| [Objective]The absorption and biophysical-chemical interactions at the nanobiological interface was a huge challenge and hot topics in the research fields of biomedicine,nanobiotechnology,chemistry and materials science in recent years.Antimicrobial peptides,one of whose function could be programmed to perform a specific task of peptides and bind nanoparticles was used as a potential method of treatment.Human? defensin 3(hBD3),one of the antimicrobial peptides,had antibacterial properties and could promote regeneration.Gold nanoparticles(AuNPs)also showed promising value in tissue engineering.However,the effects of AuNPs and hBD3 on human periodontal ligament cells(hPDLCs)and chronic periodontitis were less studied.Therefore,the purpose of this dissertation was to explore the effect of hBD3 and AuNPs on the osteogenic differentiation of hPDLCs and the effect on the treatment of chronic periodontitis under inflammatory microenvironments.[Methods]1.Chemical reduction method was used to prepare AuNPs of 45nm.hPDLCs were cultured with E.coli-LPS(1 ?g/mL),hBD3(5 ?g/mL)and AuNPs(10 ?M).2.The effect of cell viability on cell viability was examined by CCK-8.Transmission electron microscopy(TEM)was used to observe the uptake of AuNPs.Alkaline phosphatase(ALP)staining and alizarin red staining were used to observe the osteogenic differentiation of hPDLCs.Real-time PCR and western blot were performed to evaluate the osteogenic differentiation-related gene and protein expression of ALP,collagenase-I(COL-1)and runt-related transcription factor 2(Runx-2)and Wnt/p-catenin signaling pathway related gene and protein expression.Then the Wnt inhibitor ICG-001 was added to hBD3-AuNPs-treated hPDLCs.We use the western blot,real-time PCR,ALP staining and quantitation to evaluate the mechanism of the osteogenic differentiation.3.Ligating the maxillary second molar with 4-0 silk thread was used to establish periodontitis models in rats.The experimental group were given hBD3(5 ?g/mL)and AuNPs(10 ?M)by local injection of gingival.The control group were given physiological saline with or without ligation by local injection of gingival.After 2 weeks,collecting the maxilla and serum of each rat.Then Micro-CT scanning,HE staining,Masson staining,TRAP staining and Enzyme-Linked Immunosorbent Assay(ELISA)was used to analyze.[Results]1.In the inflammatory microenvironments stimulated by E.coli-LPS,we found that AuNPs and hBD3 increased the proliferation of hPDLCs slightly.2.Under the stimulation of E.coli-LPS,hBD3 and AuNPs could significantly enhance the ALP activity and mineralized nodule formation;Meanwhile,real-time PCR and western blot results also showed that hBD3 and AuNPs could significantly up-regulate the osteogenic differentiation-related gene and protein expression of ALP,COL-1 and Runx-2.Moreover,hBD3 combined AuNPs activated the Wnt/?-catenin signaling pathway and up-regulated the gene and protein expression of ?-catenin and cyclin D1.After ICG-001 was added,the expression of cyclin D1 and ?-catenin decreased significantly.In addition,under the stimulation of E.coli-LPS,the formation of ALP and mineralized nodules in the group of hBD3 and AuNPs was also inhibited,and the expression of osteogenic gene of ALP,COL-1,and Runx2 were also down-regulated.3.hBD3 combined AuNPs reduced the alveolar bone loss.HE staining showed that the effect of hBD3 and AuNPs could significantly reduce the pathological changes of gingival epithelial spine thickening and epithelial hyperplasia.Furthermore,the TRAP staining demonstrated that hBD3 combined AuNPs could reduce the number of osteoclast in alveolar bone.Moreover,lower IL-6 and TNF-? expression,higher IFN-y expression were observed in the hBD3-AuNPs-treated group.[Conclusions]1.hBD3 combined AuNPs could significantly promote the osteogenic differentiation of hPDLCs in inflammatory microenvironments via activating the Wnt/?-catenin signaling pathway.2.hBD3 combined AuNPs could reduce the extent of inflammation in periodontal tissues and slow down the progression of periodontitis. |
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