RESUMEN
Epigenetic modifications affect cell differentiation via transcriptional regulation. G9a/EHMT2 is an important epigenetic modifier that catalyzes the methylation of histone 3 lysine 9 (H3K9) and interacts with various nuclear proteins. In this study, we investigated the role of G9a in osteoclast differentiation. When we deleted G9a by infection of Cre-expressing adenovirus into bone marrow macrophages (BMMs) from G9afl/fl (Ehmt2fl/fl) and induced osteoclastic differentiation by the addition of macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL), the number of TRAP-positive multinucleated osteoclasts significantly increased compared with control. Furthermore, the mRNA expression of osteoclast markers, TRAP, and cathepsin K, and to a lesser extent, NFATc1, a critical transcription factor, increased in G9a KO cells. Infection of wild-type (WT) G9a-expressing adenovirus in G9a KO cells restored the number of TRAP-positive multinucleated cells. In G9a KO cells, increased nuclear accumulation of NFATc1 protein and decreased H3K9me2 accumulation were observed. Furthermore, ChIP experiments revealed that NFATc1 binding to its target, Ctsk promoter, was enhanced by G9a deletion. For in vivo experiments, we created G9a conditional knock-out (cKO) mice by crossing G9afl/fl mice with Rank Cre/+ (Tnfrsf11aCre/+) mice, in which G9a is deleted in osteoclast lineage cells. The trabecular bone volume was significantly reduced in female G9a cKO mice. The serum concentration of the C-terminal telopeptide of type I collagen (CTX), a bone-resorbing indicator, was higher in G9a cKO mice. In addition, osteoclasts differentiated from G9a cKO BMMs exhibited greater bone-resorbing activity. Our findings suggest that G9a plays a repressive role in osteoclastogenesis by modulating NFATc1 function.
Asunto(s)
Resorción Ósea , Diferenciación Celular , N-Metiltransferasa de Histona-Lisina , Factores de Transcripción NFATC , Osteoclastos , Osteogénesis , Animales , Factores de Transcripción NFATC/metabolismo , Factores de Transcripción NFATC/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , Ratones , Osteoclastos/metabolismo , Resorción Ósea/metabolismo , Osteogénesis/fisiología , Ratones Noqueados , Ligando RANK/metabolismo , Ratones Endogámicos C57BL , Células CultivadasRESUMEN
OBJECTIVES: The tongue comprises multiple tissues of different embryonic origins, including pharyngeal arch, somite, and cranial neural crest (CNC). However, its developmental regulatory mechanisms, especially those involving epigenetic modifiers, remain poorly understood. This study examined the roles of the epigenetic modifier G9a in murine tongue development. METHODS: We deleted G9a using Sox 9 (SRY-related HMG-box gene 9)-Cre recombinase, which acts in tongue progenitor cells, including CNC-derived cells, to generate G9a conditional knockout (cKO) mice. Histochemical and immunohistochemical analyses were conducted on sections prepared from tongue tissues of control and cKO mice. RESULTS: Cre-dependent LacZ reporter mice, generated by crossing Rosa-LacZ mice with sox9-Cre mice, revealed Cre recombinase activity in the mucosal epithelium and tongue connective tissue of the embryonic tongue. Tongue volume was significantly reduced on embryonic day 17.5 (E17.5) and postnatal day 0 (P0) in cKO mice. Histological sections showed that the lingual mucosal epithelium was thinner in cKO mice. Reduced G9a levels were accompanied by decreased levels of a G9a substrate, dimethylated lysine 9 in histone H3, in the embryonic tongue. BrdU injection at E16.5 revealed reduced numbers of BrdU-positive cells in the mucosal epithelium and underlying connective tissue at E17.5 in cKO mice, indicating suppression of cell proliferation in both tissues. Investigation of keratin 5 and 8 protein localization showed significantly suppressed expression in the lingual mucosal epithelium in cKO mice. CONCLUSIONS: G9a is required for proper proliferation and differentiation of sox9-expressing tongue progenitor cells and is thereby involved in tongue development.
