Effect Of Bioaggregate On Cellular Behavior Of Osteoblasts And Periodontal Ligament Fibroblasts

An ideal root-end filling material should satisfy the following requirements:excellent biocompatibility, good sealing ability, superior antibacterial effect, satisfactory ability for tissue regeneration, sufficient radio-opacity, and desired manipulability. Because the root-end filling material in the apical cavity comes into physical contact with the surrounding cells or tissues, understanding the cell-material interfacial activity is important. For this reason, many studies have focused on the biocompatibility of aggregate materials with cells(Mitchell et al.1999, Vajrabhaya et al.2006), on the biological behavior of cells in the presence of root-end filling materials, and on the influence of these materials in terms of related cellular signal pathways. Osteoblasts and PDL fibroblasts are the major cells involved in the reaction of cells with root-end filling material. Substantial research now has been carried out on MTA regarding its reaction with osteoblasts or PDL fibroblasts. MTA is currently viewed as being biocompatible with osteoblasts or PDL fibroblasts and could have positive influences on the cellular behavior of these cells in vivo.Bioaggregate, a novel root-end filling material, is mainly composed of calcium silicate, calcium hydroxide, and hydroxyapatite. Bioaggregate has shown similar antibacterial effects and biocompatibility to those of MTA and is considered as a possible alternative to MTA. However, no studies have yet evaluated the effects of Bioaggregate on cellular behavior of osteoblasts or PDL fibroblasts. The purpose of our study was therefore to assess the cytotoxicity of Bioaggregate to these two cells and to investigate the effect of Bioaggregate on cellular behavior of these cells.Objective:The purpose of this study was to examine the cytotoxicity of Bioaggregate to osteoblasts MC3T3-E1 compared to MTA.Materials and Methods:The osteoblasts MC3T3-E1 were seeded on 96-well plates at a density of 1,000 cells per well in 100μl growth medium. After 24 hours of incubation, cells were subjected to three different treatments in 3 groups:negative control group, MTA group, and Bioaggregate group. In the MTA group or Bioaggregate group each specimen was directly supplemented into respective groups. On the 1st,2nd, and 3rd day, the cells were collected for MTT assay to test the cytotoxicity of Bioaggregate or MTA to MC3T3-E1. Statistical significance was analyzed by one-way ANOVA analysis of variance followed by a Tukey test. The data were expressed by mean±standard deviation and P<0.05 was considered as statistically significant.Result:There was no statistical difference in cell viability between the Bioaggregate group, the MTA group, and the control group throughout the culture period.Conclusion:These findings demonstrated that both Bioaggregate and MTA has no obvious cytotoxicity to osteoblasts MC3T3-E1.Objective:The purpose of this study was to examine the effect of Bioaggregate on mineral associated gene expression in osteoblasts MC3T3-E1.Materials and Methods:The osteoblasts MC3T3-E1 were seeded on 24-well plate at a density of 5×104 cells per well and incubated in 1 ml osteogenic medium. On the 1st,2nd, and 3rd day after incubation, cells were collected to examine mineralization-associated gene expression(COLⅠ,OCN,OPN) via qRT-PCR. The qRT-PCR was performed with a SYBR Green PCR kit on the ABI PRISM7500 sequence detection system. Each sample was tested in triplicate. The data for gene expression were analyzed by the△△Ct method. Statistical significance was analyzed by one-way ANOVA analysis of variance followed by a Tukey test. The data were expressed by mean±standard deviation and P<0.05 was considered as statistically significant.Result:COLⅠ. The COLⅠexpression level declined in both MTA and Bioaggregate groups compared to the control group on the 1st day. However, gene expression recovered to the control level in both the Bioaggregate and MTA group on the 2nd and 3rd day. In addition, the COLⅠexpression in the Bioaggregate group increased significantly compared to MTA group. OCN. OCN expression was substantially suppressed by Bioaggregate or MTA on the 1st day compared to the control group. On the 2nd day, OCN expression level raised in the Bioaggregate group but remained inhibited in the MTA group compared to control group. By the 3rd day, there were no longer any significant differences among three groups. OPN. OPN expression in the MTA and Bioaggregate groups decreased significantly compared to control group on the 1st day. By the 2nd day, OPN expression was similar among all groups. By the 3rd day, OPN expression in the Bioaggregate group was clearly enhanced compared to other two groups.Conclusion:These findings demonstrated that Bioaggregate had the pontential to induce mineral associated gene expression in osteoblasts MC3T3-E1.Objective:The purpose of this study was to examine the cytotoxicity of Bioaggregate to human PDL fibroblasts.Materials and Methods:The PDL fibroblasts were trypsinized with 0.25% Trypsin,1 g/L EDTA and seeded in 96-well plate at a density of 1,000 cells per well in 100μl DMEM supplemented with 10% FCS,100 U/ml penicillin,100μg/ml streptomycin. After 24 hours of incubation, the cells were divided into three groups:negative control group, MTA group, and Bioaggregate group. In the MTA and Bioaggregate group the specimens were put into each well in respective group directly. On day 1,2, and 3 post-incubation, the cells were collected for MTT assays to test the cytotoxicity of MTA and Bioaggregate. Experimental data were analyzed by one-way ANOVA analysis of variance followed by Tukey Tests. The results were expressed as means±standard deviation and P<0.05 was considered statistically significant.Result:Human PDL fibroblasts proliferated throughout the culture period in all three groups. However, the rate of proliferation of PDL fibroblasts in the MTA group was significantly lower than in the control group on days 1,2, and 3. There was also a significant decrease in proliferation in response to Bioaggregate on day 2.Conclusion:These findings showed that Bioaggregate had no cytotoxicity to human PDL fibroblasts while MTA was cytotoxic to human PDL fibroblasts. Objective:The purpose of this study was to examine the effect of Bioaggregate on osteogenic differentiation of human PDL fibroblasts.Materials and Methods:The human PDL fibroblasts were seeded on 24-well plate at the density of 5×104 each well and incubated in DMEM supplemented with 10% FCS,100 U/ml penicillin,100μg/ml streptomycin,50μg/ml ascorbic acid,10 mmol/l Na-β-glycerphosphate, and 10 nM dexamethasone to promote osteogenic differentiation of the fibroblasts. On day 3,5, and 7 after incubation, cells were collected and the selected gene expression was determined by quantitative real-time PCR. The effect of Bioaggregate and MTA on osteogenic differentiation of human PDL fibroblasts was evaluated by examination of several related genes:COLⅠ, ALP, and NID2. The housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as control gene for normalization of RNA expression. Experimental data were analyzed by one-way ANOVA analysis of variance followed by Tukey Tests. The results were expressed as means±standard deviation and P<0.05 was considered statistically significant.Results:The effect of Bioaggregate on mRNA expression of genes involved in osteogenic differentiation(COLⅠ, ALP, NID2) of fibroblasts was examined via qRT-PCR. In the Bioaggregate group, COLⅠexpression was significantly enhanced on day 5 and 7. Similarly, COLⅠexpression was also induced by MTA on day 3 and day 5. The ALP expression level was highest on day 7 in the presence of Bioaggregate, and ALP expression was substantially increased in the MTA group on day 3. NID2 expression in the Bioaggregate group was significantly higher than in the control group and this increased with increasing culture time. In contrast, NID2 expression in MTA group was obviously enhanced only on day 3.Conclusions:Our findings indicated a clear role for BA in the induction of osteogenic differentiation of human PDL fibroblasts.