Rat Incisor Research Articles

Establishment of rat models with altered anterior guidance and observation of histological changes in temporomandibular joint synovium

This study aims to establish a stable animal model with altered anterior guidance and investigate histological changes on temporomandibular joint (TMJ) synovium in rats. Thirty-two rats were divided into four groups, namely, control group, anterior-guidance-deficient group (T1), and anterior guidance inclined up to 15 degree (T2) and 30 degree (T3) groups. Metal crowns that add 15 and 30 degrees to the palatal side were cemented to the incisors of maxilla to increase the inclination of anterior guidance. The low-speed diamond wheel shorts for both upper and lower incisors of rats were applied to avoid contact between anterior teeth in all direction, thus establishing an anterior guidance deletion model. TMJ joints on one side of two animals in each group were harvested after 3, 7, 14, and 28 days. Pathological changes were investigated in the TMJ synovium using hematoxylin-eosin (HE) staining. The weight of T1 decreased briefly at first, and then increased slightly (P<0.05). The weight of T3 obviously reduced and then slowly increased, but remained below the initial weight (P<0.05). T3 manifested obvious synovial lining proliferation and vascular dilation (P<0.05). The rat model of anterior guidance inclined up to 30 degree could imitate the pathological features of TMJ synovium with excessive increase in anterior guidance. A higher inclination of anterior guidance might cause more damage of TMJ synovium. The effect of anterior guidance deletion on TMJ synovium is yet to be determined. .

Estimated Time of Biomineralization in Developing Rat Incisors

이 연구의 ëª©ì ì€ 쥐의 í•˜ì• ì ˆì¹˜ì—ì„œ ìƒë¬¼í•™ì  광화에 대한 발육 단계를 íƒ€ìž„í Œì´ë¸”ë¡œ 수립하는데 있다. 맹출 길이가 ì¸¡ì •ë˜ì—ˆê³ ì¡°ì§í•™ì  ì ˆíŽ¸ê³¼ 미세단층촬영기(microscopic computerized tomography) 단면으로 ì ˆì¹˜ 발육의 4단계 (1) preodontoblast, (2) dentin matrix secretion, (3) enamel matrix secretion, ê·¸ë¦¬ê³ (4) enamel calcification을 확인하였다. 쥐의 í•˜ì• ì ˆì¹˜ì—ì„œ 총 맹출 속도는 600 ± 70 μm/day (mean ± SD; n = 12) 였다. 법랑질 분비 길이는 ì¡°ì§í•™ì  ì ˆíŽ¸ì—ì„œ 4.59 ± 0.75 mm, ë°©ì‚¬ì„ í•™ì  단면에서 3.64 ± 0.63 mmì˜€ê³ ì´ë¥¼ 각각 속도로 환산하면 180.4 ± 30.0 hours, 145 ± 25 hours 으로 나타났다(n = 24). 이러한 결과들은 쥐의 ì ˆì¹˜ì˜ ìƒë¬¼í•™ì  광화가 일어나는 발육 4 단계가 단 ë©°ì¹ ë§Œì— 이루어진다는 것을 ì œì‹œí•œë‹¤. 이번 동물 실험연구의 결과는 발육 중인 치배의 ë¹ ë¥¸ 광물화 ê³¼ì •ì„ 치아 발육 기간의 분석을 통해 ì´í•´í• ìˆ˜ 있다는 것에 의의가 있다.

共焦点レーザ走査顕微鏡(CLSM)による低Ca食飼育ラット切歯ラベリング像の観察

共焦点レーザ走査顕微鏡(CLSM)による低Ca食飼育ラット切歯ラベリング像の観察

Effect of lipopolysaccharide stimulation on stem cell-associated marker-expressing cells.

