Effects Of LAMA3 Immobilization On Titanium Surface On The Biological Sealing Between Implant And Gingival Epithelium

The success of a dental implant treatment depends not only on the healing of hard tissues but also on the formation of soft tissues. A good biological sealing between the soft tissue and the implant could prevent oral bacteria and their products from penetrating the body and minimize the risk of peri-implantitis. Like the junctional epithelium, the peri-implant epithelium attaches to the surface of the implant via hemidesmosomes and a basal-lamina-like extracellular matrix, which is termed the internal basal lamina.However, hemidesmosomes and internal basal lamina, only formed in the apical portion of the implant-peri-implant epithelium interface and were absent in the upper-middle portion. Laminin 332 is an important component of the internal basal lamina, and the junctional epithelial cells interact with the LG domain of the α3 chain of laminin 332 via integrins α3β1 and α6β4, which is crucial for mediating cell behaviors and the formation of hemidesmosomes. In this study, Ad-LAMA3 vectors (coding LG domain of laminin α3 chain) were immobilized on a multilayer coating on the titanium surface. Then, evaluations in vitro and in vivo were taken to assess the effects of LAMA3 immobilization on titanium surface on the biological sealing between implant and gingival epithelium.The multilayer coating, consisting of chitosan, hyaluronate and collagen IV, was assembled on smooth titanium surface using a layer-by-layer technique. Surface analyses using field emission scanning electron microscopy(FSEM), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), and surface contact angle measurements confirmed the fabrication of CS/(HA/COL)n coating on titanium surface. The results revealed that 5 layers were the optimal assembled layers, and the coating could improve the hydrophilia of smooth Ti. The cell viability test indicated that the CS/(HA/COL)n coatings have good cytocompatibility before and after crosslinking.Based on the CS/(HA/COL)5 coating, the anti-adenovirus antibody was crosslinked onto the surface of the coating, and then Ad-LAMA3 vectors were immobilized on the titanium for in situ transduction. The concentration of anti-adenovirus antibody was 30μg/well, and the number of the virus was 1×108PFU after optimization.The HaCaT cells, immortalized human keratinocytes, were adopted to evaluate the biological functions of the coatings. The results revealed that LAMA3 immobilization on CS/(HA/COL)5 coatings on titanium surfaces could promote cell adhension and cell spreading in the early stage, and the expression of laminin a3, and the formation of hemidesmosomes on the interface.Lastly, the animal experiment was carried out to evaluate the effects of LAMA3 immobilization on implant surfaces on the biological sealing between implant and gingival epithelium. With the immunohistochemical analysis, transmission electron microscopy observation and penetration test of horse radish peroxidase (HRP), the results demonstrated that the LAMA3 gene coating on implant surface has the potential to accelerate the healing of peri-implant epithelium, and could promote the expression of laminin α3 and the formation of hemidesmosomes, and finally improve the biological sealing between implant and epithelium.In this study, a multilayer coating CS/(HA/COL)5 functioned with Ad-LAMA3 for in situ transduction was successfully established on titanium surface. The coating with good biocompatibility could promote the expression of laminin α3 in epithelial cells and the integration of hemidesmosomes on the interface, and efficiently improve the biological sealing between implant and gingival epithelium. The study provide a new idea and experimental evidences for the research on the interface of impant and soft tissues.