Oxyge Concentration On Decidual Stromal Cells Toward The Direction Of Osteoblast Differentiation Of Influence

Objective:It is found that bone marrow stem cells, used as seed cells for bone tissue engineering, has limitations in number, proliferation and differentiation. Thus it is difficult to apply to clinical use. Recent studies show that decidual stromal cells (DSC) can be induced in certain conditions into osteoblast. This experiment is intended to isolate human pregnancies DSC and cultured in osteogenic media in vitro. Partly decidual stromal cells differentiated to osteoblast direction. We observed normoxic and hypoxic conditions affect DSC proliferation and differentiation.Method:Abortion decidua taken from 6 to 8 weeks of pregnancy woman was isolated by mechanical grinding and enzyme digestion. After several passages of proliferation, DSCs were purified. The morphology and the characters of these cells were stable when they came to the fifth generation. They were used to our experimental osteogenic differentiation. DSCs were digested and seeded into 6-well plates, then divided into four groups:(1)normoxia group (2)normoxia and osteogenesis medium group(3) hypoxia group (4)hypoxia osteogenesis medium group. Replace the medium every two to three days for 28 days and test alkaline phosphatase (ALP) expression of each group by PNPP every 7 days. Alizarin red stain to observe mineralized nodule formation.Results:1.Decidual was isolated by mechanical grinding and enzyme digestion. After several passages of proliferation, highly puried DSCs with good cell activity were acquired. 2.Induced by Osteogenic medium, DSCs grow into a colony in three days later obviously, with greater cells of polygon, triangle, irregular shape. Cell volume increased obviously. Cytoplasm is transparent, contains a little high-density structure like small grain or cords(Fig 1,2); 10 days later,cells grow into layers with high density, nodular-like structures appear in the center(Fig 3). After induction, cell proliferation is relatively slow,90% cell fusion is reached 10～14 days later; In the control group, with the conventional culture medium (1640medium+10% FBS), the cells are smaller, spindle-like, occasional polygon-like, and cell proliferation is faster,90% cell fusion is reached about 5-7 days later.3.Microplate detection light density values (OD) values detection each ALP activity expression display:the activity of ALP of DSC normoxic osteogenesis induction group is significantly higher than another three groups(P <0.05); the activity of ALP of DSC hypoxia osteogenesis induction group is significantly higher than another two groups cultured with conventional medium (P<0.05)4.Four groups of sample are stained with alizarin red after being cultured for 7 days,14 days,21 days and 28 days, in cells concentrated area of osteogenesis induction group, mineralized bodies are formed, and is orange after alizarin red staining(Fig 4). It is more clear in normoxic osteogenesis induction group than hypoxia group, and can not see in the remaining two groups.Conclusion:1.Decidual stromal cells can be induced to differentiate into osteoblasts in certain conditions;2.Under the same certain condition, compare with the hypoxia, the normoxic group is benefical to osteogenesis.