| [Background]Periodontitis is mainly the acute or chronic inflammation of periodontal tissue induced by periodontal pathogens,and leads to the destruction of soft tissue and alveolar bone.The traditional treatment of periodontitis mostly focus on controlling inflammation rather than reconstruct natural periodontal tissue.In the research of tissue engineering and regenerative medicine,mesenchymal stem cells(MSCs)can be induced to differentiate into osteoblasts(OB),and then generate alveolar bone.However,in the environment of periodontal inflammation,tumor necrosis factor-α(TNF-α)secreted by inflammatory cells can inhibit the proliferation and differentiation of MSCs,which makes the periodontal tissue defects difficult to repair.Therefore,the key to the treatment of alveolar bone defects is to block the effect of TNF-α on MSCs and promote the osteogenic differentiation of MSCs.It is worth noting that the expression of WW domain-containing E3 ubiquitin protein ligase 1(WWP1)of MSCs increased after TNF-α stimulation.WWP1 is a multifunctional protein,which inhibits the osteogenic differentiation of cells by promoting the ubiquitination and degradation of Runx2 and junB.It has been confirmed that the expression of osteogenic related genes(ALP,OCN)in inflammatory MSCs increased after silencing WWP1 gene by RNA interference(RNAi).In periodontal tissue,there are a certain number of periodontal ligament stem cells(PDLSCs)with high proliferation,self-renewal and differentiation potential,which play an important role in the process of periodontal tissue regeneration.This suggests that we can silence WWP1 gene of inflammatory PDLSCs by RNAi to promote alveolar bone regeneration.The biggest obstacle of RNAi application in vivo is that small interfering RNAs(siRNA)is easily degraded,cell uptake rate is low,and targeted delivery is lacking.Therefore,the delivery of siRNA often depends on carrier Unfortunately,the common carriers have the disadvantages of complex preparation,high cost and high cytotoxicity.Therefore,reducing toxicity,immunogenicity,and costs of siRNA carrier materials are key goals for RNAi technology transition from bench to bed.Recently,calcium ions(Ca2+)have garnered attention as a novel,alternative material for delivering siRNA to cells.However,the tolerance for Ca2+ concentration varies in different cell types,which has limited its applications in vivo.Bovine serum albumin(BSA)can bind to Ca2+ through chelation.Moreover,BSA is a favorable coating material for nanoparticles owing to its excellent biocompatibility.Therefore,we hypothesized that coating Ca2+-siRNA with BSA helps buffer Ca2+ toxicity in vivo.Based on the above research background,we propose the design of this project:firstly,we observed the effect of TNF-α on the function of PDLSCs,and proved the effect of WWP1 on the osteogenic differentiation of PDLSCs.Secondly,a low-cost,convenient,efficient and safe carrier was constructed and combined with siRNA to form a transfection complex.Then,the biocompatibility and transfection characteristics of the transfection complex were analyzed.Finally,the role of WWP1 siRNA in promoting bone formation in vitro and in vivo was evaluated to provide a new idea for the treatment of bone defects in periodontitis by RNAi.Part Ⅰ Objective to study the effect of TNF-α on the biological behavior of PDLSCs[Purpose]To isolate and culture PDLSCs for phenotype identification,and analyze the effect of TNF-α on the biological behavior of PDLSCs.[Methods]1.We isolated stem cells from the periodontal ligament tissues of the healthy.The primary culture of PDLSCs was carried out using the method of tissue block culture,and the surface markers were detected by flow cytometry.2.Osteogenic and adipogenic induction were performed.The osteogenic ability was investigated using Alizarin red and ALP staining.The adipogenic ability was investigated using Oil red 0 staining.The mRNA expression levels of ALP,OCN,Runx2 and JunB were analyzed using RT-PCR.3.Lentiviral carriers was loaded with plasmid containing eGFP gene sequence,and PDLSCs were transfected to overexpress GFP.[Results]1.The surface markers expressed by PDLSCs were same as MSCs,namely,CD29,CD44,CD90,CD105 and CD146 were positive,and CD34 and CD45 were negative.2.TNF-α could inhibit the multi-directional differentiation ability of PDLSCs,and up-regulate the expression of WWP1 and down-regulate the expression of ALP and OCN in PDLSCs during osteogenic induction.3.Under the infection condition of MOI 80 and the screening condition of 1 μg/mL puromycin,GFP-PDLSCs cell lines with fluorescence rate above 90%were obtained.[Conclusion]1.PDLSCs had the characteristics of high proliferation,self-renewal and differentiation,but TNF-α could inhibit their multidirectional differentiation potential.2.By inducing the up-regulation of WWP1 expression and down-regulation of ALP and OCN expression,TNF could inhibit the osteogenic differentiation of PDLSCs.Part Ⅱ Preparation and characterization of BSA-Ca2+-siRN A nano particles[Purpose]To construct BSA-Ca2+-siRNA nanoparticles,and screen out the ratio of BSA/Ca2+/siRNA with high stability and siRNA loading rate.