| BackgroundAs one of the main reasons for tooth loss, periodontal disease is a chronicinfectious disease which caused the destruction of alveolar bone, periodontalligament, cementum and gingival. So the goal of periodontal therapy is tocontrol the inflammation which was caused by periodontal disease and topromote the regeneration of destructive periodontal tissue and the formationnew attachment. The initial therapy of periodontitis can effectively control theinflammation but it cannot achieve the purpose of severe periodontitis for theregeneration of periodontal tissue when the periodontal supporting tissues havegreatly loss. With the great development of the periodontal tissue engineering,the periodontal scaffolds or carrier material have been one of the hottest topicfor the treatment of periodontal disease and it would be great hope in the futurefor the clinical use.Chitosan thermosensitive hydrogel has the characteristic of body temperaturesolidification, which it would show a good fluid state when it is placed at room temperature or below, but it would become gel when the temperature is close tobody temperature (37degrees C), it can be gelated in a short time with anon-flowable coagulation. It would have great promising in the regeneration ofperiodontal tissue as periodontal scaffolds with many advantages that it wouldimprove the regeneration of periodontal tissue and has the characteristic ofexcellent biocompatibility, non-toxicity, antibacterial property, easy adhesion.However, some reports show that the conventional sterilization of chitosanthermosensitive hydrogel would cause the reduction of viscosity, molecularweight and mechanical property. It would limit its application and effectiveness.Therefore, we designed this study.Aim：To improve the sterilization method of chitosan, test its physical properties andbiocompatibility, and compare the improved method with the conventionalsterilization method of chitosan. To detect its ability of promoting periodontaltissue regeneration by animal experiments.Methods：1Comparative study of chitosan thermosensitive hydrogels by differentsterilization methods and their physical properties.1.1Preparation of chitosan thermosensitive hydrogel by two sterilizationmethods:Preparation of chitosan thermosensitive hydrogel by improved sterilizationmethod: solution A was prepared by0.2g chitosan powder was sterilized bysteam sterilization (120°C/10min).9ml hydrochloric acid solution was addedand stirred by magnetic stirrer for2h. Solution B was prepared by0.56g ofβ-glycerophosphate which was dissolved in1ml double distilled water.0.2gchitosan powder was added into9ml hydrochloric acid solution and the mixture was stirred by magnetic stirrer for2h until completely dissolved, then thesolution A was prepared after the mixture were sterilized by steam sterilization.By the same method solution B and thermo-sensitive gel were prepared.1.2The physical properties of Chitosan thermosensitive hydrogels by twosterilization methods:Chitosan thermosensitive hydrogels by two sterilization methods were bathed in37°C water, to test their gel time, viscosity by viscosity tester (Brookfield HBT),and the ultrastructure of chitosan gel which was visualized by scanning electronmicroscopy.1.3The results showed that: the gelation time of improved sterilization methodof chitosan thermosensitive hydrogel was5-6min, which was the half time ofconventional sterilization method; viscosity test results showed that regardlessof the initial viscosity to the final viscosity. The viscosity of the improved groupwas increased nearly10times than that of the conventional group; both groupshad a porous three-dimensional mesh structure, however the pore size of theimproved method group was significantly smaller than that of conventionalmethod group.2Comparative study of the biocompatibilities of chitosan thermosensitivehydrogels by two sterilization methods2.1The cultivation of human periodontal ligament cellsFresh healthy premolars were harvest from volunteers who were more than18years old. After being washed by PBS for three times, the middle of1/3root ofthe periodontal ligament was scraped, and periodontal ligament cells werecultured by enzymatic digestion method culturing. The culture medium waschanged every three days and the passage3cells were used in the experiment.2.2The growth of human periodontal ligament cells were observed on the surface of two kinds of thermosensitive gel7days later being stained by DAPI,the amount of human periodontal ligament cells which were cultured onchitosan thermosensitive hydrogel surface by two kinds of sterilization methodswere counted under fluorescence microscope and the cell compatibility by twokinds of thermosensitive gel were tested.2.3the effects of chitosan thermosensitive hydrogel extract by differentsterilization methods on the proliferation of human periodontal ligament cellsPreparation of extracts: each of5ml chitosan thermosensitive hydrogel by twokinds of sterilization methods was placed in50ml sterile reagent bottlerespectively,45ml α-MEM cell culture medium containing10%fetal bovineserum (FBS) was added into the bottle, after being soaked for24h, the culturemedium was used as the extracts in the experiment.The passage3healthy human periodontal ligament cells were cultured byChitosan thermosensitive hydrogel extracts by two different sterilizationmethods, and MTT assay was used to detect the cell proliferation.2.4resultsHuman periodontal ligament cells not only could grow well on the chitosanthermosensitive hydrogel surface which were prepared by two sterilizationmethods, but also could proliferate normally in two kinds of chitosanthermosensitive hydrogel extracts. Improved sterilization method group ofchitosan thermosensitive hydrogel and conventional sterilization method grouphad good biocompatibility.3Animal experiment3.1Experimental groups3healthy mongrel dogs, the second and third premolars from the4districts ofboth jaws were randomly selected in each dog as the experimental tooth, which were randomly divided into improved sterilization the methods of chitosanthermosensitive hydrogel group and blank control group.3.2Animal bone defect preparation and surgeryPrepare the animal model of premolar III furcation defects and grind a notch forthe histological observation marks at root surface of the alveolar bone defectarea. chitosan thermosensitive hydrogen by the improved sterilization methodwas implanted into the bone defects.12w postsurgery the tissue was harvestedand was observed by morphological and histological studies in order to test theregeneration of periodontal tissue.3.3resultssome new the alveolar bones with relatively loosen structure and periodontaltissue regeneration promoting capacity could be observed in the bone defect areaof the improved sterilization method of chitosan thermosensitive group. Whilefew new the alveolar bones were observed in the control group.ConclusionAlthough the aperture had become a little narrowed, the improvement of steriletechniques could shorten the gel time and increase the viscosity with athree-dimensional mesh structure. And it had good biocompatibility, adhesionand growth when it was co-cultured with the chitosan thermosensitive hydrogel.And it could induce the regeneration of periodontal bone tissue, so it would havemore broad application prospects than the conventional group. |
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