Preparation Of The Composite Hydrogel With Loading D-valine Microspheres And Biological Studies In Vitro

Background and Objective:Periodontitis and peri-implantitis are the main causes for teeth loss and implant failure,which seriously affect oral health.Plaque biofilm is an initiating factor for the failure,which participates in the whole process of periodontitis and peri-implantitis directly or indirectly.Hence,early inhibition of biofilm formation is the key to prevent the periodontitis and peri-implantitis.Porphyromonas gingivalis(P.gingivalis)is one of the most important pathogens in plaque biofilm,which can evade host defense mechanisms and resist changes in microenvironment and secrete a large number of virulence factors to aggravate inflammation.The results of our previous experiments have proved that specific concentrations(50-150 mM)of D-valine(D-val)could inhibit the formation of P.gingivalis biofilm.However,D-val itself has the disadvantages of high cost and insoluble in water,which limits its direct application in oral disease.Drug release system can be used to improve the bioavailability of D-val,and keep D-val concentrations within biosafety levels,it will contribute to utilization of D-val of periodontitis and periimplantitis.Chitosan(CS)has excellent biodegradable property,strong absorption and biocompatibility,is an ideal biological drug carrier.The purpose of this study is to synthesize composite hydrogels loading D-val CS microspheres,to achieve the controlled release of D-val by composite hydrogels,and to explore the biocompatibility of the composite hydrogels through biological studies in vitro,so as to provide a reliable basis for further clinical application of D-val.Methods:Chitosan(CS)microspheres loading D-val were prepared by ionic gelation method,and then the loaded D-val microspheres were incorporated into aldehyde-glucan-hydroxypropyl chitosan hydrogels to obtain the loaded microspheres composite hydrogels.Fourier transform infrared spectroscopy(FTIR)and scanning electron microscopy(SEM)were used to characterize the structure of microspheres and hydrogels,release experiments and degradation in vitro were tested.Using CCK-8 to detect the effects of composite hydrogels on the proliferation activity of L929 fibroblasts.The influence of composite hydrogels on biofilm formation of P.gingivalis was evaluated by crystal violet staining and exopolysaccharide experiments.Results:1?FTIR results showed that D-val was successfully encapsulated in CS microspheres.The CS microspheres loaded with D-val had a spherical structure with smooth surface and uniform size,the microspheres had sizes at average of 2.52±0.13?m.The encapsulation rate and drug loading rate of D-val in microspheres were 45.5% and 20.5% respectively.2?The CS microspheres loaded with D-val were dispersed in the gridded aldehyde glucan-hydroxypropyl chitosan hydrogel.The sustained release of Dval in composite hydrogels reached 13.0 days,and the composite hydrogels finally degraded completely at about 22 days.3?CCK-8 results showed that the composite hydrogel had no effect on L929 fibroblasts proliferation activity(P>0.05).4?Compared with the control group,the composite hydrogel significantly inhibited the biofilm volume of P.gingivalis)(P<0.01),and significantly inhibit its extracellular polysaccharide production(P<0.01).Conclusions:1?D-val can be loaded to obtain microspheres composite hydrogels which have regular shape of microspheres,D-val could stablely release for 13 days.2?The composite hydrogel with loading D-valine microspheres have no effect on the proliferation of L929 cells.3?The composite hydrogel with loading D-valine microspheres can reduce the amount of exopolysaccharide in P.gingivalis biofilms and inhibit the formation of P.gingivalis biofilms.