To examine the effect of inflammatory stimuli on the proliferation/migration of dental pulp stem cells by assessing the responses of stem cell-associated marker-expressing cells in rat incisors to lipopolysaccharide (LPS) stimulation invivo. The crowns of rat incisors were removed, and the coronal pulp chamber was instrumented. After haemostasis, an absorbent point soaked in LPS was inserted into the cavity, which was then sealed. At 3, 12, and 48h after LPS application, pulp tissues were subjected to double-immunoperoxidase labelling using two of the antibodies against microtubule-associated protein 1B (MAP1B), CD146 and STRO-1. For gene expression analysis, total RNA was extracted, and mRNA expression levels of stem cell factor (SCF), stromal-derived factor 1 (SDF-1), CD146 and MAP1B were analysed with real-time polymerase chain reaction. SCF and SDF-1 protein levels were also assessed by Western blot. Statistical analysis was performed by Kruskal-Wallis nonparametric analysis of variance, followed by Mann-Whitney U-tests with Bonferroni correction. The density of MAP1B+ CD146+ cells and STRO-1+ CD146+ cells in LPS-stimulated pulp tissue increased significantly at 3h and exhibited a four- to sixfold increase at 48h as compared with the density observed in normal pulp tissue (P<0.05). The expression of CD146 mRNA in LPS-stimulated pulp showed significant upregulation at 3h as compared with that observed in normal pulp tissue (P<0.05). MAP1B, SCF and SDF-1 mRNA levels also showed significant upregulation at 3 and 72h (P<0.05), and Western blot analysis revealed increases in SCF and SDF-1 following LPS stimulation. LPS-stimulated pulp tissue exhibited upregulation of stem cell differentiation/migration markers and showed increases in the number of MAP1B+ CD146+ and STRO-1+ CD146 stem-like cells.

Major salivary gland hypertrophy model in immature rats: morphometric and histochemical epithelial cell characteristics

The purpose of the study is to estimate the functional state of epithelial cells of acini and ducts of major salivary glands with hypertrophy caused by repeated incisor amputations in immature rats. Materials and methods. The experiment was carried out on immature (20 days), white male rats, divided into 3 groups: intact, control and group of rats with repeated incisor amputations. Animals were taken out in 2d, 3d, 4th, 6th, 8th, 10th and 12th weeks after the first incisor amputation. Morphofunctional state of rat major salivary glands was assessed by histological (hematoxylin and eosin), histochemistrical (Alcian blue, PAS-reaction, Brachet method) and morphometrical (acini area, intralobular ducts volume) methods. Results. Repeated incisor amputations led to the increase of acini area and the decrease of intralobular duct volume in submandibular glands in 2nd–4th weeks of the experiment. Cytoplasm pyroninophilia of submandibular gland acinar cells was less pronounced and intensity of PAS-reaction was more pronounced than in intact animals in 3rd week of the experiment. Morphological and functional changes of parotid and sublingual gland epithelial cells were not observed after repeated amputations of incisors in immature rats. Conclusion. Repeated incisor amputations in immature male rats lead to submandibular gland acinar cell hypertrophy in the early stages of the experiment (2d–4th weeks) with accumulation of glycoproteins and protein synthesis weakening in these cells. Hypertrophy of acinar cells are accompanied by retardation in the development of granular convoluted tubule cells which are the source of synthesis and secretion of the endocrine biologically active factors of submandibular glands.

Expression of Steroid Receptors in Ameloblasts during Amelogenesis in Rat Incisors.

Endocrine disrupting chemicals (EDCs) play a part in the modern burst of diseases and interfere with the steroid hormone axis. Bisphenol A (BPA), one of the most active and widely used EDCs, affects ameloblast functions, leading to an enamel hypomineralization pattern similar to that of Molar Incisor Hypomineralization (MIH). In order to explore the molecular pathways stimulated by BPA during amelogenesis, we thoroughly investigated the receptors known to directly or indirectly mediate the effects of BPA. The expression patterns of high affinity BPA receptors (ERRγ, GPR30), of ketosteroid receptors (ERs, AR, PGR, GR, MR), of the retinoid receptor RXRα, and PPARγ were established using RT-qPCR analysis of RNAs extracted from microdissected enamel organ of adult rats. Their expression was dependent on the stage of ameloblast differentiation, except that of ERβ and PPARγ which remained undetectable. An additional large scale microarray analysis revealed three main groups of receptors according to their level of expression in maturation-stage ameloblasts. The expression level of RXRα was the highest, similar to the vitamin D receptor (VDR), whereas the others were 13 to 612-fold lower, with AR and GR being intermediate. Immunofluorescent analysis of VDR, ERα and AR confirmed their presence mainly in maturation- stage ameloblasts. These data provide further evidence that ameloblasts express a specific combination of hormonal receptors depending on their developmental stage. This study represents the first step toward understanding dental endocrinology as well as some of the effects of EDCs on the pathophysiology of amelogenesis.

