Construction Of Multifuntonal Biomimetic Three-dimensional Scaffold For The Treatment Of Refractory Bone Defects

The treatment of refractory bone defects caused by trauma,infection and tumors has been a clinically significant problem.Autologous bone grafting is a gold standard for the treatment of bone defects.However,limited donor sources constrain its widespread applications.A variety of bone tissue engineering scaffolds based on polymer materials have achieved desired treatment effect on conventional bone defects.However,under the stress of some wounds and diseases,the microenvironment of the defected bone tissue is disturbed,and the therapeutic effect of conventional scaffold is often limited.The rapid development of biomaterials and regenerative medicine provides a new perspective for solving the above problems.As a typical two-dimensional material,graphene has attracted great attention in bone tissue repair due to its good osteogenic activity.In particular,graphene-based materials are easy to assemble and modify,making them a good functional platform.In this paper,graphene materials were used as the bulk unit.A series of functional biomimetic three-dimensional porous scaffolds were constructed according to the pathological characteristics of different types of refractory bone defects.The physicochemical properties,in vitro and in vivo osteogenic activities were characterized.It provides a new therapeutic approach for refractory bone defects treatment.The research content of this topic is summarized as the following three parts:(1)In order to treat large-area acute bone defects,a reduced graphene oxide/nano-hydroxyapatite(nHA@RGO)three-dimensional porous scaffold was constructed by hydrothermal reduction self-assembly of graphene oxide(GO)and nano-hydroxyapatite(nHA).By detecting the Tyndall effect of the solution before and after the reaction,it was found that the nano-hydroxyapatite was completely loaded into the formed scaffold during the reduction process.The morphology of the surface of the scaffold was observed by field emission scanning electron microscopy(FESEM).The results show that the blending amount of nano-hydroxyapatite has a great influence on the morphology of the scaffold.As the amount of nHA gradually increases from 20%to 80%,the porous structure of the scaffold gradually becomes blurred.Synchronous surface elemental analysis not only qualitatively reveals the uniform distribution of nHA on the scaffold but also quantitatively confirms the relationship between the structure of the scaffold and the nHA loading.The results of transmission electron microscopy(TEM)show that nHA may form a complex with GO before the reaction,and the amount of nHA is positively correlated with the density of nHA on GO,which provides some data support for studying the self-assembly mechanism.Further infrared spectroscopy(FTIR)and X-ray diffraction(XRD)studied the physicochemical properties of the prepared scaffolds from the composition and structure,and finally proved the successful preparation of nHA@RGO scaffold.Rat bone marrow stromal cells(rBMSC)were used as model cells.The in vitro adhesion,proliferation and osteogenesis experiments showed that 20%nHA@RGO porous scaffold had the best cell expansion and osteogenic activity.Subsequent in vivo implanting experiment indicate that the prepared scaffold has excellent compatibility not only at the cell level,but also in tissue compatibility.Finally,the in-situ regeneration ability of the 20%nHA@RGO scaffold was studied by the rabbit orthotropic skull defect model.Histological and radiographic image at different time points showed that 20%nHA@RGO significantly accelerated collagen deposition,shortened osteogenic maturity,and promoted wound healing compared to blank and pure RGO scaffold(2)Based on the research in the second chapter,using ascorbic acid as reducing agent and silver ion as precursor,a nanosilver particles incorporated nHA@RGO three-dimensional porous scaffold(AHRG)was constructed for the treatment of infectious bone defects caused by multi-drug resistant bacteria.The surface morphology of the material was observed by scanning electron microscopy(SEM).It was found that the AgNPs were uniformly distributed on the surface of the pores.The elemental surface scanning pattern analysis showed that elements such as calcium(Ca),phosphorus(P),silver(Ag)and carbon(C)were dispersed on the surface of the scaffold.The formation of AgNPs in the complex was proved in experimental results of lattice pattern and electronic energy state of XPS and SAED,respectively.The clinically collected methicillin-resistant Staphylococcus aureus was used as a model bacterium.The antibacterial properties of AHRG were studied by the plate inhibition zone method and the bacterial dynamic culture