RESUMEN

As the efficiency of the clustered regularly interspaced short palindromic repeats/Cas system is extremely high, creation and maintenance of homozygous lethal mutants are often difficult. Here, we present an efficient in vivo electroporation method called improved genome editing via oviductal nucleic acid delivery (i-GONAD), wherein one of two alleles in the lethal gene was selectively edited in the presence of a non-targeted B6.C3H-In(6)1J inversion identified from the C3H/HeJJcl strain. This method did not require isolation, culture, transfer, or other in vitro handling of mouse embryos. The edited lethal genes were stably maintained in heterozygotes, as recombination is strongly suppressed within this inversion interval. Using this strategy, we successfully generated the first Tprkb null knockout strain with an embryonic lethal mutation and showed that B6.C3H-In(6)1J can efficiently suppress recombination. As B6.C3H-In(6)1J was tagged with a gene encoding the visible coat color marker, Mitf, the Tprkb mutation could be visually recognized. We listed the stock balancer strains currently available as public bioresources to create these lethal gene knockouts. This method will allow for more efficient experiments for further analysis of lethal mutants.

Asunto(s)

Edición Génica , Ácidos Nucleicos , Animales , Sistemas CRISPR-Cas , Gónadas , Ratones , Ratones Endogámicos C3H

RESUMEN

BACKGROUND: Neuromuscular disorders (NMDs) occur in different forms and are generally diagnosed using muscle biopsy. Among the available anesthetic management options for infants with a suspected NMD are general anesthesia (GA) and regional anesthesia (RA), including spinal anesthesia (SA). Anesthesia selection is often challenging from the point of potential airway risks and anesthetic drug-related complications. CASE PRESENTATION: A 6-month-old male infant repeatedly underwent endotracheal intubation and extubation after birth because of respiratory muscle weakness and copious secretions. He was suspected of having NMD and was scheduled for muscle biopsy. His generalized hypotonia and decreased respiratory function presented a potentially difficult airway and complicated the selection of an appropriate anesthetic method. We selected SA and dexmedetomidine, which are safe for infants. CONCLUSION: We report the successful and effective anesthetic management of SA and dexmedetomidine in an infant with a suspected NMD.

RESUMEN

Osteoclasts are terminally differentiated from cells of monocyte/macrophage lineage by stimulation with TNF-related activation-induced cytokine (TRANCE) (receptor activator of NF-kappaB ligand/osteoprotegerin ligand/osteoclast differentiation factor/TNFSF11/CD254). In the present study, we attempted to determine when and how the cell fate of precursors becomes committed to osteoclasts following TRANCE stimulation. Although mouse bone marrow-derived macrophages (BMMs) were able to differentiate into either osteoclasts or dendritic cells, the cells no longer differentiated into dendritic cells after treatment with TRANCE for 24 h, indicating that their cell fate was committed to osteoclasts. Committed cells as well as BMMs were still quite weak in tartrate-resistant acid phosphatase activity, an osteoclast marker, and incorporated zymosan particles by phagocytosis. Interestingly, committed cells, but not BMMs, could still differentiate into osteoclasts even after incorporation of the zymosan particles. Furthermore, IL-4 and IFN-gamma, potent inhibitors of osteoclast differentiation, failed to inhibit osteoclast differentiation from committed cells, and blocking of TRANCE stimulation by osteoprotegerin resulted in cell death. Adhesion to culture plates was believed to be essential for osteoclast differentiation; however, committed cells, but not BMMs, differentiated into multinucleated osteoclasts without adhesion to culture plates. Although LPS activated the NF-kappaB-mediated pathway in BMMs as well as in committed cells, the mRNA expression level of TNF-alpha in the committed cells was significantly lower than that in BMMs. These results suggest that characteristics of the committed cells induced by TRANCE are distinctively different from that of BMMs and osteoclasts.

Asunto(s)

Proteínas Portadoras/metabolismo , Diferenciación Celular/inmunología , Glicoproteínas de Membrana/metabolismo , Osteoclastos/ultraestructura , Células Madre/ultraestructura , Animales , Northern Blotting , Células de la Médula Ósea/inmunología , Adhesión Celular , Células Dendríticas/citología , Citometría de Flujo , Expresión Génica , Humanos , Immunoblotting , Macrófagos/citología , Ratones , Microscopía Electrónica de Transmisión , Fagocitosis/inmunología , Ligando RANK , ARN Mensajero/análisis , Receptor Activador del Factor Nuclear kappa-B , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Zimosan/metabolismo

