Correction: The Swine Plasma Metabolome Chronicles "Many Days" Biological Timing and Functions Linked to Growth.

[This corrects the article DOI: 10.1371/journal.pone.0145919.].

Effects of LED curing light on silver diamine fluoride penetration into dentin.

This ex-vivo study investigated the effect of a light-emitting diode (LED) curing light on the depth of penetration of Silver Diamine Fluoride (SDF) into carious lesions. Twenty-four primary teeth with untreated caries lesions were allocated into groups and treated within 5 min after extraction: (1) n = 6 treated for 1 min with one drop of SDF followed by 10 sec rinse with tap water, (2) n = 6 treated for 10 sec with one drop of SDF and exposed to LED light for 20 sec (30 sec total SDF exposure) followed by 10 sec rinse with tap water, (3) n = 6 treated for 10 sec with one drop of SDF followed by a 10 sec rinse with tap water, (4) n = 3 untreated, and (5) n = 3 untreated but exposed to LED light for 20 sec. Samples were prepared, embedded, sectioned and silver penetration was measured using backscattered electron imaging in the scanning electron microscope and energy-dispersive X-ray spectroscopy analysis. Results were expressed as the average relative depth of penetration (%) = Ag depth/lesion depth × 100 from 5 sites in each lesion. Group means were compared using mixed model analysis. Mean ± standard deviation (SD) penetration was: 86.4 ± 20.7% in Group 1, 94.3 ± 13.7% in Group 2, and 26.7 ± 13.9% in Group 3. Groups 1 and 2 were statistically similar and different from Group 3 (p < 0.001). Groups 4 and 5 had no silver present. Use of LED light for 20 sec after 10 sec SDF application appears to facilitate silver penetration, similar to a 1 min SDF application. Clinical studies are needed to define the role of silver penetration in sustained caries arrest.

Long-term effects of canagliflozin treatment on the skeleton of aged UM-HET3 mice.

Sodium glucose cotransporter-2 inhibitors (SGLT2is) promote urinary glucose excretion and decrease plasma glucose levels independent of insulin. Canagliflozin (CANA) is an SGLT2i, which is widely prescribed, to reduce cardiovascular complications, and as a second-line therapy after metformin in the treatment of type 2 diabetes mellitus. Despite the robust metabolic benefits, reductions in bone mineral density (BMD) and cortical fractures were reported for CANA-treated subjects. In collaboration with the National Institute on Aging (NIA)-sponsored Interventions Testing Program (ITP), we tested skeletal integrity of UM-HET3 mice fed control (137 mice) or CANA-containing diet (180 ppm, 156 mice) from 7 to 22 months of age. Micro-computed tomography (micro-CT) revealed that CANA treatment caused significant thinning of the femur mid-diaphyseal cortex in both male and female mice, did not affect trabecular bone architecture in the distal femur or the lumbar vertebra-5 in male mice, but was associated with thinning of the trabeculae at the distal femur in CANA-treated female mice. In male mice, CANA treatment is associated with significant reductions in cortical bone volumetric BMD by micro-CT, and by quantitative backscattered scanning electron microscopy. Raman microspectroscopy, taken at the femur mid-diaphyseal posterior cortex, showed significant reductions in the mineral/matrix ratio and an increased carbonate/phosphate ratio in CANA-treated male mice. These data were supported by thermogravimetric assay (TGA) showing significantly decreased mineral and increased carbonate content in CANA-treated male mice. Finally, the sintered remains of TGA were subjected to X-ray diffraction and showed significantly higher fraction of whitlockite, a calcium orthophosphate mineral, which has higher resorbability than hydroxyapatite. Overall, long-term CANA treatment compromised bone morphology and mineral composition of bones, which likely contribute to increased fracture risk seen with this drug.

3D printed mesoporous bioactive glass, bioglass 45S5, and ß-TCP scaffolds for regenerative medicine: A comparative in vitro study.

