aluminum bone toxicity in immature rats exposed to simulated high altitude

ORIGINAL ARTICLE

Aluminum bone toxicity in immature rats exposedto simulated high altitude

Marıa del Pilar Martınez • Clarisa Bozzini •

Marıa Itatı Olivera • Ganna Dmytrenko •

Marıa Ines Conti

Received: 2 June 2010 / Accepted: 17 December 2010 / Published online: 17 February 2011

� The Japanese Society for Bone and Mineral Research and Springer 2011

Abstract Aluminum (Al) is an element to which humans

are widely exposed. Chronic administration induces a

negative effect on bone tissue, affecting collagen synthesis

and matrix mineralization. Its toxic effects are cumulative.

Hypobaric hypoxia induces stress erythropoiesis, leading to

hypertrophy of the erythropoietic marrow affecting the

bone. This study was designed to evaluate the risk of Al

bone toxicity among immature rats maintained at simulated

high altitude (SHA) by mechanical assessment of stiffness

and strength, calculation of some indicators of bone

material and geometrical properties, as well as blood

determinations. Forty growing rats were divided into con-

trol and experimental groups whether injected with vehicle

or Al, as Al(OH)3, three times a week for 3 months. Half of

each group was exposed to hypobaric conditions (HX) by

placing the animals in a SHA chamber. Both treatments

negatively affected structural properties of bones,

decreasing the maximum capacity to withstand load, the

limit elastic load and the capacity of absorbing energy in

elastic conditions. Al administration significantly depres-

sed mandible structural stiffness, although diaphyseal

stiffness was not modified. Indicators of bone material

intrinsic properties, elastic modulus and stress, were sig-

nificantly reduced by Al or HX. Treatments increased the

diaphyseal sectional bending moment of inertia, suggesting

that femur, but not mandible, compensates for the decline

in the material properties with an adaptation of its archi-

tecture to maintain structural properties. The different

biomechanical behaviors between the two kinds of bone

are probably due to their different embryological origin and

specific functions, as mandible is a bone that adjusts its

strength to biting forces, whereas femur is designed to

support load.

Keywords Aluminum intoxication � Simulated high

altitude � Bone biomechanics

Introduction

Aluminum (Al) is a nonessential element to which humans

are constantly exposed because of industrialization and

improvements in health-related technology. It is widely

used in the paper and dye industries, for water purification,

cooking pans, in cosmetic and pharmaceutical prepara-

tions, in toothpastes, in material for dental implants, in

paints and pigments, and in the textile and food processing

industries. Al toxicity can cause several clinical disorders

such as anemia, bone diseases and neurotoxicity. In bone

tissue Al exerts inhibition of bone cell proliferation,

hydroxyapatite formation and growth and suppression of

bone cell activity. These effects lead to the inhibition of

bone mineralization, decreased bone formation and lower

bone mass [1]. Because Al is sequestered in bone for long

periods of time, its toxic effects are cumulative. As a result,

even intermittent or low doses of Al add to the total load of

this toxin in the bone.

The increase in economic activities has contributed to

the fact that in South America nearly 35 million people are

M. del Pilar Martınez (&) � C. Bozzini �M. I. Olivera � M. I. Conti

Department of Physiology, Faculty of Odontology,

University of Buenos Aires, MT de Alvear 2142 3� A,

CP 1122, Buenos Aires, Argentina

e-mail: [email protected]

G. Dmytrenko

Faculty of Odontology, University of Buenos Aires,

Buenos Aires, Argentina

123

J Bone Miner Metab (2011) 29:526–534

DOI 10.1007/s00774-010-0254-4

now living at an altitude of over 2500 m, as are about

80 million people in Asia, 15 million in North America and

12 million in the Qinghai Tibetan Plateau. Therefore, an

increasing number of people are being exposed to an

environment they have not experienced before and are

subsequently undergoing physiological changes that may

pose a risk of adverse consequences [2].

Peru, Bolivia, Argentina and Chile share a geographical

area characterized by high altitudes (over 4000 m) where

mining activities that arose over the last few years have

resulted in individuals being exposed to chronic alterations

between work at high altitude and rest at sea level (chronic

intermittent hypobaric hypoxia) [3]. These individuals are

frequently exposed to Al, especially because of the daily

use of kitchenware.

