Involvement of neural crest and paraxial mesoderm in oral mucosal development and healing.

Tissue engineering therapies using adult stem cells derived from neural crest have sought accessible tissue sources of these cells because of their potential pluripotency. In this study, the gingiva and oral mucosa and their associated stem cells were investigated. Biopsies of these tissues produce neither scarring nor functional problems and are relatively painless, and fresh tissue can be obtained readily during different chairside dental procedures. However, the embryonic origin of these cells needs to be clarified, as does their evolution from the perinatal period to adulthood. In this study, the embryonic origin of gingival fibroblasts were determined, including gingival stem cells. To do this, transgenic mouse models were used to track neural crest derivatives as well as cells derived from paraxial mesoderm, spanning from embryogenesis to adulthood. These cells were compared with ones derived from abdominal dermis and facial dermis. Our results showed that gingival fibroblasts are derived from neural crest, and that paraxial mesoderm is involved in the vasculogenesis of oral tissues during development. Our in vitro studies revealed that the neuroectodermal origin of gingival fibroblasts (or gingival stem cells) endows them with multipotential properties as well as a specific migratory and contractile phenotype which may participate to the scar-free properties of the oral mucosa. Together, these results illustrate the high regenerative potential of neural crest-derived stem cells of the oral mucosa, including the gingiva, and strongly support their use in cell therapy to regenerate tissues with impaired healing.

Measurement of sodium concentration in sweat samples: comparison of 5 analytical techniques.

Sweat sodium concentration (SSC) can be determined using different analytical techniques (ATs), which may have implications for athletes and scientists. This study compared the SSC measured with 5 ATs: ion chromatography (IChr), flame photometry (FP), direct (DISE) and indirect (IISE) ion-selective electrode, and ion conductivity (IC). Seventy sweat samples collected from 14 athletes were analyzed with 5 instruments: the 883 Basic IC Plus (IChr, reference instrument), AAnalyst 200 (FP), Cobas 6000 (IISE), Sweat-Chek (IC), and B-722 Laqua Twin (DISE). Instruments showed excellent relative (intraclass correlation coefficient (ICC) ≥ 0.999) and absolute (coefficient of variation (CV) ≤ 2.6%) reliability. Relative validity was also excellent between ATs (ICC ≥ 0.961). In regards to the inter-AT absolute validity, compared with IChr, standard error of the estimates were similar among ATs (2.8-3.8 mmol/L), but CV was lowest with DISE (3.9%), intermediate with IISE (7.6%), and FP (6.9%) and highest with IC (12.3%). In conclusion, SSC varies depending on the AT used to analyze samples. Therefore, results obtained from different ATs are scarcely comparable and should not be used interchangeably. Nevertheless, taking into account the normal variability in SSC (∼±12%), the imprecision of the recommendations deriving from FP, IISE, IC, and DISE should have trivial health and physiological consequences under most exercise circumstances.

Comparison of Sodium Chloride Tablets-Induced, Sodium Chloride Solution-Induced, and Glycerol-Induced Hyperhydration on Fluid Balance Responses in Healthy Men.

Savoie, FA, Asselin, A, and Goulet, EDB. Comparison of sodium chloride tablets-induced, sodium chloride solution-induced, and glycerol-induced hyperhydration on fluid balance responses in healthy men. J Strength Cond Res 30(10): 2880-2891, 2016-Sodium chloride solution-induced hyperhydration (NaCl-SolIH) is a powerful strategy to increase body water before exercise. However, NaCl-SolIH is associated with an unpleasant salty taste, potentially dissuading some athletes from using it and coaches from recommending it. Therefore, we evaluated the hyperhydrating potential of sodium chloride tablets-induced hyperhydration (NaCl-TabIH), which bypasses the palatability issue of NaCl-SolIH without sacrificing sodium chloride content, and compared it to NaCl-SolIH and glycerol-induced hyperhydration (GIH). Sixteen healthy males (age: 21 ± 2 years; fat-free mass (FFM): 65 ± 6 kg) underwent three, 3-hour long passive hyperhydration protocols during which they drank, over the first 60 minutes, 30-ml·kg FFM of an artificially sweetened solution. During NaCl-TabIH, participants swallowed 7.5, 1 g each, sodium chloride tablets with every liter of solution. During NaCl-SolIH, an equal quantity of sodium chloride tablets was dissolved in each liter of solution. With GIH, the glycerol concentration was 46.7 g·L. Urine production, fluid retention, hemoglobin, hematocrit, plasma volume, and perceptual variables were monitored throughout the trials. Total fluid intake was 1948 ± 182 ml. After 3 hour, there were no significant differences among treatments for hemoglobin, hematocrit, and plasma volume changes. Fluid retention was significantly greater with NaCl-SolIH (1150 ± 287 ml) than NaCl-TabIH (905 ± 340 ml) or GIH (800 ± 211 ml), with no difference between NaCl-TabIH and GIH. No differences were found among treatments for perceptual variables. NaCl-TabIH and GIH are equally effective, but inferior than NaCl-SolIH. NaCl-TabIH represents an alternative to hyperhydration induced with glycerol, which is prohibited by the World Anti-Doping Agency.

