Dental pulp in mature replanted human teeth: morphological alterations and metalloproteineses-2 and -9, Annexin-5, BCL-2 and iNOS modulation.

Tooth replantation, as a treatment concept, has been subject to controversies regarding the mechanism as well as the various parameters underlying this process. This work aimed to study time-related changes in the pulp of replanted mature human premolars through the changes in the levels of certain factors involved in the underlying mechanisms of pulpal tissue healing after replantation. Eleven experimental mature teeth were extracted, immediately replanted in the original socket and left without any other intervention for 1, 2, 3 and 12 weeks before re-extraction. Three premolars served as control. All specimens were subject to histological analysis and the levels of MMP-2, MMP-9, Annexin V, iNOS and BCL-2 (anti-apoptotic family) were analyzed employing immunohistochemistry. The results showed degradation of the extracellular matrix (ECM), inflammatory cell infiltrate, loss in pulpo-dentine interface and loss of odontoblasts in the dental pulp tissue. This was accompanied by increase over time of MMP-9, Annexin V, iNOS and a decrease of BCL-2 and MMP-2, suggesting that apoptosis increased throughout the experimental period.

Expression of bone markers and micro-CT analysis of alveolar bone during orthodontic relapse.

OBJECTIVES: To investigate biological changes in alveolar bone occurring during orthodontic relapse. MATERIALS AND METHODS: Rat maxillary first molars were moved mesially for 10 days. After orthodontic tooth movement (OTM), appliances were removed, and the molars were allowed to relapse for one, three, five, seven, 14 or 21 days. Changes in 3D morphometric parameters of bone located mesial to the first molars were evaluated by micro-CT. Total RNA was isolated from the same bone site, and real-time RT-PCR was used to measure the expression of bone formation and resorption markers. RESULTS: One day after appliance removal, the molars relapsed to a mean 73% of the achieved OTM and then steadily relapsed to 93% at 21 days. Tissue mineral density and per cent bone volume increased over the experimental period. Inversely, there was a decrease in total porosity. Gene expression of OCN, Coll-I and ALP decreased during OTM, whilst as the molars relapsed showed tended to increase. Gene expression of RANKL and TRAP increased during OTM. Changes in mRNA expression of H(+)-ATPase were minor. By 21 days post-appliance removal, the remodelling process in rats appeared to have returned to control levels. CONCLUSIONS: Bone tissue reactions on a molecular level are similar during OTM and orthodontic relapse. These findings validate the importance of immediate retention following active OTM.

Reactions of periodontal ligament epithelial cell clusters and OX6-immunopositive cells to experimental tooth movement and periodontitis.

BACKGROUND AND OBJECTIVE: The aim of this study was to investigate reactions of periodontal ligament epithelial cell clusters and major histocompatibility complex class II (OX6)-immunopositive cells to simultaneously induced tooth movement and periodontitis employing Waldo's method. MATERIAL AND METHODS: Elastic gums were inserted between the right upper first and second molars of rats. Animals were killed by intracardiac perfusion on days 1, 3, 7 and 14 after the experimental procedures, and maxillary molars were decalcified and processed for OCT compound. Cytokeratin and OX6 antibodies to detect epithelial and immunocompetent cells were used for double-fluorescence immunohistochemistry. Immunostained sections of rat upper molar regions were examined with a fluorescence microscope. RESULTS: Large periodontal ligament epithelial cell clusters appeared and became contiguous with each other, and OX6-immunopositive cells surrounded the clusters over time in the periodontal ligament near the gum insertion site. In the periodontal ligament distant from the gum insertion site, epithelial cell clusters and OX6-immunopositive cells were scattered. After 14 d, thickened epithelium and elongated rete pegs were found close to large epithelial cell clusters in the periodontal ligament near the gum insertion site. CONCLUSION: These findings suggest proliferation and/or aggregation of periodontal ligament epithelial cells, and interaction between OX6-immunopositive cells and the periodontal ligament epithelial cells, in response to tooth movement and periodontal inflammation. This method may be a useful experimental model to elucidate the relationship between rete pegs and periodontal ligament epithelial cell clusters in inflammatory conditions.

Delayed recruitment of immunocompetent cells in denervated rat periodontal ligament following experimental tooth movement.

