatites; Intercellular Signaling Peptides and Proteins; Nanostructures; Stem Cell Transplantation; Tissue Engineering; Tissue Scaffolds; Vascularized Composite Allotransplantation
PubMed: 28194530
DOI: 10.1007/s10006-017-0608-3 -
Biological Trace Element Research May 2022This study was designed to determine the effects of dietary hazelnut oil (HO) and sunflower oil (SO) on the mineral composition of chicken femur and tibia bones. A...
This study was designed to determine the effects of dietary hazelnut oil (HO) and sunflower oil (SO) on the mineral composition of chicken femur and tibia bones. A 40-day trial was initiated with 600 1-day-old Ross 308 chickens. Initially, the chickens were randomly divided into four main groups of 150 each according to the proportion of SO and HO supplementation in their diets: control SO (25 g/kg SO), LHO (25 g/kg HO), HHO (50 g/kg HO), and MO (50 g/kg blend of 25 g/kg SO + 25 g/kg HO). Each group was further divided into six subgroups of 25 chickens. At the end of the trial, four chickens from each of the six subgroups were randomly selected and slaughtered. Their right tibia and right femur bones were isolated and analyzed for macro (Ca, P, Mg) and trace (Fe, Cu, Mn, Zn, Cr, Co, and Se) minerals in addition to ether extract and ash. The results suggest that the diets' fat concentration and fatty acid composition significantly affected the Ca, P, Mg, Fe, Cu, Cr, and Se composition of tibia bones (p < 0.01). The ether extract and ash contents of the femur bones were increased in both HHO and MO groups (p < 0.05), while Fe and Cr compositions were lower in all femur bones, except those from SO chickens (p < 0.05). The Se content of femur bones in the LHO group was higher than the other treatment groups (p < 0.05). In conclusion, the amount of oil and fatty acid in the diet affected the accumulation of Ca, P, Mg, Fe, Cu, and Cr minerals in the tibia bone. Moreover, the amount of Cr and Fe was lower in the femur and tibia bones in the group that received the HO supplement and all groups in which the amount of oil fed was increased.
Topics: Animal Feed; Animals; Calcification, Physiologic; Chickens; Diet; Dietary Fats, Unsaturated; Dietary Supplements; Ether; Fatty Acids; Minerals; Plant Extracts
PubMed: 34278547
DOI: 10.1007/s12011-021-02833-9 -
The Journal of Steroid Biochemistry and... Apr 2015Vitamin D has pleiotropic extra-skeletal effects which have been noted in mouse models of deletion of either the 25-hydroxy vitamin D 1α-hydroxylase enzyme, cyp27b1... (Review)
Review
Vitamin D has pleiotropic extra-skeletal effects which have been noted in mouse models of deletion of either the 25-hydroxy vitamin D 1α-hydroxylase enzyme, cyp27b1 (1OHase(-/-) mice) or of the vitamin D receptor (Vdr(-/-) mice); these may be preventable or reversible by either restoring normal signaling of the 1,25(OH)2D/VDR system, or in some cases by restoring normal mineral homeostasis. However, effects on skeletal and mineral homeostasis are clearly the major phenotype observed in humans with loss-of-function mutations in either CYP27B1 or VDR. In mouse phenocopies of these human disorders, correction of hypocalcemia and hypophosphatemia reduce elevated circulating parathyroid hormone concentrations and normalize impaired bone mineralization, but restoration of normal 1,25(OH)2D/VDR signaling may be required for optimal bone formation. Induction of high endogenous 1,25(OH)2D concentrations in genetically modified mouse models may cause increased bone resorption and decreased mineralization. Transgenic Vdr overexpression and conditional Vdr deletion in cells of the osteoblastic lineage have also provided insights into the stages of osteoblast differentiation which may mediate these actions. These anabolic and catabolic effects of the 1,25(OH)2D system on bone may therefore be a function of both the ambient concentration of circulating 1,25(OH)2D and the stage of differentiation of the osteoblast. This article is part of a Special Issue entitled '17th Vitamin D Workshop'.
Topics: 25-Hydroxyvitamin D3 1-alpha-Hydroxylase; Animals; Bone and Bones; Calcification, Physiologic; Humans; Mice; Mice, Knockout; Receptors, Calcitriol
PubMed: 25237033
DOI: 10.1016/j.jsbmb.2014.09.011 -
Journal of Biomedical Materials... May 2019In vitro synthesis of bone tissue has been paid attention in recent years; however, current methods to fabricate bone tissue are still ineffective due to some remaining...
In vitro synthesis of bone tissue has been paid attention in recent years; however, current methods to fabricate bone tissue are still ineffective due to some remaining gaps in the understanding of real in vivo bone formation process, and application of the knowledge in bone synthesis. Therefore, the objectives of this study were first, to perform a systematic and ultrastructural investigation of the initial mineral formation during intramembranous ossification of mouse calvaria from a material scientists' viewpoint, and to develop novel mineralization methods based on the in vivo findings. First, the very initial mineral deposition was found to occur at embryonic day E14.0 in mouse calvaria. Analysis of the initial bone formation process showed that it involved the following distinct steps: collagen secretion, matrix vesicle (MV) release, MV mineralization, MV rupture, and collagen fiber mineralization. Next, we performed in vitro mineralization experiments using MVs and hydrogel scaffolds. Intact MVs embedded in collagen gel did not mineralize, whereas, interestingly, MV nanofragments obtained by ultrasonication could promote rapid mineralization. These results indicate that mechanically ruptured MV membrane can be a promising material for in vitro bone tissue synthesis. © 2019 The Authors. journal Of Biomedical Materials Research Part A Published By Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1021-1030, 2019.
