| [Background]Titanium implants,anchored in jawbones through osseointegration,have been utilized in dental clinics for decades to replace lost teeth and to make maxillofacial prostheses.However,the long-term survival of an implant depends on not only bone-implant osseointegration but also soft tissue sealing around the implant.Previous studies have revealed that the peri-implant soft tissue lacks perpendicular collagen fibers and blood vessels,which is quite different from that of a natural tooth.As a consequence,the attachment of soft tissue to the surface of an implant is relatively weaker than that to a natural tooth,and it is vulnerable to the invasion of pathogenic microorganisms.The invasion of microorganisms and the formation of biofilm are crucial reasons for peri-impant inflammation.Therefore,improving biological sealing of soft tissue around the implant and enhancing the antibacterial performance of the implant represent research focus in this field.In recent years,with the development of implant surface treatment technology,a growing number of researchers have devoted themselves to the specific designs of percutaneous/transmucosal implants utilizing the implant modification methods to promote soft tissue sealing and to reduce the incidence of inflammation.For example,superparamagnetic complex was fabricated on the surface of implant to prevent soft tissue recession and to eliminate inflammatory reaction,and fibronectin was covalently immobilized on the implant to promote epithelial attachment,but the effect is still far from perfect.Compared to systemic administration,the preparation of an antibacterial sustained-release coating on the implant surface can effectively avoid high-dose antibiotic induced hepatorenal complications and bacterial resistance.In the first part of the experiment,we prepared a decomposable silicate-gentamycin(SG)nanoparticles loaded with gentamicin,and farbricated a novel SG nanoparticle/gelatin coating on the percutaneous implant.Then the in vitro and in vivo biocompatibility and the in vitro antibacterial properties of the coating were investigated.This part of the experiment provides a new strategy for preventing soft tissue infection across the implant.A tight barrier formed by the junctional epithelium on the implant played a key role in protecting the bone-embedded implant from the invasion of pathogenic oral microorganisms.In the second part of the experiment,we fabricated a Chi/Col/pLAMA3-CM coating based on plasmid transfection,and then evaluated the effect of the coating on the soft tissue closure both in vitro and in vivo.This specific design of implant surface coating may effectively promotes soft tissue attachment and improve epithelial closure.Part Ⅰ:In vitro and in vivo evaluation of the biocompatibility and antibacterial properties of silicate-gentamycin(SG)nanoparticle/gelatin coated percutaneous implants[Objectives]1.To fabricate SG nanoparticles and SG nanoparticle/gelatin coating on the percutaneous implant surface,and provide suitable research models for the in vitro and in vivo experiments.2.To evaluate the effects of SG nanoparticle/gelatin coating on the proliferation and morphology of S.aureus and human fibroblasts.3.To establish a bilateral tibial implant model in SD rats,and to evaluate the effects of SG nanoparticle/gelatin coating on the healing of peri-implant soft tissue.[Methods]1.SG nanoparticles were fabricated using a non template method.The morphology,particle size,and zeta potential of the nanoparticles were investigated using SEM,TEM,and nanoparticle size analyzer respectively.After soaking in the PBS buffer for 14 d,the released gentamycin from the nanoparticles were detected using an ELISA kit.The morphological change of the SG nanoparticles during drug release was also observed using TEM.Micro arc oxidation(MAO)was used to prepare a micro scale coating on the surface of the smooth titanium(S-Ti).SG nanoparticle/gelatin coating was fabricated by adding SG nanoparticle/gelatin suspension onto the surface of the MAO-Ti substrates,and further crosslinked with glutaraldehyde.The morphology of the S-Ti,MAO-Ti,and SG-Ti specimens were observed using SEM.The drug release properties of the SG-Ti were investigated by soaking in the PBS buffer for 28 d.2.Formation of the bacteriostatic ring was observed by incubating the S-Ti,MAO-Ti,and SG-Ti specimens on the Mueller-Hinton agar plates coated with bacterial suspension for 