RESUMEN

Osteoprotegerin (OPG)-deficient mice exhibit severe bone loss including the destruction of growth plate cartilage. Using OPG-deficient mice, we attempted to clarify the differentiation and ultrastructure of osteoclasts located on the destroyed growth plate cartilage and trabecular bone matrix in long bones. In (-/-) homozygous OPG knockout mice, adjacent to the growth plate cartilage, the formation of bone trabeculae without a calcified cartilaginous core resulted in an irregular chondrocyte distribution in the growth plate cartilage. At the metaphyseal ossification center, TRAP-positive osteoclasts showed unusual localization on both type-II collagen-positive cartilage and type-I collagen-positive bone matrix. Osteoclasts located on cartilage matrix lacked a typical ruffled border structure, but formed resorption lacunae. During growth plate cartilage destruction, osteoclasts formed ruffled border structures on bone matrix deposited on the remaining cartilage surfaces. These findings suggest that, in OPG (-/-) mice, osteoclast structure differs, depending on the matrix of either cartilage or bone. Then, we examined the effects of OPG administration on the internal trabecular bone structure and osteoclast differentiation in OPG (-/-) mice. OPG administration to OPG (-/-) mice significantly inhibited trabecular bone loss and maintained the internal trabecular bone structure, but did not reduce the osteoclast number on bone trabeculae. For most osteoclasts, OPG administration caused disappearance or reduction of the ruffled border, but induced neither necrotic nor apoptotic damages. These results suggest that OPG administration is an effective means of maintaining the internal structure and volume of trabecular bone in metabolic bone diseases by inhibition of osteoclastic bone resorption.

Asunto(s)

Matriz Ósea/ultraestructura , Huesos/ultraestructura , Diferenciación Celular/fisiología , Glicoproteínas/genética , Osteoclastos/citología , Receptores Citoplasmáticos y Nucleares/genética , Receptores del Factor de Necrosis Tumoral/genética , Fosfatasa Ácida/metabolismo , Animales , Matriz Ósea/metabolismo , Huesos/metabolismo , Cartílago/metabolismo , Cartílago/ultraestructura , Colágeno Tipo I/metabolismo , Colágeno Tipo II/metabolismo , Glicoproteínas/farmacología , Placa de Crecimiento/metabolismo , Placa de Crecimiento/ultraestructura , Isoenzimas/metabolismo , Ratones , Ratones Noqueados , Microscopía Electrónica de Transmisión , Osteoclastos/metabolismo , Osteoprotegerina , Fosfatasa Ácida Tartratorresistente

RESUMEN

We examined the differentiation and resorptive function of osteoclasts (OC) cultured on the slices of calcified bone, decalcified bone and hyaline cartilage, and found that OC differentiation depends on the co-cultured substratum, as well as osteoblast-derived factors. Bone marrow-derived macrophages (BMM) were formed from marrow cells of 5 week old ddY mice and cultured for 3 days on freeze-dried slices of calcified bone, decalcified bone or cartilage, all prepared from rabbit costal bone. BMM cultured on calcified bone slices exhibited tartrate-resistant acid phosphatase (TRAP) activity and were structurally characterized by multinucleation and ruffled border development. However, on decalcified bone slices, BMM seldom became multinucleated and exhibited weak TRAP activity. BMM cultured on cartilage slices were mononuclear, devoid of TRAP activity and structurally resembled mononuclear phagocytes. In SEM observations of co-cultured slices, resorption lacunae were formed only on calcified bone slices, and not on slices of decalcified bone and cartilage. Our results, therefore, indicated that BMM could differentiate into functional OC only on calcified bone slices, suggesting a key role of calcified components in the bone matrix for the terminal OC differentiation. Then, we cultured BMM on the same slices with yeast particles. In cultures with yeast particles, BMM exhibited intense TRAP activity, developed a ruffled border-like structure and formed resorption lacunae even on decalcified bone and cartilage slices. Vacuolar-type H+-ATPase was strongly expressed along the ruffled border membranes of these OC. Only the BMM that had not incorporated yeast particles developed a ruffled border, whereas the BMM that had incorporated yeast particles did not become multinucleated and lacked a ruffled border structure. Thus, our results further suggest that, even on uncalcified substrata, the terminal differentiation of BMM into functional OC is induced by an unidentified external stimulus, which may be contained in the cell membrane of yeast particles.