BACKGROUND: While autografts to date remain the "gold standard" for bone void fillers, synthetic bone grafts have garnered attention due to their favorable advantages such as ability to be tailored in terms of their physical and chemical properties. Bioactive glass (BG), an inorganic material, has the capacity to form a strong bond with bone by forming a bone-like apatite surface, enhancing osteogenesis. Coupled with additive manufacturing (3D printing) it is possible to maximize bone regenerative properties of the BG. OBJECTIVE: The objective of this study was to synthesize and characterize 3D printed mesoporous bioactive glass (MBG), BG 45S5, and compare to ß-Tricalcium phosphate (ß-TCP) based scaffolds; test cell viability and osteogenic differentiation on human osteoprogenitor cells in vitro. METHODS: MBG, BG 45S5, and ß-TCP were fabricated into colloidal gel suspensions, tested with a rheometer, and manufactured into scaffolds using a 3D direct-write micro-printer. The materials were characterized in terms of microstructure and composition with Thermogravimetric Analyzer/Differential Scanning Calorimeter (TGA/DSC), Fourier Transform Infrared Spectroscopy (FTIR), X-ray Diffraction (XRD), Micro-Computed Tomography (µ-CT), Scanning Electron Microscopy (SEM), Energy Dispersive X-ray Spectroscopy (EDS), and Mattauch-Herzog-Inductively Coupled Plasma-Mass Spectrometry (MH-ICP-MS). RESULTS: Scaffolds were tested for cell proliferation and osteogenic differentiation using human osteoprogenitor cells. Osteogenic media was used for differentiation, and immunocytochemistry for osteogenic markers Runx-2, Collagen-I, and Osteocalcin. The cell viability results after 7 days of culture yielded significantly higher (p < 0.05) results in ß-TCP scaffolds compared to BG 45S5 and MBG groups. CONCLUSION: All materials expressed osteogenic markers after 21 days of culture in expansion and osteogenic media.

Life history in primate teeth is revealed by changes in major and minor element concentrations measured via field-emission SEM-EDS analysis.

Overcoming the non-specificity of histological accentuated growth lines in hard tissues is an ongoing challenge. Identifying season at death and reproductive events has profound implications for evolutionary, ecological and conservation studies. Dental cementum is a mineralized tissue with yearly periodicity that continues deposition from tooth formation until death, maintaining a record spanning almost the entire life of an individual. Recent work has successfully employed elemental analysis of calcified incremental tissues to detect changes in extrinsic conditions such as diet and climate and to identify two important life-history milestones: weaning and sexual maturity. Here, we employ field-emission scanning electron microscopy and energy-dispersive X-ray analysis to measure the relative concentrations of calcium, phosphorous, oxygen, magnesium and sodium in the cementum of 34 teeth from seven male and female rhesus macaques with known medical and life-history information. We find that changes in relative magnesium concentrations correspond with reproductive events in females and breastfeeding in infants. Additionally, we observe seasonal calcium patterns in 77.3% of the samples.

Elemental composition of primary lamellar bone differs between parous and nulliparous rhesus macaque females.

Extracting life history information from mineralized hard tissues of extant and extinct species is an ongoing challenge in evolutionary and conservation studies. Primary lamellar bone is a mineralized tissue with multidien periodicity that begins deposition prenatally and continues until adulthood albeit with concurrent resorption, thus maintaining a record spanning several years of an individual's life. Here, we use field-emission scanning electron microscopy and energy-dispersive X-ray analysis to measure the relative concentrations of calcium, phosphorous, oxygen, magnesium and sodium in the femora of seven rhesus macaque with known medical and life-history information. We find that the concentration of these elements distinguishes parous from nulliparous females; that in females calcium and phosphorus are lower in bone formed during reproductive events; and that significant differences in relative magnesium concentration correlate with breastfeeding in infants.

Dental cementum virtual histology of Neanderthal teeth from Krapina (Croatia, 130-120 kyr): an informed estimate of age, sex and adult stressors.

