RESUMEN
We analyzed the contributions of structural variants (SVs) to gliomagenesis across 179 pediatric high-grade gliomas (pHGGs). The most recurrent SVs targeted MYC isoforms and receptor tyrosine kinases (RTKs), including an SV amplifying a MYC enhancer in 12% of diffuse midline gliomas (DMG), indicating an underappreciated role for MYC in pHGG. SV signature analysis revealed that tumors with simple signatures were TP53 wild type (TP53WT) but showed alterations in TP53 pathway members PPM1D and MDM4. Complex signatures were associated with direct aberrations in TP53, CDKN2A and RB1 early in tumor evolution and with later-occurring extrachromosomal amplicons. All pHGGs exhibited at least one simple-SV signature, but complex-SV signatures were primarily restricted to subsets of H3.3K27M DMGs and hemispheric pHGGs. Importantly, DMGs with complex-SV signatures were associated with shorter overall survival independent of histone mutation and TP53 status. These data provide insight into the impact of SVs on gliomagenesis and the mechanisms that shape them.
Asunto(s)
Neoplasias Encefálicas , Glioma , Neoplasias Encefálicas/genética , Proteínas de Ciclo Celular/genética , Niño , Glioma/genética , Histonas/genética , Humanos , Mutación , Proteínas Proto-Oncogénicas/genéticaRESUMEN
Patient-specific ex vivo models of human tumours that recapitulate the pathological characteristics and complex ecology of native tumours could help determine the most appropriate cancer treatment for individual patients. Here, we show that bioprinted reconstituted glioblastoma tumours consisting of patient-derived tumour cells, vascular endothelial cells and decellularized extracellular matrix from brain tissue in a compartmentalized cancer-stroma concentric-ring structure that sustains a radial oxygen gradient, recapitulate the structural, biochemical and biophysical properties of the native tumours. We also show that the glioblastoma-on-a-chip reproduces clinically observed patient-specific resistances to treatment with concurrent chemoradiation and temozolomide, and that the model can be used to determine drug combinations associated with superior tumour killing. The patient-specific tumour-on-a-chip model might be useful for the identification of effective treatments for glioblastoma patients resistant to the standard first-line treatment.
Asunto(s)
Bioimpresión/métodos , Quimioradioterapia/métodos , Glioblastoma/tratamiento farmacológico , Dispositivos Laboratorio en un Chip , Encéfalo/diagnóstico por imagen , Neoplasias Encefálicas/diagnóstico por imagen , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Combinación de Medicamentos , Evaluación de Medicamentos , Sinergismo Farmacológico , Células Endoteliales , Regulación Neoplásica de la Expresión Génica , Glioblastoma/diagnóstico por imagen , Glioblastoma/genética , Glioblastoma/patología , Humanos , Oxígeno , Temozolomida/farmacología , Microambiente Tumoral/efectos de los fármacosRESUMEN
Wnt signaling pathway plays critical roles in body axes patterning, cell fate specification, cell proliferation, cell migration, stem cell maintenance, cancer development and etc. Deregulation of this pathway can be causative of cancer, metabolic disease and neurodegenerative disease such as Parkinson`s disease. Among the core components of Wnt signaling pathway, we discovered that Dishevelled (Dsh) interacts with ULK1 and is phosphorylated by ULK1. Unexpectedly, the knockdown of ULK1 elicited a marked increase in Wnt/ß-catenin signaling. Multiple ULK1 phosphorylation sites existed on Dsh and many of them were located on the PDZ-DEP region. By using evolutionarily well conserved Drosophila Dsh, we found that S239, S247 and S254 in the PDZ-DEP region are involved in phosphorylation of Dsh by ULK1. Among these, S247 and S254 were conserved in human Dsh. When phospho-mimetic mutants (2D and 2E Dsh mutants) of these conserved residues were generated and expressed in the eyes of the fruit flies, the activity of Dsh was significantly decreased compared to wild type Dsh. Through additional alanine scanning, we further identified that S239, S247, S254, S266, S376, S554 and S555 on full length Dsh were phosphorylated by ULK1. In regards to the S266A mutation located in the PDZ domain among these phosphorylated residues, our results suggested that Dsh forms an SDS-resistant high molecular weight complex with ß-catenin and TCF in the nucleus in an S266 phosphorylation-dependent manner. Based on these results, we propose that ULK1 plays a pivotal role in the regulation of Wnt/ß-catenin signaling pathway by phosphorylating Dsh.