Reliability and validity of a low cost, pocket-sized and battery operated sodium analyzer in measuring urinary sodium concentration.

BACKGROUND: The 24-h urine collection technique is the reference method for assessing sodium intake. Frequent assessments of urinary sodium excretion improve patients' motivation in adhering to sodium-restricted diets. No portable, inexpensive, user-friendly and reliable sodium analyzers are available on the market. Allowing field practitioners access to such an instrument could facilitate patients' monitoring of urinary sodium output, potentially improving patients care and overall population health. OBJECTIVE: To determine the validity and intra- and inter-instruments reliability of two portable, easy-to-use and inexpensive sodium analyzers. METHODS: Urine samples (N= 77) were collected from 31 men and compared against reference values of an ion chromatograph. RESULTS: Both analyzers demonstrated excellent absolute (95% limits of agreement (LoA) of ± 3-4%) and relative intra-instrument reliability (intraclass correlation coefficients (ICC) of 1.00). Inter-instruments relative reliability was excellent (ICC: 0.99), whereas absolute reliability was good (95% LoA of ± 13%). Compared with results obtained by ion chromatography, relative validity was excellent for both analyzers. Absolute validity was good-to-moderate (95% LoAs ranging from ± 15% to ± 20%). CONCLUSIONS: A low cost, portable analyzer can reliably be used by field practitioners to monitor changes in urinary sodium excretion across time and provide adequate guidance for individuals on sodium-restricted diets.

Intestinal temperature does not reflect rectal temperature during prolonged, intense running with cold fluid ingestion.

It is generally assumed that intestinal temperature (Tint), as measured with a telemetric pill, agrees relatively well with rectal temperature (Trec) during exercise. However, whether Tint reflects Trec during prolonged, intense and continuous exercise when cold fluids are consumed is unknown. Therefore, we compared Trec and Tint during a half-marathon during which cold water was ingested to prevent bodyweight (BW) losses >2%. Nine endurance athletes (age 30 ± 5 years) underwent a 21.1 km running time-trial (TT) in the heat (~30 °C and 44% RH) while BW losses were maintained to ~1% with continuous cold (4 °C) water provision. Tint and Trec were monitored throughout the TT. Hypohydration level, TT time and fluid intake were 1.2 ± 0.4% BW, 93.2 ± 9.9 min and 2143 ± 264 ml, respectively. Trec was systematically higher than Tint by 0.25 °C (95% CI: 0.14-0.37 °C). Tint and Trec showed an excellent relative (r = 0.90, p < 0.01), but poor absolute agreement as reflected by a 95% limit of agreement of ±1.07 °C and a standard error of measurement of ±0.39 °C. In conclusion, Tint does not mirror Trec during prolonged, intense running with cold fluid ingestion and, therefore, these measures should not be used interchangeably under this scenario.

Sodium-induced hyperhydration decreases urine output and improves fluid balance compared with glycerol- and water-induced hyperhydration.

