Scanning electron-microscopic study of pellicle and plaque formation on tetracycline-impregnated dentin

Previous experiments have shown that tetracyclines may react with hydroxyapatite, e.g. in enamel and dentin, without losing their antimicrobial capacity. The present paper examines the pattern of pellicle and plaque formation on doxycycline-treated dentin by the use of scanning electron microscopy (SEM). From newly extracted human teeth were prepared standardized dentin slabs, half of which were soaked in aqueous solutions of doxycycline HCl, 10 mg/ml (pH 2.5) for 10 min. Seven volunteers carried doxycycline-impregnated specimens ligated to the buccal surface of a maxillary molar for 2 h, 8 h, 24 h and 120 h, respectively. Untreated control specimens were ligated to the contralateral teeth. After removal from the oral cavity, the dentin slabs were briefly rinsed in water, allowed to air dry and processed for SEM. SEM assessment of the specimens showed that doxycycline-impregnation resulted in a superficial etching of the dentin, a reduced rate of pellicle formation as well as an impairment of pellicle adhesion, and a retarded bacterial plaque formation on the dentin surfaces.     

Chemical and morphological studies of the acquired pellicle formed subgingivally on dentin in vivo

The morphological appearance and chemical composition of the subgingival pellicle were studied, using Auger analysis and scanning and transmission electron microscopy. A pellicle was formed on pieces of dentin (2 X 2 X 1 mm), prepared from freshly extracted teeth after root planing. The dentin slabs were inserted for 2 h into healthy gingival sulci. Control slabs cemented supragingivally were used for comparison. The results confirmed the presence of an organic film on the surface of all slabs. Auger analysis of the organic film showed the presence of Ca in the supragingival integument but not in the subgingival integument. The subgingival pellicle was in all cases thicker than the supragingival pellicle. The transmission and scanning electron microscopy observations confirmed the presence of a film essentially free of bacteria on the subgingival specimens and also indicated a possible morphological difference between the supra- and sub-gingival pellicle.     

An in vivo model for the identification of serum proteins in the acquired subgingival pellicle

The present study describes an in vivo model for the collection of the subgingival pellicle adsorbed to tooth surface, and the identification of some serum proteins within this layer. Clean dentin slabs were prepared from freshly extracted teeth, and then placed subgingivally for 2 h. The dentin slabs with their adsorbed pellicle layer were processed for transmission electron microscopy. Thin sections were made from the specimens, and treated with antisera to human immunoglobulins and albumin. The reactions were visualized by means of protein A-gold complex, which allowed semiquantification of the serum proteins. The indicator proteins were all identified within the pellicle material, but their amounts and distribution varied. Albumin demonstrated higher amounts in the pellicle layer than other proteins, followed by IgA, IgG, and IgM in descending order. The model described seems useful for studying the acquired subgingival pellicle under varying degrees of disease and health.     

Protective effect of the in situ pellicle on dentin erosion - an ex vivo pilot study

AIM: The acquired pellicle is well known as an anti-erosive proteinaceous layer on enamel, but its protective properties on dentin have not been investigated in detail until now. The aim of the present ex vivo study was to evaluate the erosive effects on pellicle coated dentin. METHODS: Bovine dentin slabs were exposed to the oral cavity of one subject for 120 min for in situ pellicle formation. Subsequently, the slabs were incubated with HCl (pH 2.3) in vitro for 5 min and erosive calcium-release was measured photometrically. In addition, the acid treated specimens were evaluated by transmission electron microscopy (TEM). Pellicle free samples served as controls. RESULTS: Calcium erosion from the pellicle coated dentin slabs amounted to 23.5+/-2.9 microg Ca/min (pellicle free samples: 32.2+/-4.2 microg Ca/min). The difference was statistically significant (p < or = 0.05). In pellicle coated as well as in uncoated dentin samples, TEM-evaluation showed a demineralised dentinal surface layer which thickness ranged between 3 and 6 microm. The pellicle itself was partially dissolved but not removed by hydrochloric acid treatment. CONCLUSION: The protective properties of the acquired pellicle against an erosive challenge of the dentinal surface are limited. The dentinal pellicle functions like an ion permeable network rather than a barrier.     