Asunto(s)

Huesos/fisiología , Cartílago/fisiología , Diferenciación Celular/fisiología , Osteoblastos/citología , Animales , Huesos/citología , Calcificación Fisiológica , Células Cultivadas , Microscopía Electrónica de Rastreo , Osteoblastos/fisiología , Conejos

RESUMEN

We examined the effects of long-term bisphosphonate (BP, pamidronate) administration at a therapeutic dose (1.5 mg/kg/day) on the distribution, structure, and vacuolar-type H(+)-ATPase expression of osteoclasts, and the resulting trabecular bone volume and structure in ovariectomized (OVX) mature rats. Six-month-old female rats were allocated to sham-operated control, untreated-OVX, and BP-administered OVX groups. Postoperatively, BP was administered intraperitoneally once a day to OVX rats for up to 30 days. On postoperative days 14, 30, and 60, all of the rats were killed and the distal metaphyseal area of the dissected humeri was examined. Quantitative backscattered-electron image analysis revealed that the trabecular bone volume/unit medullary area in untreated OVX rats was significantly (P < 0.05) lower than that in sham-operated controls at 30 and 60 days postoperation. BP administration significantly (P < 0.05) increased trabecular bone volume at 14, 30, and 60 days postoperation in BP-administered OVX rats compared to both sham-operated and untreated OVX rats. Compared to untreated OVX rats, the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts along the bone trabeculae in BP-administered OVX rats was not significantly decreased on days 14 and 30, but was significantly decreased on day 60. Ultrastructurally, BP administration caused the disappearance of both the ruffled border (RB) and the clear zone (CZ) structures, and decreased the expression of vacuolar-type H(+)-ATPase in most osteoclasts, but did not significantly induce apoptosis of osteoclasts detected by the terminal dUTP nick end-labeling (TUNEL) method. Our results suggest that long-term BP administration significantly reduces bone and calcified cartilage resorption through impairment of the structure and bone-resorbing function of osteoclasts, and thereby effectively maintains trabecular bone volume and structure in ovariectomy-induced acute estrogen deficiency in mature rats.

Asunto(s)

Resorción Ósea/prevención & control , Calcinosis , Cartílago , Difosfonatos/administración & dosificación , Osteoclastos/efectos de los fármacos , Ovariectomía , Fosfatasa Ácida/metabolismo , Animales , Apoptosis , Resorción Ósea/patología , Resorción Ósea/fisiopatología , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Femenino , Húmero/efectos de los fármacos , Húmero/enzimología , Húmero/patología , Húmero/ultraestructura , Procesamiento de Imagen Asistido por Computador , Isoenzimas/metabolismo , Osteoclastos/enzimología , Osteoclastos/ultraestructura , Pamidronato , Ratas , Ratas Wistar , Fosfatasa Ácida Tartratorresistente , Difracción de Rayos X

RESUMEN

We examined the biological effects of porcine enamel matrix derivative (EMD; Emdogain) on the formation of reparative dentine and dentine bridges in rat molars after pulp amputation. The pulp chambers of upper molars of Wistar rats were perforated and the amputated pulp surfaces were directly capped with either EMD or its carrier propylene glycol alginate (PGA) as control. The cavities were then restored with glass-ionomer cement. On post-amputation days 4-30, the dissected maxillae were examined by light and electron microscopy. In PGA-capped pulp, reparative dentine had been formed over the dentine walls under the prepared cavity on day 7 post-amputation and its thickness extended until day 30. On day 30, as well as reparative dentine formation, diffuse calcification had occurred beneath the amputated wound surfaces. Dentine bridge formation under the amputated coronal pulp surface was observed in 18.2% of amputated pulp on day 30. In EMD-capped pulp, reparative dentine had already been formed by odontoblast-like cells over the dentine walls, already on day 4 post-amputation, and its thickness extended until day 30. The Ca and P weight % and Ca/P ratio of reparative dentine matrix were similar to those of pre-existing dentine matrix, and these values were not different between PGA and EMD-capped pulp. Dentine bridge formation was observed in 27.3% of EMD-capped pulp on day 30. Our results suggest that EMD enhances the formation of both reparative dentine and dentine bridges during wound healing of amputated rat molar pulp.

Asunto(s)

Proteínas del Esmalte Dental , Recubrimiento de la Pulpa Dental , Dentina/fisiología , Diente Molar/fisiología , Cicatrización de Heridas , Amputación Quirúrgica , Animales , Recubrimiento de la Pulpa Dental/métodos , Exposición de la Pulpa Dental/terapia , Dentina/anatomía & histología , Dentina Secundaria/anatomía & histología , Femenino , Microscopía Electrónica , Diente Molar/cirugía , Diente Molar/ultraestructura , Odontoblastos/citología , Ratas , Ratas Wistar , Porcinos