Topics: Animals; Apatites; Biomimetic Materials; Calcification, Physiologic; Cell Line; Embryo, Mammalian; Extracellular Matrix; Female; Mice, Inbred ICR; Nanoparticles; Skull
PubMed: 30675987
DOI: 10.1002/jbm.a.36618 -
Bone Nov 2019Osteocalcin is one of the most abundant noncollagenous proteins in bone. Phenotypes of osteocalcin knock-out mice (OC-/-) may vary on different backgrounds and with sex....
Osteocalcin is one of the most abundant noncollagenous proteins in bone. Phenotypes of osteocalcin knock-out mice (OC-/-) may vary on different backgrounds and with sex. Previous studies using adult female (OC-/-) mice on a mixed genetic background (129/B6) showed osteocalcin inhibited bone formation leading to weaker bone in wild-type (OC+/+). Yet on a pure (B6) genetic background male mice revealed osteocalcin improved fracture resistance and OC-/- bones were more prone to fracture. Osteocalcin is decreased with age and in some diseases (diabetes) where bone weakness is observed. The effect of osteocalcin in adult female bone from mice on a pure B6 background is unknown. We investigated differences in bone mineral properties and bone strength in female adult (6 months) (OC+/+) and (OC-/-) mice on a pure C57BL/6J background using Fourier Transform Infrared Imaging (FTIRI), micro-computed tomography (uCT), biomechanical measurements, histomorphometry and serum turnover markers (P1NP, CTX). Similar to female age matched mice on the (129/C57) background we found B6 OC-/- mice had a higher bone formation rate, no change in bone resorption, more immature mineral, decreased crystallinity and increased trabecular bone as compared to OC+/+. In contrast, the OC-/- mice on a pure B6 background had a lower bone mineral density, lower mineral to matrix ratio resulting in reduced stiffness and weaker bone strength. Our results demonstrate some properties of the OC-/- phenotype are dependent on genetic background. This may suggest that reduced osteocalcin may contribute to fracture and weaker bone in some groups of elderly and adults with diseases where osteocalcin is low.
Topics: Animals; Bone Density; Calcification, Physiologic; Female; Mice, Inbred C57BL; Mice, Knockout; Osteocalcin; Spectroscopy, Fourier Transform Infrared; Stress, Mechanical; X-Ray Microtomography
PubMed: 31401301
DOI: 10.1016/j.bone.2019.08.004 -
Postepy Biochemii 2016Vascular calcification accompanies the pathological process of atherosclerotic plaque formation. Artery calcification results from trans-differentiation of vascular... (Review)
Review
Vascular calcification accompanies the pathological process of atherosclerotic plaque formation. Artery calcification results from trans-differentiation of vascular smooth muscle cells (VSMCs) into cells resembling mineralization-competent cells such as osteoblasts and chondrocytes. The activity of tissue-nonspecific alkaline phosphatase (TNAP), a GPI-anchored enzyme necessary for physiological mineralization, is induced in VSMCs in response to inflammation. TNAP achieves its mineralizing function being anchored to plasma membrane of mineralizing cells and to the surface of their derived matrix vesicles (MVs), and numerous important reports indicate that membranes play a crucial role in initiating the crystal formation. In this review, we would like to highlight various functions of lipids and proteins associated to membranes at different stages of both physiological mineralization and vascular calcification, with an emphasis on the pathological process of atherosclerotic plaque formation.
Topics: Animals; Calcification, Physiologic; Calcinosis; Chondrocytes; Humans; Membrane Lipids; Osteoblasts; Plaque, Atherosclerotic
PubMed: 28132453
DOI: No ID Found -
Journal of Bone and Mineral Metabolism May 2020The composite nature of bone as a material governs its structure and mechanical behavior. How the collagenous matrix mineralizes, in terms of both mineral deposition and...
INTRODUCTION
The composite nature of bone as a material governs its structure and mechanical behavior. How the collagenous matrix mineralizes, in terms of both mineral deposition and structure of the mineral crystals, is highly interesting when trying to elucidate the complex structural changes that occur during bone growth and maturation. We have previously looked at mineral deposition and structural evolution of the collagenous matrix, linking both to changes in mechanics. The purpose of this study was to provide specific information on changes in crystal size and organization as a function of growth and maturation.
MATERIALS AND METHODS
Using micro-computed tomography (µCT) and micro-focused scanning small-angle X-ray scattering (SAXS) we investigated cortical bone in two orthogonal directions relative to the long axis of the humeri of New Zealand White rabbits spanning from new-born to 6-months of age. We also investigated the changes with tissue age by looking at radial profiles of osteonal structures in the 6-months old rabbits. The findings were compared to our previous compositional, structural and mechanical data on the same sample cohort.
RESULTS
µCT showed a continuous mineral deposition up until 3-months of age, whilst the SAXS data showed an increase in both crystal thickness and degree of orientation up until 6-months of age. The osteonal profiles showed no statistically significant changes in crystal thickness.
CONCLUSIONS
Comparison to previously collected mechanical data suggests that changes are not only explained by amount of mineral in the tissue but also by the crystal dimensions.
Topics: Aging; Animals; Bone Density; Calcification, Physiologic; Cortical Bone; Female; Imaging, Three-Dimensional; Rabbits; Scattering, Small Angle; X-Ray Microtomography
PubMed: 31807903
DOI: 10.1007/s00774-019-01068-y