Study Of Ti Implant Modified By BMP-2 Based On Trehalose/Lyophilization

Objects:Titanium and its alloys are used extensively in developing dental and orthopedic implants.Numerous efforts have been made to modify the surface topography and composition of Ti implants to enhance biocompatibility and osseointegration between titanium implants and surrounding bone tissue.In this study,Lenti-BMP-2-loaded TiO₂nanotube coatings were fabricated by lyophilization in the presence of trehalose to functionalize the surface.Furthermore,high doses of bone morphogenetic protein-2?BMP-2?have been widely applied to induce bone regeneration,however,these high dosages often cause undesirable side effects.To avoid or reduce the severity of these adverse effects induced by high doses of BMP-2,we also investigated whether delivery of BMP-2 lyophilized in the presence of trehalose?Lyo/Tre?could reduce the dose of BMP-2required for bone regeneration.Materials and methods:1.TiO₂ nanotube layers were prepared by anodizing thin Ti foils?0.25 mm thick,99.5%?or Ti rods?2 mm in diameter and 8 mm in length?.Lenti-EGFP or Lenti-BMP-2 was produced by cotransfecting the 3 plasmids system of lentivirus into 293T cell.Preparations of coatings were taken.F344 rat BMSCs were cultured.The successful loading of Lenti-BMP-2 onto the TiO₂ nanotube layers was also examined.2.We characterized TiO₂ nanotube layers in terms of the following:surface morphology;Lenti-BMP-2 and trehalose release;their ability to induce osteogenesis,proliferation,and anti-inflammation in vitro.3.The osseointegration of implant was evaluated in vivo by transplanting the implant into femur defects in F344 rats.The new bone formation and mineralization around implants were analyzed by histological analysis and the mechanical properties were evaluated by push-out test.4.BMP-2 was loaded on nanotubes and scanning electron microscopy?SEM?observations were taken.5.We measured BMP-2 release characteristics,bioactivity,and stability,and determined the effects of BMP-2 on the osteogenic differrentiation of BMSCs in vitro.6.We also examined the ability of this formulation to regenerate new bone around implants in rat femur defects by micro-computed tomography?micro-CT?,sequential fluorescent labeling,and histological analysis.Results:1.The TiO₂ nanotubes prepared are uniform,with a diameter and length of approximately100 nm and 400 nm,respectively.Lenti-EGFP and Lenti-BMP-2 were successfully obtained and F344 BMSCs were cultured and proliferated successfully in vitro.The loading of Lenti-BMP-2 on TiO₂ nanotubes was also successful and the distribution was uniform.2.The anodized TiO₂ nanotube surfaces exhibited an amorphous glassy matrix perpendicular to the Ti surface.Both Lenti-BMP-2 and trehalose showed sustained release over the course of 8 days.Results from real-time quantitative polymerase chain reaction studies demonstrated that lyophilized Lenti-BMP-2/TiO₂ nanotubes constructed with trehalose?Lyo-Tre-Lenti-BMP-2?significantly promoted osteogenic differentiation of bone marrow stromal cells but not their proliferation.In addition,Lyo-Tre-Lenti-BMP-2nanotubes effectively inhibited lipopolysaccharide-induced interleukin-1?and tumor necrosis factor-?production.3.In vivo,8 weeks after implantation,Lyo-Tre-Lenti-BMP-2 TiO₂ nanotubes also significantly promoted osseointegration than the other 3 groups.4.The anodized TiO₂ nanotubes was uniform with little amorphous glassy matrix remaining on the TiO₂-BMP-2 and much more amorphous glassy matrix on the TiO₂-Lyo-Tre-BMP-2.5.Compared with absorbed BMP-2-loaded TiO₂ nanotubes?TiO₂-BMP-2?,TiO₂-Lyo-Tre-BMP-2 exhibited sustained release,consistent bioactivity,and higher stability of BMP-2 and resulted in significantly greater osteogenic differentiation of rat BMSCs.6.Eight weeks postoperation,TiO₂-Lyo-Tre-BMP-2 nanotubes,with various doses of BMP-2,regenerated significantly greater amounts of new bone than TiO₂-BMP-2nanotubes.Delivery of 0.3 and 0.5?g of BMP-2 by TiO₂-Lyo-Tre-BMP-2 resulted in bone regeneration similar to that of 0.5 and 1.0?g of BMP-2 delivered by TiO₂-BMP-2,respectively.Conclusions:1.These data indicated that the application of a combination of trehalose and lyophilization treatment to modify Ti-based implant surfaces with lentiviral particles encoding BMP-2,which can potentially promote enhanced osteogenesis and reduced inflammatory responses,simultaneously,to improve implant osseointegration.2.BMP-2 delivery using TiO₂-Lyo-Tre nanotubes promoted greater osteogenic differentiation of BMSCs in vitro and more bone regeneration in vivo than BMP-2delivery using TiO₂ nanotubes alone.Importantly,delivery of BMP-2 using Lyo/Tre-based system can reduce the BMP-2 dose required for bone regeneration around implants.