Evidence for the existence of intraepithelial nerve endings in the junctional epithelium of rat molars: an immunohistochemical study using protein gene product 9.5 (PGP 9.5) antibody

Innervation of the junctional epithelium was investigated in rat molars by means of immunohistochemistry for protein gene product 9.5 (PGP 9.5) at light and electron microscopic levels. In comparison with our previous study on same tissues using neurofilament protein (NFP)-antibody, the PGP 9.5-immuno-staining further disclosed numerous nerve fibers in the gingiva of rat molars and revealed the existence of a well-developed plexus of PGP 9.5-positive nerve fibers. The interproximal portion also contained numerous nerve fibers.
Observation of horizontal sections revealed a denser innervation toward the inner junctional gingival epithelium than the outer marginal epithelium. The nerve fibers, beaded in appearance and extending from the nerve bundles in the lamina propria, penetrated into the junctional epithelial layer and were distributed throughout the junctional epithelium, with some nerves being located near the epithelial surface. Non-neuronal cells showing PGP 9.5-immunoreactivity were absent in the junctional epithelium. In immunoelectron microscopy, the axoplasm of nerves in the gingiva was filled with electron-dense reaction products of PGP 9.5, except for the cell organellae. The nerve fibers were devoid of Schwann cell investment and terminated among the epithelial cells in the junctional epithelium, frequently beneath the epithelial surface. The intraepithelial nerve endings contained various kinds of vesicles including large-cored ones, supporting the presence of peptidergic innervation shown by previous studies. These findings confirmed the usefulness of PGP 9.5-immunohisto-chemistry for the identification of delicated nerve fibers in dental tissue, and suggested the dense network of nerve fibers that may serve as sensory receptor and other functions in the junctional epithelium.     

Effect of experimentally induced occlusal trauma on substance p expression in human dental pulp and periodontal ligament

Introduction

The purpose of this study was to quantify the effect of occlusal trauma experimentally induced with occlusal interferences on substance P (SP) expression in healthy human dental pulp and periodontal ligament.

Methods

Twenty-eight human dental pulp and periodontal ligament samples were obtained from healthy premolars in which extraction was indicated for orthodontic reasons. Before extraction, occlusal trauma was induced with experimental occlusal interferences in half of these premolars by placing a resin block over their occlusal surface and submitting patients to chew gum for 30 minutes. The remaining healthy premolars were extracted without occlusal trauma and served as a control group. All dental pulp and periodontal ligament samples were processed, and SP was measured by radioimmunoassay.

Results

There was 45% and 120% greater SP expression in dental pulp and periodontal ligament, respectively, of teeth with experimentally induced occlusal trauma. Paired t test showed statistically significant differences for both human dental pulp and periodontal ligament (P = .02 and P < .001, respectively) of teeth submitted to occlusal trauma when compared with control group values.

Conclusions

SP expression in human dental pulp and periodontal ligament increases when teeth are submitted to occlusal trauma experimentally induced with occlusal interferences.     

咬合创伤诱导海马区星形胶质细胞的上调

目的：探讨咬合创伤后海马区星形胶质细胞的变化及意义。方法：通过将大鼠左侧上颌第一磨牙咬合抬高0．5mm造成咬合创伤动物模型,应用免疫荧光染色观察咬合创伤后1、3、7d、2w、4w,5w时星形胶质细胞在海马区的分布和表达变化。结果：正常大鼠海马区仅见少量星形胶质细胞,7d组星形胶质细胞数开始增加,4W时达到高峰,5W时阳性细胞数开始下降。结论：咬合创伤引起了海马区星形胶质细胞的数量增加,海马结构参与了咬合创伤反应,其CA1、CA3区与咬合创伤所致口颌面痛及痛情绪调控关系密切。     

