Hematologic, Biochemical, and Endocrine Parameters in Horses With Tooth Resorption and Hypercementosis.

BACKGROUND: Equine odontoclastic tooth resorption and hypercementosis (EOTRH) is a frequently diagnosed condition in adult horses. The underlying etiology is still unknown. Hematologic, biochemical, and endocrine values have not been reported in EOTRH-affected horses. OBJECTIVES: The main objective of the study was to describe the hematologic, biochemical, and endocrine parameters in horses with EOTRH. STUDY DESIGN: Descriptive cross-sectional study of client-owned animals with EOTRH. METHODS: A complete blood count, biochemistry panel, and endocrine profile were performed in horses diagnosed with EOTRH. Diagnosis was based on oral and radiographic examination findings and confirmed with histopathology. RESULTS: Eighteen horses with EOTRH aged 10 to 32 years from various regions of the United States were sampled. The only consistent abnormality on the complete blood cell count and chemistry panel was hypoalbuminemia (88%). Endocrine parameters demonstrated no major abnormalities in the functioning of the thyroid and pituitary pars intermedia. The parathyroid hormone concentration was increased in 7 (47%) of 15 horses with an elevated 25-hydroxy vitamin D in 3 (17%) of 17 horses. Main Limitations: The main limitations of this study are the small sample size and lack of age-matched and management-matched control horses. CONCLUSIONS: The relevance of elevated parathyroid hormone in this study cannot be determined due to the lack of age-based controls and large population studies. With the small population evaluated in this study, there are no obvious hematological, biochemical, and endocrine changes evident. Further evaluation with signalment-matched controls will be necessary to evaluate some trends noted in the laboratory values.

Osteoclasts and odontoclasts: signaling pathways to development and disease.

Osteoclasts are cells essential for physiologic remodeling of bone and also play important physiologic and pathologic roles in the dentofacial complex. Osteoclasts and odontoclasts are necessary for tooth eruption yet result in dental compromise when associated with permanent tooth internal or external resorption. The determinants that separate their physiologic and pathologic roles are not well delineated. Clinical cases of primary eruption failure and root resorption are challenging to treat. Mineralized tissue resorbing cells undergo a fairly well characterized series of differentiation stages driven by transcriptional mediators. Signal transduction via cytokines and integrin-mediated events comprise the detailed pathways operative in osteo/odontoclastic cells and may provide insights to their targeted regulation. A better understanding of the unique aspects of osteoclastogenesis and osteo/odontoclast function will facilitate effective development of new therapeutic approaches. This review presents the clinical challenges and delves into the cellular and biochemical aspects of the unique cells responsible for resorption of mineralized tissues of the craniofacial complex.

Optimised isolation method for RNA extraction suitable for RNA sequencing from feline teeth collected in a clinical setting and at post mortem.

Advanced next generation sequencing approaches have started to reveal the cellular and molecular complexity of the microenvironment in many tissues. It is challenging to obtain high quality RNA from mineralised tissues. We developed an optimised method of RNA extraction from feline teeth collected in a clinical setting and at post mortem. Teeth were homogenised in phenol-guanidinium solution at near-freezing temperatures and followed by solid-phase nucleic acid extraction utilising a commercially available kit. This method produced good RNA yields and improved RNA quality based on RNA integrity numbers equivalent (RINe) from an average of 3.6 to 5.6. No correlation was found between RNA purity parameters measured by A260:280 or A230:260 ratios and degree of RNA degradation. This implies that RNA purity indicators cannot be reliably used as parameters of RNA integrity. Two reference genes (GAPDH, RPS19) showed significant changes in expression levels by qPCR at low and moderate RINe values, while RPL17 was stable at all RINe values tested. Furthermore, we investigated the effect of quantity and quality of RNA on the quality of the resultant RNA sequencing (RNA-Seq) data. Thirteen RNA-seq data showed similar duplication and mapping rates (94 to 95%) against the feline genome regardless of RINe values. However one low yield sample with a high RINe value showed a high duplication rate and it was an outlier on the RNA-seq multidimensional scaling plot. We conclude that the overall yield of RNA was more important than quality of RNA for RNA-seq quality control. These results will guide researchers who wish to perform RNA extractions from mineralised tissues, especially if collecting in a clinical setting with the recognised restraints that this imposes.

