Effect of Simulated Dental Pulpal Pressure Using Fetal Bovine Serum for the Bonding Performance of Contemporary Adhesive to Dentin.

This study evaluated the effect of simulated pulpal pressure (SPP) conditions and storage time on contemporary adhesive systems' microtensile bond strength (µTBS) to dentin. Extracted human molars were prepared and randomly divided into four groups according to the adhesives: Clearfil Megabond 2 (CSE), Beautibond Xtreme Universal (BXU), G2-Bond (G2B), and Scotchbond Universal Plus (SBP). Each adhesive group was further divided following the SPP conditions: control with no simulation (SPP-CTR), SPP with distilled water (SPP-DTW), and SPP with fetal bovine serum (SPP-FBS). Resin composite build-ups were prepared, and teeth were stored in water (37 °C) for 24 h (24 h) and 3 months (3 m). Then, teeth were sectioned to obtain resin-dentin bonded beams and tested to determine the µTBS. Data were analyzed using three-way ANOVA, Tukey post hoc tests (=0.05), and Weibull failure analysis. Failure mode was observed using scanning electron microscopy. The µTBS response was affected by adhesive systems, simulated pulpal pressure conditions, and storage time. SPP-CTR groups presented a higher overall bond strength than SPP-DTW and SPP-FBS, which were not significantly different from each other. Only for SBP, the SPP-FBS group showed higher µTBS than the SPP-DTW group. The Weibull analysis showed that the bonding reliability and durability under SPP-DTW and SPP-FBS were inferior to SPP-CTR, and the 24 h bonding quality of adhesives to dentin was superior to that of 3 m. SPP drastically reduced the µTBS of all adhesives to dentin regardless of solution (distilled water or fetal bovine serum). Storage after 3 m also decreased µTBS despite the SPP condition.

Green synthesis of chitosan- and fluoride-functionalized silver nanoparticles using Camellia sinensis: Characterization and dental applications.

The development of new biocompatible and eco-friendly materials is essential for the future of dental practice, especially for the management of dental caries. In this study, a novel and simple method was applied for the green synthesis of silver nanoparticles (AgNPs) from the aqueous extract of Camellia sinensis (WT) and functionalized with chitosan (CHS) and NaF. The effects of WT_AgNPs application on demineralized dentin were evaluated for potential dental applications. The WT_AgNPs showed molecular groups related to organic compounds, potentially acting as reducing and capping agents. All AgNPs presented spherical shapes with crystal sizes of approximately 20 nm. Forty human molars were assigned to control: sound (SD) and demineralised dentine (DD), and experimental groups: WT_AgNPs, WT_AgNPs_NaF, and WT_AgNPs_CHS. Then, the NPs were applied to DD to evaluate the chemical, crystallographic, and microstructural characteristics of treated-dentine. In addition, a three-point bending test was employed to assess mechanical response. The application of WT_AgNPs indicated a higher mineralisation degree and crystallites sizes of hydroxyapatite than the DD group. SEM images showed that WT_AgNPs presented different degrees of aggregation and distribution patterns. The dentine flexural strength was significantly increased in all WT_AgNPs. The application of WT_AgNPs demonstrated remineralising and strengthening potential on demineralised dentine.

Hydrothermal Transformation of Eggshell Calcium Carbonate into Apatite Micro-Nanoparticles: Cytocompatibility and Osteoinductive Properties.

