Establishment And Application Of A New Assay In Vitro Of Osteoinductivity Of Biomaterials

Large bone defects and spine fusions, bone tumors, reconstructive surgery need massive bone graft materials, but autogeneic bone graft can't provide enough bone grafts. Many osteoconductive and osteoinductive bone graft substitutive materials had been used in orthopedics. The bioactivity of some osteoinductive bone graft materials such as demineralized bone marix, composited materials containing native bone morphogenetic protein, and other bioactive materials containing recombined growth factors depends on the activity of growth factors in it, but the bioactivity of these materials is easy to be inactivated by some treatments, such as preparation, storage, sterilization and so on. It is necessary to evaluate osteoinductivity of these bioactivite materials for the effective use of bioactive materials. The traditional assay method in animal body for evaluating bioactivity of materials have many shortcomings, such as sacrificing animals, long term of assay, many interference factors to test result, standardizing and sequencing difficultly, high cost, and so on. It can't satisfy the actual need. This research will establish a new standardized assay method in vitro for evaluating osteoinductivity of bioactive materials. This assay should be convenient, repetitive, standard and low cost, and can replace the traditional assay method in vivo. This research will also attempt to assay osteoinductivity of kinds of bioactive materials and compare with each other.Objective:To establish a new standardized assay method in vitro for evaluating osteoinductivity of bioactive materials, which is convenient, repetitive and low cost, can replace the traditional assay method in vivo. Besides, this research will attempt to assay osteoinductivity of kinds of bioactive materials and compare with each other. Methods:Part one:Establishment of a new assay in vitro for evaluating osteoinductivity of bioactive materials.The goal DBM was prepared using fresh cortical bone by a serial of biological and chemical treatments. BMP-2 in the goal demineralized bone matrix (DBM) was determined by immunohistomical test. Goal DBM(group A), rhBMP-2(group B),and bovine tendon type I collagen (group C) were respectively co-incubated with C2C12 cells in a 96-well plate for 72 hours, then the C2C12 cells was lysed in 1%Triton-X100 solution,and the lysate were assayed for alkaline phosphatase(ALP) and total protein content.The absorbance of ALP and total protein was recorded. The relative ratio between absorbance of ALP and total protein can represent the activity of ALP in the unit quantitative cells. Osteocalcin (bone gla protein BGP) in the culture fluid in culture plate wells was determined by radio-immunity assay (RIA).Part two:Application of a new assay in vitro for evaluating osteoinductivity of bioactive materials. The osteoinductivity of goal DBM, OsteoSet(?), human DBM, bovine tendon type I collagen were evaluated by the new assay method, and compared with each other.Results:Part one:The mmunohistomical test indicated the goal DBM contained BMP-2. In the goal DBM and rhBMP-2 groups, the content of ALP in the C2C12 cells and BGP in the culture wells had a higher level compared with the control group. The ALP activity and BGP content in rhBMP-2 group was higher than in the goal DBM group. The linear correlation was high between the result of BGP and ALP, the correlation coefficient being 0.877(p<0.01)oPart two:Goal DBM, human DBM can promote C2C12 cells to produce more ALP and secrete more BGP significantly. The ALP mean of in the two groups was 1.668,1.108 respectively. The ALP activity in the two experimental groups differed significantly (P< 0.001). OsteoSet(?) and type I collagen can not stimulate C2C12 cells to produce more ALP than the control group (P>0.05).The ALP mean in OsteoSet(?) and typeâ… collagen groups was 0.483,0.446 respectively. The result of BGP was parallel with one of ALP. Conclusions:1,rhBMP-2 can stimulate C2C12 cells to produce more ALP and BGP which are psteoblast-specific markers. As well as in rhBMP-2 group, C2C12 cells treated with other osteoinductive bioactive materials can produce more alkaline phosphatase and BGP. The ALP activity in the control group was lower than in rhBMP-2 group and DBM group. The result indicates that the assay in vitro is precise and feasible to evaluate osteoinductivity of bioactive materials.2,The assay in vitro can evaluate osteoinductivity of kinds of bioactive materials effectively in the same term. It is easy to compare the osteoinductivity of bioactive materials effectively by quantitive indexes. The result of the evaluating osteoinductivity of different bioactive materials indicates that the osteoinductivity in goal DBM,OsteoSet(?), human DBM and bovine type I collagen differ significantly. The osteoinductivity of the goal DBM is higher than human DBM. OsteoSet(?) and bovine type I collagen have no osteoinductivity.3,The new assay in vitro method can evaluate the osteoinductivity of all kinds of osteoinductive materials effectively. It can perform batches of assay at the same time; also give quantitive information to the result. It is easy to compare osteoinductivity of batches of materials with each other. The assay in vitro is convenient, precise, repetitive, standardized and low-cost, and can replace the traditional assay method in vivo.