[Methods]1.BSA-Ca2+-siRNA nanoparticles were prepared,and the size,shape,encapsulation,and release efficiency were characterized using atomic force microscopy,scanning electronic microcopy,and gel electrophoresis.2.The binding of nanoparticles were evaluated using attenuated total reflection Fourier transform infrared spectroscopy,isothermal titration calorimetry and molecular simulation docking.3.The stability and solubility of nanoparticles were evaluated using agarose gel electrophoresis and ultraviolet spectrophotometer.[Results]1.BSA and Ca2+-siRNA could form spherical particles with a rough surface,with a charge of 4.72 ± 0.37 mV(～140 nm in diameter).2.FTIR showed that there were many C-C or C-N stretches in the structure of BSA-Ca2+-siRNA nanoparticles,and the peaks at 3430,2358 and 2155 cm-1 are the strongest;ITC showed that the dissociation constant(Kd)of nanoparticles was about 9.80 mM;three Ca2+in BSA-Ca2+interacted with siRNA directly,and the structure of the nanoparticles was stable.3.When BSA>500 μg/mL,BSA-Ca2+-siRNA nanoparticles could firmly retain and protect the siRNA cargo,and the release rates of siRNA in neutral or acidic PBS were 48%and 64%,respectively.[Conclusion]1.BSA-Ca2+-siRNA nanoparticles was a nano-sized spherical particle,and Ca2+acted as abridge to connect BSA and siRNA together,which has strong binding energy.2.BSA-Ca2+-siRNA nanoparticles had high stability,and could achieve sustained siRNA release.In acidic environment,the degradation efficiency of BSA-Ca2+-siRNA was improved.Part Ⅲ Analysis of the transfection characteristics and biological safety of BSA-Ca2+-siRNA nanoparticles[Purpose]To evaluate the transfection efficiency,cellular uptake and intracellular release pathway,and biological safety of BSA-Ca2+-siRNA nanoparticles.[Methods]1.The cellular uptake,intracellular release,and gene knockdown of nanoparticles were evaluated in PDLSCs using laser-scanning confocal microscope,Inverted fluorescence microscope and flow cytometry.2.The cytotoxicity was evaluated using CCK-8 method,and the biocompatibility was analyzed using hemolysis experiment and injection experiment in vivo.[Results]1.The BSA-Ca2+-siRNA nanoparticles were taken up by the cells,primarily through macropinocytosis,and were then released intracellularly through the acidification of endosomes/lysosomes.2.the BSA-Ca2+carrier had high transfection efficiency and biocompatibility both in vitro and in vivo.[Conclusion]BSA-Ca2+could potentially be used for siRNA delivery,which is not only highly efficient and cost-effective but also biocompatible to host tissues owing to the BSA coating.Part Ⅳ The osteogenesis of BSA-Ca2+-siRNA in vitro[Purpose]To evaluate the in vitro osteogenic differentiation of BSA-Ca2+-siRNA nanoparticles.[Methods]1.The osteogenesis of BSA-Ca2+-siWWPl nanoparticles was evaluate using histological staining observation and semi-quantitative analysis.2.The osteogenic mechanism of siWWP1 was analyzed using RT-PCR and Western blot.[Results]1.BSA-Ca2+-siWWPl nanoparticles could promote the secretion of ALP and OCN,the synthesis of COL-1 and the mineralization of extracellular matrix in PDLSCs.2.BSA-Ca2+-siWWPl nanoparticles could down-regulate the expression of WWP1 and up-regulate the expression of ALP,OCN and Runx2,but did not affect the expression of JunB.[Conclusion]BSA-Ca2+-siWWP1 nanoparticles could achieve RNAi effect,knockdown WWP1 gene,increase the expression of Runx2,and enhance the synthesis of ALP and OCN,the secretion of COL-1 and the mineralization of extracellular matrix,thus significantly promoting the osteogenic differentiation of PDLSCs in vitro.Part Ⅴ The osteogenesis of BSA-Ca2+-siRNA in vivo[Purpose]To evaluate the in vivo osseointegration of BSA-Ca2+-siRNA nanoparticles.[Methods]1.The rat periodontitis model was construct using injection of LPS and ligation,and evaluate the bone destruction and self-healing of the model using Micro-CT.2.The stability and validity of BSA-Ca2+-siRNA nanoparticles in vivo was evaluated using Cy-5.5 labeled siRNA.3.The osteogenesis of BSA-Ca2+-siWWPl nanoparticles in vivo was analyzed using histological staining and Micro-CT.[Results]1.After 28 days of modeling,the alveolar bone was obviously absorbed,and the rat periodontitis model was successfully established;after 15 days of removing inflammatory factors,new bone formation was obvious in the model,and it could be self-healing after 30 days.2.In BSA-Ca2+/siRNA group,the fluorescence signal could exist for 4 days,while in Lipo/siRNA and siRNA group,there was no obvious fluorescence signal after 3 days.3.Compared with the control group,the distance of CEJ-ABC in BSA-Ca2+/siRNA group was decreased,and the volume of alveolar bone at furcation was significantly increased;histological staining showed that there were more new bone formation around the experimental teeth,and the bone tissue was dense and continuous in BSA-Ca2+/siRNAgroup.[Conclusion]BSA-Ca2+-siWWP1 nanoparticles could achieve stable and continuous siRNA release,and significantly improve osseointegration in vivo. |
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