The evolution of modern human reproductive scheduling is an aspect of our life history that remains vastly uncomprehended. The present work aims to address this gap by validating a non-destructive cutting-edge methodology to infer adult life-history events on modern teeth with known life history and then applying it to fossil specimens. We use phase-contrast synchrotron X-ray microtomography to visualize the dental cementum of 21 specimens: nine contemporary humans; 10 Neanderthals from Krapina (Croatia, 130-120 kyr); one Neolithic Homo sapiens from Ajmana (Serbia); and one Mesolithic H. sapiens from Vlasac (Serbia). We were able to correctly detect and time (root mean square error = 2.1 years; R2 = 0.98) all reproductive (menarche, parturition, menopause) and other physiologically impactful events in the modern sample. Nonetheless, we could not distinguish between the causes of the events detected. For the fossil specimens, we estimated age at death and age at occurrence of biologically significant events. Finally, we performed an exploratory analysis regarding possible sexual dimorphism in dental cementum microstructure, which allowed us to correctly infer the sex of the Neolithic specimen, for which the true value was known via DNA analysis.

Adjuvant therapy with 1% alendronate gel for experimental periodontitis treatment in rats.

PURPOSE: The aim of this study was to evaluate the effects of locally delivered 1% alendronate (ALN) gel used as an adjunct to non-invasive periodontal therapy. METHODS: Ligature-induced periodontitis was performed in 96 rats. The ligature was tied in the cervical area of the mandibular left first molar. The animals were randomly divided into 4 groups: 1) NT, no treatment; 2) SRP, scaling and root planning; 3) SRP/PLA, SRP followed by filling the periodontal pocket with placebo gel (PLA); and 4) SRP/ALN, SRP followed by filling the periodontal pockets with 1% ALN gel. Histomorphometric (percentage of bone in the furcation region [PBF]) and immunohistochemical (receptor activator of nuclear factor-κB ligand, osteoprotegerin, and tartrate-resistant acid phosphatase) analyses were performed. Data were statistically analyzed, with the threshold of statistical significance set at P≤0.05. RESULTS: The SRP, SRP/PLA, and SRP/ALN groups presented a higher PBF than the NT group (P≤0.01) at 7, 15, and 30 days. The SRP/ALN group presented a higher PBF than the SRP/PLA group in all experimental periods, as well as a higher PBF than the SRP group at 15 and 30 days. No differences were observed in the immunohistochemical analyses (P>0.05 for all). CONCLUSIONS: Locally delivered 1% ALN gel used as an adjunct to SRP enhanced bone regeneration in the furcation region in a rat model of experimental periodontitis.

Adaptive Immune Response Associated with a Zirconium-Containing, Cemented, Total Knee Arthroplasty: A Case Report.

CASE: A 69-year-old woman underwent revision total knee arthroplasty for patellar component aseptic loosening. The periprosthetic tissue demonstrated histologic features of an adaptive immune response (aseptic lymphocyte-dominant vasculitis-associated lesion [ALVAL]). No particles of corrosion debris were identified. The inflammation seemed to be associated with zirconium oxide (ZrO2) particles added as a bone cement radio-opacifier. CONCLUSION: The factors responsible for the adaptive immune response cannot be determined with certainty; however, this is the first reported case of ALVAL associated with ZrO2-containing bone cement. Previous reports describing ALVAL around failed total knee prostheses have not included observations about the type of contrast material added to cement.

Weaning, parturitions and illnesses are recorded in rhesus macaque (Macaca mulatta) dental cementum microstructure.

Many open questions in evolutionary studies relate to species' physiological adaptations, including the evolution of their life history and reproductive strategies. There are few empirical methods capable of detecting and timing physiologically impactful events such as weaning, parturition and illnesses from hard tissue remains of either extant or extinct species. Cementum is an incremental tissue with post eruption annual periodicity, which covers the tooth root and functions as a recording structure of an animal's physiology. Here we test the hypothesis that it is possible to detect and time physiologically impactful events through the analysis of dental cementum microstructure. Our sample comprises 41 permanent and deciduous teeth from male and female rhesus macaques (Macaca mulatta) with known medical, lifestyle and life history information. We develop a semi-automated method of cementum histological analysis for the purpose of event detection and timing, aimed at significantly reducing the amount of intra- and interobserver errors typically associated with histological analyses. The results of our work show that we were able to detect known events including weaning, parturition, illness and physical trauma with high accuracy (false negative rate = 3.2%; n = 1), and to time them within an average absolute difference of 0.43 years (R2 = .98; p < .05). Nonetheless, we could not distinguish between the several types of stressful events underlying the changes in cementum microstructure. While this study is the first to identify a variety of life history events in macaque dental cementum, laying foundations for future work in conservation and evolutionary studies of both primates and toothed mammals at large, there are some limitations. Other types of analyses (possibly chemical ones) are necessary to tease apart the causes of the stressors.

