Increased Presence of Complement Factors and Mast Cells in Alveolar Bone and Tooth Resorption.

Periodontitis is the inflammatory destruction of the tooth-surrounding and -supporting tissue, resulting at worst in tooth loss. Another locally aggressive disease of the oral cavity is tooth resorption (TR). This is associated with the destruction of the dental mineralized tissue. However, the underlying pathomechanisms remain unknown. The complement system, as well as mast cells (MCs), are known to be involved in osteoclastogenesis and bone loss. The complement factors C3 and C5 were previously identified as key players in periodontal disease. Therefore, we hypothesize that complement factors and MCs might play a role in alveolar bone and tooth resorption. To investigate this, we used the cat as a model because of the naturally occurring high prevalence of both these disorders in this species. Teeth, gingiva samples and serum were collected from domestic cats, which had an appointment for dental treatment under anesthesia, as well as from healthy cats. Histological analyses, immunohistochemical staining and the CH-50 and AH-50 assays revealed increased numbers of osteoclasts and MCs, as well as complement activity in cats with TR. Calcifications score in the gingiva was highest in animals that suffer from TR. This indicates that MCs and the complement system are involved in the destruction of the mineralized tissue in this condition.

Transcriptomic profiling of feline teeth highlights the role of matrix metalloproteinase 9 (MMP9) in tooth resorption.

Tooth resorption (TR) in domestic cats is a common and painful disease characterised by the loss of mineralised tissues from the tooth. Due to its progressive nature and unclear aetiology the only treatment currently available is to extract affected teeth. To gain insight into TR pathogenesis, we characterised the transcriptomic changes involved in feline TR by sequencing RNA extracted from 14 teeth (7 with and 7 without signs of resorption) collected from 11 cats. A paired comparison of teeth from the same cat with and without signs of resorption identified 1,732 differentially expressed genes, many of which were characteristic of osteoclast activity and differentiation, in particular matrix metalloproteinase 9 (MMP9). MMP9 expression was confirmed by qPCR and immunocytochemistry of odontoclasts located in TR lesions. A hydroxamate-based MMP9 inhibitor reduced both osteoclast formation and resorption activity while siRNA targeting MMP9 also inhibited osteoclast differentiation although had little effect on resorption activity. Overall, these results suggest that increased MMP9 expression is involved in the progress of TR pathogenesis and that MMP9 may be a potential therapeutic target in feline TR.

Radiological and optical coherence tomography aspects in external root resorption.

Pathological external root resorption is a process by which the hard tooth substance is lost because of action of different local irritative or systemic factors. Six forms of external root resorption are described: surface external root resorption, inflammatory external root resorption, replacement external root resorption, invasive external cervical root resorption, ankylosis and transient apical breakdown. The objective of the study was to establish the prevalence of the pathological root resorption in a retrospective radiological study, to highlight radiological aspects of external root resorption and to describe optical coherence tomography (OCT) aspects in pathological root resorption. External root resorption prevalence in our study was 17%. Radiological exam was very useful to detect the form of root resorption but also to establish possible etiological factors. The study presents the radiological aspects of some forms of pathological external root resorption highlighted on radiography accompanied by radiolucency of the adjacent bone to resorption lesion (as in inflammatory external root resorption and in invasive cervical root resorption) or as changes of the root form, in which the missing part of the root is replaced by the bone tissue, without radiolucency (as in replacement root resorption). The comparison of the OCT aspects of the physiological root resorption in primary teeth with the OCT aspects of pathological root resorption in permanent teeth showed an obvious difference between the images of the OCT signal of the two types of resorption. The OCT signal from the cement is stronger in pathological root resorption, and the OCT signal from the dentin in teeth with inflammatory external root resorption showed a demineralized dentin with multiple heterogeneities, anfractuosities corresponding to resorption craters, with craze lines in subjacent layers of dentin.

