Bone 38 (2006) 368–377www.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 Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Ave., Toronto, Canada M5G 1X5b Department of Materials Science and Engineering, University of Toronto, Canada

c 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 2005Available 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 andstreptozotocin-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 treatedwith 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, glucoseand triglyceride assays assessed the diabetic state. Bone mineral density (BMD), mechanical testing, mineral assessment and histomorphometrymeasured the effects of diabetes on bone and the effects of BEOV on non-diabetic and diabetic bone. Diabetes decreased plasma insulin andincreased plasma glucose and triglycerides. In bone, diabetes decreased BMD, strength, mineralization, bone crystal length, and bone volume andconnectivity. Treatment was effective in incorporating vanadium into bone. In all treated groups, BEOV increased osteoid volume. In non-diabeticbone, BEOV increased cortical bone toughness, mineralization and bone formation. In controlled glucose rats, BEOV lowered plasma glucose andimproved BMD, mechanical strength, mineralization, bone crystal length and bone formation rate. In poorly controlled rats, BEOV treatmentslightly lowered plasma glucose but did not improve bone properties. These results suggest that BEOV improves diabetes-related bonedysfunction primarily by improving the diabetic state. BEOV also appeared to increase bone formation. Our study found no negative effects ofvanadium 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 potentialtreatment for diabetes since their glucose-lowering effects werefirst discovered [1]. These orally effective drugs have beenshown to reduce glucose and improve diabetes-relatedcomplications in animal models of the disease by acting asinsulin-enhancing agents. In human studies, vanadiumadministration has shown modest but positive effects inlowering blood glucose in Type 2 diabetics [2].

⁎ Corresponding author. Fax: +1 416 586 8844.E-mail addresses: facchini@mshri.on.ca (D.M. Facchini),

vioyuen@interchange.ubc.ca (V.G. Yuen), battell@interchange.ubc.ca(M.L. Battell), jmcneill@interchange.ubc.ca (J.H. McNeill),grynpas@mshri.on.ca (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, inthe form of vanadate, it replaces phosphate in the apatite lattice ofbone mineral [4]. Though its effects on the bone mineral latticeare not known, small amounts of vanadate can be accommodatedby the more crystalline synthetic hydroxyapatite lattice withoutproducing distortion or altering P–O or O–H bond strength [5].In vitro studies of vanadium drugs have shown a directstimulatory effect on osteoblast-like cells [6,7]. However, the invivo effects of vanadium incorporation in bone and exposure ofbone cells to vanadium have yet to be determined.

Since vanadium does deposit in bone, concern exists as towhat vanadium might do to bone structure and strength whengiven on a chronic basis. Bone disorder appears to affect Type 1diabetics, and this may make the incorporation of vanadium intodiabetic 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 theevidence for an osteoporosis-like condition, this bone disorderis not well characterized. Biochemical markers of boneformation are inconsistent [8,11,12], while markers of boneresorption often are increased [11,12]. Nevertheless, a bonedisorder does afflict Type 1 diabetics, and the effect ofvanadium on bone metabolism in Type 1 diabetic model is ofinterest.

In an animal model for Type 1 diabetes, streptozotocin (STZ)injection chemically destroys insulin-producing pancreatic ß-cells, mimicking the autoimmune destruction of these cells inthe human form of Type 1 diabetes. In rats, STZ results indecreased insulin secretion and hyperglycemia. The effect ofSTZ is dose-dependent, and sufficient ß-cells can remain sothat the rats are able to live without insulin supplementation[13].

As in the human form of Type 1 diabetes, STZ causes adecrease in BMD [14]. Histomorphometric evaluation hasshown that STZ decreases the amount of osteoid present andthe bone formation rate [14–16]. Furthermore, biochemicalmarkers of bone turnover are decreased [15,16]. This suggestsa state of low turnover in Type 1 diabetes, though this has notbeen shown in human studies. STZ also results in decreasedbone volume and poor trabecular connectivity [14]. A mineraldefect has also been suggested since there is a decreasedmineral crystal length [17] The Ca:P ratio is unchanged, but itis not clear whether the ash content increases or remains thesame with diabetes [16,17]. The morphological and mineralchanges with STZ likely contribute to the poor mechanicalproperties that are also observed [14,17,18]. Diabetes-inducedbone changes in the rat have been shown to be improved byinsulin [14, 15,18].