At increasing altitudes, animals and humans are subject

to many stresses: cold, dry air and reduced atmospheric

pressure are just a few of them. Hypobaric hypoxia is the

major and best known stimulus to induce stress erythro-

poiesis, a state of increased red cell production in response

to an enhanced secretion of erythropoietin, the renal hor-

mone that can be considered as a part of a feedback system

that has evolved to adjust the volume of the circulating red

cell mass to the tissue oxygen demand [4]. Under normal

conditions, the erythropoietic tissue is confined to the bone

marrow within the skeleton. During hypoxia, an increase in

erythroid precursor cells occurs, leading to hypertrophy of

the erythropoietic marrow [5]. The resulting expansion of

the marrow space may induce bone resorption and could

alter the biomechanical performance of the bone. When

adult female rats are exposed to simulated high altitude, a

negative effect on bone material quality occurs with

improvement in the diaphyseal cross-sectional design in an

attempt to maintain a constant bending strength of the

femur as a whole [6].

Bones are known to self-control their structural prop-

erties (e.g., strength and stiffness) by adapting their geo-

metric properties (e.g., cross-sectional area, moment of

inertia and cortical thickness) to the material ones (e.g.,

capacity to withstand stress and modulus of elasticity) as a

function of the mechanical use of the skeleton [7].

The resistance of the whole bone to deformation by a

load (structural stiffness), measurable as a load/deforma-

tion ratio, is usually kept high enough to avoid damage and

fracture. This is thought to be controlled by the ‘‘bone

mechanostat,’’ a feedback mechanism that optimizes the

bone’s design through a continual redistribution of miner-

alized tissue. This involves the triggering and directional

modulation of bone modeling and remodeling according to

the customary strains produced in physiological conditions

[8].

Rat models in bone studies can be extrapolated to the

human being [9], despite the well-known differences in

remodeling and maturation. Previous studies showed that

Al impaired the bone’s ability to resist loads [7] and altered

dental mineralization [10].

On the other hand, Bozzini et al. demonstrated that rats

under hypobaric conditions (HX) showed a negative effect

on their bone material properties. These considerations,

and the fact that many populations living at high altitude

are constantly exposed to Al, prompted us to investigate

the risk of Al’s bone toxicity among immature rats main-

tained at simulated high altitude (SHA) as assessed by

mechanical determinations of bone stiffness and strength,

calculation of some indicators of bone material properties

and the measurement of some biochemical variables in the

blood.

The study aimed to cover the lack of information

regarding the effects of this toxic agent on axial and

appendicular bones under the above experimental condi-

tions. In addition, a description of aluminum-induced

alterations on the mandibular patterns of deformations and

stresses determined during biting and chewing may be of

interest for odontological research.

Materials and methods

Experimental design

Forty growing female Sprague–Dawley rats, aged 21 days,

were used throughout the experiments. They were housed

in stainless-steel cages and maintained under local vivar-

ium conditions (temperature 22–23�C, 12-h on/off light

cycle). All animals were allowed free access to water and a

standard pelleted chow diet that has been shown to meet all

necessary requirements to allow normal growth rates [11].

Rats were randomly divided into 4 groups of 10 animals

each: groups 1 and 3 were given vehicle (glycerol 20%) as

controls, and groups 2 and 4 received intraperitoneal doses

of 27 mg/kg of elemental Al, as Al(OH)3 (Anedra�), 3

times a week for 3 months [12]. A control group (CNX)

and an Al-treated group (AlNX) were maintained at normal

ambient pressure [normoxic (NX) rats]. Further control

(CHX) and Al-treated animals were exposed to HX (18 h/

day) during the whole experimental period [hypoxic (HX)

rats, CHX, AlHX] by placing animals into a SHA chamber

in which the air pressure was maintained at 506 mbar using

a continuous vacuum pump and an adjustable inflow valve

[13].

Growing animals were selected in order to optimize the

incorporation of Al into cortical bone. All animals were

treated in accordance with the guidelines and regulations

for the use and care of animals of the University of Buenos

Aires Ethics Committee. At the end of the experimental

period, after obtaining blood samples by cardiac puncture,

J Bone Miner Metab (2011) 29:526–534 527

123

animals were killed by ether overdose, weighed and mea-

sured. Femurs and mandibles were properly dissected to

perform anthropometric and mechanical studies.

