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.

Genetic markers associated with bone composition in Rhode Island Red laying hens.

BACKGROUND: Bone damage has welfare and economic impacts on modern commercial poultry and is known as one of the major challenges in the poultry industry. Bone damage is particularly common in laying hens and is probably due to the physiological link between bone and the egg laying process. Previous studies identified and validated quantitative trait loci (QTL) for bone strength in White Leghorn laying hens based on several measurements, including bone composition measurements on the cortex and medulla of the tibia bone. In a previous pedigree-based analysis, bone composition measurements showed heritabilities ranging from 0.18 to 0.41 and moderate to strong genetic correlations with tibia strength and density. Bone composition was measured using infrared spectroscopy and thermogravimetry. The aim of this study was to combine these bone composition measurements with genotyping data via a genome-wide association study (GWAS) to investigate genetic markers that contribute to genetic variance in bone composition in Rhode Island Red laying hens. In addition, we investigated the genetic correlations between bone composition and bone strength. RESULTS: We found novel genetic markers that are significantly associated with cortical lipid, cortical mineral scattering, medullary organic matter, and medullary mineralization. Composition of the bone organic matter showed more significant associations than bone mineral composition. We also found interesting overlaps between the GWAS results for tibia composition traits, particularly for cortical lipid and tibia strength. Bone composition measurements by infrared spectroscopy showed more significant associations than thermogravimetry measurements. Based on the results of infrared spectroscopy, cortical lipid showed the highest genetic correlations with tibia density, which was negative (- 0.20 ± 0.04), followed by cortical CO3/PO4 (0.18 ± 0.04). Based on the results of thermogravimetry, medullary organic matter% and mineral% showed the highest genetic correlations with tibia density (- 0.25 ± 0.04 and 0.25 ± 0.04, respectively). CONCLUSIONS: This study detected novel genetic associations for bone composition traits, particularly those involving organic matter, that could be used as a basis for further molecular genetic investigations. Tibia cortical lipids displayed the strongest genetic associations of all the composition measurements, including a significantly high genetic correlation with tibia density and strength. Our results also highlighted that cortical lipid may be a key measurement for further avian bone studies.

Nanoscale Analysis of the Structure and Composition of Biogenic Calcite Reveals the Biomineral Growth Pattern.

The vast majority of calcium carbonate biocrystals differ from inorganic crystals in that they display a patent nanoroughness consisting of lumps of crystalline material (calcite/aragonite) surrounded by amorphous pellicles. Scanning transmission electron microscopy coupled with electron energy loss spectroscopy (STEM-EELS) was used to map the calcite secreted by a barnacle chemically and structurally with ultrahigh resolution (down to 1 nm). The material is composed of irregular lumps of calcite (up to two hundred nm in diameter) surrounded by relatively continuous cortexes (up to 20 nm thick) of amorphous calcium carbonate (ACC) and/or nanocalcite plus biomolecules, with a surplus of calcium relative to carbonate. We develop a model by which the separation of the crystalline and amorphous phases takes place upon crystallization of the calcite from a precursor ACC. The organic biomolecules are expelled from the crystal lattice and concentrate in the form of pellicles, where they stabilize minor amounts of ACC/nanocalcite. In this way, we change the previously established conception of biomineral structure and growth.

Genetics of tibia bone properties of crossbred commercial laying hens in different housing systems.