Localización de OPG en la pulpa dental del Ratón Albino Suizo / OPG Location in dental pulp of Albino Swiss Mice

En condiciones fisiológicas, el órgano dentinopulpar no se encuentra asociado a procesos de remodelación y resorción. La Osteoprotegerina (OPG) es una molécula del Factor de Necrosis Tumoral (TNF) que participa en la diferenciación y activación de osteoclastos, por lo que su papel en el control del proceso de reabsorción radicular interna debe ser determinado. Se tomaron secciones histológicas de 5 µm tomadas de primeros molares de 8 hemimandíbulas de ratón y fueron sometidas a proceso de inmunohistoquímica con la técnica de complejo avidina-biotina peroxidasa para la detección de OPG. Se identificó la presencia de OPG en las regiones central y periférica de la pulpa de ratones de 4 y 12 semanas. La pulpa de ratones albino suizo mostró presencia diferencial de OPG en las zonas central y periférica

Under physiological conditions, the dentinopulpar complex is not associated with remodeling and resorption. Osteoprotegerin (OPG) is a TNF molecule associated with osteoclast activation and differentiation. Therefore, OPG role in internal root resorption must be determined. We obtained histological sections of 5 µm in first molars from 8 hemimandibles of mice, which were subjected to the avidin-biotin peroxidase complex immunohistochemical procedure for detection of OPG. Differential OPG presence was identified in central and peripheral pulp in dental pulp of 4 and 12 week-old mice

Comparison of the ability of chondroitin sulfate derived from bovine, fish and pigs to suppress human osteoclast activity in vitro.

Chondroitin sulfate (CS) compounds are commonly used to manage OA symptoms. Recent literature has indicated that abnormal subchondral bone metabolism may have a role in the pathogenesis of OA. The aim of this study was to access the effects of chondroitin sulfate obtained from bovine, fish and porcine sources on human osteoclast formation and activity in vitro. Human osteoclasts were generated from blood mononuclear cells. Cells were cultured over 17 days with the addition of macrophage colony stimulating factor (M-CSF) and then stimulated with receptor activator of nuclear factor kappa B ligand from day 7. Cells were treated with the CS commencing from day 7 onwards. To assess effects on osteoclasts, tartrate resistant acid phosphatate (TRAP) expression and resorption of whale dentine assays were used. Bovine-derived CS consistently suppressed osteoclast activity at concentrations as low as 1 µg/ml. Fish and porcine CS was less consistent in their effects varying with different donor cells. All CS compounds had little effect on TRAP activity. mRNA analysis using real-time PCR of bovine CS treated cells indicated that the inhibition of activity was not due to inhibition of the late stage NFATc1 transcription factor (p > 0.05). These results are consistent with CS inhibition of mature osteoclast activity rather than the formation of mature osteoclasts. It would appear that there are differences in activity of the different CS compounds with bovine-derived CS being the most consistently effective inhibitor of osteoclast resorption, but the results need to be confirmed.

Increased vitamin D-driven signalling and expression of the vitamin D receptor, MSX2, and RANKL in tooth resorption in cats.

Tooth resorption occurs in 20-75% of cats (Felis catus). The aetiology is not known, but vitamin D is suggested to be involved. Vitamin D acts through a nuclear receptor (VDR) and increases the expression of receptor activator of nuclear factor-kappaB ligand (rankl) and muscle segment homeobox 2 (msx2) genes. Mice lacking the muscle segment homeobox 2 (msx2) gene show decreased levels of rankl, suggesting an interaction among VDR, MSX2, and RANKL. Here, we investigated the expression of VDR, MSX2, and RANKL proteins, and the activity of the VDR-mediated signalling pathway (using the quantitative polymerase chain reaction on VDR target genes), in tooth resorption, and measured the serum levels of vitamin D metabolites in cats. Tooth resorption was categorized into either resorptive or reparative stages. In the resorptive stage, odontoclasts expressed MSX2 and RANKL (100% and 88%, respectively) and fibroblasts expressed VDR and MSX2 (both at 100%), whereas fibroblasts expressed RANKL in only 29% of the sites analysed. In the reparative stage, cementoblasts expressed VDR, MSX2, and RANKL, whereas fibroblasts expressed VDR and MSX2, but not RANKL. The vitamin D status did not differ between the groups, based on the serum levels of 25-hydroxycholecalciferol. However, increased expression of VDR protein, and the relative gene expression levels of 1alpha-hydroxylase and the VDR-target gene, 24-hydroxylase, indicated the involvement of an active vitamin D signalling in the pathophysiology of tooth resorption in cats.

Dental pulp and periodontal ligament cells support osteoclastic differentiation.

Odontoclasts and cementoclasts are considered to play major roles in the internal resorption of dentin and the external resorption of tooth roots. In this study, we evaluated the osteoclast-inducing ability of human dental pulp and periodontal ligament cells, which are mesenchymal cells in dental tissues. These cells expressed RANKL and OPG mRNA constitutively. As osteoclast precursors, CD14(+) monocytes derived from human peripheral blood were isolated, and incubated together with human dental pulp or periodontal ligament cells. Both cell types spontaneously induced the differentiation of CD14(+) monocytes into osteoclasts without osteotropic factors. These results suggest that dental pulp and periodontal ligament cells are involved in regulating the differentiation and function of osteoclasts.