General, femoral and mandibular anthropometry

Body weight and length were registered at the beginning of

the experiment (day 0) and once a week during the

experimental period. Body length was determined by

measuring the distance between the nose tip and the last

hairs of the tail’s base with a scaled ruler.

Both femurs were dissected, avoiding periosteal dam-

age, weighed and measured from the tip to the greater

trochanter to the distal surface of the lateral-medial con-

dyles with a digital sliding caliper [14]. The mandibles

were excised, cleaned of adherent soft tissue, weighed and

split at the midline suture. Mandibular area, as an indi-

cator of the mandibular growth, was estimated directly

from the right hemimandible by taking measurements (to

the nearest 0.05 mm) with digital calipers [15] with

modifications [17] from the triangle formed by the three

points: (I) the most anterior inferior bone point of

the interdental spine, (II) the most posterior point of

the angular process and (III) the most superior point

of the coronoid process (Fig. 1).

Femurs and mandibles were stored at 4�C wrapped in

gauze soaked with Ringer’s solution in sealed plastic bags

to prevent desiccation. Before analysis each bone was

thawed at room temperature.

Blood determinations

The hematocrit and reticulocyte values were determined to

assess the degree of anemia. Uremia and creatinine blood

levels were assayed by kinetic UV and Jaffe kinetic-

colorimetric methods, respectively, in order to evaluate

renal function.

Mechanical testing of bones

Mechanical properties of the left femur and hemimandible

of each animal were determined by a three-point bending

mechanical test on an Instron Universal Testing Machine

Model 4442 (Canton, MA) [16]. Femurs were placed on

two supports (L = 13 mm span) and centered along its

length. The load was applied perpendicularly to the long

axis of the bone at a 5 mm/min speed until fracture.

Mandibles were placed on the same supports (L = 11 mm

span) with the lateral aspect facing down and centered

along its length. Load was applied transversely to the

mandible axis at a point immediately posterior to the distal

surface of the third molar until fracture (Fig. 1). The

deflection of the arch formed by the deforming bones was

measured throughout the assay by cross-head travel

allowing for machine compliance. The load/deflection

(W/d) curves (Fig. 2) showed, successively, the linearly

elastic (Hookean) behavior followed by the non-linear

(non-Hookean) behavior, separated by the yield point (y),

until fracture [7, 17]. From the load/deflection curves, the

following whole-bone (structural) properties were mea-

sured: (a) limit (yield) elastic load, Wy; (b) limit (yield)

elastic deflection, dy; (c) structural (diaphyseal) stiffness

(load/deflection ratio) during the elastic behavior, Wy/dy;

(d) elastic absorption of energy (energy absorption capacity

by the whole bone during the elastic period), EAC =

Wydy/2; (e) maximal load supported (ultimate load,

‘‘fracture’’ load, ‘‘diaphyseal strength’’), Wf.

Evaluation of geometrical properties (bone architecture)

was performed by using an Isomet low-speed diamond saw

(Buehler, Lake Bluff, IL). A 2-mm cross-section slide was

Fig. 1 Schematic representation of femur (left) and medial aspect of

the right mandible (right) showing the load (W) applied to perform the

three-point bending mechanical test on an Instron Universal Testing

Machine Model 4442. Mandible also shows significant bony points:

I (the most anterior inferior bone point of the interdental spine), II (the

most posterior point of the angular process), III (the most superior

point of the coronoid process), IV (the most anterior superior bone

point of the interdental spine), V (immediately anterior to the

anterior surface of the first molar), VI (immediately posterior to

the posterior surface of the third molar) and VII (the furthest point on

the articular surface of the condyle)