RESUMEN

The differentiation and functions of osteoclasts (OC) are regulated by osteoblast-derived factors such as receptor activator of NFKB ligand (RANKL) that stimulates OC formation, and a novel secreted member of the TNF receptor superfamily, osteoprotegerin (OPG), that negatively regulates osteoclastogenesis. In examination of the preosteoclast (pOC) culture, pOCs formed without any additives expressed tartrate-resistant acid phosphatase (TRAP), but showed little resorptive activity. pOC treated with RANKL became TRAP-positive OC, which expressed intense vacuolar-type H(+)-ATPase and exhibited prominent resorptive activity. Such effects of RANKL on pOC were completely inhibited by addition of OPG. OPG inhibited ruffled border formation in mature OC and reduced their resorptive activity, and also induced apoptosis of some OC. Although OPG administration significantly reduced trabecular bone loss in the femurs of ovariectomized (OVX) mice, the number of TRAP-positive OC in OPG-administered OVX mice was not significantly decreased. Rather, OPG administration caused the disappearance of ruffled borders and decreased H(+)-ATPase expression in most OC. OPG deficiency causes severe osteoporosis. We also examined RANKL localization and OC induction in periodontal ligament (PDL) during experimental movement of incisors in OPG-deficient mice. Compared to wild-type OPG (+/+) littermates, after force application, TRAP-positive OC were markedly increased in the PDL and alveolar bone was severely destroyed in OPG-deficient mice. In both wild-type and OPG-deficient mice, RANKL expression in osteoblasts and fibroblasts became stronger by force application. These in vitro and in vivo studies suggest that RANKL and OPG are important regulators of not only the terminal differentiation of OC but also their resorptive function. To determine resorptive functions of OC, we further examined the effects of specific inhibitors of H(+)-ATPase, bafilomycin A1, and lysosomal cysteine proteinases (cathepsins), E-64, on the ultrastructure, expression of these enzymes and resorptive functions of cultured OC. In bafilomycin A1-treated cultures, OC lacked ruffled borders, and H(+)-ATPase expression and resorptive activity were significantly diminished. E-64 treatment did not affect the ultrastructure and the expression of enzyme molecules in OC, but significantly reduced resorption lacuna formation, by inhibition of cathepsin activity. Lastly, we examined the expression of H(+)-ATPase, cathepsin K, and matrix metalloproteinase-9 in odontoclasts (OdC) during physiological root resorption in human deciduous teeth, and found that there were no differences in the expression of these molecules between OC and OdC. RANKL was also detected in stromal cells located on resorbing dentine surfaces. This suggests that there is a common mechanism in cellular resorption of mineralized tissues such as bone and teeth.

Asunto(s)

Resorción Ósea , Glicoproteínas/metabolismo , Osteoclastos/fisiología , Receptores Citoplasmáticos y Nucleares/metabolismo , Fosfatasa Ácida/biosíntesis , Fosfatasa Ácida/efectos de los fármacos , Pérdida de Hueso Alveolar/patología , Animales , Apoptosis , Diferenciación Celular , Células Cultivadas , Fémur/patología , Fémur/ultraestructura , Humanos , Inmunohistoquímica , Isoenzimas/biosíntesis , Isoenzimas/efectos de los fármacos , Ratones , Microscopía Electrónica , Osteoclastos/efectos de los fármacos , Osteoclastos/patología , Osteoclastos/ultraestructura , Osteoprotegerina , Ovariectomía , Ligamento Periodontal/patología , Receptores del Factor de Necrosis Tumoral , Especificidad de la Especie , Fosfatasa Ácida Tartratorresistente , Técnicas de Movimiento Dental , Diente Primario/fisiología

RESUMEN

We report the effects of specific and potent inhibitors of vacular-type H(+)-ATPase and lysosomal cysteine proteinases, cathepsins, on the ultrastructure, expression of these enzymes, and resorptive functions of cultured osteoclasts. Osteoclasts were formed by co-culture of marrow cells and calvarial primary osteoblasts of ddY mice. Formed osteoclasts were cultured on dentine slices for 6-48 hr with either an H(+)-ATPase inhibitor, bafilomycin A1, or a cysteine proteinase inhibitor, E-64. In control cultures with no additive, osteoclasts were structurally characterized by the development of ruffled borders and clear zones, and formed many resorption lacunae on dentine slices. Both H(+)-ATPase and cathepsin K were strongly expressed in the ruffled borders of these osteoclasts. In bafilomycin A1-treated cultures, osteoclasts lacked ruffled borders, and resorption lacuna formation was markedly diminished. This effect of bafilomycin A1 on osteoclast structure was reversible by removal of the compound. Bafilomycin A1 treatment altered the subcellular localization and decreased the expression of H(+)-ATPase molecules. H(+)-ATPase expression was observed throughout the cytoplasm, but not along the plasma membranes facing dentine slices. On the other hand, E-64 treatment did not affect the ultrastructure of osteoclasts and the expression of enzyme molecules. Although E-64 showed no effect on demineralization of dentine slices, it dose-dependently reduced resorption lacuna formation. Our results suggest that 1) bafilomycin A1 dose-dependently inhibits resorption lacuna formation via inhibition of ruffled border formation, 2) H(+)-ATPase expression is closely associated with the cytoskeleton of osteoclasts, and 3) E-64 treatment decreases the depth of resorption lacunae, by inhibition of secreted cathepsin K activity, but does not impair ruffled border formation and the associated expression of H(+)-ATPase and cathepsin K in osteoclasts.

