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.

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.

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.

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.

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.

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.

[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.

Ameloclasia. La resorción del esmalte. Casos clínicos / Ameloclasia. Enamel resorption. Clinical cases

La ameloclasia en dientes definitivos erupcionados es un tema sujeto a controversia. Se analiza una serie de casos clínicos con historia, exploración, radiografía y fotografía. En uno de ellos se realizó además un TAC y en otro un análisis histopatológico y estudios con SEM, ESEM y EDS. Se han observado resorciones macroscópicas y microscópicas del esmalte tanto en dientes definitivos incluidos como en erupcionados. Se discuten y justifican los resultados obtenidos en el marco del fenómeno de la inflamación/reparación, extrayendo conclusiones al respecto

Ameloclasia in permanent erupted teeth is a controversial matter. A few clinical cases are presented with dental records including exploration, radiographs and clinical pictures. In one of the cases a histologic study was done and evaluations with SEM, ESEM and EDS were carried out. Macroscopic and microscopic resoptions were observed in the enamel, not only in included teeth but also in erupted teeth. The results obtained are discussed and several conclusions are drawn

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.

A treatment decision-making model for infraoccluded primary molars.

The objective of this paper is to clarify the literature's findings and provide guidelines for decision-making during the long-term treatment planning of infraoccluded primary molars. This paper presents a synopsis of findings concerning prevalence, aetiology, diagnostic methods, diagnostic criteria and treatment options. Treatment decisions are mainly guided by the clinical assessment of the presence or absence of succedaneous tooth, evaluation of onset, time of diagnosis, resorption rate, rate of progression of infraocclusion, risk of adverse effects over time, and predictive clinical patterns of infraoccluded primary molars. These models are a result of a comprehensive review of the literature and presents a compilation of findings in a format that is of practical use to the clinician.

Reabsorçäo dentária fisiológica: estudo dos mecanismos (Apoptose) e das moléculas desencadeadoras (Bmp-4 e Gelatinase B) em dentes decíduos de gatos / Physiologic tooth resorption: mechanism study (apoptosis) and of molecules (Bmp 4 and Gelatinase B) in cats' deciduous teeth

A reabsorçäo dentária fisiológica ou rizólise em dentes decíduos, representa um fenômeno natural e programado pelo organismo, seguido pela substituiçäo da dentiçäo temporária pela permanente. Para estudar os mecanismos e mediadores bioquímicos envolvidos na induçäo da reabsorçäo fisiológica foram utilizados caninos e incisivos decíduos de gatos, em diferentes estádios de rizólise. Os animais utilizados tinham aproximadamente dois meses de vida pós-natal. O caráter programado da reabsorçäo dentária fisiológica motivou o estudo da participaçäo da apoptose neste processo. A apoptose foi evidenciada pela técnica TUNEL modificada nos cementoblastos, fibroblastos do tecido pulpar, do ligamento periodontal e nos restos epiteliais de Malassez dos dentes decíduos. A partir desta marcaçäo buscou-se identificar os prováveis mediadores bioquímicos indutores da apoptose nestas células, utilizando-se da hibridizaçäo in situ com radiosótopos. Uma das proteínas envolvidas na induçäo da apoptose em eventos fisiológicos, a proteína morfogenética óssea 4 (BMP-4), foi detectada nos cementoblastos e células do ligamento periodontal do dente decíduo. Topograficamente observou-se uma similaridade entre a expressäo de Bmp-4 e a técnica TUNEL modificada em cortes idênticos. Uma intensa marcaçäo de Bmp-4 foi também observada nos ameloblastos do dente permanente. Para a análise da participaçäo dos clastos na rizólise, foi avaliada via hibridizaçäo in situ, a expressäo de gelatinase B, uma metaproteinase da matriz extracelular sintetizada por estas células...

Cytodifferentiation of the odontoclast prior to the shedding of human deciduous teeth: an ultrastructural and cytochemical study.

