Study On Phenomenon Of Bone-formation From Rabbit Disc Nucleus Pulpous Cell Induced By Growth Differentiation Factor-5and Basic Fibroblast Growth Factor

Background: Lumbar olisthe is one of the common problems of Orthopaedics, withmajor clinical manifestations featured by repeated lumbago, and it seriously affects thepatients’ life quality. Most of the patients need surgical correction, however thematerials used for spinal fusion operation have different shortcomings. It is expected toreach the purpose of spinal fusion if ossification of interverterbral disc is made throughmanual intervention. According to a research, GDF-5plays an important role in thegrowth of cartilage and long bone, it can induce a variety of cells differentiate intochondroblast and osteoblast which indicates that it can induce the differentiation fromsimilar cartilage cell and nucleus pulposus cell into osteoblast.Objective: By establishing the vitro culture model of the rabbit disc nucleus pulposuscells, which provides a observation of cytology characteristics and biological propertyof the nucleus pulposus cells, then giving the rhGDF-5and bFGF manual intervention,this experiment discussed the influence on phenotype and differentiation characteristicsof nucleus pulposus cells from rhGDF-5and bFGF. Plus, it provided theoretical basisfor directly inducing the vertebra fusion of nucleus pulposus cells into bone.Method:1. Take the nucleus pulposus out from the healthy rabbit, use Ⅱ-typecollagenase digestion to isolate the original generation nucleus pulp, then subculture bymonolayer culture and trypsin digestion method together with the adoption of selectionplating technique to purify.2. Observe the changes of bone-formation phenotype andcytology characteristics induced by cell factor rhGDF-5and bFGF of nucleus pulposuscell. There are four cell culture groups based on the culture condition: Groups A、B、C and D. Group A: DMEM/15%FBS culture solution; Group B: DMEM/15%FBSculture solution+rhGDF-5; Group C: DMEM/15%FBS+bFGF; Group D: DMEM/15%FBS+rhGDF-5+bFGF.3. Observe Cell morpha by an optical microscope,Measure growth curve by MTT, Immunohistochemical observation of the expression ofcollagen typeⅠand type II, Radioimmunoassay (RIA) measurement of the expressionof osteocalcin, Alizarin red staining, etc..Results: Results: The nucleus pulposus cell is shaped like short spindle, polygon andspiral shape after fusion when first adherence. After subculturing it is like long spindleor star along with the chemotaxis growth and polarity arrangement during later culturingperiod. Approximately cells appear stagnant growth, namely aging phenomenon in thefifth generation. rhGDF-5has the function to restrain cell proliferation, enhance theexpression of osteocalcin, promote the deposition of calcium salt to form calcium nodes.Instead, bFGF can strengthen cell proliferation and the expression of type I collagen,promote the deposition of calcium salt to form calcium nodes but it has no significanteffect on the osteocalcin expression. However, the joint use of rhGDF-5and bFGF canpromote cells proliferation, enhance the expression of type I collagen and osteocalcinand improve the deposition of calcium salt. The effect of this situation excels that ofusing either cell factor alone. The expression of type Ⅱ collagen was negative in allthe four groups.Conclusion: Conclusion:1. the experiment successfully established vitro culture modelof rabbit nucleus pulposus cells. The primary cultures of nucleus pulposus cells wasmainly cartilage cell phenotype，which gradually appears dedifferentiation of fibroblast.When the cells comes to the fifth generation, they appear aging phenomenon.2.rhGDF-5can stably induce ossification of vitro nucleus pulposus cells and bFGF can enhanceeffect on it.