Lack of relationship between glycemic control and bone mineral density in type 2 diabetes mellitus

We assessed the effect of chronic hyperglycemia on bone mineral density (BMD) and bone remodeling in patients with type 2 diabetes mellitus. We investigated 42 patients with type 2 diabetes under stable control for at least 1 year, 22 of them with good metabolic control (GMC: mean age = 48.8 ± 1.5 years, 11 females) and 20 with poor metabolic control (PMC: mean age = 50.2 ± 1.2 years, 8 females), and 24 normal control individuals (CG: mean age = 46.5 ± 1.1 years, 14 females). We determined BMD in the femoral neck and at the L2-L4 level (DEXA) and serum levels of glucose, total glycated hemoglobin (HbA1), total and ionic calcium, phosphorus, alkaline phosphatase, follicle-stimulating hormone, intact parathyroid hormone (iPTH), 25-hydroxyvitamin D (25-OH-D), insulin-like growth factor I (IGFI), osteocalcin, procollagen type I C propeptide, as well as urinary levels of deoxypyridinoline and creatinine. HbA1 levels were significantly higher in PMC patients (12.5 ± 0.6 vs 7.45 ± 0.2% for GMC and 6.3 ± 0.9% for CG; P < 0.05). There was no difference in 25-OH-D, iPTH or IGFI levels between the three groups. BMD values at L2-L4 (CG = 1.068 ± 0.02 vs GMC = 1.170 ± 0.03 vs PMC = 1.084 ± 0.02 g/cm²) and in the femoral neck (CG = 0.898 ± 0.03 vs GMC = 0.929 ± 0.03 vs PMC = 0.914 ± 0.03 g/cm²) were similar for all groups. PMC presented significantly lower osteocalcin levels than the other two groups, whereas no significant difference in urinary deoxypyridine was observed between groups. The present results demonstrate that hyperglycemia is not associated with increased bone resorption in type 2 diabetes mellitus and that BMD is not altered in type 2 diabetes mellitus.

Mesenchymal stem cells from patients with chronic myeloid leukemia do not express BCR-ABL and have absence of chimerism after allogeneic bone marrow transplant

Bone marrow is a heterogeneous cell population which includes hematopoietic and mesenchymal progenitor cells. Dysregulated hematopoiesis occurs in chronic myelogenous leukemia (CML), being caused at least in part by abnormalities in the hematopoietic progenitors. However, the role of mesenchymal stem cells (MSCs) in CML has not been well characterized. The objectives of the present study were to observe the biological characteristics of MSCs from CML patients and to determine if MSCs originate in part from donors in CML patients after bone marrow transplantation (BMT). We analyzed MSCs from 5 untreated patients and from 3 CML patients after sex-mismatched allogeneic BMT. Flow cytometry analysis revealed the typical MSC phenotype and in vitro assays showed ability to differentiate into adipocytes and osteoblasts. Moreover, although some RT-PCR data were contradictory, combined fluorescence in situ hybridization analysis showed that MSCs from CML patients do not express the bcr-abl gene. Regarding MSCs of donor origin, although it is possible to detect Y target sequence by nested PCR, the low frequency (0.14 and 0.34%) of XY cells in 2 MSC CML patients by fluorescence in situ hybridization analysis suggests the presence of contaminant hematopoietic cells and the absence of host-derived MSCs in CML patients. Therefore, we conclude that MSCs from CML patients express the typical MSC phenotype, can differentiate into osteogenic and adipogenic lineages and do not express the bcr-abl gene. MSCs cannot be found in recipients 12 to 20 months after BMT. The influence of MSCs on the dysregulation of hematopoiesis in CML patients deserves further investigation.

