The effects of vanadium treatment on bone in diabetic and non-diabetic rats

Bone 38 (2006) 368 – 377 www.elsevier.com/locate/bone

The effects of vanadium treatment on bone in diabetic and non-diabetic rats D.M. Facchini a,b , V.G. Yuen c , M.L. Battell c , J.H. McNeill c , M.D. Grynpas a,b,⁎ a

c

Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Ave., Toronto, Canada M5G 1X5 b Department of Materials Science and Engineering, University of Toronto, Canada Division of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, University of British Columbia, Canada Received 24 March 2005; revised 19 August 2005; accepted 19 August 2005 Available online 26 October 2005

Abstract Vanadium-based drugs lower glucose by enhancing the effects of insulin. Oral vanadium drugs are being tested for the treatment of diabetes. Vanadium accumulates in bone, though it is not known if incorporated vanadium affects bone quality. Nine- to 12-month-old control and streptozotocin-induced diabetic female Wistar rats were given bis(ethylmaltolato)oxovanadium(IV) (BEOV), a vanadium-based anti-diabetic drug, in drinking water for 12 weeks. Non-diabetic rats received 0, 0.25 or 0.75 mg/ml BEOV. Groups of diabetic rats were either untreated or treated with 0.25–0.75 mg/ml BEOV as necessary to lower blood glucose in each rat. In diabetic rats, this resulted in a Controlled Glucose group, simulating relatively well-managed diabetes, and an Uncontrolled Glucose group, simulating poorly managed diabetes. Plasma insulin, glucose and triglyceride assays assessed the diabetic state. Bone mineral density (BMD), mechanical testing, mineral assessment and histomorphometry measured the effects of diabetes on bone and the effects of BEOV on non-diabetic and diabetic bone. Diabetes decreased plasma insulin and increased plasma glucose and triglycerides. In bone, diabetes decreased BMD, strength, mineralization, bone crystal length, and bone volume and connectivity. Treatment was effective in incorporating vanadium into bone. In all treated groups, BEOV increased osteoid volume. In non-diabetic bone, BEOV increased cortical bone toughness, mineralization and bone formation. In controlled glucose rats, BEOV lowered plasma glucose and improved BMD, mechanical strength, mineralization, bone crystal length and bone formation rate. In poorly controlled rats, BEOV treatment slightly lowered plasma glucose but did not improve bone properties. These results suggest that BEOV improves diabetes-related bone dysfunction primarily by improving the diabetic state. BEOV also appeared to increase bone formation. Our study found no negative effects of vanadium accumulation in bone in either diabetic or non-diabetic rats at the dose given. © 2005 Elsevier Inc. All rights reserved. Keywords: Vanadium; Diabetes; Bone mineral; Mechanical loading; Histomorphometry

Introduction Vanadium-based drugs have been of interest as a potential treatment for diabetes since their glucose-lowering effects were first discovered [1]. These orally effective drugs have been shown to reduce glucose and improve diabetes-related complications in animal models of the disease by acting as insulin-enhancing agents. In human studies, vanadium administration has shown modest but positive effects in lowering blood glucose in Type 2 diabetics [2]. ⁎ Corresponding author. Fax: +1 416 586 8844. E-mail addresses: [email protected] (D.M. Facchini), [email protected] (V.G. Yuen), [email protected] (M.L. Battell), [email protected] (J.H. McNeill), [email protected] (M.D. Grynpas). 8756-3282/$ - see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2005.08.015

Vanadium from such drugs accumulates in bone [3], where, in the form of vanadate, it replaces phosphate in the apatite lattice of bone mineral [4]. Though its effects on the bone mineral lattice are not known, small amounts of vanadate can be accommodated by the more crystalline synthetic hydroxyapatite lattice without producing distortion or altering P–O or O–H bond strength [5]. In vitro studies of vanadium drugs have shown a direct stimulatory effect on osteoblast-like cells [6,7]. However, the in vivo effects of vanadium incorporation in bone and exposure of bone cells to vanadium have yet to be determined. Since vanadium does deposit in bone, concern exists as to what vanadium might do to bone structure and strength when given on a chronic basis. Bone disorder appears to affect Type 1 diabetics, and this may make the incorporation of vanadium into diabetic bone different from that into non-diabetic bone. Patients with Type 1 diabetes have been shown to have

