| Titanium and its alloy were widely used in clinic hard tissue replacement(orthopedics and dentistry)on account of their high mechanical strength,good biocompatibility and corrosion resistance.However,titanium based implants still exisit lossening and dislocation phenomena,resulting in implantation failure.The leading cause of implant failure is the deficient or poor structural and functional connection(so-called ―osseointegration‖)between titanium implants surface and bone tissue in the early post-implantation period.Therefore,to address the clinic challenge of implant failure issues caused by a lack of osseointegration,it is urgent to develop new ideas and technologies,so as to enhance the bone formation on titanium based implants surface,and to accelerate the early bone healing precess.Titanium based implants confront complex microenvironmental factors(extracellular matrix,reactive oxygen spieces,blood vessel formation etc.)during the bone fracture healing process after implantation.It is desirable for surface modification of an implant to endow it with biofunctionality,to mediate bone healing microenvironment,and to improve the bone formation of the implant,while not change its original physical properties.The above tenet is essentially important for the development of novel biofunctionalized bone replacements either from science view or clinical transformation.The study aims at mimicking multilayered structure of natural bone tissue.A biomimetic bioactive multilayered interface having osteogenesis,antioxidant and angiogenesis properties was constructed on the surface of titanium substrates via layer-by-layer(LBL)assembly technique,which improved the bone healing process at the interface of titanium based imaplants and bone tissue,and enhanced the bone formation of the implant.Meanwhile,the underlying molecular mechanism was also investigated.The main contents and conclusions of this study are presented as follows:(1)Investigation on the construction of catechol-chitosan(Chi-C)based multilayer structure and its improvement for bone healingThe chitosan-chitosan(Chi-C)and hydroxyapatite nanofibers(HA)were successfully synthesized.After that,we selected the Chi-C and gelatin(Gel)as polyelectrolytes,and HA nanofibers as embedding component,to construct biomimetic multilayer structure on Ti substrates via a layer-by-layer(LBL)assembly technique.The water contact angle measurement,field emission scanning electron microscopy(SEM)and atomic force microscopy(AFM)were applied to characterize the materials surface properties.Next,primary osteoblasts were seeded onto the titanium substrates and evaluated the the infulences of materials on cellular functions.The osteoblasts displayed well adhesion and spreading on the modified titanium substrate as proofed by the cytoskeleton staining and scanning electron microscopy observation.Besides,the multilayer structure of Chi-C/Gel/HA nanofibers on Ti substrates promoted osteoblasts proliferation and differentiation,which was evidenced by cellular viability,high expression levels of alkaline phosphatase activity,collagen secretion,ECM mineralization and osteogenesis-related genes expression in vitro.The bone healing effects were further evaluated by titanium implantation in a rabbit femoral defect model.The in vivo experiments of X-ray and μCT imaging,push out test and histochemistry(H&E,Masson)staining further confirmed that Chi-C multilayered implant had great potential for improved early bone regeneration,which was also coupled by improved angiogenesis.(2)Investigation on anti-oxidative effects and mechanisms of Chi-C based multilayer structureIn this section,the in vitro osteoblasts oxidative stress model was constructed by using H2O2 exposure method.The anti-oxidative protective potential of structure and underlying molecular mechanism of were also investigated.The initial cellular adhesion results showed that Chi-C containing multilayer on Ti surface effectively attenuated the oxidative stress induced osteoblasts adheison injury.Intracellular ROS measurement results indicated that Chi-C containing multilayer coudle reduce the ROS level;qRT-PCR and Western Blotting results indicated that H2O2 exposure resulted in the oxidative stress injury,such as decreased expression of cell adhesive and anti-apoptotic related genes and improved levels of pro-apoptotic critical executioners.However,the Chi-C multilayer films could effectively protect osteoblasts from ROS damage via ROS scavenging.(3)Investigation on the effect of surface modified titanium on the co-regulation of neo-angiogenesis and osteogenesis in vitroAdipose-derived mesenchymal stem cells(Ad-MSCs)and human umbilical vein endothelial cells(HUVECs)were seeded onto surface modified titanium substrates and investigate the regulation effects of neo-angiogenesis and osteogenesis respectively.The results showed that multilayer structure could effectively promote the adhesion,spreading,migration,proliferation and osteoblastic differentiation of Ad-MSCs,which was closely related to adhesion and migration-related proteins,as well as SDF-1/CXCR4 axis signaling pathways.Meanwhile,this structure up-regulated the vascular-related factor VEGF expression,SDF-1 gene expression and paracrine level.HUVECs experiments showed that the multilayer-modified titanium substrates significantly improved cellular adhesion,spreading and migration of HUVECs and up-regulated expression levels of the related proteins.In addition,we used tranwell system to co-culture Ad-MSCs and HUVECs.The results indicated that the paracrine effects of Ad-MSCs could enhance the migration and tube formation of HUVECs.The up-regulated secretion of SDF-1 in HUVECs on modified titanium substrate could promote the migration and osteogenic differentiation of Ad-MSCs in co-culture system.(4)Investigation on the effect of surface modified titanium on the co-regulation of neo-angiogenesis and osteogenesis in vivoIn this section,the subcutaneous implantation model and the femoral defect implantation model were firstly constructed.Then,histopathological evaluation was performed.The results showed that the multilayer-modified titanium substrates enhanced the cell proliferation,mesenchymal stem cell recruitment and pro-vascularization at the bone-titanium implant interface at the early stage of injury repair(7d).After implantation for 28 days,much more bone-like tissue and higher level of late bone development markers(OCN and OPN)expression were observed at the surface of multilayer modified titanium implant.These results confirm that either subcutaneous transplantation(ectopic bone formation)or femoral transplantation(bone formation in situ)surface modified titanium could significant improve the bone formation of titanium implant.In summary,in view of the the structural features of bone tissue and multiple complex microenvironmental factors during the bone fracture healing,this study constructed a biomimetic hierarchical structure on titanium substrates with improved bone healing via layer-by-layer assembly technique.The multilayer structure improved cell adhesion,proliferation and cellular functions through ROS scavenging in fracture healing local microenvironment.Meanwhile,we found that multilayer structure not only directly regulated the osteogenesis or angiogenesis via modulating the biological functions of Ad-MSCs or HUVECs seeding on titanium substrates,but also indirectly regulated the biological functions of Ad-MSCs(or HUVECs)in co-culture system via paracrine effects of HUVECs(or Ad-MSC)cultured on the substrates,which having great scientific values for modulation of bone healing local microenvironment and improvement of bone formation of titanium implants.This study not only provides a new strategy for the fabrication of high-performance titanium implants,but also primarily reveals the effects of mediating microenvironment on bone healing,as well as its underlying molecular mechanism,which is expected to provide new theoretical support for the improvement of bone injury repair strategy. |
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