Elsevier

Journal of Biomechanics

Volume 43, Issue 6, 19 April 2010, Pages 1097-1103
Journal of Biomechanics

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure on bone material properties

https://doi.org/10.1016/j.jbiomech.2009.12.011Get rights and content

Abstract

Dioxins are known to decrease bone strength, architecture and density. However, their detailed effects on bone material properties are unknown. Here we used nanoindentation methods to characterize the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on nanomechanical behaviour of bone matrix.

Pregnant rats were treated with a single intragastric dose of TCDD (1 μg/kg) or vehicle on gestational day 11. Tibias of female offspring were sampled on postnatal day (PND) 35 or 70, scanned at mid-diaphysis with pQCT, and evaluated by three-point bending and nanoindentation.

TCDD treatment decreased bone mineralization (p<0.05), tibial length (p<0.01), cross-sectional geometry (p<0.05) and bending strength (p<0.05). Controls showed normal maturation pattern between PND 35 and 70 with decreased plasticity by 5.3% and increased dynamic hardness, storage and complex moduli by 26%, 13% and 12% respectively (p<0.05), while similar maturation was not observed in TCDD-exposed pups.

In conclusion, for the first time, we demonstrate retardation of bone matrix maturation process in TCDD-exposed animals. In addition, the study confirms that developmental TCDD exposure has adverse effects on bone size, strength and mineralization. The current results in conjunction with macromechanical behaviour suggest that reduced bone strength caused by TCDD is more associated with the mineralization and altered geometry of bones than with changes at the bone matrix level.

Introduction

Dioxins are persistent, accumulative and highly potent environmental contaminants that are ubiquitously present in the environment and in human tissues. Humans are exposed to dioxins mainly via diet, especially meat, fatty milk and fish products. Because dioxins are lipophilic compounds, they transfer from adipose tissue to mother's milk, and therefore a child can get even 20–25% of mother's dioxin burden (Tuomisto, 2001). Huge variety exists in dioxin effects from mild biochemical changes (Okey et al., 2005) to immunotoxicity, cancer, metabolic disorders and lethal wasting syndrome (Pohjanvirta and Tuomisto, 1994). However, developmental defects are potentially the most significant human health risks of dioxins, because they have been observed at very low exposure levels and they are potentially permanent (Alaluusua et al., 1999; Alaluusua and Lukinmaa, 2006; Gray et al., 1997; Kattainen et al., 2001; Mably et al., 1992; Miettinen et al., 2005). Teeth (Alaluusua et al., 1999; Alaluusua and Lukinmaa, 2006; Kattainen et al., 2001; Miettinen et al., 2002), bones (Miettinen et al., 2005) and the reproductive system (Gray et al., 1997; Mably et al., 1992) were shown to be sensitive to dioxin exposure especially in the developmental stage.

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) is the most potent dioxin congener and the best-known experimental model compound for the group. Most dioxin-induced effects are mediated via the aryl hydrocarbon receptor (AHR), which is a ligand-activated transcription factor. AHR is expressed in osteoblasts (Gierthy et al., 1994; Ilvesaro et al., 2005; Ryan et al., 2007) and osteoclasts (Ilvesaro et al., 2005), and in differentiating osteoblasts the expression peaks after the matrix maturation stage and before the initiation of mineralization (Ryan et al., 2007). Low concentrations of TCDD have been recently shown to interfere with the differentiation of both osteoblasts and osteoclasts in vitro (Korkalainen et al., 2009), and thereby bone formation and remodelling. In differentiating osteoblasts, TCDD decreases the expression of RUNX2, alkaline phosphatase and osteocalcin (Korkalainen et al., 2009; Ryan et al., 2007), whereas in osteoclasts it decreases the number and resorbing activity of the cells (Korkalainen et al., 2009).

Previous experimental studies have indicated that bone length, cross-sectional geometry and mechanical properties are all affected by exposure to TCDD (Jämsä et al., 2001; Miettinen et al., 2005). However, any possible changes of bone material properties at the matrix level after TCDD exposure are not known. Based on cellular studies it is reasonable to expect that the bone material properties may also change as the bone material properties are the ultimate consequence of cellular activity. We know that fetal and juvenile bone matrix has high water content, and a low mineral to organic ratio. During normal maturation the water content decreases and the mineral to organic ratio increases in the bone matrix (Boivin and Meunier, 2003). These compositional changes are accompanied by changes in the shape and size of the bone mineral crystallites as they gradually mature (Su et al., 2003).

Modern nanoindentation instruments make it possible to examine the material properties at the bone matrix level. To determine if the TCDD-induced decrease in bone strength is due to the changes observed macroscopically, or to changes in material properties at the bone matrix level, we did nanoindentation testing to examine this nanomechanical behaviour of bone matrix. Putative changes in bone matrix mechanical behaviour may underpin the decreased bone strength observed at the whole bone level of TCDD-exposed animals, and thereby elucidate the characteristics of dioxin-induced bone toxicity.

Section snippets

TCDD

TCDD was purchased from the UFA Oil Institute (Ufa, Russia) and was over 99% pure as analyzed by gas chromatography-mass spectrometry. It was dissolved in corn oil (Sigma Chemical). Solutions were mixed in a magnetic stirrer and ultrasonicated for 20 min before dosing.

Animal treatment

The study protocol was reviewed and approved by the Animal Experiment Committee of the University of Kuopio (Kuopio, Finland). Significant decrease in bone geometry and mechanical strength without maternal toxicity was reported

Bone densitometry

Developmental exposure to TCDD caused reduction in bone mineralization, cortical BMC being 16% lower at PND 35 (p=0.006) and 11% lower at PND 70 (p=0.004) in the exposed animals compared to controls (Table 1). Cortical BMD was reduced by 0.9% at PND 70 (p=0.035), while the decrease at PND 35 was not significant. The tibial length and cross-sectional geometry were decreased by TCDD treatment at both PND 35 and PND 70, and the decrease was somewhat more noticeable at PND 35.

Macromechanical testing

TCDD decreased bending

Discussion

Recent advances in the nanoindentation technology have made it possible to analyse static, dynamic and creep behaviour of bone material at the matrix level. Our data showed for the first time that combined in utero and lactational TCDD exposure results in disturbed age-dependent maturation process causing the tibias of pups to be more ductile, softer, and less able to store energy than control bones (Fig. 4). Analysis of the bone matrix material properties in conjunction with the mineral

Conclusions

TCDD exposure in utero and via lactation has significant adverse effects on bone size, strength and mineralization. For the first time, this study demonstrates retardation of bone maturation process as evaluated by nanoindentation at the bone matrix level. Tibias of offspring of TCDD-exposed dams were more ductile, softer, and less able to store energy than control bones. The study suggests that the reduced bone strength is more associated with the mineralization level and altered bone

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgments

We thank Janne Korkalainen, Ulla Naukkarinen, Anna-Maija Ruonala, Arja Moilanen and Christina Trossvik for excellent technical assistance. This study was financially supported by the European Commission (BONETOX, QLK4-CT-2002-02528). Mikko A.J. Finnilä was partly supported by the National Graduate School of Musculoskeletal Disorders and Biomaterials. Timo Jämsä and Juha Tuukkanen were partly supported by the Academy of Finland. The nanoindentation tests were carried out in the Biomechanics

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