Apoptosis in the human periodontal membrane evaluated in primary and permanent teeth

Abstract

Objective. Recent studies revealed a highly innervated layer in close proximity to the root surface in the periodontal membrane of human teeth. Persistence of the epithelial cells of Malassez along root surfaces without resorption has also been demonstrated. It is hypothesized that resorption is connected to apoptosis of the epithelial cells of Malassez. The purpose of this study is to localize cells undergoing apoptosis in the periodontal membrane of human primary and permanent teeth. Materials and methods. Human primary and permanent teeth were examined immunohistochemically for apoptosis and epithelial cells of Malassez in the periodontal membrane. All teeth examined were extracted in connection with treatment. Results. Apoptosis was seen in close proximity to the root surface and within the epithelial cells of Malassez. This pattern of apoptotis is similar in the periodontal membrane in primary and permanent teeth. Conclusions. The inter-relationship between apoptotis and root resorption cannot be concluded from the present study. Apoptosis seen in close proximity to the root surface presumably corresponds to the highly innervated layer of the periodontal membrane. The function of this layer still needs to be elucidated.     

Immunolocalization of RANK and RANKL along the root surface and in the periodontal membrane of human primary and permanent teeth

Abstract

Objective. Root resorption, impaired tooth eruption and early tooth loss have been described in relation to diseases that involve defects in the RANK-RANKL-OPG-expression. The aim of the present immunhistochemical study was to localize and compare the reactions for RANK and membrane-bound RANKL along root surfaces and in the periodontal membrane in close proximity to the root surface of human primary and permanent teeth. Materials and methods. The material comprised extracted human teeth (11 primary teeth and six permanent teeth) from 10 different patients. Paraffin sections were prepared of each tooth and sections of each tooth were immunohistochemically stained with antibodies specific for membrane-bound RANKL and RANK. Results. The root surface and the periodontal membrane in close proximity to the root surface did not show immunoreactivity for RANKL. RANKL was only located in odontoblasts and in cells along denticles in one primary tooth. RANK was located in mononuclear cells in the pulp and in multinucleated odontoclasts along resorbed root surfaces and along resorbed dentin surfaces in the pulp in primary teeth and one permanent tooth. Conclusions. This study demonstrated RANK positivity in resorption areas in primary and permanent teeth. RANKL was positive in the pulp of one primary tooth. RANK expression in odontoclasts and RANKL expression in the pulp may indicate that RANK/RANKL play a role during resorption.     

Denervation resulting in dento-alveolar ankylosis associated with decreased Malassez epithelium

Inferior alveolar nerve denervation causes appreciable decreases in the distribution of epithelial rests of Malassez. To explore roles of the Malassez epithelium, we attempted to evaluate possible changes in dento-alveolar tissues surrounding this epithelium by experimental denervation. We found that denervation led to dento-alveolar ankylosis with a decrease in the width of the periodontal spaces. Interestingly, with regeneration of the Malassez epithelium 10 weeks after the denervation, the periodontal space width showed a correspondingly significant increase. These findings suggest that the Malassez epithelium may be involved in the maintenance of periodontal space and that sensory innervation might be indirectly associated with it. In addition, it is of interest that denervation activated root resorption of the coronal root surface and that the consequently resorbed lacunae were repaired by cellular cementum. It is suggested that Malassez epithelium may negatively regulate root resorption and induce acellular cementum formation.     

Role of the epithelial cell rests of Malassez in the development,maintenance and regeneration of periodontal ligament tissues

Periodontitis is a highly prevalent inflammatory disease that results in damage to the tooth‐supporting tissues, potentially leading to tooth loss. Periodontal tissue regeneration is a complex process that involves the collaboration of two hard tissues (cementum and alveolar bone) and two soft tissues (gingiva and periodontal ligament). To date, no periodontal‐regenerative procedures provide predictable clinical outcomes. To understand the rational basis of regenerative procedures, a better understanding of the events associated with the formation of periodontal components will help to establish reliable strategies for clinical practice. An important aspect of this is the role of the Hertwig's epithelial root sheath in periodontal development and that of its descendants, the epithelial cell rests of Malassez, in the maintenance of the periodontium. An important structure during tooth root development, the Hertwig's epithelial root sheath is not only a barrier between the dental follicle and dental papilla cells but is also involved in determining the shape, size and number of roots and in the development of dentin and cementum, and may act as a source of mesenchymal progenitor cells for cementoblasts. In adulthood, the epithelial cell rests of Malassez are the only odontogenic epithelial population in the periodontal ligament. Although there is no general agreement on the functions of the epithelial cell rests of Malassez, accumulating evidence suggests that the putative roles of the epithelial cell rests of Malassez in adult periodontal ligament include maintaining periodontal ligament homeostasis to prevent ankylosis and maintain periodontal ligament space, to prevent root resorption, to serve as a target during periodontal ligament innervation and to contribute to cementum repair. Recently, ovine epithelial cell rests of Malassez cells have been shown to harbor clonogenic epithelial stem‐cell populations that demonstrate similar properties to mesenchymal stromal/stem cells, both functionally and phenotypically. Therefore, the epithelial cell rests of Malassez, rather than being ‘cell rests’, as indicated by their name, are an important source of stem cells that might play a pivotal role in periodontal regeneration.     

