The Mechanism Of Silver Nanoparticles On The Osteogenic Differentiation In Human Periodontal Fibroblasts:an In Vitro Study

Objective: With the development of social economy and the improvement of living standard,more and more patients seek orthodontic treatment to improve the function and aesthetics of the maxillofacial.In orthodontic treatment,proper mechanical force is applied to the teeth,which first leads to the remodeling of periodontal tissues,then to the alveolar bone,and finally to the movement of teeth for the purpose of correction.The periodontal ligament(PDL)is a specialized connective tissue that is located between the cementum of the root and alveolar bone and related to tooth support,nutrition,sensory,and repairation.Fibroblasts are the major component of PDL cells and have stem cell potential to differentiate into osteoblasts,cementoblast-like cells,and adipocytes.Periodontal fibroblast cells(PDLFs)are the primary type of the periodontal ligament cells.PDLFs have the potential of osteogenesis and are the first cells to receive orthodontic treatment.They start bone reconstruction through the transmission of some biochemical signals(cytokines,prostagnins and neurotransmitters).Therefore,the changes in the number and biological functions of human periodontal fibroblasts(hPDLFs)have become a common concern of orthodontists.Silver nanoparticles(AgNPs)are zero-valent silver ions with diameters ranging from 1 to 100 nm.They have broad-spectrum antimicrobial activity and do not develop drug resistance.AgNPs have been widely used in the medical field,especially in plastic surgery implants.Some studies have shown that AgNPs can promote osteoblastic differentiation of mammalian cells.It has also been shown that differentiation of fibroblasts into myofibroblasts by AgNPs can promote wound contraction.Currently,the application of AgNPs in the treatment of dental diseases are mostly based on its antibacterial effect.In this study,the mechanism of AgNPs on the osteoblastic differentiation in hPDLFs were researched in vitro cell experiments.It will provide an important reference for how to improve the efficiency of alveolar bone remodeling on the premise of safety and effectiveness,thus accelerating the orthodontic tooth movement and shortening the treatment course.Methods:1.The human periodontal ligament fibroblasts(hPDLFs)were cultured,and cytokeratin and vimentin were identified by immunocytochemical staining.AgNPs were prepared through chemical reduction.The hPDLFs were treated with different concentrations(0,25,50,100,250 and 500 ?M).MTT was used to detect the changes in cell viability and determine the appropriate action of AgNPs,and the effect of AgNPs on cell morphology was also observed.2.The hPDLFs were treated with different concentrations of AgNPs(0,25,50,100)and osteogenesis was induced.Alkaline phosphatase(ALP)activity assay was used to determine the changes in ALP activity in hPDLFs under different concentrations of AgNPs.Alizarin red staining was used to identify the osteogenic differentiation of hPDLFs cells in 21 days.Detection of osteogenic differentiation markers such as ALP,collagen-I,Runx2,OCN and OSX by Real-time PCR and Western blot.3.The hPDLFs were treated with 100 ?M AgNPs for 1-7 days,and the expression of the total Akt and p-Akt protein,RhoA activity and TAZ protein were detected by Western blot.After pretreatment with PI3K/Akt inhibitor(LY294002),the activity of ALP was detected by the kit,and the expression of ALP,Runx2 and collagen-I was detected by Real-time PCR.Pretreatment with Rho inhibitor(C3 toxin)was added to detect ALP activity.Real-time PCR and Western blot were used to observe the expression of ALP,Runx2 and collagen-I in each group.Transient hPDLFs of siTAZ and siCon were established.Osteogenesis was induced by adding AgNPs.ALP activity was detected by the kit.RhoA activation and expression of ALP,Runx2 and collagen-I were detected respectively.Results:1.Immunocytochemical staining showed that the cultured cells were negative for keratin staining and positive for vimentin staining,which could be identified as fibroblasts(hPDLFs).MTT results showed that 250 and 500 ?M AgNPs significantly inhibited the viability of hPDLFs cells.In Addition,100 ?M AgNPs treatment had no effect on cell morphology.2.Compared with the non-AgNPs treatment group,the ALP activity in hPDLFs increased significantly 7 days after incubation with 100 ?M AgNPs(P < 0.01).Theexpression of ALP,Runx2,OCN,OSX and collagen-I in hPDLFs increased in a concentration-dependent manner 7 days after incubation with 25 ?M-100 ?M AgNPs,which was consistent with the Western blot results.3.The 3rd-7th day 100 ?M AgNPs mediated the up-regulation of Akt phosphorylation in hPDLFs cells,which was reversed by PI3K/Akt inhibitor(LY294002).In addition,LY294002 pretreatment inhibited the up-regulation of ALP activity mediated by AgNPs on the 7th day,and reversed the up-regulation of ALP,Runx2 and collagen-I mRNA expression mediated by AgNPs.In comparison,5d-7d 100?M AgNPs mediated the activation of RhoA and the up-regulation of TAZ expression in hPDLFs cells.This phenomenon was reversed by C3 toxin,an inhibitor of RhoA signaling pathway.C3 pretreatment inhibited the increase of ALP activity induced by AgNPs on the 7th day,and reversed the up-regulation of TAZ,Runx2,collagen-I gene and protein expression mediated by AgNPs and the up-regulation of collagen-I.siTAZ transfection decreased the expression of TAZ in hPDLFs,but did not affect the activation of RhoA mediated by AgNPs.In addition,siTAZ transfection reversed the increase of ALP activity and ALP gene expression induced by AgNPs.Similarly,siTAZ transfection also eliminated the increase of Runx2 and collagen-I mRNA and protein levels induced by AgNPs.Conclusions:1.AgNPs with 25-100 ?M concentration were identified as an acceptable concentration for hPDLFs,which could be used in subsequent experiments.2.The study confirmed the impact of AgNPs on the osteogenic differentiation of hPDLFs.3.PI3K/Akt signalling pathway may be involved in the process of AgNPs regulating the osteogenic differentiation of periodontal ligament fibroblasts.In addition,AgNPs exerts the molecular mechanism of adjusting the osteogenic differentiation of periodontal ligament fibroblasts through RhoA/TAZ axis.