Osteoporosis and bone fractures are a severe problem for the welfare of laying hens, with genetics and environment, such as housing system, each making substantial contributions to bone strength. In this work, we performed genetic analyses of bone strength, bone mineral density, and bone composition, as well as body weight, in 860 commercial crossbred laying hens from 2 different companies, kept in either furnished cages or floor pens. We compared bone traits between housing systems and crossbreds and performed a genome-wide association study of bone properties and body weight. As expected, the 2 housing systems produced a large difference in bone strength, with layers housed in floor pens having stronger bones. These differences were accompanied by differences in bone geometry, mineralization, and chemical composition. Genome scans either combining or independently analyzing the 2 housing systems revealed no genome-wide significant loci for bone breaking strength. We detected 3 loci for body weight that were shared between the housing systems on chromosomes 4, 6, and 27 (either genome-wide significant or suggestive) and these coincide with associations for bone length. In summary, we found substantial differences in bone strength, content, and composition between hens kept in floor pens and furnished cages that could be attributed to greater physical activity in pen housing. We found little evidence for large-effect loci for bone strength in commercial crossbred hens, consistent with a highly polygenic architecture for bone strength in the production environment. The lack of consistent genetic associations between housing systems in combination with the differences in bone phenotypes could be due to gene-by-environment interactions with housing system or a lack of power to detect shared associations for bone strength.

Antimicrobial defenses of table eggs: Importance of antibacterial proteins in egg white as a function of hen age in an extended production cycle.

The importance of egg natural defences to prevent bacterial contamination and their relation with hen age in extended production cycles were evaluated. Egg-white from eggs of different hen age groups (up 100-weeks-old) and lines (Hy-Line white and brown) were inoculated with Gram-positive Staphylococcus aureus or Gram-negative Salmonella Typhimurium, ranging from 103-106 CFU/mL. Our results show that concentrations of egg-white lysozyme and, particularly, ovotransferrin are important to modulate bacterial survival in a dose-dependent matter. Depending on protein concentration, their effect ranges from bactericidal to bacteriostatic, with a threshold for bacterial contamination that depends also on hen age and line. The concentrations of lysozyme and ovotransferrin increased with hen age (up to 2 and 22 w/w% of total protein, respectively), and eggs laid by older hens exhibited the greatest potential to prevent the growth of the highest Salmonella inoculum (106 CFU/mL). Salmonella-penetration experiments demonstrated that non-contaminated eggs display significantly higher concentrations of antimicrobial proteins. However, eggs from older hens needed a higher concentration of these proteins (>20% ovotransferrin) to prevent bacterial contamination, showing that antimicrobial protein concentrations in egg-whites was not the only factor influencing bacterial contamination. Finally, this study demonstrated that egg-white of eggs produced by old hens are less prone to contamination by Salmonella.

Crystallographic control of the fabrication of an extremely sophisticated shell surface microornament in the glass scallop Catillopecten.

The external surface microornament of the glass scallops Catillopecten natalyae and malyutinae is made by calcitic spiny projections consisting of a stem that later divides into three equally spaced and inclined branches (here called aerials). C. natalyae contains larger and smaller aerials, whereas C. malyutinae only secreted aerials of the second type. A remarkable feature is that aerials within each type are fairly similar in size and shape and highly co-oriented, thus constituting a most sophisticated microornament. We demonstrate that aerials are single crystals whose morphology is strongly controlled by the crystallography, with the stem being parallel to the c-axis of calcite, and the branches extending along the edges of the {104} calcite rhombohedron. They grow epitaxially onto the foliated prisms of the outer shell layer. The co-orientation of the prisms explains that of the aerials. We have developed a model in which every aerial grows within a periostracal pouch. When this pouch reaches the growth margin, the mantle initiates the production of the aerial. Nevertheless, later growth of the aerial is remote, i.e. far from the contact with the mantle. We show how such an extremely sophisticated microornament has a morphology and co-orientation which are determined by crystal growth.

Properties, Genetics and Innate Immune Function of the Cuticle in Egg-Laying Species.