Fig. 2 Diagram of a load (W)/deformation (d) curve showing the

elastic (Hookean behavior) and plastic (non-Hookean behavior)

phases, separated by the yielding point

528 J Bone Miner Metab (2011) 29:526–534

123

cut from the regularized fracture section in order to per-

form micromorphometrical determinations of the vertical

(load direction) and horizontal (right angle to load direc-

tion) outer and inner diameters of the elliptic-shaped

fracture sections (B = vertical outer diameter, H = hori-

zontal outer diameter, b = vertical inner diameter,

h = horizontal inner diameter) using a stereomicroscope

(Stemi DV4 Stereomicroscope, Carl Zeiss Micro Imaging,

Gottingen, Germany) and a digital caliper (Digimess,

Geneva, Switzerland). This procedure enabled calculation

of the following cross-sectional (geometric) properties:

(a) the entire cross-sectional area (CSA, mm2, p/4 BH); the

cross-sectional medullar area (CSMA, mm2, p/4 bh) and

the cross-sectional cortical area [CSCA, mm2 (CSA -

CSMA)], indicators of the amount of bone mass, and

(b) the second moment of inertia of the cross section in

relation to the horizontal axis (CSMI, mm4, p [B3H -

b3h]/64), a measure of the architectural efficiency of the

cortical design in relation to the distance at which the cor-

tical tissue is distributed from the bending axis in the cross-

section concerning the kind of deformation [18].

Expression of structural properties in relation to geo-

metric properties indirectly allowed calculation of the

following material or intrinsic properties of the bone,

which are independent of its size and shape: (a) Young’s

modulus of elasticity of the bone mineralized tissue

(E = Wy L3/48 dy CSMI, N mm-2, an estimator of the

intrinsic stiffness of the ‘‘solid’’ bone tissue) and (b) limit

elastic stress (stress = LB Wy/8 CSMI, N mm-2, B being

the maximal anterior–posterior diameter of the bone cross

section) representing the load supported per unit of cortical

bone CSA at the end of the elastic period, ‘‘bone tissue

strength’’ [19].

Bone ash determinations

After mechanical testing, femurs and hemimandibles were

dissecated for bone ash determination in a muffle furnace at

600�C for 18 h.

Al and calcium contents in these ashes were determined

by spectrophotometric atomic absorption (Varian AA 475).

Statistical analysis

Data were analyzed by one-way analysis of variance

(ANOVA), followed by Student–Newman–Keuls multiple

comparison test. Analyses were performed using the Soft-

ware package Instat and Prism V.3 (GraphPad Software

Inc., San Diego, CA). A P value less than 0.01 was

considered statistically significant. This protocol was

reviewed and approved by the Institutional Ethics Review

Committee.

Results

General anthropometry, aluminum accumulation,

hematological and metabolic parameters

Al administration did not affect body and femoral weight,

although they were significantly depressed by long-term

exposure to HX, as was previously reported by Bozzini

et al. [6] (Table 1).

Femoral length was reduced by HX and further dimin-

ished by Al treatment. The hemimandible area was unaf-

fected by Al administration and SHA exposure, although

the hemimandible weight was depressed when both treat-

ments were performed simultaneously. Al content in

femoral and mandibular ashes was significantly higher in

Al-treated than in control rats. Bone ash Ca content (mg/g

ashes) did not significantly differ between control and Al or

HX-treated rats. The mean hematocrit value was depressed

and the percent reticulocyte value was increased by addi-

tional Al administration. As expected, hypoxia exposure

significantly enhanced both parameters. Blood uremia and

creatinine levels ranked among normal values, indicating

no renal failure after treatments.

Whole-bone structural properties

Al treatment decreased the ultimate load (Fig. 3a, e), the

limit elastic load (Fig. 3b, f) and the elastic absorption of

energy (Fig. 3d, h) of both kinds of bones. However, it

reduced the structural stiffness (load/deformation ratio)

only in the mandibles (Fig. 3c, g).

HX treatment reduced the ultimate strength (Fig. 3a, e),

the limit elastic load (Fig. 3b, f) and the elastic absorption

of energy (Fig. 3d, h) more severely than Al treatment did

in both types of bones, but reduced the structural stiffness

only in the femur diaphyses (Fig. 3c, g).

Combined Al ? HX treatment showed intermediary

effects on bone structural properties, similar to those of HX

alone (Fig. 3).

Geometrical properties

Al improved cross-sectional geometry (cortical area and

design) in the femurs (Fig. 4b, c).

The cross-sectional moment of inertia was improved by

HX only in the femurs (Fig. 4c), whereas the medullar area

was enhanced in both types of bones (Fig. 4a, d).