24 h.Antibacterial rate of each specimen was investigated by incubating the specimens in the bacterial suspension for 24 h,and the attached bacteria were cultivated on the Mueller-Hinton agar plates for colony formation counting.Bacterial viability was investigated by incubating the specimens in the bacterial suspension for 24 h and stained with a LIVE/DEAD BacLightTM Bacterial Viability Kit.The morphology of bacteria attached on each specimens were observed using SEM.Primary human skin fibroblasts were isolated and cultured for further study.After seeding the cells on the S-Ti,MAO-Ti,and SG-Ti specimens for 2 h and 24 h,the morphology of the attached cells were observed using SEM.The proliferation of the cells cultured in the specimen extracts diluted to different concentrations were evaluated using a CCK-8 kit.3.Soft tissue healing of the S-Ti,MAO-Ti,and SG-Ti implants were evaluated by scoring the soft tissue reaction four weeks post surgery.Sections of tibiae block containing the implants were then harvested.After that,the position and direction of the implants in the rats’ tibiae were examined using Micro-CT.Further,VG staining were used to evaluate the soft and hard tissue formation of the implants.Finally,the integration between the soft tissue and implants were evaluated using SEM and EDX.[Results]1.SG nanoparticles were spherical in shape,with a diameter of 298.1 ± 12.2 nm.The zeta potential of the nanoparticles was-42.64 ± 1.10 mV.A prolonged release pattern was observed for gentamycin released from the SG nanoparticles.29.40%of the gentamycin was released for the first 12 h.Overall,88.22%of the accumulated drug release was achieved following 14 d of investigation.After soaking in the PBS buffer for 3 d,a hollow feature appeared in a few nanoparticles.The number of hollow nanoparticles increased in the following days,and some nanoparticles even collapsed.The MAO-Ti surface was porous with volcanic pores of 1-5 μm in diameter.The pores were filled with SG nanoparticles,and the crosslinked gelatin could be seen between the nanoparticles in the SG-Ti.Moreover,gentamycin were slowly released from the SG-Ti for 28 d.2.The bacteriostatic ring of the SG-Ti specimen was 16.7 ± 1.4 mm in diameter.In contrast,no bacteriostatic ring could be found around the S-Ti and MAO-Ti specimens.The number of the bacteria colony formed in the SG-Ti group was significantly less compared to that of the MAO-Ti and S-Ti groups.The SG-Ti possessed a positive and high AR value of 86.35%.However,the AR value of the MAO-Ti specimens was-270.32%.The immunofluorescence staining showed that the number of viable S.aureus attached to the SG-Ti specimen was far less than that of the S-Ti and MAO-Ti specimens.Meanwhile,dead S.aureus were only observed on the SG-Ti specimen.SEM scanning showed that the number of bacteria attached on the S-Ti and MAO-Ti specimens was significantly more than the SG-Ti specimens.A few bacteria in direct contact with the SG nanoparticles exhibited perforated and deformed membranes.As to the cytocompatibility of the specimens,human fibroblasts were firmly attached and well spread after seeding on the three groups of the specimens for 24 h.They all showed a fusiform morphology,with some filopodia and lamellipodia extensions.The CCK-8 test revealed that there was no significant difference in cytotoxicity among different extract concentrations for each of the specimens.3.While soft tissue healed well in the S-Ti and SG-Ti groups,soft tissue recession was observed in the MAO-Ti group four weeks post implant surgery.The soft tissue reaction score of the MAO-Ti group was relatively higher than that of the S-Ti and SG-Ti groups,however,there was no significant difference between the S-Ti and SG-Ti groups.Micro-CT scanning showed that all the remaining implants were in correct positions/directions.Relatively good osseointegration achieved in all groups,but obvious fibrous capsules were found in the MAO-Ti group.While there was no obvious gap between the implant and soft tissue in the S-Ti and SG-Ti groups,the MAO-Ti group had a broad gap between the micro arc oxidation coating and surrounding soft tissue.The results of EDX showed that the S-Ti group had a Ti element peak related to the titanium implant substrates and C,H,O element peaks related to the soft tissue;the MAO-Ti group had a Ti element peak related to the titanium implant substrates,S and P element peaks related to the micro arc oxidation coating and C,H,O element peaks related to the soft tissue;and the SG-Ti group had a Ti element peak related to the titanium implant substrates,a Si element peak related to the SG nanoparticles,S and P element peaks related to the micro arc oxidation coating,and C,H,O element peaks related to the soft tissue.