BMP4 promote chondrogenic differentiation of human periosteum-derived cells in vitro / 中华显微外科杂志

ABSTRACT

Objective To explore biological characteristics of chondrogenic differentiation of human periosteum-derived cells and the role of BMP4 in chondrogenic differentiation of these cells.Methods From October 2009 to September 2012,periosteum was obtained from tibia of patients undergoing leg amputation surgery,and isolated periosteum-derived cells by tissue culture method.Cells were cultured in DMEM/F12 containing 10％ fetal bovine serum,and morphology of cells were observed under inverted microscope.Periosteum-derived cells growth and the effect of BMP4 on cells growth examined by cell count using trypan blue,and cells growth curve was made.Experiment was divided into control group,chondrogenic differentiation group and BMP4 group,cells were expanded and differentiated in the presence or absence of BMP4 and complete medium.Then toluidine and immunohistochemical staining analyzed proteoglycan and collagenⅡ expression of these cells after 14 and 21 days.The expression of aggrecan,collagen Ⅱ and SOX9 mRNA of these cells using real-time PCR.Results (1) Periosteumderived cells adhered to growth in vitro,the shape of cell presented fibroblast-like morphology changing into polygonal after 1 week and round cell formation after 2 weeks chondrrogenic differemtiation.Growth curve showed that the passage 3 and 9 cells had similar reproductive activity.The passage 3 cells were positive for CD90 (21.07％) and CD105 (25.84％).(2)Toluidine bule staining and type Ⅱ collagen immunohistochemical staining showed BMP4 group (40.29 ± 4.29,56.74 ± 5.12) and chondrogenic differentiated group (19.27 ± 3.71,38.31 ± 4.25) ccould secrete proteoglycan and collagen Ⅱ,control group were negative (10.24 ± 1.21,15.28 ± 2.23),BMP4 group were significantly than chondrogenic differentiated group.(3) The expression of aggrecan,collagen Ⅱ and SOX9 mRNA of BMP4 group(25.76 ±0.57,6.48 ±0.48,2.91 ±0.18)were significantly higher than that of control group(2.37 ±0.24,1.12 ± 0.31,1.07 ± 0.22)and chondrogenic differentiated group(11.12 ± 0.38,2.24 ± 0.41,1.54 ± 0.35)using real-time PCR.Conclusion Periosteum-derived cells have strong proliferative,and have good potentials of differentiating into chondroblasts like mesenchymal stem cells.BMP4 can promote chondrogenic differentiation of periosteum-derived cells in vitro cultures.