Maternal vitamin D depletion disrupts neonatal skeletal development in mice

Harriet Buckley; Stephanie Borg; Kirsty Nicholson; Mark Kinch; David Hughes; Tim Skerry; Nick Bishop

doi:10.1530/boneabs.4.LB1

Fractures in infancy raise the spectre of child abuse; it has been suggested that lack of vitamin D could result in bone abnormalities that could predispose to fractures. We utilised a mouse model system to investigate whether vitamin D deficiency in utero alters early bone growth and development.

C57BL/6 female mice received vitamin D deficient or replete diet for 6 weeks, then mated and continued on their respective diets until weaning. Pups were culled at Day 0 (D0) or Day 22 (D22). A Skyscan 1172 desktop micro CT machine quantified total bone volume per tissue volume (BV/TV) at D0 and cortical morphometry at D22. D0 pups were skeletally stained using alcian blue/ alizarin red whole mount stain. Haematoxylin and Eosin (H+E) staining and Osteomeasure histomorphometry analysis were used to visualise and quantify growth plate zone depths.

At D0 no significant differences were observed between antenatal deplete and replete weights 1.35 g±0.10 vs 1.35 g±0.10. At D22 deficient pups weighed significantly less than replete 6.35 g±0.87 vs 9.01 g±0.29 (P=0.0001).

Skeletal staining showed vitamin D replete D0 pups to have a greater area of ossified bone tissue within the axial and appendicular skeleton compared to deplete pups.

Micro CT show pups exposed to vitamin D deficiency in utero to have a reduced BV/TV. Skeletal staining also showed reduced areas of ossified bone tissue within the skeleton. Pup weights and OBN/BP and OCN/BP remained unchanged at D0. At D22 however large significant changes were found in weight, cortical morphometry, epiphyseal growth plate structure and endosteal OBN/BP suggesting vitamin D deficiency in utero to have negative impacts on early stage bone growth and development which are exacerbated with age.

Table 1
	Vitamin D deficient		Vitamin D replete
	Mean	S.D.	Mean	S.D.	P-value for difference
D0 BV/TV %	1.93	0.22	3.22	1.07	0.03
D22 cortical volume %	0.11	0.04	0.19	0.03	0.004
D22 cortical thickness %	0.06	0.01	0.08	0.01	0.01
D22 cortical porosity %	8.88	1.99	4.8	0.01	0.002
D22 growth plate depth μm	79	0.013	61	0.003	0.007
D22 hypertrophic zone μm	37	0.008	17	0.001	<0.001
D22 ossification zone μm	9	0.000	15	<0.001	<0.001
No significant differences were observed between resting and proliferating zone depths at D22.

Disclosure: The authors declared no competing interests.

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