Using online game-based platforms to improve student performance and engagement in histology teaching.

BACKGROUND: Human morphology is a critical component of dental and medical graduate training. Innovations in basic science teaching methods are needed to keep up with an ever-changing landscape of technology. The purpose of this study was to investigate whether students in a medical and dental histology course would have better grades if they used gaming software Kahoot® and whether gamification effects on learning and enjoyment. METHODS: In an effort to both evoke students' interest and expand their skill retention, an online competition using Kahoot® was implemented for first-year students in 2018 (n = 215) at the University of Eastern Finland. Additionally, closed (160/215) or open-ended (41/215) feedback questions were collected and analyzed. RESULTS: The Kahoot® gamification program was successful and resulted in learning gains. The overall participant satisfaction using Kahoot® was high, with students (124/160) indicating that gamification increased their motivation to learn. The gaming approach seemed to enable the students to overcome individual difficulties (139/160) and to set up collaboration (107/160); furthermore, gamification promoted interest (109/160), and the respondents found the immediate feedback from senior professionals to be positive (146/160). In the open-ended survey, the students (23/41) viewed collaborative team- and gamification-based learning positively. CONCLUSION: This study lends support to the use of gamification in the teaching of histology and may provide a foundation for designing a gamification-integrated curriculum across healthcare disciplines.

Determinants of serum 25-hydroxyvitamin D concentration in Finnish children: the Physical Activity and Nutrition in Children (PANIC) study.

We studied vitamin D intake, serum 25-hydroxyvitamin D (S-25(OH)D) concentration, determinants of S-25(OH)D and risk factors for S-25(OH)D <50 nmol/l in a population sample of Finnish children. We studied 184 girls and 190 boys aged 6-8 years, analysed S-25(OH)D by chemiluminescence immunoassay and assessed diet quality using 4-d food records and other lifestyle factors by questionnaires. We analysed the determinants of S-25(OH)D using linear regression and risk factors for S-25(OH)D <50 nmol/l using logistic regression. Mean dietary intake of vitamin D was 5·9 (sd 2·1) µg/d. Altogether, 40·8 % of children used no vitamin D supplements. Of all children, 82·4 % did not meet the recommended total vitamin D intake of 10 µg/d. Milk fortified with vitamin D was the main dietary source of vitamin D, providing 48·7 % of daily intake. S-25(OH)D was <50 nmol/l in 19·5 % of children. Consumption of milk products was the main determinant of S-25(OH)D in all children (standardised regression coefficient ß=0·262; P<0·001), girls (ß=0·214; P=0·009) and boys (ß=0·257; P=0·003) in multivariable models. Vitamin D intake from supplements (ß=0·171; P=0·035) and age (ß=-0·198; P=0·015) were associated with S-25(OH)D in girls. Children who drank ≥450 g/d of milk, spent ≥2·2 h/d in physical activity, had ≥13·1 h/d of daylight time or were examined in autumn had reduced risk for S-25(OH)D <50 nmol/l. Insufficient vitamin D intake was common among Finnish children, one-fifth of whom had S-25(OH)D <50 nmol/l. More attention should be paid to the sufficient intake of vitamin D from food and supplements, especially among children who do not use fortified milk products.

Wnt and steroid pathways control glutamate signalling by regulating glutamine synthetase activity in osteoblastic cells.