When both treatments were assayed together, a strong

trend to enhance the diaphyseal bone marrow cavity was

observed (Fig. 4a).

Photographs of transverse slices of the longitudinal sec-

tions of femur and the mandibular interradicular bone of one

animal per group selected randomly are shown in Fig. 5.


123

Table 1 General anthropometry, aluminum accumulation, hematological and metabolic parameters

Variable CNX AlNX CHX AlHX

General anthropometry

Body weight (g) 273.89 ± 30.22a 278.77 ± 33.07a 247.97 ± 14.96b 249.87 ± 12.75b

Body length (cm) 22.95 ± 0.55a 23.05 ± 0.59a 22.78 ± 0.60a 23.42 ± 1.66a

Bone anthropometry

Femoral weight (g) 0.93 ± 0.08a 0.95 ± 0.08a 0.85 ± 0.05b 0.83 ± 0.05b

Femoral length (mm) 32.11 ± 0.04a 32.43 ± 0.03a 31.43 ± 0.49b 30.70 ± 0.30c

Hemimandible weight (g) 0.53 ± 0.05a 0.488 ± 0.02a 0.49 ± 0.03a 0.462 ± 0.01b

Hemimandible area (mm2) 134.46 ± 8.01a 135.61 ± 10.56a 129.81 ± 5.40a 131.38 ± 2.85a

Hematology parameters

Hematocrit (%) 53.50 ± 3.12a 45.00 ± 5.00b 70.28 ± 8.50c 63.40 ± 7.70c

Reticulocyte (%) 1.25 ± 0.21a 1.80 ± 0.22b 1.63 ± 0.30b 2.22 ± 0.24c

Metabolic indicators

Bone ash Ca (mg g-1) 353 ± 9a 349 ± 8a 348 ± 7a 340 ± 19a

Bone ash Al (mg g-1) 7 ± 1a 98 ± 12b 6 ± 1a 92 ± 13b

Uremia (mg dl-1) 43.5 ± 4.9a 45.5 ± 3.8a 46.0 ± 4.1a 48.0 ± 5.2a

Creatinine (mg dl-1) 0.65 ± 0.20a 0.65 ± 0.17a 0.75 ± 0.22a 0.70 ± 0.16a

Values are mean ± SD of 10 rats. Equal letters indicate no significant differences. A significant difference between groups was chosen as

p \ 0.01 determined by ANOVA followed by Student–Newman–Keuls multiple comparison test

CNX Normoxic control rats, AlNX aluminum-treated normoxic rats, CHX hypoxic control rats, AlHX aluminum-treated hypoxic rats

CNx AlNx CHx AlHx0

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100

150

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200

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Fig. 3 Changes in whole-bone structural properties: ultimate load,

Wf (a, e); limit elastic load, Wy (b, f); structural stiffness, Wy/dy (c,

g); energy-absorption capacity, EAC (d, h) at the end of the

experimental period. Values are mean ± SD of 10 rats. Equal lettersindicate no significant differences. A significant difference between

groups was chosen as p \ 0.01, determined by ANOVA followed by

Student–Newman–Keuls multiple comparison test. CNX Normoxic

control rats, AlNX aluminum-treated normoxic rats, CHX hypoxic

control rats, AlHX aluminum-treated hypoxic rats


123

Material properties

The indicators of bone material (intrinsic) proper-

ties, Young’s modulus of elasticity and the stress

produced at the yield point, were significantly reduced

in both bones by Al or by HX. The combined treat-

ment showed no additive features in either bone

(Fig. 6).

CNx AlNx CHx AlHx0

2

4

6

a a

c

b

fem

oral

cro

ss s

ectio

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med

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2

4 a aaa

hem

iman

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sect

iona

lm

omen

t of i

nert

ia, m

m4 F

Fig. 4 Changes in geometrical

properties: cross-sectional

medullar area (a, d), cross-

sectional cortical area (b, e) and

cross-sectional moment of

inertia (c, f) at the end of the

experimental period. Values are

mean ± SD of 10 rats. Equalletters indicate no significant

differences. A significant

difference between groups was

chosen as p \ 0.01 determined

by ANOVA followed by

Student–Newman–Keuls

multiple comparison test. CNXNormoxic control rats, AlNXaluminum-treated normoxic

