Roles Of A New Drug-delivery Abutment And TNFR1-PLAD In The Prevention And Treatment Of Peri-implant Infections

Part ?: Roles of a new drug-delivery abutment in the prevention and treatment of peri-implant infections1.Preparation and surface analysis of the drug-delivery abutmentAim: The aim of the study is to design and manufacture the drug-delivery abutment and analysis the surface feature of it.Materials and methods: The drug-delivery abutment is composed of three parts,including the cylinder-shaped drug chamber,the closure to seal the drug chamber and the connection part.The drug chamber is a hollow cylinder with evenly distributed drug delivery holes in the peripheral wall.The lower connector connects with the implant through its threaded structure.According to the different sizes(0.3 mm,0.5 mm and 0.8 mm)of drug delivery hole,drug-delivery abutment and normal healing abutment were manufactured using medical pure titanium sticks.Scanning probe microscopy(SPM)was used to detect the surface morphology and roughness of the two kinds of abutment.Results: The surface of the drug-delivery abutment presented typical titanium metal surface morphology,similar to the normal healing abutment.And the surface roughness of both kinds of abutment was less than 0.2 micron with no significant difference between the two groups.Conclusion: In this study,we designed and manufactured the drug-delivery abutment with different size of drug delivery holes and normal healing abutment.The surface roughness of both abutments meets the requirement for the further use in dental implant.2.The bacteriostatic effect of the drug delivery system on the normal oral bacteria and peri-implant infection related pathogenAim: The aim of the study is to evaluate the bacteriostatic effect of the drug delivery system on the normal oral bacteria and peri-implant infection related pathogen in vitro.Materials and methods: Streptococcus mutans,Streptococcus sanguinis,Porphyromonas gingivitis were cultured and identified by 16 s r RNA sequencing method.Bacterial suspension was evenly smeared on the surface of the medium plate,and the center of the plate was perforated.The drug-delivery abutment with different size of drug delivery holes loaded with minocycline ointment was placed in it.After 24 hours,the formation of inhibition zones on the plate was observed and the diameters of inhibition zones was recorded and compared.Taking Streptococcus mutans and Streptococcus sanguinis as the bacteria to be tested,the drug-delivery abutment with 0.5mm drug delivery holes loaded with minocycline ointment was placed in the plate,and the plate was replaced every day to observe the formation of inhibition zones at different observation points(1-7 days).Results: Inhibition zones were found surrounding all the tested drug-delivery abutments in all three bacterial strains.As the drug delivery hole size of the abutments increased,the diameters of the inhibition zones increased.Inhibition zones were found at different observation points(1-7 days).Conclusion: The drug-delivery abutment could effectively deliver the drugs to the surrounding tissues through the delivery holes in vitro with sustained drug delivery ability.3.The influence of the drug delivery system on plaque accumulation at early stage of osseointegrationAim: The aim of this study is to evaluate the plaque accumulation on the surface of drug-delivery abutments at early stage of osseointegration in vivo.Materials and methods: Three 18 months-old Beagle dogs were involved in this study.The third and fourth premolars and the first molar of the mandible were extracted bilaterally from all three dogs.After 12 weeks,three bone-level implants were placed in the edentulous area along the crestal alveolar ridge of each side of the mandible.A total of 18 abutments were evenly divided into three groups: drug-delivery abutments(the diameter of the drug delivery holes was 0.5 mm)loaded with minocycline ointment(group: DDA + minocycline);drug-delivery abutments without any drugs(group: DDA);normal healing abutments(group: control).The 18 abutments were connected to the implants and the mucoperiosteal faps were adjusted and sutured.A soft diet was applied to the dogs and no plaque-control program was initiated.Seven days after surgery,plaque formation on the three groups of abutments was observed and quantitatively detected by Basic Fuchsin staining.Results: Seven days after the placement of implants and abutments,a significant decrease of stained areas was vividly observed on the abutment of DDA + minocycline group compared with the control group and DDA group.The result of quantitative measurement of Basic Fuchsin in stained plaque was in accordance with the apparel observation.No significant difference was found between control group and DDA group.Conclusion: At the early stage of implant placement and healing phase of the implant surgery,drug-delivery abutment can effectively release drugs to the peri-implant tissues to reduce plaque accumulation,which may be applied to the prevention of peri-implant inflammatory diseases.4.The influence of the drug delivery system on progress of peri-implant inflammation in experimental peri-implant infection modelAim: The aim of this study is to investigate the influence of the drug delivery system on progress of peri-implant inflammation in in vivo experimental peri-implant infection model.Materials and methods: 12 weeks after implant placement,establishment of the experimental peri-implant infection model was initiated by silk ligation in three Beagle dogs.At the same time,drug-delivery abutments(the diameter of the drug delivery holes was 0.5 mm)loaded with minocycline ointment(group: DDA + minocycline)and normal healing abutments(group: control)were connected to the implant.Clinical measurements were performed