Asunto(s)

Catepsinas/metabolismo , Cisteína Endopeptidasas/metabolismo , Leucina/análogos & derivados , Lisosomas/enzimología , Osteoclastos/efectos de los fármacos , Osteoclastos/enzimología , ATPasas de Translocación de Protón Vacuolares/metabolismo , Animales , Animales Recién Nacidos , Células de la Médula Ósea/metabolismo , Catepsina K , Catepsinas/antagonistas & inhibidores , Catepsinas/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Cisteína Endopeptidasas/efectos de los fármacos , Inhibidores de Cisteína Proteinasa/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Leucina/farmacología , Lisosomas/ultraestructura , Macrólidos/farmacología , Ratones , Ratones Endogámicos , Osteoclastos/ultraestructura , ATPasas de Translocación de Protón Vacuolares/efectos de los fármacos

RESUMEN

Using 3-day-old newborn rats, we examined the differentiation processes of osteoclasts associated with the destruction of the femoral growth plate cartilage and primary trabecular bone. In the growth plate cartilage, thin mineralized areas were detected solely in the longitudinal septal cartilage matrix in the hypertrophic zone, but the transverse septal cartilage matrix between adjacent chondrocytic lacunae within a row of chondrocytes remained unmineralized. The longitudinal septal cartilage between adjacent rows of chondrocytes appeared to persist, forming the walls of opened lacunar canals. Consistent with the removal of the transverse septal cartilage matrix, the longitudinal canals of opened chondrocytic lacunae were deeply invaded by capillary vessels, mononuclear cells and multinucleated pre-osteoclasts lacking a ruffled border. CD34-positive endothelial cells of capillary vessels deeply penetrated into the transverse septal cartilage matrix facing the medullary cavity and the opened chondrocytic lacunae. ED1-positive monocytes/macrophages were distributed at the chondro-osseous junction, but they were distant from the erosive front of the transverse septa. Tartrate-resistant acid phosphatase-positive multinucleated pre-osteoclasts lacking a ruffled border and differentiated osteoclasts with a ruffled border were localized mainly at two locations: the chondro-osseous junction and the growth front of primary bone trabeculae. Osteoclasts were located on the type-I collagen-positive bone trabeculae close to the growth plate, but they appeared to be distant from the type-II collagen-positive cartilage matrix. Even within opened chondrocytic lacunae, when osteoclasts were distant from the cartilage and bone matrix, they lacked polarized cytoplasmic organization and a ruffled border. The osteoclasts located in the remaining septal cartilage also exhibited neither a ruffled border nor a clear zone. Osteoclasts with a prominent ruffled border and clear zone were located in bone matrix covering the remaining septal cartilage. These results suggest that osteoclasts require hydroxyapatite crystals and bone matrix constituents for ruffled border formation and are not involved in resorption of the unmineralized transverse and mineralized longitudinal septal cartilage without covering bone matrix at the chondro-osseous junction.

Asunto(s)

Fémur/crecimiento & desarrollo , Placa de Crecimiento/citología , Osteoclastos/citología , Animales , Animales Recién Nacidos , Antígenos CD34/metabolismo , Desarrollo Óseo , Huesos/citología , Huesos/fisiología , Diferenciación Celular , Colágeno Tipo I/metabolismo , Endotelio Vascular , Fémur/citología , Inmunohistoquímica , Microscopía Electrónica , Ratas , Ratas Sprague-Dawley