Asunto(s)
Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Proteínas Dishevelled/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Vía de Señalización Wnt , Células Cultivadas , Células HEK293 , Humanos , FosforilaciónRESUMEN
High-bone-mass (HBM)-causing missense mutations in the low density lipoprotein receptor-related protein-5 (Lrp5) are associated with increased osteoanabolic action and protection from disuse- and ovariectomy-induced osteopenia. These mutations (e.g., A214V and G171V) confer resistance to endogenous secreted Lrp5/6 inhibitors, such as sclerostin (SOST) and Dickkopf homolog-1 (DKK1). Cells in the osteoblast lineage are responsive to canonical Wnt stimulation, but recent work has indicated that osteoclasts exhibit both indirect and direct responsiveness to canonical Wnt. Whether Lrp5-HBM receptors, expressed in osteoclasts, might alter osteoclast differentiation, activity, and consequent net bone balance in the skeleton, is not known. To address this, we bred mice harboring heterozygous Lrp5 HBM-causing conditional knock-in alleles to Ctsk-Cre transgenic mice and studied the phenotype using DXA, µCT, histomorphometry, serum assays, and primary cell culture. Mice with HBM alleles induced in Ctsk-expressing cells (TG) exhibited higher bone mass and architectural properties compared to non-transgenic (NTG) counterparts. In vivo and in vitro measurements of osteoclast activity, population density, and differentiation yielded significant reductions in osteoclast-related parameters in female but not male TG mice. Droplet digital PCR performed on osteocyte enriched cortical bone tubes from TG and NTG mice revealed that ~8-17% of the osteocyte population (depending on sex) underwent recombination of the conditional Lrp5 allele in the presence of Ctsk-Cre. Further, bone formation parameters in the midshaft femur cortex show a small but significant increase in anabolic action on the endocortical but not periosteal surface. These findings suggest that Wnt/Lrp5 signaling in osteoclasts affects osteoclastogenesis and activity in female mice, but also that some of the changes in bone mass in TG mice might be due to Cre expression in the osteocyte population.
Asunto(s)
Huesos/metabolismo , Catepsina K/metabolismo , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Mutación/genética , Absorciometría de Fotón , Alelos , Animales , Biomarcadores/sangre , Células de la Médula Ósea/metabolismo , Resorción Ósea/sangre , Resorción Ósea/patología , Huesos/diagnóstico por imagen , Diferenciación Celular , Femenino , Integrasas/metabolismo , Masculino , Ratones Transgénicos , Tamaño de los Órganos/genética , Osteoclastos/metabolismo , Osteoclastos/patología , Osteogénesis/genética , Periostio/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Recombinación Genética/genética , Transgenes , Microtomografía por Rayos XRESUMEN
The WNT pathway has become an attractive target for skeletal therapies. High-bone-mass phenotypes in patients with loss-of-function mutations in the LRP5/6 inhibitor Sost (sclerosteosis), or in its downstream enhancer region (van Buchem disease), highlight the utility of targeting Sost/sclerostin to improve bone properties. Sclerostin-neutralizing antibody is highly osteoanabolic in animal models and in human clinical trials, but antibody-based inhibition of another potent LRP5/6 antagonist, Dkk1, is largely inefficacious for building bone in the unperturbed adult skeleton. Here, we show that conditional deletion of Dkk1 from bone also has negligible effects on bone mass. Dkk1 inhibition increases Sost expression, suggesting a potential compensatory mechanism that might explain why Dkk1 suppression lacks anabolic action. To test this concept, we deleted Sost from osteocytes in, or administered sclerostin neutralizing antibody to, mice with a Dkk1-deficient skeleton. A robust anabolic response to Dkk1 deletion was manifest only when Sost/sclerostin was impaired. Whole-body DXA scans, µCT measurements of the femur and spine, histomorphometric measures of femoral bone formation rates, and biomechanical properties of whole bones confirmed the anabolic potential of Dkk1 inhibition in the absence of sclerostin. Further, combined administration of sclerostin and Dkk1 antibody in WT mice produced a synergistic effect on bone gain that greatly exceeded individual or additive effects of the therapies, confirming the therapeutic potential of inhibiting multiple WNT antagonists for skeletal health. In conclusion, the osteoanabolic effects of Dkk1 inhibition can be realized if sclerostin upregulation is prevented. Anabolic therapies for patients with low bone mass might benefit from a strategy that accounts for the compensatory milieu of WNT inhibitors in bone tissue.
