The Study Of Tea Polyphenol On The Inhibition Of Artificial Caries On Root Surface In Vitro

Purpose Dental caries is a chronic disease with progressive destruction of the hard tissue, the cause of which is multiple and the bacteria is the most important one. It is a common and frequently-occurring disease. Root caries are the caries that located on the surface of the teeth root which usually occurred in the elderly. It is one of the principal reasons why the tooth are broken in the neck and lost in the elderly. The third national oral epidemiological survey showed that the incidence of the elderly between 65 and 74 suffering from root surface is 98.4%, while on the contrary only 9 to 22 percent of them are treatable. China is facing an aging population problem. How to prevent the elderly from having the root surface caries has become a social issue. Most of the root surface caries destroy the cementum in the CEJ, and involve in the dentin directly or indirectly.Researching on the plaque of the healthy root surface and the root surface with caries, the Nordic scholars put forward a"Polymicrobial etiology Hypothesis". Up to now, Streptococcus mutans is considered as the most significant pathogen in the dental caries. The long term clinical observasion and deep-going research tell us that there are disadvantages in some traditional medicines (for example: the remineralization medicines) used for the prevention of root surface caries. More and more researchers have been focusing on the natural medicine recently. Tea Polyphenols(TP) is multi-hydroxy phenolic compounds in tea, especially green tea, which weighs 20% to 30% in the dry tea. The main components of TP is catechins whose weight possession is 80%.TP is not only a natural antibacterial agent, but also a highly effective low toxicity of the free radical scavenger and anti-oxidants. It has been applied to the treatment of oral diseases in recent years. For example, it can eliminate bad smell, promote oral mucosal ulcer healing, inhibit carcinogenesis of oral mucosa, and so on. Therefore, this study was designed to investigate the effects of TP on the prevention of artificial dental root caries, and to seek rationale for better clinical application.Method 1 The preparation of tooth in vitro: 60 molars migration three-degree in patients with severe periodontitis are selected for this study, which with the standard is that caries-free and the surface of cementum is lubricous. Calculus and pericementum on the root surface are eliminated , the specimens are washed in the ultrasonic cleaner for five minutes and dried naturally. Choose the medial surface or distal surface as the test area, each specimen is coated with acid-resistant nail varnish, leaving a rectangular window of 5mm×5mm on surface near CEJ area. Divide them into four groups randomly and then disinfect them in 75% alcohol solution and store them in 4℃refrigeratory for use.2 The preparation of bacterium suspension: inoculate the international standard strains of Streptococcus mutans IngbrittC in BHI (Brain Heart Infusion) medium, then resuscitate it under anaerobic conditions for 48hours. Choose a little colony and identificate that it is S. mutans and grows well. After subcultivating it in the BHI medium for 24 hours in anaerobic environment 37℃condition, adjust the bacterium suspension to OD=1.0 under UV spectrophotometer with germfree physiological saline solution.3 The preparation of TP solution: dissolve the TP powder in BHI medium, duplicate according to 1:2 gradient. 64mg/ml, 32mg/ml, 16mg/ml, 8mg/ml, 4mg/ml, 2mg/ml, 1mg/ml, 0.5mg/ml are prepared.4 Experiment of bacteriostasis in vitro: ager diffusion method is applied to measure the diameters of bacteriostatic ring surrounding every agent with caliper rule.5 The formation of artificial root surface caries: Put all the specimens in the bacterium suspension in anaerobic environment 37℃condition for 48 hours. The Calcium ion concentration in the bacterium is compared to find whether there are significant differences in the four groups.Determinate the Calcium ion concentration: determinate the Calcium ion concentration of the solution. (condition: Japan's Hitachi Z2000 atomic absorption spectrophotometer, Ca2+ hollow cathode lamp, lamp current 75mA, spectral bandwidth of 2.6nm, the air 1.60kg/cm2, acetylene 0.26kg/cm2).6 Treat teeth in vitro with drugs: Put the decalcified specimens into the fresh bacterium suspension, and replace the bacterium suspension every 48 hours. In the meantime, rub the surface of window areas with MIC of TP solution, 380mg/ml Ag(NH4)2F4,20mg/ml NaF, and deionized water respectively (three times a day, each 5 minutes). Determinate the concentration of each decalcifying fluid at the point of 48h , 96h and 144h. At last, two specimens are selected randomly from each group to be observed by scanning electron microscope.7 Scanning Electron Microscope observation: Put the specimens into 2% glutaral for fixing for 24 hours after rinsing them with PBS balanced solution, then in osmic acid for 2 hours. Poach them with PBS balanced solution again, dehydrate them with 75% to absolute alcohol gradually, and dry at critical point, spray gold. Observe the structural changes in the surface of window areas by the scanning electron microscope .8 Statistical analysis: Data with x±s, use the statistical software package SPSS13.0 to single-factor analysis of variance.Result 1 To contrast with the negative control group, the groups treated with TP above the concentration 4mg/ml can restrain the growth of S. mutans effectively. The TP group of 4mg/ml is the very concentration of MIC. The diameters of bacteriostatic ring are in the trend of increasing by the upgrade of the TP concentration from 4mg/ml to 32mg/ml.2 After the early artificial root caries model is prepared, the concentration of Calcium ion in bacterium suspension of each group is measured. There is no statistically significant difference among the four groups (P>0.05).There are statistically significant differences in concentration of Calcium ion among the negative control group ,the 48h group, the 96h group and the 144h group, and there are statistically significant differences in concentration of Calcium ion among the 48h group, the 96h group and the 144h group (P<0.05). The antimicrobial effect of 380mg/ml Ag(NH4)2F4 is the strongest, The antimicrobial effect of 20mg/ml NaF is the weakest, and the antimicrobial effect of 4mg/ml TP is between them. The concentration of Calcium ion in bacterium suspension of TP group is in the trend of decreasing by time.3 The root surface of D'w group is ruptured seriously. It takes on the appearance of honeycomb, with demineralization of the hard tissue and destruction of matrix fiber. Among the three treated groups, the 380mg/ml Ag(NH4)2F4 group is lightly destroyed, while the 20mg/ml NaF group is serious. The lamellar structure can be observed in 20mg/ml NaF group, with a small amount of pores, which is lesser than the 20mg/ml NaF and D'w groups.Conclusion Contrasted with the control group and the other two treated groups, we find that tea polyphenol can restrain the growth of streptococcus mutans and the demineralization of cementum. So, we can draw the conclusion that tea polyphenol can restrain the development of root surface caries at early stage.