Glutamate signalling has recently been found functional also outside the central nervous system, especially in bone. Glutamate is converted to glutamine by glutamine synthetase (GS), which is therefore able to regulate intracellular concentrations of glutamate. We previously characterized the induction of GS expression by glucocorticoids (GCs) in human osteoblast-like cells. Besides this observation, the mechanisms controlling GS in bone are unknown. Therefore, the aim of our present study was to investigate further the regulation of GS in osteoblastic cells. We observed that vitamin D inhibited basal and, even more efficiently, GC-stimulated GS activity by affecting both the mRNA and protein levels of the enzyme in human MG-63 osteoblast-like cells. In osteoblasts derived from rat bone marrow stem cells (rMSCs), GS activity was induced accordingly by the osteogenic culture conditions including GCs. Also in these primary cells, vitamin D clearly inhibited GS activity. In addition, the canonical Wnt signalling pathway was characterized as a negative regulator of GS activity. All these changes in GS activity were reflected on the intracellular glutamate concentration. Our results provide novel evidence that GS activity and expression are regulated by several different signalling pathways in osteoblastic cells. Therefore, GS is a strategic enzyme in controlling glutamate concentration in bone environment: GCs decreased the amount of this signalling molecule while vitamin D and Wnt signalling pathway increased it. Interestingly, GS activity and expression declined rapidly when the rMSC derived osteoblasts began to mineralize. Due to its downregulation during osteoblast mineralization, GS could be held as a marker for osteoblast development. Further supporting this, GS activity was stimulated and intracellular glutamate concentration maintained by the N-methyl-d-aspartate (NMDA) type glutamate receptor antagonist MK801, which inhibited osteogenic differentiation of the rMSCs. GS, a novel target for both steroidal and Wnt pathways in bone, might be a central player in the regulation of osteoblastogenesis and/or intercellular signal transmission. Therefore, the proper understanding of the interplay of these three signalling cascades, i.e., steroidal, Wnt, and glutamate signalling, gives vital information on how bone cells communicate together aiming to keep bone healthy.

Genome-wide microarray analysis of MG-63 osteoblastic cells exposed to ultrasound.

It is well documented that low intensity pulsed ultrasound can be clinically used to accelerate bone fracture healing. Additionally, in vitro studies have shown that ultrasound can, for instance, increase mineralization, collagen production and alkaline phosphatase activity in osteoblasts. Despite the extensive research on the subject, the exact mechanism of ultrasound effect on bone cell gene regulation has not yet been deduced. In this study, we made an effort to reveal the features of genome-wide transcriptional response of osteoblast-type cells to ultrasound. MG-63 osteoblastic cell transcriptome was analyzed with whole genome microarray either 6 or 24 h after 30 min long exposure to 1.035 MHz pulsed ultrasound with three different acoustic pressures. Special attention was paid to the experimental design to minimize thermal effects and unwanted reflections of ultrasound. Microarray analysis suggested that ultrasound affects the genes involved with cellular membranes, and regulation of transcription as well. Several plasma membrane solute carriers were also regulated by ultrasound. It also changed the transcript level of several transcription factors belonging to the zinc finger proteins. However, ultrasound did not clearly promote genes involved with osteoblast differentiation.

Body fat mass, lean body mass and associated biomarkers as determinants of bone mineral density in children 6-8years of age - The Physical Activity and Nutrition in Children (PANIC) study.

Lean body mass (LM) has been positively associated with bone mineral density (BMD) in children and adolescents, but the relationship between body fat mass (FM) and BMD remains controversial. Several biomarkers secreted by adipose tissue, skeletal muscle, or bone may affect bone metabolism and BMD. We investigated the associations of LM, FM, and such biomarkers with BMD in children. We studied a population sample of 472 prepubertal Finnish children (227 girls, 245 boys) aged 6-8years. We assessed BMD, LM, and FM using whole-body dual-energy x-ray absorptiometry and analysed several biomarkers from fasting blood samples. We studied the associations of LM, FM, and the biomarkers with BMD of the whole body excluding the head using linear regression analysis. LM (standardized regression coefficient ß=0.708, p<0.001), FM (ß=0.358, p<0.001), and irisin (ß=0.079, p=0.048) were positive correlates for BMD adjusted for age, sex, and height in all children. These associations remained statistically significant after further adjustment for LM or FM. The positive associations of dehydroepiandrosterone sulphate (DHEAS), insulin, homeostatic model assessment for insulin resistance (HOMA-IR), leptin, free leptin index, and high-sensitivity C-reactive protein and the negative association of leptin receptor with BMD were explained by FM. The positive associations of DHEAS and HOMA-IR with BMD were also explained by LM. Serum 25-hydroxyvitamin D was a positive correlate for BMD adjusted for age, sex, and height and after further adjustment for FM but not for LM. LM and FM were positive correlates for BMD also in girls and boys separately. In girls, insulin, HOMA-IR, leptin, and free leptin index were positively and leptin receptor was negatively associated with BMD adjusted for age, height, and LM. After adjustment for age, height, and FM, none of the biomarkers was associated with BMD. In boys, leptin and free leptin index were positively and leptin receptor was negatively associated with BMD adjusted for age, height, and LM. After adjustment for age, height and FM, 25(OH)D was positively and IGF-1 and leptin were negatively associated with BMD. FM strongly modified the association between leptin and BMD. LM but also FM were strong, independent positive correlates for BMD in all children, girls, and boys. Irisin was positively and independently associated with BMD in all children. The associations of other biomarkers with BMD were explained by LM or FM.