Analysis of calcium, phosphorus, and carbon concentrations during developmental calcification of dentin and enamel in rat incisors using scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDX)

ObjectiveThe study was designed to investigate the concentrations of calcium (Ca), phosphorus (P), and carbon (C) during developmental calcification of dentin and enamel in rat incisors. MethodsMandibular incisors from eight 2-week-old male Wistar rats were analyzed by scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDX). We analyzed data on the elements in the course of developmental processes in dentin and enamel and along the vertical line of the matrix between odontoblasts and ameloblasts. ResultsThe dentin concentrations of Ca and P and the Ca/P ratio were the lowest, while the C concentration was the highest in initial dentin. The Ca and P concentrations were the lowest, whereas the C concentration was the highest in predentin along the vertical line; the Ca/P ratio did not show any differences. The concentrations of Ca and P increased, while the C concentration decreased during early maturation and more so in late maturation in developing enamel, while the Ca/P ratio increased during late maturation. The Ca and P concentrations and the Ca/P ratio were the highest, while the C concentration was the lowest in enamel adjacent to the junction with dentin on the vertical line. ConclusionsDuring tooth development, the initial dentin matrix may possess distinctive mineral characteristics as compared with other parts of dentin and predentin. Elemental composition of the mineral in enamel may change during late maturation. Our results are suggestive of degradation of organic components during developmental calcification in dentin and enamel.

Doxycycline reduces the expression and activity of matrix metalloproteinase‐2 in the periodontal ligament of the rat incisor without altering the eruption process

Doxycycline is an antibiotic agent that inhibits the activity of matrix metalloproteinases (MMPs) present in the extracellular matrix. In this study, the rat incisor was submitted to a hypofunctional condition, and the effects of doxycycline (80 mg/kg/d) on the expression and activity of MMP-2, as well as on eruption rate, were determined in the odontogenic region and in the periodontal ligament for 14 d. Rats were distributed into four groups: normofunctional (NF); doxycyline normofunctional (DNF); hypofunctional (HP); and doxycyline hypofunctional (DHP). The left lower incisors of 10 rats were shortened every 2 d, using a high-rotation drill, to produce the HP and DHP groups, after starting doxycycline treatment (80 mg/kg) by gavage. Eruption was measured using a millimeter ocular, from the gingival margin to the top of the tooth in the HP and DHP groups, and also by a mark made in the tooth previously, in the NF and DNF groups. The hemimandibles were removed and the teeth were extracted to collect the periodontal and odontogenic tissues for immunohistochemical analyses and zymography. The eruption rates were higher in the HP and the DHP groups than in the NF and DNF groups, respectively (p < 0.05). In the odontogenic region, neither of the treatments changed the expression and activity of MMP-2. In the HP group, the shortening treatment decreased the expression, but not the activity, of MMP-2, while doxycycline was able to inhibit the increase of expression and activity of MMP-2. We conclude that the inhibition of MMP-2 by doxycycline, during incisor shortening, was not enough to alter the eruption rate, which suggests that MMP-2 may have an important role in the turnover of extracellular matrix of the periodontal ligament during the tooth-eruption process.

CD271 Negative Human Dental Pulp Cells Yield Significantly More Adherent Colony Forming Cells than the Positive Phenotype

Cell surface markers for isolating proliferative human dental pulp stromal cells are currently lacking. Other tissues containing mesenchymal stromal cells have been studied in greater depth and candidate markers for cell isolation identified, one such marker being CD271. Previous reports suggest CD271 as a marker for isolating dental pulp stromal cells from rat incisors. We aimed to study the utility of CD271 as a marker for isolating human dental pulp stromal cells. CD271 positive cells from both third molar dental pulp and bone marrow mononuclear cells were isolated by magnetic separation followed by in vitro expansion. Phenotypic analysis was performed by flow cytometry. Our data showed that although CD271 is present in the adult molar dental pulp, significantly greater numbers of colonies are produced from the CD271 negative fraction. The opposite was seen with bone marrow mononuclear cells; all colony forming cells were derived from the CD271 positive fraction. Phenotypic analysis of expanded CD271 negative cells showed that these cells are identical to dental pulp stromal cells isolated by non-selective plastic adherence. We conclude that, in contrast to bone marrow, CD271 is not a positive selection marker for the predominant colony forming cell types from human molar dental pulp.

