Growth of dental plaque on hydroxyapatite splints

A technique was developed for growing dental plaque in vivo on an artificial surface which contains hydroxyapatite. Splints made of hydroxyapatite crystals embedded in an epoxy resin were adapted and cemented to the buccal surfaces of maxillary teeth in human test subjects. The splints were left in place for 24 or 48 hours. Observations in scanning and transmission electron microscopes revealed that plaque accumulated both in epoxy resin and hydroxyapatite areas of the splint surface. An extensive network of thread-like structures connected the individual organisms to each other and to the underlying pellicle layer. This layer generally was somewhat thicker on the crystal surfaces than on the epoxy resin, possibly due to enhanced absorption of glycoproteins to the high energy surface of the hydroxyapatite crystals. Advantages of the splint method are: 1) Easy and rapid technique unrestricted by the presence of cavities or available teeth for extraction. 2) Plaque development may occur on hydroxyapatite surfaces which can be readily sectioned for transmission electron microscopy without decalcification. 3) Sequential colonization can be studied by removing segments of the splint at different time intervals.     

Scanning and transmission electron microscope study of pellicle morphogenesis.

The morphology of pellicle formation was studied using correlated transmission and scanning electron microscopy. Pellicle developed on hydroxyapatite/epoxy resin splints attached to the buccal surfaces of molars and premolars in six young individuals. Splint segments were removed at intervals of 2, 4, 6, 12, 24, and 48 h. Measurements of pellicle thickness revealed that during the first 12 h considerably more material formed on the apatite surfaces than on the epoxy resin areas of the splints. This difference was equalized in 24- and 48-h specimens. Three types of pellicles could be distinguished on the basis of morphologic criteria. A globular pellicle, characterized by a consistent presence of globules of varying size and configuration, seemed to predominate in most subjects. These globules could not be mistaken for bacteria due to their dimensions, contour, and lack of cell walls. A fibrillar pellicle consisted of 3- to 7-nm-wide fibrils and frequently included some finely globular particles. A granular pellicle displayed a relatively even surface contour and did not exhibit globular or fibrillar structures. In contrast to the two other types, the granular pellicle contained distinct laminations. Apparently, the varying morphology of the early pellicle is a reflection of its complex chemical composition.     

Scanning and transmission electron microscope study of pellicle morphogenesis

abstract – The morphology of pellicle formation was studied using correlated transmission and scanning electron microscopy. Pellicle developed on hydroxyapatite/epoxy resin splints attached to the buccal surfaces of molars and premolars in six young individuals. Splint segments were removed at intervals of 2 , 4, 6, 12, 24, and 48 h. Measurements of pellicle thickness revealed that during the first 12 h considerably more material formed on the apatite surfaces than on the epoxy resin areas of the splints. This difference was equalized in 24- and 48-h specimens. Three types of pellicles could be distinguished on the basis of morphologic criteria. A globular pellicle., characterized by a consistent presence of globules of varying size and configuration, seemed to predominate in most subjects. These globules could not be mistaken for bacteria due to their dimensions, contour, and lack of cell walls. A fibrillar pellicle consisted of 3- to 7-nm-wide fibrils and frequently included some finely globular particles. A granular pellicle displayed a relatively even surface contour and did not exhibit globular or fibrillar structures. In contrast to the two other types, the granular pellicle contained distinct laminations. Apparently, the varying morphology of the early pellicle is a reflection of its complex chemical composition.     

Formation of an experimental dental cuticle

abstract – Slices of dental enamel, cementum and dentin, and segments of epoxy resin splints with exposed synthetic hydroxyapatite crystals were incubated in human serum overnight. Observations by light and electron microscopy revealed that a cuticular structure with staining reactions and ultrastructural appearance similar to those of dental cuticle had formed on the various surfaces. The results support the hypothesis that the dental cuticle in vivo may consist of serum components which have been adsorbed to the tooth surface as a result of gingival exudation.     