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Epigénesis Genética , Lengua , Animales , Ratones , Bromodesoxiuridina/metabolismo , Diferenciación Celular/fisiología , Epitelio/metabolismo , Lengua/metabolismoRESUMEN
Transplantation and replantation of teeth are effective therapeutic approaches for tooth repositioning and avulsion, respectively. Transplantation involves transplanting an extracted tooth from the original site into another site, regenerating tissue including the periodontal ligament (PDL) and alveolar bone, around the transplanted tooth. Replantation places the avulsed tooth back to its original site, regenerating functional periodontal tissue. In clinical settings, transplantation and replantation result in favorable outcomes with regenerated PDL tissue in many cases. However, they often result in poor outcomes with two major complications: tooth ankylosis and root resorption. In tooth ankylosis, the root surface and alveolar bone are fused, reducing the PDL tissue between them. The root is subjected to remodeling processes and is partially replaced by bone. In severe cases, the resorbed root is completely replaced by bone tissue, which is called as "replacement resorption." Resorption is sometimes accompanied by infection-mediated inflammation. The molecular mechanisms of ankylosis and root resorption remain unclear, although some signaling mechanisms have been proposed. In this mini-review, we summarized the biological basis of repair mechanisms of tissues in transplantation and replantation and the pathogenesis of their healing failure. We also discussed possible therapeutic interventions to improve treatment success rates.
Asunto(s)
Resorción Radicular , Anquilosis del Diente , Avulsión de Diente , Humanos , Ligamento Periodontal/patología , Resorción Radicular/etiología , Resorción Radicular/patología , Anquilosis del Diente/complicaciones , Anquilosis del Diente/patología , Avulsión de Diente/complicaciones , Avulsión de Diente/patología , Avulsión de Diente/terapia , Reimplante Dental/efectos adversosRESUMEN
Tooth ankylosis is a pathological condition of periodontal ligament (PDL) restoration after tooth replantation. Platelet-derived growth factor-BB (PDGF-BB) has been proposed as a promising factor for preventing tooth ankylosis. Using rat tooth replantation model, we investigated whether PDGF-BB accelerates the repair of PDL after tooth replantation without ankylosis, and its molecular mechanisms. In PDGF-BB pretreated replanted teeth (PDGF-BB group), ankylosis was markedly reduced and functionally organized PDL collagen fibers were restored; the mechanical strength of the healing PDL was restored to an average of 76% of that in non-replanted normal teeth at 21 days. The numbers of PDGF-Rß- and BrdU-positive cells in the periodontal tissues of the PDGF-BB group were greater than those of atelocollagen pretreated replanted teeth (AC group). Moreover, in the PDGF-BB group, the periodontal tissues had fewer osteocalcin-positive cells and decreased number of nuclear ß-catenin-positive cells compared to those in the AC group. In vitro analyses showed that PDGF-BB increased the proliferation and migration of human periodontal fibroblasts. PDGF-BB downregulated mRNA expressions of RUNX2 and ALP, and inhibited upregulatory effects of Wnt3a on ß-catenin, AXIN2, RUNX2, COL1A1, and ALP mRNA expressions. These findings indicate that in tooth replantation, topical PDGF-BB treatment enhances cell proliferation and migration, and inhibits canonical Wnt signaling activation in bone-tooth ankylosis, leading to occlusal loading of the PDL tissues and subsequent functional restoration of the healing PDL. This suggests a possible clinical application of PDGF-BB to reduce ankylosis after tooth replantation and promote proper regeneration of PDL.