The eggshell is a biomineral consisting of CaCO3 in the form of calcite phase and a pervading organic matrix (1-3.5 wt.%). Transforming eggshell calcite particles into calcium phosphate (apatite) micro-nanoparticles opens the door to repurposing the eggshell waste as materials with potential biomedical applications, fulfilling the principles of the circular economy. Previous methods to obtain these particles consisted mainly of two steps, the first one involving the calcination of the eggshell. In this research, direct transformation by a one-pot hydrothermal method ranging from 100-200 °C was studied, using suspensions with a stoichiometric P/CaCO3 ratio, K2HPO4 as P reagent, and eggshells particles (Ø < 50 µm) both untreated and treated with NaClO to remove surface organic matter. In the untreated group, the complete conversion was achieved at 160 °C, and most particles displayed a hexagonal plate morphology, eventually with a central hole. In the treated group, this replacement occurred at 180 °C, yielding granular (spherulitic) apatite nanoparticles. The eggshell particles and apatite micro-nanoparticles were cytocompatible when incubated with MG-63 human osteosarcoma cells and m17.ASC murine mesenchymal stem cells and promoted the osteogenic differentiation of m17.ASC cells. The study results are useful for designing and fabricating biocompatible microstructured materials with osteoinductive properties for applications in bone tissue engineering and dentistry.

Apatite-coated outer layer eggshell membrane: A novel osteoinductive biohybrid composite for guided bone/tissue regeneration.

Hybrid biomimetic materials aim to replicate the organic-inorganic constructs of mineralized tissues. During eggshell formation, the outer surface of the eggshell membrane (ESM) promotes calcium carbonate nucleation, while the inner one prevents mineralization toward the egg white and yolk. In the current study, the outer surface of the ESM acted as a heteronucleant in calcium phosphate precipitation by the vapor diffusion sitting drop method, while the inner one remained unmineralized. The aim was to fabricate a 2D biomaterial with dual functions, osteoinductive on one side and protective against cell invasion on the other side. The microstructural, physicochemical, morphological, and mechanical properties of the mineralized ESM were characterized by XRD, TGA, XPS, FTIR/Raman, HR-SEM, and mechanical testing techniques. The cytocompatibility and osteoinductive ability were assessed by biological assays of cell viability, proliferation, and osteogenic differentiation on human mesenchymal stromal cells (hMSCs). Results indicate that the outer surface of the ESM induces the heterogeneous precipitation of carbonate-apatite phase depicting biomimetic features. In addition, the apatite/ESM shows a much higher cytocompatibility than the pristine ESM and promotes the osteogenic differentiation of hMSCs more efficiently. Overall, the apatite/ESM composite exhibits compositional, crystalline, mechanical, and biological properties that resemble those of mineralized tissues, rendering it an approachable and novel material especially useful in guided tissue/bone regeneration.

Analysis of topography, flexural strength, and microstructure of a lithium-disilicate glass-ceramic after surface finishing.

Glass-ceramic restorations often require adjustments using coarse diamond burs (D) which have the potential to introduce critical defects at the surface from which a crack can initiate and propagate until fracture. Surface finishing using fine and ultra-fine burs (F), polishing (P), or glazing (G) have the potential to reduce or eliminate these defects. PURPOSE: This study investigated the influence of finishing, polishing, and glazing on the roughness, flexural strength, and microstructure of a lithium disilicate glass-ceramic (LD). MATERIALS AND METHODS: LD CAD-CAM blocks were cut into 96 discs (1.2 mm x 12 mm). After crystallization, a layer of glaze was applied to the discs surface. The specimens were randomly divided into eight groups (n=12) for different surface treatments: G- glaze (control), GD, GDG, GDP, GF GFG, GFP, and GDFP. Mean roughness (Ra), mean square height (Rq) and maximum roughness (Rz) were measured. The biaxial flexural strength test was performed in a universal testing machine at 0.5 mm/min. Microstructural analysis was performed using X-ray diffraction patterns (XRD). Differences on the roughness obtained in distinct groups was analyzed by Kruskal-Wallis and Bonferroni tests (α = 0.05). The flexural strength tests were compared by one-way ANOVA. RESULTS: Glazed groups (G, GDG and GFG) presented the lowest surface roughness, lower crystallinity, and higher flexural strength than the other groups. Although polishing and glaze presented similar surface roughness, polishing protocol decreased the flexural strength compared to the control group G. CONCLUSION: clinically adjusted LD glass-ceramic restorations should be reglazed whenever possible to improve strength.

Evaluation of the remineralizing capacity of silver diamine fluoride on demineralized dentin under pH-cycling conditions.

