Reference intervals of 24 trace elements in blood, plasma and erythrocytes for the Slovenian adult population.

OBJECTIVES: The aim of the present study was to establish the population- and laboratory-specific reference intervals (RIs) for the Slovenian adult population for 24 trace elements (TEs) in blood, plasma and erythrocytes and to evaluate the impact of gender, age, seafood consumption, smoking habits and amalgam fillings on TEs levels. METHODS: TEs (Mn, Co, Cu, Zn, Se and Mo, Li, Be, V, Cr, Ni, Ga, As, Rb, Sr, Ag, Cd, Sn, Cs, Au, Hg, Tl, Pb and U) were determined in 192 a priori selected blood donors (107 women and 85 men, aged 18-65 years), using inductively coupled plasma mass spectrometry (ICP-MS) with the Octopole Reaction System. Participants filled out a questionnaire, and RIs were established according to the Clinical and Laboratory Standards Institute (CLSI) guidelines for TEs. RESULTS: Uniform RIs for non-essential and gender-specific for essential TEs in blood, plasma and erythrocytes were established. In our population, higher blood and plasma Cu, and erythrocyte Mn levels in women were found. In men, blood Zn, plasma Zn, Mn and Se, and erythrocyte Cu levels were higher. Zn levels were higher in 30-39 years age group. Pb and Sr increased with age. Smoking positively affected Cd, Pb, Cs and Rb; seafood consumption increased As, Hg and Zn; and amalgam increased Hg, Ag and Cu levels. CONCLUSIONS: Essential TEs were inside recommended levels, and the non-essential ones were far below critical levels. Established RIs will provide an important foundation for clinical diagnostics, safety erythrocyte transfusions assessment, toxicology and epidemiological studies.

TBP, PPIA, YWHAZ and EF1A1 Are the Most Stably Expressed Genes during Osteogenic Differentiation.

RT-qPCR is the gold standard and the most commonly used method for measuring gene expression. Selection of appropriate reference gene(s) for normalization is a crucial part of RT-qPCR experimental design, which allows accurate quantification and reliability of the results. Because there is no universal reference gene and even commonly used housekeeping genes' expression can vary under certain conditions, careful selection of an appropriate internal control must be performed for each cell type or tissue and experimental design. The aim of this study was to identify the most stable reference genes during osteogenic differentiation of the human osteosarcoma cell lines MG-63, HOS, and SaOS-2 using the geNorm, NormFinder, and BestKeeper statistical algorithms. Our results show that TBP, PPIA, YWHAZ, and EF1A1 are the most stably expressed genes, while ACTB, and 18S rRNA expressions are most variable. These data provide a basis for future RT-qPCR normalizations when studying gene expression during osteogenic differentiation, for example, in studies of osteoporosis and other bone diseases.

Glucocorticoid Receptor Regulates TNFSF11 Transcription by Binding to Glucocorticoid Responsive Element in TNFSF11 Proximal Promoter Region.

Glucocorticoid osteoporosis is a serious side effect of long term glucocorticoid uptake and it is caused by osteoblast apoptosis and imbalance in the major bone remodeling pathway RANK/RANKL/OPG. The impact of glucocorticoid on the maintenance of RANK/RANKL/OPG is well explored; dexamethasone was shown to disturb the ratio between OPG and RANKL level by decreasing the expression level of OPG and increasing level of RANKL. Here, were aimed to decipher whether glucocorticoid receptor directly influences RANKL promoter activity and its transcriptional regulation. We demonstrate that overexpression of glucocorticoid receptor (GR) NR3C1 increased RANKL promoter activity in human osteosarcoma, cervical cancer (2-fold) and adenocarcinoma cells (4.5-fold). Mutational analysis revealed that +352 site in the RANKL promoter is functional glucocorticoid responsive element (GRE) since the effect of GR on RANKL promoter activity was diminished by mutation at this site. Overexpression of NR3C1 upregulated RANKL mRNA expression 1.5-fold in human A549 and HOS cells. On the other hand silencing of NR3C1 caused slight decrease in RANKL mRNA level, suggesting that NR3C1 directly accounts for RANKL transcriptional regulation. Using electrophoretic mobility shift assay we demonstrate that NR3C1 binds to the proximal RANKL promoter region. Our study provides evidences that NR3C1 directly upregulates RANKL transcription in human cell lines and connects the missing link in the mechanism of RANK/RANKL/OPG imbalance of glucocorticoid induced osteoporosis.

