Early life influences on skeletal growth - effects of vitamin D and mechanical loading

I propose to determine whether early life vitamin D deficiency alters later skeletal responsiveness to loading in a model system.

Fractures in childhood are common and their incidence is increasing. Fractures are more common in children with smaller, narrower bones and lower bone mass for body size. Tracking of fracture risk would be a logical consequence of bone size tracking into adult life. Some studies show evidence of earlier fracture being associated with later bone size and mass. Other studies based on adult recall of childhood fracture do not support the tracking of fracture risk, but those studies recalled rates of fracture are much lower (4-5 fold) than those now reported in the UK in children.

Regular loading of bone during childhood, particularly before puberty, has been shown to produce a sustained increase in bone mass and may reduce the risk of fracture both in childhood and in later life. Studies in both those participating in regular sporting activities such as gymnastics that create large strains, and in school-based exercise programmes, have shown biologically relevant increases in bone size and mass at the hip, in the spine and in the whole body. Such increases persist from childhood into post-pubertal life and have biological relevance in the prevention of adult osteoporotic disease.

The contribution of vitamin D to fracture rates during childhood has been difficult to assess; a recent study of vitamin D supplementation in infancy showed a trend towards increased bone volume with higher vitamin D doses up to age 3 months. Current Department of Health advice is targeted at the prevention of rickets, rather than altering fracture risk either during childhood or later. The traditional view of vitamin D in relation to skeletal health is that it enhances the absorption of calcium. Lower calcium intake is associated with an increased risk of fracture.

The roles of both diet and mechanical loading are therefore of considerable interest in respect of determining their likely contribution to skeletal health and the prevention of fracture in childhood. The timing of loading interventions is thought to be important - prepubertal loading may result in post-pubertal benefit for bone size and mass. What has not been addressed is the impact of the timing of interventions in respect of vitamin D.

The suggestion that early life events could influence later growth and development was first advanced by McCance in the early 1960s. The concept has developed further and is now an established field of investigation - the Developmental Origins of Adult Health and Disease. Observational studies in humans suggest that infants of mothers with lower vitamin D levels during pregnancy may have narrower bones, and that this alteration in bone size persists into later childhood, increasing their risk of fracture.

The persistence of the effects of lower vitamin D in pregnancy on the skeleton at least to age 8-9 years suggests that the normal response of the skeleton to factors likely to influence its size and shape post-natally is abrogated to some extent. The periods of pregnancy and early post-natal life represent an important opportunity for intervention that could improve skeletal health across the life course.

I therefore propose to study the effect of reducing vitamin D intake during pregnancy and early life on the skeleton's response to mechanical loading using an animal model system. The study will help us understand the extent to which diet in early life affects our body's ability to respond appropriately later on, and if successful, may help us reduce the risk of fractures both in childhood and later years.

2 breeding female C57BI6 mice will be fed a Vitamin D deficient diet and housed in UVB-free lighting or a matched diet with Vitamin D3 and standard UVB lighting from age 4 weeks, and mated at age 10 weeks, continuing that diet throughout gestation. After weaning at 21 days 96 pups will be fed on either a Vitamin D deficient (under UVB-free light; dam=D replete/deficient; n=24/group) or replete diet (dam=D replete/deficient; n=24/group) until testing at age 10 or 20 weeks.
Tibial loading will be performed under isoflurane anaesthesia by intermittent cyclical loading, i.e. 40 cycles daily of axial compression of the tibia 6 times over 2 weeks (weeks 8-10 or 18-20; n=12/group). Peak load magnitudes will achieve peak compressive strains on the tibial surface of 1200 microstrain (submaximal) and 600 microstrain (sub-threshold). Each group will go for microCT/histology (n=6) or indentation (n=6).
Tibiae will be fixed in formalin, decalcified in EDTA and embedded in paraffin wax. Sections will be stained with either haematoxylin and eosin (H&E) or tartrate-resitant acid phosphatase (TRAP) to identify osteocytes. Immunohistochemistry will be performed using relevant primary mono/polyclonal antibodies by light microscopy using an ABC/DAB kit.
MicroCT: Bone structure will be analysed using a microCT scanner (Skyscan). After image reconstruction and analysis a 3D micro finite element model of the tibia will be derived. Bone material properties will be estimated from microCT-equivalent bone density and fabric measurements. Boundary conditions will be defined by loading and strain and stress distributions calculated.
Microindentation properties of mouse bones will be measured ex vivo using a BioDent Hfc Reference Point Indenter. Bones will be tested at 5 locations along the shaft; reference force of 270-310N, testing protocol of 2N at 2Hz over 5 cycles. Calculation of indentation distances and elastic properties will be provided through built-in BioDent software.

Our approach will have initial impact via its novelty. Previous studies have focused on calorie or protein restriction simulating the effects of general placental insufficiency. This study will develop a model system to specifically determine the effects of prenatal vitamin D on the developing skeleton. Immediate impact will be upon other scientists working in similar or related fields. In particular those working in the fields of nutrition; mechanical stimulation of the musculoskeletal system; and developmental origins of health and disease. Investigators will also be interested in the use of novel techniques such as microindentation and in vivo microCT. The wider scientific community will benefit as these novel technologies are an improvement on the current state of the art. For example, microindentation can only currently be done ex vivo. These techniques are likely to become more widely used over the next 5 years and will be further developed via take up by other investigators. There will be an economic benefit to the manufacturer of the microindentation device as the methodologies for the use of the instrument are refined and promulgated in terms of the sales of both the instruments and the indentation probes.

In the commercial sector, animal trials companies will also be impacted via the methodology developed in this project. The techniques employed will give them a greater understanding of whether conditions used for the housing of pregnant mice for research need to be amended in the light of the results of the study. Longer term, food and food supplement manufacturers may also benefit from enhanced market opportunities as the result of an evidential based approach that demonstrates the requirement for the maintenance of sufficient vitamin D intake.

As this study is to be undertaken in a mouse model the relevance of this work to Government and Regulatory bodies is likely to be initially limited. However, if the work proposed is successful it would provide a basis for investigators to justify, to both parties, the requirement for further studies that build upon our findings and extend the work to human subjects. Such studies may then influence and inform policy makers and regulatory bodies in terms of guidelines and public health policy designed to improve skeletal health via interventions related to the role of exercise and vitamin D intake.

Longer term impacts to the wider public will clearly occur. The fundamental aim of this project is intrinsically linked to the nation's health. The system developed will enable assessment of the mechanisms underpinning the early skeletal response to vitamin D and determine whether post-natal intervention can reduce any adverse effects. Such outcomes, as previously mentioned, could have direct bearing on future public health policies and guidance in this area and ultimately on an individual's quality of life.