Asunto(s)
Anquilosis , Anquilosis del Diente , Animales , Anquilosis/patología , Becaplermina/farmacología , Subunidad alfa 1 del Factor de Unión al Sitio Principal , Ligamento Periodontal , Proteínas Proto-Oncogénicas c-sis/farmacología , ARN Mensajero/farmacología , Ratas , Anquilosis del Diente/patología , Reimplante Dental , beta CateninaRESUMEN
The methyltransferase G9a was originally isolated as a histone methyltransferase that catalyzes the methylation of histone 3 lysine 9 (H3K9) to a dimethylated state (H3K9me2). Recent studies have revealed that G9a has multiple functions in various cells, including osteoblasts. Here, we investigated G9a function during cranial bone formation. Crossing Sox9-cre with G9aflox/flox (fl/fl) mice generated conditional knockout mice lacking G9a expression in Sox9-positive neural crest-derived bone cells. Sox9-Cre/G9afl/fl mice showed severe hypo-mineralization of cranial vault bones, including defects in nasal, frontal, and parietal bones with opened fontanelles. Cell proliferation was inhibited in G9a-deleted calvarial bone tissues. Expression levels of bone marker genes, i.e., alkaline phosphatase and osteocalcin, were suppressed, whereas Runx2 expression was not significantly decreased in those tissues. In vitro experiments using G9a-deleted calvarial osteoblasts showed decreased cell proliferation after G9a deletion. In G9a-deleted osteoblasts, expression levels of fibroblast growth factor receptors and several cyclins were suppressed. Moreover, the expression of bone marker genes was decreased, whereas Runx2 expression was not altered by G9a deletion in vitro. G9a enhanced the transcriptional activity of Runx2, whereas siRNA targeting G9a inhibited the transcriptional activity of Runx2 in C3H10T1/2 mesenchymal cells. We confirmed the direct association of endogenous Runx2 with G9a. Chromatin immunoprecipitation experiments showed that G9a bound to Runx2-target regions in promoters in primary osteoblasts. Furthermore, Runx2 binding to the osteocalcin promoter was abrogated in G9-deleted osteoblasts. These results suggest that G9a regulates proliferation and differentiation of cranial bone cells through binding to and activating Runx2.
Asunto(s)
Subunidad alfa 1 del Factor de Unión al Sitio Principal , Osteogénesis , Animales , Diferenciación Celular , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , N-Metiltransferasa de Histona-Lisina , Ratones , Osteoblastos , Regiones Promotoras Genéticas , CráneoRESUMEN
Little is known about the molecular mechanisms of enthesis formation in mature animals. Here, we report that annexin A5 (Anxa5) plays a critical role in the regulation of bone ridge outgrowth at the entheses. We found that Anxa5 is highly expressed in the entheses of postnatal and adult mice. In Anxa5-deficient (Anxa5-/- ) mice, the sizes of bone ridge outgrowths at the entheses of the tibias and femur were increased after age 7 weeks. Bone overgrowth was not observed at the fibrous enthesis where the fibrocartilage layer does not exist. More ALP-expressing cells were observed in the fibrocartilage layer in Anxa5-/- mice than in wild-type (WT) mice. Calcein and Alizarin Red double labeling revealed more mineralized areas in Anxa5-/- mice than WT mice. To examine the effects of mechanical forces, we performed tenotomy in which transmission of contractile forces by the tibial muscle was impaired by surgical muscle release. In tenotomized mice, bone overgrowth at the enthesis in Anxa5-/- mice was decreased to a level comparable to that in WT mice at 8 weeks after the operation. The tail-suspended mice also showed a decrease in bone overgrowth to similar levels in Anxa5-/- and WT mice at 8 weeks after hindlimb unloading. These results suggest that bone overgrowth at the enthesis requires mechanical forces. We further examined effects of Anxa5 gene knockdown (KD) in primary cultures of osteoblasts, chondrocytes, and tenocytes in vitro. Anxa5 KD increased ALP expression in tenocytes and chondrocytes but not in osteoblasts, suggesting that increased ALP activity in the fibrocartilaginous tissue in Anxa5-/- mice is directly caused by Anxa5 deletion in tenocytes or fibrocartilage cells. These data indicate that Anxa5 prevents bone overgrowth at the enthesis, whose formation is mediated through mechanical forces and modulating expression of mineralization regulators. © 2018 American Society for Bone and Mineral Research.