Comprehensive analysis of skeletal muscle- and bone-derived mesenchymal stem/stromal cells in patients with osteoarthritis and femoral neck fracture.

BACKGROUND: Mesenchymal stem/stromal cells (MSCs) can replenish the aged cells of the musculoskeletal system in adult life. Stem cell exhaustion and decrease in their regenerative potential have been suggested to be hallmarks of aging. Here, we investigated whether muscle- and bone-derived MSCs of patients with osteoarthritis and osteoporosis are affected by this exhaustion, compared to healthy donors. METHODS: Patients with primary osteoarthritis, femoral neck fractures due to osteoporosis, and healthy donors (controls) were included. MSCs were isolated from the skeletal muscle and subchondral bone from each patient and compared using ex vivo and in vitro analyses, including immunophenotyping, colony-forming unit fibroblast assays, growth kinetics, cell senescence, multilineage potential, and MSC marker gene expression profiling. RESULTS: Freshly isolated cells from muscle from patients with osteoarthritis showed a lower proportion of CD45/CD19/CD14/CD34-negative cells compared to patients with osteoporosis and healthy donors. Freshly isolated muscle cells from patients with osteoarthritis and osteoporosis also showed higher clonogenicity compared to healthy donors. MSCs from both tissues of osteoarthritis patients showed significantly reduced osteogenesis and MSCs from the bone also reduced adipogenesis. Chondrogenic pellet diameter was reduced in bone-derived MSCs from both patient groups compared to healthy donors. A significant positive correlation was observed between adipogenesis and CD271 expression in muscle-derived MSCs. CD73 was significantly lower in bone-derived MSCs from osteoarthritis patients, compared to osteoporosis patients. Gene expression profiling showed significantly lower expression of MSC marker gene leptin receptor, LEPR, previously identified as the major source of the bone and adipocytes in the adult bone marrow, in bone-derived MSCs from patients with osteoarthritis in comparison with osteoporotic patients and healthy donors. CONCLUSIONS: Our results show deficient ex vivo and in vitro properties of both skeletal muscle- and bone-derived MSCs in osteoarthritis and osteoporosis patients, compared to healthy donors. In bone-derived MSCs from patients with osteoarthritis, we also identified a lower expression of the leptin receptor, a marker of MSCs that present a major source of MSCs in the adult bone marrow. This suggests that exhaustion of skeletal muscle- and bone-derived MSCs is a hallmark of osteoarthritis and osteoporosis, which defines the need for further clinical trials of stem cell transplantation in these patients.

Increased Exhaustion of the Subchondral Bone-Derived Mesenchymal Stem/ Stromal Cells in Primary Versus Dysplastic Osteoarthritis.

Mesenchymal stem/ stromal cell (MSC) exhaustion has been suggested to be a hallmark of aging. Osteoarthritis has a complex etiology that comprises several factors. Dysplasia has been shown to be an individual risk factor for osteoarthritis. Subchondral bone changes are often the first detectable alterations in osteoarthritis. In this study, we aimed to determine whether skeletal MSCs are differentially affected in patients with primary versus dysplastic osteoarthritis. Patients undergoing hip arthroplasty due to primary osteoarthritis (n = 11) and osteoarthritis with hip dysplasia (n = 10) were included in the study. Femoral head subchondral bone was used for isolation of MSCs. The cells were compared using detailed ex-vivo and in-vitro analyses, which included immunophenotyping, colony-forming-unit fibroblast assay, growth kinetics, senescence, multilineage potential, immunophenotyping, and MSC marker-gene expression profiling. Isolated cells from primary osteoarthritis patients showed decreased viability in comparison with those from dysplasia patients, with similar mesenchymal fractions (i.e., CD45/ CD19/ CD14/ CD34-negative cells). In-vitro expanded MSCs from primary osteoarthritis patients showed reduced osteogenic and chondrogenic potential in comparison with dysplasia patients. There were no differences in clonogenicity, growth kinetics, senescence, adipogenic potential, and immunophenotype between these groups. Gene expression profiling showed well-known marker of bone marrow MSCs, the leptin receptor, to be significantly lower for primary osteoarthritis patients. Our study shows that the pathology of primary osteoarthritis is accompanied by bone MSC exhaustion, while biomechanical dysfunction associated with hip dysplasia can induce secondary osteoarthritis without this MSC impairment. Our study suggests that subchondral bone MSC exhaustion is implicated in the pathology of primary osteoarthritis.