OBJECTIVE: (1) to determine the effects of the silver diamine fluoride (SDF) and sodium fluoride (NaF) in demineralized dentin exposed to an acid challenge by pH-cycling, (2) to evaluate the remineralizing capacity of SDF/NaF products based on the physicochemical and mechanical properties of the treated dentin surfaces. METHODOLOGY: In total, 57 human molars were evaluated in different stages of the experimental period: sound dentin - negative control (Stage 1), demineralized dentin - positive control (Stage 2), and dentin treated with SDF/NaF products + pH-c (Stage 3). Several commercial products were used for the SDF treatment: Saforide, RivaStar, and Cariestop. The mineral composition and crystalline and morphological characteristics of the dentin samples from each experimental stage were evaluated by infrared spectroscopy (ATR-FTIR), X-ray diffraction, and electron microscopy (SEM-EDX) analytical techniques. Moreover, the mechanical response of the samples was analyzed by means of the three-point bending test. Statistics were estimated for ATR-FTIR variables by Wilcoxon test, while the mechanical data analyses were performed using Kruskal-Wallis and Mann Whitney U tests. RESULTS: Regarding the chemical composition, we observed a higher mineral/organic content in the SDF/NaF treated dentin + pH-c groups (Stage 3) than in the positive control groups (Saforide p=0.03; Cariestop p=0.008; RivaStar p=0.013; NaF p=0.04). The XRD results showed that the crystallite size of hydroxyapatite increased in the SDF/NaF treated dentin + pH-c groups (between +63% in RivaStar to +108% in Saforide), regarding the positive control. SEM images showed that after application of the SDF/NaF products a crystalline precipitate formed on the dentin surface and partially filled the dentin tubules. The flexural strength (MPa) values were higher in the dentin treated with SDF/NaF + pH-c (Stage 3) compared to the positive control groups (Saforide p=0.002; Cariestop p=0.04; RivaStar p=0.04; NaF p=0.02). CONCLUSIONS: The application of SDF/NaF affected the physicochemical and mechanical properties of demineralized dentin. According to the results, the use of SFD/NaF had a remineralizing effect on the dentin surface even under acid challenge.

Eggshell Membrane as a Biomaterial for Bone Regeneration.

The physicochemical features of the avian eggshell membrane play an essential role in the process of calcium carbonate deposition during shell mineralization, giving rise to a porous mineralized tissue with remarkable mechanical properties and biological functions. The membrane could be useful by itself or as a bi-dimensional scaffold to build future bone-regenerative materials. This review focuses on the biological, physical, and mechanical properties of the eggshell membrane that could be useful for that purpose. Due to its low cost and wide availability as a waste byproduct of the egg processing industry, repurposing the eggshell membrane for bone bio-material manufacturing fulfills the principles of a circular economy. In addition, eggshell membrane particles have has the potential to be used as bio-ink for 3D printing of tailored implantable scaffolds. Herein, a literature review was conducted to ascertain the degree to which the properties of the eggshell membrane satisfy the requirements for the development of bone scaffolds. In principle, it is biocompatible and non-cytotoxic, and induces proliferation and differentiation of different cell types. Moreover, when implanted in animal models, it elicits a mild inflammatory response and displays characteristics of stability and biodegradability. Furthermore, the eggshell membrane possesses a mechanical viscoelastic behavior comparable to other collagen-based systems. Overall, the biological, physical, and mechanical features of the eggshell membrane, which can be further tuned and improved, make this natural polymer suitable as a basic component for developing new bone graft materials.