Cleidoic eggs possess very efficient and orchestrated systems to protect the embryo from external microbes until hatch. The cuticle is a proteinaceous layer on the shell surface in many bird and some reptile species. An intact cuticle forms a pore plug to occlude respiratory pores and is an effective physical and chemical barrier against microbial penetration. The interior of the egg is assumed to be normally sterile, while the outer eggshell cuticle hosts microbes. The diversity of the eggshell microbiome is derived from both maternal microbiota and those of the nesting environment. The surface characteristics of the egg, outer moisture layer and the presence of antimicrobial molecules composing the cuticle dictate constituents of the microbial communities on the eggshell surface. The avian cuticle affects eggshell wettability, water vapor conductance and regulates ultraviolet reflectance in various ground-nesting species; moreover, its composition, thickness and degree of coverage are dependent on species, hen age, and physiological stressors. Studies in domestic avian species have demonstrated that changes in the cuticle affect the food safety of eggs with respect to the risk of contamination by bacterial pathogens such as Salmonella and Escherichia coli. Moreover, preventing contamination of internal egg components is crucial to optimize hatching success in bird species. In chickens there is moderate heritability (38%) of cuticle deposition with a potential for genetic improvement. However, much less is known about other bird or reptile cuticles. This review synthesizes current knowledge of eggshell cuticle and provides insight into its evolution in the clade reptilia. The origin, composition and regulation of the eggshell microbiome and the potential function of the cuticle as the first barrier of egg defense are discussed in detail. We evaluate how changes in the cuticle affect the food safety of table eggs and vertical transmission of pathogens in the production chain with respect to the risk of contamination. Thus, this review provides insight into the physiological and microbiological characteristics of eggshell cuticle in relation to its protective function (innate immunity) in egg-laying birds and reptiles.

Parasitism by metacercariae modulates the morphological, organic and mechanical responses of the shell of an intertidal bivalve to environmental drivers.

Environmental variation alters biological interactions and their ecological and evolutionary consequences. In coastal systems, trematode parasites affect their hosts by disrupting their life-history traits. However, the effects of parasitism could be variable and dependent on the prevailing environmental conditions where the host-parasite interaction occurs. This study compared the effect of a trematode parasite in the family Renicolidae (metacercariae) on the body size and the shell organic and mechanical characteristics of the intertidal mussels Perumytilus purpuratus, inhabiting two environmentally contrasting localities in northern and central Chile (ca. 1600 km apart). Congruent with the environmental gradient along the Chilean coast, higher levels of temperature, salinity and pCO2, and a lower pH characterise the northern locality compared to that of central Chile. In the north, parasitised individuals showed lower body size and shell resistance than non-parasitised individuals, while in central Chile, the opposite pattern was observed. Protein level in the organic matter of the shell was lower in the parasitised hosts than in the non-parasitised ones regardless of the locality. However, an increase in polysaccharide levels was observed in the parasitised individuals from central Chile. These results evidence that body size and shell properties of P. purpuratus vary between local populations and that they respond differently when confronting the parasitism impacts. Considering that the parasite prevalence reaches around 50% in both populations, if parasitism is not included in the analysis, the true response of the host species would be masked by the effects of the parasite, skewing our understanding of how environmental variables will affect marine species. Considering parasitism and identifying its effects on host species faced with environmental drivers is essential to understand and accurately predict the ecological consequences of climate change.

Bone quality and composition are influenced by egg production, layer line, and oestradiol-17ß in laying hens.

Keel bone fractures are a serious animal welfare problem in laying hens. The aim of the current study was to assess the influence of egg production, oestradiol-17ß, and selection for high laying performance on bone quality. Hens of two layer lines differing in laying performance (WLA: 320 eggs per year, G11: 200 eggs per year) were allocated to four treatment groups. Group S received a deslorelin acetate implant that suppressed egg production. Group E received an implant with the sexual steroid oestradiol-17ß. Group SE received both implants and group C did not receive any implant. In the 63rd week of age, composition and characteristics of the tibiotarsi were assessed using histological analysis, three-point bending test, thermogravimetric analysis, infrared spectroscopy, and two-dimensional X-ray diffraction, respectively. Non-egg-laying hens showed a higher total bone area and a higher relative amount of cortical bone compared to egg-laying hens. Hens of layer line G11 showed a higher relative amount of medullary bone and a higher degree of mineralization of the cortical bone compared to hens of layer line WLA. These differences in bone composition may explain different susceptibility to keel bone fractures in non-egg-laying compared to egg-laying hens as well as in hens of layer lines differing in laying performance. The effect of exogenous oestradiol-17ß on bone parameters varied between the layer lines indicating a genetic influence on bone physiology and the way it can be modulated by hormone substitution.