Sex-determining region Y (SRY) attributes to gender differences in RANKL expression and incidence of osteoporosis.

Receptor activator of nuclear factor κB ligand (RANKL) plays a crucial role in bone metabolism. RANKL gene misregulation has been implicated in several bone and cancer diseases. Here, we aimed to identify novel transcription regulators of RANKL expression. We discovered that transcription factors, sex-determining region Y (SRY) and c-Myb, regulate RANKL expression. We demonstrated that c-Myb increases and male-specific SRY decreases RANKL expression through direct binding to its 5'-proximal promoter. These results are corroborated by the gene expression in human bone samples. In osteoporotic men, expression of RANKL is 17-fold higher, which correlates with the drastically reduced expression (200-fold) of Sry, suggesting that in osteoporotic men, the upregulation of RANKL is caused by a decrease of Sry. In healthy men, the expression of RANKL is 20% higher than that in healthy women. Our data suggest that gender differences in RANKL expression and bone quality could be due to the sex-specific transcription factor SRY.

Skeletal-muscle-derived mesenchymal stem/stromal cells from patients with osteoarthritis show superior biological properties compared to bone-derived cells.

Mesenchymal stem/stromal cells (MSCs) are being exploited for patient-derived stem-cell therapies. As the biological properties of MSCs derived from skeletal muscle of osteoarthritis patients are poorly understood, the aim of this study was to compare muscle MSCs with well-recognized bone and bone marrow-derived MSCs from these patients. Paired samples of skeletal muscle and trabecular bone tissue were obtained from 21 patients with osteoarthritis. Isolated cells were compared using ex vivo immunophenotyping and detailed in vitro analyses. These included the colony forming unit fibroblast assay, growth kinetics, senescence, multilineage potential, immunophenotyping, and MSC marker gene expression profiling. Freshly isolated MSCs from muscle showed improved viability over bone-derived MSCs, with similar mesenchymal fraction. Muscle-derived MSCs showed superior clonogenicity, higher growth rates, and lower doubling times. Muscle-derived MSCs also showed superior osteogenic and myogenic properties and a positive correlation between CD271 expression and adipogenesis. Senescence rate as well as adipogenic and chondrogenic potentials were similar. Skeletal muscle-derived MSCs of osteoarthritis patients have superior clonogenicity and growth kinetics compared to bone-derived MSCs, making them a good candidate for autologous stem-cell therapies. Moreover, the positive correlation between CD271 and adipogenesis suggest that CD271 expressing muscle MSCs might contribute to muscle steatosis observed in osteoarthritis.

Human Skeletal Muscle-Derived Mesenchymal Stem/Stromal Cell Isolation and Growth Kinetics Analysis.

The most studied sources of mesenchymal stem/stromal cells (MSCs) are bone marrow and adipose tissue. However skeletal muscle represents an interesting source of diverse subpopulations of MSCs, such as paired box 7 (Pax-7)-positive satellite cells, fibro-/adipogenic progenitors, PW1-positive interstitial cells and others. The specific properties of some of these muscle-derived cells have encouraged the development of cell therapies for muscle regeneration. However, the identity and multilineage potential of the diverse muscle-resident cells should first be evaluated in vitro, followed by in vivo clinical trials to predict their regenerative capacity. Here, we present protocols for the isolation of MSCs from skeletal muscle using enzymatic digestion and mechanical trituration. We also provide a method to determine their specific growth rate, a feature that is of particular interest when designing cell therapies.

Increased Plasma Cathepsin s at the Time of Percutaneous Transluminal Angioplasty is Associated with 6-months' Restenosis of the Femoropopliteal Artery.

BACKGROUND: We tested the hypothesis that increased levels of cathepsin S and decreased levels of cystatin C in plasma at the time of percutaneous transluminal angioplasty (PTA) are associated with the occurrence of 6-months' restenosis of the femoropopliteal artery (FPA). METHODS: 20 patients with restenosis and 24 matched patients with patent FPA after a 6-months follow-up were in - cluded in this study. They all exhibited disabling claudication or critical limb ischemia and had undergone technically successful PTA. They were all receiving statins and ACE in hi - bitors (or angiotensin II receptor antagonist) before the PTA and the therapy did not change throughout the observational period. Plasma concentrations of C-reactive protein were < 10 mg/L and of creatinine within the reference range at the time of the PTA. Plasma concentration and activity of cathepsin S, together with its potent inhibitor cystatin C, were measured the day before and the day after the PTA. RESULTS: The increased plasma concentration and activity of cathepsin S at the time of PTA was associated with the occurrence of 6-months' restenosis of FPA, independently of established risk factors (lesion complexity, infrapopliteal run-off vessels, type of PTA, age, gender, smoking, diabetes, lipids) and of cystatin C. Plasma cystatin C concentration was not associated with restenosis and did not correlate with cathepsin S activity and concentration in the plasma. CONCLUSION: Increased level of plasma cathepsin S at the time of PTA is associated with 6-months' restenosis of PTA, independently of established risk factors.