[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.

Mesenchymal dental pulp cells attenuate dentin resorption in homeostasis.

Dentin in permanent teeth rarely undergoes resorption in development, homeostasis, or aging, in contrast to bone that undergoes periodic resorption/remodeling. The authors hypothesized that cells in the mesenchymal compartment of dental pulp attenuate osteoclastogenesis. Mononucleated and adherent cells from donor-matched rat dental pulp (dental pulp cells [DPCs]) and alveolar bone (alveolar bone cells [ABCs]) were isolated and separately cocultured with primary rat splenocytes. Primary splenocytes readily aggregated and formed osteoclast-like cells in chemically defined osteoclastogenesis medium with 20 ng/mL of macrophage colony-stimulating factor (M-CSF) and 50 ng/mL of receptor activator of nuclear factor κB ligand (RANKL). Strikingly, DPCs attenuated osteoclastogenesis when cocultured with primary splenocytes, whereas ABCs slightly but significantly promoted osteoclastogenesis. DPCs yielded ~20-fold lower RANKL expression but >2-fold higher osteoprotegerin (OPG) expression than donor-matched ABCs, yielding a RANKL/OPG ratio of 41:1 (ABCs:DPCs). Vitamin D3 significantly promoted RANKL expression in ABCs and OPG in DPCs. In vivo, rat maxillary incisors were atraumatically extracted (without any tooth fractures), followed by retrograde pulpectomy to remove DPCs and immediate replantation into the extraction sockets to allow repopulation of the surgically treated root canal with periodontal and alveolar bone-derived cells. After 8 wk, multiple dentin/root resorption lacunae were present in root dentin with robust RANKL and OPG expression. There were areas of dentin resoprtion alternating with areas of osteodentin formation in root dentin surface in the observed 8 wk. These findings suggest that DPCs of the mesenchymal compartment have an innate ability to attenuate osteoclastogenesis and that this innate ability may be responsible for the absence of dentin resorption in homeostasis. Mesenchymal attenuation of dentin resorption may have implications in internal resorption in the root canal, pulp/dentin regeneration, and root resorption in orthodontic tooth movement.

Ameloblasts: secretory and resorptive functions.

The sequence of steps which an ameloblast passes through in forming enamel appears remarkably similar in amphibian and man. Despite considerable improvements in understanding the early events in the life cycle that lead to the intracellular synthesis and secretion of enamel matrix by the cell, little is known about the cell's functional role at later stages of the life cycle when the extracellular enamel matrix undergoes classical maturation in becoming fully mineralized.

The submucosal implantation of human allogeneic decalcified dentin.

Decalcified human dentin was implanted into the buccal oral mucosa of 10 human volunteers and was later recovered for histologic examination. Extensive resorption took place. Bone did not form. Human dentinal matrix is not a bone inducer. Differences between the two methods of decalcification were not relevant.

Characteristics of periodontal mechanoreceptors supplying reimplanted canine teeth in cats.

These characteristics were investigated 12 weeks and one year after tooth reimplantation. Electrophysiological recordings were made from single mechanosensitive units dissected from the inferior alveolar nerve and each unit was characterized by applying forces to the crown of the tooth. The characteristics were compared with those of receptors innervating normal teeth. The periodontal mechanoreceptors supplying teeth reimplanted 12 weeks earlier responded to applied forces over a narrower range of directions, had higher force thresholds, lower discharge frequencies and adapted more rapidly than controls. One year after reimplantation their characteristics were nearer to normal but they remained significantly different from those of control units. It seems likely that the altered receptor characteristics could have resulted from a combination of changes in tooth mobility, disorganisation of the collagen matrix and direct injury to the nerve terminals.

Hard tissue repair in idiopathic external resorption of teeth.