[Conclusion]In the present study,gentamycin loaded SG nanoparticles were successfully fabricated and could release drug for up to 14 d.Further,the SG nanoparti cl e/gelatin coating were fabricated using the crosslinking method,and the sustained release of gentamycin was prolonged to 28 d.The SG nanoparticle/gelatin coating had an antibacterial activity against S.aureus,and was biocompatibility to the human fibroblasts and peri-implant soft tissues.It was revealed that the SG nanoparticle/gelatin coating could be used for the improvement and optimization of the percutaneous implants in a relatively simple,safe,and practical way.Part Ⅱ:The effect of Chi/Col/pLAMA3-CM coated mucosal implant on gingival epithelial closure[Objectives]1.To fabricate Chi/Col/pLAMA3-CM coating on the permucosal implant surface,and to provide suitable research models for the in vitro and in vivo experiments.2.To evaluate the effect of Chi/Col/pLAMA3-CM coating on the adhesion,morphology,proliferation,and cellular uptake of the plasmid;and to evaluate the effect of the coating on the expression of the adhesion related genes and proteins as well as formation of hemidesmosomes(HDs).3.To establish an immediate implant model in SD rats and to evaluate the effect of Chi/Col/pLAMA3-CM coating on the expression of adhesion related proteins and formation of HDs.[Methods]1.Titanium dioxide nanotubes(NT)were fabricated on the surface of the S-Ti specimen by anodization,and Chi/Col-Ti and Chi/Col/pLAMA3-Ti were fabricated by cathodic electrodepositing the Chi/Col or Chi/Col/pLAMA3 composites on the surface of the NT-Ti.SEM,AFM,and contact angle test were used to investigate the morphology and wettability of the four groups of the titanium specimens(S-Ti,NT-Ti,Chi/Col-Ti,and Chi/Col/pLAMA3-CM-Ti).After soaking in the PBS buffer,the cumulative release of pLAMA3-CM from the Chi/Col/pLAMA3-CM-Ti specimen were detected within 28 d.The morphological change of the coating were also investigated during the release of the plasmid using SEM.2.hGECs were isolated,cultured,and identified for further use.Cell attachment was investigated by seeding hGECs on the four groups of specimens(S-Ti,NT-Ti,Chi/Col-Ti,and Chi/Col/pLAMA3-CM-Ti)for 4 h and observed using CLSM.Cell morphology was conducted by seeding hGECs on the S-Ti,NT-Ti,and Chi/Col-Ti specimens for 2 h and 6 h,and seeding on the Chi/Col/pLAMA3-CM-Ti specimen for 2 h,6 h,12 h and 3 d and then observed using SEM.Cell proliferation was evaluated by seeding hGECs on the four groups of specimens for 4 h,1 d,3 d,5 d,and 7 d,then further detected using a CCK-8 kit.Cellular uptake of the pLAMA3-EGFP-CM by hGECs was performed by seeding cells on the specimens for 48 h and observed using CLSM.The expression levels of the adhesion related genes(LAMA3 and ITGB4)and proteins(Laminin-5 α3 and Integrin β4)were evaluated using qRT-PCR and immunofluorescence staining respectively.Formation of HDs in hGECs was observed using TEM.3.Immediate implant surgery was conducted on the right maxillary of the SD rats.Sections of the maxillary block containing the implants were then harvested four weeks post surgery.After that,the position and direction of the implants in the rats’ maxillae were examined using Micro-CT.Further,VG staining were used to examine the tissue formation of the implants.After demineralization,H&E staining was performed to evaluate the healing of the implants,and immunofluorescence staining was conducted to evaluate the expression level of Laminin-5 α3 and Integrin β4 across the peri-implant soft tissue.Further,TEM was used to observe HD formation.Finally,DAB reagent was used to detect the infiltration of HRP across the peri-implant soft tissue.