| Purpose: Severe local infection and bone defects caused by prosthesis joint infection(PJI)are serious clinical challenges.Traditional treatment requires multiple debridement operations and many systemic antibiotics,which quickly cause severe toxic side effects and make it challenging to eradicate infection.Increasing bacterial drug resistance has also become a problem that clinicians must face.Considering how to treat bone defects effectively,kill drug-resistant bacteria,and maintain effective local drug concentration,tissue engineering to prepare multifunctional biocomposite scaffolds has become a new research direction.However,the feasibility of using a 3D-printed porous tantalum biocomposite scaffold(p Ta)to treat periprosthetic infections has yet to be systematically explored.Further research is needed on simplifying material preparation,improving drug loading efficiency and therapeutic effect,and activating the inherent immune and antimicrobial mechanism.Methods: In this study,PDLLA-PEG-PDLLA(PLEL)injectable temperature-sensitive hydrogel was used as a carrier to load vancomycin onto the surface and interior of p Ta.The biocomposite scaffolds were characterized by SEM,particle size analysis,Zeta potential detection,XRD,FTIR,and other materials to detect the synthesis of scaffolds.The drug release performance of biocomposite scaffolds was characterized by testing the drug release curve.In addition,the mechanical properties of the biocomposite scaffolds were tested.The bacteriostatic ring,bacteriostatic counting,SEM,crystal violet,and bacterial live/dead staining assay characterized the scaffolds’ anti-MRSA and anti-bacterial biofilm properties.The in vitro biocompatibility of the materials was characterized by CCK-8 assay,SEM,Ed U cell proliferation assay,and live/death staining assay.The in vitro osteogenic properties of the materials were characterized by phalloidine/DAPI,ALP,alizarin red,and Sirius red staining.A femoral bone defect model of SD-rat was established,and a biocomposite scaffold was implanted.X-Ray,μ-CT,toluidine,Van Gieson,calcein/alizarin red staining,SEM,and EDS were used to observe the in vivo osteogenic properties of hard tissue sections.In vivo biocompatibility was evaluated for critical organs,blood routine,and liver and kidney function tests.Subsequently,a mouse subcutaneous infection model was established,and the antibacterial properties of the biocomposite scaffolded in vivo were characterized by bacterial counting,crystal violet,Giemsa,and H&E staining.The gene sequencing of the surrounding tissue of the mouse subcutaneous infection model interfered with biocomposite scaffolds was detected and finally verified by an immunofluorescence experiment in tissue sections.Results: The macroscopic structure of 3D printed p Ta showed a uniform porous morphology with interconnected pores.When the drugloaded hydrogel was loaded on the surface and inside of the scaffold,it was observed that the hydrogel uniformly covered the wire surface of the scaffold.SEM showed that the surface of p Ta was rough,and there was a thick hydrogel coating on the scaffold surface after drug loading,which gradually degraded with the sustained release of the drug.Particle size analysis showed that the average particle size of PLEL hydrogel was32.45 nm,and the PDI was 0.196.The average particle size of VAN was502.2nm,and the PDI was 0.604.The Zeta potential was 16.3m V for VAN,0.936 m V for PLEL,and 3.37 m V for VAN-PLEL.XRD and FTIR showed that vancomycin formed a stable cross-link with p Ta through PLEL,which made the drug loading and encapsulation efficiency of VAN-PLEL@p Ta biocomposite scaffolds reach 6.45% and 90.43%,respectively.UVspectrum and SEM showed that vancomycin had a stable drug release property,and the drug sustained-release time was up to 14 days and remained above the MIC.It was also observed that the drug release rate was affected by the concentration of the hydrogel,the drug concentration,and the PH environment.The mechanical test results showed that the elastic modulus of the biocomposite scaffold reached 83.64±3.98 Mpa,which met the needs of human cancellous bone.In vitro antibacterial experiments showed that the average diameter of the inhibition zone of VAN-PLEL@p Ta sustained-release solution at different time points(1,5,7,10 days)was 17.7mm,14 mm,12.3mm,and 12 mm,respectively.However,the average diameter of the inhibition ring of the VANPLEL@p Ta drug-loaded scaffold was 20 mm,and the bacterial survival rate was reduced to 4.4%.Crystal violet staining,SEM,and Syto9/PI staining showed that VAN-PLEL@p Ta had a strong killing effect on MRSA biofilm.In vitro cell experiments,CCK-8,SEM,Ed U,live/dead cell staining,phalloidin /DAPI,ALP,alizarin red,and sirius red staining showed that VAN@p Ta and VAN-PLEL@p Ta biocomposite scaffolds had similar biocompatibility and osteogenic mineralization properties as p Ta.In vivo imaging experiments showed that VAN-PLEL@p Ta had a BV/TV ratio of 7.5±0.4% and 20.8±1.1% at 4 and 8 weeks,respectively,similar to that of p Ta and VAN@p Ta.Toluidine,Van Gieson,and alizarin red staining were used to observe the bone ingrowth effect in hard tissue sections.SEM and EDS analysis showed that the Ca/P ratios of p Ta,VAN@p Ta,and VAN-PLEL@p Ta were 1.99±0.05,1.93±0.05,1.94±0.03 at four weeks and1.75±0.07,1.75±0.07,1.76±0.05 at eight weeks.It was further confirmed that p Ta,VAN@p Ta,and VAN-PLEL@p Ta had the same trend of bone ingrowth inside the scaffold.The results of the staining of vital organs,blood routine,and liver and kidney function tests in vivo indicated that VAN-PLEL@p Ta had good biocompatibility.In vivo antibacterial experiments showed that the biocomposite scaffolds VAN@p Ta and VANPLEL@p Ta could effectively inhibit MRSA with a bacterial count of6.0±2.8 and 5.7±3.9,and effectively kill the biofilm,which had the same trend in Giemsa and H&E staining.m RNA sequencing results showed that the biocomposite scaffold might activate the anti-inflammatory effect of the body,and immunofluorescence analysis showed that it plays an important role by activating macrophage polarization.Conclusion: 3D-printed porous tantalum scaffold p Ta was successfully prepared with VAN-PLEL@p Ta biocomposite scaffold containing vancomycin and PLEL injectable thermosensitive hydrogel,which has sustained drug release ability and meets the mechanical properties of cancellous bone.With good biocompatibility and integration performance of bone growth in vivo and in vitro,it can kill MRSA and bacterial biofilms by continuously releasing antibiotics and activating innate immunity.Therefore,VAN-PLEL@p Ta biocomposite scaffolds may become an exciting tissue engineering material,providing a new idea for clinical application. |
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