Asunto(s)
Anexina A5/metabolismo , Desarrollo Óseo , Huesos/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Anexina A5/deficiencia , Cartílago/crecimiento & desarrollo , Diferenciación Celular , Condrocitos/metabolismo , Fémur/crecimiento & desarrollo , Fémur/metabolismo , Miembro Posterior/metabolismo , Ratones Noqueados , Osteoblastos/metabolismo , Tendones/crecimiento & desarrollo , Tenocitos/metabolismo , Tibia/crecimiento & desarrollo , Tibia/metabolismo , Soporte de PesoRESUMEN
This study was designed to evaluate the volume of alveolar bone augmentation after immediate implant placement using demineralized bone. We examined the collagen matrix of demineralized bone and biologically active substances contained therein. Rat maxillary first molars were extracted, and the animals were divided into five groups as follows: tooth extraction only, implant into the mesial root socket, implant and other root sockets covered with demineralized bone sheet, implant and other root sockets filled with demineralized bone powder under the sheet, and implant and other root sockets covered with demineralized bone sheet from which proteins were extracted. We ascertained whether biologically active substances are contained in extracted proteins. Biologically active substances were detected in extracted proteins. Conditions using demineralized bone sheet with biologically active substances significantly augmented the height of the alveolar bone. Such resorbable membranes containing biologically active substances hold promise as clinical agents for bone augmentation upon implantation.
Asunto(s)
Implantes Dentales , Proceso Alveolar , Aumento de la Cresta Alveolar , Animales , Implantación Dental Endoósea , Ratas , Alveolo Dental/cirugíaRESUMEN
The aim of the present study was to evaluate the gingival connective tissue response to screw-type titanium implants coated with Type I collagen nanofibers, which were prepared using the electrospray deposition method. Implants were immediately inserted into the socket of maxillary first molars after the extraction. Undecalcified sections after 4 weeks implantation were histologically observed. Better contact of the gingival connective tissue was generally observed around the collagen nanofiber-coated implants than titanium and non-fibrous collagen-immobilized implants. Gingival connective tissue to implant contact was significantly greater with the collagen nanofiber-coated implants than with the titanium and collagen-immobilized implants at the distal side, but not at the mesial side. Polarized light microscopy revealed that some birefringent collagen fiber bundles are oriented perpendicularly to the implant surfaces in the gingival connective tissue adjacent to the collagen nanofiber-coated implants. Collagen nanofiber-coating may have a possibility for improving gingival connective tissue response to titanium implants.
Asunto(s)
Tejido Conectivo/fisiología , Implantación Dental Endoósea/métodos , Implantes Dentales , Encía/fisiología , Alveolo Dental/cirugía , Animales , Materiales Biocompatibles Revestidos , Diseño de Prótesis Dental , Maxilar , Microscopía Electrónica de Rastreo , Microscopía de Polarización , Diente Molar , Nanofibras , Proyectos Piloto , Ratas , TitanioRESUMEN
Development of regenerative therapies for damaged tendons remains a great challenge, largely because of lack of information regarding the mechanisms responsible for differentiation of tenocytes. Mouse tenocytes have not been fully characterized owing to the absence of efficient and reproducible methods for their in vitro expansion without losing phenotypic features. The objective of the study was to establish an improved and reliable method for stable primary culture of mouse tenocytes by using collagen gel. Achilles and tail tendon tissues were harvested and embedded in collagen gel. After 10 days of continuous culture, the gel was digested and cells were passaged on tissue culture-treated plastic dishes. Mouse tenocytes cultured in collagen gel exhibited significantly shorter doubling time and higher numbers of proliferation when maintained on the plastic dishes compared with those cultured without using gel. Transmission electron microscopic analyses showed that cultured tenocytes retained some morphological features of tenocytes in tendon tissues, such as cell-cell junctional complex formation, well-developed rough endoplasmic reticulum, and mitochondria in their cytoplasm. mRNA expression of tenocyte markers (tenomodulin, type I collagen, periostin, and scleraxis) was higher in cells cultured in collagen gel than in those cultured in the absence of gel. Our results show that tenocytes cultured using the collagen gel method express typical lineage markers and exhibit improved growth characteristics, thus providing a stable platform for studying molecular mechanisms that control their differentiation.
