RESUMEN
Calcium phosphate cements (CPC) are used as bone void filler in various orthopedic indications; however, there are some major drawbacks regarding mixing, transfer, and injection of traditional CPC. By using glycerol as mixing liquid, a premixed calcium phosphate cement (pCPC), some of these difficulties can be overcome. In the treatment of vertebral fractures the handling characteristics need to be excellent including a high radio-opacity for optimal control during injection. The aim of this study is to evaluate a radiopaque pCPC regarding its resorption behavior and biocompatibility in vivo. pCPC and a water-based CPC were injected into a Ø 4-mm drilled femur defect in rabbits. The rabbits were sacrificed after 2 and 12 weeks. Cross sections of the defects were evaluated using histology, electron microscopy, and immunohistochemical analysis. Signs of inflammation were evaluated both locally and systemically. The results showed a higher bone formation in the pCPC compared to the water-based CPC after 2 weeks by expression of RUNX-2. After 12 weeks most of the cement had been resorbed in both groups. Both materials were considered to have a high biocompatibility since no marked immunological response was induced and extensive bone ingrowth was observed. The conclusion from the study was that pCPC with ZrO(2) radiopacifier is a promising alternative regarding bone replacement material and may be suggested for treatment of, for example, vertebral fractures based on its high biocompatibility, fast bone ingrowth, and good handling properties.
Asunto(s)
Materiales Biocompatibles/farmacología , Cementos para Huesos/farmacología , Fosfatos de Calcio/farmacología , Medios de Contraste/farmacología , Ensayo de Materiales , Animales , Biomarcadores/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Femenino , Fémur/efectos de los fármacos , Fémur/patología , Fémur/ultraestructura , Regulación de la Expresión Génica/efectos de los fármacos , Inmunohistoquímica , Inflamación/patología , ConejosRESUMEN
Notch signalling, via its downstream mediators HES1 and HES5, regulates development of several different tissues. In vitro studies suggest that these genes are also involved in chondrogenesis and endochondral bone formation. In order to investigate the importance of HES1 and HES5 for these developmental processes, mice lacking chondrogenic expression of HES1 and HES5 were constructed by interbreeding HES5(-/-) mice homozygous for the floxed HES1 allele (HES1(flox/flox)) with COL2A1-Cre transgenic mice, creating conditional HES1;HES5 double mutant mice. The formation of cartilage and endochondral bone was studied in these mice using histological and immunohistochemical stainings, including Alcian Blue van Gieson, Safranin-O, modified Mallory Aniline Blue, tartrate-resistant acid phosphatase and collagen type II stainings. The mice were also studied using several different morphometrical analyses and the differentiation potential of the chondrocytes was evaluated in vitro. Unexpectedly, the conditional HES1;HES5 double mutant mice did not display impaired development of cartilage or endochondral bone. Lack of altered phenotype in the conditional HES1;HES5 double mutant mice can be explained either by the HES1 and HES5 genes not being involved in cartilage and endochondral bone development or by functional redundancy between the genes belonging to the family of HES genes: that is, disruption of one gene could be compensated for by the activity of another. Our results further shed light on the compensatory reserves available during the developing cartilage and bone.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Desarrollo Óseo/fisiología , Cartílago/fisiología , Condrogénesis/fisiología , Proteínas de Homeodominio/fisiología , Proteínas Represoras/fisiología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Desarrollo Óseo/genética , Cartílago/crecimiento & desarrollo , Cartílago/metabolismo , Condrocitos/metabolismo , Condrocitos/patología , Colágeno Tipo II/genética , Colágeno Tipo II/fisiología , Femenino , Expresión Génica , Regulación de la Expresión Génica , Genotipo , Proteínas de Homeodominio/genética , Masculino , Ratones , Ratones Transgénicos , Proteínas Represoras/genética , Factor de Transcripción HES-1RESUMEN
