The effect of a milk lipid fraction on bone properties of growing female rats and the growth and function of bone cells : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Human Nutrition at Massey University, Manawatu, Palmerston North, New Zealand

Objective: To investigate the effects of a milk lipid fraction (MLF) on the accruel of bone mass in growing rats by evaluating the effects of MLF on growth and bone parameters such as bone mineral content (BMC), bone mineral density (BMD) and biomechanical properties in growing rats and the effects of MLF on the development and activity of bone cells, including osteoblasts and osteoclasts. Methods: There were one hundred and eight 3-month old female Sprague-Dawley rats were randomly allocated into three groups: a control group (n=48), a low-dose MLF group (n=30) fed with 250 mg/rat/day of MLF, and a high-dose MLF group (n=30) fed with 500 mg/rat/day of MLF. Forty-five rats (n=15 for each group) were selected to terminate at month 3 for biomechanical testing while sixty-three continued into a second arm of the trial after ovariectomy. Body composition and bone parameters of animals (n=108) were measured in vivo by Dual Energy X-Ray Absorptiometry (DEXA) at baseline and week 12 of the study. Length and diaphyseal width and thickness of the left femur were measured. The three-point bending test was used to evaluate the biomechanical characteristics of the left femur of rat. The effect of MLF on proliferation, differentiation and mineralization of murine osteoblasts were investigated using the osteoblastic cell line MC3T3-E1. The cells were cultured with 0-1,000 µg/ml or 0-100 µg/ml MLF for 5, 9 and 24 days, respectively for proliferation, differentiation and mineralization. Cell proliferation was determined using the methyl-thiazolyl tetrazolium (MTT) assay. The differentiation of osteoblasts was detected using the alkaline phosphatase (ALP) activity assay. Mineralized nodules were examined using an Alizarin red histochemistry assay. The effect of MLF on RANKL-induced osteoclastogenesis was evaluated in the murine monocytic cell line RAW264.7. The cells were cultured with 0-100 µg/ml MLF for five days. Osteoclastogenesis was determined using the tartrate-resistant acid phosphatase (TRAP) staining assay and counting numbers of TRAP-positive multinucleated cells. Results: Rats fed with the high-dose MLF diet had a significant increase in body lean mass compared with those fed with the control and low-dose MLF diets. The high-dose MLF group also had significant gains in BMC at the femur and in BMD at the femur and lumbar spine compared with the control group. There were no significant differences in dimensional and biomechanical results among groups. The MLF significantly increased the proliferation of MC3T3-E1 at 0.1, 1.0 and 100 µg/ml. There was a dose-dependent, but not significant increase in the differentiation of osteoblasts cultured with MLF for 9 days. After 24-days of cell culture, the MLF at the low concentrations of 0.1 and 1.0 µg/ml led to non-significant increase in calcium deposition by the differentiated osteoblasts. MLF at 10 and 100 µg/ml significantly inhibited calcium nodule formation. RANKL-stimulated osteoclastogenesis was significantly increased in RAW 264.7 cells cultured with the MLF at concentrations up to 10 µg/ml. Conclusion: These results indicated that oral administration of MLF to the growing rats improved bone accrual and has a favourable effect on achievement of peak bone mass. The MLF increased the proliferation of MC3T3-E1 pre-osteoblast cell line, but there was no effect on osteoblast differentiation and the higher concentration of MLF may have inhibited the function of mature osteoblast. Additionally, the MLF stimulated osteoclastogenesis from RAW264.7 cells. Further studies are required to investigate some of the contradictory findings presented in this report