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Tendón Calcáneo/citología , Geles/farmacología , Cultivo Primario de Células/métodos , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/biosíntesis , Moléculas de Adhesión Celular/biosíntesis , Proliferación Celular , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Células Cultivadas , Colágeno Tipo I/biosíntesis , Retículo Endoplásmico Rugoso/fisiología , Proteínas de la Membrana/biosíntesis , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de Transmisión , Mitocondrias , Traumatismos de los Tendones/terapia , Uniones Estrechas/fisiologíaRESUMEN
Bisphosphonates (BPs) are a major class of antiresorptive drug, and their molecular mechanisms of antiresorptive action have been extensively studied. Recent studies have suggested that BPs target bone-forming cells as well as bone-resorbing cells. We previously demonstrated that local application of a nitrogen-containing BP (N-BP), alendronate (ALN), for a short period of time increased bone tissue in a rat tooth replantation model. Here, we investigated cellular mechanisms of bone formation by ALN. Bone histomorphometry confirmed that bone formation was increased by local application of ALN. ALN increased proliferation of bone-forming cells residing on the bone surface, whereas it suppressed the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts in vivo. Moreover, ALN treatment induced more alkaline phosphatase-positive and osteocalcin-positive cells on the bone surface than PBS treatment. In vitro studies revealed that pulse treatment with ALN promoted osteocalcin expression. To track the target cells of N-BPs, we applied fluorescence-labeled ALN (F-ALN) in vivo and in vitro. F-ALN was taken into bone-forming cells both in vivo and in vitro. This intracellular uptake was inhibited by endocytosis inhibitors. Furthermore, the endocytosis inhibitor dansylcadaverine (DC) suppressed ALN-stimulated osteoblastic differentiation in vitro and it suppressed the increase in alkaline phosphatase-positive bone-forming cells and subsequent bone formation in vivo. DC also blocked the inhibition of Rap1A prenylation by ALN in the osteoblastic cells. These data suggest that local application of ALN promotes bone formation by stimulating proliferation and differentiation of bone-forming cells as well as inhibiting osteoclast function. These effects may occur through endocytic incorporation of ALN and subsequent inhibition of protein prenylation.
Asunto(s)
Alendronato/farmacología , Conservadores de la Densidad Ósea/farmacología , Osteoblastos/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Prenilación de Proteína/efectos de los fármacos , Reimplante Dental , Fosfatasa Ácida/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Técnicas In Vitro , Isoenzimas/metabolismo , Modelos Animales , Osteoblastos/citología , Osteoblastos/metabolismo , Osteocalcina/metabolismo , Ratas , Fosfatasa Ácida TartratorresistenteRESUMEN
The ß-galactosidase gene (lacZ) of Escherichia coli is widely used as a reporter gene. The expression of lacZ can be detected by enzyme-based histochemical staining using chromogenic substrates such as 5-bromo-4-chloro-3-indolyl-ß-D: -galactoside (X-gal). Because the enzymatic activity of lacZ is vulnerable to high temperatures and acid treatment for demineralization, detection of lacZ on paraffinized sections is difficult, especially for hard tissues, which require demineralization before sectioning in paraffin. To circumvent this problem, whole-mount X-gal staining before sectioning is performed. However, detection of lacZ activity in the center of larger portions of hard whole adult tissues is challenging. In this study, focusing on fixation procedures, we determined the conditions conducive to improved detection of lacZ activity in deeper areas of whole tissues. We used an annexin a5 (Anxa5)-lacZ reporter mouse model in which the Anxa5 expression in hard tissue is indicated by lacZ activity. We found that lacZ activity could be detected throughout the periodontal ligament of adult mice when fixed in 100% acetone, whereas it was not detected in the periodontal ligament around the root apex fixed in glutaraldehyde and paraformaldehyde. This staining could not be detected in wild-type mice. Acetone maintains the lacZ activity within 48 h of fixation at both 4°C and at room temperature. In conclusion, acetone is the optimal fixative to improve permeability for staining of lacZ activity in large volumes of adult hard tissues.