Epigenetic enzymes influenced by oxidative stress and hypoxia mimetic in osteoblasts are differentially expressed in patients with osteoporosis and osteoarthritis.

Epigenetic mechanisms including posttranslational histone modifications and DNA methylation are emerging as important determinants of bone homeostasis. With our case-control study we aimed to identify which chromatin-modifying enzymes could be involved in the pathology of postmenopausal osteoporosis and osteoarthritis while co-regulated by estrogens, oxidative stress and hypoxia. Gene expression of HAT1, KAT5, HDAC6, MBD1 and DNMT3A affected by oxidative stress and hypoxia in an in vitro qPCR screening step performed on an osteoblast cell line was analysed in trabecular bone tissue samples from 96 patients. Their expression was significantly reduced in patients with postmenopausal osteoporosis and osteoarthritis as compared to autopsy controls and significantly correlated with bone mineral density and several bone histomorphometry-derived parameters of bone quality and quantity as well as indicators of oxidative stress, RANK/RANKL/OPG system and angiogenesis. Furthermore, oxidative stress increased DNA methylation levels at the RANKL and OPG promoters while decreasing histone acetylation levels at these two genes. Our study is the first to show that higher expression of HAT1, HDAC6 and MBD1 is associated with superior quantity as well as quality of the bone tissue having a more favourable trabecular structure.

Genetic effects on bone health.

PURPOSE OF REVIEW: In recent years, the lower costs of arrays and sequencing technologies, and the better availability of data from genome-wide association studies (GWASs) have led to more reports on genetic factors that are associated with bone health. However, there remains the need for a summary of the newly identified genetic targets that are associated with bone metabolism, and the status of their functional characterization. RECENT FINDINGS: GWASs revealed dozens of novel genetic loci that are associated with bone mineral density (BMD). Some of these targets have been functionally characterized, although the vast majority have not. Glypican 6, a membrane surface proteoglycan involved in cellular growth control and differentiation, was identified as a novel determinant of BMD and represents a possible drug target for treatment of osteoporosis. Pathway analysis also showed that cell-growth pathways and the SMAD proteins associated with low BMD. SUMMARY: Hits that were significantly associated with BMD in different studies represent likely candidates (e.g. SOST, WNT16, ESR1 and RANKL) for functional characterization and development of osteoporosis treatments. Indeed, currently available treatment for osteoporosis (antibody against RANKL) appeared a significant target in four recent GWAS studies indicating their applicability and importance for future treatment development.

Mesenchymal Stem Cells in the Musculoskeletal System: From Animal Models to Human Tissue Regeneration?

The musculoskeletal system includes tissues that have remarkable regenerative capabilities. Bone and muscle sustain micro-damage throughout the lifetime, yet they continue to provide the body with the support that is needed for everyday activities. Our current understanding is that the regenerative capacity of the musculoskeletal system can be attributed to the mesenchymal stem/ stromal cells (MSCs) that reside within its different anatomical compartments. These MSCs can replenish various tissues with progenitor cells to form functional cells, such as osteoblasts, chondrocytes, myocytes, and others. However, with aging and in certain disorders of the musculoskeletal system such as osteoarthritis or osteoporosis, this regenerative capacity of MSCs appears to be lost or diverted for the production of other non-functional cell types, such as adipocytes and fibroblasts. In this review, we shed light on the tissue sources and subpopulations of MSCs in the musculoskeletal system that have been identified in animal models, discuss the mechanisms of their anti-inflammatory action as a prerequisite for their tissue regeneration and their current applications in regenerative medicine. While providing up-to-date evidence of the role of MSCs in different musculoskeletal pathologies, in particular in osteoporosis and osteoarthritis, we share some thoughts on their potential as diagnostic markers in musculoskeletal health and disease.

The role of artichoke leaf tincture (Cynara scolymus) in the suppression of DNA damage and atherosclerosis in rats fed an atherogenic diet.