BACKGROUND: In human deciduous teeth, odontoclastic resorption takes place at the pulpal surface of the coronal dentine prior to shedding, and this resorption shows clear time-related histological changes (Sahara et al., 1992). METHODS: Using this phenomenon as an observation system, we examined the cytodifferentiation of human odontoclasts by light and electron microscopy. For a histochemical marker of odontoclast differentiation and function, tartrate-resistant acid phosphatase (TRAP) activity was determined by light and electron microscopic enzyme histochemistry. RESULTS: As root resorption neared completion, TRAP-positive mononuclear cells were initially detected in the pulp chamber. They had abundant mitochondria, small lysosomes, and moderately developed rough endoplasmic reticulum throughout their cytoplasm. In these mononuclear cells, TRAP activity was localized in compartments of the biosynthetic pathway, i.e., in cisternae of the endoplasmic reticulum and Golgi lamellae, as well as small lysosomes. The TRAP-positive mononuclear cells first made contact with the predentine surface by their elongated cellular processes. After attachment, they spread out along the predentine surface and developed specialized membrane structures, clear zones, and ruffled borders. Next, they fused with each other on the predentine surface and formed typical multinucleate odontoclasts. After termination of their resorption function, the odontoclasts lost their ruffled borders and became detached from the resorbed surface. Most of the detached odontoclasts had numerous large pale vacuoles and secondary lysosomes and appeared to be in the process of degeneration. CONCLUSIONS: The present study demonstrates that: (1) odontoclasts differentiated from TRAP-positive mononuclear cells, which presumably originate from circulating progenitor cells, (2) membrane specialization of odontoclasts, i.e., development of a clear zone and ruffled border, is induced following their contact with the resorption surface, (3) multinucleation of odontoclasts takes place only after their attachment to the resorption surface, (4) mature multinucleate odontoclasts can resorb predentine as well as dentine in the same way as osteoclasts resorb bone, and (5) at the end of the resorption, odontoclasts gradually lose their ruffled borders and become detached from the resorbed surface.

The rate of osteoclastic destruction of calcified tissues is inversely proportional to mineral density.

This study examined the relative ease with which three dissimilar mineralized tissues from one individual organ were resorbed by osteoclasts in vitro. Cells released from the long bones of prehatch chicks by agitating fragments of the chopped bones in medium were cultured for 24 hours on slices cut from an Elephas maximus molar so that enamel, dentine, and coronal cementum were present in bands on the surface of the slice. The resultant pits were measured using a video-rate, line-confocal reflection light microscope system. Variations in tissue mineralization were characterized by analysis of digital backscattered electron images. The enamel pits were smaller than the dentine and the cementum pits, but the dentine and cementum pits were not significantly different from each other. The sizes of the pits correlated with the relative mineral densities of the three tissues, showing that the rate of osteoclastic destruction of calcified tissues is inversely proportional to mineral density. This indicates that the initial step in osteoclasis, the removal of the mineral phase, determines the volume removed and is the rate-limiting step.

[Dysostosis cleidocranialis--a case report]. / Dysostosis cleidocranialis--prikaz slucaja.

An 18-year female patient with dysostosis cleidocranialis, treated for dental anomalies characteristic of her basic disease since the age of 12, is described. Delayed resorption of deciduous teeth and eruption of permanent teeth were recorded. At the age of 12, the patient had still had all deciduous teeth, with the exception of lower incisors and right upper mesial incisor. The presence of 11 succedaneous teeth, 7 in the maxilla and 4 in the mandible, had been recorded by X-ray. The treatment performed step by step is presented. Firstly, deciduous teeth were extracted, and a prosthesis for both the maxilla and mandible were constructed to stimulate eruption of permanent teeth. All supernumerary teeth from the jaws were also surgically removed. After the permanent tooth eruption had been accomplished, orthodontic treatment of these teeth was required and it has still been successfully performed. Thus, a conclusion is made that the treatment of dental anomalies should start at the time of normally expected deciduous tooth exfoliation.