咬合创伤诱导海马区星形胶质细胞的上调

目的：探讨咬合创伤后海马区星形胶质细胞的变化及意义。方法：通过将大鼠左侧上颌第一磨牙咬合抬高0．5mm造成咬合创伤动物模型,应用免疫荧光染色观察咬合创伤后1、3、7d、2w、4w,5w时星形胶质细胞在海马区的分布和表达变化。结果：正常大鼠海马区仅见少量星形胶质细胞,7d组星形胶质细胞数开始增加,4W时达到高峰,5W时阳性细胞数开始下降。结论：咬合创伤引起了海马区星形胶质细胞的数量增加,海马结构参与了咬合创伤反应,其CA1、CA3区与咬合创伤所致口颌面痛及痛情绪调控关系密切。     

Altered expression level of calbindin D28k in the periodontal ligament of rat molar in response to changes in occlusal force

The present immunohistochemical study was designed to investigate the alteration in the expression level of calbindin D28k in the periodontal ligament of the rat molar in response to changes in occlusal force to clarify the physiological role(s) of this protein in the ligament. In normal periodontal ligament of the lower first molar, immunoreactivity for calbindin D28k was found in the spindle-shaped cells, presumably fibroblasts, at the alveolar portion of the ligament at the distal side of the mesial root and mesial side of the distal root. Following the overload of occlusal force to the upper first molar by bite-raising, the number and immunoreactivity of the positive cells in the periodontal ligament of the lower first molar increased gradually. A more significant increase was detected at 7 d following the bite-raising compared to the normal animals. When occlusal force was removed by the extraction of the upper first molar, the expression level of calbindin D28k in the periodontal ligament of the lower first molar rapidly decreased, however a subsequent gradual increase was recognized. Statistical analysis of the spatial immunoreactivity of calbindin D28k in the periodontal ligament was performed and showed statistically significant differences. The present results suggest that calbindin D28k may play important roles in the homeostasis and cytoprotection of the periodontal fibroblasts against occlusal force.     

咬合创伤对三叉神经脊束核尾侧亚核c-fos的影响

目的:观察三叉神经脊束核尾侧亚核(Sp5C)中c-fos(核磷酸蛋白原癌基因)对咬合创伤的反应,探讨神经元在咬合创伤中的功能变化。方法:将直径0.5mm方丝粘固于大鼠左侧上颌第一磨牙,形成左侧磨牙早接触的创伤咬合,观察咬合创伤后1,3,7d,2w,4w,5w时Sp5C中c-fos蛋白的表达变化。结果:正常大鼠Sp5C中偶见极少量c-fos阳性神经元细胞,咬合创伤后7d,c-fos阳性神经元明显增加,2w时达到高峰,4w时开始下降。结论:咬合创伤刺激诱导了Sp5C中神经元的激活。     

Reinnervation in the canine periodontal ligament of replanted teeth using an antibody to protein gene product 9.5: an immunohistochemical study

Abstract— The re-innervation process in the periodontal ligament of replanted canine teeth was examined by immunohistochemistry for protein gene product 9.5 (PGP 9.5), a general marker for neurons, and by electron microscopy. Within 1 week of replantation, the periodontal fibers had regenerated, filling the narrow spaces between the alveolar bone and the root surface around the cervical and apical regions. Near the root apex, however, no PGP 9.5-im-munopositive nerve fibers were found in the regenerated periodontal ligament except for those in the alveolar half of the ligament. At 2 weeks after replantation, many nerve fibers positive for PGP 9.5 had ascended the periodontal ligament from the thick nerve bundles located near the root apex. Fine nerve endings showing complicated ramification were also present in the apical region. By 3 or 4 weeks after replantation, the vascular network was regenerated and principal periodontal fibers were re-established throughout the entire length of the periodontal ligament. The extensively ramified PGP 9.5-immunopositive structures had increased in thickness and density and showed characteristic treelike profiles by 3 weeks. Electron microscopy confirmed that most of these structures were Ruffini-like endings, and demonstrated that such nerve terminals were almost regenerated by 4 weeks post-replantation. These results indicated that, in the periodontal ligament of replanted canine teeth, the regeneration of the nerve fibers including mechanosensory receptors first showed signs of regeneration by 2 weeks following tooth replantation and proceeded rapidly thereafter. Regeneration of the periodontal ligament including fiber architecture as well as vascular and neural elements was almost complete by 4 weeks after replantation.     