Plasticity in organic composition maintains biomechanical performance in shells of juvenile scallops exposed to altered temperature and pH conditions.

The exposure to environmental variations in pH and temperature has proven impacts on benthic ectotherms calcifiers, as evidenced by tradeoffs between physiological processes. However, how these stressors affect structure and functionality of mollusk shells has received less attention. Episodic events of upwelling of deep cold and low pH waters are well documented in eastern boundary systems and may be stressful to mollusks, impairing both physiological and biomechanical performance. These events are projected to become more intense, and extensive in time with ongoing global warming. In this study, we evaluate the independent and interactive effects of temperature and pH on the biomineral and biomechanical properties of Argopecten purpuratus scallop shells. Total organic matter in the shell mineral increased under reduced pH (~ 7.7) and control conditions (pH ~ 8.0). The periostracum layer coating the outer shell surface showed increased protein content under low pH conditions but decreasing sulfate and polysaccharides content. Reduced pH negatively impacts shell density and increases the disorder in the orientation of calcite crystals. At elevated temperatures (18 °C), shell microhardness increased. Other biomechanical properties were not affected by pH/temperature treatments. Thus, under a reduction of 0.3 pH units and low temperature, the response of A. purpuratus was a tradeoff among organic compounds (biopolymer plasticity), density, and crystal organization (mineral plasticity) to maintain shell biomechanical performance, while increased temperature ameliorated the impacts on shell hardness. Biopolymer plasticity was associated with ecophysiological performance, indicating that, under the influence of natural fluctuations in pH and temperature, energetic constraints might be critical in modulating the long-term sustainability of this compensatory mechanism.

Impact of Different Layer Housing Systems on Eggshell Cuticle Quality and Salmonella Adherence in Table Eggs.

The bacterial load on the eggshell surface is a key factor in predicting the bacterial penetration and contamination of the egg interior. The eggshell cuticle is the first line of defense against vertical penetration by microbial food-borne pathogens such as Salmonella Enteritidis. Egg producers are increasingly introducing alternative caging systems into their production chain as animal welfare concerns become of greater relevance to today's consumer. Stress that is introduced by hen aggression and modified nesting behavior in furnished cages can alter the physiology of egg formation and affect the cuticle deposition/quality. The goal of this study was to determine the impact of caging systems (conventional, enriched, free-run, and free-range), on eggshell cuticle parameters and the eggshell bacterial load. The cuticle plug thickness and pore length were higher in the free-range eggs as compared to conventional eggs. The eggshells from alternative caging (enriched and free-range) had a higher total cuticle as compared to conventional cages. A reduction in bacterial cell counts was observed on eggshells that were obtained from free-range eggs as compared to the enriched systems. An inverse correlation between the contact angle and Salmonella adherence was observed. These results indicate that the housing systems of layer hens can modify the cuticle quality and thereby impact bacterial adherence and food safety.

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.

The argonaut constructs its shell via physical self-organization and coordinated cell sensorial activity.

The shell of the cephalopod Argonauta consists of two layers of fibers that elongate perpendicular to the shell surfaces. Fibers have a high-Mg calcitic core sheathed by thin organic membranes (>100 nm) and configurate a polygonal network in cross section. Their evolution has been studied by serial sectioning with electron microscopy-associated techniques. During growth, fibers with small cross-sectional areas shrink, whereas those with large sections widen. It is proposed that fibers evolve as an emulsion between the fluid precursors of both the mineral and organic phases. When polygons reach big cross-sectional areas, they become subdivided by new membranes. To explain both the continuation of the pattern and the subdivision process, the living cells from the mineralizing tissue must perform contact recognition of the previously formed pattern and subsequent secretion at sub-micron scale. Accordingly, the fabrication of the argonaut shell proceeds by physical self-organization together with direct cellular activity.