rats, CHX hypoxic control rats,

AlHX aluminum-treated

hypoxic rats

PROXIMAL FEMUR

HYPOXIA

ALUMINUM +

+ +

+

MANDIBULAR INTERRADICULAR

BONE

Cross sectional medullar area

Cross sectional cortical area

Fig. 5 Photographs of transverse slices of the longitudinal sections of

femur and the mandibular interradicular bone of one animal per group

selected randomly (columns from left to right represent: CNX, AlNX,

CHX and AlHX groups). Resected hemimandibles stained with H&E

were observed under a stereomicroscope (95). Longitudinal sections

of femurs were observed under a magnifying glass


123

Discussion

The process of growth and maturation may be influenced

by several environmental factors. Among these, the effect

of Al or hypobaric hypoxia on both the sizes and biome-

chanical quality of bones are of particular relevance to the

understanding of the pathogenesis of toxic Al effects in

populations living and working at high altitude.

Within the above-mentioned restrictions, some of our

results could be extrapolated to the human skeleton [9]. We

had previously demonstrated in our laboratory that the

experimental time must extend over 120 days of postnatal

life of the animal to be certain that bone has achieved its

maximum size and optimal biomechanical quality [20].

Thus, the present study was performed in rats for a 3-month

period.

The mechanical properties of bone strongly depend on

the intrinsic mechanical quality of its constitutive sub-

stance (material properties) and the amount and spatial

distribution of the mineralized tissue (geometrical proper-

ties) [18, 21]. The effects of Al intoxication and chronic

hypoxia may affect either of these factors. Previously

reported studies performed on the normal rat [7] have

shown that Al treatment reduces femoral mineralization

and Young’s elastic modulus, with no visible effects on the

net bone mass. An improvement of the spatial distribution

of the available cortical tissue was observed. This pre-

sumably adaptive response was proposed to have main-

tained a normal structural (diaphyseal) stiffness, but could

not have provided a complete compensation of the

impaired diaphyseal strength.

It was demonstrated that the erythropoietic marrow is

concentrated in the spine, pelvis and proximal metaphyses

of leg bones [22, 23]. Increased densities of erythroid

elements in the marrow must result in the expansion of

bone marrow cavities and a consequent displacement of the

mineralize tissue [24]. This has been observed during stress

erythropoiesis, which leads to an impairment in the struc-

tural strength of femur diaphyses as well as the bone

material quality in growing rats [25].

In our rats, stress erythropoiesis was caused by chronic

SHA exposure. The marked increase in the rate of eryth-

ropoiesis in response to treatment was clearly demonstrated

by the intense reticulocytosis observed.

Bone ash analysis showed that the administered Al was

deposited in the skeleton in significant amounts. As

expected, a decrease in the hematocrit level was observed

in Al intoxicated rats not exposed to hypobaric hypoxia.

The interpretation of Al intoxication effects on bone

biomechanics under either normal or stimulated erythro-

poiesis requires the analysis of all material and structural

properties and cross-sectional geometry of the studied

bones.

Al treatment impaired bone material properties in both

types of bones. However, cortical area and design were

improved only in the femurs. Pre-yield and ultimate

strength and toughness were reduced in both bones, but

structural stiffness was only affected in the mandibles. The

effects induced in the femurs confirm previous results and

suggest that the spatial distribution of the available cortical

tissue assessed by the CSMI in Al-treated rats could have

been positively adapted as a natural reaction of bone cells

to the impairment in bone material stiffness. In those

studies the analysis of the negative relationship between

the CSMI as a geometric indicator and E as a material

indicator shows precisely this phenomenon. This type of

correlation can be regarded as a ‘‘distribution/quality’’