Effect of silicone oil on protein adsorption to hydroxyapatite in vitro and on pellicle formation in vivo

Abstract – Silicone oil has been introduced in a dentifrice for smokers because of its effect as a polishing agent. Silicone oils are hydrophobic in character and have low surface tensions and good wetting properties. Due to the low surface tension, silicone oils may spread readily on solid surfaces and cover them with a thin, water-repellant film. Introduced via dentifrices silicone oil may thus well be able to adsorb to enamel surfaces and to interfere with surface characteristics such as protein adsorption. The aim of the present study was to examine the effect of silicone oil on protein adsorption to hydroxyapatite (HA) in vitro and on pellicle formation in vivo. The effect on protein adsorption to HA in vitro was studied by adsorption of albumin to either untreated or silicone oil treated HA powders. Ion exchange chromatography was also used with either untreated or silicone oil treated HA as bed materials. The effect on pellicle formation in vivo was studied using enamel fragments carried in the mouth to acquire pellicle material. The chemical composition of the acquired pellicle was studied by collection and chemical analysis of pellicle material formed on enamel surfaces in vivo. The study showed that silicone oil treated HA took up less protein and that the adsorbed protein was bound to hydroxyapatite by a different mechanism as compared to untreated controls. The results indicated that hydrophobic interactions could be involved in binding of proteins to silicone oil treated hydroxyapatite. Silicone oil treated enamal fragments carried in the mouth showed a slower rate of pellicle formation as compared to untreated fragments. The amino acid composition of the acquired pellicle collected in vivo from silicone oil treated enamel surfaces was also different from pellicle material collected from untreated enamel.     

Effect of silicone oil on protein adsorption to hydroxyapatite in vitro and on pellicle formation in vivo

Silicone oil has been introduced in a dentifrice for smokers because of its effect as a polishing agent. Silicone oils are hydrophobic in character and have low surface tensions and good wetting properties. Due to the low surface tension, silicone oils may spread readily on solid surfaces and cover them with a thin, water-repellent film. Introduced via dentifrices silicone oil may thus well be able to adsorb to enamel surfaces and to interfere with surface characteristics such as protein adsorption. The aim of the present study was to examine the effect of silicone oil on protein adsorption to hydroxyapatite (HA) in vitro and on pellicle formation in vivo. The effect on protein adsorption to HA in vitro was studied by adsorption of albumin to either untreated or silicone oil treated HA powders. Ion exchange chromatography was also used with either untreated or silicone oil treated HA as bed materials. The effect on pellicle formation in vivo was studied using enamel fragments carried in the mouth to acquire pellicle material. The chemical composition of the acquired pellicle was studied by collection and chemical analysis of pellicle material formed on enamel surfaces in vivo. The study showed that silicone oil treated HA took up less protein and that the adsorbed protein was bound to hydroxyapatite by a different mechanism as compared to untreated controls. The results indicated that hydrophobic interactions could be involved in binding of proteins to silicone oil treated hydroxyapatite. Silicone oil treated enamel fragments carried in the mouth showed a slower rate of pellicle formation as compared to untreated fragments. The amino acid composition of the acquired pellicle collected in vivo from silicone oil treated enamel surfaces was also different from pellicle material collected from untreated enamel.     

Ultrastructural investigation of pellicle morphogenesis at two different intraoral sites during a 24-h period

The purpose of the present in vivo study was to examine salivary pellicle formation on enamel surfaces at two different intraoral sites for periods of 1 min up to 24 h by means of transmission electron microscopy. Bovine enamel specimens were attached to the buccal and lingual surfaces of the upper first molars in three subjects using removable intraoral splints. Specimens were carried over periods of 1, 10, 30 and 60 min, 2, 6 and 24 h and were processed for transmission electron microscopy. After 1 min, an electron dense pellicle layer, 10–20 nm thick, was observed on the enamel surfaces. The subsequent adsorption of salivary biopolymers was governed by local influences of the oral cavity. Specimens located on the lingual aspect were covered within 2 h by a 20- to 80-nm-thick, homogeneous, predominantly granular-structured pellicle. The thickness of the surface coatings that were adsorbed on lingually carried specimens increased to 100–200 nm after 24 h. In contrast, on the buccally mounted specimen surfaces, a variably structured pellicle with granular and globular components could be detected after intraoral exposure for 2 h. The thickness of the 2-h buccal pellicles ranged between 200 and 700 nm. After 24 h, the buccally positioned specimens were covered by a dense globular pellicle layer varying in thickness from 1000 to 1300 nm. It is suggested that in vivo pellicle formation is initiated by adsorption of an electron-dense layer of salivary proteins. Further adsorption of salivary biopolymers leads to the formation of an outer loosely arranged pellicle layer. Under oral conditions, the locally available salivary biopolymers and the influence of locally effective shearing forces are of significance for the ultrastructural pattern and extent of pellicle formation. Received: 8 March 1999 / Accepted: 19 April 1999     

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.     