Impact of marked weight loss induced by bariatric surgery on bone mineral density and remodeling

Data about the impact of bariatric surgery (BS) and subsequent weight loss on bone are limited. The objective of the present study was to determine bone mineral density (BMD), bone remodeling metabolites and hormones that influence bone trophism in premenopausal women submitted to BS 9.8 months, on average, before the study (OGg, N = 16). The data were compared to those obtained for women of normal weight (CG, N = 11) and for obese women (OG, N = 12). Eight patients in each group were monitored for one year, with the determination of BMD, of serum calcium, phosphorus, magnesium, parathyroid hormone, 25-hydroxyvitamin D, insulin-like growth factor-I (IGF-I) and osteocalcin, and of urinary calcium and deoxypyridinoline. The biochemical determinations were repeated every three months in the longitudinal study and BMD was measured at the end of the study. Parathyroid hormone levels were similar in the three groups. IGF-I levels (CG = 332 ± 62 vs OG = 230 ± 37 vs OGg = 128 ± 19 ng/mL) were significantly lower in the operated patients compared to the non-operated obese women. Only OGg patients presented a significant fall in BMD of 6.2% at L1-L4, of 10.2% in the femoral neck, and of 5.1% in the forearm. These results suggest that the weight loss induced by BS is associated with a significant loss of bone mass even at sites that are not influenced by weight overload, with hormonal factors such as IGF-I being associated with this process.

Bone metabolism and vascular calcification

Osteoporosis and atherosclerosis are chronic degenerative diseases which have been considered to be independent and whose common characteristic is increasing incidence with age. At present, growing evidence indicates the existence of a correlation between cardiovascular disease and osteoporosis, irrespective of age. The morbidity and mortality of osteoporosis is mainly related to the occurrence of fractures. Atherosclerosis shows a high rate of morbidity and especially mortality because of its clinical repercussions such as angina pectoris, acute myocardial infarction, stroke, and peripheral vascular insufficiency. Atherosclerotic disease is characterized by the accumulation of lipid material in the arterial wall resulting from autoimmune and inflammatory mechanisms. More than 90% of these fatty plaques undergo calcification. The correlation between osteoporosis and atherosclerosis is being established by studies of the underlying physiopathological mechanisms, which seem to coincide in many biochemical pathways, and of the risk factors for vascular disease, which have also been associated with a higher incidence of low-bone mineral density. In addition, there is evidence indicating an action of antiresorptive drugs on the reduction of cardiovascular risks and the effect of statins, antihypertensives and insulin on bone mass increase. The mechanism of arterial calcification resembles the process of osteogenesis, involving various cells, proteins and cytokines that lead to tissue mineralization. The authors review the factors responsible for atherosclerotic disease that correlate with low-bone mineral density.

Analysis of the diagnostic presentation profile, parathyroidectomy indication and bone mineral density follow-up of Brazilian patients with primary hyperparathyroidism

Primary hyperparathyroidism is an endocrine disorder with variable clinical expression, frequently presenting as asymptomatic hypercalcemia in Western countries but still predominantly as a symptomatic disease in developing countries. The objective of this retrospective study was to describe the diagnostic presentation profile, parathyroidectomy indication and post-surgical bone mineral density follow-up of patients with primary hyperparathyroidism seen at a university hospital. We found 115 patients (92 women, median age 56 years) with primary hyperparathyroidism diagnosed during the last 20 years. We defined symptomatic patients based on the presence of any classical symptom affecting bone, kidney or the neuromuscular system. Surgical criteria followed the guidelines of the National Institutes of Health regarding asymptomatic primary hyperparathyroidism. Symptomatic patients and patients meeting surgical criteria for parathyroidectomy were 66 and 93% of the sample, respectively. Median calcium and parathyroid hormone values were 11.9 mg/dL and 189 pg/mL, respectively. After surgical treatment, 97% of patients were cured, with increases in bone mineral density of 19.4% in the lumbar spine and 15.7% in the femoral neck 3 years after surgery. Greater bone mass increases were detected in pre-menopausal women, men, and in symptomatic and younger patients, both in the lumbar spine and femoral neck. Our results support the previous findings of a predominantly symptomatic disease with a presentation profile that could be mainly related to a delayed diagnosis. Nevertheless, genetic and racial backgrounds, and nutritional factors such as calcium and vitamin D deficiency may play a role in the clinical presentation of primary hyperparathyroidism of Brazilian patients.