Asunto(s)
Coloración y Etiquetado/métodos , beta-Galactosidasa/metabolismo , Animales , Anexina A5/genética , Anexina A5/metabolismo , Operón Lac , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
A non-linear visco-elastic constitutive model is adopted to describe the relaxation phenomena of the periodontal ligament (PDL). The introduction of a non-linear formulation of visco-elasticity is necessary because experimental data from the literature referring to animal models show that the relaxation rate depends on the level of strain applied. In particular, the percentage of relaxation increases with decrease of the applied strain. The constitutive model is consistent with the non-linear elastic behavior of the PDL in the case of high rate loading and large strains attained by the tissue. A hyperelastic formulation is adopted for the elastic behavior of the PDL and this formulation is developed adopting suitable measures of stress and strain. The anisotropy of the tissue induced by specific spatial orientation of collagen fibers is included in the model. With respect to recent numerical formulation proposed to describe the non-linear visco-elasticity of the PDL, the proposed model has the advantage of being more consistent with the micro-structural configuration of the tissue and the large strains it can undergo. The results obtained show that a reasonable description of the PDL relaxation phenomena can be obtained by assuming that relaxation times are independent of strain, whereas the relative stiffness results are dependent on strain applied through an exponential function.
Asunto(s)
Modelos Anatómicos , Ligamento Periodontal/anatomía & histología , Elasticidad , HumanosRESUMEN
The mechanical strength of the periodontal ligament (PDL) was first measured as force required to extract a tooth from its socket using human specimens. Thereafter, tooth-PDL-bone preparations have extensively been used for measurement of the mechanical response of the PDL. In vitro treatments of such specimens with specific enzymes allowed one to investigate into the roles of the structural components in the mechanical support of the PDL. The viscoelastic responses of the PDL may be examined by analysis of the stress-relaxation. Video polarised microscopy suggested that the collagen molecules and fibrils in the stretched fibre bundles progressively align along the deformation direction during the relaxation. The stress-relaxation process of the PDL can be well expressed by a function with three exponential decay terms. Analysis after in vitro digestion of the collagen fibres by collagenase revealed that the collagen fibre components may play an important role in the long-term relaxation component of the stress-relaxation process of the PDL. The dynamic measurements of the viscoelastic properties of the PDL have recently suggested that the PDL can absorb more energy in compression than in shear and tension. These viscoelastic mechanisms of the PDL tissue could reduce the risk of injury to the PDL.
RESUMEN
To determine whether fish intestinal epithelial cells (IECs) contribute to mucosal immunity, we established a method for isolating IECs from the rainbow trout Oncorhynchus mykiss and examined cytokine production in these cells. Components of the intestinal epithelium were released by incubation of intestinal pieces with 1mM dithiothreitol (DTT)/ethylenediamine tetraacetic acid (EDTA). The IEC-rich fraction (purity >90%; survival rate approximately 95%) was obtained by centrifugation on a 35%/40% Percoll gradient, followed by magnetic cell sorting using an anti-trout IgM antiserum. The gene expression profiles of 14 cytokines in trout IECs were investigated after culturing the cells for 6h with or without the pathogenic bacterium Aeromonas salmonicida. Trout IECs could produce several cytokines, of which IL-1beta and TNFalpha2 were upregulated when the cells were stimulated with live A. salmonicida. Immunohistochemical analyses with the anti-trout TNF antibody confirmed that the TNF protein was present in the IECs of trout that were intra-anally challenged with live A. salmonicida. These results show that trout IECs are an important trigger of the intestinal immune system. Further, formalin-killed A. salmonicida, conditioned medium of this bacterium, or live nonpathogenic Escherichia coli could not upregulate the expression of these cytokines. These results indicate that the production of inflammatory cytokines by IECs is caused by the adhesion of A. salmonicida, but is not due to only simple ligand-receptor interactions between the surface molecules of IECs and the bacterium or in response to bacterial secretions.
