| Objective:Diabetic periodontitis is a common disease in human beings.How to effectively treat periodontal inflammation and accelerate periodontal tissue regeneration in diabetic patients is a major medical problem in clinic.At present,the commonly used mechanical debridement assisted antibiotic periodontal local sustained-release preparations are limited due to their limited scope of action,easy to produce bacterial resistance and fixed administration time and dose.Glucose-sensitive smart hydrogel can adjust the drug release rate with the change of glucose concentration,which has become a research hotspot in exploring new drug delivery carriers for diabetic complications.Glucose-sensitive hydrogels fabricated by immobilization of glucose oxidase(GOx)on p H-sensitive hydrogels(such as chitosan)have attracted much attention because of their low toxicity and simple preparation pproces.Hydrogels prepared from chitosan(CS)have many advantages,such as good biocompatibility,biodegradability,natural safety,and being able to form composite materials with other materials,which have wide application value in modern biomedicine.However,the traditional physical adsorption method of glucose oxidase immobilized on chitosan has the shortcoming of weak binding force and unstable enzyme action,which limits it’s application of such materials.In this study,methacrylic anhydride(MA)was grafted onto chitosan to obtain photocrosslinked chitosan hydrogel(CM)with low cytotoxicity.Then,CM hydrogel was immersed in tannic acid(TA)solution to form CM / TA composite hydrogel.Finally,glucose oxidase and CM / TA hydrogel were covalently crosslinked by N-hydroxysuccinimide(NHS)/ 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDC)mediated chemical reaction to form NE / GOx composite hydrogel film with glucose sensitivity and anti-inflammatory properties.The related physical and chemical properties,glucose sensitivity,in vitro cell compatibility and antimicrobial and anti-inflammatory properties were comprehensively tested to study the feasibility of the composite hydrogel film as a periodontal local sustained release preparation.Methods:In this study,chitosan hydrogel was prepared by modified photocrosslinked ethylene chitosan and loaded with TA to form composite hydrogel.The interaction between CM and TA was analyzed by FTIR.GOx was immobilized in the composite hydrogel by NHS / EDC-mediated chemical reaction,and the possible chemical bonds between GOx and chitosan were speculated by FTIR.Then,the adhesion and stability of GOx on the hydrogel film prepared with or without NHS / EDC immobilized enzyme were compared to evaluate the effectiveness of chemical immobilization of GOx.Subsequently,a series of glucose-responsive NE / GOx hydrogel membranes with chemical immobilization enzymes were prepared.The pore size,expansion rate and drug release rate of the hydrogel membrane soaked in different concentrations of glucose were evaluated by SEM,expansion rate test and drug release rate,and the effect of glucose change in the environment on the performance of NE / GOx hydrogel membrane was discussed.And the changes of mechanical properties of this hydrogel films immersed in different concentrations of TA were also discussed.The mouse preosteoblast MC3T3-E1(MC3T3)was inoculated on the composite hydrogel membrane.The adhesion and diffusion of MC3T3 were observed by SEM and immunofluorescence microscopy.The proliferation of MC3T3 was evaluated by CCK-8,and the compatibility of the composite hydrogel membrane was evaluated.Finally,the effect of TA released by NE / GOx hydrogel membrane on TNF-α,IL-6 and NO related inflammatory factors of mouse macrophage strain RAW264.7(RAW264.7)was determined by in vitro periodontitis model.And the inhibitory effect of TA on Porphyromonas gingivalis was evaluated by antibacterial activity test.Results:1、Through FTIR detection,it was found that there was hydrogen bond between CM and TA,and GOx was successfully immobilized in the composite hydrogel film.And it was speculated that GOx was covalently bonded with chitosan through amide bond.In addition,through the GOx adhesion and stability test,it was found that the fixed GOx content on the NHS / EDC activated composite hydrogel film was higher than that on the unactivated film,and the stability of the fixed GOx was markedly higher than that on the unactivated membrane.After 5 days stability test,the amount of GOx shedding was393.45 ug / ml in Con / GOx hydrogel membrane,but only 170.14 ug / ml in NE / GOx hydrogel membrane.And the fixed GOx in Con / GOx group continued to fall off,while the NE / GOx group has begun to remain stable.2、Through the expansion rate test,it was found that the expansion rate of NE / GOx gradually increased with the increase of GC in the solution.Then SEM images showed that the average pore size of NE / GOx hydrogel membrane gradually increased with the increase of GC after 72 h immersion in different GC solutions.Image J analysis showed that the pore size of the sample gradually increased from 0 mmol / L(102.308 μm)to25 mmol / L(414.558 μm).Finally,the drug release results showed that the release rate of TA in NE / GOx hydrogel membrane increased significantly with the increase of GC.These results indicate that glucose can effectively stimulate the further expansion of NE / GOx hydrogel membrane,increase the internal pore size and drug release.NE /GOx hydrogel membrane has obvious glucose sensitivity.In addition,the tensile test results of universal mechanical tester showed that the addition of TA significantly improved the tensile strength of the composite hydrogel film.3、CCK-8 results showed that although the addition of TA may temporarily slow down cell metabolism,the content of cells was still increasing with time.Combined with the observation of the adhesion of MC3T3 on hydrogel membrane in vitro,it can be known that the NE/GOx composite hydrogel membrane is suitable for cell growth and has good cell compatibility.At the same time,the model of periodontitis in vitro was established by RAW264.7 stimulated by Pg-LPS,and the effects of TA released from NE/GOx hydrogel film on the changes of TNF-α,IL-6 and NO were detected.The results showed that TA could inhibit the level of TNF-α,IL-6 and NO in macrophages in a certain concentration-dependent manner,showing an effective anti-inflammatory activity.Finally,The results of antibacterial activity test showed that TA had inhibitory effect on the growth of Porphyromonas gingivalis,especially at high glucose concentration.Conclusion:In this study,photocrosslinked chitosan hydrogel was synthesized from chitosan and loaded with TA to form composite hydrogel.GOx was immobilized in the hydrogel by NHS / EDC-mediated coupling chemistry to obtain NE / GOx glucose-sensitive hydrogel membrane with good glucose sensitivity,biocompatibility and anti-inflammatory properties.The related test results showed that NE / GOx hydrogel membrane could respond to glucose stimulation,rapidly and correspondingly change its internal pore size,and then control the release rate of TA loaded in it.The membrane has good cell affinity and cytocompatibility,which is applicable for adhesion and proliferation of cell on the film.NE / GOx hydrogel membrane conditioned medium showed good antibacterial and anti-inflammatory ability.The results show that NE / GOx hydrogel membrane has good glucose response and cytocompatibility,and is expected to be further developed as a periodontal local sustained release material that can promote tissue recovery. |
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