BACKGROUND: Notch signalling controls differentiation and proliferation in various cell types and is associated with several diseases. We investigated the localization and regulation of several Notch markers in human osteoarthritic (OA) cartilage as well as identified genes controlled by Notch signalling. METHODS: Immunolocalization and real-time PCR analysis of Notch markers in healthy and OA articular cartilage were performed. Genes regulated by Notch signalling were studied using microarray. Cytokine-induced transcription of Notch markers was analyzed using real-time PCR and its effect on cellular localization of the intracellular domain of Notch1 (NICD1) was investigated using immunohistochemistry, subcellular fractionation, and transfection. The effect of NFkappaB activation on HES5 transcription was studied using the NFkappaB inhibitor pyrrolidine dithiocarbamate. RESULTS: Notch signalling was activated in OA cartilage and Notch1, Jagged1, and HES5 were abundantly expressed compared to healthy cartilage. Notch signalling regulated the expression of several genes associated with OA, like interleukin-8, lubricin, CD10, matrix metalloproteinase-9, and bone morphogenetic protein-2. Cytokines significantly affected the expression of several Notch markers and repressed expression of HES5, but did not affect the cellular localization of NICD1. CONCLUSION: Notch signalling is dysregulated in OA, inducing and repressing transcription of genes that could potentially partly contribute to the OA phenotype.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas de Unión al Calcio/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de la Membrana/metabolismo , Osteoartritis/metabolismo , Receptor Notch1/metabolismo , Proteínas Represoras/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Western Blotting , Proteínas de Unión al Calcio/genética , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Condrocitos/patología , Técnica del Anticuerpo Fluorescente , Expresión Génica/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Interleucina-1beta/farmacología , Proteína Jagged-1 , Proteínas de la Membrana/genética , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Osteoartritis/genética , Osteoartritis/patología , Receptor Notch1/genética , Proteínas Represoras/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Serrate-Jagged , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
BACKGROUND: The INK4A tumor suppressor gene plays a crucial role in the regulation of the G1 cell cycle phase. It encodes two transcripts, p16 and p14 alternate reading frame (ARF), involved in retinoblastoma protein (pRb)- and p53- cell growth control pathways, respectively. METHODS: To define the role of gene status and molecule expression involved in the INK4A regulatory system, immunohistochemistry, immunoblotting, and polymerase chain reaction (PCR) analysis were performed on 35 primary high grade osteosarcomas (OS). RESULTS: Although p16 and p14ARF proteins were found negative or weakly detectable in 60% and 57% of the cases respectively, INK4A gene analysis of exons 1alpha, 1beta and 2 did not reveal any deletion or mutation. However, methylation status of the 5'CpG promoter region, assessed by methylation-specific PCR, was found in 12 out of 21 OSs with negative or weak p16 expression. A statistical analysis based on pRb/p16 and p53/p14ARF staining status showed that pRb and p16 co-expression was inversely correlated to tumor relapse and was a marker for a more favorable prognosis. A statistically significant inverse correlation was found between wt-p53 and p14ARF expression. In the group of wt-p53 tumors, the loss of p14ARF was associated with a decreased expression of p21 protein, suggesting a down-regulation of the transcriptional activity of p53. CONCLUSIONS: The current results suggest that, in OS, the altered expression of INK4A products plays a primary role in the deregulation of both pRb and p53 cell growth control pathways, contributing to tumor pathogenesis and development.
Asunto(s)
Biomarcadores de Tumor/análisis , Neoplasias Óseas/genética , Neoplasias Óseas/fisiopatología , Transformación Celular Neoplásica , Inhibidor p16 de la Quinasa Dependiente de Ciclina/farmacología , ADN de Neoplasias/genética , Proteínas Fúngicas , Regulación Neoplásica de la Expresión Génica , Osteosarcoma/genética , Osteosarcoma/fisiopatología , Adolescente , Adulto , Niño , Inhibidor p16 de la Quinasa Dependiente de Ciclina/biosíntesis , Exones , Femenino , Humanos , Immunoblotting , Inmunohistoquímica , Masculino , Metilación , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Pronóstico , Regiones Promotoras Genéticas , Serina Endopeptidasas/biosíntesis , Transcripción Genética , Proteína p53 Supresora de Tumor/biosíntesisRESUMEN
The locus encoding the tumor suppressor p16 has been found to code for a second, different protein. This protein, p14(ARF), has been shown to protect p53 from degradation. Like p16, its gene is often altered in different cancers. In this study, the first unique exon, exon 1 beta, of p14(ARF), has been studied in 22 chondrosarcoma tissues using polymerase chain reaction, DNA sequencing and methylation-specific polymerase chain reaction. One chondrosarcoma was found to have exon 1 beta homozygously deleted, but neither mutations nor methylations were found in any of the chondrosarcomas. This indicates that genetic changes of p14(ARF) are a rare event in chondrosarcoma.
