Study On The Performance Of DDM Released BMP-2

Objective:The aim was to study the release of BMP-2 by decalcificated dentin matrix(DDM)and its chemotaxis to cells,and provide theoretical support for DDM as a kind of bone graft materials.Methods:1.Collect the teeth,remove the soft tissue attached to the teeth,enamel,cementum and pulp,crushing,screening of particle size 100～400 m dentin particles,degreasing;demineralizating,after incubation by PBS with acidiodine acetic acid,ultrasonic cleaning with distilled water 3 times,drying,stored Preparated DDM at-20.At the same time,Urist physicochemical method was used to extract the BMP-2 in the dentin of 5g,and the content of BMP-2 was detected by enzyme linked immunosorbent assay(ELISA).2.The homemade DDM(group A)and finished product DDM(group B)were placed in DMEM,and the pure DMEM was set up as group C.Group A and group B were experimental groups,group C was blank control group,Leaching for 1,2,3,5,7,9,13days.BMP-2 were detected by enzyme linked immunosorbent assay(ELISA).3.The third part:Mice osteogenesis precursor cells MC3T3-E1 was sub-cultured.After being cultured to the third generation,the cells as well as lower chamber homemade DDM(group D),finished product DDM(group E),Bio-Oss(group F)and medium containing no material(group G)were jointly cultured with Transwell Chambers.Group D and group E were the experimental group,while F group was the control group,and group G was blank control group.On the first,second and the third day of the serum-free culture,the release quantity of BMP-2 and collagen in the medium of lower chamber was detected with enzyme-linked immunoassay method,and then compared with the release quantity of BMP-2 and collagen of the first three days in the second part of the experiment.Besides,the crystal violet staining was conducted on Transwell chamber.The quantity of cellscrossing Transwell chamber was observed with fluorescence microscope.Statistical analysis was implemented on all the data with SPSS 19.0 softwareResults:1.Chalky DDM with even particle size of 100～400μm was successfully prepared.BMP-2 was successfully extracted from dentine.The 5g average content of BMP-2 extracted from 5g of dentine was 588.32±14.51pg,namely,0.12ng/g.2.The concentration of BMP-2 in the leaching solution of experimental groups first rose,and then dropped and finally became stable with the passage of time.The concentration of BMP-2 in the leaching solution on the first,second,third,fifth,seventh,ninth,eleventh and the thirteenth day was respectively detected.Then,pairwise comparison was carried out on the results with multivariate analysis SNK-q.For the comparison between group A/B and group C,P<0.05,showing there were statistically significant differences.In addition,for the comparison between group A and group B,P<0.05,indicating there were statistically significant differences.The concentration of Collagen Type I in the leaching solution on the first,second,third,fifth,seventh,ninth,eleventh and the thirteenth day was respectively detected.Pairwise comparison was carried out on the results with multivariate analysis SNK-q.For the comparison between groups,P<0.05,showing there were statistically significant differences.3.The concentration of BMP-2 in the lower chamber medium showed gradually rising trend with the passage of time.The concentration of BMP-2 in the lower chamber medium of each group on the first,second and the third day was respectively detected.Then,pairwise comparison was carried out on the statistical results with multivariate analysis SNK-q.For the comparison between group D and group E/F/G,P<0.05,showing there were statistically significant differences.In addition,for the comparison between group E and group D/F/G,P<0.05,indicating there were statistically significant differences.Besides,the comparison results between group G and group G showed P>0.05,suggesting there was no statistically significant difference.According to the comparison results between the groups of data with the data of the second part,BMP-2 concentration in the homemade DDM group presented gradually rising trend over time in the two experiments.Moreover,the comparison results between experiment 2 and experiment 3 on the first and the third day showed P<0.05,suggesting there were statistically significant differences,while the comparison results between experiment 2 and experiment 3 on the second day showed P>0.05,suggesting there was no statistically significant difference.The concentrationof Collin the leaching solution of each group on the first,second and the third day was respectively detected.Pairwise comparison was carried out on the results with multivariate analysis SNK-q.For the comparison between group D and group E/F/G,P<0.05,showing there were statistically significant differences;for the comparison between group F and group G,P>0.05,indicating there was no statistically significant difference.According to the comparison results between the groups of data with the data of the second part on the first,second and the third day,the concentration of Collin the homemade DDM group presented gradually rising trend over time in the two experiments.Moreover,the comparison results between experiment 2 and experiment 3 on the first and the third day showed P<0.05,suggesting there were statistically significant differences.The quantity of cells of each group crossing Transwell chamber was counted on the first,second and the third day respectively.Pairwise comparison was carried out on the statistical results with multivariate analysis SNK-q.For the comparison between group D and group E/F/G,P<0.05,showing there were statistically significant differences.Furthermore,for the comparison between group E and group D/F/G,P<0.05,indicating there were statistically significant differences.For the comparison between group F and group G,P>0.05,indicating there was no statistically significant difference.Conclusions:1.The experiment showed there was BMP-2 in dentin.DDM included about 0.12ng/g of BMP-2.2.DDM could release BMP-2.When DDM released BMP-2 at the approximate stable state in the experiment,the final content of BMP-2 in the homemade group was 58±1.78pg/ml,and the final concentration of the finished product group was 6.42±1.25 pg/ml.3.The co-culture with cell affected the release of BMP-2 in DDM in the experiment.4.DDM had certain chemotaxis for derived osteogenesis cells.Therefore,it can be used as bone graft material.