RESUMEN

Osteoprotegerin (OPG) is an osteoblast-derived secreted member of the tumour necrosis factor receptor superfamily that inhibits osteoclastogenesis. Mice that are OPG-deficient have severe bone loss, including growth plate cartilage destruction. Using OPG-deficient mice as a useful animal model, we attempted to clarify differentiation and ultrastructural features of osteoclasts located on destructed growth plate cartilage and trabecular bone matrix. In the humerus and femur of OPG homozygous (-/-) mice, adjacent to the growth plate cartilage, bone trabeculae without a calcified cartilage core were characteristically formed at the metaphyseal side of the medullary cavity, which resulted in an irregular chondrocyte distribution and arrangement in growth plate cartilage. During growth plate cartilage destruction, osteoclasts positive for tartrate-resistant acid phosphatase showed unusual localization on both type-II collagen-positive cartilage and type-I collagen-positive trabecular bone matrix at the ossification centre of the epiphyseal/metaphyseal border. Although multinucleated osteoclasts were distributed within open lacunar canals in the growth plate, those on uncalcified cartilage matrix lacked a ruffled border. Facing the calcified cartilage matrix within lacunar canals, osteoclasts showed irregularly formed ruffled borders. After growth plate destruction, a thin bone layer was deposited on the remaining cartilage surfaces by invading osteoblasts. Osteoclasts formed prominent ruffled border structures on bone matrix, deposited on the remaining growth plate cartilage. These results suggest that, in OPG (-/-) mice, terminal osteoclast differentiation requires the presence of newly produced bone matrix, as the coupled phenomenon of bone formation and resorption, as well as osteoblast-derived cytokines.

Asunto(s)

Fémur/crecimiento & desarrollo , Glicoproteínas/deficiencia , Placa de Crecimiento/fisiología , Húmero/crecimiento & desarrollo , Osteoclastos/citología , Osteogénesis , Receptores Citoplasmáticos y Nucleares/deficiencia , Animales , Resorción Ósea , Cartílago , Diferenciación Celular , Colágeno Tipo I , Colágeno Tipo II , Fémur/ultraestructura , Glicoproteínas/genética , Húmero/ultraestructura , Ratones , Microscopía Electrónica , Osteoprotegerina , Receptores Citoplasmáticos y Nucleares/genética , Receptores del Factor de Necrosis Tumoral

RESUMEN

The differentiation and functions of osteoclasts (OCs) are regulated by osteoblast-derived factors such as receptor activator of NF kappa; B ligand (RANKL) that stimulates OC formation and a novel secreted member of the TNF receptor superfamily, osteoprotegerin (OPG), that negatively regulates osteoclastogenesis. Preosteoclasts (pOCs) treated with RANKL became TRAP-positive OCs, which express vacuolar-type H (+) -ATPase and exhibit resorptive activity. Such effects of RANKL on pOCs are inhibited by OPG. OPG inhibits ruffled border formation in mature OCs, reduces their resorptive activity, and induces apoptosis.

RESUMEN

Osteoprotegerin (OPG) is a novel osteoblast-derived secreted member of the tumour necrosis factor receptor superfamily that inhibits osteoclastogenesis. We examined the effects of OPG administration on the distribution, ultrastructure and vacuolar-type H+-ATPase expression of osteoclasts and resulting trabecular bone loss in the femurs of ovariectomized (OVX) mice. Two-month-old female ddY mice were allocated to the following groups: (1) pretreatment base-line controls; (2) untreated sham-operated controls; (3) untreated OVX; and (4) OPG-administered OVX mice. Postoperatively, OPG (0.3 mg kg(-1) day(-1)) was intraperitoneally administered daily to OVX mice for 7 days. On postoperative day 7, all mice were sacrificed, and the dissected femurs were examined by means of light and immunoelectron microscopy and quantitative backscattered-electron image analysis. Backscattered-electron examination revealed that trabecular bone area/unit medullary area in untreated OVX mice was significantly lower than that of base-line control and sham-operated control mice. Compared with untreated OVX mice, OPG administration to OVX mice significantly increased trabecular bone area, which was similar to that of sham-operated control mice. Surprisingly, the number of TRAP-positive osteoclasts along the trabecular bone surfaces in OPG-administered OVX mice was not significantly decreased compared with that of sham-operated control and untreated OVX mice. Ultrastructurally, OPG administration caused disappearance of ruffled borders in most osteoclasts, but induced neither necrotic nor apoptotic changes. In addition, the expression of vacuolar-type H+-ATPase in osteoclasts was decreased by OPG administration. Our results suggest that low-dose OPG administration significantly reduces trabecular bone loss in OVX mice via impairment of the structure and bone resorbing activity of osteoclasts.