Serum 25-Hydroxyvitamin D, Plasma Lipids, and Associated Gene Variants in Prepubertal Children.

Context: The associations of serum 25-hydroxyvitamin D [25(OH)D] with plasma lipids remain controversial in children. Objective: To examine the associations and interactions of 25(OH)D and related gene variants with lipids in children. Design: Cross-sectional. Setting: Kuopio, Finland. Participants: Population sample of 419 prepubertal white children aged 6 to 8 years. Main Outcome Measures: 25(OH)D, total cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, and triglycerides. Results: Serum 25(OH)D was negatively associated with total cholesterol (ß = -0.141, P = 0.004), LDL cholesterol (ß = -0.112, P = 0.023), HDL cholesterol (ß = -0.150, P = 0.002), and triglycerides (ß = -0.104, P = 0.035) adjusted for age and sex. Associations of 25(OH)D with total cholesterol, LDL cholesterol, and HDL cholesterol remained after adjustment for adiposity, physical activity, sedentary behavior, diet, daylight time, and parental education. Children in the highest quartile of 25(OH)D had the lowest total cholesterol (P = 0.022) and LDL cholesterol (P = 0.026) adjusted for age and sex. Cytochrome P450 family 2 subfamily R member 1 (CYP2R1) rs12794714, CYP2R1 rs10741657, and vitamin D binding protein (DBP) rs2282679 were associated with 25(OH)D adjusted for age and sex. CYP2R1 rs12794714 was associated with total cholesterol and LDL cholesterol and C10orf88 rs6599638 with HDL cholesterol adjusted for age, sex, and 25(OH)D. The gene variants did not explain or modify the associations of 25(OH)D with lipids. Conclusions: 25(OH)D was independently and inversely associated with total cholesterol, LDL cholesterol, and HDL cholesterol. CYP2R1 rs12794714, CYP2R1 rs10741657, and DBP rs2282679 were associated with 25(OH)D. CYP2R1 rs12794714 was associated with total cholesterol and LDL cholesterol and chromosome 10 open reading frame 88 (C10orf88) rs6599638 with HDL cholesterol independent of 25(OH)D. None of the gene variants modified the associations of 25(OH)D with lipids. Further studies are needed to detect the mechanisms for the associations of 25(OH)D with lipids.

Student-focused virtual histology education: Do new scenarios and digital technology matter?

This article was migrated. The article was marked as recommended. Innovative changes have become a critical part of teaching when resources are limited. In this study, we examined whether the student-oriented teaching method, when powered by virtual microscopy, improves histology learning compared to traditional microscope-based studies. Anonymous and voluntary post-course surveys were administered to students and essays were processed for content analysis. Google Analytics was used to obtain accurate Internet usage monitoring for WEBMICROSCOPE®. Using SPSS statistics, the examination scores for 2016 were compared to those of previous year, when the course was taught with a traditional-microscope-based model. The results demonstrated that the new teaching scenario was an effective tool, based on the mean examination scores in 2016 compared to the identical groups in 2015. The survey analysis showed that the students benefited more from using WEBMICROSCOPE® and that they frequently gained access to the Web server when they were not in class. The new scenario helped clarify the concept of histology for most of the students and was generally appreciated during teamwork-based histology classes. Students perceived that the use of the digital technology significantly influenced their confidence in learning the fundamentals of histology. In addition, changing to the new teaching scenario powered by WEBMICROSCOPE® improved the students' motivation to participate in discussions and better understand the concept of Histology between the 2015 and 2016 academic years. Finally, these changes all had a positive impact on the students' attention and satisfaction.