Desorption of acquired enamel pellicle in vivo by pyrophosphate

Soluble pyrophosphate (PP) has been introduced in dentifrices to inhibit the formation of dental calculus. The mechanism of inhibition is probably an adsorption of the pyrophosphate ions to the Ca-sites on the enamel surfaces and a blocking of the active sites for crystal growth. It has been shown in a recently published study that PP reduced the protein adsorption to hydroxyapatite (HA) in vitro and also inhibited the pellicle formation in vivo. The aim of the present study was to examine the desorption potential of pyrophosphate on the acquired enamel pellicle in vivo. Enamel fragments were carried in the mouth to collect pellicle material and some of the enamel surfaces were then treated with PP. Pellicle formation was examined by SEM of the enamel surfaces. The results showed that pyrophosphate desorbed the acquired enamel pellicle effectively. The clinical consequences of this effect is unknown, but it could possibly explain some aspects of hypersensitivity of teeth observed in some individuals using dentifrices containing PP.     

Desorption of acquired enamel pellicle in vivo by pyrophosphate

Abstract – Soluble pyrophosphate (PP) has been introduced in dentifrices to inhibit the formation of dental calculus. The mechanism of inhibition is probably an adsorption of the pyrophosphate ions to the Ca-sites on the enamel surfaces and a blocking of the active sites for crystal growth. It has been shown in a recently published study that PP reduced the protein adsorption to hydroxyapatite (HA) in vitro and also inhibited the pellicle formation in vivo. The aim of the present study was to examine the desorption potential of pyrophosphate on the acquired enamel pellicle in vivo. Enamel fragments were carried in the mouth to collect pellicle material and some of the enamel surfaces were then treated with PP. Pellicle formation was examined by SEM of the enamel surfaces. The results showed that pyrophosphate desorbed the acquired enamel pellicle effectively. The clinical consequences of this effect is unknown, but it could possibly explain some aspects of hypersensitivity of teeth observed in some individuals using dentifrices containing PP.     

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.     

Formation and photocatalytic decomposition of a pellicle on anatase surfaces

The acquired dental pellicle plays a critical role in the adhesion and detachment of dental plaque bacteria. It has been reported that titanium dioxide biomaterials decompose single-protein films by photocatalysis. However, it is not known whether this can also be achieved with complex structured pellicle films. This in vitro study investigated in real-time the formation and photocatalytic decomposition of human pellicle at anatase-saliva interfaces. Nanostructured polycrystalline anatase layers were deposited on titanium-coated quartz crystals by magnetron-sputtering, serving as a model for titanium implant surfaces. The quartz crystals were used as acoustic sensors in a quartz-crystal microbalance (QCM) system with dissipation. In situ UV irradiation of pellicle-covered anatase caused a statistically significant decrease of the adsorbed salivary mass. In contrast, photocatalytic decomposition of pellicle could not be observed on reference titanium surfaces. Wettability characterization revealed superhydrophilicity of anatase upon UV irradiation, whereas titanium was unaffected. XPS measurements provide further information concerning the decomposition of the salivary films. The results suggest that the photocatalytic activity of polycrystalline anatase-modified biomaterial surfaces is able to decompose complex structured macromolecular pellicle films. Therefore, this study opens the way to surface modifications supporting therapeutic approaches of biofilm removal.     

Intrinsic enzymatic crosslinking and maturation of the in situ pellicle

AIM: The acquired enamel pellicle is a proteinaceous layer formed on all solid substrata exposed to the oral cavity. It has been supposed that the pellicle undergoes maturation after protein adsorption. The aim of the present study was to investigate enzyme activities with an impact on intrinsic maturation processes in in situ formed pellicles. METHODS: Bovine enamel specimens were exposed to the oral cavity in six subjects to allow in situ pellicle formation over 3, 30 and 120 min. The slabs were fixed on the buccal and palatal surfaces of individual splints fixed with silicone impression material. After rinsing with deionised water, the pellicle samples were tested fluorimetrically for transglutaminase, protease and elastase activity. Phosphatase activities were tested photometrically. Separate samples were used for each of the enzymes tested. RESULTS: Transglutaminase was detected in in situ pellicle (16.7+/-21.2 mU/cm(2)) as was alkaline phosphatase activity (0.87+/-0.99 mU/cm(2)). For both enzymes, there was no correlation of enzyme activities with time or localisation of pellicle formation. Acidic phosphatase- and protease-activities were not detectable. Only traces of elastase activity were found in 57% of the samples. CONCLUSION: Transglutaminase and phosphatase activity are detectable within in situ pellicle. Enzymatic crosslinking and dephosphorylation appear more important for intrinsic maturation of the acquired enamel pellicle than proteolysis.     