Enamel multidien biological timing and body size variability among individuals of Chinese Han and Tibetan origins.

AIMS: To measure the number of days of enamel formation between periodic striae of Retzius growth lines, the Retzius periodicity (RP), and to compare this multi-day, or multidien rhythm, to body height and weight among people from Beijing, China and Lhasa, Tibet/China. SUBJECTS AND METHODS: Subjects requiring dental extractions from clinics in Beijing, China (N = 338) and Lhasa, Tibet/China (N = 227) provided a tooth and body size information. Multiple observers examined histological sections of the teeth and recorded RP. RP values were statistically compared to body height and weight. RESULTS: In Beijing and Lhasa samples, respectively, average height was 166.38 and 165.70 cm, average weight was 59.53 and 66.53 kg, and average RP was 7.47 and 7.69 d. Statistically significant differences were found between Beijing and Lhasa weight and RP means. Correlations for height and weight against RP were significant, but only comparatively strong for height. CONCLUSIONS: Supporting the negative correlation presented in previous studies, RP is negatively associated with height and weight among a large intraspecific sample of people from Beijing and Lhasa. RP represents a metabolic-mediated multidien biological timing mechanism responsible for the rate of cell proliferation and maintenance of the body.

In vitro shear bond strength of 2 resin cements to zirconia and lithium disilicate: An in vitro study.

STATEMENT OF PROBLEM: There is little evidence on how the multiple layers of zirconia (ZrO2) or glazed material will affect the shear bond strength (SBS) of different resin cements. PURPOSE: The purpose of this in vitro study was to compare the SBS of 2 resin cements with the different layers of a monolithic polychromatic ZrO2 ceramic, both glazed and nonglazed, and a lithium disilicate (LDS) ceramic. MATERIAL AND METHODS: One hundred and sixty-eight composite resin cylinders and 48 monolithic polychromic ZrO2 plates were prepared. Twenty-four were milled and sintered, and 24 were milled and sintered and had a glaze cycle applied with no liquid glaze. These plates and 12 LDS plates were mounted in autopolymerized acrylic resin. Bonding surfaces were polished, airborne-particle abraded, and cleaned ultrasonically. The different layers (cubic, hybrid, and tetragonal) of the ZrO2 plates were identified and marked. The plates were assigned to 2 cement groups: a self-adhesive, autopolymerized resin cement, and a dual-polymerizing, adhesive resin cement (DPRC). Bonding surfaces were cleaned and treated according to the cement manufacturer's instructions. Three composite resin cylinders were luted to the ZrO2 plates at the appropriate layer, and 2 cylinders were luted to each LDS plate. The specimens were stored in a moist environment for 24 hours at 37 °C. The SBS test was performed with a universal testing machine. Visual inspections of the debonded surfaces were compared under magnification. The data were analyzed with a 2-way ANOVA and a subsequent Student t test (α=.05). RESULTS: The 2-way ANOVA found no difference among luting agent and LDS and ZrO2. The SBSs of the nonglazed tetragonal and cubic layer to the ZrO2 were higher than to the surface exposed to a glazing cycle (P=.001). The bonded surfaces were examined tactilely and under ×3.5 magnification, followed by light and scanning electron microscopy and recorded as either adhesive, cohesive, or mixed. Almost all failures in the glazed ZrO2 were mixed and cohesive. However, more adhesive failures were observed in the DPRC group of nonglazed ZrO2. CONCLUSIONS: No differences were found between the 2 luting agents for the LDS. For the ZrO2 cubic and tetragonal layers, the DPRC had higher bond strengths to the nonglazed surfaces.

MH-ICP-MS Analysis of the Freshwater and Saltwater Environmental Resources of Upolu Island, Samoa.