Parathyroid hormone intermittent administration promotes delay on rat incisor eruption

ObjectiveThis study evaluated the influence of parathyroid hormone (PTH) (1–34) intermittent administration on rat eruption rates of lower incisors under normo, hyper and hypofunctional conditions, Sharpey fibers insertion, and alveolar bone formation. Materials and methodsWistar male rats received PTH (1–34) three times a week during the entire experimental period, 31days. Control animals received the same concentration of the vehicle solution during the same period. Three injections of alizarin were also performed. The experiment evaluated the eruptive rate, the alveolar bone formation and also the morphology, and the area density of Sharpey fibers. After the sacrifice, the mandibles were dissected and samples were prepared for fluorescence and scanning electron microscopy observations. ResultsPTH-treated animals showed significantly reduced eruption rates in all different functional conditions. Analysis evidenced that PTH-treated rats present an increase in bone formation and area density of the Sharpey fibers. ConclusionWe concluded that the PTH (1–34) intermittent administration reduced the eruptive process rates, through bone formation enhancement and increase in the area density of Sharpey fibers.

The Novel NF‐κB Inhibitor, MTI‐II Peptide Anti‐Inflammatory Drug, Suppresses Inflammatory Responses in Odontoblast‐Like Cells

Regulation of inflammation is important for pulp wound healing, including protective responses by odontoblast-like cells. However, methods for directly regulating pulp inflammation have not yet been described. The inflammatory response is mediated by a transcription factor, nuclear factor-κB (NF-κB), which activates inflammatory cytokines including tumor necrosis factor (TNF)-α. Macromolecular translocation inhibitor II (MTI-II) was previously demonstrated as an enhancer of the transcriptional activity of glucocorticoid-bound glucocorticoid receptor. Recently, a MTI-II peptide anti-inflammatory drug (MPAID) was bioengineered from the structure of MTI-II as an inhibitor of NF-κB transactivation. Here, we examined the effects of MTI-II and MPAID on the inflammatory responses of odontoblast-like cells. TNF-α inhibited alkaline phosphatase (ALP) activity, a marker of odontoblast/osteogenic differentiation, and induced NF-κB transcriptional activity in KN-3 cells, which are odontoblast-like cells derived from dental papilla cells of rat incisors, without affecting their viability. Exogenous expression of MTI-II suppressed the NF-κB transcriptional activity induced by TNF-α or overexpression of p65 (a main subunit of NF-κB) in the cells, whereas it failed to inhibit degradation of IκBα and nuclear translocation of p65 after TNF-α treatment, suggesting that MTI-II inhibits NF-κB transcriptional activity by modulating the duration of DNA binding by p65. MPAID also inhibited TNF-α-induced NF-κB transcriptional activity, the mRNA expression of IL-6 and IL-8, and IL-6 production. Furthermore, pretreatment of the cells with MPAID restored the inhibitory effect of TNF-α on ALP activity. These results suggest that MPAID may be able to regulate the inflammatory response and maintain a protective response of dental pulp. J. Cell. Biochem. 117: 2552-2558, 2016. © 2016 Wiley Periodicals, Inc.

Nanosilver coated orthodontic brackets: in vivo antibacterial properties and ion release.

Silver nanoparticles are currently utilized in the fields of dentistry. The aim of this study was to evaluate the antibacterial properties and ion release of nanosilver coated orthodontic brackets compared to conventional brackets. Nanosilver coating process was applied to standard orthodontic brackets placed on the mandibular incisors of Wistar Albino rats in the study group and conventional brackets in the control group. Dental plaque, mucosal vestibular smears, saliva, and blood samples were collected from rats at various days. The amounts of nanosilver ions in blood and saliva were measured and microbiological evaluation was made for Streptococcus mutans. For testing cariogenicity, all rats were sacrificed at the end of 75 days under anaesthesia. Teeth were stained using a caries indicator, then the caries ratio was assessed. Nanosilver coated orthodontic bracket favoured the inhibition of S.mutans on Day 30 and reduction of caries on the smooth surfaces. The nanosilver amounts in the saliva and serum samples were significantly higher in the study group on Day 7. It is suggested that nanosilver coated orthodontic brackets, as an antibacterial agent without patient compliance, could be helpful for the prevention of white spot lesions during fixed orthodontic treatment.