Compositional analysis of human acquired enamel pellicle by mass spectrometry

Relatively little is known about the formation of the acquired enamel pellicle other than that it involves the selective adsorption of specific proteins from oral fluids. Previous studies on the identification of pellicle components have relied largely on immunological or enzymatic detection and have been hampered by the fact that only minute quantities of pellicle can be removed from tooth surfaces. The present work describes an improved method of harvesting pellicle that combines mechanical and chemical removal; this approach was used to investigate systematically the desorption of in vitro pellicle components with different solutions. Eleven major in vitro pellicle proteins were identified by using a combination of electrophoretic separation and matrix-assisted laser desorption/ionization-reflectron time-of-flight mass spectrometry. A similar analysis of in vivo-formed pellicle revealed the presence of intact statherin, lysozyme, albumin and amylase. Further analysis of in vivo pellicle by liquid chromatography-electrospray ionization mass spectrometry suggested the presence of numerous low molecular-weight fragments of precursor proteins. The protein composition of in vitro whole-salivary pellicle adsorbed to hydroxyapatite and that of in vivo enamel pellicle differed for proline, the result of a reduction in the content of acidic proline-rich proteins in the in vivo samples. Unique features of the oral environment such as enzymatic activities or mineral surface properties may account for these differences between in vivo and in vitro pellicle formation.     

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.     

Effect of two organic phosphonates on protein adsorption in vitro and on pellicle formation in vivo

Abstract – Organic phosphonates have been introduced in dentifrices to reduce the formation of dental calculus. They may conceivably act as calcium sequestrants or crystal growth inhibitors, interfering directly with the calcium ions on the hydroxyapatite (HA) and enamel surfaces. The aim of the present study was to examine the effect of two organic phosphonates on protein adsorption to hydroxyapatite in vitro and on pellicle formation in vivo. The effect on protein adsorption in vitro was studied by adsorption of albumin to either untreated or phosphonate treated HA powder. Ion exchange chromatography was also performed with columns with untreated and phosphonate treated HA as bed materials and with linear gradients of either phosphate or phosphonates. The effect on pellicle formation in vivo was studied by scanning electron microscopy on untreated and phosphonate treated enamel fragments which had been carried in the mouth to acquire pellicle materials. The present study showed that phosphonate-treated HA took up less protein. The adsorbed protein was, furthermore, less, firmly bound to phosphonate treated hydroxyapatite. Phosphonate-treated enamel fragments carried in the mouth also exhibited a slower rate of pellicle formation as compared to the untreated enamel fragments.     

Protective effect of the in situ formed short-term salivary pellicle

Salivary pellicle, as previously investigated, protects the enamel surface after certain processes of maturation against the influence of acidic agents. The aim of the present study was to investigate the protective effect of the short-term salivary pellicle formed in situ over periods of 3, 60 and 120 min. Six human volunteers used intraoral acrylic splints with bovine enamel samples fixed at the buccal and palatal sites of the maxillary first molars and second premolars. Enamel specimens (n = 252) with and without pellicle were immersed for 60 s in 1.0% citric acid solution under agitation. Knoop surface hardness (KHN) of uneroded polished enamel was measured as a baseline and estimated immediately after erosive treatment reflecting the microhardness loss (DeltaKHN). The amounts of calcium dissolved from the eroded enamel surface were analysed by atomic absorption spectroscopy and scored in mg/l per 10 mm2 of enamel surface area. In addition, the scanning electron microscope was used for the micromorphological examination of the erosive alterations of the enamel surface. The average microhardness loss values after erosion of the enamel samples with buccally/palatally formed pellicle layers were measured as 139.1/144.9 DeltaKHN for 3 min pellicle, 145.9/146.9 DeltaKHN for 60 min pellicle and 141.7/138.6 DeltaKHN for 120 min pellicle. Calcium release values from the specimens with buccal/palatal pellicles were amounted to 15.0/14.9, 16.5/15.9 and 15.3/17.4 mg/l per 10 mm2 for 3, 60 and 120 min-old pellicles, respectively. No significant differences were related to the pellicle formation time and intraoral site (buccal or palatal) in all tested series (ANOVA, P < 0.05). However, significant protection of the enamel surface provided by the pellicle layer was observed on all pellicle-covered surfaces if compared to the non-covered enamel samples (calcium release: 25.6 mg/l per 10 mm2; microhardness loss 187.0 DeltaKHN). These data were in accordance with the morphologic alterations caused by citric acid on the pellicle-covered and pellicle non-covered specimens. It could be concluded that salivary pellicle formed in situ within a period of 3 min offers protection of enamel against citric acid. However, pellicle does not completely inhibit the erosive action of citric acid under the conditions of the present study.     