system.It was found that when the loading of AgNPs in the scaffold reach to 4%,the scaffold had better biocompatibility and antibacterial ability.This antibacterial effect was further confirmed in the bacterial biofilm inhibition test.In addition,the combination with RGO also resulted in a strong sustained antibacterial effect of the prepared AHRG scaffold.The composite scaffold still had some antibacterial activity even after continuous elution for 2 weeks in neutral phosphate buffered saline(PBS,pH=7.0).The extract of the material was co-cultured with rBMSCs,and the cell cytotoxicity and the extracellular lactate dehydrogenase were detected.The leaching solution of the material was co-cultured with rBMSCs,and the cell cytotoxicity of the prepared composite scaffold was confirmed by the detection of cellular respiratory chain activity and extracellular lactate dehydrogenase.The antibacterial and osteogenic activities of 4%AHRG in vivo were studied using a rabbit radius osteomyelitis model.The changes of high-sensitivity C-reactive protein(CRP)and white blood cells(WBC)at different time points were used as indicators to investigate the inhibition of infection in vivo after composite implantation.At the same time,high-resolution micro-CT was used to evaluate and compare the bone repair process.Finally,combined with the results of histological analysis,it was confirmed that the prepared 4%AHRG porous scaffold has good antibacterial activity in vivo and promotes healing of infectious bone defects.Moreover,no recurrence of infection was observed within 3 months from the end of treatment,indicating its eradication to multidrug-resistant bacteria colonized in the body.(3)Inspired by the successful application of graphene composites in the previous two chapters,a graphene composite scaffold with strong photothermal conversion efficiency was constructed to synergistically treat tumorous bone defects through the photothermal effect and osteogenic activity of the materials.Specifically,a graphene porous scaffold with uniform pore size was prepared by chemical vapor deposition(CVD)using nickel(Ni)foam as a template.The scaffold was then coated by hydroxyapatite by a modified semi-dry electrochemical deposition method.Different from the composite materials used in the previous two chapters,the scaffold prepared in this chapter is grown from a layer of graphene without any structural defects,so its conductivity and photothermal conversion efficiency are significantly improved compared with RGO.Due to the good electrical conductivity of the prepared stent,during the semi-dry electrochemical deposition process,the current passes through the porous graphene stent to which the electrolyte is adsorbed.This accelerates the mineralization rate and also improves the distribution of deposits in the scaffold,thereby overcoming the problem of uneven distribution caused by excessive growth of the deposited layer at the electrode/electrolyte interface.The morphology and phase structure of the material were analyzed by SEM and XRD.It was found that the time of electrodeposition and the applied voltage on the graphene scaffold had a great influence on the structure of the deposit.When the applied voltage was 10 V,deposition When the time is 4 minutes(min),a HA@G stent with a clear channel structure and uniform surface mineralization can be obtained.In vitro cell experiments showed that graphene after HA coating can effectively improve the osteogenic differentiation of rBMSC.In particular,the HA@G scaffold can rise to 50 ~oC within X min under 980nm near-infrared light at 0.6 W/cm~2,indicating its excellent photothermal conversion capability.In vivo thermal imaging confirmed the results of the in vitro experiments,and also found that the temperature of the non-tumor parts of the mouse body did not significantly fluctuate under the light source parameters,revealing the safety of the stent in vivo application.Finally,the in vivo anti-tumor and osteogenic effects of HA@G were verified by mouse tumor and rat skull defect models,respectively.In summary,this thesis is mainly aimed at the treatment challenge of various refractory bone defects.Taking graphene-based materials as the basic unit,biomimetic mineralized three-dimensional porous scaffolds with specific functions were prepared by various assembly and modification.The effects of implantation on the pathological environment of the defect site were investigated.The in vitro and in vivo osteogenic activity of the prepared scaffold was evaluated.This thesis provides several new approaches and research perspectives for the treatment of refractory bone defects.It also provides a preliminary theoretical basis for understanding the mechanism of graphene-based materials in various osteogenic microenvironments.