Toshio Izumi

Immunohistochemical study on the immunocompetent cells of the pulp in human non-carious and carious teeth

The condition of the pulp tissue was classified into seven groups according to the depth of carious lesions from stage (S) 0 (non-carious teeth) to S6 (exposed pulp). A substantial change in the infiltration of immunocompetent cells occurred between S3 and S4; all types were markedly increased in S4 as compared to S3, with a remarkable increase in the number of helper T lymphocytes, B-lineage cells, neutrophils and macrophages. Therefore, the pulpal immune reaction to carious stimuli could be classified into early (S1-S3) and advanced phases (S4-S6). In the early phase a cellular immunoresponse would be induced by T-lineage cells, and in the advanced phase the humoral immunoresponse is furthered by B-lineage cells concomitant with the destruction of pulp tissue by proteolytic enzymes released from infiltrating neutrophils and macrophages. Human dental pulp is thus equipped with a functional immune response that is sufficient as a biodefensive mechanism. Dental caries should be treated before S4.

Pro-inflammatory Cytokines Induce Suppressor of Cytokine Signaling-3 in Human Periodontal Ligament Cells

INTRODUCTION Periapical inflammation is initiated by insult to the dental pulp and mediated by inflammatory cytokines in the periodontal tissue. On the other hand, the destruction of tissue can be prevented by the suppression of pro-inflammatory cytokine activity. The balance between these cytokines and their counterregulatory molecules has been suggested to regulate tissue destruction. Suppressors of cytokine signaling (SOCS) proteins are known to suppress inflammatory cytokine signaling via the classic negative feedback loop. However, the mechanism by which they are induced by inflammatory cytokines and regulated during the development of periodontal disease remains to be clarified. We investigated the effects of inflammatory cytokines on SOCS protein expression and their signaling pathways in human periodontal ligament (PDL) cells. METHODS We examined the effect of inflammatory cytokines on SOCSs expression and its signaling pathway in human PDL cells using reverse transcription- and real-time polymerase chain reaction, Western blot methods. Furthermore, we also examined whether these cytokines-induced SOCS-3 suppress chemokines secretion using ELISA methods. RESULTS We found that inflammatory cytokines interleukin (IL)-1beta and IL-6 induced expression of SOCS-3 but not that of SOCS-2 in human PDL cells. IL-1beta and IL-6 simultaneously induced IL-8 and monocyte chemoattractant protein-1 secretion in PDL cells, whereas SOCS-3 overexpression suppressed secretion of these chemokines through inhibition of phosphorylation in downstream signaling. CONCLUSION The results suggest that pro-inflammatory cytokines induced SOCS-3 expression. The SOCS-3 induction suggests playing an important role in negative feedback, suppressing serious destruction of periodontal tissue in apical periodontitis through a chemokine-dependent mechanism.

An immunohistochemical and ultrastructural study of vasomotor nerves in the microvasculature of human dental pulp

Immunohistochemical methods for S-100 protein and neurone-specific enolase showed two types of nerve endings around the pulp microvasculature: the free endings, which comprise the major neural component and are distributed in all types of microvessel such as arterioles, venules and capillaries; and the varicose endings. The varicose ending was a less frequent, minor component observed only in the arterioles. Both immunohistochemical and ultrastructural observations confirmed that the varicose endings were the terminal axons of efferent vasomotor nerves. Further extensive ultrastructural examinations on the vasomotor nerves added the following new findings to our previous reports. Vasomotor nerves sometimes ramified into more than two terminal axons around arterioles, and most of these ramified axons ended in the adventitia-media junction of the arteriolar wall; however, nerve endings occasionally penetrated into the media. These findings suggest an intimate structural association between vasomotor nerves and arterioles in regulating the arteriolar microcirculation in human dental pulp by stimulating smooth-muscle cells not only of the outermost but also of the inner layers. Furthermore, the deep penetration of terminal axons into the arteriolar wall seems to provide effective regulation of pulpal blood flow under physiological and pathological conditions.

Biological Behavior of Human Dental Pulp Cells in Response to Carious Stimuli Analyzed by PCNA Immunostaining and AgNOR Staining

The change in proliferative and metabolic activities of human dental pulp cells responding to carious stimuli was studied by means of immunohistochemical staining for proliferating cell nuclear antigen (PCNA) and silver-binding nucleolar organizer region (AgNOR) staining. We classified the pulp tissues into five groups according to the progression of dental caries, ranging from grade 0 (the pulp of noncarious teeth) to grade 4 (the pulp of perforated carious teeth). PCNA-positive pulp cells were detected only in advanced dental caries (grades 3 and 4), and the difference in immuno-positive rate was significant between the two grades (p <0.001). However, the mean number of AgNORs per nucleus increased even in the early phase of dental caries, significant differences being detected between grades 1 and 2 (p<0.005), 2 and 3 (p<0.005), and 3 and 4 (p<0.001). Our data suggested that the metabolic activity of dental pulp cells was enhanced in the early phase of dental caries. However, proliferation of pulp cells occurred later in small degrees during fully developed caries such as grades 3 and 4. The slow and weak response in cellular proliferation might contribute to the usual fragility of the pulp to various assaults including caries or pulpitis.