Asunto(s)
Aeromonas salmonicida/inmunología , Citocinas/biosíntesis , Enfermedades de los Peces/inmunología , Forunculosis/veterinaria , Infecciones por Bacterias Gramnegativas/veterinaria , Mucosa Intestinal/inmunología , Oncorhynchus mykiss/inmunología , Animales , Adhesión Bacteriana , Citocinas/inmunología , Enfermedades de los Peces/microbiología , Forunculosis/inmunología , Perfilación de la Expresión Génica , Infecciones por Bacterias Gramnegativas/inmunología , Inmunidad Mucosa/inmunología , Mucosa Intestinal/microbiología , Oncorhynchus mykiss/genética , Oncorhynchus mykiss/microbiologíaRESUMEN
Bromodeoxyuridine (BrdU) is used to label synthesizing DNA and to chase label-retaining cell (LRC). As stem cells divide slowly in adult tissues, they can be visualized as LRCs. In order to identify LRCs in hard tissue, we examined optimal conditions of fixation, demineralization, and DNA denaturation/antigen retrieval for immunohistochemistry of BrdU in hard tissues including bone, tooth, and periodontal ligament. Mice were subcutaneously injected with BrdU (50 microg/g body weight) twice a day from the postnatal day 11 to day 15 and sacrificed at 2 h after the last injection. Dissected maxillae were fixed (Bouin's solution or 4% paraformaldehyde), demineralized (Morse's solution or EDTA), and embedded in paraffin. Antigen retrieval procedures were performed before incubation with primary antibody. When sections were treated with HCl for DNA denaturation, the staining intensity of BrdU positive cells was not affected by difference of fixatives. Higher sensitivity was obtained by demineralization with Morse than with EDTA. Although heat-induced antigen retrieval techniques in citrate buffer (pH 6.0) showed as well or better sensitivity than acid pretreatment, heating caused tissue damage specifically to tooth dentine and the surrounding tissue. When the LRCs at four weeks after the last injection of BrdU were compared, much more LRCs were observed in specimen demineralized with Morse than with 10% EDTA. Our data suggest that demineralization with Morse with Bouin fixative plus HCl pretreatment gives rise to the optimal results for BrdU immunodetection in hard tissue.
Asunto(s)
Huesos/citología , Bromodesoxiuridina/química , ADN/química , Inmunohistoquímica/métodos , Ligamento Periodontal/citología , Diente/citología , Animales , Anticuerpos/química , Anticuerpos/inmunología , Antígenos/química , Antígenos/inmunología , Antígenos/metabolismo , Huesos/química , Huesos/inmunología , Huesos/metabolismo , Bromodesoxiuridina/inmunología , Bromodesoxiuridina/metabolismo , Ácido Cítrico/química , ADN/inmunología , ADN/metabolismo , Ácido Edético/química , Calor , Concentración de Iones de Hidrógeno , Ratones , Ratones Endogámicos ICR , Ligamento Periodontal/inmunología , Ligamento Periodontal/metabolismo , Fijación del Tejido/métodos , Diente/inmunología , Diente/metabolismoRESUMEN
The aims of this study are to observe microscopic changes in the periodontal ligament (PDL) collagen fibres after collagenase treatment, to analyse stress-relaxation behaviour of PDL specimens treated with collagenase, and to elucidate the contribution of the collagen component to the viscoelastic behaviour of the PDL. Transverse sections of rat mandibular first molars (n=24) were treated in vitro with 0, 8, 16, or 24 units of bacterial collagenase for 4h at 37 degrees C. Histological specimens were then prepared, and image analyses were done for polarised light microscopic appearances of collagen fibres. Further, stress-relaxation tests were performed for PDL specimens treated with 8 units of collagenase (n=7) and control specimens (n=7). Image analysis showed that higher concentrations of collagenase reduced greater area occupied by the PDL collagen fibres and birefringent retardation of the fibres. The amount of stress-relaxation during 600 s was 1.37 times greater in the collagenase-treated specimens than in the controls. The observed values of the stress-relaxation process were well described by a function with three exponential decay terms. The relaxation parameters of the first and second terms did not show significant differences, but those of the third term did so between the collagenase-treated and control specimens. The ratio and relaxation time of the third term for the collagenase-treated specimens were significantly less than those for the controls. These findings suggest that in the long-term relaxation component of the stress-relaxation process of the PDL the viscoelastic properties of the collagen fibres may play an important role.