relationship [7]. Mandibular bone was significantly less

affected than femoral bone. Whether all mandibular bone

and the axial and peripheral skeleton respond similarly to

systemic bone loss remains controversial. It was previously

shown that mechanical loading during mastication influ-

ences bone mass and architecture of the mandibular alve-

olar bone. In humans, trabecular bone of the alveolar

processes is approximately two times thicker than that of

the proximal tibia [26]. Mandibular bone arises from a

different embryonic germ layer (neuroectoderm) than bone

CNx AlNx CHx AlHx0

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1600

2400a

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b

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CNx AlNx CHx AlHx0

50

100

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CNx AlNx CHx AlHx0

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500

750a

b b

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CNx AlNx CHx AlHx0

10

20

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40 a

bbb

D

hem

iman

dibl

e lim

it el

astic

stre

ss, N

.mm

-2

Fig. 6 Changes in material properties: Young’s modulus of elastic-

ity, E (a, c), and limit elastic stress (b, d) at the end of the

experimental period. Values are mean ± SD of 10 rats. Equal lettersindicate no significant differences. A significant difference between

groups was chosen as p \ 0.01 determined by ANOVA followed by

Student–Newman–Keuls multiple comparison test. CNX Normoxic

control rats, AlNX aluminum-treated normoxic rats, CHX hypoxic

control rats, AlHX aluminum-treated hypoxic rats


123

of the axial and appendicular skeleton, which arises from

mesoderm. Mavropoulos et al. [27] hypothesized that the

mechanical loading of the alveolar processes during mas-

tication may protect the alveolar bone from the detrimental

effects observed in other skeletal sites. A larger trabecular

thickness and a smaller bone surface fraction may blunt the

deterioration of the trabecular microarchitecture in the case

of mandibular bone. The different embryological origin of

the two skeletal bones may also play a role in the observed

distinct response of the cortical area and design of the

mandible under Al intoxication.

HX treatment impaired bone material properties in both

bones. It improved the cross-sectional geometry (as

assessed by the CSMI) and structural stiffness in the

femurs, and reduced the pre-yield and ultimate strength and

toughness of both bones more severely than Al treatment

did. In the HX rats the lesser ability of the bones to with-

stand loads could have resulted from an impairment in

either the bone material properties or the cortical and/or

trabecular bone mass. This would support our previous

findings [6], suggesting that what we had previously

hypothesized concerning the mandibular response under Al

intoxication may be also valid under hypoxic conditions.

Although combined Al ? HX treatment showed no

interaction on bone material properties, we observed a

trend to being mutual agonists to enhance the bone marrow

cavity and to neutralize mutually their eventually positive

effects on bone geometry. The combined effects on bone

structural properties were either intermediary or similar to

those induced by HX alone. The mechanically assessed

structural damage induced by the combined treatment

could have also reflected progress in microfractures that

eventually favors bone fatigue [18]. As the ash bone cal-

cium concentration showed no differences between treat-

ments, the induced impairment in bone structural properties

could be ascribed to an alteration in collagen fibers or

microstructural crystal arrangements. The enlargement of

the cross-sectional medullar area may be justified,

according to Quarles [28], because Al has been shown to

accumulate into larger areas in those bones that previously

suffered alterations in mineralization. It is thus proposed

that Al exposure can impair a previous or simultaneous

alteration of bone tissue structure. Precisely, stress eryth-

ropoiesis, which induces femoral endostic cortical resorp-

tion, is exacerbated in the presence of Al.

In conclusion, our results suggest that either chronic Al

intoxication or exposure to SHA induced negative effects

on femoral material quality and significantly enhanced its

moment of inertia. This is consistent with the proposition

that whole bones self-regulate their stiffness and strength

by adapting their geometric properties to their material

ones by a feed-back controlled system regulating bone

modeling during growth [7]. However, when both

treatments were assayed together, the geometric adaptation

of cortical tissue was less effective. Furthermore, mandi-

bles did not respond to the decline in the material proper-

ties with an adaptation of its architecture to maintain

structural properties. As the pathological effects of metals

on bone are slow, longer studies are necessary to evaluate

the risk of Al intoxication in our experimental model on a

bone with the peculiar characteristics of the mandible that

is subjected to forces produced by the mandibular muscles

and by reaction to forces applied to the temporal-mandib-

ular joints and teeth.

Acknowledgments The authors acknowledge the collaboration of

physiology laboratory technicians Graciela M. Champin and Elsa

Lingua, Department of Physiology, School of Dentistry, University of

Buenos Aires. This investigation was supported by research grants

from the University of Buenos Aires (UBACyT O-407).

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aluminum bone toxicity in immature rats exposed to simulated high altitude

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