Conflicting reports exist regarding the influences ofimpaired insulin secretion and metabolic control on bonemetabolism [9,10]. In vitro studies have shown an influence ofglucose and insulin on osteoblast- and osteoclast-like cells.Insulin receptors have been found in more mature osteoblastcell lines [19], and insulin itself has been shown to increasecollagen synthesis in UMR106 osteoblast-like cells [20]. Invivo, local insulin treatment to the hemicalvaria in non-diabetic mice increased osteoid volume and the number ofosteoblasts [21]. Cultured osteoclast-like cells have beenshown to express insulin receptors as well, and insulin wasfound to decrease pit formation in a dose-dependent manner[22]. High glucose levels were found to cause decreasedcalcium uptake, irregular nodule morphology and decreasedosteoblast differentiation during in vitro bone mineral noduleformation [23]. High extracellular glucose levels have alsobeen found to cause changes in osteoblastic gene expression[15], inhibit osteoblast-like cell growth [24] and promoteosteoclastic bone resorption [25].

In this study, we assessed the effects of BEOV, an orallyeffective vanadium-containing anti-diabetic drug [26], on bonequality (mechanical, mineral, histological and structuralproperties) in non-diabetic and STZ-diabetic rats. The effects ofdiabetes on bone and the effects of BEOVon non-diabetic anddiabetic bone were examined.

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 experimentalgroups: Control (n = 10), control-low dose (Low Dose, n = 10), control-highdose (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 diabeticanimals with blood glucose <20 mM following treatment were considered tohave Controlled Glucose (n = 10), while those with blood glucose >20 mMwere considered to have Uncontrolled Glucose (n = 12). Control and diabeticuntreated animals were housed 2 rats/cage. All treated rats were housedindividually for fluid monitoring purposes. Rats were allowed ad libitumaccess to food and drinking solution and were housed on a 12 h light:darkschedule at constant temperature and humidity. All animals were cared for inaccordance with the principles and guidelines of the Canadian Council onAnimal Care.

Treatment regime

BEOV was administered in the drinking water at concentrations rangingfrom 0.125 to 0.75 mg/ml for a 12-week period. Low Dose animals received amaximum concentration of 0.25 mg/ml. High Dose animals received amaximum concentration of 0.75 mg/ml. Diabetic-treated animals receivedconcentrations as tolerated by the individual rat to lower blood glucose. Bodyweight and fluid intake were measured daily and used to calculate vanadiumdose in mmol/kg/day. Solutions were freshly prepared every 2 days.

Blood was collected weekly from the tail vein, centrifuged at10,000 × g × 25 min and the plasma analyzed for plasma glucose using aBeckman Glucose Analyzer 2. At termination, blood was collected fordetermination of plasma insulin using a radioimmunoassay kit (Linco Research,Inc.) and triglyceride levels by a colorimetric assay (Roche). Rats were killedwith an overdose of pentobarbital 100 mg/kg by intraperitoneal injection. Thefemurs, tibias, humeri and vertebrae were dissected, cleaned of adherent softtissue and stored at −20°C for future analysis.

Bone mineral density

BMD of the excised right femur and fifth lumbar vertebrae was determinedby DEXA using a dedicated small animal densitometer (PIXImus/GE). Analuminum/Lucite phantom (GE) was used to calibrate the machine [27].

Mechanical tests

The mechanical failure properties of the femur and vertebra weredetermined using an Instron 4465 Materials Testing System (Instron Corp.)and custom-built torsion testing apparatus. In all experiments, force anddeformation data were collected using LabView 5.0 data acquisition software(National Instruments). In these experiments, the point of failure was definedas a drop in load of >10%.

The diaphysis of the left femur was tested to failure in three-point bendingaccording to the procedure described previously [28]. Briefly, samples weresubjected to a preload of 1 N and then deformed at a rate of 1 mm/min untilfailure. The diaphysis of the right femur was tested to failure in torsion accordingto the procedure described previously [29]. Briefly, samples were deformed at arate of 35°/min until failure. The body of the fifth lumbar vertebra was tested tofailure in unconfined compression using a procedure similar to that describedpreviously [30]. Briefly, a preload of 1 N was applied to the sample and thendeformed 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 gradesof 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 stainedwith Goldner’s Trichrome or Von Kossa for use in static histomorphometric andconnectivity analyses.