[Results]1.A well-distributed nanotubes with average diameter of 100 nm were formed on the surface of the S-Ti specimen after anodization,and Chi/Col and Chi/Col/pLAMA3-CM coatings were uniformly immobilized on the surface of the NT-Ti substrates through cathodic electrophoretic deposition.The Sa value and contact angle of the coated specimens(Chi/Col-Ti and Chi/Col/pLAMA3-CM-Ti)were relatively lower and higher compared to those of the NT-Ti specimen respectively.However,the contact angle of the coated specimens were significantly lower than that of the S-Ti specimen.SEM scanning showed that the Chi/Col/pLAMA3-CM coating degraded slowly in PBS buffer and released pLAMA3-CM for at least 28 d,and up to 67.78%of the plasmids were released at the end of the investigation.2.Primary hGECs positively expressed the epithelial cell marker Cytokeratin 10 were successfully isolated and cultured.CLSM investigation showed that the number of the attached cells on the NT-Ti specimen was significantly decreased compared to that on the S-Ti specimen.However,coating the NT-Ti with either Chi/Col or Chi/Col/pLAMA3-CM increased the number of attached cells compared to the S-Ti specimen.SEM scanning showed that the hGECs on the Chi/Col/pLAMA3-CM-Ti specimen were spherical with clear filopodia extensions after culturing for 2 h.The number of extensions increased from 2 h to 6 h.Well-flattened cells with both filopodia and lamellipodia were observed at 12 h.Finally,the surface of the specimen was fully covered with cells with extended filopodia and lamellipodia across different directions on d 3.Although similar cell morphology and filopodia extensions were observed on the S-Ti specimen after culturing for 2 and 6 h,the cells on the NT-Ti specimen were poorly stretched and displayed an elongated and irregular shape with very few filopodia.Moreover,spherical cells could be found on the Chi/Col-Ti specimen and a few filopodia were observed after 6 h.CCK-8 assay showed that there was no significant difference of viability among the hGECs seeded on the four types of specimens.As shown by the green fluorescence under CLSM,pLAMA3-EGFP-CM were phagocytized by hGECs when seeded on the Chi/Col/pLAMA3-EGFP-CM-Ti specimen.qRT-PCR assay showed that the LAMA3 and ITGB4 expression levels were decreased in cells cultured on the NT-Ti specimen compared with those cultured on the S-Ti.However,coating the NT-Ti specimen with Chi/Col led to increased expression levels of both genes in the hGECs.Interestingly,the Chi/Col/pLAMA3-CM coating further increased the cellular expression levels of both genes compared to those cells seeded on the Chi/Col-Ti specimen.The immunofluorescence staining showed that cells on the Chi/Col/pLAMA3-CM-Ti specimen presented relatively higher protein expression level of Laminin-5 α3 than that on the other three groups.As to Integrin β4,hGECs on the NT-Ti specimen presented the lowest expression level.TEM observation showed that the number of HDs formed in the Chi/Col/pLAMA3-CM-Ti group was significantly higher than that S-Ti and Chi/Col-Ti groups.3.The Micro-CT scanning showed that all the implants were in correct positions/directions four weeks post surgery.The VG and H&E staining showed that both soft and hard tissues had formed around the S-Ti,Chi/Col-Ti,and Chi/Col/pLAMA3-CM-Ti implants.However,obvious fibrous capsules with marked inflammatory cell infiltration could be observed across the NT-Ti implants.The immunofluorescence staining showed that the expression levels of Laminin-5 α3 and Integrin β4 in the Chi/Col/pLAMA3-CM-Ti group were significantly higher than those in other groups.Significant assembled HDs were found in the peri-implant epithelia attached to either Chi/Col/pLAMA3-CM-Ti or Chi/Col-Ti.Although HDs formation was found in the S-Ti group,it was seldom observed in the NT-Ti group.While the peri-implant tissues in the Chi/Col/pLAMA3-CM-Ti group were HRP-negative,HRP-positive areas were clearly found in both epithelial and connective tissues in the NT-Ti and Chi/Col-Ti groups.The peri-implant soft tissues in the S-Ti group were slightly HRP-positive.[Conclusion]The Chi/Col/pLAMA3-CM coating had relatively low surface roughness,high hydrophilicity and could release pLAMA3-CM continuously for 28 d.The in vitro experiments showed that Chi/Col/pLAMA3-CM coating could promote the adhesion and proliferation of hGECs,and promote cellular uptake of pLAMA3-CM,expression of adhesion related genes and proteins,and formation of HDs.The in vivo experiments showed that the coating could promote the expression of adhesion related proteins and formation of HDs of the peri-implant soft tissues.In summary,Chi/Col/pLAMA3-CM coating was beneficial to soft tissue sealing across the permucosal implants and has great potential in the application of implant surface modification. |
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