Fabrication,bacteriostasis And Osteointegration Performances Researches Of The Porous Tantalum Scaffolds Loaded With Vancomycin

Infected bone defects(IBDs)remain a tremendous challenge facing the orthopaedic surgeons.The routine clinical treatments for IBDs are thorough debridement,high-dose antibiotics administration,and implantation of scaffolds,including Papineau technique,vascularized fibular graft,Masquelet membrane induction technique,and Ilizarov bone transfer technique.Overusing of high-dose antibiotics would lead to systemic side effect and the emergence of drug-resistance bacteria,and the second-stage surgery would cause additional trauma and economic burden to the patients,making the efficacy unsatisfactory.Therefore,it takes great significance and necessity to develop a scaffold with long-acting and sustained antibiotics releasing for repairing IBDs.In this study,basing on the customizable additively-manufactured porous tantalum(Ta),a novel antibiotic-loaded composite scaffold was fabricated.Vancomycin(Van)was incorporated into the poly(lactic-co-glycolic acid)(PLGA)microspheres through the double emulsion method,and then the microspheres were loaded into the porous Ta via gelatin methacryloyl(GelMA)hydrogel,constructing the novel Ta/Gel/PLGA/Van composite scaffolds.The porous Ta reconstructed the defect structurally,simultaneously,the antibacterial drugs were continuously and slowly released from the composite scaffolds to eliminate pathogenic bacteria in the infectious fields,rebuilding the well-developed osteogenic microenvironment and promoting osseointegration.The novel scaffolds were characterized by physiochemical tests such as scanning electron microscopy(SEM).The bacteriostasis and osteointegration properties were tested by in-vitro/vivo experiments.The results suggested that the novel scaffolds introduced in this study possessed excellent antibacterial activity,and good osteointegration properties.The novel Ta/Gel/PLGA/Van composite scaffolds had great potential for clinical application,which would provide new idea for the treatment of IBDs.This study mainly included the following three parts:Part Ⅰ.Fabrication and Characterization of the Ta/Gel/PLGA/Van Composite ScaffoldsObjective:To select the most suitable antibiotic through comparing the influence of Gentamicin(Gen),Van and Norfloxacin(Nor)on rat-derived bone marrow mesenchymal stem cells(BMSCs),preparing the novel Ta/Gel/PLGA/Van composite scaffolds and characterizing the physicochemical properties.Methods:The cellular viability and proliferation were detected using CCK8 kit and EDU/DAPI fluorescence labelling.Osteogenic activities were evaluated via ALP staining,Sirius red staining,alizarin red staining and qRT-PCR.The drug-loaded PLGA microspheres were prepared through the double emulsion method,and the distribution,morphology and particle sizes were detected using SEM.The drug-loading rate and encapsulation efficiency were tested using the ultraviolet spectrophotometer.In-vitro sustained-releasing test was performed to detect the releasing feature of Vancomycin.Loading the drug-loaded PLGA microspheres into porous Ta using GelMA hydrogel to construct the novel Ta/Gel/PLGA/Van composite scaffolds.Results:The results of CCK8 and EDU/DAPI staining indicated that the cellular viability of the Van-treated group was not significantly different from that of the control group,both of which were much better than those of the Gen-and Nor-treated groups.ALP staining,alizarin red staining,Sirius red staining and qRT-PCR suggested that Van showed no significant effect on the osteogenic differentiation of BMSCs.The Van-loaded PLGA microspheres were stable and well-distributed,with the particle size being about 34.84±1.04μm.The encapsulation efficiency and drug-loading rate were 46.66±3.48%and 16.97±0.84%respectively.The effective releasing duration of Van was over two weeks.PLGA microspheres distributed in the Ta/Gel/PLGA/Van composite scaffolds evenly.Conclusions:Van possessed relatively better biocompatibility and was the most suitable for the fabrication of the antibacterial composite scaffolds.The Van-loaded PLGA microspheres and Ta/Gel/PLGA/Van composite scaffolds were successfully manufactured,which were stable.Part Ⅱ.Study on the In-vitro Properties of the Ta/Gel/PLGA/Van Composite ScaffoldsObjective:This part aimed to evaluate the in-vitro biocompatibility,osteogenic performance and antibacterial properties of the newlydeveloped Ta/Gel/PLGA/Van composite scaffolds.Methods:The biocompatibility for BMSCs were evaluated using CCK8,EDU/DAPI fluorescence staining,living/dead cellular staining,cytoskeleton staining and SEM observation.Osteogenic performance was evaluated by ALP staining,alizarin red staining,Sirius red staining and qRT-PCR.The bacteriostatic activity of the newly-developed composite scaffolds for MRSA was evaluated via the bacterial counting,bacteriostasis circle test,living/dead bacterial staining and SEM observation.Results:After co-cultured with the composite scaffolds,the proliferating activity of BMSCs were similar with those in the control group.Osteogenic activity of BMSCs co-cultured with the newly-developed composite scaffolds was excellent.The new scaffolds and corresponding releasing liquid could give rise to obvious bacteriostasis circles,and destroy the morphology of MRSA with the bacteriostatic efficiency up to 95%and the effective duration over two weeks.Conclusions:The Ta/Gel/PLGA/Van composite scaffolds introduced in this study were well biocompatible and beneficial for osteogenic differentiation,and possessed potent bacteriostatic activity in vitro.Part Ⅲ.Study on the In-vivo Properties of the Ta/Gel/PLGA/Van Composite ScaffoldsObjective:This part further explored the in-vivo biosafety,osteointegration and bacteriostasis properties of the newly-developed Ta/Gel/PLGA/Van composite scaffolds.Methods:After implantation into the bone defects for 2,4,and 8 weeks,the in-vivo biocompatibility was evaluated using hematological indexes and HE staining of the critical organs.X-ray,micro-CT scanning,osteogenic fluorescence labelling,V-G staining,toluidine blue staining,HE staining,SEM and EDS detection were applied to detect the ingrowth of new bone tissue inside the composite scaffolds and integration with the host bone tissue.Masson trichrome staining and immunohistochemistry were performed to evaluate the new bone formation and expression of osteogenesis-related genes in the tissues around the scaffolds.Giemsa staining and bacterial counting tests were performed to detect the inhibitory effect of the newly-developed scaffolds on MRS A in vivo.Results:At different time points,there was no significant difference regarding the hematological indexes as well as critical organs staining among the control group,Ta group,Ta/Gel group,Ta/Gel/PLGA group and Ta/Gel/PLGA/Van group.The results of fluorescence labelling and VG staining,et al.showed that considerable new bone grew into the scaffolds,which increased significantly with time prolonging.Masson staining and immunohistochemical results indicated that well osteogenic status was observed in the tissues surrounding the scaffolds.Giemsa staining and in-vivo bacterial counting indicated that the bacteria of the Ta/Gel/PLGA/Van composite scaffolds group were significantly less than the control group.Conclusions:The newly-developed Ta/Gel/PLGA/Van composite scaffolds possessed well in-vivo biocompatibility,osteointegration properties and potent bacteriostatic activity.