The role of dental pulp cells in resorption of deciduous teeth.

OBJECTIVE: To address the question whether dental pulp cells of exfoliating human deciduous teeth have some roles for controlling or regulating the root resorption via secreting key molecules (OPG, RANKL, CSF-1, TGFbeta, MCP-1 and Cbfa-1) in osteoclastogenesis, we used a sensitive reverse transcriptase polymerase chain reaction (RT-PCR) method for detection of mRNA expressions for the cytokines listed. STUDY DESIGN: The dental pulps were retrieved from incisor and molar teeth in the late stage of shedding (n = 30) and from sound premolar teeth extracted for orthodontic reasons (control group; n = 30). The RT-PCR assays were used to identify targeted gene expression. RESULTS: Of the cytokines examined, RANKL and CSF-1 expressions showed significantly higher occurrence in deciduous dental pulps than in permanent teeth pulpal tissues (P < .040). CONCLUSIONS: The findings may suggest an interactive role for pulp tissue cells in the physiologic root resorption process. The cells of dental pulp may have some cytokine-producing cells which mediate monocyte-macrophage lineage to form osteo/odontoclasts, and the RANKL/RANK system might be involved in human deciduous teeth resorption.

[Effect of extracellular zinc on osteoclastic resorption in dental mineralized tissues].

OBJECTIVE: To investigate the bone resorption caused by osteoclasts and modulating functions of zinc ion on dental slices. METHODS: Osteoclasts were separated from long-limb bones of neonatal rabbits, cultured with de-activated human tooth slices and glass slices. The cells in the experiment group were treated with 1x10(-14)mol/L-1x10(-4)mol/L zinc+10% (volume fraction) fetal calf serum (FCS)+alphaMEM, while those in the control group were grown in 10%FCS+alphaMEM. Osteoclasts on glass slices were stained by TRAP staining. The absorption pits on tooth slices were observed by inverted phase contrast microscope. The resorbing activity was evaluated with the concentration of calcium in the supernatant liquid of osteoclasts. The ratio between the concentration of calcium in the experiment group and that of the control group was termed the resorption index. RESULTS: The isolated cells were multinuclear and showed positive in cytoplasma by TRAP staining. Usually, osteoclasts resorbed tooth slices first on the cementum and dentin, which had lower content of mineralized tissue. Compared with those on bone slices, the lacunae on the dental slices appeared less in amount, less in area and shallower in depth. They often showed shallow pits in a large area. Microscopy showed that the number and area of absorption pits formed on treated tissues were less than those on the control tissues. The content of calcium in the supernatant liquid increased at the concentrations of 1x10(-4)-1x10(-14)mol/L zinc, especially in the group of 1x10(-8)mol/L, 1x10(-10) mol/L, 1x10(-14)mol/L zinc on the 3rd day (P<0.05). But they were reversed on the 7th day, except in the group of 1x10(-14)mol/L zinc. At the end of culture, the resorption indexes of 1x10(-4)-1x10(-7)mol/L, 1x10(-9)mol/L, 1x10(-12)mol/L and 1x10(-13)mol/L group were lower than 1, but those of 1x10(-8)mol/L, 1x10(-10)mol/L, 1x10(-11)mol/L and 1x10(-14)mol/L group were higher than 1. CONCLUSION: The effect of zinc ion on osteoclastic resorption in dental slices is associated with phase and dosage closely.

Cementum-like tissue deposition on the resorbed enamel surface of human deciduous teeth prior to shedding.