三叉神经脊束核尾侧亚核星形胶质细胞对咬合创伤的反应

目的：观察大鼠咬合创伤后三叉神经脊束核尾侧亚核（Sp5C）内星形胶质细胞的反应,探讨星形胶质细胞在咬合创伤中的作用。方法：用方丝将大鼠左侧上颌第一磨牙咬合抬高0.5mm,形成左侧磨牙早接触的创伤咬合。粘固后1,3,7,14,30d取三叉神经脊束核尾侧亚核,免疫荧光染色观察星形胶质细胞在三叉神经脊束核尾侧亚核的表达变化。结果：对照组见少量胶质原纤维酸性蛋白（GFAP）阳性星形胶质细胞呈散在分布,咬合创伤3d时,实验侧GFAP阳性面积增加,荧光强度增强;7d时达到高峰,14d组GFAP阳性反应产物开始减少。结论：咬合创伤激活了星形胶质细胞,提示星形胶质细胞在口颌面痛产生及维持中发挥作用。     

Epithelial and PGP9.5-immunoreactive cells of Malassez epithelium in the periodontal ligament of cats: a transmission electron microscopic study

Objective. The aim of the study was to investigate the ultrastructural features of Malassez epithelium (ME) containing protein gene product 9.5 (PGP9.5)-immunoreactive (IR) cells in the cat periodontal ligament (PDL). Material and methods. Specimens from the teeth and tooth-supporting tissues of four mature cats of both sexes, 18 to 24 months of age, were used. The fixed jaws were decalcified in EDTA. Frozen sagittal sections 20 µm thick were immunostained for PGP9.5, and the ME, containing IR cells in the PDL, were evaluated under a transmission electron microscope. Results. Several epithelial cells and PGP9.5 IR cells formed clusters and were enveloped by a basal lamina and separated from the surrounding connective tissue. A large nucleus and scanty cytoplasm were observed in most of the ME cells, which contained abundant keratin filaments and mitochondria. Caveolae-like structures and vesicles were found in the periphery of the ME. The small cytoplasmic processes of some of the epithelial cells extended toward the surrounding connective tissues. The cytoplasmic matrix of one type of cell comprising the ME showed immunoreactivity for anti-PGP9.5 antibody. The IR cell in the cell clusters was connected to adjacent epithelial cells and extended cytoplasmic processes toward the adjacent epithelial cells. The IR cell contained keratin filaments and abundant densely cored vesicles approximately 100–250 nm in diameter. Conclusions. The findings of the study suggest endocytotic capabilities of the epithelial cells and neuroendocrine functions of the IR cells. It is possible that the two different cell types react to extrinsic stimuli and interact with cells comprising the clusters and cords in the PDL. These ultrastructural evidences may imply functional heterogeneity of the ME in the PDL.     

咬合创伤下大鼠磨牙牙周膜中转化生长因子β1表达的变化

目的：了解咬合创伤下转化生长因子β1(transforming growth factorβ1 TGFβ1)在大鼠磨牙牙周膜中表达的变化，探讨咬合创伤下TGFβ1在牙周组织改建中的作用。方法：建立咬合创伤动物模型，利用HE染色法和免疫组织化学技术，观察大鼠磨牙在咬合创伤后12小时、3天、7天、14天、30天的不同时期内牙周膜中TGFβ1表达的变化，并运用图像分析技术将结果数值化。结果：咬合创伤下，牙周膜中TGFβ1的表达在12小时组、3天组与正常咬合力组相比无显著差异；7天组、14天组与正常咬合力组相比有显著差异，并高于后者；30天组与正常咬合力组相比无显著差异。结论：TGFβ1可能在咬合创伤下牙周组织的改建中起了重要的作用。     

