Dental pulp inflammatory/immune response to a chitosan-enriched fibrin hydrogel in the pulpotomised rat incisor.

Current pulpotomy is limited in its ability to induce regeneration of the dental-pulp (DP) complex. Hydrogels are reported to be well-suited for tissue engineering and are unlikely to induce an inflammatory response that might damage the remaining tissue. The present study investigated the molecular and cellular actors in the early inflammatory/immune response and deciphered M1/M2 macrophage polarisation to a chitosan-enriched fibrin hydrogel in pulpotomised rat incisors. Both fibrin and fibrin-chitosan hydrogels induced a strong increase in interleukin-6 (IL-6) transcript in the DP when compared to the DP of untreated teeth. Gene expression of other inflammatory mediators was not significantly modified after 3 h. In the viable DP cell population, the percentage of leukocytes assessed by flow cytometry was similar to fibrin and fibrin-chitosan hydrogels after 1 d. In this leukocyte population, the proportion of granulocytes increased beneath both hydrogels whereas the antigen-presenting cell, myeloid dendritic cells, T cells and B cells decreased. The natural killer (NK) cell population was significantly decreased only in DPs from teeth treated with fibrin-chitosan hydrogel. Immunolabeling analysis of the DP/hydrogel interface showed accumulation of neutrophil granulocytes in contact with both hydrogels 1 d after treatment. The DP close to this granulocyte area contained M2 but no M1 macrophages. These data collectively demonstrated that fibrin-chitosan hydrogels induced an inflammatory/immune response similar to that of the fibrin hydrogel. The results confirmed the potential clinical use of fibrin-chitosan hydrogel as a new scaffold for vital-pulp therapies.

[Bone density changes in the apical area after rapid orthodontic extrusion of subgingivally fractured tooth].

OBJECTIVE: To evaluate the bone density changes in the apical area of subgingivally fractured tooth after rapid orthodontic extrusion. METHODS: Twelve fractured incisors in 11 patients extended 2 - 5 mm below the gingival line were selected. Two weeks after root canal therapy, the subgingival fragment was lifted up and the fracture line was brought 1.5 - 2.0 mm above the level of the gingival line by means of edgewise fixed appliance. After the extrusion completed, the tooth had been stabilized and held for 6 months. The CT Analyser software was used to measure the bone density changes in the apical area on radiographs once a month. Changes of relative value in bone density was quantitatively analyzed. RESULTS: The average period of extrusion was 11 days. The relative value of bone density in the apical region was -39.6% immediately after extrusion and continuously increased afterwards. In the third month, the value (18.5%) changed most rapidly (P < 0.01). CONCLUSIONS: Under the continuous and proper tooth axial extrusion force, the tooth moved rapidly and steadily. The bone density in the apical area approached normal value within 3 months after treatment.

A flow cytometric analysis of the biodefensive response of deciduous tooth pulp to carious stimuli during physiological root resorption.

The present study observed biodefensive responses of deciduous tooth pulp to advancing caries lesions and analysed the role of physiological root resorption on pulpal defense potential. For this purpose, immunocompetent cell content of deciduous tooth pulp was examined using flow cytometry. A total of 49 deciduous incisor and molar teeth at various stages of physiological root resorption (carious or non-carious) to be extracted for clinical reasons were used in this study. Teeth were classified according to carious lesion depth and root resorption stage. CD3+ lymphocytes were observed to be most prevalent in the pulp and to show remarkable increase along with increase in carious lesion depth. Numbers of CD8+ lymphocytes also increased significantly as carious lesions approached the pulp. However, increase in the number of CD3+ and CD8+ cells did not significantly alter CD4+/CD8+ ratios. The study also found that while B-lymphocytes increased significantly in association with root resorption, there were no significant differences in B/CD3+ lymphocyte ratios. Thus, there was no evidence of irreversible pulpal pathosis in any groups. It can be concluded that pulp maintains its healing and defense capacity against advancing carious lesion and progressive root resorption in deciduous teeth.

