Experimental Study Of Composite Osteogenic Inducer Promoted Rat Bone Marrow Stromal Cells To Differentiate Into Osteoblast In Vitro

Objective: To study Composite osteogenic inducer whether canpromote rat bone marrow stromal cells (BMSCs) to differentiate intoosteoblast by different concentrations of ALN with the sameconcentration of conventional osteogenic inducer (10-8mol/Ldexamethasone,10.0mmol β-glycerin sodium,50mg/L ascorbic acid)，what kind of concentrations have a ideal effect if it can be strengthened.This study will discuss how to improve the efficiency of BMSCs，whichis the one of the seed cells of the bone tissue engineering in vitro. Theexpression of bone morphogenetic protein2(BMP-2) was detectedduring the BMSCs differentiation into osteoblast. This study will offerbasic preparation for the future clinical research.Methods: Two healthy SD rats (2months old, male) were executedby cutting off the heads to obtain its femur and tibia sterilely, get theBMSCs purified by gradient centrifugation and adherent culture，and thenprepare single cell suspension. Inoculate it into L-DMEM with10%FBS.Through continuous subculture, non-adherent cells and unrelated cellswere removed further. When adherent cells reached about80%of thebottle’s bottom, added0.25%trypsin to digestion and subcultured until the third generation. Take the cells with1×106/L concentration, identifyits phenotype by CD44and CD45, make sure it is the rat BMSCs anddescribe the growth curve of it. The experimental cells (1×108/Lconcentration) were divided into seven groups (A to G), A is the blankgroup which continue to add in L-DMEM with10%FBS. B to G areexperimental groups which were added the same concentrationosteoblast-induced fluid with10-8mol/L dexamethasone,10.0mmol/Lβ-glycerin, sodium and50mg/L ascorbic acid. Then the C group is addedwith10-6mol/L ALN, D is added with10-7mol/L ALN. E to G isseparately added with10-8mol/L,10-9mol/L，10-10mol/L ALN. All of thegroups continue culture, change the medium every2-3days and observethe shapes of cells with inverted microscope.1,2and3weeks later, usingwestern-blot test the expression of the BMP-2separately, then analysesthe data by t-test.Results: Using gradient centrifugation with adherent culture can getpurified BMSCs. The adherent cells morphology showed oval-shaped,polygonal and short spindle-shaped after48h. Scattered adherent cellsshow spindle-shaped after72h and show the formation of cloning after10-14days. Cell arrangement was disorder and often connected throughthe protruding between cells in the early times while about3weeks laterthere was a dense layer of adherent cells. Subculture by this way, cellswere further purified and showed homogeneous of a long spindle-shaped arrangement of swirling, and the growth rate accelerated. The growthcurve showed that the cells will be logarithmic growth after4days，thenit will reach the peak after about7days. The expression of CD44wasactive（93.9%），CD45was negative（5.5%）. All of BMSCs grew wellwhen L-DEME was added in the osteoblast-induced fluid, at the sametime, the cells of E group became mast and polygonal, which wassurrounded by a mass of itself secretions. Little part of cells in A groupbecame mast and polygonal, which was surrounded by a little of itselfsecretion. The order of the expression of BMP-2，which was tested bywestern-blot at three different times，showed E>D>F>C>G>B>A.Conclusion:1. Purified BMSCs could be obtained by gradientcentrifugation with adherent culture.2. ALN with normalosteoblast-induced fluid have non-poisonous to BMSCs.3. The ALNwith10-8mol/L concentration，which was added in normal osteogenicinducer，have the best effect to promote BMSCs to differentiate intoosteoblast.