Histomorphometry

Static histomorphometry was performed on Goldner trichrome-stainedsections (5 μm) of the proximal tibia. Goldner trichrome stains the mineralizedbone green and the osteoid red. Measurements of the trabecular bone were takenfrom a 5 mm2 area in the central region. The area of measurement was located 1mm distal to the growth plate. A semiautomated image analysis system(Bioquant, R&M Biometrics) was used to determine the following statichistomorphometry parameters: osteoid volume (OV/BV), osteoid surface (OS/BS), osteoid thickness (O.Th) and eroded surface (ES/BS), as well as trabecularbone volume (BV/TV). All measurements and calculations were done followingthe 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 μmfollowing the Goldner trichrome-stained sections. Measurements of trabecularbone were taken from the same area as in the static histomorphometric analysis.Sections were examined under UV light for tetracycline labeling. Measurementswere taken using a semiautomated image analysis system (Bioquant, R&MBiometrics). Dynamic histomorphometric parameters, including mineralizingsurface (MS/BS), mineral apposition rate (MAR), bone formation rate (BFR)and mineralization lag time (Mlt) were determined. All measurements andcalculations 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 theproximal tibia. Von Kossa stains the mineralized bone in black. Sections werescanned to and analyzed using a Quantimet 570 (Leica) image processing andanalysis system. After thresholding the gray-level images, binary images wereobtained and then skeletonized. Strut analysis was then performed to determineconnectivity parameters, including number of nodes (NN) and number of endpoints (NE) [32].

Mineralization

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

Mineral chemistry

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

Bone crystal size

Humeri powder was analyzed by X-ray diffraction (XRD) with a RigakuMultiflex X-ray powder diffractometer according to a procedure described

previously [36]. The corrected full width at half the maximum height of theapatite 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],

D ¼ 57:3KkB1=2cosh

where 57.3 is a conversion factor from degrees to radians, λ is the X-raywavelength, 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 thecrystal habit, for apatite K = 0.9, reflecting the elongated crystals of bone.Each sample was analyzed three times, and the average of the threemeasurements was taken.

Statistical analysis

Results are presented as mean ± SEM unless otherwise noted. Body weight,fluid intake, vanadium dose, plasma glucose and plasma insulin data wereanalyzed by GLM-ANOVA. Bone vanadium content was analyzed using a one-way ANOVA. Triglyceride data were analyzed using a two-way ANOVA. ANewman–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 bycomparing Control, Low Dose and High Dose groups using a one-way ANOVAfollowed by a Fisher PLSD post hoc test, P < 0.05. To assess the effects ofdiabetes on the properties of bone, Control and Diabetic groups were comparedby Student’s t test. The effects of BEOVon the properties of diabetic bone weredetermined by comparing Control, Diabetic, Controlled Glucose andUncontrolled Glucose groups using a one-way ANOVA followed by a FisherPLSD post hoc test, P < 0.05.

Correlations were determined using multiple regression analysis followed byNewman–Keuls ad hoc test.

Results

Animal model and treatment

The body weight and fluid intake of the rats in the variousgroups at the end of the treatment period are shown in Table 1.No significant difference was noted in weight between thevarious control groups; however, the diabetic animals that weretreated with BEOV weighed less than the other groups in theexperiment. As expected, in rats made diabetic with STZ, thefluid intake of the diabetic rats was very high; this was partiallycorrected by the vanadium treatment. The average vanadiumdose over the weeks of the treatment is shown in Table 1. Theaverage vanadium dose of the control rats receiving the higherconcentration of BEOV (High Dose) was greater than the groupreceiving the lower concentration (Low Dose). Because thediabetic groups drank more fluid than the control rats, theyingested a greater average vanadium dose. Among the diabetic-treated rats, the more severely diabetic group (UncontrolledGlucose) consumed more fluid than the Controlled Glucosegroup and had a higher average dose of vanadium. The bonevanadium content (Table 1) was greater in the High Dose,Controlled Glucose and Uncontrolled Glucose groups than inthe 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 resultin the destruction of about 90% of the ß-cells in the pancreas.

