Effect And Mechanism Of Atmospheric Room Temperature Plasma On Osteogenic Differentiation Of Human Periodontal Ligament Stem Cells

Objective:Human periodontal ligament stem cells(hPDLSCs)are isolated from human periodontal ligament tissues.The cell has high osteogenic activity,and is one of the most promising cells to restore periodontal bone defect.Atmospheric room temperature plasma(ARTP)has many functions,such as disinfection and sterilization,promoting cell proliferation,inducing apoptosis of cancer cells.It can cause biological effect by impacting on the change of intracellular ROS.The purpose of this study was to investigate the effect of ARTP on the osteogenic differentiation of hPDLSCs and the relationship between the effect of ARTP on intracellular ROS and the osteoblastic differentiation of hPDLSCs,so as to explore the effect and possible mechanism of ARTP on the osteogenic differentiation of hPDLSCs.Methods:(1)Human periodontal ligament cells were primarily cultured by using tissue explant method,and hPDLSCs were isolated by using limiting dilution technique.The colony forming efficiency of hPDLSCs was assayed by cristal violet staining,the growth curve was determined by CCK-8 kit,the differentiation potential was examined by osteogenic and adipogenic induction,and the surface markers were detected by flow cytometry.(2)HPDLSCs were treated by ARTP and then were induced into osteoblasts.Cells were grouped as follows:the blank group,ARTP-treated in 30 s,1 min and 2 min.The alkaline phosphatase(ALP)of hPDLSCs was assayed by ALP activity kit at the 7th and 14th day;on day 21,the mineralization was detected by alizarin red S staining and semi-quantitative analysis;and the expression levels of osteogenic differentiation-related genes were assessed by RT-qPCR on 3 and 7 days.(3)HPDLSCs were pretreated by ROS scavenger N-acetylcysteine(NAC)to remove and inhibit the production of intracellular ROS,and then were treated by ARTP.Cells were divided into 4 groups:(i)the blank group,(ii)NAC group,(iii)ARTP-treated group,(iv)NAC+ARTP treated group.After the above treatment,cells were cultured in osteogenic differentiation medium.The total amount of ROS in the hPDLSCs was assayed by ROS detection kit;the ALP activity of hPDLSCs was assayed by ALP activity kit on day 7 and 14;the mineralization was detected by alizarin red staining and semi-quantitative analysis on day 21;and the expression levels of osteogenic differentiation-related genes were assessed by RT-qPCR on day 3 and 7.Results:(1)The primary cultured hPDLSCs presented spindle,triangular or polygon shapes.The clone formation rate was about 44%.The growth curve of hPDLSCs was similar to“S”shape.The cells could be differentiated into osteoblast and adipocyte.The flow cytometry showed as follows:CD90(+),CD146(+),CD34(-),CD45(-).(2)Compared with cells treated by ARTP in 0 s,30 s and 2 min,the ALP activity was enhanced when hPDLSCs were treated in 1 min at 7th and 14th day(P<0.05),the number of mineralized nodules were increased at 21th day(P<0.05),and RT-qPCR results showed that the mRNA expression levels of RUNX2,ALP and COL-I were also increased at 3rd and 7th day(P<0.05).(3)In the pretreatment of hPDLSCs by ROS scavenger,compared with the controlled group,the intracellular ROS levels increased when cells were only treated by ARTP,while the levels were decreased in NAC group.In NAC+ARTP group,the ROS levels decreased as compared to ARTP-treated group.Compared with ARTP-treated group,the ALP activity of cells was decreased in NAC+ARTP group at 7th and 14th day(P<0.05),the number of mineralized nodules were significantly diminished at 21th day(P<0.05),and the expression levels of RUNX2,ALP and COL-I were also decreased at 3rd and 7th day(P<0.05).Conclusions:(1)The primary cultured hPDLSCs had good proliferation,satisfactory differentiation potential,and highly expressed stem cell surface markers.(2)Treatment of hPDLSCs with ARTP for a certain period of time(1 min)could enhance the expression of related osteogenic genes in cells,and increase the ALP activity and mineralization ability of cells,therefore promoting the osteogenic differentiation of cells.(3)ROS plays an important role during the process of osteoblastic differentiation of hPDLSCs treated by ARTP.