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Anabolizantes/administración & dosificación , Glicoproteínas/antagonistas & inhibidores , Hiperostosis/tratamiento farmacológico , Osteogénesis/efectos de los fármacos , Sindactilia/tratamiento farmacológico , Vía de Señalización Wnt/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales , Animales , Anticuerpos Neutralizantes/administración & dosificación , Proteínas Morfogenéticas Óseas/genética , Modelos Animales de Enfermedad , Femenino , Fémur/citología , Fémur/diagnóstico por imagen , Fémur/patología , Marcadores Genéticos/genética , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Hiperostosis/diagnóstico por imagen , Hiperostosis/genética , Hiperostosis/patología , Péptidos y Proteínas de Señalización Intercelular/genética , Mutación con Pérdida de Función , Masculino , Ratones , Osteocitos , Columna Vertebral/citología , Columna Vertebral/diagnóstico por imagen , Columna Vertebral/patología , Sindactilia/diagnóstico por imagen , Sindactilia/genética , Sindactilia/patología , Resultado del Tratamiento , Regulación hacia Arriba/efectos de los fármacos , Microtomografía por Rayos XRESUMEN
Approximately 10% of all bone fractures do not heal, resulting in patient morbidity and healthcare costs. However, no pharmacological treatments are currently available to promote efficient bone healing. Inhibition of Ca2+ /calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) reverses age-associated loss of trabecular and cortical bone volume and strength in mice. In the current study, we investigated the role of CaMKK2 in bone fracture healing and show that its pharmacological inhibition using STO-609 accelerates early cellular and molecular events associated with endochondral ossification, resulting in a more rapid and efficient healing of the fracture. Within 7 days postfracture, treatment with STO-609 resulted in enhanced Indian hedgehog signaling, paired-related homeobox (PRX1)-positive mesenchymal stem cell (MSC) recruitment, and chondrocyte differentiation and hypertrophy, along with elevated expression of osterix, vascular endothelial growth factor, and type 1 collagen at the fracture callus. Early deposition of primary bone by osteoblasts resulted in STO-609-treated mice possessing significantly higher callus bone volume by 14 days following fracture. Subsequent rapid maturation of the bone matrix bestowed fractured bones in STO-609-treated animals with significantly higher torsional strength and stiffness by 28 days postinjury, indicating accelerated healing of the fracture. Previous studies indicate that fixed and closed femoral fractures in the mice take 35 days to fully heal without treatment. Therefore, our data suggest that STO-609 potentiates a 20% acceleration of the bone healing process. Moreover, inhibiting CaMKK2 also imparted higher mechanical strength and stiffness at the contralateral cortical bone within 4 weeks of treatment. Taken together, the data presented here underscore the therapeutic potential of targeting CaMKK2 to promote efficacious and rapid healing of bone fractures and as a mechanism to strengthen normal bones. © 2018 American Society for Bone and Mineral Research.
