Experimental Study Of 3D Printed Vancomycin Hydrochloride Loaded PGCL@Ti₃C₂ Multifunctional Scaffold In The Treatment Of Acute Infected Bone Defects

Background:Trauma and surgical reconstruction can cause acute infected bone defects.Despite the presence of many organisms associated with skeletal infections,Staphylococcus aureus remains the most prevalent and devastating causative pathogen.In particular,the emergence of methicillin-resistant Staphylococcus aureus（MRSA）remains a great challenge in clinical treatment.Systemic and topical antibioticsarecommonanti-infective strategies.However,the disruption of blood vessels and the damage of bone in the lesion site result in ineffectiveness of drug concentration.Although conventional topical antibiotics are widely used,secondary operation is needed to retrieve these materials after antibiotic release.Allografts and autografts suffer from problems with lacking bone sources and inducing immune rejection.Hence,medical treatment requires advanced therapeutic platforms with dual functions of antibacterial properties and osteogenesis.One-time implants do not require following surgery,streamline the process of treatment,and overcomes the disadvantages of traditional treatment.Several scaffolds with dual functions have been developed.The implantable biomaterials are typically a combination of functional components that have osteogenic activity and antibacterial performances.However,it seems to be also critical to regulate hyperinflammatory responses in the infected state.Therefore,biological components with immunomodulatory functions are gradually applied to bone tissue engineering.Biodegradable and biocompatible polymers are widely studied due to their excellent properties.Polyethylene glycol-co-caprolactone（PGCL）has adjustable mechanical properties and biodegradation rate,It exhibits excellent elasticity and plasticity,which cause it easily process into bone tissue engineering scaffolds and fill irregular infected bone defects.In recent years,2D nanomaterials have been increasingly utilized in a wide spectrum of biomedical applications.Ti₃C₂（MXene）nanosheets have good physicochemical properties.Several studies have reported that Ti₃C₂ has antibacterial and osteogenic effects.However,there are few researches on its immune regulation.Fused deposition modelling（FDM）is one of most popular 3D printing techniques of thermoplastic polymers.It have various advantageous properties such as simple operation and low material loss.This technique can create precise geometries and internal structures based on image data.Purpose:3D printed vancomycin hydrochloride loaded PGCL@Ti₃C₂ multifunctional scaffold using FDM-3D printing technology for constructing an antibacterial,anti-inflammatory and pro-osteogenic platform,and optimize treatment strategies for acute infected bone defects.Methods:1.PGCL were synthesized through a ring-opening polymerization ofε-caprolactone（CL）and glycolide（GA）2.3D printed PGCL@Ti₃C₂ scaffold containing 1%,5%,10%mass fraction of Ti₃C₂ and PGCL@Van scaffold containing 1%,3%,5%mass fraction of Van using FDM-3D printing technology.The antibacterial property and biocompatibility are characterized by antibacterial experiment and cell culture.The primary objective was to determine the optimal ratio for multifunctional scaffold.Subsequently,pure PGCL,PGCL@5%Ti₃C₂,PGCL@5%Van,and PGCL@5%Ti₃C₂/5%Van scaffolds are prepared by the same approach and physically characterized.3.The method aimed to evaluate the in vitro antibacterial properties（bacte riostatic ring）and biological activity（cell adhesion,osteogenic differentiation,a nti-inflammatory activities）of different groups of scaffolds with pure PGCL,P GCL@5%Ti₃C₂,PGCL@5%Van,PGCL@5%Ti₃C₂/5%Van.4.Different scaffolds were implanted in an acute infected bone defect mod el in rat tibia,including pure PGCL,PGCL@5%Ti₃C₂,PGCL@5%Van,PGCL@5%Ti₃C₂/5%Van.These assays were performed at different time points to ev aluate antimicrobial,anti-inflammatory and osteogenic properties in vivo,includ ing routine blood tests,Micro-CT,H&E and Masson staining of tissue sections,rapid Gram staining and immunofluorescence staining.5.Differentially expressed genes in cells grown by PGCL@Ti₃C₂ and PGCL scaffolds were selected for transcriptomic analysis.These differential genes was analyzed by clustering to study osteogenic differentiation and anti-inflammatory related signaling pathways.Results:1.The successful synthesis of polymer PGCL was confirmed by characterization.2.The optimum mass fraction of Ti3C2 and Van was 5%by evaluating the antibacterial properties and biocompatibility of different groups of scaffolds.Vancomycin hydrochloride loaded PGCL@Ti3C2 multifunctional scaffold exhibited desirable material properties（including pore size,porosity）,good hydrophilicity,and significant advantages in biodegradation and in vitro mineralization.3.The PGCL@5%Ti₃C₂/5%Van multifunctional scaffold with ideal antibacterial effect and biocompatibility exhibits excellent osteogenic differentiation and anti-inflammatory activity in vitro experiments.4.In vivo animal experiments showed that the PGCL@5%Ti₃C₂/5%Van multifunctional scaffold has good antibacterial,anti-inflammatory and bone repair effects by blood tests,imaging and histological evaluation.5.Genomic analysis of PGCL@Ti₃C₂and pure PGCL differentially expressed m RNA,Ti₃C₂ was found to possibly upregulate Tnn and Di O₂ genes,and participate in ECM-receptor interaction pathway,Wnt/β-catenin pathway and parathyroid hormone（PTH）pathway for osteogenic differentiation.In addition,Di O₂regulate the thyroid hormone T3 that involved in inflammatory response,which inhibit IL-1βand COX2 gene expression through activation of LXRα.Conclusion:1.3D Printed PGCL@5%Ti₃C₂/5%Van multifunctional scaffold exhibits excellent bio-compatibility in vivo and in vitro,which plays an important role in efficient antibacterial properties,anti-inflammation and osteogenic activity.It could treat acute infected bone defects in rats.2.Ti₃C₂ might be involved in osteogenesis and immune regulation through upregulation of Tnn and Di O₂ genes by genomic analysis of PGCL@Ti₃C₂ and pure PGCL differential genes...