Study On Potential Of Bone-formation From Rabbit Anulus Flbrosus Cells Induced By Bone Morphogenetic Proteins-6and Basic Ifbroblast Growth Factor

Background:Our country has entered the aging society, degenerative spine, which ismajorly composed of prolapse of lumbar intervertebral disc and lumbar olisthe, hasbecome common and frequently occurring disease.The major clinical manifestationsof the two disease are repeated lumbago and sciatica,and it seriously affects thepatients’ work and life.At present, the materials used for spinal fusion operation havedifferent shortcomings. If ossification of interverterbral disc was made throughmanual intervention,we could not noly stabilize the spinal columnand treat lumbarolisthe,but also prevent the nucleus pulposus from protruding backword into spinalcanal.Extensive literature has confirmed that BMP-2,-4,-7can induce ectopicossification effect.However, recent study have shown that BMP-6hassimilarfunctionality,and more efficient than BMP-2.Objective: By establishing the vitro culture model of the rabbit intervertebral discannulus fibrosus cells, which provides a observation of cytology characteristics andbiological property of the annulus fibrosus cells.Study of the stimulating effect ofosteogenesis potential on rabbitannulus fibrosus cells which has been induced byBMP-6and b-FGF two kinds of growth factors.Method:1. Take the annulus fibrosus out from the healthy rabbit, use Ⅱ-typecollagenase digestion to isolate the original generation annulus fibrosus, thensubculture by monolayer culture and trypsin digestion method together with theadoption of selection plating technique to purify.2. Observe the changes ofbone-formation phenotype and cytology characteristics induced by cell factorrhGDF-5and bFGF of annulus fibrosus cell. There are four cell culture groups based on the culture condition: Groups A、B、C and D. Group A: DMEM/10%FBS culturesolution; Group B: DMEM/10%FBS culture solution+b-FGF; Group C: DMEM/10%FBS+rhBMP-6; Group D: DMEM/10%FBS+rhBMP-6+b-FGF.3. Observecell morpha by an optical microscope, and measure growth curve by MTT. Observethe expression of osteopontin by applying immunohistochemical techniques.UseWestern blotting techniques to determine the amount of osteopontin.Results:The firstly adherent annulus fibrosus cell mostly appeared asround,with thepseudopodia being stretching out, which is shaped like sector.With the increasing ofadherent cells, The morphology of the cell gradually extend for the spindle. Aftersubculturing it is like long spindle or polygonal along with the chemotaxis growth andpolarity arrangement during later culturing period. The cell bodybecome longer,andcytoplasm become less,appearing stagnant growth. rhBMP-6has the function toenhance the expression of osteopontinsignificantly, but has little contribution to cellproliferation. Instead, b-FGF can strengthen cell proliferation and the express lessosteopontin than rhBMP-6. However, the joint use of rhBMP-6and b-FGF canpromote cells proliferation and enhance the expression of osteopontin The effect ofthis situation excels that of using either cell factor alone.Conclusion:1.the experiment successfully established vitro culture model of rabbitannulus fibrosus cells. The primary cultures of annulus fibrosus cells was mainlyfibroblastcell phenotype. When the cells comes to the fourth generation, they appearaging phenomenon.2.rhBMP-6can stably induce ossification of vitro annulus fibrosuscells and bFGF can enhance effect on it.