A Study Of Biomineralization And Inhibition Of Dental Calculus

Dental calculus is a mineralized deposit on the teeth that persists in the periodontal tissue for a long time and can lead to periodontal disease as well as some systemic chronic diseases.Dental calculus consists of organic matrix and inorganic components.The organic matrix includes some proteins,lipids,carbohydrates,etc.The bacteria in the oral cavity are involved in the formation of dental calculus in at least the following two aspects:on the one hand,it can form a dental plaque biofilm,which is conducive to the accumulation of mineral ions,and on the other hand metabolism creates a slightly alkaline environment conducive to mineral deposition.Calcium phosphate is the main inorganic component of dental calculus.In the process of dental calculus maturation,under the influence of various factors in the environment,the calcium phosphate phase will undergo a kinetically favorable transformation—from calcium hydrogen phosphate dihydrate precipitate（DCPD）into hydroxyapatite（HAP）,this calcium phosphate phase transformation will be accompanied by changes in dental calculus’color,hardness,and degree of adhesion to the tooth surface.In modern medical science,dental calculus can be removed by physical means such as hand curette,ultrasonic wave,there are also chemicals on the market that can remove dental calculus or reduce plaque formation.Although these methods are usually effective in removing calculus,they also have a short action time and may not be effective in removing specific parts.Therefore,it is necessary to develop a new strategy to prevent or treat dental calculus with less side effects by reducing the formation of dental plaque and inhibiting the mineralization of calcium phosphate by studying the biomineralization mechanism of dental calculus.The main research contents of this thesis are as follows:（1）Establishment of in vitro simulated dental calculus system.Selected mineralization system:the same volume of 0.1 M Ca（NO₃）₂ and 0.06 M NH₄H₂PO₄through chemical precipitation to achieve the synthesis of DCPD and HAP.The conditions for synthesizing DCPD were p H 6-9 reaction for 2 hours;the conditions for synthesizing HAP were p H 10-13 reaction for 8 hours.（2）Analysis of influencing factors of mineralization in simulated dental calculus system.The simulated environment of saliva,urea,Si O₂,and bacteria was designed,and it was found that they all promoted the mineralization of DCPD to HAP to varying degrees.Gram-negative bacteria contained more phospholipids in chemical composition,that the mineralization time was faster.Finally,two existing systems were selected.Through biofilm experiments and mineralization experiments,it was found that the biofilm removal effect was not ideal,and the system would be mineralized within 2 hours.（3）New strategies for biological control of dental calculus.Two key processes of dental calculus formation（the conversion of DCPD to HAP and the formation of bacterial biofilms）are regulated by rationally designing protein.The recombinant protein DspB（D₃E₂）₂ was constructed by fusingβ-N-acetylglucosaminidase（Dispersing B,DspB）with calcium phosphate binding peptide（D₃E₂）₂.First,the induced expression of DspB（D₃E₂）₂ was achieved,and the optimal conditions for induction were obtained:the final concentration of 0.2 m M IPTG,induction at 37°C for 3 hours,followed by purification and ultrafiltration concentration to obtain a large amount of target protein.It was proved by enzyme activity experiment and biofilm experiments that the modified protein still maintained biological activity,and the maximum enzyme activity was at p H6.0 and 30°C.Through infrared analysis,fluorescence analysis,adsorption and desorption experiments and biofilm experiments,it was proved that DspB（D₃E₂）₂can be successfully immobilized on HAP and can remove biofilms more efficiently.It was verified by scanning electron microscopy and X-ray diffraction that DspB（D₃E₂）₂ could slow down the mineralization process of DCPD to HAP.