Asunto(s)

Glicoproteínas/uso terapéutico , Osteoclastos/patología , Osteoporosis/prevención & control , Inhibidores de la Síntesis de la Proteína/uso terapéutico , Receptores Citoplasmáticos y Nucleares/uso terapéutico , Animales , Modelos Animales de Enfermedad , Fémur/efectos de los fármacos , Fémur/patología , Fémur/ultraestructura , Ratones/cirugía , Microscopía Electrónica , Osteoclastos/enzimología , Osteoclastos/ultraestructura , Osteoporosis/patología , Osteoporosis/fisiopatología , Osteoprotegerina , Ovariectomía , Receptores del Factor de Necrosis Tumoral , ATPasas de Translocación de Protón Vacuolares/metabolismo

RESUMEN

The differentiation and functions of osteoclasts (OCs) are regulated by osteoblast-derived factors. Receptor activator of NFkB ligand (RANKL) is one of the key regulatory molecules in OC formation. Osteoprotegerin (OPG) is a novel secreted member of the TNF receptor superfamily that negatively regulates osteoclastogenesis and binds to RANKL. We examined the biological actions of macrophage-colony-stimulating factor (M-CSF), RANKL, and OPG on the differentiation of OCs isolated from cocultures of mouse osteoblastic cells and bone marrow cells. Preosteoclasts (pOCs) and OCs were characterized by their ultrastructure and the expression of OC markers such as tartrate-resistant acid phosphatase (TRAP) and vacuolar-type H(+)-ATPase. pOCs formed without any additives expressed TRAP, but showed little resorptive activity on cocultured dentine slices. TRAP-positive pOCs treated with M-CSF began to fuse with each other, but lacked a ruffled border (RB) and showed almost no resorptive activity. pOCs treated with RANKL became TRAP-positive multinucleated cells, which expressed intense vacuolar-type H(+)-ATPase along the RB membranes and exhibited prominent resorptive activity. Such effects of RANKL on pOCs were completely inhibited by the addition of OPG. OPG inhibited RB formation in mature OCs and reduced their resorptive activity, and also induced apoptosis of some OCs. These results suggest that 1) RANKL induces differentiation of functional OCs from pOCs, 2) M-CSF induces macrophage-like multinucleated cells, but not OCs, 3) OPG inhibits RB formation and resorptive activity in mature OCs, 4) OPG also induces apoptosis of OCs, and 5) RANKL and OPG are, therefore, important regulators of not only the terminal differentiation of OCs but also their resorptive function.

Asunto(s)

Proteínas Portadoras/farmacología , Glicoproteínas/farmacología , Glicoproteínas de Membrana/farmacología , Osteoblastos/citología , Animales , Biomarcadores , Células de la Médula Ósea/citología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Células Cultivadas , Aparato de Golgi/ultraestructura , Inmunohistoquímica , Factor Estimulante de Colonias de Macrófagos/farmacología , Ratones , Microscopía Electrónica , Osteoprotegerina , ATPasas de Translocación de Protón/análisis , Ligando RANK , Receptor Activador del Factor Nuclear kappa-B , Receptores Citoplasmáticos y Nucleares , Receptores del Factor de Necrosis Tumoral , Vacuolas/enzimología , Vacuolas/ultraestructura

RESUMEN

Osteoprotegerin (OPG) is a novel secreted member of the tumor necrosis factor (TNF) receptor superfamily that negatively regulates osteoclastogenesis. The receptor activator of the NFKB ligand (RANKL) is one of the key regulatory molecules in osteoclast formation and binds to OPG. In this study, it was suggested that OPG and RANKL are involved in alveolar bone remodeling during orthodontic tooth movement. We examined RANKL localization and osteoclast induction in periodontal tissues during experimental movement of incisors in OPG-deficient mice. To produce orthodontic force, an elastic band was inserted between the upper right and left incisors for 2 or 5 days, and the dissected maxillae were examined for cytochemical and immunocytochemical localization of tartrate-resistant acid phosphatase (TRAP), vacuolar-type H(+)-ATPase, and RANKL. Compared to wild-type OPG (+/+) littermates, TRAP-positive multinucleated cells were markedly induced in the periodontal ligament (PDL) on the compressed side and in the adjacent alveolar bone of OPG-deficient mice. These multinucleated cells exhibited intense vacuolar-type H(+)-ATPase along the ruffled border membranes. Because of accelerated osteoclastic resorption in OPG-deficient mice, alveolar bone was severely destroyed and partially perforated at 2 and 5 days after force application. In both wild-type and OPG-deficient mice, RANKL expression became stronger at 2 and 5 days after force application than before force application. There was no apparent difference in intensity of RANKL expression between OPG (+/+) littermates and OPG-deficient mice. In both wild-type and OPG-deficient mice, expression of RANKL protein was detected in osteoblasts, fibroblasts, and osteoclasts mostly located in resorption lacunae. These results suggest that during orthodontic tooth movement, RANKL and OPG in the periodontal tissues are important determinants regulating balanced alveolar bone resorption.

