| Objectives:The purpose of this study was to prepare a novel temperature-sensitive gel triblock copolymer-poly(5-ethylene glycol ketal-?-caprolactone-?-caprolactone)-polyethylene glycol-poly(5-ethylene glycol ketal-?-caprolactone-?-caprolactone)(PECT),simultaneously loaded with ibuprofen(IBU)and basic fibroblast growth factor(bFGF)to make a drug delivery system.It was tested for phase transition behavior,rheological behavior,observed the internal morphology of gel,and analyzed for drug release patterns in vitro.The gel system was studied to promote the proliferation and adhesion of gingival fibroblasts and the effect of reducing inflammation,and to promote the healing of soft tissue around the implant and prevent the occurrence of peri-implantitis,and to explore the feasibility of its application to local application around the implant.Methods:1.bFGF + IBU / PECT gel synthesized by ring-opening copolymerization of polycaprolactone and 5-ethylene ketal-?-caprolactone(TOSUO)in the presence of polyethylene glycol using nanoprecipitation technique.Phase transition behavior of bFGF + IBU / PECT aqueous dispersion was studied by vial inversion method.Flow of bFGF + IBU / PECT aqueous dispersion(25wt%)was studied using a rheometer(Stress Tech,Reologica Instruments AB)behavior.The internal morphology of the gel was observed by Scanning Electron Microscope(SEM).The release of bFGF was simulated by labeled bovine serum albumin-FITC(BSA-FITC)and the drug release profile was determined in vitro by an ultraviolet(UV)spectrophotometer and a high performance liquid chromatograph.The release process and mechanism of bFGF +IBU / PECT gel were further studied using Transmission Electron Microscope(TEM)and Dynamic Light Scattering(DLS).2.Patients who underwent periodontal extension in the Department of Periodontology,Stomatological Hospital of Tianjin Medical University(this project has been approved by the Ethics Committee of Tianjin Medical UniversityStomatological Hospital,ethical number: TMUhMEC2018102).Taking its healthy gum tissue,human gingival fibroblasts were cultured by tissue culture primary culture method.The morphology of the cultured cells were observed under a microscope,and the third-generation well-grown HGFs were identified by immunohistochemistry.Subsequent cell experiments were performed on 4th to 6th generation HGFs.3.The in vitro cell assay was used to evaluate the effect of bFGF + IBU / PECT gel on HGFs proliferation and adhesion.Preparation of smooth titanium sheets,the effect of bFGF + IBU / PECT nanoparticle dispersion on the morphology and number of early adhesion of HGFs on titanium surface was observed by SEM.The proliferation of HGFs by bFGF + IBU / PECT nanoparticle dispersion and the long-term effects of bFGF + IBU / PECT gel on the proliferation of HGFs were detected by CCK-8.Immunofluorescence was used to detect the protein expression level of adhesion-related gene vinculin on titanium sheets.Real-time quantitative PCR was used to detect the mRNA level of vinculin on each group of titanium sheets.4.HGFs were stimulated with Porphyromonas gingivalis-Lipopolysaccharide(Pg-LPS)to establish a model of cellular inflammation.The same concentration of IBU aqueous solution and bFGF + IBU / PECT nanoparticle dispersion were separately added in vitro,and co-cultured with HGFs under inflammatory conditions.The supernatants of each group were collected at 10 h,24h,48 h and 72 h,and the concentrations of prostaglandin E-2(PGE2)were detected by enzyme-linked immunosorbent Assays(ELISA).Results:1.The bFGF + IBU / PECT sustained-release gel was successfully prepared by nanoprecipitation technology and stored as lyophilized powder.The results of the vial inversion experiment showed that bFGF + IBU / PECT lyophilized powder was dissolved in double distilled water to prepare bFGF + IBU / PECT NPs dispersion.It was in the solution at room temperature,and the solution can occur phase transition and becomes a gel state when the vial was placed in a 37°C incubator for one minute.Rheology studies show that as the temperature increases,the storage modulus of thegel increases and is greater than the loss modulus.SEM and DLS results of gel show bFGF + IBU / PECT gel is loose and porous,which is beneficial to drug release.The drug release curve shows that the sustained release of hydrophobic drug IBU can reach 30 days,and the hydrophilic drug BSA-FITC can reach more than 10 days without obvious sudden release.The results of TEM of gel nanoparticle solution indicate that the bFGF + IBU / PECT nanoparticles have uniform particle size and no obvious polymerization.2.The culture results of human gingival fibroblasts showed that HGFs was successfully cultured from tissue blocks by primary culture of tissue blocks.The microscopy was used to observe cells,and the primary cells can be seen around the tissue block after 4-7 days of primary culture.After passage,the cell morphology was similar to that of the primary cells,showing a long spindle shape,and the cells were arranged radially.The cell source was identified by immunohistochemistry.The results showed that the cells were negative for anti-keratin staining and positive for anti-wave protein staining.It was confirmed that the cells cultured in this experiment were mesoderm-derived HGFs.3.The SEM results of HGFs on the surface of titanium sheets showed that compared with the control group,the bFGF and bFGF + IBU / PECT nanoparticle dispersion was more conducive to the proliferation of HGFs and the extension of pseudopods during early adhesion.The results of CCK-8 cell proliferation experiments showed that bFGF + IBU / PECT nanoparticle dispersion can promote the proliferation of HGFs well,and the effect of gel on HGFs proliferation can last ?7 days.Immunofluorescence results show that bFGF and bFGF + IBU / PECT nanoparticle dispersion on titanium conducive has more intracellular adhesion-related gene,vinculin,and more excellent cell morphology.Real-time quantitative fluorescent PCR detection of vinculin mRNA expression levels showed that the expression of vinculin in cells on the titanium plate of bFGF + IBU / PECT nanoparticle dispersion was higher.4.ELISA results showed that after treatment of HGFs with 1?g/ml Pg-LPS,the concentration of PGE2 in the LPS group was significantly higher than that in the control group(p<0.05),indicating that the cell inflammation model was successfullyestablished.IBU aqueous solution and bFGF + IBU / PECT nanoparticle dispersion were co-cultured with HGFs under inflammatory conditions at different time points.The concentration of PGE2 in IBU group and bFGF + IBU / PECT nanoparticle dispersion group were lower than that of the LPS group at 10,24,48,72.(p<0.05).In addition,the PGE2 levels in the bFGF + IBU / PECT nanoparticle dispersion group were lower than those in the IBU group at each time point(p<0.05),and the PGE2 concentration became lower and lower with the prolongation of the culture time.Conclusions:1.This study successfully prepared bFGF + IBU / PECT gel by using PECT temperature sensitive gel simultaneously loaded bFGF and IBU,which has good temperature sensitivity and can achieve good sustained release.2.bFGF + IBU / PECT gel can promote the proliferation of HGFs and its adhesion on the surface of titanium,and has a good anti-inflammatory effect in vitro.3.The bFGF + IBU / PECT gel designed in this study provides a new way to prevent the occurrence of peri-implantitis. |
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