COL1A1 Sp1 polymorphism associates with bone density in early puberty.

Optimal acquisition of bone mass in puberty is a key determinant of the lifetime risk of osteoporosis and has a strong genetic basis. We investigated the relationship between the COL1A1 Sp1 polymorphism and BMD in early puberty, and how the genotypes relate to bone size and geometry as well as bone turnover and material properties in 247 10- to 13-year-old girls. Bone properties were measured using DXA, pQCT, and ultrasound. Also, serum P1NP, OC, B-ALP, and TRACP 5b were assessed. Our results showed that girls with the TT genotype had significantly lower BMC and BMD of the total body, lumbar spine, and proximal femur, as well as BUA at the calcaneus, than those with the GT and GG genotype. They also had significantly lower B-ALP, as well as P1NP/TRACP 5b and (OC + B-ALP)/TRACP 5b, compared to the others. These findings indicate that the COL1A1 polymorphism is associated with low bone properties in early puberty and suggest a possible physiological effect on collagen metabolism and bone turnover. This information may contribute to the identification of children at risk for suboptimal acquisition of peak bone mass and may ultimately be of value in the planning of early preventive strategies for osteoporosis.

Estrogen receptor alpha genotype confers interindividual variability of response to estrogen and testosterone in mesenchymal-stem-cell-derived osteoblasts.

Hormone replacement therapy is effectively used to prevent postmenopausal bone loss. Variation in response to the therapy is, however, frequently seen. In addition, the direct effects of sex steroids on isolated human bone marrow stromal cells have been reported to vary depending on the donor, but the biological mechanisms are not understood. The aim of this study was to investigate the effects of 17beta-estradiol (E2) and testosterone in human-bone-marrow-derived mesenchymal stem cell (MSC) cultures from both female and male donors of various ages. The osteoblast differentiation capacity and activity of the MSCs were quantified in vitro by measuring alkaline phosphatase activity and calcium deposition. We show here that also the osteoblast responses of MSCs to sex hormones vary widely depending on the donor. When the results from all donors were analyzed together, treatment with E2 increased calcium deposition significantly by MSCs of both sexes but ALP activity only in the male MSCs. Testosterone had no effect on ALP activity nor calcium deposition in either sex. To further characterize the individual variation, we investigated estrogen receptor alpha PvuII restriction site polymorphism with PCR. Restriction fragment-length polymorphism was assigned as P or non-P, P signifying the absence of the restriction site. Our results indicate that higher basal osteoblast differentiation capacity of MSCs is associated with the presence of the P allele in females, whereas higher response to sex steroids treatment is associated with the non-P allele. These results could help explain the contradictory effects of E2 on osteoblasts in vitro and might also provide new insights to understanding the differences in responses to hormone replacement therapy.

Effects of calcium, dairy product, and vitamin D supplementation on bone mass accrual and body composition in 10-12-y-old girls: a 2-y randomized trial.

BACKGROUND: Little is known about the relative effectiveness of calcium supplementation from food or pills with or without vitamin D supplementation for bone mass accrual during the rapid growth period. OBJECTIVE: The purpose was to examine the effects of both food-based and pill supplements of calcium and vitamin D on bone mass and body composition in girls aged 10-12 y. DESIGN: This placebo-controlled intervention trial randomly assigned 195 healthy girls at Tanner stage I-II, aged 10-12 y, with dietary calcium intakes <900 mg/d to 1 of 4 groups: calcium (1000 mg) + vitamin D3 (200 IU), calcium (1000 mg), cheese (1000 mg calcium), and placebo. Primary outcomes were bone indexes of the hip, spine, and whole body by dual-energy X-ray absorptiometry and of the radius and tibia by peripheral quantitative computed tomography. RESULTS: With the use of intention-to-treat or efficacy analysis, calcium supplementation with cheese resulted in a higher percentage change in cortical thickness of the tibia than did placebo, calcium, or calcium + vitamin D treatment (P = 0.01, 0.038, and 0.004, respectively) and in higher whole-body bone mineral density than did placebo treatment (P = 0.044) when compliance was >50%. With the use of a hierarchical linear model with random effects to control for growth velocity, these differences disappeared. CONCLUSIONS: Increasing calcium intake by consuming cheese appears to be more beneficial for cortical bone mass accrual than the consumption of tablets containing a similar amount of calcium. Diverse patterns of growth velocity may mask the efficacy of supplementation in a short-term trial of children transiting through puberty.