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

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

Control Low Dose High Dose Diabetic ControlledGlucose

UncontrolledGlucose

Body weight (g) 384 ± 12 348 ± 10 348 ± 9 348 ± 9 308 ± 8 a,b 309 ± 10 a,b

Fluid intake (ml/day) 66 ± 18 49 ± 4 41 ± 6 242 ± 7 a 104 ± 16 a 178 ± 18 a

Vanadium dose (mmol/kg/day) N/A 0.1 ± 0.01 c 0.3 ± 0.04 c N/A 0.4 ± 0.07 c 0.6 ± 0.07 c

Bone vanadium content (μg/g) N/A 1.7 ± 0.1 6.5 ± 0.6 d N/A 8.9 ± 1.6 d 9.8 ± 1.4 d

Plasma glucose (mM) 6.6 ± 0.1 6.4 ± 0.2 6.2 ± 0.1 31.4 ± 1.0 a 15.9 ± 1.3 a,b 24.3 ± 1.0 a,b

Plasma insulin (ng/ml) 1.5 ± 0.1 2.0 ± 0.3 1.6 ± 0.3 0.3 ± 0.1 a 0.9 ± 0.2 a,b 0.3 ± 0.1 a

Plasma triglycerides (mM) 0.9 ± 0.1 0.8 ± 0.1 1.2 ± 0.2 10.7 ± 1.6 a 2.0 ± 0.4 b 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.

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We have previously shown [38] that a difference of 1 or 2%in the number of surviving β-cells will result in a varyingdegree of severity of diabetes such that rats with just slightlymore surviving ß-cells will respond to treatment withvanadium, while rats with fewer surviving cells will be moreseverely diabetic and unable to respond to treatment withvanadium. This proposed survival of the ß-cells is reflected inthe plasma insulin content at termination, which is shown inTable 1. The group of diabetic-treated rats which hadmarkedly improved plasma glucose (Controlled Glucose,15.9 ± 1.3 mM glucose, average of weeks 3 to 11 of theexperiment) also had a plasma insulin level which wassignificantly greater than the group of diabetic rats whichresponded less well to vanadium treatment (UncontrolledGlucose, 24.3 ± 1.0 mM glucose, average of weeks 3 to 11 ofthe experiment). Vanadium is an insulin-enhancing agent asopposed to an insulin mimic. When there is very little insulinbeing produced, the rats have insufficient insulin to respondwell to vanadium. Plasma triglycerides are a sensitive indicatorof diabetic status. As seen in Table 1, the triglycerides of thediabetics were considerably higher than the controls. Treatmentwith BEOV normalized the triglyceride values in theControlled Glucose group, which had lower glucose levels, andpartially normalized the levels in the Uncontrolled Glucosegroup.

Bone mineral density

The results of BMD of the fifth lumbar vertebrae and thefemur are shown in Fig. 1. Supplementation of controls with ahigh or low dose of BEOV did not affect vertebral or femoralBMD when compared to untreated controls. Diabetes caused adecrease in vertebral and femoral BMD compared to control.Treatment of diabetic rats with BEOV did not affect vertebralBMD but did show an improvement in femoral BMD in thegroup in which glucose was controlled.

Mechanical testing

Three-point bending and torsion tests were performed onfemurs to determine the mechanical properties of cortical bone

in tension/compression and shear, respectively. The results ofthe 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 BEOVcompared to untreated diabetic. Table 2 also shows the results ofthe torsion test of the femurs. Supplementing rats with a low orhigh dose of BEOV caused an increase in toughness whencompared to untreated controls but did not change othertorsional properties.

The mechanical properties of trabecular bone weredetermined by vertebral compression. The results from thevertebral compression testing are shown in Table 2.Supplementing control rats with BEOV did not changecompressive properties compared to untreated control. Incontrast to cortical bone where diabetes induced changes in

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

Control Low Dose High Dose Diabetic ControlledGlucose

UncontrolledGlucose

Three-point bendingStrength (MPa) 161.5 ± 5.0 170.2 ± 4.8 159.6 ± 4.8 136.4 ± 6.1 a 155.7 ± 6.8 b 155.8 ± 5.1 b

Modulus (GPa) 3.52 ± 0.19 3.60 ± 0.03 3.37 ± 0.07 3.15 ± 0.22 3.50 ± 0.18 3.48 ± 0.17Toughness (mJ/mm3) 5.77 ± 0.76 7.18 ± 0.57 6.91 ± 0.71 5.27 ± 0.64 6.41 ± 0.48 5.87 ± 0.35

TorsionStrength (MPa) 58.7 ± 1.9 63.0 ± 2.6 61.6 ± 1.9 52.7 ± 2.9 56.7 ± 2.6 56.7 ± 3.5Modulus (MPa) 243.1 ± 11.3 232.3 ± 20.4 232.4 ± 19.0 232.0 ± 19.0 252.3 ± 10.4 262.1 ± 24.8Toughness (mJ/mm3) 0.59 ± 0.04 0.77 ± 0.08 a 0.77 ± 0.07 a 0.62 ± 0.03 0.57 ± 0.05 0.64 ± 0.06