The potential for dental plaque to protect against erosion using an in vivo-in vitro model--a pilot study

BACKGROUND: Tooth erosion is a problem for professional wine tasters (exogenous erosion from frequent exposure to wine acids) and for people with gastro oesophageal reflux disease (GORD) and bulimia who experience frequent reflux of gastric contents into the mouth (endogenous erosion from mainly HCl). The objective in this study was to determine whether plaque/pellicle could provide teeth with any protection from two common erosive acids, using an in vivo-in vitro technique. METHODS: Tiles of human tooth enamel and root surfaces were prepared from six extracted, unerupted third molar teeth and sterilized. Mandibular stents were prepared for six volunteer subjects and the tiles bonded to the buccal flanges of these stents. They were worn initially for three days to permit a layer of pellicle and plaque to form over the tile surfaces, and for a further 10 days of experimentation. Following cleaning of the plaque/ pellicle layer from the tiles on the right side flange, all the tiles were submerged in either 0.06M HCl or white wine for an accumulated time of 600 and 1500 minutes, respectively. Depths of erosion were determined using light microscopy of sections of the enamel and root tiles. SEM of the lesion surfaces was carried out to investigate the nature of erosive damage and of plaque/pellicle remnants. RESULTS: Retained plaque was found to significantly inhibit dental erosion on enamel, from contact with both HCl and wine, compared with that resulting following its removal. However, it was found to provide no significant protection on root surfaces. SEM analysis of the tile surfaces revealed marked etching of enamel on the cleaned surfaces, and considerable alteration to the appearance of remaining plaque and pellicle on most surfaces. CONCLUSION: Within the limitations of numbers of specimens, dental plaque/pellicle provided a significant level of protection to tooth enamel against dental erosion from simulated gastric acids and from white wine, using an in vivo-in vitro model. It was unable to provide any significant protection to root surfaces from these erosive agents. Possible reasons for this difference are explored.     

Submicron hiati in acid-etched dentin are artifacts of desiccation.

OBJECTIVES: The submicron hiatus represents a potential space between the base of the collagen network and the mineralized dentin when dentin is acid-etched for bonding. These spaces were observed in SEM studies after acid-etched dentin specimens were critical point dried or dehydrated in hexamethyldisilasane. However, they have never been identified in TEM studies of dentin hybrid layers. This study critically examined the cause of submicron hiati formation using a silver staining technique to measure nanoleakage. METHODS: Two multi-step, total-etch adhesives (One-Step, Bisco; Single Bond, 3M) and two single-step, self-etching adhesives (Prompt L-Pop, ESPE; One-Up Bond F, Tokuyama) were examined. Flat dentin surfaces were bonded with these adhesives and a lining composite. In each adhesive group, 0.8mm thick slabs from the same bonded tooth were coated with nail varnish applied 1mm from the bonded interfaces. The varnish was either left to dry completely for 10min before immersing in 50wt% silver nitrate (AgNO(3)) for 24h (group D), or painted on blotted tooth slabs that were immediately dropped into the AgNO(3) solution (group M). After developing, undemineralized, unstained, epoxy resin-embedded sections were prepared for transmission electron microscopy (TEM) to identify the amount and distribution of silver uptake. RESULTS: Nanoleakage patterns were observed in all adhesive-bonded teeth, regardless of brand. Fine reticular silver deposits were also found in the underlying undemineralized dentin. In group D, submicron hiati were seen as tunnels of heavy silver deposits beneath hybrid layers. Specifically, a hiatus occurred between the undemineralized intertubular dentin and a cohesively fractured layer of the same matrix that was attached to the base of the hybrid layer. Hiati were completely absent in group M, regardless of the brand of adhesive. SIGNIFICANCE: Submicron hiati are artifacts created by desiccation during specimen processing, and should be referred to as such in future studies of bonded dentin interfaces.     