Expression of receptor activator of nuclear factor kappa B ligand relates to inflammatory bone resorption, with or without occlusal trauma, in rats

BACKGROUND AND OBJECTIVE: Receptor activator of nuclear factor kappa B ligand (RANKL) is an important factor in osteoclast differentiation, activation and survival; however, its involvement in inflammatory bone resorption, with or without occlusal trauma, is unclear. The purpose of the present study was to investigate the distribution of RANKL-expressing cells in rat periodontium during lipopolysaccharide-induced inflammation with or without occlusal trauma. MATERIAL AND METHODS: Lipopolysaccharide was injected into rat gingiva of the lower left first molar to induce inflammation. In addition, the occlusal surface of the upper left first molar of rat was raised by placing a gold inlay to induce occlusal trauma in the lower left first molars. The distribution of RANKL-expressing cells was immunohistochemically observed. RESULTS: In the inflammatory model, many osteoclasts were observed at the apical inter-radicular septum on day 5 and they were reduced by day 10. On the other hand, in the inflammatory model with occlusal trauma, many osteoclasts were still observed on day 10. RANKL expression was similar to the changes in osteoclast number. The expression of RANKL increased in endothelial cells, inflammatory cells and periodontal ligament cells. CONCLUSION: These findings clearly demonstrated that RANKL expression on endothelial cells, inflammatory cells and periodontal ligament cells is involved in inflammatory bone resorption and the expression is enhanced by traumatic occlusion. These results suggest that RANKL expression on these cells is closely involved in the increase of osteoclasts induced by occlusal trauma.     

Re-innervation in the canine periodontal ligament of replanted teeth using an antibody to protein gene product 9.5: an immunohistochemical study

The re-innervation process in the periodontal ligament of replanted canine teeth was examined by immunohistochemistry for protein gene product 9.5 (PGP 9.5), a general marker for neurons, and by electron microscopy. Within 1 week of replantation, the periodontal fibers had regenerated, filling the narrow spaces between the alveolar bone and the root surface around the cervical and apical regions. Near the root apex, however, no PGP 9.5-immunopositive nerve fibers were found in the regenerated periodontal ligament except for those in the alveolar half of the ligament. At 2 weeks after replantation, many nerve fibers positive for PGP 9.5 had ascended the periodontal ligament from the thick nerve bundles located near the root apex. Fine nerve endings showing complicated ramification were also present in the apical region. By 3 or 4 weeks after replantation, the vascular network was regenerated and principal periodontal fibers were re-established throughout the entire length of the periodontal ligament. The extensively ramified PGP 9.5-immunopositive structures had increased in thickness and density and showed characteristic tree-like profiles by 3 weeks. Electron microscopy confirmed that most of these structures were Ruffini-like endings, and demonstrated that such nerve terminals were almost regenerated by 4 weeks post-replantation. These results indicated that, in the periodontal ligament of replanted canine teeth, the regeneration of the nerve fibers including mechanosensory receptors first showed signs of regeneration by 2 weeks following tooth replantation and proceeded rapidly thereafter. Regeneration of the periodontal ligament including fiber architecture as well as vascular and neural elements was almost complete by 4 weeks after replantation.     

Regeneration of nerve fibres in the peri-implant epithelium incident to implantation in the rat maxilla as demonstrated by immunocytochemistry for protein gene product 9.5 (PGP9.5) and calcitonin gene-related peptide (CGRP)

