Study On The Scavenging Effects Of D-arginine-enhanced α-amylase On The Biofilm On Implant Surfaces

Dental implants have become the primary protocol for the treatment of dentition defects and edentulous patients nowadays.Nevertheless,the clinical effect and long-term stability of implants could be greatly reduced by peri-implant mucositis and its secondary peri-implantitis,which are urgent clinical problems.The formation of plaque biofilm around the neck of implants is the initiating factor for peri-implant mucositis.However,there is no putative method that could effectively scavenge the biofilm within peri-implantitis in clinical practice yet.Hence,strategies for eradicating the whole biofilm should be essential for the treatment of peri-implantitis.The extracellular matrix accounts for about 90%of the total amount of biofilm,and extracellular polysaccharides（EPS）are the main component of the extracellular matrix.Thus,eliminating EPS could become a potential strategy against biofilm.Actinomyces viscosus（A.viscosus）and Streptococcus sanguinis（S.sanguinis）are recognized as the pivotal bacteria related to the formation of biofilm at the initial as well as the early stages of peri-implantitis.Both of them could secrete a large amount of EPS,which promotes the colonization of a variety of bacteria and the maturation of biofilm.We speculate that certain specific glycosidases may effectively hydrolyze the EPS upon the implant surface,combined with small molecule compounds such as D-amino acids,thus achieving the target of scavenging the biofilm on implant surfaces.In chapter 2,in order to investigate the effects of D-arginine（D-Arg）on enhancing the ability ofα-Amylase（Amy）to disperse biofilms,single strains and hybrid biofilms of A.viscosus and S.sanguinis were respectively established.A.viscosus biofilm was cultured to screen glycosidases that could disperse the biofilm,and their cytotoxicity was subsequently evaluated.The effects of D-Arg combined with Amy on biofilm EPS,biofilm thickness,biofilm morphology,and extracellular matrix structure were explored,respectively,which could comprehensively assess the dispersing effects of Amy+D-Arg on A.viscosus biofilm.Afterward,the dispersing effects of Amy+D-Arg on S.sanguinis biofilm as well as the hybrid biofilm were further studied.The results showed that 0.5%Amy could significantly disperse A.viscosus biofilm,but with significant toxicity.While Amy≤0.05%has little effect on biofilm.Intriguingly,8 m M D-arginine drastically enhanced the eradication of A.viscosus biofilm biomass by 0.01%Amy with biosafety in 30 min,which manifested as hydrolyzation of EPS,drastically decrease of biofilm thickness,disappearance of biofilm morphology,and the wreck of the extracellular matrix structure.Moreover,0.01%Amy+8 m M D-Arg could also effectively disperse S.sanguinis biofilm and the hybrid biofilm of A.viscosus and S.sanguinis.In chapter 3,the mechanism of D-Arg enhancing the catalytic activity of Amy was investigated.The active ingredients were determined by a heat inactivation test.By comparing the enhancing effects of D-Arg and Ca²⁺on Amy hydrolyzing polysaccharides,the intensity of their catalytic activity on Amy was evaluated.In addition,molecular dynamics simulations and molecular docking were conducted to elucidate the mechanism of D-Arg enhancing Amy.The results showed that D-Arg and Ca²⁺had a synergistic enhancing effect on the hydrolytic activity of Amy.D-Arg manifested a better effect on enhancing the catalytic activity of Amy than Ca²⁺.In addition,D-Arg could transform Amy into a Ca²⁺-independent type.D-Arg enhanced the catalytic activity of Amy by shortening the distance among the catalytic triad and stabilizing the structure of calcium-binding regions.In chapter 4,the scavenging effects of the 0.01%Amy+8 m M D-Arg method on biofilm on SLA titanium surface were studied in vitro,compared with titanium brush mechanical cleaning and glycine air abrasion.The results showed that the contact angles of the titanium brush group had the most significant increase.Massive mud-like spots or scratches on the surface were observed under SEM.Similarly,the contact angles of the glycine group also increased,and granular material remained on the surface under SEM and CLSM.The macrophages culturing on the surfaces of the titanium brush group and the glycine group polarized toward the pro-inflammatory type.The 0.01%Amy+8 m M D-Arg method noninvasively and effectively scavenged the biofilm on the SLA surface and promoted macrophages to polarize toward the anti-inflammatory type in vitro.In chapter 5,the titanium plate was placed into a rabbit proximal tibia to construct a model of bone defect with peri-implantitis,which was then utilized for the study of the osteogenesis effect of 0.01%Amy+8 m M D-Arg method.Micro CT showed that extensive trabecular bone structures were formed on the decontaminated surface of the control group（clean surface）and the 0.01%Amy+8 m M D-Arg group at 8 weeks.On the contrary,the titanium brush group and the glycine group lacked similar structures,and the bone volume/tissue volume as well as the bone-implant contact ratios of the two groups were also lower than those of the control group.Methylene blue-acid fuchsin staining showed that the control group and the 0.01%Amy+8 mm D-Arg group formed un-interfered osseointegration on the titanium surface.While thin connective tissue capsules were found to separate titanium surfaces from the newly formed bone both in the titanium brush group and the glycine group.Moreover,the histological bone-implant contact ratios of the titanium brush group and the glycine group were significantly lower than the control group and the 0.01%Amy+8 m M D-Arg group.A number of cord-like particles between the newly formed bone and the titanium surface were detected under high magnification in the titanium brush group and the glycine group.These suggest that compared with titanium brush mechanical cleaning and glycine air abrasion,the 0.01%Amy+8 m M D-Arg method can effectively promote re-osseointegration on the decontaminated implant surface in vivo.Moreover,this method is non-invasive to the implant surface and will not lead to the residue of redundant materials,thus possessing potential application prospects.