Morphological and functional changes in rat incisor triggered by chronic exposure to fluoride

Ultrafast laser photoinscription and laser-based refractive index engineering develop towards a unique way for three-dimensional optical design inside bulk materials for the conception of embedded photonic applications. The specific optical functions for the light guiding elements, notably their single mode characteristics and the accessible spectral domains, depend on the achieved refractive index contrast in the material transparency window and on the characteristic dimensions of the optical modification. We give here an overview of several laser processing options, developed to increase the effective index area and contrast using pulse engineering methods in space and time, and optical design involving focal shaping, tubular concepts, evanescently coupled waveguide arrays, or structured waveguides.

Isolation and characterization of dental epithelial cells derived from amelogenesis imperfecta rat.

Disruption of the third zinc finger domain of specificity protein 6 (SP6) presents an enamel-specific defect in a rat model of amelogenesis imperfecta (AMI rats). To understand the molecular basis of amelogenesis imperfecta caused by the Sp6 mutation, we established and characterized AMI-derived rat dental epithelial (ARE) cells. ARE cell clones were isolated from the mandibular incisors of AMI rats, and amelogenesis-related gene expression was analyzed by reverse transcription polymerase chain reaction (RT-PCR). Localization of wild-type SP6 (SP6WT) and mutant-type SP6 (SP6AMI) was analyzed by immunocytochemistry. SP6 transcriptional activity was monitored by rho-associated protein kinase 1 (Rock1) promoter activity with its specific binding to the promoter region in dental (G5 and ARE) and non-dental (COS-7) epithelial cells. Isolated ARE cells were varied in morphology and gene expression. Both SP6WT and SP6AMI were mainly detected in nuclei. The promoter analysis revealed that SP6WT and SP6AMI enhanced Rock1 promoter activity in G5 cells but that enhancement by SP6AMI was weaker, whereas no enhancement was observed in the ARE and COS-7 cells, even though SP6WT and SP6AMI bound to the promoter in all instances. ARE cell clones can provide a useful in vitro model to study the mechanism of SP6-mediated amelogenesis imperfecta.

Effect of dietary protein or calcium supplement on the expression of collagen I and dentine phosphoprotein of rats with dental fluorosis.

This study aims to assess the roles of dietary protein (Pr) and calcium (Ca) levels associated with excessive fluoride (F) intake and the impact of Pr, Ca, and F on expression of collagen I (COL I) and dentine phosphoprotein (DPP) in rat incisors. Seventy-two rats were randomly allotted to six groups (NC, NC + F, LPrLCa, LPrLCa + F, HPrLCa + F and LPrHCa + F). F, Pr, and Ca were orally administrated to rats for 120 days. The genes and proteins expression levels were quantified. We found that levels of COL1A1 and DSPP gene were respectively decreased by 41.9% and 43.1% in the NC + F group compared with NC group, were respectively decreased by 47.9% and 40.8% in the LPrLCa + F group compared with LPrLCa group, were respectively increased by 71.8% and 39.5% in the HPrLCa + F group and increased by 26.5% and 21.1% in the LPrHCa + F group compared with LPrLCa + F group. Meanwhile, the proteins' expression display with the genes' expression changes were consistent. This study demonstrated that the gene and protein expressions of COL I and DPP were reduced by F both in normal nutrition and malnutrition, and it was reversed by Pr and Ca supplementation, with the effect of Pr being greater than that of Ca.

Micro-CT evaluation of the radioprotective effect of resveratrol on the mandibular incisors of irradiated rats

Micro-CT evaluation of the radioprotective effect of resveratrol on the mandibular incisors of irradiated rats

In vivo evaluation of the effects of hydraulic calcium silicate dental cements on plasma and liver aluminium levels in rats.