Evaluation of the remineralizing capacity of silver diamine fluoride on demineralized dentin under pH-cycling conditions

Abstract Objective (1) to determine the effects of the silver diamine fluoride (SDF) and sodium fluoride (NaF) in demineralized dentin exposed to an acid challenge by pH-cycling, (2) to evaluate the remineralizing capacity of SDF/NaF products based on the physicochemical and mechanical properties of the treated dentin surfaces. Methodology In total, 57 human molars were evaluated in different stages of the experimental period: sound dentin - negative control (Stage 1), demineralized dentin - positive control (Stage 2), and dentin treated with SDF/NaF products + pH-c (Stage 3). Several commercial products were used for the SDF treatment: Saforide, RivaStar, and Cariestop. The mineral composition and crystalline and morphological characteristics of the dentin samples from each experimental stage were evaluated by infrared spectroscopy (ATR-FTIR), X-ray diffraction, and electron microscopy (SEM-EDX) analytical techniques. Moreover, the mechanical response of the samples was analyzed by means of the three-point bending test. Statistics were estimated for ATR-FTIR variables by Wilcoxon test, while the mechanical data analyses were performed using Kruskal-Wallis and Mann Whitney U tests. Results Regarding the chemical composition, we observed a higher mineral/organic content in the SDF/NaF treated dentin + pH-c groups (Stage 3) than in the positive control groups (Saforide p=0.03; Cariestop p=0.008; RivaStar p=0.013; NaF p=0.04). The XRD results showed that the crystallite size of hydroxyapatite increased in the SDF/NaF treated dentin + pH-c groups (between +63% in RivaStar to +108% in Saforide), regarding the positive control. SEM images showed that after application of the SDF/NaF products a crystalline precipitate formed on the dentin surface and partially filled the dentin tubules. The flexural strength (MPa) values were higher in the dentin treated with SDF/NaF + pH-c (Stage 3) compared to the positive control groups (Saforide p=0.002; Cariestop p=0.04; RivaStar p=0.04; NaF p=0.02). Conclusions The application of SDF/NaF affected the physicochemical and mechanical properties of demineralized dentin. According to the results, the use of SFD/NaF had a remineralizing effect on the dentin surface even under acid challenge.

Physicochemical and Mechanical Effects of Commercial Silver Diamine Fluoride (SDF) Agents on Demineralized Dentin.

PURPOSE: To investigate the effects of four commercial silver diamine fluoride (SDF) agents on the chemical composition and microstructural properties of dentin, and its relation to the bond strength of two adhesives. MATERIALS AND METHODS: Ninety human molars were randomly divided into sound dentin (negative control), demineralized dentin (positive control), and four experimental groups (n = 15) according to the SDF treatments (Cariestop [Biodinamica Quimica y Farmaceutica], RivaStar 1 [SDI], RivaStar 2 [SDI], and Saforide [Tokyo Seiyaku Kasei]). ATR-FTIR, x-ray diffraction, and SEM techniques were employed to characterize the compositional, crystalline, and microstructural properties of the samples. The microtensile bond strength test evaluated the bonding performance of two adhesives in demineralized dentin treated with SDF agents. RESULTS: Regarding the chemical composition, all SDF-treated groups showed a significantly higher phosphate:organic matrix ratio than the demineralized dentin group (p < 0.05). The XRD analyses revealed that the crystallite size for hydroxyapatite crystals increased on the surface areas (deep, medium, and superficial dentin) for all experimental groups compared to demineralized dentin (p < 0.05). SEM images showed that the behavior of the agents used differs on each surface treated. Bond strength values were adversely affected with both adhesive systems in the four experimental groups (p < 0.05). CONCLUSIONS: The application of SDF agents resulted in the formation of different crystalline phases of silver salts and the increase of mineralization of the pretreated demineralized dentin. However, SDF application showed a negative effect on the bond strength of the adhesives.

Proanthocyanidin-functionalized hydroxyapatite nanoparticles as dentin biomodifier.