Asunto(s)
Callo Óseo/enzimología , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/metabolismo , Curación de Fractura/fisiología , Proteínas Hedgehog/metabolismo , Osteogénesis/fisiología , Transducción de Señal/fisiología , Animales , Bencimidazoles/farmacología , Colágeno Tipo I/metabolismo , Curación de Fractura/efectos de los fármacos , Masculino , Ratones , Naftalimidas/farmacología , Osteogénesis/efectos de los fármacosRESUMEN
OBJECTIVE: Cranial defects can result from trauma, infection, congenital malformations, and iatrogenic causes and represent a surgical challenge. The current standard of care is cranioplasty, with either autologous or allogeneic material. In either case, the intrinsic vascularity of the surrounding tissues allows for bone healing. The objective of this study was to determine if mechanotransductive gene manipulation would yield non-weight-bearing bone regeneration in a critical size calvarial defect in mice. METHODS: A mouse model of Sost deletion in Sost knockout (KO) mice was created in which the osteocytes do not express sclerostin. A critical size calvarial defect (4 mm in diameter) was surgically created in the parietal bone in 8-week-old wild-type (n = 8) and Sost KO (n = 8) male mice. The defects were left undisturbed (no implant or scaffold) to simulate a traumatic calvariectomy model. Eight weeks later, the animals were examined at necropsy by planimetry, histological analysis of new bone growth, and micro-CT scanning of bone thickness. RESULTS: Defects created in wild-type mice did not fill with bone over the study period of 2 months. Genetic downregulation of sclerostin yielded animals that were able to regenerate 40% of the initial critical size defect area 8 weeks after surgery. A thin layer of bone covered a significant portion of the original defect in all Sost KO animals. A statistically significant increase in bone volume (p < 0.05) was measured in Sost KO mice using radiodensitometric analysis. Immunohistochemical analysis also confirmed that this bone regeneration occurred through the Wnt pathway and originated from the edge of the defect; BMP signaling did not appear to be affected by sclerostin. CONCLUSIONS: Mechanical loading is an important mechanism of bone formation in the cranial skeleton and is poorly understood. This is partially due to the fact that it is difficult to load bone in the craniomaxillofacial skeleton. This study suggests that modulation of the Wnt pathway, as is able to be done with monoclonal antibodies, is a potentially efficacious method for bone regeneration that requires further study.
Asunto(s)
Desarrollo Óseo/genética , Regeneración Ósea/genética , Deleción Cromosómica , Modelos Animales de Enfermedad , Glicoproteínas/genética , Mecanotransducción Celular/genética , Cráneo/crecimiento & desarrollo , Proteínas Adaptadoras Transductoras de Señales , Animales , Regulación hacia Abajo/genética , Péptidos y Proteínas de Señalización Intercelular , Masculino , Ratones , Ratones Noqueados , Osteocitos/fisiología , Microtomografía por Rayos XRESUMEN
DJ-1 is one of the causative genes for early onset familiar Parkinson's disease (PD) and is also considered to influence the pathogenesis of sporadic PD. DJ-1 has various physiological functions which converge on controlling intracellular reactive oxygen species (ROS) levels. In RNA-sequencing analyses searching for novel anti-oxidant genes downstream of DJ-1, a gene encoding NADP+-dependent isocitrate dehydrogenase (IDH), which converts isocitrate into α-ketoglutarate, was detected. Loss of IDH induced hyper-sensitivity to oxidative stress accompanying age-dependent mitochondrial defects and dopaminergic (DA) neuron degeneration in Drosophila, indicating its critical roles in maintaining mitochondrial integrity and DA neuron survival. Further genetic analysis suggested that DJ-1 controls IDH gene expression through nuclear factor-E2-related factor2 (Nrf2). Using Drosophila and mammalian DA models, we found that IDH suppresses intracellular and mitochondrial ROS level and subsequent DA neuron loss downstream of DJ-1. Consistently, trimethyl isocitrate (TIC), a cell permeable isocitrate, protected mammalian DJ-1 null DA cells from oxidative stress in an IDH-dependent manner. These results suggest that isocitrate and its derivatives are novel treatments for PD associated with DJ-1 dysfunction.