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decreased bone mineral density (BMD) [8–10]. Despite the evidence for an osteoporosis-like condition, this bone disorder is not well characterized. Biochemical markers of bone formation are inconsistent [8,11,12], while markers of bone resorption often are increased [11,12]. Nevertheless, a bone disorder does afflict Type 1 diabetics, and the effect of vanadium on bone metabolism in Type 1 diabetic model is of interest. In an animal model for Type 1 diabetes, streptozotocin (STZ) injection chemically destroys insulin-producing pancreatic ßcells, mimicking the autoimmune destruction of these cells in the human form of Type 1 diabetes. In rats, STZ results in decreased insulin secretion and hyperglycemia. The effect of STZ is dose-dependent, and sufficient ß-cells can remain so that the rats are able to live without insulin supplementation [13]. As in the human form of Type 1 diabetes, STZ causes a decrease in BMD [14]. Histomorphometric evaluation has shown that STZ decreases the amount of osteoid present and the bone formation rate [14–16]. Furthermore, biochemical markers of bone turnover are decreased [15,16]. This suggests a state of low turnover in Type 1 diabetes, though this has not been shown in human studies. STZ also results in decreased bone volume and poor trabecular connectivity [14]. A mineral defect has also been suggested since there is a decreased mineral crystal length [17] The Ca:P ratio is unchanged, but it is not clear whether the ash content increases or remains the same with diabetes [16,17]. The morphological and mineral changes with STZ likely contribute to the poor mechanical properties that are also observed [14,17,18]. Diabetes-induced bone changes in the rat have been shown to be improved by insulin [14, 15,18]. Conflicting reports exist regarding the influences of impaired insulin secretion and metabolic control on bone metabolism [9,10]. In vitro studies have shown an influence of glucose and insulin on osteoblast- and osteoclast-like cells. Insulin receptors have been found in more mature osteoblast cell lines [19], and insulin itself has been shown to increase collagen synthesis in UMR106 osteoblast-like cells [20]. In vivo, local insulin treatment to the hemicalvaria in nondiabetic mice increased osteoid volume and the number of osteoblasts [21]. Cultured osteoclast-like cells have been shown to express insulin receptors as well, and insulin was found to decrease pit formation in a dose-dependent manner [22]. High glucose levels were found to cause decreased calcium uptake, irregular nodule morphology and decreased osteoblast differentiation during in vitro bone mineral nodule formation [23]. High extracellular glucose levels have also been found to cause changes in osteoblastic gene expression [15], inhibit osteoblast-like cell growth [24] and promote osteoclastic bone resorption [25]. In this study, we assessed the effects of BEOV, an orally effective vanadium-containing anti-diabetic drug [26], on bone quality (mechanical, mineral, histological and structural properties) in non-diabetic and STZ-diabetic rats. The effects of diabetes on bone and the effects of BEOV on non-diabetic and diabetic bone were examined.

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Materials and methods Animal model Sixty-four (64) female Wistar rats (9–12 months of age, retired breeders, Charles River Laboratories) were randomly divided into 5 experimental groups: Control (n = 10), control-low dose (Low Dose, n = 10), control-high dose (High Dose, n = 10), Diabetic (n = 12) and treated diabetic (n = 22). Diabetes was induced by a single tail vein injection of 45 mg/ml STZ in 0.9% NaCl under light halothane anesthesia. Only those animals with blood glucose >13 mM 3 days post-STZ injection were taken as diabetic. Treated diabetic animals with blood glucose <20 mM following treatment were considered to have Controlled Glucose (n = 10), while those with blood glucose >20 mM were considered to have Uncontrolled Glucose (n = 12). Control and diabetic untreated animals were housed 2 rats/cage. All treated rats were housed individually for fluid monitoring purposes. Rats were allowed ad libitum access to food and drinking solution and were housed on a 12 h light:dark schedule at constant temperature and humidity. All animals were cared for in accordance with the principles and guidelines of the Canadian Council on Animal Care.

Treatment regime BEOV was administered in the drinking water at concentrations ranging from 0.125 to 0.75 mg/ml for a 12-week period. Low Dose animals received a maximum concentration of 0.25 mg/ml. High Dose animals received a maximum concentration of 0.75 mg/ml. Diabetic-treated animals received concentrations as tolerated by the individual rat to lower blood glucose. Body weight and fluid intake were measured daily and used to calculate vanadium dose in mmol/kg/day. Solutions were freshly prepared every 2 days. Blood was collected weekly from the tail vein, centrifuged at 10,000 × g × 25 min and the plasma analyzed for plasma glucose using a Beckman Glucose Analyzer 2. At termination, blood was collected for determination of plasma insulin using a radioimmunoassay kit (Linco Research, Inc.) and triglyceride levels by a colorimetric assay (Roche). Rats were killed with an overdose of pentobarbital 100 mg/kg by intraperitoneal injection. The femurs, tibias, humeri and vertebrae were dissected, cleaned of adherent soft tissue and stored at −20°C for future analysis.

Bone mineral density BMD of the excised right femur and fifth lumbar vertebrae was determined by DEXA using a dedicated small animal densitometer (PIXImus/GE). An aluminum/Lucite phantom (GE) was used to calibrate the machine [27].