Paullinia cupana Mart var. sorbilis, guaraná, reduces cell proliferation and increases apoptosis of B16/F10 melanoma lung metastases in mice

We showed that guaraná (Paullinia cupana Mart var. sorbilis) had a chemopreventive effect on mouse hepatocarcinogenesis and reduced diethylnitrosamine-induced DNA damage. In the present experiment, we evaluated the effects of guaraná in an experimental metastasis model. Cultured B16/F10 melanoma cells (5 x 10(5) cells/animal) were injected into the tail vein of mice on the 7th day of guaraná treatment (2.0 mg P. cupana/g body weight, per gavage) and the animals were treated with guaraná daily up to 14 days until euthanasia (total treatment time: 21 days). Lung sections were obtained for morphometric analysis, apoptotic bodies were counted to calculate the apoptotic index and proliferating cell nuclear antigen-positive cells were counted to determine the proliferation index. Guaraná-treated (GUA) animals presented a 68.6% reduction in tumor burden area compared to control (CO) animals which were not treated with guaraná (CO: 0.84 ± 0.26, N = 6; GUA: 0.27 ± 0.24, N = 6; P = 0.0043), a 57.9% reduction in tumor proliferation index (CO: 23.75 ± 20.54, N = 6; GUA: 9.99 ± 3.93, N = 6; P = 0.026) and a 4.85-fold increase in apoptotic index (CO: 66.95 ± 22.95, N = 6; GUA: 324.37 ± 266.74 AB/mm², N = 6; P = 0.0152). In this mouse model, guaraná treatment decreased proliferation and increased apoptosis of tumor cells, consequently reducing the tumor burden area. We are currently investigating the molecular pathways of the effects of guaraná in cultured melanoma cells, regarding principally the cell cycle inhibitors and cyclins.

Comparison of bone quantity by ultrasound measurements of phalanges between white and black children living in Paraná, Brazil, with Europeans

The objective of this study was to determine bone quantity by ultrasound measurements of the proximal finger phalanges (AD-SoS = amplitude-dependent speed of sound) of healthy Brazilian schoolchildren living in Paraná, Brazil and to compare these values with European populations. The sample was composed of 1356 Brazilian schoolchildren of both genders (660 males, 696 females), aged 6 to 11 years, divided into white (840) and black (516) groups and compared to age- and gender-matched Europeans. AD-SoS of the schoolchildren increased significantly with age for both genders. Significantly higher AD-SoS values were observed for the white children (1916 ± 58) compared to their black counterparts (1898 ± 72) and for the female gender (1920 ± 61) compared to the male gender (1898 ± 66). Overall, the AD-SoS outcomes for females were similar to those of European studies. However, the AD-SoS of the Brazilian schoolchildren of both genders and skin colors was lower than that reported for children in Poland. AD-SoS outcomes for Brazilian schoolboys were similar to those obtained in Italian studies and were lower than those of the Spanish children. In conclusion, Brazilian schoolchildren of both genders and skin colors showed lower bone quantities than Polish children and Spanish males, and levels similar to Italian children and Spanish females.