The response of nerve fibres in the peri-implant epithelium to titanium implantation was investigated with an experimental model using rat maxilla and immunohistochemical techniques. The latter employed antibodies to protein gene product 9.5 (PGP9.5), and to calcitonin gene-related peptide (CGRP). In control rats without an implantation, a dense innervation of PGP9.5- and CGRP-positive nerve fibres was recognized throughout the junctional epithelium, as has been previously reported. A titanium-implantation induced a remarkable inflammatory reaction, as well as the destruction of covering epithelial cells. By 3-5 days post-implantation, inflammatory reaction showed a tendency to disappear, and the peri-implant epithelium showed proliferation and down-growth along the implant. At this stage, no nerve fibres were found around the peri-implant epithelium. At 10 days, a few nerve fibres reached the basal cell layers of the peri-implant epithelium, and entered it 15 days after implantation when the peri-implant epithelial cells showed morphological features roughly resembling those of normal junctional epithelial cells. At the complete osseointegration stage (days 20-30), the PGP9.5- and CGRP-positive nerve fibres, thin and beaded in appearance, were found distributed in the peri-implant epithelium. After 20 days, the numerical density of the intraepithelial nerves in the peri-implant epithelium appeared the same as, or less than, that in the normal junctional epithelium. These findings indicate that the peri-implant epithelium shows the same innervation as that in normal junctional epithelium, and that the intraepithelial nerve fibres in the peri-implant epithelium might have diverse functions, which have been suggested in the literature.     

Odontoblast responses to GaAlAs laser irradiation in rat molars: an experimental study using heat-shock protein-25 immunohistochemistry

Pulpal responses to gallium-aluminum-arsenide (GaAlAs) laser irradiation applied to the tooth remains to be elucidated. This study aimed to evaluate the effect of the GaAlAs laser on odontoblasts using immunohistochemistry for heat-shock protein (HSP)-25, which labels mature and newly differentiated odontoblasts. The mesial surface of the upper right first molar of 8-wk-old Wistar rats was lased at an output power of 0.5-1.5 W for 180 s. The animals were perfusion-fixed at intervals of 6 h to 30 d after irradiation. At 6 h to 7 d, the intensity of HSP-25-immunoreactivity was found to be disturbed in the coronal odontoblast-layer in an energy-dependent manner. At 30 d, tertiary dentin with/without bone-like tissue was formed abundantly in the dental pulp. Statistical analysis revealed that the area occupied by the new hard tissues was significantly wider in 1.5 W-lased specimens than in 0.5 W-lased specimens. An intense HSP-25 immunoreactivity was seen in the odontoblasts underlying the tertiary dentin, whereas immunoreactivity was weak around the bone-like tissue. It was concluded that the GaAlAs laser may induce the formation of tertiary dentin by influencing the secretory activity of odontoblasts. However, higher energies may cause irreversible changes to the pulp, often leading to the formation of an intrapulpal bone-like tissue.     

Validity of a laser fluorescence system (DIAGNOdent) for detection of occlusal caries in third molars: an in vitro study

The aim of this study was to compare the validity of the measurements of the laser fluorescence device, KaVo DIAGNOdent, with the result of polarized light microscopy in the detection of occlusal fissure caries in extracted third molars. Ten impacted, surgically removed, and 25 extracted third molars with macroscopically intact occlusal surfaces were selected. The DIAGNOdent measurements of the occlusal test site were recorded by two observers at intervals of 2 days. The teeth were then sectioned at the specified test sites for histological examinations. Prepared specimens were evaluated under the polarized light microscopy and all images were scored with the caries classification of D1 (sound and fissure lesion in the half of the outer enamel), D2 (enamel decay) and D3 (dentin decay) level (gold standard). The kappa value for the inter-observer repeatability was calculated and the value 0.83 for the first measurements and 0.67 for the second measurement were obtained,respectively. Inter-observer correct classification percentages were 88.5 and 77.1 for the first and second measurements, respectively. The kappa value for intra-observer repeatability was 0.79 for the first observer and 0.75 for the second observer. Intra-observer correct classification percentage values were 85.7 and 82.8 for the first and second observer, respectively. Value of specificity for the detection of enamel caries at D1 level was 0.74 and sensitivity values at D2 and D3 levels were 0.66 and 1.00, respectively. The present study indicates that the DIAGNOdent provides not only almost perfect agreement but also sufficient repeatability at D1, D2, D3 levels and better specificity at D1 level as well as lower sensitivity at D2 level and excellent sensitivity at D3 level.     