Asunto(s)
Proteínas de Drosophila/genética , Isocitrato Deshidrogenasa/genética , Degeneración Nerviosa/genética , Proteínas del Tejido Nervioso/genética , Enfermedad de Parkinson/genética , Animales , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Drosophila melanogaster/genética , Regulación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Isocitratos/metabolismo , Mitocondrias/genética , Mitocondrias/patología , NADP/genética , Factor de Transcripción NF-E2/genética , Degeneración Nerviosa/fisiopatología , Estrés Oxidativo/genética , Enfermedad de Parkinson/patologíaRESUMEN
Sclerostin (Sost) is a negative regulator of bone formation that acts upon the Wnt signaling pathway. Sost is mechanically regulated at both mRNA and protein level such that loading represses and unloading enhances Sost expression, in osteocytes and in circulation. The non-coding evolutionarily conserved enhancer ECR5 has been previously reported as a transcriptional regulatory element required for modulating Sost expression in osteocytes. Here we explored the mechanisms by which ECR5, or several other putative transcriptional enhancers regulate Sost expression, in response to mechanical stimulation. We found that in vivo ulna loading is equally osteoanabolic in wildtype and Sost-/- mice, although Sost is required for proper distribution of load-induced bone formation to regions of high strain. Using Luciferase reporters carrying the ECR5 non-coding enhancer and heterologous or homologous hSOST promoters, we found that ECR5 is mechanosensitive in vitro and that ECR5-driven Luciferase activity decreases in osteoblasts exposed to oscillatory fluid flow. Yet, ECR5-/- mice showed similar magnitude of load-induced bone formation and similar periosteal distribution of bone formation to high-strain regions compared to wildtype mice. Further, we found that in contrast to Sost-/- mice, which are resistant to disuse-induced bone loss, ECR5-/- mice lose bone upon unloading to a degree similar to wildtype control mice. ECR5 deletion did not abrogate positive effects of unloading on Sost, suggesting that additional transcriptional regulators and regulatory elements contribute to load-induced regulation of Sost.
Asunto(s)
Adaptación Fisiológica/fisiología , Elementos de Facilitación Genéticos/fisiología , Glicoproteínas/deficiencia , Osteocitos/fisiología , Osteogénesis/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Fenómenos Biomecánicos/fisiología , Proteínas Morfogenéticas Óseas/deficiencia , Proteínas Morfogenéticas Óseas/genética , Femenino , Glicoproteínas/genética , Péptidos y Proteínas de Señalización Intercelular , Ratones , Ratones Noqueados , Ratones Transgénicos , ARN no Traducido/genéticaRESUMEN
ß-Catenin (ßcat) is a major downstream signaling node in canonical Wingless-related integration site (Wnt) signaling pathway, and its activity is crucial for canonical Wnt signal transduction. Wnt signaling has recently been implicated in the osteo-anabolic response to PTH, a potent calcium-regulating factor. We investigated whether ßcat is essential for the anabolic action of intermittent PTH by generating male mice with adult-onset deletion of ßcat in a subpopulation of bone cells (osteocytes and late-stage osteoblasts), treating them with an anabolic regimen of PTH, and measuring the skeletal responses. Male (10kb)Dmp1-CreERt2 transgenic mice that also harbored floxed loss-of-function ßcat alleles (ßcat(f/f)) were induced for Cre activity using tamoxifen, then injected daily with human PTH 1-34 (30 µg/kg) or vehicle for 5 weeks. Mice in which ßcat was deleted showed either total lack of bone mineral density (BMD) gain, or BMD loss, and did not respond to PTH treatment. However, bone mass measurements in the trabecular compartment of the femur and spine revealed PTH-induced bone gain whether ßcat was deleted or not. PTH-stimulated increases in periosteal and cancellous bone formation rates were not impaired by ßcat deletion, but resorption markers and cortical porosity were significantly increased in induced mice, particularly induced mice treated with PTH. These results suggest that ßcat is required for net-positive BMD effects of PTH therapy but that the anabolic effects per se of PTH treatment might not require osteocytic/osteoblastic ßcat.
