Demineralized Dentin Matrix Effect The Growth Behavior Of A Human Osteoblast-like Cell Line (SaoS-2) In Vitro

[Objective]This study investigated the effect of Demineralized Dentin Matrix on adhesion, proliferation,and differentiation of osteoblast in vitro, to privide theoretical support demineralized demtin matrix as a bone graft material.[Methods]In this study,we choose human osteosarcoma cell line(SaoS-2) as a osteoblast model for research, after culture for 5 generation,by co-culture of SaoS-2 cells and Commercialization DDM(Group A)、the homemade DDM(Group B)、Bio-Oss(Group C)and culture medium(Group D), Group A and Group B were experimental groups, the positive control group was Group C, Group D was the blank control group.The cell adhesion level was evaluated by means of scanning electronic microscopy after culturing for 5、10 days; the cell proliferation level was tested according to CCK-8 assay every day; ALP activity was evaluated by quantitative ALP assay after culturing for 1、3、5、7 days. All the datas were used SPSS 17.0 software to statistical analysis.[Results]1.The result of scanning elcttron microscope observationsWith the incubation time prolonged, the cells have a tendency to increase on the surface of the four materials. The morphology of Saos-2 cells adhered on the surface of homemade DDM was full, its cell density was the highest, cell pseudopods were long and stretched well,closely attached to the surfaces of homemade DDM, single-celllayer is formed; the density of SaoS-2 cells adhered on the surface of Bio-Oss(L) took the second place.the cells were thin and scattered,the pseudopods were small and short,no celllayer formed; the number of SaoS-2cells adhered on the surface of commercialization DDM and Bio-Oss(S) was minimum, but the cell morphology on the surface of the commercialization DDM is full, the cell pseudopods were much longer and attached more closely to the surface of commercialization DDM.2.The result of CCK-8 assayThe cell numbers showed a general increasingtrend over time.The values of absorbance of each group measured for 7 consecuyive days were as follows (x±s): Group A:0.2379±0.0348,0.4233±0.0519,0.5181±0.0208,0.07547±0.0496, 1.0893±0.1694,1.1852±0.1194,1.4772±0.0892; Group B:0.2662±0.0316, 0.3924±0.0490,0.5419±0.0255,0.7671±0.0246,1.0941±0.1441,1.1687±0.1518. 1.2780±0.0299; Group C:0.2518±0.0447,0.4844±0.0519,0.5332±0.0270,0.7529 ±0.0275,0.8637±0.0263,0.9343±0.0303,1.0636±0.0681; Group D:0.3408 ±0.0161,0.4269±0.0210,0.5339±0.0332,0.7562±0.0347,0.8462±0.0388, 0.9126±0.0433,1.0842±0.0806. Using multivariate of variabce SNK-q pairwise comparison of group,on the 1st day,the difference between D and A、D and B、D and C was statistically significant(P<0.05), there was no significant difference between the other groups(P>0.05); on the 2nd day, the difference between C and B、C and A was statistically significant(P<0.05), there was no significant difference between the other groups(P>0.05); on the 3rd and 4th day,there was no significant between the four groups(P>0.05); on the 5th and 6th day, in addition to the differences between A D and C、B and A was not statistically significant(P>0.05), the difference between other groups was statistically significant (P<0.05); on the 7th day, in addition to the difference between D and C was not statistically significant(P>0.05), the difference between other groups was statistically significant (P<0.05).3.The result of quantitative ALP activity assayThe ALP activity in each group presented a rising trend as time went by. The concentration value of ALP measured on the 1st、3rd、5th and 7th days were as follows (U/L x±s):Group A:47.1880±1.9008、58.5380±2.0180、68.6894±3.3221、 80.9660±1.7922; Group B:49.7165±1.6292、50.8420±1.6211、58,7380±2.5587、 79.1720±1.9733; Group C:95.4340±1.6505、102.2680±1.7504、111.8239±3.6653、 150.1260±2.5671;Group D:75.5301±3.0229、76.9180±1.7332、76.3880±1.3551、 81.4980±1.6653.Using multivariate of variabce SNK-q pairwise comparison of group, on the 1st day, in addition to the difference between B and A was not statistically significant(P>0.05), the difference between other groups was statistically significant (P<0.05); on the 3rd and 5th day, There were significant differences between each groups (P<0.05); on the 7th day, in addition to the difference between D and B> D and A、B and A was not statistically significant(P>0.05), the difference between other groups was statistically significant (P<0.05).[Conclusions]1. Scanning electron microscope observations, the cell adhesion property of homemade DDM was stronger than large particles Bio-Oss; the cell adhesion property of small particles and commercialization DDM is poor.2. In this experiment,DDM could promote proliferation of osteoblasts, the role of commercialization DDM is greater than homemade DDM; Bio-Oss couldn’t promote proliferation of osteoblast.3. In this experiment,Bio-Oss could promote osteogenic differentiation of osteoblasts; DDM couldn’t promote osteogenic of osteoblast.4. DDM displayed a great biocompatibility,it could be used as a bone graft material.