The Effect Of Different Cross Linkers On Bonding Strength And Cytotoxicity Of Mussel-inspired Tissue Bioadhesives

Objective:This study was aimed to evaluate the effect of different cross linkers on bonding strength and cytotoxicity of mussel-inspired tissue bioadhesives.Methods:Firstly,DAH-iCMBAs pre-polymer was synthesized by polycondensation of citric acid(CA),poly(ethylene glycol)(PEG),dopamine,1,6-diaminohexane and 2,2-dimethylol propionic acid according to our previous work,which the PEG molecular weight of 400 was selected,0.5 eq.[to PEG]of dopamine,0.1 eq.[to PEG]of 1,6-diaminohexane.Secondly,evaluating the effect of different cross linkers on the bond strength of mussel biomimetic adhesives,the adhesion strengths of different formulations were determined by lap shear strength test.The sodium periodate(PI),ferric chloride and sodium hydroxide(Fe3+),silver nitrate(SN)were respectively cross-linking DAH-iCMBAs prepolymer,and FG was used as a control.Then,the cytotoxicity of DAH-iCMBAs prepolymer and sol content or leachable fraction and degradation products of crosslinked DAH-iCMBAs were studied using Counting Kit-8(CCK-8)against NIH/3T3 cells.The cytotoxicity test of the prepolymer used DAH-iCMBAs prepolymer as the experimental,hydroxyethyl methylacrylate(HEMA)as a positive control,injectable citrate-based mussel-inspired tissue bioadhesives(iCMBAs)prepolymer as a negative control and the complete medium as the blank control.Cytotoxicity of sol content or leachable fraction of crosslinked DAH-iCMBAs,referred to as sol-cytotoxicity.The PI,Fe3+,SN respectively cross-linking DAH-iCMBAs prepolymer were usd as the experimental,Fibrin glue(FG)as a positive control and the complete medium as the blank control.The cytotoxicity of degradation products was also evaluated.The PI,Fe3+,SN respectively cross-linking DAH-iCMBAs prepolymer were usd as the experimental,the poly(lactic-co-glycolic acid)(PLGA)as a positive control and the complete medium as the blank control.Finally,for Live/Dead assays,the culture mediums of NIH/3T3 was replaced by 200 ml sol content or leachable fraction of crosslinked DAH-iCMBAs or pure cell culture mediums,respectively.Live/Dead cell viability test were conducted on day-1,-3,and-5,respectively with a Live/Dead viability/cytotoxicity kit.All the cytotoxicity tests were conducted in triplicate.Results:Based on the results of previous experiments,the structure of 1,6-diaminohexane and the structure of catechol to be linked to the main chain of citric acid and poly(ethylene glycol)were confirmed by by Proton Nuclear Magnetic Resonance spectroscopy(1H NMR),Fourier Transform Infra Red(FT-IR)spectroscopy and UV-VIS spectroscopy.In the lap shear strength test,when the volume ratio of ferric chloride solution to the DAH-iCMBAs prepolymer solution is 1:2,the lap shear strength of the crosslinked DAH-iCMBAs is the largest;PI crosslinking DAH-iCMBAs has no significant difference with Fe3+crosslinking DAH-iCMBAs in the lap shear strength,which were far superior to SN cross-linking DAH-iCMBAs and FG.And SN crosslinking DAH-iCMBAs is superior to FG,but SN crosslinking DAH-iCMBAs has no significant difference with FG.In the cytotoxicity experiment,the cell activity of NIH/3T3 was more than 85%after iCMBAs prepolymer and DAH-iCMBAs prepolymer were diluted 10-fold.The cell activity of iCMBAs prepolymer was slightly better than DAH-iCMBAs prepolymer.The cytotoxicity of sol content or leachable fraction and degradation products of crosslinked DAH-iCMBAs,after 10-fold dilution,the NIH/3T3 cell activity reached 70%or more.In addition,by observing sol content or leachable fraction of crosslinked DAH-iCMBAs cultured cells with the prolongation of the culture time.The effect of sol content or leachable fraction on cell proliferation,after diluted 10-fold,the cultured cells were proliferative over time.Compared to the control,it's not clearly show that sol content or leachable fraction of crosslinked DAH-iCMBAs interfered with cell proliferation rate.Conclusion:At appropriate concentrations,different cross linkers did not interfere with the cell proliferation activity of DAH-iCMBAs;in the lap shear strength test,PI,Fe3+can promote the bonding strength of the DAH-iCMBAs prepolymer after cross-linking,while the strength after cross-linking by SN is only slightly stronger than FG,so PI,Fe3+ are the wonderful cross linkers and have good biocompatibility,which can provide a theoretical basis for further experiments.