right before the ligation and the second,the forth and the eighth week after the ligation,including the BOP index,PPD,volume of peri-implant crevicular fluid(PICF),activity of the aspartate aminotransferase(AST)and alkaline phosphatase(ALP)in PICF,level of Tumor Necrosis Factor alpha(TNF-?)and Interleukine-1 beta(IL-1?)in PICF.The distance between the implant shoulder and the marginal bone level(MBL)next to the implant was determined by radiographic measurement right before the ligation and eight weeks after the ligation.Progress of peri-implant inflammation was evaluated by the results of clinical and radiographic measurements.Results: During the silk ligation,BOP,PPD,volume of PICF,AST,ALP,TNF-alpha and IL-1beta levels in PICF of the DDA + minocycline group and the control group showed an increasing trend.Compared with the control group,the increase of BOP,PPD,PICF volume,and AST and IL-1beta levels in the DDA+minocycline group was significantly decreased.X-ray examination showed bone resorption in both groups,and there was no significant difference in bone resorption level between the two groups.DDA+minocycline group showed a relatively slower rate of deterioration of the mucosal inflammation and probing depth in the experimental peri-implant lesions.Conclusion: It is suggested that this drug-delivery abutment could effectively deliver medications into peri-implant tissues to relieve peri-implant inflammation in the experimental peri-implant infection model,thus reveal its possible application in treatment of peri-implant infections.Part ?: A Preliminary study on the possibility of TNFR1-PLAD as an anti-TNF drug in the treatment of peri-implant infections1.Construction of p CDH-TNFR1-PLAD plasmid and the over-expression of TNFR1-PLAD gene in human gingival fibroblastsAim: The aim of the study is to observe the over-expression of TNFR1-PLAD gene in human gingival fibroblasts(HGF)by transfecting the p CDH-TNFR1-PLAD plasmid into HGF with lentivirus.Materials and methods: HGF were cultured with tissue culture method and identified by immunohistochemical staining.The p CDH-TNFR1-PLAD over-expression plasmid was constructed by inserting synthetic TNFR1-PLAD gene fragment into p CDH plasmid with restriction enzyme digestion and confirmed by DNA sequencing analysis.HGF were transfected by lentiviruses packaged in a three-plasmid system.Expression of TNFR1-PLAD was detected by PCR and Western Blot in the p CDH-TNFR1-PLAD transfected HGF(test group),p CDH transfected HGF and non-virus transfected HGF(control group).Results: Fluorescence microscopy showed that p CDH-TNFR1-PLAD was successfully transfected into HGF.The expression of TNFR1-PLAD gene in the test group was significantly higher than that in the control groups according to results of ordinary PCR and q RT-PCR.The results of Western Bolt showed that TNFR1-PLAD protein was detected in pCDH-TNFR1-PLAD transfected cells.Conclusion: The p CDH-TNFR1-PLAD plasmid was successfully constructed and sustained over-expression of TNFR1-PLAD in human gingival fibroblasts(HGF)was achieved by lentiviral vector.2.Human gingival fibroblasts response to over-expressed TNFR1-PLAD in inflammatory environmentAim: The aim of this study is to investigate the influence of over-expressed TNFR1-PLAD on HGF in inflammatory environment in vitro.Materials and methods: Three groups of HGF were stimulated with TNF-alpha(10ng/ml),including the p CDH-TNFR1-PLAD transfected HGF(test group),p CDH transfected HGF and non-virus transfected HGF(control group).Five minutes later,total protein of the cells of each group was extracted.Western Blot was used to detect the expression levels of p65,p-p65 and p-I?B?.After 3 hours,RNA was collected and IL-6 and IL-8 expression were detected by q RT-PCR.After 72 hours,the cell supernatants were collected and the contents of IL-6 and IL-8 in the supernatants were detected by ELISA.Results: With the stimulation of TNF-alpha,the levels of p-p65 and p-I?B? in p CDH-TNFR1-PLAD transfected HGF group were significantly decreased,suggesting that the activation level of NF-?B pathway was significantly inhibited.The results of PCR and ELISA showed that the levels of IL-6 and IL-8 in p CDH-TNFR1-PLAD transfected HGF were significantly lower than those in the control group.Conclusion: TNFR1-PLAD can specifically block TNFR1 signal transduction and inhibit the activation of NF-?B pathway and the secretion of related inflammatory factors in HGF under inflammation.It may be used as a new TNF inhibitor in the control and treatment of periodontal and peri-implant inflammation.3.Preparation and evaluation of chitosan-PLAD sustained-release microspheresAim: The aim of the study is to prepare and evaluate the character of the chitosan-PLAD microspheres with sustained-release ability.Materials and methods: Chitosan-PLAD microspheres were prepared by complex coacervation method with 0.02% chitosan solution prepared by 5 m M acetic acid-sodium acetate buffer solution and 100?g/ml plasmid solution prepared by 50 M sodium sulfate solution.Laser dynamic analyzer was used to analyze the size of microspheres.The release dose of microspheres in vitro was quantitatively analyzed by lysozyme degradation method.The degradation performance of microspheres in vitro was also tested.Results:The average particle size of chitosan-PLAD microspheres prepared by complex agglutination method was 811.4+85.93 nm.The encapsulation efficiency of the microspheres was 92.7 ± 2.2%,and the drug-loading rate was 18.5 ± 1.7%.The microspheres exhibit good release performance and slow degradation.Conclusion: Chitosan-PLAD plasmid microspheres were successfully prepared by complex agglutination method.In vitro tests showed that the microspheres were with suitable particle size,good drug delivery performance and slow degradation characteristics.