Postnatal expression of calretinin-immunoreactivity in periodontal Ruffini endings in the rat incisor: a comparison with protein gene product 9.5 (PGP 9.5)-immunoreactivity.

The postnatal expression of immunoreactivity for calretinin, one of the calcium binding proteins, and for protein gene product 9.5 (PGP 9.5), a general neuronal marker, was investigated in mechanoreceptive Ruffini endings in the periodontal ligament of the rat incisor. Age-related changes in the expression of these two proteins in periodontal nerves were further quantified with a computerized image analysis. At 1 day after birth, a few PGP 9.5-immunoreactive nerve fibers and a still smaller number of calretinin-positive fibers were found in the periodontal ligament: they were thin and beaded in appearance and no specialized nerve terminals were recognized. Tree-like terminals, reminiscent of immature Ruffini endings, were recognizable in 4-day-old rats by PGP 9.5-immunohistochemistry, while calretinin-immunostaining failed to reveal these specialized endings. At postnatal 7-11 days when PGP 9.5-immunostaining could demonstrate typical Ruffini endings, calretinin-immunopositive nerve fibers merely tapered off without forming the Ruffini type endings. A small number of Ruffini endings showing calretinin-immunoreactivity began to occur in the periodontal ligament at 24-26 days after birth when the occlusion of the first molars had been established. At the functional occlusion stage (60-80 days after birth), the Ruffini endings showing calretinin-immunoreactivity drastically increased in number and density, but less so than those positive for PGP 9.5-immunoreaction. The delayed expression of calretinin suggests that the function of the periodontal Ruffini endings is established after the completion of terminal formation because Ca2+, which binds to calcium binding proteins including calretinin with high affinity, plays an important role in mechano-electric transduction.

Loss of deciduous teeth and germs of permanent incisors in a 4-year-old child. An atypic prepubertal periodontitis? A clinical, microbiological, immunological and ultrastructural study.

A 4-year-old child was referred, in April 1988, to Rennes Dental School (France) for deciduous tooth mobility with premature loss of 4 deciduous teeth and germs of 2 permanent incisors. Microbiological examinations by culture revealed the presence of the periodontal pathogen Actinobacillus actinomycetemcomitans. Immunofluorescence of plaque samples revealed the presence of Porphyromonas gingivalis that had not been isolated by culture. Neutrophil functions were within normal ranges. Transmission electron microscopy of gingiva showed a disorganised epithelium. The connective tissue was infiltrated by inflammatory cells. The basement membranes were normal, but the connective tissue-epithelium interface was mainly composed of short rete pegs. Scanning electron microscopy of extracted deciduous teeth revealed lack of cementum, lacunae in the cementum and lack of fibrillar insertion on the middle part of the root. Skin lesions, mainly situated on face, were observed. Treatment was by extraction of mobile deciduous teeth combined with 3-week courses of metronidazole. Clinical and microbiological follow-up was continued over a 7-year period. No periodontal lesions have been detected since eruption of the permanent teeth. The present subgingival and lingual microflora (December 1995) is composed of bacteria associated with periodontal health. However, the future appearance of a hitherto undetected systemic disease is still possible.

Dendritic cells: a novel cellular component of the rat incisor enamel organ appearing in the late stages of enamel maturation.

Immunocompetent cells in the enamel organ of rat incisors were examined immunohistochemically using OX6, ED1, and ED2 monoclonal antibodies known to recognize the Class II MHC molecules, a monocyte-macrophage lineage, and residential macrophages, respectively. The OX6 immunopositive cells (MHC cells) were located exclusively in the enamel maturation zone. MHC cells increased in number in the incisal direction and occasionally extended cytoplasmic processes deep into the ameloblast layer. Migration of MHC cells in the ameloblast layer were also encountered. MHC cells lacked phagolysosomes and could be distinguished from typical macrophages. ED2 immunopositive cells were not seen in the enamel organ. ED1 positive cells displayed identical localization to MHC cells except that some appeared in the transitional zone. MHC cells could not be seen in the enamel organ of rat molar tooth germs. Our data confirmed the presence of a large population of "dendritic" immunocompetent cells in the enamel organ of rat incisors and characterized the ultrastructural features of these cells. Biological significance of the immunocompetent cells in the enamel organ during amelogenesis needs to be clarified.

