| Objective: The purpose of this study was to prepare the composite porous scaffold loaded with β-cyclodextrin/chlorhexidine inclusion complex(PLA/nHA/β-CD-CHX),and to explore the slow-release performance,osteogenic induction and antibacterial performance of the scaffold in depth,in order to provide research basis and theoretical basis for the construction of a practical bone tissue engineering scaffold.Methods:1.Preparation and characterization of β-cyclodextrin/chlorohexidine inclusion conjugate(β-CD-CHX): β-CD-CHX,a slow release carrier for chlorohexidine(CHX),was prepared by the saturated aqueous solution method;the structures of CHX,β-cyclodextrin(β-CD)and β-CD-CHX were characterized by scanning electron microscopy(SEM),energy dispersive spectroscopy(EDS),X-ray diffraction(XRD)and Fourier transform infrared spectroscopy(FTIR).2.Preparation of PLA/nHA/β-CD-CHX composite porous scaffold and its performance study: According to the digital design program,Polylactic acid/nano-hydroxyapatite(PLA/nHA)loaded β-CD-CHX composite porous scaffold(PLA/nHA/β-CD-CHX)was printed layer by layer by 3D printing technology.The PLA/nHA/β-CD-CHX was characterized by SEM,EDS,XRD,FTIR,X-ray photoelectron spectroscopy(XPS);the control release effect of PLA/nHA/β-CD-CHX on CHX was investigated by in vitro release assay;The biocompatibility of PLA/nHA/β-CD-CHX to mouse embryonic osteoblast precursor cells(MC3T3-E1)was evaluated by CCK-8 method,4’,6-diamidino-2-phenylindole(DAPI)and rhodamine staining,calcein-propidium iodide(AM-PI)living dead cell staining;the growth and adhesion ability of MC3T3-E1 on PLA/nHA/β-CD-CHX was detected by DAPI staining and SEM;alkaline phosphatase(ALP)staining and its activity quantification,alizarin red staining and mineralization deposition quantification were used to test the scaffold’s ability to induce osteogenesis;the antibacterial properties of PLA/nHA/β-CD-CHX were verified using bacterial dynamic OD growth curves and antibacterial biofilm formation method.Results:1.SEM,EDS,FTIR and XRD confirmed the successful synthesis of β-CD-CHX.β-CD-CHX is a fine needle-shaped crystalline particle,which is distinctly different from the morphology of β-CD and CHX.2.PLA group,PLA/nHA group and PLA/nHA/β-CD-CHX group all showed porous structure with uniform pore size and about 300 μm pore spacing.Compared with PLA group,the surface of PLA/nHA group and PLA/nHA/β-CD-CHX group were rougher.EDS,FTIR,XRD,XPS confirmed the successful printing of PLA/nHA/β-CD-CHX scaffold and that β-CD-CHX was uniformly distributed on the scaffold.In vitro release assay results showed that the PLA/nHA/β-CD-CHX group exhibited a more optimal slow release compared to β-CD-CHX.The cumulative release of CHX in PLA/nHA/β-CD-CHX lasted for more than 2 months,reaching 70% in the second month,while pure β-CD-CHX showed 90% release in the first month only.The results of the cell proliferation assay showed that: DAPI and rhodamine staining observed a much higher number of MC3T3-E1 cells in the PLA/nHA and PLA/nHA/β-CD-CHX groups than in the PLA and control groups,a result also demonstrated by the CCK-8 assay,with higher OD values in both the PLA/nHA and PLA/nHA/β-CD-CHX groups than in the PLA and control groups(P<0.05),but there was no statistical difference between the OD values of the PLA/nHA and PLA/nHA/β-CD-CHX groups(P>0.05).The results of cell adhesion assay showed that the cells on PLA/nHA and PLA/nHA/β-CD-CHX groups spread over a larger area compared to PLA,and were widely distributed on the inner and outer surfaces of the porous scaffolds.After AM-PI live/dead staining,a large number of green fluorescently labelled live cells were found on PLA/nHA/β-CD-CHX group,and almost no red fluorescently labelled dead cells.The ALP activity of PLA/nHA/β-CD-CHX group was 1.59-fold higher(P<0.01)than that of PLA and control groups after days 7 and 14 of induction of MC3T3-E1 cells by each scaffold group,while qualitative staining and quantitative assay analysis of PLA/nHA and PLA/nHA/β-CD-CHX groups based on ALP activity showed no statistical difference(P>0.05);after 21 days of induction,the results of alizarin red staining showed more calcium deposits in the PLA/nHA and PLA/nHA/β-CD-CHX groups compared to the PLA and control group,and the quantitative analysis was consistent with the qualitative analysis regarding the staining of calcium nodules.In the antibacterial assay,the PLA/nHA/β-CD-CHX group had significantly lower OD values than the PLA and PLA/nHA groups at 1,2,3,4,5,6,9 and12 h,and after 12 h,there were bacteria growth in the PLA/nHA/β-CD-CHX group,no bacterial biofilm was formed,but dense bacterial biofilm was formed in the control group,PLA group and PLA/nHA group.Conclusions:1.The 3D printed PLA/nHA/β-CD-CHX composite porous scaffold designed digitally can meet the morphological and structural requirements of tissue engineered bone construction.2.PLA/nHA/β-CD-CHX composite porous scaffold has excellent slow-release,osteogenesis-inducing and antibacterial properties.It is expected to provide a new strategy for repairing large jaw defects at risk of infection. |
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