The elemental composition of freshwater and saltwater samples around the South Pacific island of Upolu, Samoa has been investigated together with other indicators of water quality. Up to 69 elements from Li (3) to U (92) are measured in each sample, analyzed by Mattauch-Herzog-inductively coupled plasma-mass spectrometry (MH-ICP-MS). One hundred and seventy-six samples were collected from surface freshwater sources (24 rivers, two volcanic lakes, one dam) and from seawater sources from the surface to 30 m depth (45 inner reef, reef, and outer reef locations) around Upolu Island, including river mouths and estuaries. Principal component and hierarchical clustering correlation analyses were performed on quantile normalized log transformed elemental composition data to identify groups of samples with similar characteristics and to improve the visualization of the full spectrum of elements. Human activities, such as the use of herbicides and pesticides, may relate to observed elevated concentrations of some elements contained in chemicals known to have deleterious obesogenic effects on humans that may also cause coral reef decline. Furthermore, the salinity of some saltwater samples tested were very high, possibly due to climate variability, which may additionally harm the health and biodiversity of coral reefs.

A comprehensive survey of Retzius periodicities in fossil hominins and great apes.

Recent studies have provided great insight into hominin life history evolution by utilizing incremental lines found in dental tissues to reconstruct and compare the growth records of extant and extinct humans versus other ape taxa. Among the hominins, studies that have examined Retzius periodicity (RP) variation have come to contradictory conclusions in some instances. To clarify RP variation among hominins and better place this variation in its broader evolutionary context, we conduct the most comprehensive analysis of published RP values for hominins and great apes to date. We gathered all available data from the literature on RP data from extant humans, great apes, and fossil hominins and assessed their variation using parametric and nonparametric analyses of variance. We also performed phylogenetic generalized least-squares regressions of RP data for these taxa as well as a larger set of hominoids for which RP data have been published against data for body mass, encephalization, and mean semicircular canal radius (a proxy for metabolic rate). Our results show that modern humans have a mean RP significantly differing from that of other hominins. Pongo also is significantly different from nearly all other taxa in all analyses. Our results also demonstrate that RP variation among hominins scales with respect to body mass, encephalization, and semicircular canal radius similarly to other hominids but that modern humans and Pongo stand out in this regard. Operating within the hypothesis that RP reflects autonomic biorhythms that regulate multiple life history variables, our results reinforce the idea that Homo sapiens has evolved a life history distinct from other hominins, even from other members of Homo, and suggest that many of these life history differences may be driven by hypothalamic output from the brain.

Parturitions, menopause and other physiological stressors are recorded in dental cementum microstructure.

The life history pattern of recent humans is uniquely derived in many of its aspects including an extended post-reproductive lifespan combined with short interbirth intervals. A number of theories have been proposed to explain the evolution of this unusual pattern. However most have been difficult to test due to the fragmentary nature of the hominin fossil record and the lack of methods capable of inferring such later life history events. In search of a method we tested the hypothesis that the physiologically impactful events of parturition and menopause are recorded in dental cementum microstructure. We performed histomorphological analyses of 47 teeth from 15 individuals with known life history events and were able to detect reproductive events and menopause in all females. Furthermore, we found that other stressful events such as systemic illnesses and incarceration are also detectable. Finally, through the development of a novel analytical method we were able to time all such events with high accuracy (R-squared = 0.92).

Short and long period growth markers of enamel formation distinguish European Pleistocene hominins.

Characterizing dental development in fossil hominins is important for distinguishing between them and for establishing where and when the slow overall growth and development of modern humans appeared. Dental development of australopiths and early Homo was faster than modern humans. The Atapuerca fossils (Spain) fill a barely known gap in human evolution, spanning ~1.2 to ~0.4 million years (Ma), during which H. sapiens and Neandertal dental growth characteristics may have developed. We report here perikymata counts, perikymata distributions and periodicities of all teeth belonging to the TE9 level of Sima del Elefante, level TD6.2 of Gran Dolina (H. antecessor) and Sima de los Huesos. We found some components of dental growth in the Atapuerca fossils resembled more recent H. sapiens. Mosaic evolution of perikymata counts and distribution generate three distinct clusters: H. antecessor, Sima de los Huesos and H. sapiens.

Faster growth corresponds with shallower linear hypoplastic defects in great ape canines.