Prior to the shedding of human deciduous teeth, odontoclastic resorption takes place at the pulpal surface of the coronal dentin, and this resorption occasionally extends coronally from the dentinoenamel junction into the enamel. After the end of resorption, however, the resorbed enamel surface is repaired by the deposition of a cementum-like tissue. Using this phenomenon as an observation model, in this study we examined the sequence of cellular and extracellular/matrix events involved in the enamel resorption repair by light and electron microscopy. As the odontoclast terminated its resorption activity, it detached from the resorbed enamel surface; thereafter, numerous mononuclear cells were observed along the resorbed enamel surface. Most of these mononuclear cells made close contact with the resorbed enamel surface, and coated pits or patches were observed on their plasma membrane facing this surface. Furthermore, they frequently contained thin needle- or plate-like enamel crystals in their cytoplasmic vacuoles as well as secondary lysozomes. Following the disappearance of these monononuclear cells, the resorbed enamel surface now displayed a thin coat of organic matrix. Ultrastructurally, this organic layer was composed of a reticular and/or granular organic matrix, but contained no collagen fibrils. Energy-dispersive X-ray microanalysis of this thin organic layer in undecalcified sections revealed small spectral peaks of Ca and P. Cementum-like tissue initially formed along this thin organic layer, increased in width, and appeared to undergo mineralization as time progressed. The results of our observations demonstrate that regardless of type of matrix of dental hard tissues, tooth repair may be coupled to tooth resorption, and suggest that mononuclear cells and an organic thin layer found on the previously resorbed enamel surface may play an important role in the repair process initiated after resorption of the enamel.

Elemental composition of teeth with and without odontoclastic resorption lesions in cats.

OBJECTIVE: To determine elemental composition of teeth with and without odontoclastic resorption lesions (ORL) in cats. SAMPLE POPULATION: Normal teeth from 22 cadaver cats and ORLaffected teeth from 21 cats admitted to the veterinary hospital for dental treatment. PROCEDURE: An electron microprobe was used to analyze weight percentages of calcium, phosphorus, fluorine, sodium, magnesium, sulfur, potassium, and iron in enamel, dentin, and cementum. RESULTS: Calcium and phosphorus were the most abundant elements. Fluorine, sodium, and magnesium combined were < 5% and sulfur, potassium, and iron combined were < 0.1% of total elemental composition. In enamel of normal teeth, a significant sex-by-jaw location interaction was seen in mean (+/- SD) phosphorus content, which was higher in mandibular teeth of females (1764+/-0.41%) but lower in mandibular teeth of males (16.71+/-0.83%). Mean iron content in dentin of normal teeth was significantly lower in mandibular teeth than maxillary teeth (0.014+/-0.005% vs 0.023+/-0.019%). Mean enamel sodium content was significantly higher (0.77+/-0.046% vs 0.74+/-0.025) and mean enamel iron content was significantly lower (0.017+/-0.008% vs 0.021+/-0.005%) in ORLaffected teeth, compared with normal teeth. In cementum, mean fluorine content was significantly lower (2.98%+/-0.27 vs 2.99+/-0.20%) and mean magnesium content was significantly lower (0.54+/-0.13% vs 0.60+/-0.13%) in ORL affected teeth, compared with normal teeth. CONCLUSIONS AND CLINICAL RELEVANCE: Results of our study establish baseline mineral content of enamel, dentin, and cementum for normal teeth in cats. Minimal differences in mineral content of enamel and cementum of normal and ORL-affected teeth were detected.

Expression of cathepsin K mRNA and protein in odontoclasts after experimental tooth movement in the mouse maxilla by in situ hybridization and immunoelectron microscopy.

This study demonstrated the simultaneous expression of cathepsin K (CK) mRNA by in situ hybridization and CK protein by immunoelectron microscopy in odontoclasts in mouse maxillae after experimental tooth movement. On the pressure side (the area under pressure during tooth movement), CK mRNA was detected in odontoclasts in resorption lacunae in the tooth root, in osteoclasts in bone resorption lacuane, and in fibroblasts in the periodontal ligament. Using electron microscopy, CK protein was detected at the apex of odontoclasts, intracellularly in vesicles and granules, and extracellularly in irregularly shaped vacuoles (extracellular spaces), on the plasma membrane of the ruffled border, and on and between typical striated type I collagen fibrils in the lacunae. These vesicles and granules appeared to fuse with irregular vacuoles containing CK-positive fragmented fibril-like structures close to the ruffled border. In the basolateral portion of odontoclasts, small amounts of CK-positive rough endoplasmic reticulum (ER) were found. CK-positive intracellular vacuoles (not extracellular spaces) also appeared to fuse with the vesicles and granules. However, these fused organelles rarely contained fragmented fibril-like structures. They are probably endolysosomes. The distribution of CK in odontoclasts was similar to that previously seen in osteoclasts. Furthermore, CK-positive fibril-like structures were found in the vacuoles of fibroblasts. These results indicated that during tooth movement CK is synthesized in odontoclasts on the pressure side and secreted into the tooth resorption lacunae. Therefore, CK may take part in the degradation of the dentin matrix (type I collagen fibrils and non-collagenous protein) of the tooth root, and in the subsequent intracellular degradation of endocytosed fragmented fibril-like structures in endolysosomes.