Finite element analysis suggests functional bone strain accounts for continuous post-eruptive emergence of teeth

ObjectiveTo explore the possible role of functional stress in driving continuous post-eruptive emergence of teeth.DesignA two dimensional finite element analysis model was established with a single mandibular premolar subjected to sagittal bending. Equivalent strain was charted for the inner and outer surfaces of the lamina dura, because bone deposition and resorption of this structure is confined to surface osteoblasts and osteoclasts. Bone disuse resorption was assumed to take place at equivalent strain values below 0.0008, while deposition was above 0.002. Strain in the periodontal ligament and principal stress throughout the model were also characterized.ResultsStrain analysis indicated bone maintenance for the lamina dura throughout most of the root length, but in both the apical and upper root regions, resorption was predicted for the outer surface, and bone deposition was predicted for the inner surface of the lamina dura. Strain in the periodontal ligament varied little with the exception of a marked increase close to the crown. Principal stress analysis revealed compression of the lower model border, with areas of increasing tension towards the upper model border.ConclusionsStrain from functional forces may continuously drive post-eruptive emergence of teeth through bony remodelling of the lamina dura, lifting teeth by both raising the apical lamina dura, and narrowing the upper root space to accommodate tapering root form. Such strain-driven bone turnover may contribute to pre-eruptive movement of teeth.     

Induction of MMP‐2 at the interface between epithelial cells and fibroblasts from human periodontal ligament

Shimonishi M, Takahashi I, Terao F, Komatsu M, Kikuchi M. Induction of MMP‐2 at the interface between epithelial cells and fibroblasts from human periodontal ligament. J Periodont Res 2010; 45: 309–316. © 2009 John Wiley & Sons A/S Background and Objective: MMP‐2 can degrade type IV collagen and MMP‐14 can activate pro MMP‐2. The present study was undertaken to examine the expression of MMP‐2 and MMP‐14 with respect to interaction between the cells of the epithelial rests of Malassez and fibroblasts from human periodontal ligament. Material and Methods: Explants of human periodontal ligament tissues produced outgrowths containing both putative epithelial rests of Malassez cells and human periodontal ligament fibroblasts after incubation in a modified serum‐free medium. The distribution and expression of MMP‐2 and MMP‐14 were analysed using immunohistochemistry, in situ hybridization and RT‐PCR analysis. The conditioned media and cell extracts were collected for western blot analysis for MMP‐2. Results: Putative epithelial rests of Malassez cells at the interface between the cells of the epithelial rests of Malassez and fibroblasts expressed MMP‐2 and MMP‐14 strongly. However, in situ hybridization analysis revealed that human periodontal ligament fibroblasts expressed MMP‐2 mRNA while putative epithelial rests of Malassez cells expressed MMP‐14 mRNA at the interface. The RT‐PCR analysis showed that the expression of MMP‐2 mRNA was significantly higher when putative epithelial rests of Malassez cells and human periodontal ligament fibroblasts were cultured together than when cultured alone. Western blot analysis showed that the active form of MMP‐2 was detected at higher levels in the conditioned medium of the co‐cultured cells. Conclusion: These findings indicate that putative epithelial rests of Malassez cells stimulate the production of MMP‐2 in human periodontal ligament fibroblasts. Up‐regulated proMMP‐2 bound by MMP‐14 expressed in epithelial rests of Malassez cells can degrade matrix molecules, such as type IV collagen, in the basal membrane between putative epithelial rests of Malassez cells and human periodontal ligament fibroblasts.     