It has previously been shown that the number of mononuclear phagocytic cells in the periodontal ligament (PDL) of orthodontically moved rat molars is significantly increased (p < or = 0.05) at 3, 7, and 14 days compared with the controls. Since these changes coincide with increased density of peptidergic nerve fibers, it was of particular interest to investigate a possible relation between the immunocompetent cells and sensory nerve fibers in the PDL of experimentally moved and denervated rat molars. Twenty-two young animals had the first right mandibular molar moved mesially, 7, 14, and 21 days after ipsilateral inferior alveolar nerve axotomy. The left side served as unoperated control. An immunohistochemical procedure was carried out on alternate, serial, cryostat sections with antibodies against CDllb (macrophages, dendritic cells) and class II major histocompatibility complex (MHC) molecules (RT1B). At 7 and 14 days, the number of CD11b+- and RT1B-expressing cells in the denervated PDL showed no significant difference compared with the contralateral side. However, at 21 days, when periodontal tissue re-innervation is established, the number of the investigated immunocompetent cells in the PDL of the denervated and experimentally moved mandibular molars demonstrated a significant difference compared with the contralateral and control molars (p < or = 0.05). It can be concluded that axotomy of the inferior alveolar nerve delays the recruitment of macrophage-like and class II MHC molecule-expressing cells in the PDL of orthodontically moved rat molars. The results further indicate that sensory nerve fibers interact with immunocompetent cells and participate in their mobilization to locally inflamed tissues.

Neurokinin-1 receptor expression in the mature dental pulp of rats.

Substance P induces inflammatory reactions in peripheral tissues including the dental pulp, but its regulatory effects in target tissues are dependent on receptor signalling. Here the expression of the substance-P receptor neurokinin-1 (NK1) in the mature molar pulp of the rat was examined in order to localize the main target areas for substance P. A polyclonal antibody directed against the C-terminal of the receptor was used, and immunohistochemistry was performed by the avidin-biotin peroxidase complex method. The results showed that the NK1 receptor was intensely expressed along vessel-like structures in the odontoblast and subodontoblast layer. A granulated and diffusely distributed NK1-receptor labelling was found along larger blood vessels in the root pulp and pulp proper. NK1 receptor-positive cells were frequently observed in the cell-rich zone beneath the odontoblast layer. The results indicate that, in the mature rat molar pulp, the main targets for substance P acting through the NK1 receptors are tissues related to blood vessels in the odontoblast and subodontoblast area. Furthermore, the expression of NK1 receptors on cells located in the subodontoblast area could indicate that substance P also affects cell functions in this area.

Neural modulation of inflammatory reactions in dental tissues incident to orthodontic tooth movement. A review of the literature.

This article reviews the current knowledge of the biological aspects of dental tissue changes incident to orthodontic tooth movement. The inflammatory nature of these tissue changes was first recognized in the early 1970s, and since then a number of morphological and quantitative investigations have been published in support of this view. The studies dealing with vascular and cellular dental tissue changes, as well as those concerned with inflammatory mediators present at sites of orthodontic tooth movement are systematized and presented accordingly. Special emphasis is placed upon the role of the sensory nerve fibres and their neuropeptides in the control, and development of an inflammatory process, i.e. their role in tooth movement.

Effect of inferior alveolar nerve axotomy on periodontal and pulpal blood flow subsequent to experimental tooth movement in rats.

The aims of this study were to evaluate the effect of inferior alveolar nerve (IAN) axotomy on periodontal (PDL) and pulpal blood flow incident to experimental tooth movement and to investigate whether nerve fiber regeneration coincides with blood flow changes. The first right mandibular molar was moved mesially for 3, 7, and 14 days after ipsilateral IAN axotomy in 29 rats. Four rats served as unoperated controls. At the end of each experimental period fluorescent microspheres (FM) were injected into the left ventricle and thereafter counted in serial sections in the PDL and pulp of the right and left first mandibular molars. The number of FM per tissue volume was taken as a measure of blood flow. Re-innervation of nerve fibers was mapped immunohistochemically 7, 14, and 21 days after IAN axotomy in 9 rats that had no orthodontic appliance. The statistical analysis showed no significant differences in the number of FM/mm3 PDL between the denervated and the contralateral side at 3 and 7 days. At 14 days the PDL on the denervated side showed a significant increase in the number of FM/mm3, coinciding with the initial periodontal nerve fiber re-innervation. In the pulp no significant differences were found between the denervated and the contralateral, innervated side in any experimental period. It can be concluded that IAN axotomy postpones an increase in periodontal blood flow until a sensory tissue re-innervation is established, thus indicating that neurogenic mechanisms play an important role in the development of the inflammatory reaction induced by experimental tooth movement.