Our aim was to test whether the presence of three hydraulic calcium silicate dental cements--MTA Angelus, MTA Fillapex, and Theracal LC--in the dental extraction socket of an in vivo model, would affect the levels of aluminium (Al) in the plasma and liver. Following anesthesia, the right upper incisor of each male Wistar albino rat was extracted and polyethylene tubes filled with MTA Angelus, MTA Fillapex, or Theracal LC were inserted into the depth of the extraction socket and gingival tissue was sutured. The rats were killed 7, 30, or 60 d after the operation. Blood and liver samples were obtained from the rats before they were killed, and the levels of Al were measured by atomic absorption spectrometry. Plasma Al levels were higher in the rats in which the mineral trioxide aggregate (MTA) cements were implanted, especially MTA Angelus and MTA Fillapex, compared with control rats. In liver samples, however, the differences in Al level were not statistically significant. Our results show that Al might have been released into the circulation from the three dental cements tested, especially MTA Angelus and MTA Fillapex. Further research should be carried out on the possible biological effects of Al liberated from dental cements.

Effect of caffeic acid phenethyl ester on bone formation in the expanded inter-premaxillary suture.

BackgroundNarrow maxilla is a common problem in orthodontics and dentofacial orthopedics. To solve this problem, a procedure called rapid maxillary expansion (RME) has been used. However, relapse tendency is a major problem of RME. Although relapse tendency is not clearly understood, various treatment procedures and new applications have been investigated. The present study aimed to investigate the possible effectiveness of caffeic acid phenethyl ester (CAPE) on new bone formation in rat midpalatal suture after RME.Materials and methodsTwenty male Sprague Dawley rats were used in this study. The animals were randomly divided into two groups as control and CAPE group. In the CAPE group, CAPE was administered systemically via intraperitoneal injection. RME procedure was performed on all animals. For this purpose, the springs were placed on the maxillary incisors of rats and activated for 5 days. After then, the springs were removed and replaced with short lengths of rectangular retaining wire for consolidation period of 15 days. At the end of the study, histomorphometric analysis was carried out to assess new bone formation.ResultsNew bone formation was significantly greater in the CAPE group than the control group (P<0.05). CAPE enhances new bone formation in midpalatal suture after RME.ConclusionThese results show that CAPE may decrease the time needed for retention.

Dental development and microstructure of bamboo rat incisors

Bone adapts to habitual loads by remodeling to resist stresses that would otherwise break it. The question of whether the same holds for teeth, however, remains unanswered. We might expect species with ever-growing dentitions to alter enamel histology in response to diet. In this study we fed bamboo rats (Rhizomys sinensis) different foods to assess effects on enamel microstructure. Results indicate that gnawing fracture-resistant items (i.e., bamboo) produces substantively different dental microstructures than does eating less mechanically-challenging ones (i.e., potato). Bamboo induces a structured, anisotropic pattern of rods that strengthens incisor enamel, whereas potato produces less structured, weaker enamel. Blood tests suggest that these differences are not related to nutrient variation. Rather, these ever-growing teeth evidently require a specific mechanical environment to develop normal dental microstructure.

Effects of Fluoride on the Expression of Beclin1 and mTOR in Ameloblasts

Exposure to high levels of fluoride (F^-) can result in dental fluorosis in different individuals, but the mechanism of dental fluorosis remains unclear. Autophagy is a highly conserved intracellular digestion process that degrades damaged organelles and protein aggregates. This study examined the effect of sodium fluoride (NaF) on the expression of Beclin1 and mTOR to elucidate the development mechanisms of dental fluorosis. HAT-7 cells were incubated with various concentrations of NaF, and autophagic vacuoles were studied by transmission electron microscopy. At both mRNA and protein level, expression of Beclin1, which is required for autophagosome formation and decreases the expression of mTOR, an autophagy-related complex, was increased at 1.2 mmol/l NaF compared to baseline (0 mmol/l NaF). Additionally, immunohistochemical analysis was performed on paraffin-embedded rat incisor sections to identify the expression of Beclin1 and mTOR proteins in vitro. Highly significant differences were detected compared to controls. In summary, our results demonstrate unequivocally that excessive amounts of fluoride cause autophagy of HAT-7 cells, indicating that autophagy is involved in dental fluorosis.