Deeper or more 'severe' linear enamel hypoplasia (LEH) defects are hypothesized to reflect more severe stress during development, but it is not yet clear how depth is influenced by intrinsic enamel growth patterns. Recent work documented inter- and intraspecific differences in LEH defect depth in extant great apes, with mountain gorillas having shallower defects than other taxa, and females having deeper defects than males. Here, we assess the correspondence of inter- and intraspecific defect depth and intrinsic aspects of enamel growth: enamel extension rates, outer enamel striae of Retzius angles, and linear enamel thickness. Thin sections of great ape canines (n = 40) from Gorilla beringei beringei, Gorilla gorilla gorilla, Pan troglodytes, and Pongo spp. were analyzed. Enamel extension rates were calculated within deciles of enamel-dentine junction length. Linear enamel thickness and the angle of intersection between striae of Retzius and the outer enamel surface were measured in the imbricational enamel. Mountain gorillas have faster enamel extension rates and shallower striae angles than the other taxa examined. Mountain gorillas have thinner imbricational enamel than western lowland gorillas and orangutans, but not chimpanzees. In the combined-taxon sample, females exhibit larger striae angles and thicker imbricational enamel than males. Enamel extension rates are highly negatively correlated with striae angles and LEH defect depth. Enamel growth variation corresponds with documented inter- and intraspecific differences in LEH defect depth in great ape canines. Mountain gorillas have shallower striae angles and faster extension rates than other taxa, which might explain their shallow LEH defect morphology and the underestimation of their LEH prevalence in previous studies. These results suggest that stressors of similar magnitude and timing might produce defects of different depths in one species or sex vs. another, which has implications for interpretations of stress histories in hominins with variable enamel growth patterns.

Parenchymal and stromal tissue regeneration of tooth organ by pivotal signals reinstated in decellularized matrix.

Cells are transplanted to regenerate an organs' parenchyma, but how transplanted parenchymal cells induce stromal regeneration is elusive. Despite the common use of a decellularized matrix, little is known as to the pivotal signals that must be restored for tissue or organ regeneration. We report that Alx3, a developmentally important gene, orchestrated adult parenchymal and stromal regeneration by directly transactivating Wnt3a and vascular endothelial growth factor. In contrast to the modest parenchyma formed by native adult progenitors, Alx3-restored cells in decellularized scaffolds not only produced vascularized stroma that involved vascular endothelial growth factor signalling, but also parenchymal dentin via the Wnt/ß-catenin pathway. In an orthotopic large-animal model following parenchyma and stroma ablation, Wnt3a-recruited endogenous cells regenerated neurovascular stroma and differentiated into parenchymal odontoblast-like cells that extended the processes into newly formed dentin with a structure-mechanical equivalency to native dentin. Thus, the Alx3-Wnt3a axis enables postnatal progenitors with a modest innate regenerative capacity to regenerate adult tissues. Depleted signals in the decellularized matrix may be reinstated by a developmentally pivotal gene or corresponding protein.

Differential regulation of Ca2+ influx by ORAI channels mediates enamel mineralization.

Store-operated Ca2+ entry (SOCE) channels are highly selective Ca2+ channels activated by the endoplasmic reticulum (ER) sensors STIM1 and STIM2. Their direct interaction with the pore-forming plasma membrane ORAI proteins (ORAI1, ORAI2, and ORAI3) leads to sustained Ca2+ fluxes that are critical for many cellular functions. Mutations in the human ORAI1 gene result in immunodeficiency, anhidrotic ectodermal dysplasia, and enamel defects. In our investigation of the role of ORAI proteins in enamel, we identified enamel defects in a patient with an ORAI1 null mutation. Targeted deletion of the Orai1 gene in mice showed enamel defects and reduced SOCE in isolated enamel cells. However, Orai2-/- mice showed normal enamel despite having increased SOCE in the enamel cells. Knockdown experiments in the enamel cell line LS8 suggested that ORAI2 and ORAI3 modulated ORAI1 function, with ORAI1 and ORAI2 being the main contributors to SOCE. ORAI1-deficient LS8 cells showed altered mitochondrial respiration with increased oxygen consumption rate and ATP, which was associated with altered redox status and enhanced ER Ca2+ uptake, likely due to S-glutathionylation of SERCA pumps. Our findings demonstrate an important role of ORAI1 in Ca2+ influx in enamel cells and establish a link between SOCE, mitochondrial function, and redox homeostasis.