Antibacterial Properties Of Titanium Surface Planted With Chitosan Deirvatives

BackgroundWith the implant materials beening widely used in orthopedic surgery， infectionof the implant materials has become a difficult issue. Host immunosuppression overorthopedic implant surface, the surrounding tissue damaged by wear particles,bacterial resistance, biofilm formation, result in the extremely difficult treatment ofthis type of infection. It also leads to prolonged hospitalization, increased patientsuffering and the risk of complications and death, and also huge expenditure ofmedical resources. Many studies focus on the implant surface modification, bu oftenresult in poor antibacterial capacity, bacterial resistance and osseointegration defects.Novel antibacterial plant surfaces,which has good antibacterial ability andbiocompatibility, could preventing bacterial adhesion and biofilm formation, andeven promote bone integration, will be a certain amount of research and applicationprospects.Part ⅠObjectivesSynthesis of different degree of substitution of chloro acetyl thioureacarboxymethyl chitosan derivative (CCMC), to detect its antibacterial properties,cytotoxicity, and the impact of the proliferation and differentiation of bone marrow mesenchymal stem cells (MSCs).MethodsInhibition zone test and the inhibition rate test were used to determine theabilities of in vitro antibacterial properties of CCMC against Staphylococcus aureus,Staphylococcus epidermidis. MTT assay was used to determine the cytotoxic towardsMC3T3-E1cells. MSCs proliferation was also determined by the MTT assey.Alkaline phosphatase activity and osteocalcin content were measured for determiningMSCs differentiation.ResultsAlong with the increase of the degree of substitution of CCMC, the antibacterialactivity gradually increased. CCMC(0.88) had an in vitro cytotoxicity towards toMC3T3-E1cells, while the other two did not. CCMC(0.24) and CCMC(0.53) couldpromote the proliferation and differentiation of MSCs, while CCMC(0.88) had acertain inhibition on it.Part ⅡObjectivesTo determine the minimum inhibitory concentrationand the minimumbiofilm eradication concentrations of CCMC, as well as the impact of biofilmquantity and biofilm activity against Staphylococcus aureus and Staphylococcusepidermidis biofilm inhibition rate. MethodsBacterial quantitative method was used to determine the minimum inhibitoryconcentration of different degrees of substitution of CCMC. The minimum biofilmeradication concentration was determined also. The biofilm of each degree ofsubstitution CCMC inhibition rates were determined against three kinds of bacterials.The impact of CCMC on Staphylococcus epidermidis biofilm activity was determinedby the method of immunofluorescence assay.ResultsAs higher degree of substitution of CCMC, the minimum inhibitoryconcentration and the the minimum biofilm eradication concentration graduallydecreases. As in the same concentration, the inhibition rate of the CCMC againstthree biofilm increased along with the degree of substitution of CCMC. The degree ofsubstitution of0.88and0.53CCMC were effectively in a small concentration ininhibiting biofilm growth. Higher degree of substitution of CCMC had a strongerinhibition capacity on Staphylococcus epidermidis biofilm activity. With the increaseof the concentration of each substituted degrees CCMC, the inhibitory capacity of thebiofilm activity were stronger.Part ⅢObjectivesTitanium alloy surface covalently bonded with the0.53degree of substitution ofthe CCMC (Ti-CCMC). Through the comparison with the surface of titanium (Ti),the ability to inhibit the biofilm, the impact on the adhesion and proliferation ofosteoblasts were determined. A New Zealand white rabbit infection model was established. local impact of bone tissue and systemic impact of the animals weredetermined.MethodsImmunofluorescence assay was used to compare the ability of Ti-CCMC and Tiagainst biofilm activity.Bacteria quantitative method were used to compare the impactof Ti-CCMC and Ti on adhesion of Staphylococcus epidermidis and MC3T3-E1celladhesion and proliferation. Quantitative detection method was used for thedetermination of the bacterial content in the bone tissue around the titanium rod. Therabbits weight changes, Blood bacterial culture, X-ray examination, were used tocompare the differences in the two groups of rabbits. Differences between the twogroups of rabbits middle femur medullary cavity were compared between the twogroups of bacteria local migration by the method of gram stain.ResultsTi-CCMC had a significantly ability of inhibiting biofilm activity. Ti-CCMCcould also inhibit epidermal aureus adhesion.The Ti-CCMC could promote celladhesion and proliferation of MC3T3-E1.Rabbits in the Ti-CCMC group had nobacterial growth in the bone tissue surounding the rod. Rabbits weighing between thetwo groups showed no significant difference between the two groups. PostoperativeX-ray examination revealed the femur bone destruction in the Ti group, but theTi-CCMC group had no obvious abnormalities, osteolysis or Radiolucent line. Themiddle femur medullary cavity Gram stain in the Ti groups showed bacteria whichwere stained in blue.ConlusionsWith the good antibacterial ability, biocompatibility and bone integrationcapabilities. Chitosan acyl thiourea derivatives can be used as a new material of orthopedic implant surface. It deserves further research and has an applicationprospects.