CompressionStrength (MPa) 25.2 ± 1.5 27.6 ± 1.4 23.3 ± 1.3 15.6 ± 1.1 a 19.3 ± 1.5a,b 15.3 ± 1.1 a

Modulus (MPa) 255.1 ± 19.0 304.9 ± 5.0 256.3 ± 13.7 159.5 ± 16.5 a 182.0 ± 16.6 a 159.0 ± 14.4 a

Toughness (mJ/mm3) 1.77 ± 0.16 1.66 ± 0.16 1.57 ± 0.02 1.19 ± 0.11 a 1.42 ± 0.15 1.22 ± 0.16 a

a P < 0.05 vs. Control.b P < 0.05 vs. Diabetic.

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strength during the three-point bending test, in trabecular bonediabetes caused decreases in compressive strength, modulus andtoughness when compared to control. Treating diabetics withBEOV to control glucose increased compressive strengthcompared to untreated diabetics and increased toughness to alevel that was not different from untreated control. In rats whereBEOV treatment failed to control glucose levels, compressivestrength was not improved relative to untreated diabetics.

Histomorphometry

The results for the trabecular bone structure are shown inTable 3. There were no changes in trabecular bone volume whencontrols were supplemented with a low or high dose of BEOVcompared to untreated controls. Diabetes caused a dramaticdecrease in trabecular bone volume, and treating diabetics withBEOV did not improve this parameter (Fig. 2). Table 3 also

Table 3Static histomorphometry, dynamic histomorphometry and strut analysis results

Control Low Dose High Do

Trabecular bone structure and connectivityBV/TV (%) 30.2 ± 3.5 30.3 ± 2.3 25.9 ± 2N.N (mm−2) 8.27 ± 0.91 6.48 ± 0.50 a 6.48 ± 0NE (mm−2) 12.4 ± 0.9 12.3 ± 1.3 11.5 ± 1

Bone remodelingOV/BV (%) 0.19 ± 0.04 0.46 ± 0.07 a 0.39 ± 0OS/BS (%) 3.05 ± 0.76 5.89 ± 0.68 6.22 ± 0O.Th (μm) 3.76 ± 0.18 4.35 ± 0.28 4.26 ± 0ES/BS (%) 0.53 ± 0.09 0.48 ± 0.12 0.38 ± 0

Dynamic histomorphometryMS/BS (%) 3.27 ± 0.46 7.27 ± 1.89 a 7.65 ± 1MAR (μm/day) 1.27 ± 0.06 1.31 ± 0.08 1.30 ± 0BFR/BS (μm/day) 0.042 ± 0.006 0.117 ± 0.031 a 0.105 ± 0Mlt (day) 3.83 ± 0.84 3.31 ± 0.70 3.15 ± 0

a P < 0.05 vs. Control.b P < 0.05 vs. Diabetic.

shows the trabecular bone remodeling parameters. In all animalsthat received BEOV, there were increases in osteoid volume andosteoid surface compared to their untreated counterparts.Diabetes also caused an increase in osteoid volume whencompared to controls. In diabetic rats where BEOV failed tocontrol glucose, increased osteoid thickness and decreasederoded surface were observed.

The dynamic histomorphometric parameters are shown inTable 3. Supplementing controls with BEOV caused an increasein mineralizing surface and bone formation rate compared tountreated controls. Diabetes caused an increase in mineralizationlag time. Treating diabetic rats with BEOV to control glucoseimproved these properties and also increased the bone formationrate and mineralizing surface. In those rats where BEOVtreatment failed to control glucose, there were no changes indynamic histomorphometric parameters compared to untreateddiabetics.