A histopathological study has been made of 18 erupted and eight unerupted teeth which showed signs of idiopathic external resorption. The findings support the generally accepted theory that osteoclasts are actively involved in the resorptive process, there was no evidence that mononuclear cells were active in this process. The concept that metaplastic hard tissue was deposited on resorbed dentine surfaces, possibly as a means to shield the pulp from the external environment could not be supported from the evidence from this study. When measured, the length of resorption on which metaplastic hard tissues had been deposited, expressed as a percentage of the resorbed area ranged from zero to 69.3 per cent in the 18 erupted teeth. The percentage of metaplastic tissue replacing resorbed tissue in the eight unerupted teeth examined ranged from zero to 55.6 per cent. This study has provided some information on the type of cell responsible for idiopathic external resorption and these findings have been discussed in the light of the results of research of others in this field. The role of metaplastic hard tissue and its function have been evaluated.

Pathophysiological and therapeutic aspects of dentoalveolar resorption.

Both physiological and pathological forms of bone and tooth resorption are seen in the dentoalveolar complex, and much of the non-restorative component of a clinician's work is occupied by problems related to loss of bone or dentine. Bone resorption is also a feature of many systemic diseases, some of which can affect the alveolar bone. The aim of this report is to summarize some recent work in which the dentoalveolar complex has been used as a model system to study physiological, pathological and therapeutic aspects of resorption of bone and dentine.

Finite element analysis of equine incisor teeth. Part 2: investigation of stresses and strain energy densities in the periodontal ligament and surrounding bone during tooth movement.

This study investigated the hypothetical contribution of biomechanical loading to the onset of equine odontoclastic tooth resorption and hypercementosis (EOTRH) and to elucidate the physiological age-related positional changes of the equine incisors. Based on high resolution micro-computed tomography (µCT) datasets, 3-dimensional models of entire incisor arcades and the canine teeth were constructed representing a young and an old incisor dentition. Special attention was paid to constructing an anatomically correct model of the periodontal ligament (PDL). Using previously determined Young's moduli for the equine incisor PDL, finite element (FE) analysis was performed. Resulting strains, stresses and strain energy densities (SEDs), as well as the resulting regions of tension and compression within the PDL and the surrounding bone were investigated during occlusion. The results showed a distinct distribution pattern of high stresses and corresponding SEDs in the PDL and bone. Due to the tooth movement, peaks of SEDs were obtained in the PDL as well as in the bone on the labial and palatal/lingual sides of the alveolar crest. At the root, highest SEDs were detected in the PDL on the palatal/lingual side slightly occlusal of the root tip. This distribution pattern of high SEDs within the PDL coincides with the position of initial resorptive lesions in EOTRH affected teeth. The position of high SEDs in the bone can explain the typical age-related alteration of shape and angulation of equine incisors.

Osteoclast progenitors from cats with and without tooth resorption respond differently to 1,25-dihydroxyvitamin D and interleukin-6.

Both vitamin D and inflammatory cytokines can stimulate osteoclast formation and activity. We studied the effect of 1,25-dihydroxycholecalciferol (1,25(OH)(2)D), and interleukin-6 (IL-6), on the formation and activity of feline osteoclasts, using peripheral blood mononuclear cells (PBMCs) from cats with and without tooth resorption (TR(+) and TR(-)) as a source of osteoclast precursors. The formation of osteoclast-like cells (defined as multinucleated, tartrate-resistant acid phosphatase-positive cells) was assessed at 7 and 14 days. In the presence of M-CSF and RANKL, with and without IL-6, more osteoclasts were formed from TR(-) PBMCs than from TR(+) PBMCs on plastic. More osteoclasts were formed from TR(+) PBMCs on bone slices in the presence of M-CSF/RANKL with 1,25(OH)(2)D. This opposite effect may be due to a higher expression of the vitamin D receptor (VDR) in TR(+) osteoclasts and precursors on bone. Formation of resorption pits was analyzed and confirmed with scanning electron microscopy. In conclusion, we propose that TR(+) PBMCs when cultured on bone are sensitive to 1,25(OH)(2)D, whereas the differentiation of TR(-) PMBCs on bone seem more sensitive to IL-6, suggesting that osteoclast precursors from cats with and without tooth resorption respond differently to osteoclast stimulating factors.