Asunto(s)
Neoplasias Óseas/genética , Condrosarcoma/genética , Proteínas/genética , Secuencia de Bases , Neoplasias Óseas/metabolismo , Condrosarcoma/metabolismo , ADN de Neoplasias/análisis , Exones/genética , Humanos , Datos de Secuencia Molecular , Proteínas/metabolismo , Homología de Secuencia de Ácido Nucleico , Proteína p14ARF Supresora de TumorRESUMEN
Perinatal asphyxia still constitutes a clinical hazard associated with considerable neurologic morbidity. Several growth factors, including insulin-like growth factor-I (IGF-I), have been reported to have a neuroprotective effect in experimental models of hypoxic ischemia (HI). In the present study, we have applied solution hybridization for quantification of the time course for mRNA expression of IGF-I, IGF-I receptor, and growth hormone (GH) receptor after HI in 7-d-old rats. There was a significant increase in IGF-I mRNA in the damaged hemisphere 72 h (1.19 +/- 0.28 vs 0.48 +/- 0.02 amol/microg DNA, p < 0.05) and 14 d (0.61 +/- 0.18 vs 0.19 +/- 0.05 amol/microg DNA, p < 0.05) after HI. In the contralateral hemisphere, both IGF-I and GH receptor mRNA had increased by 14 d after the insult (0.36 +/- 0.042 vs 0.13 +/- 0.011, p < 0.05, and 0.31 +/- 0.013 vs 0.11 +/- 0.004 amol/microg DNA, p < 0.001, respectively). There were no changes in IGF-I receptor mRNA throughout the study period. We have also evaluated the neuroprotective effect of GH after HI in neonatal rats. GH administered s.c. after HI in daily doses of 50 and 100 mg/kg provided a moderate neuroprotection of 20%. These results suggest a role for the GH/IGF-I axis in the neurochemical process leading to HI brain injury.
Asunto(s)
Isquemia Encefálica/metabolismo , Hormona del Crecimiento/metabolismo , Hipoxia/metabolismo , Factor I del Crecimiento Similar a la Insulina/biosíntesis , Receptor IGF Tipo 1/biosíntesis , Receptores de Somatotropina/biosíntesis , Animales , Isquemia Encefálica/fisiopatología , Hipoxia/fisiopatología , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Ratas , Ratas WistarRESUMEN
Induction of two-kidney, one clip hypertension (renal hypertension) is characterized by a slow increase in left ventricular tension and aortic wall stress, as opposed to aortocaval fistula or shunt volume overload, which induces a marked and rapid onset of wall stress in the caval vein and right ventricle. In the present study, we applied hemodynamic challenge to study the growth response involving gene expression of insulin-like growth factor-I (IGF-I) and growth hormone receptor (GH-R) mRNA in aorta and caval vein. Volume overload and pressure overload were induced in Wistar rats by means of shunt and renal hypertension, respectively. Systolic pressure was measured before excision of the great vessels, which was performed between 2 and 12 days postoperatively. Aortic and caval vein IGF-I and GH-R mRNA expressions were measured by means of a solution hybridization assay, and the caval vein was analyzed for IGF-I protein by immunohistochemistry. In the volume-distended but not pressurized caval vein in shunt rats, verified by telemetry recordings, there was an eightfold increase in IGF-I and 3.5-fold increase in GH-R mRNA at day 4 versus control. The IGF-I protein appeared to be localized in smooth muscle cells. In the aorta of the renal hypertension group, changes were of a slower onset. At day 7, there was a fourfold increase in IGF-I and five-fold increase of GH-R mRNA expressions versus sham-operated rats. Both the shunt caval vein and renal hypertension aorta showed evidence of a structural adaptation of the growth response. The present study suggests that acute elevation in vascular wall stress is an important triggering factor for overexpression of IGF-I and GH-R mRNA in great vessels. The growth hormone/insulin-like growth factor axis may be an important link in mediating structurally adaptive growth responses in the blood vessel wall.
