| Objectives The aim of this study was to find a simple,efficient and biological modification method for the surface of medical devices.By simply mixing natural phlorotannin with the electropositive drug chlorhexidine,a material independent multifunctional coating was developed with rapid coating yield,high drug loading efficiency and good physiological stability.Experiments have shown that the coating has good antibacterial,biocompatibility and osteogenic properties,as well as the potential for secondary modification.Methods1.Preparation and characterization of physical and chemical properties of PL@CHX coating: After preparation of the coating by the liquid phase deposition method,Scanning electron microscopy(SEM),water contact Angle(WCA),atomic force microscopy(AFM),X-ray photoelectron spectroscopy(XPS),solution Zeta potential detection,surface film Zeta potential detection,attenuated total reflectionFourier transform infrared spectroscopy(ATR-FTIR),quartz crystal microbalance and dissipation monitoring(QCM-D)were used.2.The antibacterial properties of PL@CHX coatings were evaluated in vitro by turbidimetry,zone of inhibition(ZOI)and agar plating methods.For in vivo analysis,bare Ti rods and the PL@CHX-Ti rods were implanted into the bone marrow cavity of SD rats and removed after 1 day and 1 week.The bacteria isolated from the surface of the titanium rod were plating and pressed to culture to explore the antibacterial ability of PL@CHX-Ti.3.Evaluation of the stability of PL@CHX coating: Titanium discs with prepared PL@CHX coatings were immersed in PBS for 1,7,14,21,28,35,42,49,and 56 days.SEM images of the Ti surface were taken,contact angles were measured,and antibacterial activity was evaluated to measure its structural stability and antimicrobial stability.4.Biocompatibility evaluation of PL@CHX coating: The effect of PL@CHX coating on cell adhesion in vitro was explored by using mouse derived MC3T3-E1 preosteoblasts and bone marrow mesenchymal stem(BMSC)cells SD rats cultured on bare Ti surface and PL@CHX-Ti surface.The antibacterial effect was further evaluated by comparing with the minimum inhibitory concentration(MIC)of CHX.The antibacterial ability and cell compatibility were evaluated by co-culture with MC3T3-E1 cells after pre-colonization and biofilm formation of Staphylococcus aureus.The cytotoxicity was quantitatively evaluated by MTT colorimetric assay and CCK-8 assay.5.To evaluate the effect of the coating in vivo,the screw was implanted into the lateral condyle of the femur of SD rats,and CT films were taken.The rats were sacrificed after 4 and 8 weeks of implantation,and the femur was taken for micro-CT scanning and hard tissue sectioning.HE staining and toluidine blue staining were used to evaluate the histocompatibility in vivo.6.Material-independent evaluation of PL@CHX coating: The above method was used to prepare PL@CHX coating on the surface of silicon wafer(Si),gold wafer(Au),copper foil(Cu),aluminum foil(Al),stainless steel foil(SS),nitrocellulose film(NC),nylon film(PA),glass film(Glass),PET film,and polytetrafluoroethylene film(PTFE).WAC and SEM photographs were taken to measure that.7.Gene sequencing of the coating: The functional gene distribution characteristics of Control group and PL@CHX group were explored by RNA sequencing.The genes related to osteogenesis were selected for q RT-PCR reaction to explore the osteogenesis in vivo.Results1.Preparation and characterization of PL@CHX coatings The PL@CHX coating was prepared on the surface of Ti discs by liquid phase deposition.SEM results showed that the surface of bare Ti was rough and scale-like.Compared with bare Ti,the sheet roughness of PL-Ti was slightly reduced.However,the surface roughness of PL@CHX-Ti plate group was blurred,and new particulate matter was formed on its surface.The results of AFM for the roughness change of its surface are also consistent with those of SEM mentioned above.The water contact Angle results showed that the water contact angle of PL-Ti(19.20°)was significantly lower than that of bare Ti(53.47°).With the increase of CHX concentration,the water contact Angle value of the PL@CHX-Ti gradually increased.ATR-FTIR spectra showed that the original functional groups of the polyphenols were basically unchanged,but the Cl peak appeared.XPS analysis showed that the Ti peak of PL@CHX-Ti disappeared,the N peak increased,and the Cl peak appeared compared with bare Ti.These results clearly indicate that the PL@CHX coating was successfully prepared to the surface of Ti.The Zeta potential of PL and CHX mixed solution was detected,and the results showed that after adding CHX solution,the potential of negatively charged PL solution gradually increased.The zeta potential of PL gradually increased from-48.612 m V to 8.93 m V.The zeta potential values of the PL@CHX group also increased with increasing CHX concentration.Combined with the results of the membrane potential of the coating surface,it can also be proved that the membrane potential is negatively increased after the preparation of pure PL coating on the surface of the titanium sheet.However,the negative charge on the surface of the titanium plate with PL@CHX coating decreased.With the increase of CHX concentration in PL@CHX group,the surface membrane potential of the titanium sheet gradually approached neutral from negative charge.2.In vitro antibacterial properties of PL@CHX-TiStaphylococcus aureus(S.aureus)and Escherichia coli(E.coli)were selected as model bacteria because they are the two pathogens responsible for peri-implant