CONTEXT: Polyphenols and flavonoids in artichoke leaf tincture (ALT) protect cells against oxidative damage. OBJECTIVES: We examined ALT effects on deoxyribonucleic acid (DNA) damage and lipid profiles in rat plasma and gene expression in rat aorta [haemeoxygenase-1 (HO1), haemeoxygenase-2 (HO2), NADPH oxidase 4 (NOX-4), monocyte chemoattractant protein-1 (MCP-1) and nuclear factor (erythroid-derived 2)-like 2 (Nrf2)]. MATERIALS AND METHODS: Eighteen male Wistar albino rats were divided into three groups (n = 6/group): The control group (CG) was fed with standard pellet chow for 11 weeks; the AD group was fed for a similar period of time with pellet chow supplemented with 2% cholesterol, 3% sunflower oil and 1% sodium cholate. The ADA group was fed with pellet chow (for 1 week), the atherogenic diet (see above) for the following 4 weeks and then with ALT (0.1 mL/kg body weight) and atherogenic diet for 6 weeks. According to HPLC analysis, the isolated main compounds in ALT were chlorogenic acid, caffeic acid, isoquercitrin and rutin. RESULTS: Normalized HO-1 [0.11 (0.04-0.24)] and MCP-1 [0.29 (0.21-0.47)] mRNA levels and DNA scores [12.50 (4.50-36.50)] were significantly lower in the ADA group than in the AD group [0.84 (0.35-2.51)], p = 0.021 for HO-1 [0.85 (0.61-3.45)], p = 0.047 for MCP-1 and [176.5 (66.50-221.25)], p = 0.020 for DNA scores. HO-1 mRNA was lower in the ADA group than in the CG group [0.30 (0.21-0.71), p = 0.049]. CONCLUSIONS: Supplementation with ALT limited the effects of the atherogenic diet through reduced MCP-1 expression, thereby preventing oxidative damage.

VEGF-A is associated with early degenerative changes in cartilage and subchondral bone.

Paired cartilage and subchondral bone of subjects with no clinical history of joint disorders were analyzed to determine whether antioxidant enzymes, inflammatory cytokines and growth factors can be linked to a pre-osteoarthritis. Tissue explants were phenotyped according to Osteoarthritis Research Society International grading and micro-computed tomography, and also screened for the expression of several markers using quantitative polymerase chain reaction. The expression of these same genes was measured in SW1353 cells treated with hydrogen peroxide, to gain insight into the pathways involved with oxidative stress responses. Vascular endothelial growth factor A (VEGF-A) was up-regulated in the cartilage samples that showed early cartilage or bone degeneration. Oxidative stress in chondrocytes provoked up-regulation of interleukin-1ß, interleukin-6, aggrecan, and SRY-box containing gene 9. Our results confirm the hitherto evidence of the deteriorating effects of the oxidative stress on cartilage and suggest the link between VEGF-A and pre-osteoarthritis.

The impact of gene polymorphisms in angiotensin receptor 1 and aldosterone synthase in peritoneal dialysis patients.

AIM: Longevity of peritoneal membrane is an important issue in patients treated with peritoneal dialysis (PD). In our study, we studied the impact of angiotensin receptor 1 (AGT R1) and aldosterone synthase (CYP11B2) gene polymorphism on peritoneal concentrations of interleukin-6 (PI-IL-6), vascular endothelial growth factor (PI-VEGF), plasminogen activator inhibitor-1 (PI-PAI-1), transforming growth factor-ß (PI-TGF-ß), and cancer antigen-125 (PI-CA-125) as known markers of peritoneal fibrosis. The single nucleotide polymorphism rs5186 (A1166C) in AGT R1 gene is located in 3' untranslated region (UTR) of the gene, while polymorphism rs1799998 (T -344 C) in CYP11B2 gene is located in the promoter region of the gene. METHODS: We compared marker concentrations in patients with genotype DD vs. Dd and dd for AGT R1 and patients with genotype HH vs. Hh and hh for CYP11B2. RESULTS: The results show that polymorphism of CYP11B2 gene is associated with serum concentration of aldosterone. Patients with genotype HH had statistically significantly lower serum concentration of aldosterone (p = 0.04). These patients also showed a trend to a lower rate of production of I-IL-6 (p = 0.07), which correlated with lower concentrations of PAI-1 (p = 0.002) and VEGF (p = 0.005). AGT R1 gene polymorphism did not show any association with studied variables. CONCLUSIONS: Our findings suggest the possibility of genetic predisposition for development of peritoneal fibrosis that could be important for identification of patients with an "unfavorable" genotype, which could lead to customized prescription of appropriate therapy and personalized patient management.
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Risk factors for symptomatic osteonecrosis in childhood ALL: A retrospective study of a Slovenian pediatric ALL population between 1970 and 2004.