OBJECTIVE: This study evaluated the potential combined effects of nanohydroxyapatite and proanthocyanidin on the remineralization and collagen stabilization of demineralized dentin. METHODS: Seventy-five coronal dentin beams (6 × 1 × 1 mm3) were randomly allocated into five experimental groups (n = 15): Sound (no treatment), Control (pH-cycling), nHAp (nanohydroxyapatite), nHAp_PA (Proanthocyanidin-functionalized nanohydroxyapatite), and PA (proanthocyanidin) treatments. The sound group (negative control) were immersed in distilled water over the experimental period. The remaining groups were submitted to a pH-cycling process for 14 days. Following the de-re mineralization process, specimens corresponding to the control group (positive control) were immersed in distilled water whereas the test groups were immersed in 1 mL of respective solution treatment (nHAp, nHAp_PA, or PA) for 1 min. The dentin samples were analyzed to determine their chemical composition (ATR-FTIR and Thermogravimetric) and mineralogical (XRD) characteristics as well as their mechanical response, obtained by three-point bending test. RESULTS: Higher phosphate content (v4 PO4: ATR-FTIR) and amount of mineral (XRD) was observed in the nHAp_PA group. Furthermore, a larger induction of collagen cross-links (ATR-FTIR) and %Organic Matter (TGA) would indicate the PA incorporation and the achievement of dentin matrix stability. These effects on dentin properties were related to increasing flexural strength (MPa), demonstrating that 15% w/v nHAp_PA treatment improved the mechanical properties of the samples. SIGNIFICANCE: nHAp_PA shows significant potential for promoting remineralization while improving collagen stability into demineralized dentin in a clinically feasible period of 1 min.

Citrate Stabilizes Hydroxylapatite Precursors: Implications for Bone Mineralization.

Mineralization of hydroxylapatite (HAp), the main inorganic phase in bone, follows nonclassical crystallization routes involving metastable precursors and is strongly influenced by organic macromolecules. However, the effect of small organic molecules such as citrate on the formation of HAp is not well constrained. Using potentiometric titration experiments and titration calorimetry, in combination with a multianalytical approach, we show that citrate stabilizes prenucleation species as well as a liquid-like calcium phosphate precursor formed before any solid phase nucleates in the system. The stabilization of a liquid-like precursor phase could facilitate infiltration into the cavities of the collagen fibrils during bone mineralization, explaining the enhancement of collagen-mediated mineralization by citrate reported in previous studies. Hence, citrate can influence bone mineralization way before any solid phase (amorphous or crystalline) is formed. We also show that HAp formation after amorphous calcium phosphate (ACP) in the absence and presence of citrate results in nanoplates of about 5-12 nm thick, elongated along the c axis. Such nanoplates are made up of HAp nanocrystallites with a preferred c axis orientation and with interspersed ACP. The nanoplatelet morphology, size, and preferred crystallographic orientation, remarkably similar to those of bone HAp nanocrystals, appear to be an intrinsic feature of HAp formed from an amorphous precursor. Our results challenge current models for HAp mineralization in bone and the role of citrate, offering new clues to help answer the long-standing question as to why natural evolution favored HAp as the mineral phase in bone.

Characterization of an Experimental Two-Step Self-Etch Adhesive's Bonding Performance and Resin-Dentin Interfacial Properties.

This study evaluated an experimental two-step self-etch adhesive (BZF-29, BZF) by comparing it with a reference two-step self-etch adhesive (Clearfil Megabond 2, MB) and a universal adhesive (G-Premio Bond, GP) for microtensile bond strength (µTBS) and resin-dentin interfacial characteristics. Twenty-four human third molars were used for the µTBS test. Bonded peripheral dentin slices were separated to observe the resin-dentin interface and measure the adhesive layer thickness with SEM. µTBS data of the central beams were obtained after 24 h and 6 months of water storage. Fracture modes were determined using a stereomicroscope and SEM. Nine additional third molars were used to determine the elastic modulus (E) employing an ultra microhardness tester. Water storage did not affect µTBS of the tested adhesives (p > 0.05). µTBS of BZF and MB were similar but significantly higher than GP (p < 0.05). BZF achieved the highest adhesive layer thickness, while GP the lowest. E of BZF and MB were comparable but significantly lower than GP (p < 0.05). Except for GP, the predominant fracture mode was nonadhesive. The superior bonding performance of BZF and MB could be attributed to their better mechanical property and increased adhesive thickness imparting better stress relief at the interface.