An immunohistochemical study of HLA-DR and α1-antichymotrypsin-positive cells in the pulp of human non-carious and carious teeth

The condition of the pulp tissue was classified into seven groups according to the progression of carious lesions from stages S0 (non-carious teeth) to S6 (exposed pulp). There was a small number of anti-HLA-DR antibody-positive cells in the pulp of the early carious teeth, and a markedly increased number at S5 and S6. The recruitment of a large number of anti-HLA-DR cells concomitant with a marked increase of other kinds of immunocompetent cells in the pulp of late-stage caries might indicate the occurrence of antigen presentation followed by both cell-mediated and humoral immune reactions. The number of anti-alpha 1-antichymotrypsin (ACT) antibody-positive macrophages showed a proportional increase with the development of caries, and these cells may be involved in protecting against the tissue damage caused by proteases released from inflammatory cells, as well as having a defensive role by phagocytosis of toxic micro-organisms and damaged tissue residues. Thus anti-HLA-DR and anti-ACT antibody-positive cells might participate in both an efficient immune system and a tissue-protective mechanism in the human dental pulp.

Ultrastructure of the neuromuscular junction of vasomotor nerves in the microvasculature of human dental pulp

Vasomotor nerves in human dental pulp were more closely related to arterioles than to venules. Most were composed of unmyelinated fibres, which were mainly adrenergic. They appeared close to arterioles that were surrounded by a few layers of contractile smooth-muscle cells. The smaller arterioles with a diameter of 10-15 microns received a more intimate innervation by vasomotor nerves than did the larger. These vessels occasionally showed much narrower neuromuscular junctions than previously reported. Most of these nerve fibres were identified as adrenergic by the presence of chromaffin-positive synaptic vesicles detected by ultrastructural enzyme histochemistry. Their function appeared to be to regulate the blood flow and/or the blood pressure by stimulating smooth-muscle cells, resulting in contraction and a change in the calibre of the vessels. Capillaries and venules, which have a higher permeability, received weaker innervation by the vasomotor nerves than did arterioles. The intimate relation between vasomotor nerves and arterioles is related to the function of dental pulp in normal and pathological conditions.

Fibrinogen/fibrin and fibronectin in the dentin-pulp complex after cavity preparation in rat molars.

OBJECTIVE The purpose of this study was to examine changes in distribution of fibrinogen/fibrin and fibronectin in the dentin-pulp complex after cavity preparation. STUDY DESIGN Class V cavity preparations were prepared on maxillary first molars of 12 rats. The dentin and pulps were observed histologically and immunohistochemically for fibrinogen and fibronectin at 6 hours and 1, 2, and 3 days after the preparation. RESULTS At 6 hours and 1 day after cavity preparation, positive staining for fibrinogen was noted in the exudative lesion and in the dentinal tubules under the cavity preparation. Fibronectin staining in the exudate showed a pattern with close similarity to the fibrinogen staining. At 3 days after cavity preparation, the irregularly shaped predentin under the cavity preparation showed strong positive staining for fibronectin. CONCLUSIONS Fibrinogen/fibrin and fibronectin are present during the healing process of the dentin-pulp complex after cavity preparation.

Short-term histomorphological effects of Er:YAG laser irradiation to rat coronal dentin-pulp complex

OBJECTIVE The objective of the study was to examine the morphological changes of neural elements in dentin-pulp complex ultrastructurally after Er:YAG laser irradiation and elucidate the mechanism of pain reduction in cavity ablation. STUDY DESIGN The Er:YAG laser was applied at occlusal surfaces of upper and lower first molar cusps of 6 rats, and shallow cavities were ablated. The dentin and pulps were examined with light and electron microscopes at 6 hours after the irradiation. Teeth, without laser irradiation, from three rats were used as controls. RESULTS Disruption of nerve terminals in the dentinal tubules, degeneration of nerve terminals between odontoblasts, and disruption of the myelin sheath in the pulp core were demonstrated with electron microscope. CONCLUSION Some Er:YAG laser beams could penetrate to deeper areas than ablated area, and damage of nerve fibers and terminals might be a mechanism of pain reduction in cavity ablation with Er:YAG laser.

Ultrastructural relation between nerve terminals and dentine bridge formation after pulpotomy in human teeth

Close association between nerve terminals and preodontoblasts, odontoblasts and predentine was observed during healing after pulpotomy. The nerve terminals frequently contained large numbers of synaptic vesicles. Terminals with many vesicles tended to be fewer in the predentine than in the odontoblastic layer. The distribution of terminals was more dense at the stage before the regenerated odontoblasts became arranged regularly beneath the predentine. It is suggested that these terminals have some efferent role(s), especially during collagen synthesis at the early stage of dentinogenesis. The nerves may release their abundant synaptic vesicles, in addition to serving a sensory role for monitoring the increased sensitivity in the injured areas.

Electron microscopic study on nerve terminals during dentin bridge formation after pulpotomy in dog teeth

This study was designed to examine the relation between pulpal nerves and the differentiation of pulpal cells into preodontoblasts and odontoblasts during the healing process after pulpotomy. A total of 36 upper and lower teeth obtained from six adult dogs were used. The pulp chamber was opened with a sterile diamond bur, the coronal pulp was exposed, and the whole surface of the amputated pulp was capped with calcium hydroxide. The interval between pulpotomy and extraction was 5, 7, 10, 15, 20, 30, 40, 50, and 60 days, and then specimens were examined ultrastructurally. Close contact between fibroblast-like cells/osteoblast-like cells and nerve terminals at the calcification front was observed in the early healing process after pulpotomy, suggesting a close relation between nerve fibers and pulpal cell differentiation.