Women with primary ovarian insufficiency have lower bone mineral density

The aim of the present study was to assess the prevalence of osteoporosis in a sample of 32 patients with spontaneous primary ovarian insufficiency (POI) in comparison to reference groups of 25 pre- and 55 postmenopausal women. Hip (lumbar) and spinal bone mineral density (BMD) measurements were performed by dual-energy X-ray absorptiometry in the three groups. The median age of POI patients at the time of diagnosis was 35 years (interquartile range: 27-37 years). The mean ± SD age of postmenopausal reference women (52.16 ± 3.65 years) was higher than that of POI (46.28 ± 10.38 years) and premenopausal women (43.96 ± 7.08; P = 0.001) at the time of BMD measurement. Twenty-seven (84.4%) POI women were receiving hormone replacement therapy (HRT) at the time of the study. In the postmenopausal reference group, 30.4% were current users of HRT. Lumbar BMD was significantly lower in the POI group (1.050 ± 0.17 g/cm²) compared to the age-matched premenopausal reference group (1.136 ± 0.12 g/cm²; P = 0.040). Moreover, 22 (68.7%) POI women had low bone density (osteopenia/osteoporosis by World Health Organization criteria) versus 47.3% of the postmenopausal reference group (P = 0.042). In conclusion, the present data indicate that BMD is significantly lower in patients with POI than in age-matched premenopausal women. Also, the prevalence of osteopenia/osteoporosis is higher in POI women than in women after natural menopause. Early medical interventions are necessary to ensure that women with POI will maintain their bonemass.

Co-culture of chondrocytes and bone marrow mesenchymal stem cells in vitro enhances the expression of cartilaginous extracellular matrix components

Chondrocytes and bone marrow mesenchymal stem cells (BMSCs) are frequently used as seed cells in cartilage tissue engineering. In the present study, we determined if the co-culture of rabbit articular chondrocytes and BMSCs in vitro promotes the expression of cartilaginous extracellular matrix and, if so, what is the optimal ratio of the two cell types. Cultures of rabbit articular chondrocytes and BMSCs were expanded in vitro and then cultured individually or at a chondrocyte:BMSC ratio of 4:1, 2:1, 1:1, 1:2, 1:4 for 21 days and cultured in DMEM/F12. BMSCs were cultured in chondrogenic induction medium. Quantitative real-time RT-PCR and Western blot were used to evaluate gene expression. In the co-cultures, type II collagen and aggrecan expression increased on days 14 and 21. At the mRNA level, the expression of type II collagen and aggrecan on day 21 was much higher in the 4:1, 2:1, and 1:1 groups than in either the articular chondrocyte group or the induced BMSC group, and the best ratio of co-culture groups seems to be 2:1. Also on day 21, the expression of type II collagen and aggrecan proteins in the 2:1 group was much higher than in all other groups. The results demonstrate that the co-culture of rabbit chondrocytes and rabbit BMSCs at defined ratios can promote the expression of cartilaginous extracellular matrix. The optimal cell ratio appears to be 2:1 (chondrocytes:BMSCs). This approach has potential applications in cartilage tissue engineering since it provides a protocol for maintaining and promoting seed-cell differentiation and function.

A Brazilian family with hereditary inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia

Inclusion body myopathy associated with Paget disease and frontotemporal dementia (IBMPFD) is a progressive and usually misdiagnosed autosomal dominant disorder. It is clinically characterized by a triad of features: proximal and distal myopathy, early onset Paget disease of bone (PDB), and frontotemporal dementia (FTD). It is caused by missense mutations in the valosin-containing protein (VCP) gene. We describe here the clinical and molecular findings of the first Brazilian family identified with IBMPFD. Progressive myopathy affecting the limb girdles was detected by clinical examination followed by muscle biopsy and creatine kinase measurement. PDB was suggested after anatomopathological bone examination and FTD was diagnosed by clinical, neuropsychological and language evaluations. Brain magnetic resonance revealed severe atrophy of the anterior temporal lobes, including the hippocampi. A R93C mutation in VCP was detected by direct sequencing screening in subject W (age 62) and in his mother. Four more individuals diagnosed with "dementia" were reported in this family. We also present a comprehensive genotype-phenotype correlation analysis of mutations in VCP in 182 patients from 29 families described in the literature and show that while IBM is a conspicuously penetrant symptom, PDB has a lower penetrance when associated with mutations in the AAAD1 domain and FTD has a lower penetrance when associated with mutations in the Junction (L1-D1) domain. Furthermore, the R93C mutation is likely to be associated with the penetrance of all the clinical symptoms of the triad.

