| Objectives The aim of this study was the exploration of novel bioactive materias——hyaluronic acid(HA)and the Ag-doped sol-gel derived bioactive glass with hyaluronic acid(HA/Ag-BG).The biological properties of the HA and HA/Ag-BG were evaluated in culture with human dental pulp stem cells(DPSCs).The cell proliferation,cell differentiation potential into odontoblasts and the antibacterial properties against Streptococcus mutans(S.mutans)and Lactobacillus casei(L.casei)was measured.It could potentially obtain an available dental regenerative materials.Methods HA and HA/Ag-BG extracts at 1/20 dilutions were screened as the optimal concentration for promoting the activity of human DPSCs in previous experiments of the research.This study was to investigate the effects of HA and HA/Ag-BG on proliferation of human DPSCs,differentiation into odontoblasts and antibacterial properties.Three parts were included in this study: 1 The effects of HA and HA/Ag-BG on proliferation of human DPSCs were evaluated.The Ag-BG was incorporated into HA at ratio 1:1 in wt%.Undiluted extracts of HA and HA/Ag-BG were collected.The HA and HA/Ag-BG extracts at 1/20 dilutions were diluted with Alpha-MEM medium containing 10% FBS and 1% PS.Human DPSCs were cultured and seeded into 96-well plates,and all inoculated cells were randomly divided into three groups.The original medium was discarded after the cells adhered onto the 96-well plates.The extract-free medium(untreated),HA extract and HA/Ag-BG extract were added to the three groups to continue culture.After 1,3,5 and 7 days of culture,the cell proliferation was evaluated using the cell counting kit-8(CCK-8)assay respectively.2 The effects of HA and HA/Ag-BG on differentiation of human DPSCs into odontoblastic cells were evaluated.Differentiation cell culture media were prepared as control group,HA group and HA/Ag-BG group.Human DPSCs were cultured and seeded into 6-well plates,then all inoculated cells were randomly divided into three groups.Each group of cells were incubated with the tooth slice and cultured to 21 days in three groups of differentiation cell culture media.Total RNA was isolated from each group of cells after 21 days,and the relative expression levels of odontogenic differentiation markers ALP,DMP1,DSPP,and RUNX2 were detected by Real-time PCR.3 The antibacterial properties of HA and HA/Ag-BG against Streptococcus mutans and Lactobacillus casei were evaluated.Equal volumes of HA extract,HA/Ag-BG extract and PBS,which was used as control,were mixed separately with Streptococcus mutans and Lactobacillus casei and then incubated for 24 h at 37°C.Each mixture was diluted serially to a gradient of 10-1,10-2,10-3,10-4,and 10-5,and subsequently was plated on agar plates.All plates were incubated for 48 h at 37°C,and the colony forming units were counted.Results 1 The test of CCK-8 results: When human DPSCs were cultured in the regular medium,HA extract and HA/Ag-BG extract on the 1st and 3rd day,there was no statistically significant in proliferation activity among the three groups of cells(P>0.05);When the culture was continued until the 5th and 7th day,there were no statistically significant both the HA group and the HA/Ag-BG group compared to the control group separately(P>0.05),however,the HA/Ag-BG group showed higher proliferative activity compared to the HA group(P<0.05).2 The detection of Real-time PCR results: The expression levels of odontogenic differentiation markers ALP,DMP1,DSPP,and RUNX2 were up-regulated significantly in the differentiation medium of HA group compared to the HA/Ag-BG group(P<0.05);At the same time,the expression levels of odontogenic differentiation markers DSPP and RUNX2 were up-regulated significantly in the differentiation medium of HA group compared to the control group(P<0.05);However,the expression levels of odontogenic differentiation markers DMP1 in the HA/Ag-BG group was down-regulated significantly compared to the control group(P<0.05),there was no statisticant difference among the expression of ALP,DSPP,and RUNX2 compared to the control group.3 The results of antibacterial experiment: The mixture of HA group and HA/Ag-BG group showed significant decrease compared to PBS group followed by counting the colony forming units whether in S.mutans or in L.casei(P<0.05).Conclusions 1 HA/Ag-BG expressed a higher ability to promote the proliferation of human DPSCs than HA.2 HA expressed a higher ability to promote the differentiation of human DPSCs into odontoblasts than HA/Ag-BG.3 HA and HA/Ag-BG could effectively inhibit the growth of Streptococcus mutans and Lactobacillus casei. |
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