Adenovirus-mediated VEGF-A gene transfer induces bone formation in vivo.

Osteoporosis is a major problem in elderly population. We tested the hypothesis whether vascular endothelial growth factor (VEGF-A) gene transfer is an appropriate way to enhance bone formation and recruitment of osteoblasts in vivo. Adenovirus vectors containing VEGF-A or lacZ cDNAs (1.4x10(10) pfu) were injected locally into right distal femurs of New Zealand White rabbits. Saline was injected into all contralateral distal femurs. One and three weeks after the gene transfers femurs were collected for analyses. X-Gal staining showed that up to 20% of the bone marrow cells were transfected although gene transfer also resulted in biodistribution of the vector and expression of the transgene in liver and spleen. Trabecular bone hard tissue histomorphometry of the distal femurs was performed to analyze the effect of gene transfer on bone turnover. When compared with unilateral lacZ transfected trabecular bone at one-week and three-week time points, VEGF-A gene transfer significantly increased bone formation parameters, such as osteoblast number, osteoid volume, and bone volume. Also, bone resorption surface was greatly reduced. It is concluded that injection of adenovirus vector can transfect bone marrow cells in vivo with a relatively high efficiency. Our results suggest that adenovirus-mediated VEGF-A gene transfer induces bone formation via increasing osteoblast activity and may be useful for the treatment of osteoporosis and other diseases that require efficient osteogenic therapy.

Association between exercise and pubertal BMD is modulated by estrogen receptor alpha genotype.

UNLABELLED: Genetic and environmental factors contribute to bone mass, but the ways they interact remain poorly understood. This study of 245 pre- and early pubertal girls found that the PvuII polymorphism in the ER-alpha gene modulates the effect of exercise on BMD at loaded bone sites. INTRODUCTION: Impaired achievement of bone mass at puberty is an important risk factor for the development of osteoporosis in later life. Genetic, as well as environmental, factors contribute to bone mass, but the ways they interact with each other remain poorly understood. MATERIALS AND METHODS: We investigated the interaction between a PvuII polymorphism at the ER-alpha gene and physical activity (PA) on the modulation of bone mass and geometry in 245 10- to 13-year-old pre- and early pubertal Finnish girls. Level of PA was assessed using a questionnaire. Bone properties were measured using DXA and pQCT. The analyses were controlled for the effects of Tanner stage and body size index. RESULTS: Girls with heterozygote ER-alpha genotype (Pp) and high PA had significantly higher bone mass and BMD, as well as thicker cortex, at loaded bone sites than their low-PA counterparts. No differences were found in bone properties of the distal radius, which is not a weight-bearing bone. Bone properties did not differ in either homozygote groups (PP and pp) regardless of the PA level. CONCLUSIONS: These findings suggest that the PvuII polymorphism in the ER-alpha gene may modulate the effect of exercise on BMD at loaded bone sites. The heterozygotes may benefit most from the effect of exercise, whereas neither of the homozygote groups received any significant improvement from high PA. Furthermore, high PA may hide the genetic influence on bone. Indeed, it seems that one may compensate one's less favorable Pp genotype by increasing leisure PA at early puberty.

Relation of androgen receptor gene polymorphism to bone mineral density and fracture risk in early postmenopausal women during a 5-year randomized hormone replacement therapy trial.