Mechanical tests The mechanical failure properties of the femur and vertebra were determined using an Instron 4465 Materials Testing System (Instron Corp.) and custom-built torsion testing apparatus. In all experiments, force and deformation data were collected using LabView 5.0 data acquisition software (National Instruments). In these experiments, the point of failure was defined as a drop in load of >10%. The diaphysis of the left femur was tested to failure in three-point bending according to the procedure described previously [28]. Briefly, samples were subjected to a preload of 1 N and then deformed at a rate of 1 mm/min until failure. The diaphysis of the right femur was tested to failure in torsion according to the procedure described previously [29]. Briefly, samples were deformed at a rate of 35°/min until failure. The body of the fifth lumbar vertebra was tested to failure in unconfined compression using a procedure similar to that described previously [30]. Briefly, a preload of 1 N was applied to the sample and then deformed at a rate of 1 mm/min until failure occurred.

Specimen processing Undecalcified proximal tibiae were cut coronally using a low-speed saw (Buehler Ltd.) to expose marrow. Samples were fixed in 70% ethanol,

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dehydrated in ascending grades of acetone and infiltrated with ascending grades of Spurr/acetone before being embedded and polymerized in Spurr at 50°C. Upon completion, thin sections were cut on a rotary microtome (Model 2050, Reichert-Jung) equipped with a tungsten carbide knife. Sections were stained with Goldner’s Trichrome or Von Kossa for use in static histomorphometric and connectivity analyses.

previously [36]. The corrected full width at half the maximum height of the apatite peaks (B1/2) were measured and were used to calculate the “D” values, which are related to the crystal size/strain, by the Scherrer equation [37],

Histomorphometry

where 57.3 is a conversion factor from degrees to radians, λ is the X-ray wavelength, B1/2 is the full width at half maximum of the (002) and (130) peaks, and θ is the diffraction angle. K is a constant that depends on the crystal habit, for apatite K = 0.9, reflecting the elongated crystals of bone. Each sample was analyzed three times, and the average of the three measurements was taken.

Static histomorphometry was performed on Goldner trichrome-stained sections (5 μm) of the proximal tibia. Goldner trichrome stains the mineralized bone green and the osteoid red. Measurements of the trabecular bone were taken from a 5 mm2 area in the central region. The area of measurement was located 1 mm distal to the growth plate. A semiautomated image analysis system (Bioquant, R&M Biometrics) was used to determine the following static histomorphometry parameters: osteoid volume (OV/BV), osteoid surface (OS/ BS), osteoid thickness (O.Th) and eroded surface (ES/BS), as well as trabecular bone volume (BV/TV). All measurements and calculations were done following the American Society for Bone Mineral (ASBMR) nomenclature and guidelines [31]. Dynamic histomorphometric evaluation was performed on undecalcified, unstained sections (7 μm) of proximal tibia, which were cut at a depth of 14 μm following the Goldner trichrome-stained sections. Measurements of trabecular bone were taken from the same area as in the static histomorphometric analysis. Sections were examined under UV light for tetracycline labeling. Measurements were taken using a semiautomated image analysis system (Bioquant, R&M Biometrics). Dynamic histomorphometric parameters, including mineralizing surface (MS/BS), mineral apposition rate (MAR), bone formation rate (BFR) and mineralization lag time (Mlt) were determined. All measurements and calculations were done following the American Society for Bone Mineral (ASBMR) nomenclature and guidelines [31].

Image analysis Image analysis was performed on Von-Kossa-stained sections (5 μm) of the proximal tibia. Von Kossa stains the mineralized bone in black. Sections were scanned to and analyzed using a Quantimet 570 (Leica) image processing and analysis system. After thresholding the gray-level images, binary images were obtained and then skeletonized. Strut analysis was then performed to determine connectivity parameters, including number of nodes (NN) and number of end points (NE) [32].

Mineralization The degree of mineralization of bone was assessed by the density fractionation method of Grynpas et al. [33]. Briefly, humeri were ground into powder and sorted into incremental density fractions (<2.0–>2.25 g/cm3) by a stepwise centrifugation method. The contribution of each fraction relative to the original weight of unfractionated bone was calculated to determine a mineralization profile for each group. Shifts in mineralization profiles between groups were evaluated by comparing the logit functions of these profiles [34].

Mineral chemistry Bone calcium, magnesium, sodium and fluoride concentrations, as well as vanadium uptake by bone, were determined by Instrumental Neutron Activation Analysis (INAA). Approximately 250 mg of tibia bone powder was analyzed by the INAA at the nuclear reactor facility of McMaster University (Hamilton, Canada). Samples were irradiated for 20 s, followed by a 6-s delay before counting, and were then counted for 90 s [35]. The detection limit for vanadium was 0.7 ppm.

