Study On The Fabrication Of Biofunctional Titanium-Based Implant And Its Effects On Bone Formation

Titanium and titanium alloys were widely used in clinical hard tissue repair and replacement,mainly due to their good mechanical property,biocompatibility and corrosion resistance.Studies have shown that the biological inertness of the titanium implant surface reduced its osseointegration with surrounding bone tissue,which resulted in the increasing probability of loosening,displacement and secondary revision.This defect is especially obvious in osteoporotic pathological conditions.Therefore,how to optimize and maintain a good interface performance of bone-titanium implants is the key to guarantee the biological functions of implants for a long term.Herein,a series of titanium-based implants were constructed by anodization,layer-by-layer self-assembly,and plasma spraying,which possessed the abilities of the reduction of osteoclasts absorption,the promotion of osteoblasts differentiation,and the recruitment of endogenous MSCs.Their biological performances were analyzed and evaluated in vitro and in vivo.The main research contents of this thesis are listed as follows:1.Raloxifene(Ral)loading Ti O₂ nanotubes with hyaluronic acid-alendronate(HA-Aln)multilayered film promote osseointegration under osteoporosisTo enhance the local osseointegration of titanium-based implants under osteoporotic conditions,Ti O₂ nanotube arrays(TNT)serving as nanoreserviors to load the anti-osteoporosis drug raloxifene(Ral)were constructed on the titanium surface by anodization(TNT/Ral).Then,TNT/Ral substrates were covered with a hybrid multilayered coating composed of alendronate grafted hyaluronic acid(HA-Aln),chitosan(Chi)and gelatin(Gel)via a spin-assisted layer-by-layer self-assembly(LBL)technique.The composite structure(TNT/Ral/LBL-Aln)was characterized by field emission scanning electron microscopy(SEM),atomic force microscope(AFM)and X-ray photoelectron spectroscopy(XPS),respectively.The release test indicated the composite multilayers on Ral-loaded Ti O₂ nanotube substrates(TNT/Ral)could prevent the burst release of Ral from Ti O₂ nanotube arrays and maintain a stable Ral concentration at the implant sites,which played an essential role in inhibiting the activity of osteoclasts in the osteoporotic environment.In vitro experiments confirmed that the TNT/Ral/LBL-Aln system had a higher alkaline phosphatase(ALP)activity,mineralization capability of osteoblasts,and while inhibiting the resorption of osteoclasts effectively.In vivo,Micro-CT and histological staining results showed that TNT/Ral/LBL-Aln implants could significantly enhance the formation of new bone around the implants and promote osteointegration in osteoporotic rabbits.2.Construction of chemokine substance P(SP)multilayer film on titanium substrates and its effects on MSCs recruitment and osteointegrationMSCs were recruited to the injury site by the elevated concentrations of chemokines after bone injury and promote in-situ bone tissue regeneration.Inspired by this,an LBL multilayered film containing Chi,Gel and three different concentrations of chemokine SP were constructed on Ti substrates(Ti/LBL-SP_L,Ti/LBL-SP_M and Ti/LBL-SP_H)through LBL technique in this study.The SP released from Ti/LBL-SP could effectively recruit surrounding MSCs towards the implants after the implantation surgery,which overcome the inherent defects of insufficient tissue MSCs during the early tissue repair process.SEM,AFM,and contact angle experiments suggested that the multilayer film was successfully constructed.The release results showed that SP could be continuously released from the multilayer film for nearly 2 weeks.In vitro experiments confirmed that pseudopod of MSCs in Ti/LBL-SP group increased.In addition,the migration ability of MSCs in Ti/LBL-SP group was significantly enhanced in wound healing and transwell experiments,while showing a certain degree of concentration dependence.It was demonstrated that the matrix metalloproteinases(MMP2 and MMP9)secretion levels in the Ti/LBL-SP group were notably increased.However,there was no significant difference among all groups in the MSCs proliferation and differentiation assays.After implantation,immunofluorescence staining confirmed that the Ti/LBL-SP group could significantly enhance MSCs recruitment.Micro-CT scan analysis,histological staining(H&E staining,Masson's trichrome staining)showed that Ti/LBL-SP implants significantly promoted the formation of new bone.3.Construction of chemokine substance P-embedded biomimetic multilayer onto an alkaline magnesium silicate-titanium bioactive coating for in situ mesenchymal stem cells recruitment and bone regenerationMSCs-mediated biological processes(such as recruitment,proliferation,and osteogenic differentiation at the site of injury)play an irreplaceable role in enhancing in-situ bone tissue repair and regeneration of during bone injury.In this study,we constructed a magnesium silicate(Mg Si O₃,named as Mg Si)alkaline ceramic coating on the titanium surface by a plasma spray approach,and covered it with an LBL multilayered film structure which intercalated by chemokine SP(Mg Si/LBL-SP).The upper-layer embedded with exogenous chemokine SP could effectively recruit sufficient MSCs in the initial phase of bone healing,while the bottom Mg Si coating released Mg and Si O₃^2-ions continuously,and creating an alkaline environment to promote the further proliferation and osteogenic differentiation of MSCs.SEM,white light interferometer,X-ray diffraction(XRD)and XPS experiments showed that Mg Si/LBL-SP samples were constructed successfully.The detection Mg ions release and p H value showed that the ion release and p H change of Mg Si coating could be delayed after multilayer film coverage.It was demonstrated that the SP released from Mg Si/LBL-SP substrates could effectively enhance MSCs recruitment in vitro and in vivo by transwell assay,MMP2 secretion detection and immunofluorescence staining.Meanwhile,the Mg Si/LBL-SP group also significantly improved the proliferation and differentiation of MSCs in vitro.In vivo experiments demonstrated that the Mg Si/LBL-SP implants displayed the highest stimulation of cell proliferation activity and apoptosis inhibition.After implantation for a month,H&E staining,Masson's trichrome staining and Micro-CT results suggested that the Mg Si/LBL-SP implants had the strongest ability to facilitate new bone formation.