Before 2010, which is the year the World Anti-Doping Agency banned its use, glycerol was commonly used by athletes for hyperhydration purposes. Through its effect on osmoreceptors, we believe that sodium could prove a viable alternative to glycerol as a hyperhydrating agent. Therefore, this study compared the effects of sodium-induced hyperhydration (SIH), glycerol-induced hyperhydration (GIH) and water-induced hyperhydration (WIH) on fluid balance responses. Using a randomized, double-blind and counterbalanced protocol, 17 men (21 ± 3 years, 64 ± 6 kg fat-free mass (FFM)) underwent three 3-h hyperhydration protocols during which they ingested, over the first 60-min period, 30 mL/kg FFM of water with (i) an artificial sweetener (WIH); (ii) an artificial sweetener + 7.45 g/L of table salt (SIH); or (iii) an artificial sweetener + 1.4 g glycerol/kg FFM (GIH). Changes in body weight (BW), urine production, fluid retention, hemoglobin, hematocrit, plasma volume, and perceptual variables were monitored throughout the 3-h trials. After 3 h, SIH was associated with significantly (p < 0.05) lower hemoglobin, hematocrit (SIH: 43.1% ± 2.8%; GIH: 44.9% ± 2.4%), and urine production, as well as greater BW, fluid retention (SIH: 1144 ± 294 mL; GIH: 795 ± 337 mL), and plasma volume (SIH: 11.9% ± 12.0%; GIH: 4.0% ± 6.0%) gains, compared with GIH and WIH. No significant differences in heart rate or abdominal discomfort were observed between treatments. In conclusion, our results indicate that SIH is a superior hyperhydrating technique than, and proves to be a worthwhile alternative to, GIH.

Ameloblastin as a putative marker of specific bone compartments.

Ameloblastin (AMBN), a member of the enamel matrix protein family, has been recently identified as integral part of the skeleton beyond the enamel. However, the specific role of endogenous AMBN in bone tissue is not fully elucidated. This study aims at investigating mRNA expression of AMBN in wild-type mice in different bone sites from early embryonic to adult stages. AMBN mRNA expression started at pre-dental stages in mouse embryos (E10.5) in both head and body parts. Using laser capture microdissection on 3-day-old mice, we showed an unambiguous mRNA expression of AMBN in extra-dental tissue (mandible bone). Screening of AMBN mRNA expression in adult mice (15-week-old) revealed that mRNA expression of AMBN varied according to the bone site; a higher mRNA levels in mandibular and frontal bone compartments were observed when compared to tibia and occipital bones. These results strongly suggest that AMBN expression may be regulated in a site-specific manner and identify AMBN as a putative in vivo marker of the site-specific fingerprint of bone organs.

Tracking endogenous amelogenin and ameloblastin in vivo.

Research on enamel matrix proteins (EMPs) is centered on understanding their role in enamel biomineralization and their bioactivity for tissue engineering. While therapeutic application of EMPs has been widely documented, their expression and biological function in non-enamel tissues is unclear. Our first aim was to screen for amelogenin (AMELX) and ameloblastin (AMBN) gene expression in mandibular bones and soft tissues isolated from adult mice (15 weeks old). Using RT-PCR, we showed mRNA expression of AMELX and AMBN in mandibular alveolar and basal bones and, at low levels, in several soft tissues; eyes and ovaries were RNA-positive for AMELX and eyes, tongues and testicles for AMBN. Moreover, in mandibular tissues AMELX and AMBN mRNA levels varied according to two parameters: 1) ontogenic stage (decreasing with age), and 2) tissue-type (e.g. higher level in dental epithelial cells and alveolar bone when compared to basal bone and dental mesenchymal cells in 1 week old mice). In situ hybridization and immunohistodetection were performed in mandibular tissues using AMELX KO mice as controls. We identified AMELX-producing (RNA-positive) cells lining the adjacent alveolar bone and AMBN and AMELX proteins in the microenvironment surrounding EMPs-producing cells. Western blotting of proteins extracted by non-dissociative means revealed that AMELX and AMBN are not exclusive to mineralized matrix; they are present to some degree in a solubilized state in mandibular bone and presumably have some capacity to diffuse. Our data support the notion that AMELX and AMBN may function as growth factor-like molecules solubilized in the aqueous microenvironment. In jaws, they might play some role in bone physiology through autocrine/paracrine pathways, particularly during development and stress-induced remodeling.

Half-marathon running performance is not improved by a rate of fluid intake above that dictated by thirst sensation in trained distance runners.

