Construction Of Protein Based Antioxidative Coatings And Their Osteogenesis Studies

Bone regeneration and repair has become a worldwide issue at present.Moreover,population aging,osteoporosis,glycuresis and other factors further aggravate the occurrence of bone injury.It is still a grand challenge to enhancing osseointegration between implant and bone tissue in orthopedic clinic.There is plenty of research showing that oxidative stress is closely related to many traumatic or pathological fractures and seriously influence the surgery effectiveness.Therefore,design and fabrication of bone repair coatings with antioxidative function gradually becomes an important research aspect in the field of bone tissue regeneration.In order to enhance osseointegration of implant under oxidative stress,a variety of drug-loaded protein coatings with antioxidative and osteogenic functions were constructed via drug loading process of protein and layer by layer self-assembly technology.Based on drug release and the optimization of coatings’antioxidative units,the transformation from“oxidative stress”to“oxidative promotion”was realized,which has important guiding significance for the clinical treatment of bone defects under oxidative stress.The main research contents are summarized as follows:（1）In this study,bovine serum albumin（BSA）was used as a carrier to combine baicalein（BAI）,a flavonoid drug with bone promoting effect,to construct drug-loaded protein（BAI-BSA）via desolvent method.High performance liquid chromatography and other tests’results showed that BAI could combine with BSA effectively.In cell experiments,the drug loaded protein showed good biocompatibility,which had no cytotoxicity to osteoblasts and erythrocytes.Moreover,BAI-BSA contributed to enhancing the activity of alkaline phosphatase（ALP）and promoting the formation of mineralized calcium nodules.In terms of antioxidative function,BAI-BSA exhibited the effect of free radical scavenging,which could scavenge both ABTS^+·and DPPH^·.So,BAI-BSA proteins possess antioxidative function and promoting effects for osteogenic mineralization,which provides an important basis for the further construction of antioxidaive coating for bone repair.（2）Inspired by the biomimetic chemistry of mussel,tannic acid（TA）was used as an antioxidative unit to construct（TA/BAI-BSA）₄ coatings via layer by layer self-assembly technology.Moreover,the biocompatibility of TA based coatings and their effects on cell adhesion,proliferation and mineralization of osteoblasts were studied.The results showed that TA based coatings with drug loading could effectively maintain normal cell proliferation,promote cell adhesion of osteoblasts and reduce the level of ROS in osteoblasts under oxidative stress.In addition,the drug loaded coating showed almost no hemolytic effect on red blood cells.In vitro mineralization experiment,（TA/BAI-BSA-10）₄coating could significantly promote the formation of mineralized calcium nodules of osteoblasts.However,the osteogenic function of this drug-loaded coating was greatly affected by the long-term treatment of oxidative stress,and its antioxidative effect needs to be further improved.（3）Inspired by the phenomenon of“protein corona”,ceria nanozymes（CeO₂NZs）and BAI-BSA were used to construct（CeO₂NZs/BAI-BSA）₄ coatings via layer by layer self-assembly.The results showed that CeO₂NZs and BAI-BSA with different drug/carrier ratios could effectively combine and assemble alternately.In addition,in cell experiment,CeO₂NZs based coatings with drug loading could promote cell adhesion and decrease the negative effect of long-term oxidation treatment on osteogenic mineralization of osteoblasts.In particular,（CeO₂NZs/BAI-BSA-10）₄ coating possesses excellent blood compatibility,which can significantly inhibit platelet adhesion and blood fibrinogen combination.So,this drug-loaded coating showed high selectivity for the adhesion of osteoblasts and platelets,which may accelerate the repair of bone injury in blood environment in vivo.（4）In order to improve the antioxidative function of CeO₂NZs,poly-tannic acid（PTA）modified CeO₂NZs（PTA/CeO₂NZs）were prepared by regulating the oxidation polymerization of TA.The results showed that PTA/CeO₂NZs prepared at p H=5 had“core-shell structure”,and the thickness of PTA coating was about 1 nm.In particular,the nano PTA“shell”significantly enhanced the stability of CeO₂NZs in PBS solution.Moreover,this coating can effectively improve the biocompatibility of CeO₂NZs with promoting cell proliferation of osteoblasts and reduce the hemolysis rate of red blood cells.In addition,PTA surface modification significantly improved the free radical scavenging effect and superoxide dismutase（SOD）like activity of CeO₂NZs,and exhibited better antioxidative effect at cellular level.（5）For promoting the antioxidative function and osteogenic activity of BAI-BSA based coatings,（PTA-CeO₂NZs/BAI-BSA）₄coatings were further constructed via layer by layer self-assembly method in this study.Compared with CeO₂NZs based coatings,PTA/CeO₂NZs based coatings exhibited significantly stronger free radical scavenging activity,which can greatly reduce the negative effect of oxidative stress and maintain cell viability.In addition,（PTA-CeO₂NZs/BAI-BSA-10）₄ coating owns special“nanopore structure”,which could activate the formation of filopodia of osteoblasts and enhance the level of anti-oxidative stress.In particular,the drug-loaded coating could accelerate the osteogenic mineralization of osteoblasts under oxidative stress,showing an“oxidation promoting”effect.Moreover,this coating could enhance the osseointegration between titanium alloy scaffold and bone tissue as well.On the other hand,the drug-loaded coating also exhibited good blood compatibility,which could effectively inhibit the adhesion of platelets and blood fibrinogen.So,PTA-CeO₂NZs/BAI-BSA composite coatings possess great osteogenic and antioxidative functions,which has important application prospects in the field of bone repair under oxidative stress.