Asunto(s)
Aorta/metabolismo , Regulación de la Expresión Génica , Hipertensión/metabolismo , Receptor IGF Tipo 1/genética , Receptores de Somatotropina/genética , Venas Cavas/metabolismo , Animales , Aorta/cirugía , Derivación Arteriovenosa Quirúrgica/efectos adversos , Hemodinámica , Hipertensión/etiología , Hipertensión/fisiopatología , Hipertensión Renal/metabolismo , Hipertensión Renal/fisiopatología , Inmunohistoquímica , Masculino , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Ratas , Ratas Wistar , Receptor IGF Tipo 1/análisis , Receptor IGF Tipo 1/biosíntesis , Receptores de Somatotropina/análisis , Receptores de Somatotropina/biosíntesis , Ribonucleasas/genética , Venas Cavas/cirugíaRESUMEN
Excess levels of glucocorticoids are known to cause osteoporosis. It is speculated that the effect of glucocorticoids could be mediated via regulation of IGF-I. The aims of the present study were to detect and quantify the expression of IGF-I and/or IGF-II mRNA transcripts in human osteoblast-like cells and to investigate whether glucocorticoids regulate the expression of IGF-I mRNA transcripts in human osteoblast-like cells. Cultures of human osteoblast-like cells from trabecular bone were established. The IGF-IA and IGF-IB transcripts were detected in human osteoblast-like cells from seven out of nine patients while the IGF-II transcript was detected in human osteoblast-like cells from eight out of nine patients, as determined by RT-PCR assays. Human osteoblast-like cells, as well as human muscle tissue, expressed approximately 1/10 of the IGF-I mRNA levels found in liver, as determined by RNase protection solution hybridization assay. The IGF-I mRNA levels did not decrease with age in the human osteoblast-like cells and no difference was seen between males and females. However, cortisol (10(-6) mol/l) decreased IGF-I mRNA levels. In summary, the present study has shown that human osteoblast-like cells express IGF-I and IGF-II mRNA transcripts and that cortisol down-regulates the IGF-I mRNA levels, indicating that some of the inhibitory effect of glucocorticoids on bone formation in humans is mediated via a reduced autocrine/paracrine expression of IGF-I.
Asunto(s)
Hidrocortisona/farmacología , Factor I del Crecimiento Similar a la Insulina/metabolismo , Osteoblastos/metabolismo , ARN Mensajero/metabolismo , Adolescente , Adulto , Secuencia de Bases , Células Cultivadas , Niño , Preescolar , Cartilla de ADN/genética , Regulación hacia Abajo , Femenino , Expresión Génica/efectos de los fármacos , Humanos , Lactante , Factor I del Crecimiento Similar a la Insulina/genética , Factor II del Crecimiento Similar a la Insulina/genética , Masculino , Datos de Secuencia Molecular , Osteoblastos/efectos de los fármacos , Reacción en Cadena de la PolimerasaRESUMEN
The aim of the present study was to investigate the role of insulin-like growth factor I in the development of cardiac hypertrophy in two-kidney, one clip hypertension by relating growth hormone receptor and insulin-like growth factor I receptor mRNA levels to insulin-like growth factor I gene transcription using a solution hybridization/RNase protection assay. Two-kidney, one clip hypertension was induced in male Wistar rats, and experiments were performed 2, 4, 7, and 12 days after surgery. Systolic blood pressure was elevated 2, 7, and 12 days after clipping (P < .001). Left ventricular weights were increased 2, 4, 7, and 12 days after surgery (P < .01). Associated with the rise in blood pressure, left ventricular insulin-like growth factor I mRNA was increased 2, 7, and 12 days after surgery (P < .01). Furthermore, growth hormone receptor and insulin-like growth factor I receptor gene expression increased specifically in the left ventricle of renal hypertensive rats (P < .05 and P < .001, respectively). Left ventricular growth hormone receptor mRNA peaked 7 days after induction of renal artery stenosis. These results show that insulin-like growth factor I, growth hormone receptor, and insulin-like growth factor I receptor mRNA increase in the pressure-overloaded left ventricle of two-kidney, one clip rats, suggesting a role for insulin-like growth factor I and the growth hormone/insulin-like growth factor I axis in the development of cardiac hypertrophy.