Intraoral bacterial growth on tetracyclineimpregnated dentin

Abstract — Previous studies have shown that tetracyclines react with enamel and dentin in vitro to form slightly soluble compounds wish a pronounced and long-lasting antimicrobial capacity. The purpose of the present project was to study the effect of doxycycline-impregnation of dentin on the growth of oral microorganisms in vivo. Slabs of native human dentin were immersed in aqueous solutions of doxycycline HC1, 10 mg/ml, for 10 min, and were subsequently ligated to orthodontic brackets placed bucally on maxillary molars in seven individuals. Untreated native dentin specimens were similarly placed on contralateral teeth. Test and control specimens were removed from the oral cavity after 2 h, 24 h or 120 h, respectively. The individual specimens were then placed in 0.5-ml saline and agitated mechanically for 30 s. The resulting bacterial suspensions were plated onto blood agar and various selective growth media. Based on CFUcounts obtained after incubation of the plates, doxycycline-treated dentin specimens showed a significantly lower number of viable microorganisms after 2 h and 24 h in the oral cavity than did the control specimens. The bacterial growth was also less pronounced in the test specimens after 120 h. The effect of doxycycline was similar for all the cultivable bacteria. No significant difference in yeast growth was seen between test and control specimens.     

Comparison of microtensile bond strength to enamel and dentin of human, bovine, and porcine teeth

PURPOSE: To determine the bond strengths promoted by an adhesive system to human, bovine, and porcine enamel and dentin, and compare their etched micromorphology by scanning electron microscopy. MATERIALS AND METHODS: Thirty sound freshly extracted teeth were used in this study: ten human third molars, ten bovine incisors, and ten porcine molars. The crowns of human (H), bovine (B), and porcine (P) teeth were ground with 600-grit SiC paper to expose either enamel (E) or mid-depth dentin (D) surfaces. After application of the adhesive resin, composite crowns approximately 8 mm high were built up with TPH Spectrum composite. After 24 h of water storage, specimens were serially sectioned in the buccal-lingual direction to obtain 0.8 mm slabs, which were trimmed to an hourglass shape of approximately 0.8 mm2 at the bonded interface. Specimens were tested in tension in a universal testing machine (0.5 mm/min). Results were statistically analyzed with ANOVA and Tukey's test at the 95% confidence level. RESULTS: Tukey's test showed significant differences between bond strengths obtained on enamel and dentin (p < 0.05). However, there were no statistically significant differences in microTBS between human, bovine, and porcine teeth. SEM observations revealed a similar dentinal morphology for the three species. However, porcine enamel specimens presented a very different distribution of enamel prisms. CONCLUSION: Bovine teeth proved to be possible substitutes for human teeth in either dentin or enamel bond testing. However, even though porcine teeth provided enamel and dentin bond strengths similar to human and bovine teeth, enamel morphology presented a very different configuration.     

Early plaque colonization on human cementum

We have described the morphology of developing plaque on cementum in an in vivo human model. Slabs of cementum obtained from sound teeth, rendered anorganic with 5% sodium hypochlorite, were glued to orthodontic brackets and positioned on the upper canines, premolars and molars in 8 volunteers. The brackets were removed after 2, 4, 8, 24 h and processed for scanning electron microscopy (SEM). Within 2 h, a thin pellicle covered the cementum surface, with few micro-organisms detectable. At 4 and 8 h, coccoid plaques were present. Filaments inserted perpendicularly to the plaque surface were seen at 24 h. The results indicate that early bacterial colonization of human cementum is a selective process, mediated by an organic pellicle and mainly involves cocci.     

Protective effect of the dental pellicle against erosive challenges in situ

The acquired dental pellicle helps prevent erosion, but the protection level is unknown. This in situ study tested whether a two-hour pellicle protects against different erosive challenges by orange juice. Subjects wore palatal appliances loaded with either enamel or dentin specimens. Pellicle was allowed to form, or not (control), on the surfaces of the specimens intra-orally for 2 hrs before the erosive challenges of 0 (control), 10, 20, and 30 minutes' duration. Specimens were randomly removed from the appliances after each challenge. Percentage of surface microhardness change (%SMC) was determined for the enamel specimens, and that of mineral loss and lesion depth for the dentin specimens. Enamel specimens with the pellicle showed a significantly lower %SMC, only after the 10-minute challenge. No protection was found for dentin. It was concluded that the acquired pellicle reduced dental erosion, but that this effect was limited to the less severe erosive challenge on enamel surfaces.     

