Evaluation On The Anti-bacterial Activities And Osteogensis Propertiies Of 3D-printed PLGA/HA/HACC Porous Scaffold

OBJECIVESegmental bone defects at high risk of infection are extremely difficult to be treated in clinical orthopedics.Bone substitutes with osteoconductivity only can not achieve satisfactory therapeutic effects.In this study,HACC-loaded composite composed of polylactide-co-glycolide(PLGA)and hydroxyapatite(HA)were fabricated using three-dimensional(3D)printing and chemical grafting technologies.A series of in vitro and in vivo experiments were conducted to evaluate its physical and biological properties.Our study aimed at developing a new dual functional bone scaffold with good antibacterial and osteoconductive properties.METHODS1.Composite PLGA/HA/HACC scaffolds were prepared using 3D-printing and chemical grafting technologies.The biomechanical properties,surface morphology,chemical composition,drug loading efficacy,in vitro degradation and drug release performance were studied to determine the physicochemical characteristics of the scaffolds.2.The surface charge distribution of several pathogenic bacterial strains in orthopedics was investigated,and the minimum inhibitory concentrations(MICs)of HACC against those strains were also determined.The in vitro antibacterial properties of the scaffolds were evaluated by investigating the effects of scaffolds on the bacterial adherence and biofilm formation.The effects of the scaffolds on ATP leakage and virulence genes expression closely associated with the bacterial adherence,biofilm formation and antibiotic-resistance after co-incubation were also investigated,in order to find the potential antibacterial mechanisms of the grafted HACC in scaffolds.3.Human bone marrow mesenchymal stem cells(hBMSCs)were co-cultured with the scaffolds,and the cell adherence,proliferation,spreading,cell damage and apoptosis and osteogenic differentiation on the material surfaces were systematically investigated to assess the in vitro bone bioactivities of the scaffolds..4.A dorsum subcutaneous implantation model was established to evalute the anti-infective property and biocompatibility of the scaffolds.Furthermore,an infected segmental femur defect model was established to evaluate the anti-infective and bone repair efficacy of the scaffolds.RESULTS1.The mechanical properties of the scaffolds,which in the range of human cancellous to cortical bone,were not significantly influenced by the grafted HACC.The loading amount of HACC in P/H and P/HA/H scaffolds were 5%and 4%,respectively.The incorporated HA in scaffolds could both alleviate the acidic micro-environment and degradation rate of PLGA during in vitro degradation.Meanwhile,the surface grafted HACC could release gradually with the degradation of PLGA.2.The tested bacterial strains were all sensitive to HACC,and their surface were negative charged at different levels.The bacterial adherence and biofilm formation on P/H and P/HA/H scaffolds were significantly inhibited,and virulence genes,such as atlE,clfA,icaAD and MecA,were also down-regulated.Meanwhile,a significant increased ATP release due to the cell membranes disruption of tested bacteria was found in the microenvironment of HACC-grafted scaffolds.3.The hBMSC attachment,proliferation,spreading and osteogenic differentiation on the surface of P/HA or P/HA/H scaffolds were significantly enhanced compared with that of P or P/H scaffolds.All four scaffolds showed relatively good cytocompatibility.4.In vivo investigation demonstrated that HACC-grafted scaffolds(P/H and P/HA/H)could evidently suppress subcutaneous implant-related infection.In addition,HA-incorporated scaffolds(P/HA or P/HA/H)could obviously promote tissue integration and neovascularization in the pore structure of scaffolds compared with P or P/H scaffolds.Finally,the infected bone defects were effectively repaired in P/HA/H group only.CONCLUSIONSThe mechanical properties of the HACC-grafted scaffolds were in the range of human cancellous to cortical bone.Both in vitro and in vivo studies demonstrated that porous PLGA/HA/HACC composite scaffold could significantly inhibit the bacterial adhesion and biofilm formation,thus effectively prevent the development of bone infection.Meanwhile,it significantly promoted hBMSC adherence,proliferation,spreading and osteogenic differentiation,as well as and the repairing of infected segmental bone defects in vivo.Thus,this innovative 3D-printed porous scaffolds will be promising bone substitutes in the treatment of contaminated or infected bone defects.