se Diabetic ControlledGlucose

UncontrolledGlucose

.3 16.1 ± 1.4 a 15.6 ± 2.0 a 13.8 ± 2.4 a

.79 a 3.61 ± 0.35 a 3.27 ± 0.52 a 2.59 ± 0.49 a

.0 15.5 ± 1.2 a 15.9 ± 1.3 a 15.1 ± 1.1 a

.07 a 0.42 ± 0.03 a 0.93 ± 0.14 a,b 0.89 ± 0.13 a,b

.96 a 5.55 ± 0.41 9.1 ± 0.94 a,b 8.67 ± 1.32 a,b

.21 3.90 ± 0.18 4.11 ± 0.12 4.31 ± 0.27

.08 0.41 ± 0.10 0.40 ± 0.09 0.30 ± 0.07

.12 a 2.61 ± 0.46 6.28 ± 0.94 b 4.20 ± 0.65

.06 1.18 ± 0.08 1.24 ± 0.05 1.18 ± 0.05

.020 a 0.031 ± 0.006 0.100 ± 0.018 a,b 0.049 ± 0.008

.40 9.46 ± 1.84 a 5.40 ± 1.00 b 9.24 ± 1.28 a

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

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

373D.M. Facchini et al. / Bone 38 (2006) 368–377

Image analysis

The results of the trabecular bone connectivity are shown inTable 3. Supplementing controls with BEOV caused adecrease in the number of nodes compared to untreatedcontrols. Diabetes caused a decrease in the number of nodesand an increase in the number of end points compared tocontrols. Treatment of the diabetic rats with BEOV did notimprove trabecular bone connectivity parameters.

Mineralization

The results of the degree of mineralization are shown inFig. 3. As shown in Fig. 3A, treating control rats with ahigh dose of BEOV caused a shift in the mineralizationprofile to a denser mineral when compared to controls.Diabetes caused a shift in the mineralization profile to a lessdense mineral when compared to controls (Fig. 3B).Compared to untreated diabetics, treating diabetics withBEOV to control glucose caused a shift in the mineralizationprofile to a denser mineral that was similar to the untreatedcontrol (Fig. 3C).

Mineral chemistry

Mineral chemistry was determined using InstrumentalNeutron Activation Analysis. The vanadium content in bone isshown in Table 1. Those animals with higher vanadium dosehad higher vanadium content in their bones. There was nodetectable vanadium in untreated diabetic and untreated control.

There were no changes in the Ca, Na, Mg or F concentrationsbetween 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 controlswith a low or high dose of BEOV did not alter the crystal lengthor the crystal cross-section when compared to untreated controls.Diabetes caused a decrease in the crystal length compared tocontrol. Treating diabetics to control glucose restored the crystallength.

Relationships among parameters studied

It was not clear from the literature whether exposure to anincreased dose of vanadium would increase the amountincorporated into bone. Therefore, we examined the regressionbetween the average daily dose of BEOV (in mmol/kg) and the

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

Group Length (002)(nm)

Cross-section(310) (nm)

Control 12.95 ± 0.38 5.49 ± 0.05Low Dose 12.72 ± 0.38 5.48 ± 0.06High Dose 13.02 ± 0.46 5.61 ± 0.06Diabetic 11.75 ± 0.16 a 5.64 ± 0.06Controlled Glucose 12.34 ± 0.44 5.57 ± 0.04Uncontrolled Glucose 11.60 ± 0.14 a 5.63 ± 0.06a P < 0.05 vs. Control.

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

Correlation Group R2 Value P Value

Comparison of vanadium dose and bone vanadium contentVanadium dose vs.bone vanadium content

Control treated 0.67 0.00002Diabetic treated 0.40 0.002

Comparison of bone strength and bone vanadium contentCortical bone strength vs.bone vanadium content

Control treated 0.03 0.48Diabetic treated 0.01 0.68

Comparison of bone strength and plasma glucose levelCortical bone strength vs.plasma glucose level

Control treated 0.013 0.67Diabetic treated 0.22 0.048

Trabecular bone strengthvs. plasma glucose level

Control treated 0.007 0.77Diabetic treated 0.16 0.042

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amount of vanadium in the bone at termination (Table 5). Wefound that there was an increase in the amount of vanadium inthe bone of the control animals that had a higher dose of BEOV(Table 5). In the diabetic rats, the amount of vanadium in thebone increased with an increased dose of BEOV (Table 5). Thisis true even though the average vanadium content was notsignificantly different between the two treated diabetic groups(Table 1).

We also examined the strength of both cortical bone (threepoint bending) when compared to the amount of vanadiumincorporated into the bone (Table 5). There was no correlationbetween bone vanadium content and cortical bone strength ineither control or diabetic rats.

Plasma glucose levels were compared to strength in cortical(three-point bending) and trabecular bone (compression) (Table5). Not surprisingly, there were no correlations between thesetwo parameters in the control animals since there was only amodest variability in the glucose levels. On the other hand, thebones from treated diabetic animals clearly showed an increasein strength as the glucose values fell.