Non-destructive visualisation of protective proteins in the in situ pellicle

Several salivary anti-microbial and buffering components are part of the acquired in vivo pellicle. The purpose of the present in situ study was to visualise these proteins within the in situ formed pellicle and to investigate their distribution with respect to pellicle formation time and intra-oral localisation. Bovine enamel slabs were fixed on individual splints. They were carried by 6 subjects buccally and palatally in the region of the upper first molar teeth over 30 and 120 min, respectively, for in situ pellicle formation. After intra-oral exposure, enamel specimens were processed for transmission electron microscopy. Secretory immunoglobulin A (sIgA), lactoferrin, lysozyme, carbonic anhydrase (CA) I and II were visualised successfully in the in situ pellicle layer by gold immuno-labelling. All components were found to be distributed randomly within all layers of the pellicle. Significantly higher amounts of the proteins were detected after 120 min of formation time. Furthermore, significantly more labelled lactoferrin and lysozyme were found on buccal surfaces compared with palatal sites. For CA I, CA II and sIgA, no significant influence of the localisation was detected. All investigated anti-bacterial and buffering proteins are distributed randomly in the in situ formed pellicle layer and thus could contribute to its protective properties as an early defence barrier.     

Influence of salivary pellicle formation time on enamel demineralization – an in situ pilot study

This study assessed the protective potential of salivary pellicles formed in situ over periods ranging from 2 to 24 h. Pellicles were produced on enamel slabs mounted on the palatal aspect of removable acrylic splints and exposed to the oral environment in three subjects for 2, 6, 12 and 24 h. Enamel specimens with and without pellicles were immersed in citric acid (1%) for 60 s, and the amount of dissolved calcium was measured by atomic absorption spectroscopy. In addition, specimens were processed for transmission electron microscopy (TEM). Mean values (standard deviations) for calcium release (mg/l related to the specimen's surface area of 5×5 mm²) were: 2-h pellicle 6.94 (1.55); 6-h pellicle 6.69 (2.05); 12-h pellicle 6.57 (2.31); 24-h pellicle 5.71 (2.46); enamel without pellicle 8.95 (1.66). There were no significant differences in calcium release that were dependent on pellicle formation time, but in comparison to enamel specimens without pellicle, significantly less (p <0.05) demineralization of the enamel was observed in pellicle-covered specimens. TEM showed that the pellicle was partly, but not completely dissolved following acid exposure. It is concluded that even a 2-h in-situ-formed pellicle layer protects the enamel surface to a certain extent against demineralization.     

Influence of in vivo formed salivary pellicle on enamel erosion.

This study assessed the protective effect of the salivary pellicle formed in vivo during 24 h or 7 days against demineralization of bovine enamel caused by citric acid. In addition, the influence of acid treatment on the behavior of the pellicle was investigated. Enamel specimens with and without in vivo pellicles were immersed in citric acid (0.1, 1.0%) over 30, 60, and 300 s, and processed for scanning (SEM) and transmission electron microscopy (TEM), as well as for measurement of surface microhardness (SMH). Specimens coated with the in vivo formed pellicles revealed less extensive erosive demineralization of the enamel surface compared to uncovered enamel specimens. SEM analysis and SMH results did not indicate distinct differences between erosive surface alterations on enamel slabs covered with 24-hour pellicles and on those covered with 7-day pellicles. TEM analysis showed that the pellicle layer was dissolved in part from the enamel surface due to acid exposure. However, pellicle residues could be detected by TEM in all specimens, even after 5-min exposure to 1.0% citric acid. It is concluded that the in vivo salivary pellicle can resist the acidic action to some extent and provides protection to the underlying enamel surface against erosive destruction caused by short-term action of citric acid.     

Root surface characteristics associated with subgingival placement of monolithic tetracycline-impregnated fibers.

The purpose of this investigation was to inspect and characterize the subgingival root surface after a 10-day exposure in vivo to 25% tetracycline hydrochloride by weight in an ethylene vinyl acetate copolymer fiber matrix with and without root planing therapy. The root surfaces were examined by fluorescent-light microscopy (FLM), scanning electron microscopy (SEM), and energy dispersive spectroscopy (EDS). Thirty-two teeth were selected for study, 4 from each of 8 patients. The teeth of each patient were randomly assigned to one of four treatment groups: non-treated control (C), scaling and root planing only (RP), tetracycline-impregnated fiber only (F), and scaling and root planing with tetracycline-impregnated fiber application (RP/F). SEM revealed a visible reduction in the subgingival microbial flora in both the F and RP/F treatment groups in comparison with the C group specimens. Many of the residual bacteria observed in F and RP/F specimens appeared non-viable, exhibiting obvious loss of membrane integrity. In contrast, the RP specimens exhibited randomly distributed areas of residual subgingival plaque and calculus with newly developing plaque fronts; the plaque fronts undoubtedly having formed during the 10 days post-therapy. All RP/F specimens exhibited an incomplete removal of adsorbed root surface pellicle and demineralization of the subsequently exposed root surface. EDS analysis of large crystals adhering to root surfaces of F and RP/F specimens revealed high chloride peaks, suggesting the presence of residual tetracycline. FLM examination of F and RP/F treated specimens showed a superficial penetration of tetracycline into the root surface of about 10 microns. Areas of demineralized root showed slight tetracycline penetration into exposed dentinal tubules.     