Immunohistochemical localization of insulin-like growth factor-II and its binding protein-6 in human epithelial cells of Malassez

So-called epithelial rests of Malassez are derived from the Hertwig's root sheath and are located in the periodontal ligament, with still unknown functions. Different pathological conditions may lead to proliferation of these otherwise non-proliferative cell clusters. The insulin-like growth factor (IGF) system is an important growth factor system controlling proliferation and differentiation. In our study on Malassez cells from extracted human deciduous teeth, we investigated their structure by means of light and electron microscopy. Although they appeared as cellular clusters with a uniform epithelial phenotype, immunohistochemical analyses of components of the IGF system revealed an unique pattern: weak immunoreactivity could be seen for IGF-II while among all IGF binding proteins (IGFBPs) only IGFBP-6 and weakly IGFBP-4 were detectable in epithelial cells of Malassez. Since IGFBP-6 has a very high affinity for IGF-II and can inhibit its functions, we discuss that, in the normal periodontal ligament, autocrine IGFBP-6 may function as an antiproliferative molecule suppressing mitogenic effects of IGFs on Malassez cells.     

Epithelial–mesenchymal interactions induce enamel matrix proteins and proteases in the epithelial cells of the rests of Malassez in vitro

Epithelial–mesenchymal interactions influence morphogenesis and cell differentiation in periodontal tissue regeneration. The current study examined the expression of amelogenin, ameloblastin, matrix metallopeptidase‐20 (MMP‐20), and kallikrein‐4 (KLK‐4) and their effects on the interactions between the epithelial cells of Malassez and periodontal ligament fibroblasts. Explants of human periodontal ligament tissues produced outgrowths containing both the epithelial cells of Malassez and periodontal ligament fibroblasts after incubation in a modified serum‐free medium. Both the epithelial cells and fibroblasts were co‐cultured in the same dish. The distribution and expression of all four factors were evaluated using immunohistochemistry, in‐situ hybridization and RT‐PCR analysis. The epithelial cells of Malassez were cultured separately and were used as the control. Immunohistochemical analysis revealed weak expression of amelogenin, ameloblastin, MMP‐20 and KLK‐4 in epithelial cells of Malassez co‐cultured with periodontal ligament fibroblasts. in‐situ hybridization and RT‐PCR confirmed significant mRNA expression of these factors in co‐cultured cells compared with control cells. MMP20 mRNA was not expressed in control cells. These results suggest that the epithelial–mesenchymal interactions promote differentiation of human epithelial cells of Malassez and that the induction of enamel matrix proteases facilitates the degradation of enamel matrix proteins.     

Successive removal of periodontal tissues

Abstract The aim of the present study was to compare periodontal healing after successive removal of periodontal tissue components, from the alveolar bone to the dentin surface. The prevailing tissue reaction when adhering PDM was left on the exposed roots was that most of the bone tissue that had been removed from the buccal surfaces had regenerated and the integrity of the PDM between the new alveolar bone and cementum surface had been reestablished. On exposed etched cementum surfaces, 2 prevailing healing results were recorded. In half the number of the roots, the root surfaces were covered by connective tissue with fibers running parallel to the root surfaces in a capsule-like arrangement. The other prevailing reaction was a thin epithelial cell-lining running parallel to the root surfaces in close contact or partly penetrating the adjacent connective tissue. On exposed denuded dentin surfaces, gingival retraction was a constant finding, associated with an epithelial cell-lining of varying thickness sometimes with rete pegs and cyst-like formations. Gingival retraction was also a constant finding on etched dentin surfaces. This was associated with pathological pockets outlined by epithelial cell-layers of varying thicknesses. The significance of these findings were discussed with special emphasis on dynamics of recurrent periodontitis.     

Root surface resorption, repair, and periodontal attachment following rapid maxillary expansion in man

This study was designed to investigate human repair and reattachment of principal periodontal fibers in areas of resorption on anchor premolar root surfaces following rapid maxillary expansion. Maxillary first premolar teeth were obtained from patients requiring rapid maxillary expansion. Extraction of the teeth was scheduled after periods of retention varying between 14 and 53 weeks. The roots of the teeth were examined via light microscopy and scanning electron microscopy. Extensive root resorption characterized the buccal surfaces of anchor premolars. Repair of the resorptive defects was found to occur exclusively with cellular cementum. Anchor teeth retained for longer periods, up to the maximum of 53 weeks of retention in this study, generally demonstrated more advanced repair. Topographically, Sharpey fiber holes indicative of principal periodontal fiber insertion were found in repair cementum. However, these depressions were neither numerous nor consistent in their presence and location. In human teeth, periodontal attachment to resorbed and repairing surfaces was shown to be present. SEM studies of histologic sections revealed that periodontal fibers and fiber bundles inserted directly into the repair cellular cementum matrix, irrespective of the site of the lesion on the root.     

