Chitosan-quaternized Chitosan/nHA Thermosensitive Hydrogel In The Treatment Of Infected Bone Defect

Background:Infected bone defects have always been an intractable problem in orthopedics clinics,the contamination at the defect site causes limited blood supply,which seriously damages bone regeneration and destroys the osteogenic microenvironment.The current clinical treatment standard comprises controlling infection and bone regeneration.After the infection is completely controlled,the local bone defect can be repaired next.The clinical treatment of infection consists of debridement of the infected site followed by systemic application of antibiotics.However,to avoid bone nonunion and limb dysfunction,complete debridement with normal bone tissue preserving is a surgical challenge.Moreover,the formation of bacterial biofilm in the infected area often leads to the failure of pathogenic reduction caused by insufficient antibiotic concentration.Although autogenous bone graft was considered as the gold standard in clinic treatment,it still faced many complications such as insufficient donor source.Allograft was also limited by immunologic rejection and reinfection.In the infected bone defect,implants and extracellular matrix provided the colonization site for bacteria.If bacteria colonized first,the process of bone regeneration would be severely interfered.Conversely,if osteoblasts first attached to the surface of the implants and subsequently mature and differentiate,the bacterial colonization can be inhibited and thus promoted bone repair.Therefore,constructing a functional biomaterial that can simultaneously anticontamination and promote bone regeneration will have important clinical significance for the treatment of infected bone defects.Objective:Chitosan(CS)is a natural cationic polymer.The positively charged chitosan molecule can change the cell permeability by direct contact with the negatively charged bacterial molecule on the cell surface to exert the antibacterial effect.At the same time,chitosan has good biocompatibility and biodegradability,and is currently one of the commonly used biomaterials in bone tissue engineering.Hydroxypropyltrimethyl ammonium chloride chitosan(HACC)is a kind of quaternized chitosan(QCS)prepared by introducing quaternary ammonium groups into the free hydroxyl or amino groups of chitosan.After modification,quaternized chitosan has better water solubility than chitosan,so its antibacterial performance under physiological conditions is significantly improved.Although the corresponding high antibacterial activity can be obtained with the increase of the degree of substitution of quaternized chitosan,it will also be accompanied by significant cytotoxicity,which is not conducive to the proliferation and differentiation of osteoblasts.In this study,chitosan and quaternized chitosan were mixed in different proportions and prepared into temperature-sensitive hydrogel to reduce the cytotoxicity of quaternized chitosan while retaining its high antibacterial property.Nano-hydroxyapatite(n HA)was mixed to further improve the osteointegration ability of the hydrogel and thus constructed a bioactive interface with the dual functions of antibacterial and bone regeneration,thus improving the microenvironment of the infected bone defect area.Method:1.At 4℃,2 w/v% chitosan and quaternized chitosan solution(85%/15%,90%/10%)was mixed with β-glycerophosphate(β-GP)in a certain proportion to obtain a temperature-sensitive hydrogel respectively,and then n HA was added to obtain a composite system.2.The surface morphology of the hydrogel was observed by a scanning electron microscope,and the absorption peaks of each component of the hydrogel were determined by Fourier transform infrared spectroscopy.The elastic modulus of the hydrogel was determined by flow deformation experiment.3.The in vitro studies were divided into five groups: control group(CON),chitosan/nano-hydroxyapatite hydrogel group(C/H),85%chitosan and 15%quaternized chitosan/nano-hydroxyapatite hydrogel group(C85-Q15/H),90%chitosan and10%quaternized chitosan/nano-hydroxyapatite hydrogel group(C90-Q10/H),and the quaternized chitosan/nano-hydroxyapatite hydrogel group(Q/H).The biocompatibility of the hydrogel was verified through cell count experiment and dead cell/live cell staining experiment after co-culturing with mouse embryonic osteoblast precursor cells(MC3T3-E1);The effects of the composite system on the osteogenic differentiation and the expression of genes of MC3T3-E1 were evaluated by alizarin red staining,alkaline phosphatase quantitative analysis and staining and real-time quantitative polymerase chain reaction.The antibacterial properties of each hydrogel were compared by live/dead bacterial staining and plate spread counting after co-culturing with E.Coli and S.aureus.4.A rabbit infected radius bone defect model was established in vivo,which was divided into three groups: blank control group(CON),C/H,and C85-Q15/H.Each hydrogel was implanted into the infected radial defect of rabbits,and the effects of the composite hydrogel on the osteogenic microenvironment under the infected state were studied by X-ray,Micro-CT,histological staining analysis.Results:1.The chitosan-based composite hydrogel was successfully prepared.The results of scanning electron microscope showed that the pore size of C/H hydrogel and C85-Q15/H hydrogel was about 300μm and different mixing ratios have no effect on the pore size.The rheological test results indicated that the hydrogel had good elastic modulus and could maintain certain mechanical strength.2.In the in vitro experiment,although the pure Q/H had excellent antibacterial ability,it had a high cytotoxicity,while the hydrogels of other groups all showed good biocompatibility and antibacterial ability.The hydrogel of the C/H,C90-Q10/H,and C85-Q15/H groups all promoted the osteogenic differentiation of MC3T3-E1 and upregulated osteogenic related genes,and the effect in the C85-Q15/H group was the best.3.In vivo,we successfully established the rabbit infected radial defect.After injection each hydrogel into the bone defect for 8 and 12 weeks,the X-ary,Micro-CT and histological staining results showed that compared with the CON group,the C/H and C85-Q15/H groups both significantly promoted bone regeneration at the defect area,and C85-Q15/H group was better than C/H group.Giemsa staining also confirmed that the C85-Q15/H group had the best antibacterial activity in vivo.ConclusionIn this research,we have synthesized chitosan-quaternized chitosan thermosensitive hydrogel combining n HA based on the chitosan hydrogel.Compared to the pure chitosan hydrogel,this blended hydrogel,not only the antibacterial property of quaternized chitosan under high degree of substitution is retained,but also the toxic effect on cells is substantially weaken.The composite hydrogel can promote bone regeneration in an infected microenvironment and up-regulate that expression of osteogenic gene such as RUNX2,OCN.