Influence of de-remineralization process on chemical, microstructural, and mechanical properties of human and bovine dentin.

OBJECTIVES: This study compared the chemical composition, microstructural, and mechanical properties of human and bovine dentin subjected to a demineralization/remineralization process. MATERIALS AND METHODS: Human and bovine incisors were sectioned to obtain 120 coronal dentin beams (6 × 1 × 1 mm3) that were randomly allocated into 4 subgroups (n = 15) according to the time of treatment (sound, pH-cycling for 3, 7, and 14 days). Three-point bending mechanical test, attenuated total reflectance-Fourier transform infrared (ATR-FTIR), thermogravimetric (TG), and X-ray diffraction (XRD) techniques were employed to characterize the dentin samples. RESULTS: Regarding chemical composition at the molecular level, bovine sound dentin showed significantly lower values in organic and inorganic content (collagen cross-linking, CO3/amide I, and CO3/PO4; p = 0.002, p = 0.026, and p = 0.002, respectively) compared to humans. Employing XRD analyses, a higher mineral crystallinity in human dentin than in bovines at 7 and 14 days (p = 0.003 and p = 0.009, respectively) was observed. At the end of the pH-cycling, CI (ATR-FTIR) and CO3/PO4 ratios (ATR-FTIR) increased, while CO3/amide I (ATR-FTIR), PO4/amide I (ATR-FTIR), and %mineral (TG) ratios decreased. The extension by compression values increased over exposure time with significant differences between dentin types (p < 0.001, in all cases), reaching higher values in bovine dentin. However, flexural strength (MPa) did not show differences between groups. We also observed the correlation between compositional variables (i.e., PO4/amide I, CI, and %mineral) and the extension by compression. CONCLUSIONS: Human and bovine dentin are different in terms of microstructure, chemical composition, mechanical strength, and in their response to the demineralization/remineralization process by pH-cycling. CLINICAL RELEVANCE: These dissimilarities may constitute a potential limitation when replacing human teeth with bovines in in vitro studies.

Multi-Scale Approach for the Evaluation of Bone Mineralization in Strontium Ranelate-Treated Diabetic Rats.

Long-term diabetes mellitus can induce osteopenia and osteoporosis, an increase in the incidence of low-stress fractures, and/or delayed fracture healing. Strontium ranelate (SrR) is a dual-action anti-osteoporotic agent whose use in individuals with diabetic osteopathy has not been adequately evaluated. In this study, we studied the effects of an oral treatment with SrR and/or experimental diabetes on bone composition and biomechanics. Young male Wistar rats (half non-diabetic, half with streptozotocin/nicotinamide-induced diabetes) were either untreated or orally administered 625 mg/kg/day of SrR for 6 weeks. After sacrifice, femora from all animals were evaluated by a multi-scale approach (X-ray diffraction, Fourier transform infrared spectroscopy, inductively coupled plasma optical-emission spectrometry, static histomorphometry, pQCT, and mechanical testing) to determine chemical, crystalline, and biomechanical properties. Untreated diabetic animals (versus untreated non-diabetic) showed a decrease in femoral mineral carbonate content, in cortical thickness and BMC, in trabecular osteocyte density, in maximum load supported at rupture and at yield point, and in overall toughness at mid-shaft. Treatment of diabetic animals with SrR further affected several parameters of bone (some already impaired by diabetes): crystallinity index (indicating less mature apatite crystals); trabecular area, BMC, and vBMD; maximum load at yield point; and structural elastic rigidity. However, SrR was also able to prevent the diabetes-induced decreases in trabecular osteocyte density (completely) and in bone ultimate strength at rupture (partially). Our results indicate that SrR treatment can partially but significantly prevent some bone structural mechanical properties as previously affected by diabetes, but not others (which may even be worsened).

Effects of chronic lead exposure on bone mineral properties in femurs of growing rats.