In women, the influence of androgens on bone health is not clear. It has been suggested that the androgen receptor (AR) genotype is associated with bone mineral density and serum androgen levels in pre- and perimenopausal women, but the association between AR genotype, bone mineral density, and fracture risk has not been studied in postmenopausal women. Therefore, we studied whether AR polymorphism affects bone mineral density, bone mineral density change, or fracture risk in a 5-year randomized hormone replacement therapy (HRT) trial on 331 early postmenopausal women (mean baseline age, 52.7 +/- 2.3 years). The participants consisted of two treatment groups: the HRT group (n = 151) received a sequential combination of 2 mg estradiol valerate and 1 mg cyproterone acetate with or without vitamin D3, 100-300 IU + 93 mg calcium as lactate/day, and the non-HRT group (n = 180) received 93 mg calcium alone or in combination with vitamin D3, 100-300 IU/day for 5 years. Bone mineral density was measured from lumbar spine and proximal femur (DXA) before and after the 5-year trial. All new symptomatic, radiographically defined fractures were recorded during the follow-up. The length of CAG repeat in exon 1 of AR gene was evaluated after polymerase chain reaction (PCR) amplification. The subjects were divided into three repeat groups according to AR alleles. None of the baseline characteristics were associated with AR gene polymorphism and HRT treatment. The polymorphism did not influence the calculated annual changes of lumbar or femoral neck bone mineral density during the 5-year follow-up in the HRT (p = 0.926 and 0.146, respectively) or non-HRT (p = 0.818 and 0.917, respectively) groups. In all, 28 women sustained 33 fractures during the follow-up. Thus, the numbers of fractures were limited. The AR repeat length variation was not significantly associated with fracture risk in the HRT or non-HRT groups (p = 0.632 and 0.459, respectively; Cox proportional hazards model). In conclusion, AR gene polymorphism was not associated with baseline bone mineral density, 5-year bone mineral density change, or fracture risk in early postmenopausal Finnish women.

Relation of PvuII site polymorphism in the COL1A2 gene to the risk of fractures in prepubertal Finnish girls.

Genetic susceptibility to fractures may be detectable in early childhood. We evaluated the associations between the polymorphic PvuII site of the COL1A2 gene and bone properties assessed by different modalities (dual-energy X-ray absorptiometry; peripheral quantitative computed tomography; gel coupling scanning quantitative ultrasonometry; ultrasound bone sonometry), bone turnover markers, and the occurrence of fractures in 244 prepubertal Finnish girls. Tanner stage and physical characteristics did not differ significantly among girls with different COL1A2 genotypes. The polymorphism was not significantly associated with different bone properties or any of the bone turnover markers when girls at Tanner stage I (prepuberty) and stage II (early puberty) were considered together, but there was a significant association with spine bone mineral content (BMC) and bone mineral density (BMD), as well as with speed of sound (SOS) (P < 0.05), when girls at Tanner stage I were considered separately, as a purpose to avoid the confounding effect that the pubertal growth spurt has on skeletal development. The distribution of fractures was different between the three genotype groups (P = 0.023). The P alleles were over-represented in girls who had been fractured at least once; 88% of them had at least one copy of the P allele (either PP or Pp). Girls with the PP genotype had 4.9 times higher relative risk for fractures than girls with the pp genotype (95% CI, 1.4 to 17.4; P = 0.015). No significant difference was found between fractured and nonfractured girls in anthropometric measurements, physical activity, or bone mass. However, BMD of the spine and SOS at the radius and tibia were significantly lower in the fractured girls. We conclude that the COL1A2 polymorphism is associated with nonosteoporotic fractures in prepubertal girls independently of bone density.

Association of low 25-hydroxyvitamin D concentrations with elevated parathyroid hormone concentrations and low cortical bone density in early pubertal and prepubertal Finnish girls.