Asunto(s)

Glicoproteínas/deficiencia , Osteoclastos/fisiología , Periodoncio/fisiología , Receptores Citoplasmáticos y Nucleares/deficiencia , Receptores del Factor de Necrosis Tumoral/deficiencia , Técnicas de Movimiento Dental , Fosfatasa Ácida/análisis , Adenosina Trifosfato/análisis , Animales , Remodelación Ósea/fisiología , Proteínas Portadoras/análisis , Glicoproteínas/genética , Técnicas para Inmunoenzimas , Incisivo/fisiología , Isoenzimas/análisis , Glicoproteínas de Membrana/análisis , Ratones , Ratones Endogámicos , Ratones Noqueados , Osteoclastos/química , Osteoclastos/citología , Osteoprotegerina , Periodoncio/química , Periodoncio/citología , Ligando RANK , Receptor Activador del Factor Nuclear kappa-B , Receptores Citoplasmáticos y Nucleares/genética , Receptores del Factor de Necrosis Tumoral/genética , Fosfatasa Ácida Tartratorresistente

RESUMEN

The effects of enamel matrix derivative (EMD; Emdogain) on new trabecular bone induction after pure bioinert titanium (Ti) implantation in the rat femur were examined by means of routine light and transmission electron microscopy, immunohistochemistry, and backscattered electron image analysis. Newly designed mini-Ti implants (3.5 mm in length and 1.6 mm in diameter) were placed in the corticotrabecular area of the femur with either EMD or its carrier, propylene glycol alginate, as control. On post-implantation days 4, 7, 14, and 30, the dissected femur was examined in the transverse direction through Ti implants. In both control and EMD-applied femurs, trabecular bone formation was recognized over the implant surfaces and within medullary cavities even at 4 days post-implantation. These newly formed bone trabeculae around the Ti implants were immunoreactive for bone sialoproteins as a bone matrix marker, and osteoclastic bone resorption became evident in these bone trabeculae after 7 days post-implantation. Although trabecular bone area around the implants was markedly decreased at 30 days post-implantation compared with those at 14 days, the trabecular bone areas in EMD-applied femurs were significantly greater than those in propylene glycol alginate-applied femurs at both 14 and 30 days post-implantation. Our results suggest that EMD is an effective biological matrix for enhancing new trabecular bone induction and resulting attachment of orthopedic prostheses to the recipient bone.

Asunto(s)

Desarrollo Óseo/fisiología , Esmalte Dental/química , Implantes Experimentales , Titanio , Animales , Microanálisis por Sonda Electrónica , Electrones , Femenino , Fémur/citología , Fémur/crecimiento & desarrollo , Inmunohistoquímica , Ratas , Ratas Wistar , Dispersión de Radiación

RESUMEN

Enamel matrix derivative (EMD: Emdogain) has been reported to stimulate the biosynthesis and regeneration of trabecular bone. To address whether the biological action of EMD is dependent on the local environment of osseous tissue, circular perforations were made in parietal bones and immediately filled with either EMD or its carrier, propylene glycol alginate (PGA), as control. On post-operative days 4-60, the dissected bones were examined by various histological techniques. New bone matrix, which was immunoreactive for bone sialoprotein (BSP), was formed from the periosteum at the peripheral area of perforations. Different from the findings reported in injured long bones, mineralized tissue was produced in the regenerating connective tissue within bone defects. This mineralized tissue was hardly immunostained for BSP, contained few collagen fibres, and lacked osteocytic lacunae and layers of osteoblasts and osteoid. Energy-dispersive X-ray analysis showed that Ca and P weight % and Ca/P molar ratio of this mineralized tissue were similar to or slightly higher than those in the pre-existing parietal bones. In addition, most multinucleated cells located in mineralized tissue lacked a ruffled border structure and showed weak immunoreaction for the lysosomal cysteine proteinase, cathepsin K, whereas those located in the bone matrix exhibited ruffled borders and strong cathepsin K expression. However, multinucleated cells located in both tissues were strongly stained for tartrate-resistant acid phosphatase. The volume fraction of such mineralized tissue appeared to be higher in EMD-applied bones than in PGA-applied controls. The mineralized tissue-forming stromal cells within bone defects appeared to show greater accumulation in EMD-applied bones than in PGA-applied controls. Our results suggest that the bioactive effects of EMD on bone wound healing and mineralized tissue formation depend, at least in part, on the local osseous environment where EMD has been applied.

Asunto(s)

Desarrollo Óseo/fisiología , Proteínas del Esmalte Dental/farmacología , Esmalte Dental/química , Hueso Parietal/efectos de los fármacos , Cicatrización de Heridas/efectos de los fármacos , Animales , Regeneración Ósea , Calcificación Fisiológica/efectos de los fármacos , Microscopía Electrónica , Osteoblastos/química , Osteoblastos/ultraestructura , Hueso Parietal/anomalías , Hueso Parietal/fisiopatología , Ratas , Ratas Wistar