Light and ultrastructural studies of human chronic periapical lesions

Forty clinically chronic periapical granulomas and cysts and their corresponding teeth were removed. Diagnosis was by light and transmission electron microscopy. Ultrastructural features of the root surfaces concerned and the corresponding soft tissue were detected by scanning electron microscopy. Chronic periapical inflammation had caused root resorption which affected the cementum and/or the dentin. There were no ultrastructural differences between granuloma- and cyst-induced root resorption. Resorption lacunae were devoid of epithelial and connective tissue attachment. However, root surface exposed to granulomas and cysts also indicated spontaneous cementum repair. Moreover, at their periphery, cellular debris, cells defined as fibroblasts, and cell projections and fibrils could be seen which were continuations of the periodontal ligament.     

The effects of exclusion of epithelium from healing periodontal pockets

Epithelium was excluded experimentally from artificially induced periodontal pockets by cutting off the crowns of vital teeth and covering the roots with mucoperiosteal flaps. This procedure was carried out on 58 teeth in eight rhesus monkeys and specimens recovered at from five to 36 weeks postoperatively. Thirty-eight (66%) of the teeth remained completely covered by soft tissue for the duration of the experiment. Up to 4.4 mm (mean = 0.9 mm) of new attachment with cementum and attached collagen fibers was found on both the sides of the roots and the cut ends, but root resorption and downgrowth of epithelium over root surfaces were about equally common. Thus, even when epithelium is excluded from healing periodontal pockets, reattachment with new cementum and attached collagen fibers is not consistently achieved, even after periods of 36 weeks. Root resorption and ankylosis are major complications. Although most new attachment appears to result from cells originating in the periodontal ligament, it can also arise from cells from the gingival connective tissue.     

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 the 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 are 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.     

Role of reduced enamel epithelium in root resorption

BackgroundThe precise mechanism of root resorption in human primary teeth is unclear. However, research has suggested that the enamel organ and dental follicle of the successive (permanent) teeth are important for this process. This review focuses on the mechanism of root resorption.HighlightImpaction of the maxillary permanent canines is occasionally seen. Computer tomography studies have shown that these canines cause root resorption in 12.5% of the neighboring central and lateral incisors. In such cases, enlargement of the radiolucent areas around the canine crowns is frequently seen. These radiolucent areas include dental follicles and reduced enamel epithelium, which are composed of degraded ameloblasts and cells of the papillary layer. Root resorbing factors, expressed from the reduced enamel epithelium, are likely to induce the odontoclastic root resorption. Physiological root resorption of the primary teeth is generally milder than this pathological root resorption, but involvement of the reduced enamel epithelium of the permanent tooth can be also proposed.ConclusionThis review highlights the role of the reduced enamel epithelium in root resorption under both pathological and physiological conditions.     

Comparison of Orthodontic Root Resorption of Root-filled and Vital Teeth Using Micro–computed Tomography

ObjectiveTo evaluate the difference in orthodontic root resorption between root-filled and vital teeth.Material and MethodsSixteen individuals who required bilateral premolar tooth extraction due to orthodontic treatment and had a previously root-filled premolar tooth on one side were included in the study. The experimental group consisted of root-filled premolar teeth, and the control group consisted of contralateral vital premolar teeth. A 150-g buccally directed force was applied to these teeth using 0.017 × 0.025-inch TMA cantilever springs. The premolars were extracted 8 weeks after the application of force. Images were obtained using micro–computed tomography. Resorption measurements were obtained using the Image J program.ResultsThe mean values for resorption were 0.08869 mm³ for the root-filled teeth and 0.14077 mm³ for the contralateral teeth, indicating significantly less resorption for the root-filled teeth compared with the contralateral teeth after the application of orthodontic force (P = .003). In both groups, the most resorption was seen on the cervical-buccal and apical-lingual surfaces. The mean resorption value of the cervical region was 0.06305 mm³ in the control group and 0.0291 mm³ in the experimental group, and the difference was statistically significant (P = .002).ConclusionsRoot-filled teeth showed significantly less orthodontic root resorption than vital teeth.     