Developmental changes of the vasculature in the periodontal ligament of rat molars: a scanning electron microscopic study of microcorrosion casts

Vascularization of the periodontal ligament was examined in developing upper first molars of rats from 5 to 30 d after birth with light and scanning electron microscopy. Formation of the vascular network in the periodontal ligament (PDL) started with the beginning of root formation. The PDL vessels derived from the basal region of the tooth germ ran parallel to the long axis of the root and connected with the vascular network of the enamel organ at the cervical end. The boundary of these 2 networks was initially indistinct but became clearer with the progress of root formation. The PDL vessels further elongated longitudinally and connected with each other by lateral branches to form a coarse mesh. Other vessels derived from the alveolar bone via Volkman's canals also contributed to the vascular construction of the PDL. The vessels from the alveolar bone provided branches to the existing mesh of the PDL. Consequently, the vascular network of the PDL consisted of vessels from 2 sources: 1 derived from the basal region of the tooth germ, and the other from the alveolar bone. The density of the vascular network reduced with the progress of root formation, especially at the middle part of the root, but the mesh at the apical region maintained a basket-like structure.     

Role of receptors of advanced glycation end‐products (RAGE) in type 2 diabetic and non‐diabetic individuals with chronic periodontal disease: an immunohistochemical study

Aim: The relationship between diabetes and periodontal disease is well established. It has been shown that advanced glycation end‐products might exert noxious effects on several tissues of the body through its receptor. Evidence for the role of receptors of advanced glycation end‐products in periodontal disease for diabetes is limited, and their presence in human gingival tissues has been demonstrated in few studies. In this study, we demonstrate the presence of receptors of advanced glycation end‐products in patients with chronic periodontitis, with and without type 2 diabetes. Methods: Gingival biopsies from 19 patients with both type 2 diabetes and chronic periodontitis, and 18 healthy controls with chronic periodontitis, were immunohistochemically stained for receptors of advanced glycation end‐products. Results: On immunohistochemical analysis, positive staining for receptors of advanced glycation end‐products was seen in the endothelium and the basal and spinous layers of the inflamed gingival epithelium in both type 2 diabetes and non‐diabetes tissue, with a statistically‐significant difference between both groups (P < 0.05). Conclusions: There was a significant difference in receptors of advanced glycation end‐product immune reactivity between both groups. Receptors of advanced glycation end‐product increase in type 2 diabetes gingival tissue might indicate possible involvement of this receptor in periodontal destruction in individuals with type 2 diabetes.     

The role of lipopolysaccharide‐binding protein in innate immunity: a revisit and its relevance to oral/periodontal health

Lipopolysaccharide (LPS)‐binding protein (LBP) functions as an acute phase protein and plays a key role in the innate immune response to bacterial challenge. It is a potential acute‐phase biomarker in monitoring the progress of severe sepsis, infectious endocarditis and cardiovascular disease. LBP is mainly synthesized in hepatocytes and generates binding of bacteria and/or their products such as LPS to cell surface receptors, thereby initiating an innate host response. Interestingly, LBP has a dual role depending on its relatively low or high concentrations, and augments or downregulates the innate host defense accordingly. Emerging evidence indicates that LBP can be produced by non‐hepatocytes, including respiratory type II epithelial cells, intestinal epithelial cells and human gingival epithelia. These findings suggest that LBP formation at extrahepatic cells may be crucial in containing microbial in situ challenge constantly, critically contributing to tissue homeostasis. This review provides an update on the characteristics and novel functions of LBP as well as its gene polymorphisms and potential use as a biomarker in assessing common infectious and inflammatory diseases such as periodontal disease. This paper highlights the expression profiles of LBP in human oral/gingival cells, how its expression could be modulated by periodontopathogens such as Porphyromonas gingivalis, as well as the relevant regulation mechanisms and signaling pathways involved. The critical roles of LBP in periodontal homeostasis and perspectives for its clinical application are discussed.