Bone crystal size Humeri powder was analyzed by X-ray diffraction (XRD) with a Rigaku Multiflex X-ray powder diffractometer according to a procedure described

D¼

57:3Kk B1=2 cosh

Statistical analysis Results are presented as mean ± SEM unless otherwise noted. Body weight, fluid intake, vanadium dose, plasma glucose and plasma insulin data were analyzed by GLM-ANOVA. Bone vanadium content was analyzed using a oneway ANOVA. Triglyceride data were analyzed using a two-way ANOVA. A Newman–Keuls ad hoc test was used to determine significance, P < 0.05. The effects of BEOV on the properties of non-diabetic bone (BMD, mechanical, histomorphometric and mineral properties) were determined by comparing Control, Low Dose and High Dose groups using a one-way ANOVA followed by a Fisher PLSD post hoc test, P < 0.05. To assess the effects of diabetes on the properties of bone, Control and Diabetic groups were compared by Student’s t test. The effects of BEOV on the properties of diabetic bone were determined by comparing Control, Diabetic, Controlled Glucose and Uncontrolled Glucose groups using a one-way ANOVA followed by a Fisher PLSD post hoc test, P < 0.05. Correlations were determined using multiple regression analysis followed by Newman–Keuls ad hoc test.

Results Animal model and treatment The body weight and fluid intake of the rats in the various groups at the end of the treatment period are shown in Table 1. No significant difference was noted in weight between the various control groups; however, the diabetic animals that were treated with BEOV weighed less than the other groups in the experiment. As expected, in rats made diabetic with STZ, the fluid intake of the diabetic rats was very high; this was partially corrected by the vanadium treatment. The average vanadium dose over the weeks of the treatment is shown in Table 1. The average vanadium dose of the control rats receiving the higher concentration of BEOV (High Dose) was greater than the group receiving the lower concentration (Low Dose). Because the diabetic groups drank more fluid than the control rats, they ingested a greater average vanadium dose. Among the diabetictreated rats, the more severely diabetic group (Uncontrolled Glucose) consumed more fluid than the Controlled Glucose group and had a higher average dose of vanadium. The bone vanadium content (Table 1) was greater in the High Dose, Controlled Glucose and Uncontrolled Glucose groups than in the control group that had the lower average vanadium dose (Low Dose). Rats received STZ at a dose of 45 mg/kg body weight, which produced a diabetic state similar to that known to result in the destruction of about 90% of the ß-cells in the pancreas.

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Table 1 Body weight, fluid intake, average vanadium dose, bone vanadium content and plasma parameters in the six experimental groups

Body weight (g) Fluid intake (ml/day) Vanadium dose (mmol/kg/day) Bone vanadium content (μg/g) Plasma glucose (mM) Plasma insulin (ng/ml) Plasma triglycerides (mM)

Control

Low Dose

High Dose

Diabetic

Controlled Glucose

Uncontrolled Glucose

384 ± 12 66 ± 18 N/A N/A 6.6 ± 0.1 1.5 ± 0.1 0.9 ± 0.1

348 ± 10 49 ± 4 0.1 ± 0.01 c 1.7 ± 0.1 6.4 ± 0.2 2.0 ± 0.3 0.8 ± 0.1

348 ± 9 41 ± 6 0.3 ± 0.04 c 6.5 ± 0.6 d 6.2 ± 0.1 1.6 ± 0.3 1.2 ± 0.2

348 ± 9 242 ± 7 a N/A N/A 31.4 ± 1.0 a 0.3 ± 0.1 a 10.7 ± 1.6 a

308 ± 8 a,b 104 ± 16 a 0.4 ± 0.07 c 8.9 ± 1.6 d 15.9 ± 1.3 a,b 0.9 ± 0.2 a,b 2.0 ± 0.4 b

309 ± 10 a,b 178 ± 18 a 0.6 ± 0.07 c 9.8 ± 1.4 d 24.3 ± 1.0 a,b 0.3 ± 0.1 a 5.8 ± 1.3 a

N/A — not applicable. a P < 0.05 vs. Control. b P < 0.05 vs. Diabetic. c P < 0.05 vs. all other groups. d P < 0.05 vs. Control low dose.

We have previously shown [38] that a difference of 1 or 2% in the number of surviving β-cells will result in a varying degree of severity of diabetes such that rats with just slightly more surviving ß-cells will respond to treatment with vanadium, while rats with fewer surviving cells will be more severely diabetic and unable to respond to treatment with vanadium. This proposed survival of the ß-cells is reflected in the plasma insulin content at termination, which is shown in Table 1. The group of diabetic-treated rats which had markedly improved plasma glucose (Controlled Glucose, 15.9 ± 1.3 mM glucose, average of weeks 3 to 11 of the experiment) also had a plasma insulin level which was significantly greater than the group of diabetic rats which responded less well to vanadium treatment (Uncontrolled Glucose, 24.3 ± 1.0 mM glucose, average of weeks 3 to 11 of the experiment). Vanadium is an insulin-enhancing agent as opposed to an insulin mimic. When there is very little insulin being produced, the rats have insufficient insulin to respond well to vanadium. Plasma triglycerides are a sensitive indicator of diabetic status. As seen in Table 1, the triglycerides of the diabetics were considerably higher than the controls. Treatment with BEOV normalized the triglyceride values in the Controlled Glucose group, which had lower glucose levels, and partially normalized the levels in the Uncontrolled Glucose group.