Periodontal regeneration of transplanted rat molars after cryopreservation

The effects of cryopreservation on periodontal regeneration of transplanted rat molars were investigated histologically and histochemically in rats. Bilateral first and second maxillary molars of 4-week-old Wistar rats were gently extracted and transplanted into the abdominal subcutaneous connective tissue immediately or after cryopreservation in liquid nitrogen overnight. Donor teeth were slowly frozen by a rate-controlling freezer (program freezer) using 5% dimethylsulfoxide (DMSO) and 6% hydroxyethyl starch (HES) as cryoprotectants. One-four weeks after transplantation, they were carefully excised with the surrounding tissues. Regeneration of acellular cementum, periodontal ligament, and alveolar bone were observed 2 weeks after immediate transplantation. The pulp was repaired by the ingrowth of granulation tissue from the root apex followed by the formation of calcified tissue. The regenerated periodontal ligament was positive for alkaline phosphatase (ALP). Small or mononuclear tartrate resistant acid phosphatase (TRAP) positive cells were scattered on the newly formed alveolar bone and on the hard tissue in the pulp, but there was no external or internal progressive root resorption at 4 weeks. Cryopreserved teeth had acellular cementum with a rough surface at 1 week, but with the increase of cementoblasts and the appearance of periodontal ligament and alveolar bone, the surface became smooth at 3 weeks. Epithelial rests of Malassez (ERM) also revived. After regeneration of the periodontal tissues at 4 weeks, there was no evidence of root resorption. Although the process proceeded slowly, the cryopreserved teeth showed the periodontal regeneration substantially similar to that of the immediately transplanted teeth without progressive root resorption, indicating that they could be applicable for clinical use.     

Effect of root surface treatment with propolis and fluoride in delayed tooth replantation in rats

Abstract – Replantation is an acceptable option for treatment of an avulsed permanent tooth. Nevertheless, an extended extraoral period damages the periodontal ligament and results in external root resorption. The purpose of this study was to assess by histologic and histometric analysis, the influence of propolis 15% (natural resinous substance collected by Apis mellifera bees from various plants) and the fluoride solution used as root surface treatment on the healing process after delayed tooth replantation. Thirty Wistar (Rattus norvegicus albinus) rats were submitted to extraction of their upper right incisor. The teeth were maintained in a dry environment for 60 min. After this, the pulp was extirpated and the papilla, enamel organ and periodontal ligament were removed with scalpel. The teeth were divided into three experimental groups: Group I – teeth immersed in 20 ml of physiologic saline; Group II – teeth immersed in 20 ml of 2% acidulated phosphate sodium fluoride; Group III – teeth immersed in 20 ml of 15% propolis. After 10 min of immersion in the solutions, the root canals were dried and filled with calcium hydroxide paste and the teeth were replanted. The animals were euthanized 60 days after replantation. The results showed that similar external root resorption was seen in the propolis and fluoride groups. Teeth treated with physiologic saline tended to have more inflammatory root resorption compared with those treated with fluoride or propolis. However, the comparative analysis did not reveal statistically significant differences (P > 0.05) between the treatment modalities when used for delayed tooth replantation.     

The potential coronal migration of periodontal ligament tissue following experimental regeneration

The aim of the present study was to determine if the periodontal ligament cells can migrate onto curretted root surfaces following an experimental regeneration procedure. Buccal mucoperiosteal flaps were elevated in all three premolar regions in 5 mongrel dogs. The buccal bone was reduced to approximately 7 mm from its original level on 30 roots. Before the elevated flaps were replaced and sutured, Gore-Tex filters were adjusted to cover exposed root surfaces. No membranes were placed over 10 root surfaces, which served as controls. After 7 weeks of healing, the animals were sacrificed and all the roots were examined for histological evaluation of contralateral experimental and control teeth. With polychrome staining, the new periodontal ligament fibers of the curetted root surfaces were dyed methyl blue and the adjacent gingival collagen fibers were dyed red. On the experimental surface, coronal migration of periodontal ligament cells and connective tissue attachment were identified over long distances of curetted root surface. Periodontal ligament spaces were more prevalent in experimental sites than in control root surfaces. The results of the experiment suggest that the periodontal ligament cells actually migrated onto the curetted root surfaces when a periodontal space was created by physical barriers.     

Evaluating success of autotransplantation of embedded/impacted third molars harvested using piezosurgery: a pilot study

Objective. To evaluate the success of autogenous transplantation of embedded/impacted third molars harvested using piezosurgery. Materials and methods. This prospective pilot study enrolled 20 healthy patients with non-restorable first/second molars and a caries-free retrievable embedded/impacted third molar. Piezosurgery was used for removing inter-radicular bone at the recipient socket as well as for bone removal around the donor teeth. Results. After an average follow-up of 16.4 months (SD = 1.9), 18 cases were successful with formation of periodontal ligament around the teeth. One tooth was lost due to infection at 1 month. One patient was lost to follow-up. There was no root resorption or ankylosis in any of the cases. In six donor teeth with complete root formation, root canal treatment was carried out. All the remaining teeth responded positively with vitality testing. Conclusion. Piezosurgery is an effective device if embedded/impacted third molars are to be harvested for successful autogenous transplantation.