Asunto(s)
Proteínas de la Matriz Extracelular/genética , Eliminación de Gen , Osteogénesis , Hormona Paratiroidea/farmacología , beta Catenina/genética , Factores de Edad , Anabolizantes/metabolismo , Anabolizantes/farmacología , Animales , Densidad Ósea/efectos de los fármacos , Densidad Ósea/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutagénesis , Osteogénesis/efectos de los fármacos , Osteogénesis/genética , Hormona Paratiroidea/metabolismoRESUMEN
Since recurrence and metastasis of pancreatic cancer has a worse prognosis, chemotherapy has been typically performed to attack the remained malignant cells after resection. However, it is difficult to achieve the therapeutic concentration at the tumor site with systemic chemotherapy. Numerous local drug delivery systems have been studied to overcome the shortcomings of systemic delivery. However, because most systems involve dissolution of the drug within the carrier, the concentration of the drug is limited to the saturation solubility, and consequently cannot reach the sufficient drug dose. Therefore, we hypothesized that 3D printing of a biodegradable patch incorporated with a high drug concentration would provide a versatile shape to be administered at the exact tumor site as well as an appropriate therapeutic drug concentration with a controlled release. Here, we introduce the 3D-printed patches composed of a blend of poly(lactide-co-glycolide), polycaprolactone, and 5-fluorouracil for delivering the anti-cancer drug in a prolonged controlled manner and therapeutic dose. 3D printing technology can manipulate the geometry of the patch and the drug release kinetics. The patches were flexible, and released the drug over four weeks, and thereby suppressed growth of the subcutaneous pancreatic cancer xenografts in mice with minimized side effects. Our approach reveals that 3D printing of bioabsorbable implants containing anti-cancer drugs could be a powerful method for an effective local delivery of chemotherapeutic agents to treatment of cancers.
Asunto(s)
Antimetabolitos Antineoplásicos/administración & dosificación , Preparaciones de Acción Retardada/química , Sistemas de Liberación de Medicamentos/instrumentación , Fluorouracilo/administración & dosificación , Páncreas/efectos de los fármacos , Neoplasias Pancreáticas/tratamiento farmacológico , Poliglactina 910/química , Animales , Antimetabolitos Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Diseño de Equipo , Fluorouracilo/uso terapéutico , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Páncreas/patología , Neoplasias Pancreáticas/patología , Poliésteres/química , Impresión Tridimensional/instrumentaciónRESUMEN
Mechanical signal transduction in bone tissue begins with load-induced activation of several cellular pathways in the osteocyte population. A key pathway that participates in mechanotransduction is Wnt/Lrp5 signaling. A putative downstream mediator of activated Lrp5 is the nucleocytoplasmic shuttling protein ß-catenin (ßcat), which migrates to the nucleus where it functions as a transcriptional co-activator. We investigated whether osteocytic ßcat participates in Wnt/Lrp5-mediated mechanotransduction by conducting ulnar loading experiments in mice with or without chemically induced ßcat deletion in osteocytes. Mice harboring ßcat floxed loss-of-function alleles (ßcat(f/f)) were bred to the inducible osteocyte Cre transgenic (10)(kb)Dmp1-CreERt2. Adult male mice were induced to recombine the ßcat alleles using tamoxifen, and intermittent ulnar loading sessions were applied over the following week. Although adult-onset deletion of ßcat from Dmp1-expressing cells reduced skeletal mass, the bone tissue was responsive to mechanical stimulation as indicated by increased relative periosteal bone formation rates in recombined mice. However, load-induced improvements in cross sectional geometric properties were compromised in recombined mice. The collective results indicate that the osteoanabolic response to loading can occur on the periosteal surface when ß-cat levels are significantly reduced in Dmp1-expressing cells, suggesting that either (i) only low levels of ß-cat are required for mechanically induced bone formation on the periosteal surface, or (ii) other additional downstream mediators of Lrp5 might participate in transducing load-induced Wnt signaling.
