In vitro model for pit and fissure caries.

This paper describes the preliminary steps toward developing an in vitro model for producing pit and fissure caries. Four groups of 12 intact molars mounted in acrylic and pit and fissure areas were painted with Streptoccocus mutans-inoculated culture medium. In Group A, the bacterial inoculum was covered by agar. In Group B, carboxymethyl cellulose was used instead of agar. In Group C, agar was used and covered by a filter paper disc and collodion. Group D was the same as C except carboxymethyl cellulose was used instead of agar. Groups A and B were incubated at 37°C. Groups C and D were placed in artificial saliva and incubated in a shaking bath. The study was conducted for 8 weeks and the media were changed twice a week. Group C had the largest number of carious lesions (both microscopic and clinically). However, the difference from the other groups was not significant (P 0.05). The multifactorial nature of the dental carious process poses a great challenge to the researcher. Isolating and measuring the effects of the primary and secondary etiologic factors associated with caries is difficult. Dahl and Silverstone (1979) developed a method for pit and fissure caries in sound teeth by using acidified gels. Their histopathologic study of the carious area showed that the progression of lesions occurred in vertical depth and not in width (wall areas). Thus the cavities they observed were not similar to natural caries, wherein the cavity progression occurs in all directions. The development of a method to produce occlusal cavitation similar to that observed in clinical caries is, therefore, an urgent need. If such a procedure could be developed and standardized, the effects of proposed preventive agents applied to occlusal surfaces could be assessed by comparing cavity formation on these surfaces with those in control specimens tested with placebo agents. It was the overall goal of this study to approach the development of a method for producing occlusal cavities in sound, extracted human teeth. The specific objective of this study was to develop a method for producing in vitro occlusal caries with the following characteristics: A. The lesions should have the same characteristics as naturally occurring caries, both microscopically and clinically. B. The initiation of caries should occur as quickly as possible. Methods and Materials Forty-eight sound, extracted human first, second, and third molars from a larger population of teeth were obtained from the Department of Oral Surgery of the Indiana University School of Dentistry. The teeth had been extracted either due to periodontal or prosthodontic problems. They were kept in a 3% aqueous formaldehyde solution and stored in sterilized distilled water until use. The teeth were examined by 2 independent examiners, both clinically (visual and tactile) and microscopically. Visual-tactile examination was done with a sharp explorer #MG2 with the aid of an ordinary bright light. Microscopic evaluation was done using a binocular dissection microscope at 15x magnification. The selection by visual-tactile methods of teeth which were free of surface decalcification or cavitation was based on the criteria for determining pit and fissure carious lesions established in 1968 by the American Dental Association’s Council on Dental Research and Council on Dental Therapeutics (1968). Teeth which did not meet any of the criteria for caries were regarded as being caries-free. By sample selection the teeth were distributed into 4 groups, 12 to a group. The strata were third, second, and first molars. The roots were removed with PEDIATRIC DENTISTRY: June 1987/VoL 9 No. 2 131 a separating disc and each tooth was mounted on an individual acrylic base. A plastic cast was constructed to keep the bases always in the same position. In addition, the teeth were mounted so that the center of the occlusal surface was at the same distance from the base. In this way, approximately the same focal distance could be used for all of the specimens during microscopic evaluation at the different periods of evaluation. The teeth then were sterilized with ethylene oxide. Cariogenic Challenge The occlusal pits and fissures of each tooth were covered with a layer of artificial plaque formed by Streptococcus mutans and a solid or semi-solid medium containing sucrose. To prepare "artificial plaques" a stock culture of S. mutans 6715, grown for 18 hr in a complex medium (Jordan’s) a containing 0.25% glucose, was carefully painted with a sterilized camel hair brush over the pit and fissure area. A layer of Jordan’s medium, to which 2.0% agar b and 20.0% glycerine was added as a humectant was then applied over the initial inoculum. In some groups, 2.0% carboxymethyl cellulose (CMC)c was added to Jordan’s medium instead of agar. The 48 teeth were divided randomly into 4 groups as follows: Group A, The liquid medium, consisting of an 18-hr culture of S. mutans in 0.25% glucose-containing Jordan’s (Jordan et al. 1960) medium was carefully painted on the occlusal pit and fissure area with a sterile camel hair brush. Subsequently, the agar medium was melted and tempered to 55°C. The agar then was inoculated with i ml of 18-hr culture of S. mutans and applied to the pit and fissure area with a sterile camel hair brush. Once the agar solidified, the specimens were placed in a presterilized 600-ml beaker over a layer of wet cotton to keep the environment Jordan’s medium formula: K2H PO4 5.0 g Yeast extract 5.0 g Trypticase 5.0 g Sucrose 5.0 g Jordan’s salt mixture 0.5 ml adjust to pH of 7.2 with 0.1 N CIH q.s. to 1000 ml Jordan’s alt mixture: MgSO47H20 0.800 g FeC137H20 0.040 g MnC124H~O 0.019 g q.s. to 100 ml Jordan’s medium with agar: Same as Jordan’s medium plus: Agar 20.0 g Sucrose 50.0 g Glycerine 20.0 g Bromocresol purple 0.36 g Jordan’s medium with carboxymethyl cellulose: Same as Jordan’s medium plus: CMC 20.0 g Sucrose 50.0 g Bromocresol 0.36 g (Type 7MF, Hercules Corporation) TABLE 1. Mean Number of Affected Tooth Surfaces and Cavities Observed in Tooth Surface Microscopically After 7 and 8 Weeks Exposure to the Cariogenic Challenge Total Number of Affected Surfaces Mean Number of Cavities Per Tooth Group Week 7 Week 8 Week 7 Week 8 B ~ ~ | 0.75 0.21 | 0.75 0.21 C 11 11 |1.25 0.21 |1.58 0.28 D 10 L0.91 _+ 0.19 i_ 1.08 _+ 0.19 * Standard error of the mean (12 specimens were used per group). Values within brackets are not significantly different at P > 0.05 (chi-square test, 1-way analysis of variance, or Welch test as it may correspond). moist. The beaker was sealed and incubated at 37°C for 2 days, at which time the specimens were removed from the beaker and the teeth were brushed with a standard multi-tufted, medium-hardness, synthetic toothbrush and sterile deionized water. The empty beakers were autoclaved and the procedure repeated for another 2 days. This was repeated for 8 weeks. Evaluations were made each time the medium was changed. Any changes, both microscopic and clinical, were recorded. In addition, 2 examiners at weekly intervals observed the progression of the microscopic and clinical lesions that developed. Group B. This group was treated in the same manner as Group A except that carboxymethyl-cellulose (CMC) was used instead of agar. Group C. The specimens again were treated in the same manner as Group A except that the layer of agar was covered by a disc of sterile filter paper over which a thin layer of collodion was painted. Then the specimens were placed into a presterilized 600ml beaker containing 300 ml of sterilized artificial saliva, d Finally, the beaker was sealed with aluminum foil and adhesive tape and incubated at 37°C in a thermostatically controlled shaking bath at a speed