Treatment induced non-traumatic osteonecrosis (ON) has been reported increasingly in children treated for acute lymphoblastic leukemia (ALL). Several risk factors for ON have been identified in childhood cancer patients; however, their diagnostic and prognostic power is limited and the etiology of the disease remains unclear. Therefore, a continuous effort is focused on the identification of additional ON risk factors. We performed a retrospective study of 313 childhood ALL patients to test the association between the ON occurrence in children receiving ALL therapy and common polymorphisms in potential target genes: Thiopurine S-methyltransferase (TPMT; 460G>A, 719A>G), 5,10-methylenetetrahydrofolate reductase (MTHFR; 677C>T, 1298A>C), estrogen receptor alpha 1 (ESR1; XbaI) and collagen type I, α1 (COL1A1; Sp1). In the present cohort, higher age and more recently developed treatment protocols were independent risk factors for ON. In children >14.5 years old, TPMT genotype modulated the risk of ON. Additionally, in children <12.9 years old ESR1 genotypes were also implicated in the pathogenesis of ON. Besides greater age and more recent treatment protocols, genetic factors (polymorphisms in ESR1 and TPMT genes) were suggested to be implicated in the pathogenesis of ON and could be potentially used as genetic prognostic markers for ON.

The "Rise-Peak-Fall" Pattern of Time Dependency of the Cardiovascular Pleiotropic Effects of Treatment With Low-dose Atorvastatin, Losartan, and a Combination Thereof in Rats.

Treatment with low, subtherapeutic doses of statins and sartans expresses beneficial pleiotropic effects on the arterial wall. The present study explored whether these effects depend on treatment duration. Wistar rats were randomly divided into 4 groups and received low-dose atorvastatin, low-dose losartan, their combination, or saline (control) daily. After 4, 6, 8, and 10 weeks of treatment, the animals were anesthetized, blood samples taken, and hearts and thoracic aortas isolated. Thoracic aorta endothelium-dependent relaxation and parameters of the isolated heart exposed to ischemic-reperfusion injury were assessed along with blood serum parameters and vasoactive genes expression. Low-dose atorvastatin, losartan, and especially their combination showed the characteristic time dependency of all studied parameters (thoracic aorta relaxation, isolated heart parameters, C-reactive protein values, genes encoding endothelial nitric oxide synthase, and CD40). The peak in efficacy was observed after 6 weeks of treatment and subsequently steadily declined. The peak versus control values were significant for all measured parameters. Only a combination of atorvastatin and losartan increased nitric oxide and decreased asymmetric dimethylarginine. A characteristic time-dependent "rise-peak-fall" pattern of the cardiovascular pleiotropic effects of statins and sartans in subtherapeutic low doses was revealed. Evidently, resistance to the explored treatment occurs after a certain period.

Bone remodeling during orthodontic tooth movement in rats with type 2 diabetes.

INTRODUCTION: Type 2 diabetes is known to affect bone metabolism. In this study, we aimed to determine the effects of type 2 diabetes on bone remodeling during orthodontic tooth movement. METHODS: The 48 rats were divided into 4 groups: Wistar control group (n = 8), Goto-Kakizaki (GK) control group (n = 8), Wistar appliance group (n = 16), and GK appliance group (n = 16). The distances between the teeth were measured weekly. On day 42, maxillary alveolar bone specimens were obtained for histologic evaluation and determination of the gene expression levels of the receptor activator of nuclear factor Ò¡B (RANK), RANK ligand (RANKL), and osteoprotegerin (OPG). RESULTS: No significant difference was observed in the levels of tooth movement between the 2 appliance groups. After orthodontic force application, the alveolar bone volume and osteoblast surface in the GK rats were diminished compared with those in the Wistar rats. The increase in the osteoclast surface relative to the control groups was 2.4-fold greater in the GK rats than in the Wistar rats. Significant upregulations of the RANK and OPG gene expression levels in the Wistar appliance group were observed. The RANKL/OPG ratio was increased in the GK appliance group compared with the Wistar appliance group. CONCLUSIONS: Diminished bone formation and slightly increased bone resorption were observed during orthodontic tooth movement in the rats with type 2 diabetes.