In vitro culture and characterization of alveolar bone osteoblasts isolated from type 2 diabetics

In order to understand the mechanisms of poor osseointegration following dental implants in type 2 diabetics, it is important to study the biological properties of alveolar bone osteoblasts isolated from these patients. We collected alveolar bone chips under aseptic conditions and cultured them in vitro using the tissue explants adherent method. The biological properties of these cells were characterized using the following methods: alkaline phosphatase (ALP) chemical staining for cell viability, Alizarin red staining for osteogenic characteristics, MTT test for cell proliferation, enzyme dynamics for ALP contents, radio-immunoassay for bone gla protein (BGP) concentration, and ELISA for the concentration of type I collagen (COL-I) in the supernatant. Furthermore, we detected the adhesion ability of two types of cells from titanium slices using non-specific immunofluorescence staining and cell count. The two cell forms showed no significant difference in morphology under the same culture conditions. However, the alveolar bone osteoblasts received from type 2 diabetic patients had slower growth, lower cell activity and calcium nodule formation than the normal ones. The concentration of ALP, BGP and COL-I was lower in the supernatant of alveolar bone osteoblasts received from type 2 diabetic patients than in that received from normal subjects (P < 0.05). The alveolar bone osteoblasts obtained from type 2 diabetic patients can be successfully cultured in vitro with the same morphology and biological characteristics as those from normal patients, but with slower growth and lower concentration of specific secretion and lower combining ability with titanium than normal ones.

Prostate-specific membrane antigen can promote in vivo osseous metastasis of prostate cancer cells in mice

Reports remain insufficient on whether and how prostate-specific membrane antigen (PSMA) can influence in vivo osseous metastasis of prostate cancer (PCa). In the present study, the authors induced stable expression of PSMA in mouse PCa cell line RM-1. In vivo osseous metastasis was induced in 37 6-week-old female C57BL/6 mice weighing 22.45 ± 0.456 g. RM-1 cells were actively injected into the femoral bone cavity, leading to bilateral dissymmetry of bone density in the femoral bone. Tumor cells were also detected in bone tissue by pathological examination. The impact on bone density was demonstrated by the significant difference between animals injected with RM-PSMA cells (0.0738 ± 0.0185 g/cm²) and animals injected with RM-empty plasmid cells (0.0895 ± 0.0241 g/cm²). The lytic bone lesion of the RM-PSMA group (68.4%) was higher than that of the control group (27.8%). Immunohistochemistry showed that the expression of both vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) was distinctly higher in the RM-PSMA group than in the control group, while ELISA and Western blot assay indicated that VEGF and MMP-9 were higher in the RM-PSMA group compared to the control group (in vitro). Thus, the present study proposed and then confirmed for the first time that PSMA can promote in vivo osseous metastasis of PCa by increasing sclerotic destruction of PCa cells. Further analyses also suggested that PSMA functions positively on the invasive ability of RM-1 by increasing the expression of MMP-9 and VEGF by osseous metastases in vivo

In vitro inhibitory effects of imatinib mesylate on stromal cells and hematopoietic progenitors from bone marrow

Imatinib mesylate (IM) is used to treat chronic myeloid leukemia (CML) because it selectively inhibits tyrosine kinase, which is a hallmark of CML oncogenesis. Recent studies have shown that IM inhibits the growth of several non-malignant hematopoietic and fibroblast cells from bone marrow (BM). The aim of the present study was to evaluate the effects of IM on stromal and hematopoietic progenitor cells, specifically in the colony-forming units of granulocyte/macrophage (CFU-GM), using BM cultures from 108 1.5- to 2-month-old healthy Swiss mice. The results showed that low concentrations of IM (1.25 µM) reduced the growth of CFU-GM in clonogenic assays. In culture assays with stromal cells, fibroblast proliferation and α-SMA expression by immunocytochemistry analysis were also reduced in a concentration-dependent manner, with a survival rate of approximately 50% with a dose of 2.5 µM. Cell viability and morphology were analyzed using MTT and staining with acrydine orange/ethidium bromide. Most cells were found to be viable after treatment with 5 µM IM, although there was gradual growth inhibition of fibroblastic cells while the number of round cells (macrophage-like cells) increased. At higher concentrations (15 µM), the majority of cells were apoptotic and cell growth ceased completely. Oil red staining revealed the presence of adipocytes only in untreated cells (control). Cell cycle analysis of stromal cells by flow cytometry showed a blockade at the G0/G1 phases in groups treated with 5-15 µM. These results suggest that IM differentially inhibits the survival of different types of BM cells since toxic effects were achieved.

