We determined the effects of yolk water-soluble protein (YSP) on bone formation in pre-osteoblastic MC3T3-E1 cells. YSP (50-5,000 g/ml) increased cell proliferation and collagen content. Alkaline phosphatase (ALP) activity was also increased by YSP treatment. After enhancement of ALP activity, significant augmentation of calcification was observed. These results suggest that YSP is a promising agent for the prevention and treatment of bone loss.Key words: yolk soluble protein; MC3T3-E1 pre-osteoblastic cell; proliferation; alkaline phosphatase activity; collagen Bone is remodeled continuously during adulthood through the resorption of old bone by osteoclasts and the subsequent formation of new bone by osteoblasts.
1)These two closely coupled events are responsible for renewing the skeleton, and any imbalance between bone resorption and bone formation leads to abnormal bone metabolism. Bone loss with increasing age induces osteoporosis, and this loss is sometimes due to increased bone resorption and decreased bone formation.Eggs are thought to be a complete food and to have all the substances necessary for hatching. Egg yolk is the largest biological cell known that originates from one cell division.2) It is composed of various important chemical substances that form the basis of life. Thus, chicken egg is a storehouse of nutrients and biologically active substances, including growth promoting factors which are needed to form a chicken.2) Recently, we reported that delipidated egg yolk protein (YP) promotes longitudinal bone growth in adolescent rats.3) The active factors stimulating bone growth were concentrated in the yolk soluble protein (YSP) fraction.3) YSP promoted longitudinal bone growth, height of growth plates, and induction of bone morphogenetic protein-2 (BMP-2), which was not seen with casein.3) BMPs, mostly BMP-2 and BMP-7, play an important role in regulating osteoblast differentiation and subsequent bone formation. Hence, in present study, we investigated the effect of YSP on differentiation and mineralization using the pre-osteoblastic MC3T3-E1 cell, which has been well characterized as an in vitro model of osteoblast differentiation.
4)Egg yolk protein (YP) and yolk water-soluble protein (YSP) were prepared as described previously.3) It was donated by Pharmafoods International Co., (Kyoto, Japan). MC3T3-E1 cells were obtained from the American Type Culture Collection (Rockville, MD, USA). Cells were cultured at 37 C in a 5% CO 2 atmosphere in -modified minimal essential medium (-MEM, Gibco, MD, USA) containing 10% heat-inactivated FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin.The proliferation of the cells was determined by 3-(4.5-Dimethylthiazol-2yl)-2.5 diphenyl tetrazolium bromide (MTT) assay. After treatment with YSP (0-5,000 mg/ml) for 2 d, MTT solution (0.5 mg/ml) was added to each well, and the cells were further incubated for 4 h at 37C. Absorbance at a test wavelength of 595 nm with a reference wavelength of 690 nm was measured using a microplate reader (Biotek, VT, USA). The opt...