Ultrastructural study of early dental plaque formation

Initial Colonization and early plaque formation were studied using hydroxyapatite splint segments attached to buccal surfaces of maxillary molar and premolar teeth in six young adults given a low-sucrose diet. Segments were removed at intervals of 2, 4, 6, 12, 24 and 48 hr. Bacteria were first regularly seen in 4 or 6 hr specimens where they occurred as individual cells or as small groups of cells. No structures resembling preformed aggregates or hemispherical bacterial "globules" could be demonstrated. The bacteria most frequently attached to the pellicle surface diectly by their cell wall and gram-positive cocci were most abundant. Another mode of bacterial attachment to pellicle was by means of fine bifbrils or coarser thread-like structures. Occasionally organisms were seen attaching to apatite surfaces without interjacent pellicle material and sometimes they appeared to be completely embedded in the pellicle. Bacteria colonizing on epithelial cells regularly displayed a "fuzzy coat". Extensions of organic material from the pellicle surface sometimes made it difficult to distinguish between pellicle and plaque matrix. No clear indication of bacteria metabolizing the pellicle was seen. Trilaminar vesicles probably originating from the surface of degenerating cells were especially abundant in areas with Gramnegative bacteria. In areas with Gram-positive cells the amount of plaque matrix was greater and a number of cells displayed surface threads. The outer surface of the plaque ordinarily did not show a layer of extracellular organic material although a granular laer could be seen in local areas. These findings lend support to a concept of plaque formation as a sequential build-up resulting from selective attachment and growth of individual organisms rather than resulting from attachment of aggregates of bacteria or a passive entrapment of organisms in a matrix.     

Dental biofilms at healthy and inflamed gingival margins

Objectives: The increased plaque formation observed in gingival inflammation is not fully understood. Receptor proteins in the dental pellicle might influence bacterial adhesion and subsequent plaque formation. The purpose of the present study was to examine proteins and microorganisms in dental biofilms, at healthy and inflamed gingival margins. Material and methods: To see whether marginal inflammation affects the composition of the pellicle and the early dental plaque, samples were taken from the gingival and incisal parts of teeth in periodontally healthy subjects, both in gingival health and during experimental gingivitis. Pellicle proteins were analysed using gel-electrophoresis, immunoblotting and image analysis. Bacteria were analysed by culturing and the PCR technique. Results: During gingivitis, the rate of plaque formation increased significantly. The semiquantitative amounts of proteins and the numbers of bacteria varied considerably between individuals and surfaces. The amount of total and individual pellicle proteins and the total numbers of bacteria were, however, increased during gingivitis and the increase in proteins was statistically significant on the incisal parts of tooth surfaces. Compared to a healthy gingiva, reduced numbers for Actinomyces spp. (incisal parts only) and streptococci and increased numbers of periodontopathogens in the 4 h dental biofilms were seen at the inflamed gingiva. Conclusion: Increased gingival crevicular fluid flow during gingivitis affects pellicle formation and increased plasma proteins in the pellicle may modify bacterial attachment and early dental plaque composition.     