PURPOSE: It has been demonstrated that exercise-induced dehydration (EID) does not impair, and ad libitum drinking optimizes, cycling time-trial (TT) performance. However, the idea that EID ≥ 2 % bodyweight (BW) impairs endurance performance is well ingrained. No study has tested the impact of EID upon running TT performance. We compared the effects of thirst-driven (TD) vs. programmed fluid intake (PFI) aimed at maintaining EID-associated BW loss <2 % on half-marathon performance. METHODS: Ten trained distance runners underwent, in a randomized, crossover fashion, two, 21.1 km running TTs on a treadmill (30 °C, 42 % relative humidity) while facing a wind speed matching running speed and drinking water (1) according to thirst sensation (TD) or (2) to maintain BW loss <2 % of their pre-exercise BW (PFI), as recommended by the American College of Sports Medicine. RESULTS: Despite that PFI significantly reduced EID from 3.1 ± 0.6 (TD) to 1.3 ± 0.7 % BW (PFI), mean rectal temperature from 39.4 ± 0.4 to 39.1 ± 0.3 °C, mean body temperature from 38.1 ± 0.4 to 37.7 ± 0.2 °C and mean heart rate from 175 ± 9 to 171 ± 8 bpm, neither half-marathon time (TD 89.8 ± 7.7; PFI 89.6 ± 7.7 min) nor running pace (TD 4.3 ± 0.4; PFI 4.2 ± 0.4 min/km) differed significantly between trials. CONCLUSION: Albeit providing trivial cardiovascular and thermoregulatory advantages, in trained distance runners, PFI (1,380 ± 320 mL/h) offers no performance benefits over TD fluid intake (384 ± 180 mL/h) during a half-marathon raced under warm conditions.

Pre-exercise hyperhydration-induced bodyweight gain does not alter prolonged treadmill running time-trial performance in warm ambient conditions.

This study compared the effect of pre-exercise hyperhydration (PEH) and pre-exercise euhydration (PEE) upon treadmill running time-trial (TT) performance in the heat. Six highly trained runners or triathletes underwent two 18 km TT runs (~28 °C, 25%-30% RH) on a motorized treadmill, in a randomized, crossover fashion, while being euhydrated or after hyperhydration with 26 mL/kg bodyweight (BW) of a 130 mmol/L sodium solution. Subjects then ran four successive 4.5 km blocks alternating between 2.5 km at 1% and 2 km at 6% gradient, while drinking a total of 7 mL/kg BW of a 6% sports drink solution (Gatorade, USA). PEH increased BW by 1.00 ± 0.34 kg (P < 0.01) and, compared with PEE, reduced BW loss from 3.1% ± 0.3% (EUH) to 1.4% ± 0.4% (HYP) (P < 0.01) during exercise. Running TT time did not differ between groups (PEH: 85.6 ± 11.6 min; PEE: 85.3 ± 9.6 min, P = 0.82). Heart rate (5 ± 1 beats/min) and rectal (0.3 ± 0.1 °C) and body (0.2 ± 0.1 °C) temperatures of PEE were higher than those of PEH (P < 0.05). There was no significant difference in abdominal discomfort and perceived exertion or heat stress between groups. Our results suggest that pre-exercise sodium-induced hyperhydration of a magnitude of 1 L does not alter 80-90 min running TT performance under warm conditions in highly-trained runners drinking ~500 mL sports drink during exercise.

Nephrocalcinosis (enamel renal syndrome) caused by autosomal recessive FAM20A mutations.

BACKGROUND/AIMS: Calcium homeostasis requires regulated cellular and interstitial systems interacting to modulate the activity and movement of this ion. Disruption of these systems in the kidney results in nephrocalcinosis and nephrolithiasis, important medical problems whose pathogenesis is incompletely understood. METHODS: We investigated 25 patients from 16 families with unexplained nephrocalcinosis and characteristic dental defects (amelogenesis imperfecta, gingival hyperplasia, impaired tooth eruption). To identify the causative gene, we performed genome-wide linkage analysis, exome capture, next-generation sequencing, and Sanger sequencing. RESULTS: All patients had bi-allelic FAM20A mutations segregating with the disease; 20 different mutations were identified. CONCLUSIONS: This autosomal recessive disorder, also known as enamel renal syndrome, of FAM20A causes nephrocalcinosis and amelogenesis imperfecta. We speculate that all individuals with biallelic FAM20A mutations will eventually show nephrocalcinosis.

Expression pattern of Dlx3 during cell differentiation in mineralized tissues.