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Colágeno/química , Colagenasas/química , Ligamento Periodontal/química , Animales , Proteínas Bacterianas/química , Clostridium histolyticum/enzimología , Elasticidad , Masculino , Mandíbula/química , Mandíbula/diagnóstico por imagen , Diente Molar/química , Diente Molar/diagnóstico por imagen , Ligamento Periodontal/diagnóstico por imagen , Radiografía , Ratas , Ratas Wistar , Estrés MecánicoRESUMEN
In the present study, the mechanical response of bovine periodontal ligament (PDL) subjected to displacement-controlled tension-compression harmonic oscillations and subsequent rupture was examined. Specimens including dentine, cementum, PDL, and alveolar bone were extracted from different depths and locations of bovine first molars. They were immersed in a saline solution at room temperature and clamped on their bone and dentine extremities. The samples were tested at +/-35% of the PDL's width, with a frequency of 1 Hz. The mechanical parameters evaluated were hysteresis, phase lag, and the modulus of the stress-stretch ratio curves in tension and compression. The tensile strength and the corresponding stretch ratio were also recorded. Stress-stretch ratio curves indicated a non-linear, time-dependent response with hysteresis and preconditioning effects. The hysteresis and phase lag in compression were much higher than in tension, suggesting that the dissipated energy was higher in compression than in tension. The root depth and location did not play essential roles for the tension or compression data, with the exception of limited statistical differences for tensile strength and corresponding stretch ratio. Thus, biological variability in the specimens, as a result of different bone contours, PDL width, and fiber orientation, did not affect the energy-absorbing capacity of the PDL. The evolution of the stress rate with stress demonstrated a constant increase of stiffness with stress. The stiffness values were twofold higher in tension than in compression. The data also showed that the stiffness of the PDL was comparable with data reported for other soft tissues.
Asunto(s)
Ligamento Periodontal/fisiología , Análisis de Varianza , Animales , Bovinos , Fuerza Compresiva , Análisis del Estrés Dental , Elasticidad , Modelos Biológicos , Diente Molar/anatomía & histología , Estadísticas no Paramétricas , Estrés Mecánico , Resistencia a la Tracción , Raíz del Diente/anatomía & histología , ViscosidadRESUMEN
OBJECTIVE: The vasculature within the socket is reportedly involved in determining the position of continuously erupting teeth. Thus, loss of body fluid in anesthetized rats, which would affect the vascular physiology, should influence tooth movement. We investigated the effects of an infusion of Ringer's solution on the systemic arterial blood pressure, regional blood flow at the base of the incisor, and axial tooth movement in anesthetized rats to determine the cause of tooth displacement. MATERIAL AND METHODS: In the experimental group, the animals received intravenous infusions of Ringer's solution at 27 microl/min for 13 h. In the control group, the animals did not receive the infusion. RESULTS: The infusion of Ringer's solution suppressed an increase of the mean arterial blood pressure from 86 to 80 mmHg and a decrease of the regional blood flow from 170 to 217 mV, and increased the eruption rate from 267 to 361 microm/13 h during the experimental period. There was a positive correlation between the eruption rate and regional blood flow, and a negative correlation between the blood pressure and regional blood flow. CONCLUSIONS: These results suggest that an infusion of Ringer's solution can cause an increase in the regional blood flow, resulting in increased fluid volume, elevated intra-socket pressure, and increased eruptive movement. It is possible that the regional vascular volume and/or pressure within the socket play an important role in determining the position of the incisor.