in tension/compression and shear, respectively. The results of the three-point bending test are shown in Table 2. In bending, diabetes caused a decrease in strength compared to control. Bending strength was increased by treatment with BEOV compared to untreated diabetic. Table 2 also shows the results of the torsion test of the femurs. Supplementing rats with a low or high dose of BEOV caused an increase in toughness when compared to untreated controls but did not change other torsional properties. The mechanical properties of trabecular bone were determined by vertebral compression. The results from the vertebral compression testing are shown in Table 2. Supplementing control rats with BEOV did not change compressive properties compared to untreated control. In contrast to cortical bone where diabetes induced changes in

Bone mineral density The results of BMD of the fifth lumbar vertebrae and the femur are shown in Fig. 1. Supplementation of controls with a high or low dose of BEOV did not affect vertebral or femoral BMD when compared to untreated controls. Diabetes caused a decrease in vertebral and femoral BMD compared to control. Treatment of diabetic rats with BEOV did not affect vertebral BMD but did show an improvement in femoral BMD in the group in which glucose was controlled. Mechanical testing Three-point bending and torsion tests were performed on femurs to determine the mechanical properties of cortical bone

Fig. 1. Bone mineral density of the femur (A) and the fifth lumbar vertebrae (B). P < 0.05 vs. Control; bP < 0.05 vs. Diabetic.

a

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Table 2 Mechanical properties of bone in bending, torsion and compression Control

Low Dose

High Dose

Diabetic

Controlled Glucose

Uncontrolled Glucose

Three-point bending Strength (MPa) Modulus (GPa) Toughness (mJ/mm3)

161.5 ± 5.0 3.52 ± 0.19 5.77 ± 0.76

170.2 ± 4.8 3.60 ± 0.03 7.18 ± 0.57

159.6 ± 4.8 3.37 ± 0.07 6.91 ± 0.71

136.4 ± 6.1 a 3.15 ± 0.22 5.27 ± 0.64

155.7 ± 6.8 b 3.50 ± 0.18 6.41 ± 0.48

155.8 ± 5.1 b 3.48 ± 0.17 5.87 ± 0.35

Torsion Strength (MPa) Modulus (MPa) Toughness (mJ/mm3)

58.7 ± 1.9 243.1 ± 11.3 0.59 ± 0.04

63.0 ± 2.6 232.3 ± 20.4 0.77 ± 0.08 a

61.6 ± 1.9 232.4 ± 19.0 0.77 ± 0.07 a

52.7 ± 2.9 232.0 ± 19.0 0.62 ± 0.03

56.7 ± 2.6 252.3 ± 10.4 0.57 ± 0.05

56.7 ± 3.5 262.1 ± 24.8 0.64 ± 0.06

Compression Strength (MPa) Modulus (MPa) Toughness (mJ/mm3)

25.2 ± 1.5 255.1 ± 19.0 1.77 ± 0.16

27.6 ± 1.4 304.9 ± 5.0 1.66 ± 0.16

23.3 ± 1.3 256.3 ± 13.7 1.57 ± 0.02

15.6 ± 1.1 a 159.5 ± 16.5 a 1.19 ± 0.11 a

19.3 ± 1.5a,b 182.0 ± 16.6 a 1.42 ± 0.15

15.3 ± 1.1 a 159.0 ± 14.4 a 1.22 ± 0.16 a

a b

P < 0.05 vs. Control. P < 0.05 vs. Diabetic.

strength during the three-point bending test, in trabecular bone diabetes caused decreases in compressive strength, modulus and toughness when compared to control. Treating diabetics with BEOV to control glucose increased compressive strength compared to untreated diabetics and increased toughness to a level that was not different from untreated control. In rats where BEOV treatment failed to control glucose levels, compressive strength was not improved relative to untreated diabetics. Histomorphometry The results for the trabecular bone structure are shown in Table 3. There were no changes in trabecular bone volume when controls were supplemented with a low or high dose of BEOV compared to untreated controls. Diabetes caused a dramatic decrease in trabecular bone volume, and treating diabetics with BEOV did not improve this parameter (Fig. 2). Table 3 also

shows the trabecular bone remodeling parameters. In all animals that received BEOV, there were increases in osteoid volume and osteoid surface compared to their untreated counterparts. Diabetes also caused an increase in osteoid volume when compared to controls. In diabetic rats where BEOV failed to control glucose, increased osteoid thickness and decreased eroded surface were observed. The dynamic histomorphometric parameters are shown in Table 3. Supplementing controls with BEOV caused an increase in mineralizing surface and bone formation rate compared to untreated controls. Diabetes caused an increase in mineralization lag time. Treating diabetic rats with BEOV to control glucose improved these properties and also increased the bone formation rate and mineralizing surface. In those rats where BEOV treatment failed to control glucose, there were no changes in dynamic histomorphometric parameters compared to untreated diabetics.