infections.In order to verify the antibacterial properties of the prepared coatings,bacterial liquid turbidimetry test,agar plating test and antibacterial ring test were performed on bare Ti,PL-Ti and PL@CHX-Ti.The results of turbidimetry showed Ti in the blank control and the PL group were still turbidified after 12 hours of incubation with bacterial solution,while Ti in the experimental group were clear.The results of absorbance showed that the OD values of bare Ti and PL-Ti were significantly higher than PL@CHX-Ti.The results of the agar plates in the PL@CHX group showed no growth of S.aureus and E.coli,whereas the two bacteria on the agar plates in the other groups grew normally.The results of the inhibition ring test showed that there was no inhibition ring formed around the titanium plates in bare Ti and PL-Ti,while there was an obvious inhibition ring around the titanium plates in PL@CHX group.It can be seen that the higher the CHX concentration in PL@CHX-Ti,the larger the inhibition ring diameter.In vivo experiments also showed the same results as in vitro experiments: Ti rods from the PL@CHX and blank control groups were implanted into the femoral bone marrow cavity of male SD rats for 1 day and 1 week.It was observed that all wounds healed well without purulent discharge or other significant inflammation.After dissecting the femur,the titanium rod was removed and pressed on an AGAR plate to observe the distribution and number of bacteria.The results showed that the number of bacteria attached to the Ti rods in the PL@CHX group was much less than that bare Ti group.The culture results of bacterial extracts showed the same results.In order to evaluate the antibacterial stability of PL@CHX coating,turbidimetry test,plating plate test and antibacterial ring test were performed on the prepared titanium plates after immersion for 0,7,14,21,28,35,42,49 and 56 days.The results showed that PL@CHX experimental group had stable and obvious antibacterial effect.Even after 8 weeks of immersion in PBS solution,the inhibition ring(PL@CHX)still appeared in the experimental group.In conclusion,our PL@CHX not only has good and stable antibacterial ability in vitro,but also has the same antibacterial effect in vivo.3.Biocompatibility of PL@CHX-Ti MTT assay,CCK-8 assay and immunofluorescence assay were used to determine the cytotoxicity of PL@CHX coating on cell viability and proliferation.We found that there was no significant difference in the growth and proliferation of MC3T3-E1 cells in PL@CHX-Ti with different CHX concentrations and bare Ti.The results of fluorescent staining further showed that the MC3T3-E1 cells on the surface of PL@CHX-Ti were polygon-shaped,fully expanded,with some filopodia,and the cytoskeleton was arranged in a filamentous pattern in the same direction.The results of fluorescence staining showed that the cells in the blank control group and the pure PL group were completely contaminated and could not grow under the interference of bacteria.PL@CHX-Ti was resistant to bacterial growth,but the cell growth on the Ti surface was still good.Whether MC3T3 cells or BMSCs,PL@CHX coating had no adverse effect on cell proliferation and adhesion.CCK-8results also showed that the number of cells in the PL@CHX group was much higher than that in the CHX solution MIC group.It was further proved that PL@CHX coating further improved its cytocompatibility compared with CHX solution,so that it could play a good antibacterial property on the implant surface and has good cell compatibility.4.In vivo evaluation of PL@CHX-Ti screws Micro-CT analysis was used to evaluate the three-dimensional osteogenesis of each group.The bone volume/tissue volume ratio,bone height and bone volume ratio(BV/TV)of the experimental group were significantly higher than those of the blank control group.Toluidine blue and HE staining were used to evaluate the newly formed peri-implant bone tissue.The results showed that the degree of bone fusion in the PL@CHX group was better than that in the blank control group,with more new bone integration and no inflammatory tissue infiltration.5.Material-independence of PL@CHX coating To determine whether the PL@CHX coating can be applied to other substrates,we coated several typical material substrates,including PET,Si,Cu,SS,Al,Au,PA,PTFE,glass,and NC.Before and after coating preparation,it can be seen that the material before preparation is transparent or white,and the coating after preparation get dark.Scanning electron microscopy showed that after the coating was prepared,the pores of the porous material(PA)almost disappeared,and a large number of granular substances attached to it.Metals and materials with smooth surfaces can be seen to have a large amount of particulate matter attached.The results of water contact Angle analysis showed that the PL@CHX coating would adhere to hydrophobic surfaces such as PTFE.6.MC3T3-E1 cells were then induced to differentiate into osteoblasts,and the m RNA levels of osteogenesis-related genes,RUNX2 and OSX(master regulators of osteogenesis),were detected at day 7.Conclusion:In summary,this study demonstrates the successful development of a multifunctional conversion coating based on electrostatic binding and cation-πassembly.As a multifunctional surface modifier with negative charge,phlorotannin forms a long-term stable antibacterial PL@CHX coating by bonding the strongly positively charged CHX.It provides new solutions and concepts for treating periimplantitis in clinical practice. |
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