Lead exposure has been associated with several defective skeletal growth processes and bone mineral alterations. The aim of the present study is to make a more detailed description of the toxic effects of lead intoxication on bone intrinsic material properties as mineral composition, morphology and microstructural characteristics. For this purpose, Wistar rats were exposed (n=12) to 1000ppm lead acetate in drinking water for 90days while control group (n=8) were treated with sodium acetate. Femurs were examined using inductively coupled plasma optical emission spectrometry (ICP-OES), Attenuated Total Reflection Fourier transform infrared spectroscopy (ATR-FTIR), X-ray diffraction (XRD), and micro-Computed Tomography (µCT). Results showed that femur from the lead-exposed rats had higher carbonate content in bone mineral and (Ca2++Mg2++ Na+)/P ratio values, although no variations were observed in crystal maturity and crystallite size. From morphological analyses, lead exposure rats showed a decreased in trabecular bone surface and distribution while trabecular thickness and cortical area increased. These overall effects indicate a similar mechanism of bone maturation normally associated to age-related processes. These responses are correlated with the adverse actions induced by lead on the processes regulating bone turnover mechanism. This information may explain the osteoporosis diseases associated to lead intoxication as well as the risk of fracture observed in populations exposed to this toxicant.

Effect of 30 % hydrogen peroxide on mineral chemical composition and surface morphology of bovine enamel.

A combination of atomic absorption spectroscopy (AAS), Fourier transform infrared spectroscopy (FTIR), scanning electronic microscopy (SEM), and gas adsorption techniques was used to characterize the effect of 30 % hydrogen peroxide (HP) on enamel surface. To perform the analyses of AAS, 1 ml of 30 % HP was added to 30 mg of a bovine enamel powder sample (150-200 µm fractions) for times of 5, 20, 60, 90, and 120 min; then 5 ml of the solution was withdrawn after each time period to measure [Ca(2+)] ions. The remaining powder was recovered and analyzed by FTIR. For SEM and gas adsorption tests, 4 × 4 mm(2) enamel sectioned samples were polished and 30 % HP was applied on the surface for the same time periods. AAS data show that 30 % HP treatment mobilized calcium from the enamel at all times studied. FTIR spectra showed that the total amount of phosphate and carbonate mineral contents such as amide I decreased significantly. SEM revealed that randomly distributed areas throughout the smooth enamel surface treatment became rougher and more irregular. These alterations indicate that surface damage increases with increasing durations of HP treatment. Gas adsorption analysis proved that bleached enamel is a typically non-porous material with a small specific surface area which decreases slightly with the 30 % HP treatment. In sum, 30 % HP induced a significant alteration of the organic and mineral part of the enamel, leading to the release of calcium and a rougher, more irregular enamel surface on randomly distributed areas.

Effects of lead shot ingestion on bone mineralization in a population of red-legged partridge (Alectoris rufa).

The effect of lead (Pb) toxicity on bone mineralization was investigated in a wild population of red-legged partridge (Alectoris rufa) inhabiting a farmland area contaminated with Pb-shot from recreational hunting activities in Albacete, a southeastern province of Spain. Femora from 40 specimens of red-legged partridge were analyzed for Pb by graphite furnace atomic absorption spectroscopy (GF-AAS), and for bone composition by Fourier transform infrared (FTIR) spectroscopy and X-ray diffraction (XRD). The FTIR and DRX data of bone were analyzed in detail to determine possible alterations in bone mineral chemistry and crystallinity due to Pb toxicity. Results showed a marked decrease in the degree of mineralization as Pb concentrations in bone tissue increased while XRD analyses showed that the crystallinity of apatite crystals increased with the Pb load in bone. These load-dependent effects are indicative that Pb contamination altered bone remodeling by reducing new bone mineral formation and demonstrate that bone quality is a sensitive indicator of adverse effects on wild bird populations exposed to Pb pollution.

Chronic effects of lead (Pb) on bone properties in red deer and wild boar: relationship with vitamins A and D3.