Asunto(s)
Adaptación Fisiológica , Proteínas de la Matriz Extracelular/metabolismo , Eliminación de Gen , Osteoblastos/metabolismo , Osteocitos/metabolismo , Osteogénesis , Periostio/fisiopatología , beta Catenina/metabolismo , Absorciometría de Fotón , Alelos , Animales , Animales Recién Nacidos , Densidad Ósea , Hueso Cortical/patología , Ratones , Músculos/patología , Periostio/patología , Transgenes , Soporte de PesoRESUMEN
In cartilage tissue engineering, electromagnetic field (EMF) therapy has been reported to have a modest effect on promoting cartilage regeneration. However, these studies were conducted using different frequencies of EMF to stimulate chondrocytes. Thus, it is necessary to investigate the effect of EMF frequency on cartilage formation. In addition to the stimulation, a scaffold is required to satisfy the characteristics of cartilage such as its hydrated and dense extracellular matrix, and a mechanical resilience to applied loads. Therefore, we 3D-printed a composite construct composed of a polymeric framework and a chondrocyte-laden hydrogel. Here, we observed frequency-dependent positive and negative effects on chondrogenesis using a 3D cell-printed cartilage tissue. We found that a frequency of 45 Hz promoted gene expression and secretion of extracellular matrix molecules of chondrocytes. In contrast, a frequency of 7.5 Hz suppressed chondrogenic differentiation in vitro. Additionally, the EMF-treated composite constructs prior to implantation showed consistent results with those of in vitro, suggesting that in vitro pre-treatment with different EMF frequencies provides different capabilities for the enhancement of cartilage formation in vivo. This correlation between EMF frequency and 3D-printed chondrocytes suggests the necessity for optimization of EMF parameters when this physical stimulus is applied to engineered cartilage. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1797-1804, 2016.
Asunto(s)
Condrocitos/citología , Campos Electromagnéticos , Impresión Tridimensional , Andamios del Tejido/química , Animales , Línea Celular , Condrogénesis , Colágeno Tipo II/metabolismo , Glicosaminoglicanos/metabolismo , Humanos , Ratones Desnudos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Several studies have focused on the regeneration of liver tissue in a two-dimensional (2D) planar environment, whereas actual liver tissue is three-dimensional (3D). Cell printing technology has been successfully utilized for building 3D structures; however, the poor mechanical properties of cell-laden hydrogels are a major concern. Here, we demonstrate the printing of a 3D cell-laden construct and its application to liver tissue engineering using 3D cell printing technology through a multi-head tissue/organ building system. Polycaprolactone (PCL) was used as a framework material because of its excellent mechanical properties. Collagen bioink containing three different types of cells-hepatocytes (HCs), human umbilical vein endothelial cells , and human lung fibroblasts--was infused into the canals of a PCL framework to induce the formation of capillary--like networks and liver cell growth. A co-cultured 3D microenvironment of the three types of cells was successfully established and maintained. The vascular formation and functional abilities of HCs (i.e., albumin secretion and urea synthesis) demonstrated that the heterotypic interaction among HCs and nonparenchymal cells increased the survivability and functionality of HCs within the collagen gel. Therefore, our results demonstrate the prospect of using cell printing technology for the creation of heterotypic cellular interaction within a structure for liver tissue engineering.