Effect of experimental tooth movement on nerve fibres immunoreactive to calcitonin gene-related peptide, protein gene product 9.5, and blood vessel density and distribution in rats.

The effect of experimental tooth movement on nerve fibers immunoreactive to calcitonin gene-related peptide (CGRP) and to protein gene product (PGP) 9.5 were studied, as well as the coincidence of these responses with changes in blood vessel density and distribution in the periodontal ligament (PDL) and pulp of young Wistar rats. The first right maxillary molar was moved mesially by an orthodontic appliance for 3, 7, 14 and 21 days. Sagittal and horizontal serial sections were incubated alternately with antibodies to CGRP, PGP 9.5 and laminin. Nerve and blood vessel density and distribution between the experimental and control sides were compared in the apical and cervical PDL, and in root and coronal pulp. The most pronounced changes occurred in the 7 day group. CGRP and PGP 9.5 immunoreactive nerves in the apical PDL showed increased density, being distributed towards the alveolar bone and frequently found in bone resorption lacunae. Numerous nerve fibres were often present adjacent to hyalinized tissue, but were never found near or within root resorption lacunae. Nerve sprouting was also present both in the root and coronal pulp. Increased nerve and blood vessel density generally coincided with each other. At day 14, periodontal nerves and blood vessels were still disorganized compared with the controls. Tissues near cellular cementum and root resorption lacunae were consistently devoid of nerve fibres. After 21 days, PDL nerve and blood vessel density and distribution were nearly at control level. However, nerve fibres were regularly found inside root resorption areas. In conclusion, experimental tooth movement induces dynamic changes in density and distribution of periodontal and pulpal nerve fibres, indicating their involvement in both early stages of periodontal remodelling and later in the regenerative processes of the PDL, generally occurring in concerted action with modulation of blood vessels.

Changes in blood circulation in teeth and supporting tissues incident to experimental tooth movement.

Fluorescent microspheres (FM) were used to semi-quantify the effect of orthodontic forces on blood flow in oral tissues in young rats. Forty-five animals had an orthodontic appliance inserted on the first maxillary molar on one side exerting a mesial force of approximately 50 g. Ten animals served as unoperated controls. On days 1, 3, 7, 14, and 21 after the start of the experiment, FM were injected into the left ventricle through an abdominal approach in the experimental and control animals. FM were counted in serial sections from the jaws in the periodontal ligament, pulp, and alveolar bone in a fluorescent microscope. The number of FM per tissue volume and/or tissue weight was taken as a measure of blood flow. The experimental side had significantly lower numbers of FM/mm3 in the periodontal ligament of the first and the second molar on the first day, compared with the contralateral side. However, a steady, significant increase in the number of FM/mm3 in the periodontal and pulpal tissues, and FM/mg in the alveolar bone could be observed on the third and seventh days on the experimental side of the first, second, and third molars compared with the contralateral side, while in the later stages the values of the two sides approached each other. The results of this study indicate that a localized experimental tooth movement initiates a more generalized blood flow response in the periodontal ligament, dental pulp and alveolar bone.

Immunocompetent cells in rat periodontal ligament and their recruitment incident to experimental orthodontic tooth movement.

The aims of this study were to evaluate the number and distribution of immunocompetent cells in normal rat periodontal ligament (PDL) and to quantify their recruitment incident to experimental tooth movement. 27 young animals had the 1st right maxillary molar moved mesially by an orthodontic appliance for 1, 3, 7 and 14 days, respectively. 4 animals served as untreated controls. An immunohistochemical procedure was carried out on alternate serial cryostat sections, and monoclonal antibodies against CD11b (macrophages, dendritic cells), CD43 (lymphocytes, polymorphs), CD4 (helper T-lymphocytes), and class II MHC molecules were used. Mean counts of the immunolabeled cells in the control group showed the highest number of CD11b+ and class II molecule expressing cells, while CD4+ and CD43+ cells were scarcely found. Significant increase in the number of CD11b+, CD43+ cells and class II molecules was found in the PDL of the experimentally moved 1st molars compared with the contralateral side and the control group, while CD4+ cells showed no significant increase. CD11b+ and cells expressing class II molecules were found around hyalinized tissue, between dentin and cellular cementum and close to Malassez' epithelial cells. In conclusion, normal rat PDL has high number of macrophage and dendritic-like cells, but few lymphocytes and granulocytes. Furthermore, experimental tooth movement leads to significant recruitment of cells belonging to the mononuclear phagocytic system, but has no significant effect on the number of lymphocytes and granulocytes in the rat PDL.