Table 3 Static histomorphometry, dynamic histomorphometry and strut analysis results Control

Low Dose

High Dose

Diabetic

Controlled Glucose

Uncontrolled Glucose

Trabecular bone structure and connectivity BV/TV (%) 30.2 ± 3.5 N.N (mm−2) 8.27 ± 0.91 NE (mm−2) 12.4 ± 0.9

30.3 ± 2.3 6.48 ± 0.50 a 12.3 ± 1.3

25.9 ± 2.3 6.48 ± 0.79 a 11.5 ± 1.0

16.1 ± 1.4 a 3.61 ± 0.35 a 15.5 ± 1.2 a

15.6 ± 2.0 a 3.27 ± 0.52 a 15.9 ± 1.3 a

13.8 ± 2.4 a 2.59 ± 0.49 a 15.1 ± 1.1 a

Bone remodeling OV/BV (%) OS/BS (%) O.Th (μm) ES/BS (%)

0.46 ± 0.07 a 5.89 ± 0.68 4.35 ± 0.28 0.48 ± 0.12

0.39 ± 0.07 a 6.22 ± 0.96 a 4.26 ± 0.21 0.38 ± 0.08

0.42 ± 0.03 a 5.55 ± 0.41 3.90 ± 0.18 0.41 ± 0.10

0.93 ± 0.14 a,b 9.1 ± 0.94 a,b 4.11 ± 0.12 0.40 ± 0.09

0.89 ± 0.13 a,b 8.67 ± 1.32 a,b 4.31 ± 0.27 0.30 ± 0.07

7.27 ± 1.89 a 1.31 ± 0.08 0.117 ± 0.031 a 3.31 ± 0.70

7.65 ± 1.12 a 1.30 ± 0.06 0.105 ± 0.020 a 3.15 ± 0.40

2.61 ± 0.46 1.18 ± 0.08 0.031 ± 0.006 9.46 ± 1.84 a

6.28 ± 0.94 b 1.24 ± 0.05 0.100 ± 0.018 a,b 5.40 ± 1.00 b

0.19 ± 0.04 3.05 ± 0.76 3.76 ± 0.18 0.53 ± 0.09

Dynamic histomorphometry MS/BS (%) 3.27 ± 0.46 MAR (μm/day) 1.27 ± 0.06 BFR/BS (μm/day) 0.042 ± 0.006 Mlt (day) 3.83 ± 0.84 a b

P < 0.05 vs. Control. P < 0.05 vs. Diabetic.

4.20 ± 0.65 1.18 ± 0.05 0.049 ± 0.008 9.24 ± 1.28 a

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There were no changes in the Ca, Na, Mg or F concentrations between the various groups (data not shown). Bone crystal size Mineral crystal size was determined by X-ray diffraction, and the results are shown in Table 4. Supplementing controls with a low or high dose of BEOV did not alter the crystal length or the crystal cross-section when compared to untreated controls. Diabetes caused a decrease in the crystal length compared to control. Treating diabetics to control glucose restored the crystal length. Relationships among parameters studied It was not clear from the literature whether exposure to an increased dose of vanadium would increase the amount incorporated into bone. Therefore, we examined the regression between the average daily dose of BEOV (in mmol/kg) and the

Fig. 2. Representative Von-Kossa-stained sections of the proximal tibia of Control (A), Low Dose (B), High Dose (C), Diabetic (D), Controlled Glucose (E) and Uncontrolled Glucose (F) rats.

Image analysis The results of the trabecular bone connectivity are shown in Table 3. Supplementing controls with BEOV caused a decrease in the number of nodes compared to untreated controls. Diabetes caused a decrease in the number of nodes and an increase in the number of end points compared to controls. Treatment of the diabetic rats with BEOV did not improve trabecular bone connectivity parameters. Mineralization The results of the degree of mineralization are shown in Fig. 3. As shown in Fig. 3A, treating control rats with a high dose of BEOV caused a shift in the mineralization profile to a denser mineral when compared to controls. Diabetes caused a shift in the mineralization profile to a less dense mineral when compared to controls (Fig. 3B). Compared to untreated diabetics, treating diabetics with BEOV to control glucose caused a shift in the mineralization profile to a denser mineral that was similar to the untreated control (Fig. 3C). Mineral chemistry Mineral chemistry was determined using Instrumental Neutron Activation Analysis. The vanadium content in bone is shown in Table 1. Those animals with higher vanadium dose had higher vanadium content in their bones. There was no detectable vanadium in untreated diabetic and untreated control.

Fig. 3. Mineralization profiles of control (A), Control vs. Diabetic (B) and Diabetic (C) rats. aP < 0.05 vs. Control; bP < 0.05 vs. Diabetic.