Serum 25-hydroxyvitamin D and biochemical markers of bone metabolism in patients with juvenile idiopathic arthritis

Our objective was to evaluate the concentrations of serum 25-hydroxyvitamin D [25(OH)D], serum calcium, serum phosphorus, alkaline phosphatase, and parathormone (PTH) in patients with polyarticular juvenile idiopathic arthritis (JIA) and to associate them with disease duration and activity, bone mineral density and use of medications. In a cross-sectional and controlled study, 30 patients with polyarticular JIA were evaluated and compared to 30 healthy individuals matched for age and gender. Clinical status, anthropometry, laboratory markers in both patients and controls, and bone mineral density, only in the patients, were measured. Of the 30 patients included in the study, 23 (76.7%) were female and 16 (53.3%) non-Caucasian; mean age was 14 years (range = 4 to 20 years). Mean disease duration was 5 years (range = 1 to 12 years). The mean concentrations of serum albumin-corrected calcium (9.04 ± 0.41 mg/dL) and alkaline phosphatase (153.3 ± 100.1 IU) were significantly lower in patients with JIA than in controls (P < 0.0001 and P = 0.001, respectively). No differences in 25(OH)D, PTH or serum phosphorus were observed between JIA and control subjects. Regarding 25(OH)D concentration, 8 patients (26.7%) and 5 controls (16.7%) had 25(OH)D concentrations compatible with deficiency (lower than 20 ng/mL) and 14 patients (46.7%) and 18 controls (60%) had concentrations compatible with insufficiency (20-32 ng/mL). These values were not associated with disease activity, use of medications or bone mineral density. We observed a high frequency of 25(OH)D insufficiency and deficiency in the study sample. The compromised bone metabolism emphasizes the importance of follow-up of JIA patients.

Osteogenesis induced in goat bone marrow progenitor cells by recombinant adenovirus coexpressing bone morphogenetic protein 2 and basic fibroblast growth factor

Bone morphogenetic protein 2 (BMP2) and basic fibroblast growth factor (bFGF) have been shown to exhibit a synergistic effect to promote bone repair and healing. In this study, we constructed a novel adenovirus with high coexpression of BMP2 and bFGF and evaluated its effect on osteogenic differentiation of goat bone marrow progenitor cells (BMPCs). Recombinant adenovirus Ad-BMP2-bFGF was constructed by using the T2A sequence. BMPCs were isolated from goats by density gradient centrifugation and adherent cell culture, and were then infected with Ad-BMP2-bFGF or Ad-BMP2. Expression of BMP2 and bFGF was detected by ELISA, and alkaline phosphatase (ALP) activity was detected by an ALP assay kit. In addition, von Kossa staining and immunocytochemical staining of collagen II were performed on BMPCs 21 days after infection. There was a high coexpression of BMP2 and bFGF in BMPCs infected with Ad-BMP2-bFGF. Twenty-one days after infection, ALP activity was significantly higher in BMPCs infected with Ad-BMP2-bFGF than in those infected with Ad-BMP2. Larger and more mineralized calcium nodules, as well as stronger collagen II staining, were observed in BMPCs infected with Ad-BMP2-bFGF than in those infected with Ad-BMP2. In summary, we developed a novel adenovirus vector Ad-BMP2-bFGF for simultaneous high coexpression of BMP2 and bFGF, which could induce BMPCs to differentiate efficiently into osteoblasts.