Discussion

Animal model

As expected, STZ caused a decrease in insulin and an increasein glucose. In humans, Type 1 diabetes seems to cause increasedbone resorption, though the effects on bone formation areunclear. In rats, however, the STZ-induced diabetic bone disorderhas been described as low turnover osteoporosis [16]. Our resultsdo not support this idea of low bone turnover. In our study, STZcaused an increase in osteoid volume and surface, but no changein osteoid width, suggesting that bone formation is in factincreased. This is not, however, evidence for high turnover eithersince the other classical signs of this state that are seen inovariectomized rats, such as increased bone formation ratetogether with an increase in bone resorption, were not observed[39].

A bone disorder does persist in STZ diabetic rats as decreasedtrabecular bone volume and trabecular connectivity were seenhere and elsewhere [14]. The observed decrease in trabecularbone volume occurred in spite of the increase in bone formationseen. Thus, the net loss of bone can be attributed to anaccompanying increase in bone resorption. The increased boneresorption 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 onosteoclast-like cells, and physiological levels of insulin seem toimpair resorption [22].

A mineral defect was observed with diabetes, with a decreasein the crystal length [16,17]. In addition, the STZ-induceddiabetic state decreased the degree of mineralization, whichmay have been due to an increase in the amount of newlyformed and therefore less mineralized bone, as well as thedecreased size of mineral crystals present. These mineral defectsmay have been caused by the diabetes-induced hyperglycemiasince glucose appears to interfere with mineralization [23].

The STZ-induced diabetic state caused a decrease in BMPand decreased mechanical properties [14,17,18]. The poortrabecular structure, decreased trabecular connectivity,decreased crystal size and decreased mineralization likelycontributed to these poor mechanical properties. Our resultsindicate that, although a bone disorder persists in STZ diabeticrats, it is not necessarily low turnover and may be a morecomplex bone disorder.

Effect of BEOV on non-diabetic bone

Since vanadium is incorporated into the lattice of bonemineral when such treatment is given, we first studied theeffects of vanadium on non-diabetic bone. An increasedvanadium content in bone was observed with increasing dose ofBEOV, confirming that vanadium is incorporated into bone(Table 5). This vanadium, in the form of vanadate (VO4), waslikely incorporated into the mineral phase of bone, replacingphosphate (PO4), as has been shown in other studies [4]. Thisincorporated vanadate did not cause a phase change or changein crystal size, suggesting that the bone mineral lattice canaccommodate these small amounts of vanadate. Hydroxyapetitelattice has been shown to accommodate some vanadate withoutlattice distortion [5]. However, we found an increase inmineralization with increasing doses of BEOV. Thisobservation may have been due to an increase in the amount ofmineral per unit volume of collagen, which represents a moretightly packed mineral, by increasing the duration of

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

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mineralization, or alternatively vanadate may act like fluoride toincrease the stability of the apatite lattice [40,41].

Though trabecular bone tends to be more responsive totreatment than cortical bone, in this study, BEOVadministrationaffected cortical rather than trabecular bone mechanicalproperties. This may have resulted from the different loaddistributions in each type of bone caused by the mechanicaltests chosen, as well as the nature of bone remodeling in the rat.In torsion and bending, the mechanical properties reflect thebehavior of the outermost (in this case periosteal) surface of thematerial since it experiences the greatest force. Due to the natureof cortical bone remodeling in rats, newly formed vanadium-rich bone is expected to be concentrated in this region. Thus, ourresults may indicate that the introduction of vanadium into thebone mineral lattice at higher concentrations may increase thetoughness of bone. The fact that we observed an increase intorsional but not bending toughness may be due to the fact that,in bending, the bone is forced to fail at the point directly underthe applied force, while, under torsion, the bone is allowed tofail at the weakest point along the entire cortical shaft. Thus,torsional properties may be more sensitive to these concentratedchanges in the bone surface.