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Table 4 X-ray diffraction results showing crystal length (002) and crystal cross-section (310) Group

Length (002) (nm)

Cross-section (310) (nm)

Control Low Dose High Dose Diabetic Controlled Glucose Uncontrolled Glucose

12.95 ± 0.38 12.72 ± 0.38 13.02 ± 0.46 11.75 ± 0.16 a 12.34 ± 0.44 11.60 ± 0.14 a

5.49 ± 0.05 5.48 ± 0.06 5.61 ± 0.06 5.64 ± 0.06 5.57 ± 0.04 5.63 ± 0.06

a

P < 0.05 vs. Control.

amount of vanadium in the bone at termination (Table 5). We found that there was an increase in the amount of vanadium in the bone of the control animals that had a higher dose of BEOV (Table 5). In the diabetic rats, the amount of vanadium in the bone increased with an increased dose of BEOV (Table 5). This is true even though the average vanadium content was not significantly different between the two treated diabetic groups (Table 1). We also examined the strength of both cortical bone (three point bending) when compared to the amount of vanadium incorporated into the bone (Table 5). There was no correlation between bone vanadium content and cortical bone strength in either control or diabetic rats. Plasma glucose levels were compared to strength in cortical (three-point bending) and trabecular bone (compression) (Table 5). Not surprisingly, there were no correlations between these two parameters in the control animals since there was only a modest variability in the glucose levels. On the other hand, the bones from treated diabetic animals clearly showed an increase in strength as the glucose values fell. Discussion Animal model As expected, STZ caused a decrease in insulin and an increase in glucose. In humans, Type 1 diabetes seems to cause increased bone resorption, though the effects on bone formation are unclear. In rats, however, the STZ-induced diabetic bone disorder has been described as low turnover osteoporosis [16]. Our results do not support this idea of low bone turnover. In our study, STZ caused an increase in osteoid volume and surface, but no change in osteoid width, suggesting that bone formation is in fact increased. This is not, however, evidence for high turnover either since the other classical signs of this state that are seen in ovariectomized rats, such as increased bone formation rate together with an increase in bone resorption, were not observed [39]. A bone disorder does persist in STZ diabetic rats as decreased trabecular bone volume and trabecular connectivity were seen here and elsewhere [14]. The observed decrease in trabecular bone volume occurred in spite of the increase in bone formation seen. Thus, the net loss of bone can be attributed to an accompanying increase in bone resorption. The increased bone resorption is in agreement with the biomarker results seen in

humans, which show evidence of increased bone resorption [11,12]. Furthermore, insulin receptors have been found on osteoclast-like cells, and physiological levels of insulin seem to impair resorption [22]. A mineral defect was observed with diabetes, with a decrease in the crystal length [16,17]. In addition, the STZ-induced diabetic state decreased the degree of mineralization, which may have been due to an increase in the amount of newly formed and therefore less mineralized bone, as well as the decreased size of mineral crystals present. These mineral defects may have been caused by the diabetes-induced hyperglycemia since glucose appears to interfere with mineralization [23]. The STZ-induced diabetic state caused a decrease in BMP and decreased mechanical properties [14,17,18]. The poor trabecular structure, decreased trabecular connectivity, decreased crystal size and decreased mineralization likely contributed to these poor mechanical properties. Our results indicate that, although a bone disorder persists in STZ diabetic rats, it is not necessarily low turnover and may be a more complex bone disorder. Effect of BEOV on non-diabetic bone Since vanadium is incorporated into the lattice of bone mineral when such treatment is given, we first studied the effects of vanadium on non-diabetic bone. An increased vanadium content in bone was observed with increasing dose of BEOV, confirming that vanadium is incorporated into bone (Table 5). This vanadium, in the form of vanadate (VO4), was likely incorporated into the mineral phase of bone, replacing phosphate (PO4), as has been shown in other studies [4]. This incorporated vanadate did not cause a phase change or change in crystal size, suggesting that the bone mineral lattice can accommodate these small amounts of vanadate. Hydroxyapetite lattice has been shown to accommodate some vanadate without lattice distortion [5]. However, we found an increase in mineralization with increasing doses of BEOV. This observation may have been due to an increase in the amount of mineral per unit volume of collagen, which represents a more tightly packed mineral, by increasing the duration of

Table 5 Correlation and p values for control and diabetic treated groups Correlation

Group

R2 Value

P Value

Comparison of vanadium dose and bone vanadium content Vanadium dose vs. Control treated 0.67 bone vanadium content Diabetic treated 0.40

0.00002 0.002

Comparison of bone strength and bone vanadium content Cortical bone strength vs. Control treated 0.03 bone vanadium content Diabetic treated 0.01

0.48 0.68

Comparison of bone strength and plasma glucose level Cortical bone strength vs. Control treated 0.013 plasma glucose level Diabetic treated 0.22 Trabecular bone strength Control treated 0.007 vs. plasma glucose level Diabetic treated 0.16