BACKGROUND: Very few studies have evaluated both parathyroid hormone (PTH) and 25-hydroxyvitamin D [25(OH)D] and their effects on bone mass in children. OBJECTIVE: We studied the associations of serum 25(OH)D and intact PTH (iPTH) with bone mineral content (BMC) and bone mineral density (BMD) at different bone sites and the relation between serum 25(OH)D and iPTH in early pubertal and prepubertal Finnish girls. DESIGN: The subjects were 10-12-y-old girls (n = 193) at Tanner stage 1 or 2, who reported a mean (+/- SD) dietary calcium intake of 733 +/- 288 mg/d. 25(OH)D, iPTH, tartrate-resistant acid phosphatase 5b (TRAP 5b), urinary calcium excretion, BMC, areal BMD, and volumetric BMD were assessed by using different methods. RESULTS: Thirty-two percent of the girls were vitamin D deficient [serum 25(OH)D < or = 25 nmol/L], and 46% of the girls had an insufficient concentration (26-40 nmol/L). iPTH and TRAP 5b concentrations were significantly higher in the deficient group than in the insufficient and sufficient groups [iPTH: 43.9 +/- 15.7 compared with 38.6 +/- 11.2 pg/L (P = 0.049) and 32.7 +/- 12.1 pg/L (P < 0.001), respectively; TRAP 5b: 12.2 +/- 2.9 compared with 11.0 +/- 2.8 U/L (P = 0.009) and 10.9 +/- 1.9 U/L (P = 0.006), respectively]. The girls in the deficient group also had significantly lower cortical volumetric BMD of the distal radius (P < 0.001) and tibia shaft (P = 0.002). High iPTH concentrations were also associated with low total-body apparent mineral density and urinary calcium excretion (P < 0.007). CONCLUSIONS: Vitamin D-deficient girls have low cortical BMD and high iPTH concentrations, which are consistent with secondary hyperparathyroidism. A low vitamin D concentration accompanied by high bone resorption (TRAP 5b) may limit the accretion of bone mass in young girls.

Inhibition of MG-63 cell cycle progression by synthetic vitamin D3 analogs mediated by p27, Cdk2, cyclin E, and the retinoblastoma protein.

Progression through eukaryotic cell division cycle is regulated by synergistic activities of both positive and negative regulatory factors. The active form of vitamin D(3) (1alpha,25(OH)(2)D(3), 1,25D) and a number of its synthetic analogs have been shown to arrest cells in the G(1) phase of the cell cycle. In the present study, 1alpha,25(OH)(2)D(3) and the analogs KH1060, EB1089, and CB1093 were used to study the mechanism of the cell cycle arrest and to compare the effectiveness of these compounds in human MG-63 osteosarcoma cells. The 20-epi analogs KH1060 and CB1093, as well as the 20-normal analog EB1089, were found to be more potent than 1alpha,25(OH)(2)D(3) in inhibiting cell proliferation and arresting the MG-63 cells in the G(1) phase. These analogs were more active than 1alpha,25(OH)(2)D(3) in increasing the cyclin dependent kinase inhibitor p27 protein levels (approximately 2.3-2.5-fold compared to 1alpha,25(OH)(2)D(3)) by both increasing its formation and decreasing its degradation rate. The increased p27 formation was accompanied by stabilization of binding of nuclear proteins to the Sp1+NF-Y responsive promoter region of the p27 gene. The increase in p27 protein levels and the simultaneous decrease in cyclin E protein levels was accompanied by decreased Cdk2 kinase activity, retinoblastoma (Rb) protein hypophosphorylation and, finally, cell cycle arrest in the G(1) phase. In summary, the analogs KH1060, EB1089, and CB1093 keep Rb protein in its growth-suppressing, hypophosphorylated form and prevent cell cycle progression through the restriction point. Therefore, these synthetic vitamin D(3) analogs may be potential candidates for treating diseases, where cell cycle regulation is needed.

Nonuniform temperature rise in in vitro osteoblast ultrasound exposures with associated bioeffect.

There is a growing interest to use ultrasound to stimulate cellular material in vitro conditions for the treatment of musculoskeletal disorders. However, the beneficial effect resulting from ultrasound exposure is not accurately specified. Many in vitro ultrasound setups are very vulnerable to temperature elevation due to sound absorption, sound reflections, and inadequate heat transfer. The objective of this study is to show that temperature variations capable of modifying biological results may exist in common in vitro exposure system. Human osteoblastic MG-63 cells plated on a 24-well cell plate were treated with pulsed ultrasound in 37 °C water bath (10 min, frequency = 1.035 MHz, burst length = 200 µs, pulse repetition frequency = 1 kHz, duty cycle = 0.2, temporal-average acoustic power = 2 W, and peak pressure = 670-730 kPa) and the activation of heat-dependent canonical Wnt cell signaling was measured. The ultrasound-induced temperature rise was measured with thermocouples and infrared imaging. Chamber-to-chamber comparison showed substantial temperature variation (41.6 °C versus 49.1 °C) among the different chambers. The chamber walls were the most susceptible to heating. The variations in the chamber temperatures correlated to variations in the cell signaling levels (1.3-fold versus 11.5-fold increase). These observations underline the need for system-specific temperature measurements during in vitro exposures.