Inflammatory cytokines and the nuclear vitamin D receptor are implicated in the pathophysiology of dental resorptive lesions in cats.

Dental resorptive lesions (RL) are a common oral disease in cats (Felis catus) associated with pain and tooth destruction. The aetiology of RL in cats is unknown, but inflammation is often seen in conjunction with RL. Vitamin D involvement has been suggested because 1,25-dihydroxyvitamin D (1,25(OH)(2)D) stimulates osteoclastogenesis, through up-regulation of the nuclear vitamin D receptor (nVDR). The aim of this study is to determine the involvement of inflammatory cytokines and the possible role of vitamin D in the pathophysiology of RL using quantitative PCR. We measured the mRNA expression of cytokines with stimulatory (IL-1beta, IL-6, and TNF-alpha) and inhibitory effects (IL-10 and IFN-gamma) on osteoclastogenesis, and the mRNA expression of the receptor activator of nuclear factor-kappaB ligand (RANKL), osteoprotegerin (OPG), and nVDR in RL samples. We found increased expression of mRNA levels for inflammatory cytokines and nVDR, but not for RANKL and OPG, in tissue from RL-affected cats compared with tissue from radiological confirmed healthy controls. The mRNA levels of nVDR were positively correlated with mRNA levels of pro-inflammatory (IL-1beta, IL-6, TNF-alpha, and IFN-gamma), anti-inflammatory (IL-10), pro-resorptive (IL-1beta, IL-6, and TNF-alpha), and anti-resorptive (IFN-gamma and IL-10) cytokines in the course of resorptive lesions. These data are consistent with our view that both inflammation and an overexpression of the nVDR are likely to be involved in RL in cats.

Use of enamel matrix protein derivative before dental reimplantation: a histometric analysis.

The use of enamel matrix protein in the treatment of periodontal defects has shown a favorable action on the proliferation of periodontal ligament cells, as well as on collagen formation and mineralization. The goal was to evaluate, histologically and histometrically, periodontal tissue regeneration after dental reimplantation using enamel matrix protein derivative (Emdogain, Biora AB, Malmö, Sweden). Male rats (Albinus, Wistar), weighing between 180 and 200 g, were divided in 3 groups. Animals in group I (control) had the upper right incisors extracted, the root canal was sealed with calcium hydroxide, and teeth were reimplanted in their alveoli. Group II underwent the same procedure, but the remaining periodontal ligament was removed from the root surfaces by root planing before reimplantation. In group III,following removal of the periodontal ligament, Emdogain was applied to the root surfaces. Animals were sacrificed 7, 20, and 60 days after reimplantation, and the alveoli were fixed, processed, and stained with hematoxylin and eosin. Formation of periodontal ligament, resorption areas, and ankylosis were analyzed. The results showed that group I (control) was better than groups II and III, with statistically significant differences on days 7 and 20 after reimplantation for formation of periodontal ligament. It may be concluded that with the methodology used, Emdogain was unable to stimulate tissue repair in reimplanted teeth.

[Changes of vibration frequency due to root resorption of deciduous tooth].

The purpose of this study was to investigate the changes of vibration frequency of a deciduous tooth due to physiological root resorption. The author developed an analysis system for tooth vibration by applying an impact-hammer to the tooth. Peak frequency (Hz) of the tooth stimulated by the impact-hammer was observed by the analysis system. The following results were obtained: 1) Peak frequency of the tooth vibration shifted to a lower range according to the root resorption both in vitro (simulation model) and in vivo (15 children) and a high positive correlation was found between the peak frequency and the crown-root length ratio (r = 0.840). 2) A cause of the frequency shift to the lower range was not related to the tooth weight but to the change of tooth gravity due to the root resorption. 3) It was considered that the vibration analysis system could be useful on measuring tooth mobility in vivo and in clinic as well.