High fluoride and low calcium levels in drinking water is associated with low bone mass, reduced bone quality and fragility fractures in sheep

Abstract

Chronic environmental fluoride exposure under calcium stress causes fragility fractures due to osteoporosis and bone quality deterioration, at least in sheep. Proof of skeletal fluorosis, presenting without increased bone density, calls for a review of fracture incidence in areas with fluoridated groundwater, including an analysis of patients with low bone mass.

Introduction: Understanding the skeletal effects of environmental fluoride exposure especially under calcium stress remains an unmet need of critical importance. Therefore, we studied the skeletal phenotype of sheep chronically exposed to highly fluoridated water in the Kalahari Desert, where livestock is known to present with fragility fractures.

Methods: Dorper ewes from two flocks in Namibia were studied. Chemical analyses of water, blood and urine were executed for both cohorts. Skeletal phenotyping comprised micro-computer tomography (μCT), histological, histomorphometric, biomechanical, quantitative backscattered electron imaging (qBEI) and energy-dispersive X-ray (EDX) analysis. Analysis was performed in direct comparison with undecalcified human iliac crest bone biopsies of patients with fluoride-induced osteopathy.

Results: The fluoride content of water, blood and urine was significantly elevated in the Kalahari group compared to the control. Surprisingly, a significant decrease in both cortical and trabecular bones was found in sheep chronically exposed to fluoride. Furthermore, osteoid parameters and the degree and heterogeneity of mineralization were increased. The latter findings are reminiscent of those found in osteoporotic patients with treatment-induced fluorosis. Mechanical testing revealed a significant decrease in the bending strength, concurrent with the clinical observation of fragility fractures in sheep within an area of environmental fluoride exposure.

Conclusions: Our data suggest that fluoride exposure with concomitant calcium deficit (i) may aggravate bone loss via reductions in mineralized trabecular and cortical bone mass and (ii) can cause fragility fractures and (iii) that the prevalence of skeletal fluorosis especially due to groundwater exposure should be reviewed in many areas of the world as low bone mass alone does not exclude fluorosis.

Fig. 1

Local situation in Namibia showing groundwater wells with wind- and solar-powered pumps (a) and Dorper sheep from the southwestern Kalahari Desert (b). Fluoride content of groundwater, blood and urine samples from the sheep (c). EDX peaks indicating fluoride elevation in sheep (f) and in human (g) samples compared to their controls, respectively (d, e). Bar graphs demonstrating fluoride in weight percent in sheep (h) and human (i) specimens. (significance at *p < 0.05 or at **p < 0.01)

Fig. 2

Analyses of trabecular and cortical bone of sheep femur (a–c) and iliac crest (d–f) biopsies according to the nomenclature proposed by Parfitt and colleagues [26]. Diaphysis contact X-rays of the midshaft region and Xtreme-CT midshaft reconstruction images (a and b) show in the table section the calculated effects of cross sections of femora at midshaft scanned with μCT (c). Analyses of the iliac crest (f) performed after taking contact X-rays (upper part of d and e). Reconstructed iliac crest samples for control (d) and fluoride-exposed sheep (e) are pictured in the lower part. (significance at *p < 0.05 or **p < 0.01)

Fig. 3

Iliac crest biopsies of sheep samples were obtained, and histomorphometric analysis was performed starting with bone volume per tissue volume (BV/TV). In addition to osteoid volume per bone volume (OV/BV), osteoid surface per bone surface (OS/BS), osteoid thickness (O.Th.) and the number of buried osteoid cases per total cases, the number of osteocytes (N.Oc/B.Pm) and osteoblasts (N.Ob/B.Pm) per bone perimeter, osteoclast surface (Oc.S/BS) and osteoblast surface (Ob.S/BS) per bone surface were evaluated. The histological slides were stained with a Masson-Goldner dye (a and b) and toluidine blue dye for polarized light analysis (c and d) (significance at *p < 0.05 or at **p < 0.01)

Fig. 4

Histomorphometric analyses of iliac crest biopsies of human specimens. Osteoid volume per bone volume (OV/BV), osteoid surface per bone surface (OS/BS), osteoid thickness (O.Th) and the number of buried osteoid cases per total cases were measured. Section sign indicates reference values for healthy human controls derived from a previous study by Priemel and colleagues [50]. The histological slides were stained with a Masson-Goldner dye promoting non-mineralized tissue and buried osteoid (a and b). Polarized histological images were obtained from toluidine blue stained slides (c and d) (significance at *p < 0.05 or at **p < 0.01)

Fig. 5

Quantitative backscattered electron imaging (qBEI) analysis results and images. The images include a coloured qBEI image of bone mineralization for fluoride-exposed sheep (a iliac crest, i femur) and for human patients who had been treated with fluoride for osteoporosis (e). Mineralization mean (b), width (c) and peak (d) for sheep are also shown. Human sample results are shown in the form of bar graphs (f, g, h) and demonstrate significant differences between fluoride and control samples. The results for sheep femora are also shown (j, k, l). (significance at *p < 0.05 or at **p < 0.01)

Fig. 6

Quantitative backscattered electron imaging (qBEI) (a, b) and scanning electron microscopy (SEM) imaging (c, d) of sheep femora and quantitative analysis of canalicular connections. The number of secondary haversian canals per bone area (mm²) (N.H.Ca./B.Ar.), number of osteocytes per bone area (N.Ot/mm ²), lacunar area per micrometre (Lc. Ar. (μm²)) and the number of osteocyte canaliculi per osteocyte lacunae (N.Ot.Ca./Ot.Lc.) were evaluated. Data are shown in the table below the images (significance at *p < 0.05 or at **p < 0.01)