0.67 0.048 0.77 0.042

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mineralization, or alternatively vanadate may act like fluoride to increase the stability of the apatite lattice [40,41]. Though trabecular bone tends to be more responsive to treatment than cortical bone, in this study, BEOV administration affected cortical rather than trabecular bone mechanical properties. This may have resulted from the different load distributions in each type of bone caused by the mechanical tests chosen, as well as the nature of bone remodeling in the rat. In torsion and bending, the mechanical properties reflect the behavior of the outermost (in this case periosteal) surface of the material since it experiences the greatest force. Due to the nature of cortical bone remodeling in rats, newly formed vanadiumrich bone is expected to be concentrated in this region. Thus, our results may indicate that the introduction of vanadium into the bone mineral lattice at higher concentrations may increase the toughness of bone. The fact that we observed an increase in torsional but not bending toughness may be due to the fact that, in bending, the bone is forced to fail at the point directly under the applied force, while, under torsion, the bone is allowed to fail at the weakest point along the entire cortical shaft. Thus, torsional properties may be more sensitive to these concentrated changes in the bone surface. Increases in osteoid parameters in bone formation rate were noted, suggesting that BEOV administration increases bone formation. Osteoblasts have been found to respond to other vanadium drugs in vitro [6,7]. Vanadium may enhance the response of osteoblasts to insulin, which increases their activity and leads to increased bone formation [19,21]. Despite this increase in bone formation, no increases in bone volume were noted. Perhaps the increased bone formation was coupled with increased bone resorption. The effects of vanadium drugs on osteoclasts have not been studied. Osteoclasts have been shown to have insulin receptors and do not respond to insulin, so they may be susceptible to the insulin-enhancing actions of vanadium. However, the in vitro effects of insulin are to inhibit osteoclastic resorption, which is contrary to the speculation that vanadium increases both bone formation and resorption [22]. The changes to bone remodeling that appear to follow BEOV treatment may be responsible in part for the change in torsional toughness observed but do not seem to cause other changes to bone quality. Nonetheless, in non-diabetic bone, vanadium appears to affect bone formation and bone material properties. Effect of BEOV on diabetic bone Since diabetic bone differs from non-diabetic bone, the effects of vanadium in diabetic bone should be investigated. As expected, BEOV is effective in lowering glucose. It is most effective where there is more residual insulin, as was the case in the Controlled Glucose group. Higher doses of BEOV do not necessarily lead to the lowest glucose levels, as shown in Fig. 4. When glucose was controlled with BEOV, improved mineral properties, including crystal size and mineralization, were observed. These improvements may be due to the improvement in the diabetic state. Vanadium compounds have been shown to mimic insulin actions in different cell types [7]. Since elevated glucose has been shown to impair mineralization in vitro, the

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Fig. 4. Cumulative dose and average glucose for treated diabetic rats.

glucose-lowering actions of vanadium may be responsible for the improved mineral properties [23]. The improvement of the diabetic state by BEOV treatment seems to also contribute to the improvements in mechanical properties observed. A drop in plasma glucose level and an increase in strength in both cortical and trabecular bone in the treated DM group was observed. The lack of a correlation between bone strength and bone vanadium content in the treated diabetic rats further suggests that the primary effect being seen is an effect of improvement in glucose levels. This agrees with the improvement in mechanical properties observed by others when diabetic rats are treated with insulin [18]. This does not rule out other direct effects of vanadium on bone. The increases in bone formation rate, osteoid volume and osteoid surface in both control and diabetic rats in response to BEOV treatment suggest some direct effect on bone formation. However, this effect was not as prominent in the diabetic rats in which the glucose levels did not respond to the treatment. BEOV appears to affect bone formation in a manner that is independent of its glucose-lowering actions, and this may be related to residual insulin levels. Increased osteoid volume has been seen in non-diabetic bone when insulin was administered locally [21]. The insulin-enhancing actions of vanadium may be promoting bone formation in a similar fashion. The absence of improvement in bone architecture and connectivity parameters, despite an increase in bone formation with BEOV administration, suggests an increased osteoclast bone resorption. Vanadium may be acting to increase osteoclast activity, as appeared to be the case in the non-diabetic rats supplemented with BEOV. Interestingly, the BEOV-induced improvement in cortical bone toughness and increased mineralization seen in non-diabetic bone were not observed in diabetic bone. The detrimental effects of diabetes on these mechanical and material properties may mask these additional effects of BEOV or perhaps the actions of vanadium may differ between diabetic and nondiabetic bone. Interestingly, controlling glucose with BEOV was more effective in improving BMD and mechanical properties in the cortical bone dominant sites than in the trabecular bone dominant sites which would be expected to respond more

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rapidly to treatment. While BEOV is noted to be effective in improving diabetes-related conditions, controlling glucose with BEOV does not completely lower glucose levels nor does it improve insulin levels—two parameters that have been shown to affect bone. Perhaps the competing actions of diabetes and treatment are exaggerated in the more metabolically active trabecular bone, resulting in less dramatic improvements to bone properties than in the typically less responsive cortical bone. In conclusion, we have shown that BEOV improves diabetes-related bone dysfunction. This improvement is largely due to the glucose-lowering effect of BEOV. BEOV also appears to increase bone formation in both non-diabetic and diabetic rats and seems to affect the cortical bone material properties in non-diabetic rats. Our studies did not show any indication of negative effects of vanadium on bone at the dose administered.

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