The present study was designed to compare the expression pattern of Dlx3 in four different mineralized tissues because of: 1-its role in skeleton patterning, 2-its expression in dental epithelium and mesenchyme during morphogenesis, 3-the membranous and endochondral bone and tooth phenotype of tricho-dento-osseous syndrome related to Dlx3 gene mutation and 4-recently emerging knowledge on Dlx family members in the bone field. Ameloblasts, odontoblasts, osteoblasts and chondrocytes were analyzed in vitro and in vivo. Dlx3 transcripts were detected by RT-PCR in established model systems (microdissected dental epithelium and mesenchyme; primary cultures of rat chondrocytes), as recently performed in osteoblasts in vitro. A human 414-bp Dlx3 probe was generated. A 4.5-kb human Dlx3 sense RNA was identified in maxillo-facial samples by Northern blotting. Immunolabeling and in situ hybridization were performed in mice from Theiler stage E 14.5 until birth. In teeth, although Dlx3 was still expressed in differentiated ameloblasts, it was down regulated during odontoblast polarization. During endochondral bone formation, Dlx3 protein was detected in chondrocytes and was most strongly expressed in the prehypertrophic cartilage zone and in differentiating and differentiated osteoblasts of metaphyseal periosteum. In vitro, real-time PCR studies supported this upregulation in prehypertrophic chondrocytes, closely correlated with Ihh variations. In membranous bone, Dlx3 was present in preosteoblasts, osteoblasts and osteoid-osteocytes. The present data on Dlx3 and recently published functional studies show that this transcription factor may be instrumental during growth in the control of matrix deposition and biomineralization in the entire skeleton.

The modulation of tissue-specific gene expression in rat nasal chondrocyte cultures by bioactive glasses.

Since bone repair may occur, following endochondral ossification, we have investigated the behaviour of chondrocytes isolated from nasal septum cartilage of foetal rats and cultured up to 21 days in the presence of a melt-derived bioactive glass (Bioglass 45S5) and a less reactive glass with 60 wt% silica content (60S). In both cultures, chondrocytes proliferate and form typical cartilaginous nodules on day 5 of cultures. However, on day 12, the nodules in contact with 45S5 granules became darker than in 60S cultures, corresponding to the emergence of matrix biomineralization. Transmission electron microscopy showed a collagen-rich matrix composed of densely packed fibres and mineralized foci formed of needle-shaped crystals in contact with an electron-dense layer located at the periphery of the material. The specific activity of alkaline phosphatase was significant higher in 45S5 cultures on day 15 than in 60S cultures. Real time RT-PCR was used to monitor gene expression levels of specific chondrogenic markers. The transcription factor Sox9 was expressed throughout the culture period, but with no significant differences between the two kinds of cultures. In contrast, Runx2 expression was higher in experiment cultures on day 12. Type II collagen mRNA and aggrecan, showed an almost similar expression pattern with a strong expression at the beginning of cultures but higher in experiment cultures. Indian hedgehog was strongly expressed between day 9 and 12 with a significant stimulation in 45S5 cultures. Similarly, type X collagen mRNA seemed to be up-regulated in 45S5 cultures on day 20. In conclusion, this study shows hat 45S5 Bioglass has the ability to support the growth of chondrocytes and to stimulate some chondrogenic molecular markers.

The biomimetics of bone: engineered glass-ceramics a paradigm for in vitro biomineralization studies.

In this study, we investigated the behavior of fetal rat osteoblasts cultured up to 23 days on a bioactive apatite-wollastonite glass-ceramic (AW) and on the same material on which a carbonated apatite layer was formed by a biomimetic process (AWa). The specific activity of alkaline phosphatase activity was about 30% increased on AWa compared to AW disks at the last day of culture. Scanning electron microscopic (SEM) observations of the material surfaces after scrapping off the cell layers revealed that mineralized bone nodules remained attached to both surfaces but in larger numbers on AWa. The AWa/bone interfaces were also analyzed after fracturing the disks and by transmission electron microscopy (TEM). All these results indicated the importance of the surface composition in supporting differentiation of osteogenic cells and the subsequent apposition of bone matrix. Furthermore, prefabrication of a biological apatite layer by a biomimetic method could improve our knowledge of biomineralization processes and could find application as bone-repairing material.