Here we study the occurrence of abnormalities on bone tissue composition and turnover mechanisms through the Pb-mediated disruption of vitamins A and D in wild ungulates living in a lead (Pb)-polluted mining area. Red deer (Cervus elaphus) and wild boar (Sus scrofa) from the mining area had significantly higher liver and bone Pb levels than controls, which were associated with the depletion of liver retinyl esters and the corresponding increase of free retinol levels both in deer and boar from the mining area. Pb-exposed adult deer had lower carbonate content in bone mineral than controls, which was associated with the increased free retinol percentage. In wild boar, the degree of bone mineralization was also positively associated with higher burdens of retinyl esters. These results suggest that Pb-associated changes in bone composition and mineralization is likely influenced by the depletion of vitamin A in wildlife exposed to environmental Pb pollution.

Effects of 3,3',4,4',5-pentachlorobiphenyl (PCB126) on vertebral bone mineralization and on thyroxin and vitamin D levels in Sprague-Dawley rats.

The aim of the present study is to use Fourier transform infrared spectrometry (FTIR), and transmission electron microscopy (TEM) techniques, to make a more detailed description of toxic effects of 3,3',4,4',5-pentachlorobiphenyl (PCB126) on bone tissue at the microstructural and at the molecular level as a result of an altered bone metabolism. We have analysed potential changes on vitamin D and thyroxin serum levels since these hormones represent endocrine endpoints that are critical for bone growth and development. For this purpose Sprague-Dawley rats were exposed (n=10) to PCB126 (i.p.) for 3 months (total dose, 384microg/kg bodyweight), while control rats (n=10) were injected with corn oil (vehicle). Results from FTIR showed that vertebrae from the exposed rats had an overall lower degree of mineralization (-8.5%; p<0.05) compared with the controls. In addition, results from peripheral quantitative computed tomography (pQCT) analyses showed significant increases in the trabecular bone mineral density (+12%; p<0.05) in the exposed group compared with the controls. The TEM analyses also showed an alteration in the crystallinity properties of vertebral bone mineral with a significant decrease in the size and crystallinity of apatite crystal forming the bone tissue in the exposed vs. non-exposed rats. Serum analysis revealed lower levels of thyroid hormones, FT4 (-42%; p<0.005), TT4 (-26%; p<0.005), and vitamin D (-21%; p<0.005) in exposed group compared to control animals. The complementary techniques (TEM and FTIR) used in this study have revealed insights into possible bone mineralization alteration due to PCB126 exposure. The lowering of both the thyroxin and vitamin D serum levels might be an underlying explanation for the observed effects on bone mineralization.

Short-term exposure to dioxin impairs bone tissue in male rats.

Chronic and sub-chronic studies in rats have previously shown that dioxin-like compounds impair the bone tissue homeostasis. In the present study, tibiae and serum were analyzed to study possible effects of short term dioxin exposure on rats. Two month old (ca. 200g) male rats were injected with 50microg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) kg(-1) bw and tibiae were excised 5d following the exposure. Bone composition, dimensions and strength were analyzed by pQCT and three-point bending test on tibiae. In addition, detailed bone composition was analyzed by optical emission spectroscopy (ICP-OES) and Fourier transform infrared spectrometry (FTIR). Analysis of the serum bone biomarkers procollagen type-I N-terminal propeptide (PINP) and carboxyterminal cross linking teleopeptide (CTX) were also performed. pQCT-results showed alterations in the metaphysis, with a significant decrease in trabecular bone cross-sectional area (-19%, p<0.05) and a significant increase in total bone mineral density (+7%, p<0.05) in TCDD-exposed rats. Analyses of the bones by ICP-OES and FTIR showed that bones from exposed rats had a higher relative proportion of crystalline phosphate (+13% for a1080 and +11% for a1113, p<0.05) and lower acid phosphate content (-22% for a1145, p<0.05), resembling the composition of more mature bones. Serum analysis showed that the bone formation marker PINP was decreased (-37%, p<0.05) and that the bone resorption marker CTX was increased (+14%, p<0.05) indicating a net loss of bone tissue. In conclusion, 5d of exposure to TCDD was sufficient to negatively affect bone tissue in male rats.