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.

Research Note: Changes in eggshell quality and microstructure related to hen age during a production cycle.

We have studied in detail the changes that occur in eggshell structure and composition during a production cycle in order to better understand the deterioration of eggshell quality with hen age (at 33, 45, and 67 wk). To study changes in eggshell ultrastructure and microstructure characteristics (mammillary density, palisade layer thickness, size, and orientation of calcite crystals) and the cuticle composition, we used complementary analytical techniques such optical and electron microscopy, X-ray diffraction, and infrared spectrometry. The marked decrease in eggshell breaking strength from 5.8 Kg at 33 wk to 4.4 Kg at 67 wk (25% reduction) could not be solely explained by the modest reduction in eggshell thickness (6-10% reduction) and seems to be associate to abrupt changes in eggshell ultra- and microstructure characteristics (i.e., decreased mammillary density; increased size of crystal units), occurring in older hens. Particularly, the decrease in mammillary density reduces the attachment points of the eggshell mineral to the membranes and therefore should negatively impact eggshell mechanical properties. Also, the observed increase in the calcite crystal size making the shell could also reduce the cohesion of crystals and eggshell resistance against impacts. Additionally, there was a decrease in the amount of cuticle and internal egg quality parameters (egg albumen height) with hen age that could have a negative impact in egg safety and quality.

Relationship between Bone Quality, Egg Production and Eggshell Quality in Laying Hens at the End of an Extended Production Cycle (105 Weeks).

(1) Background: Nowadays the industry aims to improve lay persistency for extended cycles (100 weeks or longer) to make egg production more sustainable. However, intensive egg production challenges hen health, inducing severe osteoporosis and the incidence of bone fractures. In this study, the relationship between bone quality and egg production, and/or eggshell quality, was evaluated at the end of an extended laying cycle of 100 weeks, comparing groups of hens with different production and eggshell quality parameters; (2) Methods: Quality parameters of egg (as weight, egg white height), eggshell (as thickness, weight, breaking strength, elasticity and microstructure) and tibiae bone (weight, diameter, cortical thickness, ash weight, breaking strength, medullary bone) were determined; (3) Results: Hens from groups with a high egg production and good eggshell quality have poorer bone quality (lower ash weight and lesser amount of medullary bone). However, Pearson's correlation analysis shows no clear relationship between bone and egg/eggshell parameters. (4) Conclusions: Bone and egg production/eggshell quality are independent and can be improved separately. Medullary bone has an important contribution to bone mechanical properties, being important to accumulate enough bone medullary bone early in life to maintain skeletal integrity and eggshell quality in old hens.

No evidence that selection for egg production persistency causes loss of bone quality in laying hens.

BACKGROUND: The physiological adaptations that have evolved for egg laying make hens susceptible to bone fractures and keel bone damage. In modern laying hen breeds, longer periods of egg laying could result in a greater risk of poor bone quality, and selection for increased egg production has frequently been stated to be a cause. However, the existing literature does not support this hypothesis. To test the hypothesis that egg production is associated with quality, breaking strength and density of bone, genetic correlations between these traits were estimated in White Leghorn and Rhode Island Red breeds. Genetic correlations of cortical and medullary bone material chemical properties with bone quality were also estimated, in order to identify methods to improve bone quality with appropriately targeted measurement of key traits. RESULTS: Estimates of heritability for bone quality traits were moderate (0.19-0.59) for both White Leghorn and Rhode Island Red breeds, except for the keel bone trait, which had a heritability estimate equal to zero. There was no evidence for genetic or phenotypic relationships between post-peak egg production and bone quality. In the White Leghorn breed, the estimate of the genetic correlation between pre-peak production/age at first egg and bone quality was significant and negative (- 0.7 to - 0.4). Estimates of heritability of thermogravimetric measurements of tibial medullary bone mineralisation were significant (0.18-0.41), as were estimates of their genetic correlations with tibia breaking strength and density (0.6-0.9). CONCLUSIONS: The low genetic correlation of post-peak egg production with bone quality suggests that selection for increased persistency of egg production may not adversely affect bone quality. Onset of puberty and mineralisation of the medullary bone, which is a specialised adaptation for egg laying, were identified as important factors associated with the quality of the skeleton later during egg production. These are traits for which genetic, as well as environmental and management factors can positively impact the overall quality of the skeleton of laying hens.