Effect of experimentally induced marginal periodontitis and periodontal scaling on the dental pulp

Experimental breakdown of the periodontal attachment apparatus was produced in six young adult monkeys to study the effect on the tissue of the dental pulp by (1) periodontitis, (2) scaling and plaque accumulation on exposed root dentin. Periodontal tissue breakdown was induced by the placement of ligatures around the neck of 92 permanent teeth. Subsequent plaque formation caused marked loss of periodontal tissue support, which after a period of 5--7 months amounted to 30--40% of the root length. One group of teeth received no further treatment. Other teeth were subjected to scaling and root planing. Following treatment, plaque was allowed to accumulate for 2, 10, and 30 days on the freshly planed root dentin surfaces. Histologic examination revealed that in comparison to teeth with normal periodontal conditions, 57% of the teeth exposed to periodontitis exhibited pathologic pulp tissue alterations. Secondary dentin formation and/or inflammatory cell infiltrates were observed within localized areas of the pulp subjacent to root surfaces exposed to periodontal tissue destruction. The changes within the pulp were of "mild" nature and only one tooth displayed signs of total pulp necrosis. Lateral canals communicating with both the pulp cavity and the exposed root surface were never detected. Teeth subjected to scaling and subsequent plaque accumulation in comparison with teeth with periodontitis alone exhibited no obvious aggravation or increased incidence of pathologic pulp reactions. The findings show that in the monkey (1) periodontal destruction limited to the cervical half of the root and (2) plaque accumulation on exposed root dentin does not cause severe alteration in the pulp of the roots involved.     

Immediate dentin sealing improves bond strength of indirect restorations

STATEMENT OF PROBLEM: Delayed dentin sealing is traditionally performed with indirect restorations. With this technique, dentin is sealed after the provisional phase at the cementation appointment. It was demonstrated that this chronology does not provide optimal conditions for bonding procedures. Immediate dentin sealing (IDS) is a new approach in which dentin is sealed immediately following tooth preparation, before making the impression. PURPOSE: The purpose of this study was to determine whether there were differences in microtensile bond strength to human dentin using IDS technique compared to delayed dentin sealing (DDS). MATERIAL AND METHODS: Fifteen freshly extracted human molars were obtained and divided into 3 groups of 5 teeth. A 3-step etch-and-rinse dentin bonding agent (DBA) (OptiBond FL) was used for all groups. The control (C) specimens were prepared using a direct immediate bonding technique. The DDS specimens were prepared using an indirect approach with DDS. Preparation of the IDS specimens also used an indirect approach with IDS immediately following preparation. All teeth were prepared for a nontrimming microtensile bond strength test. Specimens were stored in water for 24 hours. Eleven beams (0.9 x 0.9 x 11 mm) from each tooth were selected for testing. Bond strength data (MPa) were analyzed with a Kruskal-Wallis test, and post hoc comparison was done using the Mann-Whitney U test (alpha=.05). Specimens were also evaluated for mode of fracture using scanning electron microscope (SEM) analysis. RESULTS: The mean microtensile bond strengths of C and IDS groups were not statistically different from one another at 55.06 and 58.25 MPa, respectively. The bond strength for DDS specimens, at 11.58 MPa, was statistically different (P=.0081) from the other 2 groups. Microscopic evaluation of failure modes indicated that most failures in the DDS group were interfacial, whereas failures in the C and IDS groups were both cohesive and interfacial. SEM analysis indicated that for C and IDS specimens, failure was mixed within the adhesive and cohesively failed dentin. For DDS specimens, failure was generally at the top of the hybrid layer in the adhesive. SEM analysis of intact slabs demonstrated a well-organized hybrid layer 3 to 5 microm thick for the C and IDS groups. For DDS specimens the hybrid layer presented a marked disruption with the overlying resin. CONCLUSIONS: When preparing teeth for indirect bonded restorations, IDS with a 3-step etch-and-rinse filled DBA, prior to impression making, results in improved microtensile bond strength compared to DDS. This technique also eliminates any concerns regarding the film thickness of the dentin sealant.     

Radiodensity of enamel and dentin of human, bovine and swine teeth

Several studies have aimed to evaluate the characteristics of hard dental tissues from animal species in order to adequately substitute the usage of human teeth. The purpose of this study was to evaluate the radiodensity of enamel and dentin of human, bovine and swine teeth. Five specimens of 2 mm in thickness were obtained from human, bovine and swine teeth and the radiographic images were taken positioning it on a phosphor plaque digital system, Digora (Soredex, Helsinki, Finland). The radiodensity of each specimen was obtained and data were compared by ANOVA following Tukey test (P < 0.05). The results showed that human and bovine enamel presented similar radiodensity, which was higher than the one from swine enamel; bovine and swine dentin presented similar radiodensity, and only bovine dentin presented greater similarity to human dentin. Bovine teeth seems to be more similar to human teeth in respect to radiodensity.