In vitro osteogenic differentiation of hurnan periosteal cells: gene expression and functional measurement

Abstract

Objective To study and evaluate the gene expression and function of osteoblasts differentiated from human periosteal cells induced by BMP7 in vitro.Methods The periosteal cells isolated from adult tibia periosteum were cultured by the routine method in vitro and divided into experiment group and control group.In the experiment group BMP7 and osteogenic adjuvant were added to the culture media while only osteogenic adjuvant was added in the control group.Cell proliferation was detected by CCK-8.On day 5,day 10,day 15 and day 20,the expression of the osteocalcin gene and type Ⅱ collagen were evaluated by Real TimePCR.Glycosaminoglycan was detected by toluidine blue staining.The levels of ALP,osteocalcin and osteopontin in the supematant were quantified by ELISA.ALP activity,ALP and calcium nodules were detected by colorimetry,ALP staining and Von Kossa staining,respectively.Results The expression of osteocalcin and the level of ALP,osteccalcin and osteopontin in supernatant were both increased with significant difference between the experimental group and control group ( P ＜ 0.05).The positive rate of ALP and calcium nodules staining increased with significant difference between the experimental group and control group ( P ＜ 0.05).The positive rate of toluidine blue staining and the gene expression of collagen Ⅱ in the experimental group both increased temporarily and then decreased,but with significant difference between the experimental group and control group (P ＜ 0.05). Conclusion Human periosteal cells can proliferate and differentiate towards osteoblast when induced by BMP7 in vitro.They express osteogenic specific genes and are capable of synthesizing and secreting osteogenic specific proteins.It appears that apart from a direct osteogenic differentiation pathway,a phenomenon existed that some of the periosteal cells formed chondrocytes first and then calcified into bone.A large number of osteoblasts can be made available in vitro via human periosteal cells combined with BMP7 pathway,which can be used in bone tissue engineering construction and clinical application. Key words: Cell culture techniques; Gene expression; Periosteal cells; Osteogenic differentiation; Cell function