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.

Geographical variability and parasitism on body size, reproduction and shell characteristics of the keyhole limpet Fissurella crassa (Mollusca: Vetigastropoda).

Environmental variation may alter biological interactions and their ecological consequences. For instance, in marine ecosystems hosts and parasites are subject to environmental variability across latitudinal gradients, and their co-evolutionary dynamics may be the result of the interplay with local physical-chemical variables in seawater. Thus, assessing the environmental conditions required for a host in order to improve their survival is essential to understand the host-parasite interaction and dynamics. In this study, we evaluated the impact of parasitism by Proctoeces humboldti on the body size and reproduction of the intertidal keyhole limpet Fissurella crassa collected from three populations spanning ca. 1500 km along the latitudinal gradient of the Chilean coast. In addition, for the first time, we explore whether the effect of parasitism can be extended to changes in the organic composition and mechanical properties of the host shell. Our results show that parasitism prevalence and intensity, and body size of F. crassa increased in central Chile (ca. 33°S). Unlike body size, which was greater in parasitized limpets than in non-parasitized limpets at the three study sites, reproductive performance followed this trend only in central Chile populations, with no differences between parasitized and non-parasitized limpets collected in the northern Chilean (ca. 23°S), and lower in parasitized than non-parasitized individuals from the south-central Chile (ca. 37°S). The organic composition of F. crassa shells showed significant differences between parasite conditions (e.g. polysaccharides and water decreased in parasitized limpets) and across sites (e.g. proteins levels increase in shell of parasitized limpets from central Chile, but decreased at south-central Chile). However, variability in shell mechanical properties (e.g. toughness and elastic module) do not showed significant differences across sites and parasitism condition. These results suggest the interplay of both parasitism and environmental fluctuations upon the reproductive performance and morphology of the host. In addition, our result highlight that the host may also trade-offs reproduction, growth and shell organic composition to maintain the shell functionality (e.g. protection for mechanical forces and durophagous predators).

Origin of the biphase nature and surface roughness of biogenic calcite secreted by the giant barnacle Austromegabalanus psittacus.

The calcite grains forming the wall plates of the giant barnacle Austramegabalanus psittacus have a distinctive surface roughness made of variously sized crystalline nanoprotrusions covered by extremely thin amorphous pellicles. This biphase (crystalline-amorphous) structure also penetrates through the crystal's interiors, forming a web-like structure. Nanoprotrusions very frequently elongate following directions related to the crystallographic structure of calcite, in particular, the <- 441> directions, which are the strongest periodic bond chains (PBCs) in calcite. We propose that the formation of elongated nanoprotrusions happens during the crystallization of calcite from a precursor amorphous calcium carbonate (ACC). This is because biomolecules integrated within the ACC are expelled from such PBCs due to the force of crystallization, with the consequent formation of uninterrupted crystalline nanorods. Expelled biomolecules accumulate in adjacent regions, thereby stabilizing small pellicle-like volumes of ACC. With growth, such pellicles become occluded within the crystal. In summary, the surface roughness of the biomineral surface reflects the complex shape of the crystallization front, and the biphase structure provides evidence for crystallization from an amorphous precursor. The surface roughness is generally explained as resulting from the attachment of ACC particles to the crystal surface, which later crystallised in concordance with the crystal lattice. If this was the case, the nanoprotrusions do not reflect the size and shape of any precursor particle. Accordingly, the particle attachment model for biomineral formation should seek new evidence.