The relationship between odontoblasts and immunocompetent cells during dentinogenesis in rat incisors: an immunohistochemical study using OX6-monoclonal antibody.

The relationship between odontoblasts and class II major histocompatibility complex (MHC) antigen-expressing cells in the process of dentinogenesis was studied in rat lower incisors, employing immunohistochemistry using OX6-monoclonal antibody. The dental pulp contained numerous OX6-immunopositive cells that varied in morphology from dendritic to spindle under physiological conditions. Under the electron microscope, these immunopositive cells shared common cytoplasmic features, i.e., multivesicular bodies and characteristic fine tubulovesicular structures in their cytoplasm. At the early stage of dentinogenesis, OX6-immunopositive cells, presumably of the immature type, were located in the subodontoblastic layer. During active dentin formation, the OX6-immunopositive cells increased in number and appeared in the odontoblast layer, associating intimately with fenestrated capillaries situated close to the predentin. These cells showed a dendritic appearance and possessed various sizes of multivesicular bodies and characteristic fine tubulovesicular structures, but never contained typical phagosomes. On the other hand, immunopositive macrophages characterized by typical phagosomes tended to occupy the central portion of the pulp. The results suggest that most, if not all, OX6-immunopositive cells situated deep in the odontoblast layer are dendritic cells playing a role in the defense system of the dental pulp against antigenic molecules arriving from the circulation via the fenestrated capillaries. The increasing number of OX6-immunopositive or immunonegative macrophages appearing near the incisal end of the tooth is thought to be involved in the elimination of degenerated odontoblasts.

Chondroitin sulfates in developing mouse tooth germs. An immunohistochemical study with monoclonal antibodies against chondroitin-4 and chondroitin-6 sulfates.

The role of glycosaminoglycans and proteoglycans during ontogenesis is not known. The developing tooth offers a potentially important model for studies of structure-function relationships. In this study, we have analysed the temproal and spatial expression of chondroitins of differing sulfation patterns in embryonic molars and incisors. For this purpose, we have used monoclonal antibodies (Mabs) specific for unsulfated, 4-sulfated, and 6-sulfated forms of chondroitin in conjunction with indirect immunofluorescence or immunoperoxidase labeling. Unsulfated chondroitin was not detected in embryonic teeth. Chondroitin 4- and chondroitin 6-sulfates were present in the stellate reticulum but otherwise they were confined to the dental mesenchyme. The 3B3 and MC21C-epitope, which are markers of 6-sulfated chondroitin, were uniformly distributed in the dental mesenchyme during the bud stage; they disappeared from the dental papilla of the cusps and of the anterior region of the incisor as development proceeded. These epitopes were absent from the basement membrane and from the predentin. In the odontoblastic cell lineage, the 3B3 and MC21C-epitopes were detected only between preodontoblasts at an early stage of differentiation. The monoclonal antibody 2B6 served as a probe to localize chondroitin 4-sulfate. This glycosaminoglycan was detected as early as the dental lamina stage but its expression was restricted to the basement membrane of the teeth until the late bell stage. After the onset of cusp formation, strong staining was also observed over the occlusal region of the dental papilla while the cervical region of the dental papilla remained 2B6-negative. Incisors at the bell stage exhibited a decreasing gradient of immunostaining by 2B6 from their anterior region to their posterior end. The extracellular matrix surrounding preodontoblasts reacted with 2B6 and the predentin, produced by the odontoblasts, was also intensely labeled with this antibody. Comparison between immunostaining with 3B3 and 2B6, on consecutive sections revealed a mutually exclusive pattern of distribution of the corresponding epitopes during odontogenesis. Furthermore, in the continuously growing incisor, a striking positive correlation was found between the immunostaining patterns produced by 3B3 and MC21C and the mitotic indices along the anterior-posterior axis of the tooth. Hence, sulfation of chondroitin seems developmentally regulated. We postulate that changes in the sulfation pattern of chondroitin might play a role in ontogenesis by locally altering the functional properties of the extracellular matrix.