Ultrasound-induced activation of Wnt signaling in human MG-63 osteoblastic cells.

The benefit from an ultrasound (US) exposure for fracture healing has been clearly shown. However, the molecular mechanisms behind this effect are not fully known. Recently, the canonical Wnt signaling pathway has been recognized as one of the essential regulators of osteoblastogenesis and bone mass, and thereby considered crucial for bone health. Mechanical loading and fluid shear stress have been reported to activate the canonical Wnt signaling pathway in bone cells, but previous reports on the effects of therapeutic US on Wnt signaling in general or in bone, in particular, have not been published yet. Therefore, activation of Wnt signaling pathway was assayed in human osteoblastic cells, and indeed, this pathway was found to be activated in MG-63 cells through the phosphoinositol 3-kinase/Akt (PI3K/Akt) and mTOR cascades following a single 10 min US exposure (2 W, 1.035 MHz). In addition to the reporter assay results, the Wnt pathway activation was also observed as nuclear localization of beta-catenin. Wnt activation showed also temperature dependence at elevated temperatures, and the expression of canonical Wnt ligands was induced under the thermal exposures. However, existence of a specific, non-thermal US component was evident as well, perhaps evidence of a potential dual action of therapeutic US on bone. Neither US nor heat exposures affected cell viability in our experiments. In summary, this is the first study to report that Wnt signaling cascade, important for osteoblast function and bone health, is one of the pathways activated by therapeutic US as well as by hyperthermia in human osteoblastic cells. Our results provide evidence for the potential molecular mechanisms behind the beneficial effects of US on fracture healing. Combinations of US, heat, and possible pharmacological treatment could provide useful flexibility for clinical cases in treating various bone disorders.

Calreticulin mediated glucocorticoid receptor export is involved in beta-catenin translocation and Wnt signalling inhibition in human osteoblastic cells.

Wnt signalling pathway is a multicomponent cascade involving interaction of several proteins and found to be important for development and function of various cells and tissues. There is increasing evidence that the Wnt/beta-catenin pathway constitutes also one of the essential molecular mechanisms controlling the metabolic aspects of osteoblastic cells. However, in bone, glucocorticoids (GCs) have been reported to weaken Wnt signalling. Therefore, the aim of this study was to characterize the mechanisms behind the cross-talk of these two signalling pathways in human osteoblastic cells. Based on our findings, liganded glucocorticoid receptor (GR) modulated Wnt signalling pathway by decreasing beta-catenin's nuclear accumulation and increasing its relocalization to cell membranes rather than affecting its degradation in human osteoblastic cells. The region of GR responsible for this inhibitory effect located into an area, which harbours the DNA binding as well as nuclear export domains. In further studies, a chaperone protein calreticulin (CRT), known to bind the DNA binding domain of GR and regulate receptor export, was found to be involved in the GR-mediated downregulation of Wnt signalling: GR mutants containing incomplete CRT binding sites were not able to translocate beta-catenin to cell surface. In addition, the inhibitory effect of GCs on endogenous Wnt target gene, cyclin D1, was abolished, when the expression of CRT was attenuated by the RNAi technique. Furthermore, GR and beta-catenin were shown to exist in the same immunocomplex, while interaction between CRT and beta-catenin was observed only in the presence of GR as a mediator molecule. In addition, the GR mutant lacking CRT binding ability impaired the complex formation between beta-catenin and CRT. Together with GR, beta-catenin could thus be co-transported from the nucleus in a CRT-dependent way. These observations represent a novel mechanism for GCs to downregulate Wnt signalling pathway in human osteoblastic cells. Knowledge of these molecular mechanisms is important for understanding the network of multiple signalling cascades in bone environment. Functional Wnt signalling pathway is a prerequisite for proper osteoblastogenesis, and this modulative cross-talk between the steroid pathway and Wnt cascade could therefore explain some of the two-edged effects of GCs on osteoblastic differentiation and function.