NK1, NK2, NK3 and CGRP1 receptors identified in rat oral soft tissues, and in bone and dental hard tissue cells.

The distribution of the tachykinin receptors neurokinin-1 (NK1), neurokinin-2 (NK2) and neurokinin-3 (NK3), and the calcitonin gene-related peptide-1 (CGRP1) receptor were examined in rat teeth and tooth-supporting tissues by immunohistochemical methods and light and confocal microscopy. Western blot analysis was performed to identify the NK1- and the CGRP1-receptor proteins in the dental pulp. The results showed that odontoblasts and ameloblasts, cementoblasts and cementocytes, osteoblasts and osteocytes are all supported with the tachykinin receptors NK1 and NK2, but a distinct, graded cellular labeling pattern was demonstrated. The ameloblasts were also positive for CGRP1 receptor. Blood vessels in oral tissues expressed the tachykinin receptors NK1, NK2 and NK3, and the CGRP1 receptor. Both gingival and Malassez epithelium were abundantly supplied by NK2 receptor. Pulpal and periodontal fibroblasts demonstrated NK1 and NK2 receptors. Western blot analysis identified both the NK1- and the CGRP1-receptor proteins in the dental pulp. These results clearly indicate that the neuropeptides substance P, neurokinin A, neurokinin B and CGRP, released from sensory axons upon stimulation, directly modulate the function of the different types of bone and dental hard tissue cells, and regulate functions of blood vessels, fibroblasts and epithelial cells in oral tissues.

Neuroendocrine cells in Malassez epithelium and gingiva of the cat.

Malassez epithelium has been designated as epithelial cell rests, the biological significance of which is still under debate. This study was designed to analyze Malassez epithelium for the presence of neuroendocrine cells. Gingival tissue was included as a positive control. Using immunohistochemistry, confocal and light microscopy, Malassez epithelium and gingival epithelium from mature cats (n = 5) were examined for cells containing the neuropeptides calcitonin gene-related peptide (CGRP), substance P (SP), and vasoactive intestinal peptide (VIP). Both Malassez epithelium and the basal epithelial cell layers in gingival rete pegs regularly displayed cells immunoreactive to CGRP, SP, and VIP. The immunopositive cells were most frequently present in the epithelial cell clusters and strands of Malassez located in the cervical half of thc periodontal ligament. Double immunolabeling revealed cellular co-expression of CGRP or SP with VIP, and the neuropeptides were co-localized in the cellular compartments. Labeled cells in both epithelia were occasionally supported by immunoreactive nerve fibers. This study shows that cells immunoreactive to CGRP, SP, and VIP arc located within the cat Malassez epithelium. The localization of neuroendocrine cells verifies the diversity of this epithelium and confirms that Malassez epithelium is composed of different cell types, in common with epithelia from other locations. The presence of neuroendocrine cells in Malassez epithelium strongly suggests biological functions of this tissue, and the neuropeptide content may thus indicate endocrine functions of the cells.

Merkel-like cells in Malassez epithelium in the periodontal ligament of cats: an immunohistochemical, confocal-laser scanning and immuno electron-microscopic investigation.

The cellular heterogeneity of Malassez epithelium (ME) residing in the periodontal ligament has recently been reported, and the presence and coexistence of the neuropeptides calcitonin gene-related peptide (CGRP), substance P (SP) and vasoactive intestinal peptide (VIP) in single cells in ME has been shown (1). However, the identity of these neuroendocrine cells has so far not been verified. This study was undertaken in order to elucidate the identity of the neuroendocrine cells in ME by means of transmission electron microscopy, confocal scanning microscopy and immunohistochemistry using antibodies to protein gene product (PGP) 9.5 and cytokeratin 20 (CK). Gingival tissue was included in the study as a positive control for identification of Merkel-like cells in oral epithelium. CK 20 immunopositive cells were present in both Malassez epithelium and in basal cell layers of gingival epithelium showing a distribution consistent with PGP 9.5 labelled cells in both epithelia. The results from PGP 9.5 immuno electron microscopy clearly evidenced the presence of single, intensely labelled cells and some nerve fibres invested between the Malassez epithelial cells. The conformity of the immunopositive cells in Malassez and gingival epithelium verified by double immunolabelling with PGP 9.5 and CK 20, indicates that the labelled neuroendocrine cells are identical in ME and in gingival epithelium. This demonstrates that Malassez epithelium not only exhibits neuroendocrine cells, but additionally that the neuroendocrine cells represent Merkel-like cells.