A histochemical study on lectin binding in the immature enamel and secretory ameloblasts of rat incisors.

Four lectin-fluorescent marker conjugates (Con A-F, MPA-F, PNA-F, WGA-R) were used for visualizing their binding sites on tissue sections by fluorescent microscopy. The immature enamel, Tomes' processes and distal cytoplasm of ameloblasts were stained with MPA-F and WGA-R on paraformaldehyde-fixed and paraffin-embedded tissue sections. The MPA- and WGA-binding glycoconjugates seemed to be main components of the organic enamel matrix because of the intense fluorescence from the tissue sections. The central portion of distal cytoplasm of ameloblasts was stained with PNA-F but the immature enamel and Tomes' processes were not. This suggests the PNA-binding glycoconjugates to be intermediates during the process of modifying their oligosaccharide chains or to be elements of Golgi area. Tomes' processes, the distal cytoplasm and periphery of nuclei of ameloblasts were stained with Con A-F. The Con A-binding glycoconjugates except those localizing on Tomes' processes may also be intermediates and/or intracellular elements like the PNA-binding glycoconjugates. Cells of stratum intermedium were stained with Con A-F, MPA-F and WGA-R. Cells of stellate reticulum and outer enamel epithelium were stained with Con A-F and WGA-R. It was indicated that enamel proteins, major organic components of immature enamel, are highly possible candidates for the MPA- and WGA-binding glycoconjugates.

IgE in postsecretory ameloblasts suggesting a hypersensitivity reaction at tooth eruption.

The clinical symptoms associated with the eruption of primary teeth resemble a mild hypersensitivity reaction. Light microscopic examination showed that IgE accumulated in postsecretory ameloblasts. The formation of IgE was elicited by enamel matrix proteins, which are chemotactic for mast cells.

Demonstration of an alloimmune response to embryonic enamel matrix proteins.

Young adult rabbits were immunized with enamel matrix proteins from embryonic tooth organs of the same strain of rabbit. These proteins elicited an alloimmune response as demonstrated by the specific binding of antiserum to enamel matrix which was visualized by indirect immunofluorescent microscopy. The labial and lingual surfaces of embryonic incisor tooth organs were found to share common antigenic determinants. The observations suggest that enamel proteins could possibly be autoantigens.

In vitro attachment of streptococci to the tooth surface.

The ability of Streptococcus strains to adhere to the tooth surface in vitro was investigated. Polished enamel slabs, with and without acquired pellicles, were incubated with buffer suspensions of oral streptococci, and attached bacteria were counted under a microscope using incident light. Low numbers of bacteria adhered to uncoated enamel; the presence of an acquired pellicle significantly enhanced the attachment of all strains tested. The adherence of Streptococcus sanguis was significantly greater than that of Streptococcus salivarius, and both of these strains adhered in greater numbers than did Streptococcus mutans. When bacteria were suspended in whole saliva, the adherence of S. salivarius and S. mutans was inhibited, whereas the adherence of S. sanguis was enhanced in some experiments and inhibited in others. The adherence of S. sanguis and S. salivarius was consistently inhibited by parotid fluid; this inhibitory effect persisted after thorough washing and resonication of the bacterial cells. Incubation in oral fluids was associated with the attachment of bacterial clumps to the pellicle, and parallel investigation revealed agglutination of S. sanguis and S. salivarius by whole saliva and, in particular, parotid fluid. The results are discussed in terms of surface microecology, and are related to the development of dental plaque.