Dental plaque morphology as revealed by direct observation and by replicating techniques.

The ability of four elastomer impression materials to reproduce details of bacterial plaque structures was studied by comparing areas of the replica models with corresponding areas of the replicated plaque. Plaque was grown on hydroxyapatite splint segments for 48 hours and replica models were made from three different resins. One of the resins, a methacrylate material, was not suited for this purpose due to its content of spherical particles. The polysulfide and polyether impression materials reacted with the heat curing epoxy resin, and less satisfactory results were obtained with cold curing epoxy resin. Best results were obtained by a combined use of low viscosity silicone impression materials and cold or heat curing epoxy resins as model materials. However, a considerable loss of detail occurred in the replica models compared with observations of plaque directly on splint surfaces. Outlines of individual bacteria could sometimes be seen in the models, but generally several cells seemed to fuse, and appeared like small globular structures where cells and pellicle were difficult to distinguish. The bacterial colonization started near the gingival border of the surface, initiated by attachment of individual bacteria to the pellicle surface.     

Morphologic studies on dental plaque formation.

The present summary and the following papers (I--VII) are submitted in partial fulfillment of the requirements for the degree of Doctor Odontologiae at the University of Bergen. 1: Growth of dental plaque on hydroxyapatite splints. A method of studying early plaque morphology. J. Periodontal Res. 1974, 9, 135--146. II: Pellicle formation on hydroxyapatite splints attached to the human dentition: Morphologic confirmation of the concept of adsorption. Arch. Oral Biol. 1975, 20, 739-742. III: Scanning and transmission electron microscope study of pellicle morphogenesis. Scand. J. Dent. Res. 1977, 85, 217-231. IV: Early dental plaque morphogenesis. A scanning electron microscope study using the hydorxyapatite splint model and a low-sucrose diet. J. Periodontal Res. 1977, 12, 73-89. V: Ultrastructural study of early dental plaque formation. J. Periodontal Res. 1978, 13, 391-409. VI: Dental plaque morphology as revealed by direct observation and by replicating techniques. Acta Odontol. Scand. 1978, 36, 279-288 Co-author: F. Gusberti. VII: Replica study of plaque formation on human tooth surfaces. Acta Odontol. Scand. 1979, 37, 65-72. Co-author: F. Gusberti. These papers are referred to as I-VII in the present summary. Other papers are referred to in conventional manner.     

Effect of two organic phosphonates on protein adsorption in vitro and on pellicle formation in vivo

Organic phosphonates have been introduced in dentifrices to reduce the formation of dental calculus. They may conceivably act as calcium sequestrants or crystal growth inhibitors, interfering directly with the calcium ions on the hydroxyapatite (HA) and enamel surfaces. The aim of the present study was to examine the effect of two organic phosphonates on protein adsorption to hydroxyapatite in vitro and on pellicle formation in vivo. The effect on protein adsorption in vitro was studied by adsorption of albumin to either untreated or phosphonate treated HA powder. Ion exchange chromatography was also performed with columns with untreated and phosphonate treated HA as bed materials and with linear gradients of either phosphate or phosphonates. The effect on pellicle formation in vivo was studied by scanning electron microscopy on untreated and phosphonate treated enamel fragments which had been carried in the mouth to acquire pellicle materials. The present study showed that phosphonate-treated HA took up less protein. The adsorbed protein was, furthermore, less firmly bound to phosphonate treated hydroxyapatite. Phosphonate-treated enamel fragments carried in the mouth also exhibited a slower rate of pellicle formation as compared to the untreated enamel fragments.