Increases in osteoid parameters in bone formation rate werenoted, suggesting that BEOV administration increases boneformation. Osteoblasts have been found to respond to othervanadium drugs in vitro [6,7]. Vanadium may enhance theresponse of osteoblasts to insulin, which increases their activityand leads to increased bone formation [19,21]. Despite thisincrease in bone formation, no increases in bone volume werenoted. Perhaps the increased bone formation was coupled withincreased bone resorption. The effects of vanadium drugs onosteoclasts have not been studied. Osteoclasts have been shownto have insulin receptors and do not respond to insulin, so theymay be susceptible to the insulin-enhancing actions ofvanadium. However, the in vitro effects of insulin are to inhibitosteoclastic resorption, which is contrary to the speculation thatvanadium increases both bone formation and resorption [22].

The changes to bone remodeling that appear to follow BEOVtreatment may be responsible in part for the change in torsionaltoughness observed but do not seem to cause other changes tobone quality. Nonetheless, in non-diabetic bone, vanadiumappears to affect bone formation and bone material properties.

Effect of BEOV on diabetic bone

Since diabetic bone differs from non-diabetic bone, theeffects of vanadium in diabetic bone should be investigated. Asexpected, BEOV is effective in lowering glucose. It is mosteffective where there is more residual insulin, as was the case inthe Controlled Glucose group. Higher doses of BEOV do notnecessarily lead to the lowest glucose levels, as shown in Fig. 4.

When glucose was controlled with BEOV, improved mineralproperties, including crystal size and mineralization, wereobserved. These improvements may be due to the improvementin the diabetic state. Vanadium compounds have been shown tomimic insulin actions in different cell types [7]. Since elevatedglucose has been shown to impair mineralization in vitro, the

glucose-lowering actions of vanadium may be responsible forthe improved mineral properties [23].

The improvement of the diabetic state by BEOV treatmentseems to also contribute to the improvements in mechanicalproperties observed. A drop in plasma glucose level and anincrease in strength in both cortical and trabecular bone in thetreated DM group was observed.

The lack of a correlation between bone strength and bonevanadium content in the treated diabetic rats further suggeststhat the primary effect being seen is an effect of improvement inglucose levels. This agrees with the improvement in mechanicalproperties observed by others when diabetic rats are treated withinsulin [18].

This does not rule out other direct effects of vanadium onbone. The increases in bone formation rate, osteoid volume andosteoid surface in both control and diabetic rats in response toBEOV treatment suggest some direct effect on bone formation.However, this effect was not as prominent in the diabetic rats inwhich the glucose levels did not respond to the treatment.BEOV appears to affect bone formation in a manner that isindependent of its glucose-lowering actions, and this may berelated to residual insulin levels. Increased osteoid volume hasbeen seen in non-diabetic bone when insulin was administeredlocally [21]. The insulin-enhancing actions of vanadium may bepromoting bone formation in a similar fashion.

The absence of improvement in bone architecture andconnectivity parameters, despite an increase in boneformation with BEOV administration, suggests an increasedosteoclast bone resorption. Vanadium may be acting toincrease osteoclast activity, as appeared to be the case in thenon-diabetic rats supplemented with BEOV. Interestingly, theBEOV-induced improvement in cortical bone toughness andincreased mineralization seen in non-diabetic bone were notobserved in diabetic bone. The detrimental effects ofdiabetes on these mechanical and material properties maymask these additional effects of BEOV or perhaps theactions of vanadium may differ between diabetic and non-diabetic bone.

Interestingly, controlling glucose with BEOV was moreeffective in improving BMD and mechanical properties in thecortical bone dominant sites than in the trabecular bonedominant sites which would be expected to respond more

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rapidly to treatment. While BEOV is noted to be effective inimproving diabetes-related conditions, controlling glucose withBEOV does not completely lower glucose levels nor does itimprove insulin levels—two parameters that have been shownto affect bone. Perhaps the competing actions of diabetes andtreatment are exaggerated in the more metabolically activetrabecular bone, resulting in less dramatic improvements tobone properties than in the typically less responsive corticalbone.

In conclusion, we have shown that BEOV improvesdiabetes-related bone dysfunction. This improvement is largelydue to the glucose-lowering effect of BEOV. BEOV alsoappears to increase bone formation in both non-diabetic anddiabetic rats and seems to affect the cortical bone materialproperties in non-diabetic rats. Our studies did not show anyindication of negative effects of vanadium on bone at the doseadministered.

Acknowledgments

This work was funded by the Canadian Institutes of HealthResearch. The authors thank Richard Cheung, Dr. RonHancock, Dr. Mircea Dumitriu, Katy Zhang and Maria Mendes.Diana M. Facchini is funded by the Natural Sciences andEngineering Research Council of Canada and the University ofToronto.

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