Ultrastructural features of odontoclasts that resorb enamel in human deciduous teeth prior to shedding.

Three dental hard tissues, i.e., cementum, dentin, and enamel, are resorbed by multinucleated cells referred to as "odontoclasts." These cells have morphological and functional characteristics similar to those of bone-resorbing osteoclasts. However, concerning enamel resorption, which is a process that may occur during tooth eruption, satisfactory ultrastructural data on odontoclastic resorption are still lacking. Ultrastructural and histochemical characteristics of odontoclasts resorbing enamel of human deciduous teeth prior to shedding were examined by means of light microscopy and transmission and scanning electron microscopy. Odontoclasts that that resorbed enamel were tartrate-resistant acid phosphatase (TRAP)-positive multinucleated giant cells that were essentially the same as those that resorbed dentin and cementum. Ultrastructurally, they had numerous mitochondria, lysosomes, and free polysomes in their cytoplasm. In addition, they were characteristically rich in large cytoplasmic vacuoles containing enamel crystals in the cytoplasm opposite the ruffled border. Although they extended a well-developed, ruffled border against enamel surface, a clear zone--an area typically devoid of organelles--was rarely seen in these cells. In many cases, the cells were in very close contact with the enamel surface by the peripheral part of their cytoplasm. The enamel prisms at the resorption surface contained more loosely packed and electron-lucent enamel crystals compared with those of unresorbed, intact enamel. Furthermore, numerous thin needle- or plate-like enamel crystals that were liberated from the enamel matrix were found in the extracellular channels of the ruffled border and in various-sized cytoplasmic vacuoles in their cytoplasm. The superficial layer of the enamel matrix undergoing odontoclastic resorption stained positively with toluidine blue and for TRAP activity. The results of the present study suggest that odontoclasts resorbing enamel secrete acids as well as organic components, including hydrolytic enzymes, into the resorption zone underlying their ruffled border and that they phagocytose crystals that have been liberated from the partially demineralized enamel matrix by acids, subsequently dissolving them intracellularly.

Expression of adhesion molecules during tooth resorption in feline teeth: a model system for aggressive osteoclastic activity.

Tooth resorption, a common feline dental problem, is often initiated at the cemento-enamel junction and hence is called cat 'neck' lesion. Studies have demonstrated that osteoclasts/odontoclasts are increased and activated at resorption sites, and that areas of resorption are partly repaired by formation of tissues resembling bone, cementum, and possibly dentin. However, the cellular/molecular mechanisms/factors involved in resorption and repair are unknown. In this study of tissues from cats with 'neck' lesions, we used specific antibodies and immunohistochemical analyses to examine adhesion molecules associated with mineralized tissues, bone sialoprotein (BSP) and osteopontin (OPN), and a cell-surface receptor linked with these molecules, alpha v beta 3, for their localization in these lesions. In addition, to determine general cellular activity during repair, we performed in situ hybridization using a type I collagen riboprobe. Results showed OPN localized to resorption fronts and reversal lines, while BSP was localized to reversal lines. However, some osteoclasts and odontoblasts "sat" on mineralized surfaces not associated with OPN. The cell-surface receptor, alpha v beta 3, was localized to surfaces of osteoclasts/odontoclasts. Type I collagen mRNA was expressed where osteoblasts attempted to repair mineralized tissue. In contrast, odontoblasts did not express mRNA for type I collagen. This study suggests that osteoclastic resorption is the predominant activity in 'neck' lesions and that this activity was accompanied, at least in part, by increased concentrations of OPN and an associated integrin, alpha v beta 3, at resorption sites. Lack of collagen expression by odontoblasts indicates that odontoblasts do not play an active role in attempts at repair.

Mucopolysaccharidase activity and glycosaminoglycan content in traumatized resorbing deciduous teeth.

The periodontal ligaments from traumatized deciduous teeth, while undergoing rapid resorption, were analyzed biochemically for mucopolysaccharidase activity and for total glycosaminoglycan content of dentin and cementum. Enzyme activity was present only in resorbing teeth. A concomitant 65 percent decrease in glycosaminoglycans from these teeth occurred as well.

An inhibitory principle in the matrix of periodontally diseased roots.

The inhibitory influence shown by the periodontally diseased root resides in its organic component. Its nature is uncertain and it may be an etiologic factor in periodontal destruction. The apatite structures of healthy and diseased roots are alike in being more receptive to the growth of bone and marrow than are their respective matrices.