Asunto(s)
Órganos Bioartificiales , Hígado/irrigación sanguínea , Hígado/crecimiento & desarrollo , Neovascularización Fisiológica/fisiología , Impresión Tridimensional , Ingeniería de Tejidos/instrumentación , Animales , Vasos Sanguíneos/citología , Vasos Sanguíneos/crecimiento & desarrollo , Células Cultivadas , Técnicas de Cocultivo/instrumentación , Células Endoteliales/citología , Células Endoteliales/fisiología , Diseño de Equipo , Análisis de Falla de Equipo , Femenino , Fibroblastos/citología , Fibroblastos/fisiología , Hepatocitos/citología , Hepatocitos/fisiología , Humanos , Técnicas de Cultivo de Órganos/instrumentación , Ratas , Ratas Sprague-Dawley , Andamios del TejidoRESUMEN
An electromagnetic field is an effective stimulation tool because it promotes bone defect healing, albeit in an unknown way. Although electromagnetic fields are used for treatment after surgery, many patients prefer cell-based tissue regeneration procedures that do not require daily treatments. This study addressed the effects of an electromagnetic field on adipose-derived stem cells (ASCs) to investigate the feasibility of pretreatment to accelerate bone regeneration. After identifying a uniform electromagnetic field inside a solenoid coil, we observed that a 45 Hz electromagnetic field induced osteogenic marker expression via bone morphogenetic protein, transforming growth factor ß, and Wnt signalling pathways based on microarray analyses. This electromagnetic field increased osteogenic gene expression, alkaline phosphate activity and nodule formation in vitro within 2 weeks, indicating that this pretreatment may provide osteogenic potential to ASCs on three-dimensional (3D) ceramic scaffolds. This pretreatment effect of an electromagnetic field resulted in significantly better bone regeneration in a mouse calvarial defect model over 4 weeks compared to that in the untreated group. This short-term evaluation showed that the electromagnetic field pretreatment may be a future therapeutic option for bone defect treatment.
Asunto(s)
Tejido Adiposo/citología , Campos Electromagnéticos , Curación de Fractura , Células Madre/citología , Animales , Trasplante de Células , Células Cultivadas , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Transducción de SeñalRESUMEN
Transforming growth factor-ß1 (TGFß1) induces epithelial-to-mesenchymal transition (EMT) in cultured renal tubular epithelial cells. This phenotypic transition has been known to be involved in the development of chronic kidney diseases by activating profibrotic gene expression. Since oxidative stress has been recognized as one of the contributors to this TGFß1-mediated pathology, we investigated the potential involvement of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), which is a key transcription factor for the regulation of multiple antioxidant genes, in TGFß1-stimulated EMT gene changes using the rat proximal tubular epithelial cell line NRK52E. The treatment of NRK52E with TGFß1 led to changes in EMT gene expression, including increased α-Sma and decreased E-cadherin expression. In these cells, the TGFß1 treatment decreased the transcript level of the catalytic subunit of γ-glutamate cysteine ligase (Gclc), a glutathione (GSH) biosynthetic enzyme, and reduced the total GSH content with a concomitant decrease in Nrf2 transcription activity. Accordantly, pre-incubation with the GSH precursor N-acetylcysteine attenuated TGFß1-stimulated EMT gene changes. The involvement of Nrf2 in EMT gene changes has been demonstrated using NRK52E cells with nrf2 knockdown or pharmacological activation. When the expression of Nrf2 was stably silenced in NRK52E cells using interfering RNA administration, Gclc expression was significantly reduced and the increase in the levels of α-Sma and fibronectin-1 by TGFß1 was greater than those in the nonspecific RNA control group. Conversely, Nrf2 activation and subsequent Gclc increase by Nrf2-activating sulforaphane alleviated the TGFß1-stimulated α-Sma increase and E-cadherin decrease. Collectively, these results indicate that Nrf2-GSH signaling can modulate TGFß1-stimulated EMT gene changes and further suggest a beneficial role of Nrf2 inducers in renal pathogenesis.
Asunto(s)
Células Epiteliales/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Glutatión/metabolismo , Túbulos Renales Proximales/efectos de los fármacos , Factor 2 Relacionado con NF-E2/metabolismo , Factor de Crecimiento Transformador beta1/farmacología , Animales , Línea Celular , Células Epiteliales/metabolismo , Transición Epitelial-Mesenquimal/genética , Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/metabolismo , Factor 2 Relacionado con NF-E2/genética , Ratas , Proteínas Recombinantes , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
Chronic kidney disease (CKD) is featured by a progressive decline of kidney function and is mainly caused by chronic diseases such as diabetes mellitus and hypertension. CKD is a complex disease due to cardiovascular complications and high morbidity; however, there is no single treatment to improve kidney function in CKD patients. Since biological markers representing oxidative stress are significantly elevated in CKD patients, oxidative stress is receiving attention as a contributing factor to CKD pathology. Nuclear factor erythroid-2 related factor 2 (NRF2) is a predominant transcription factor that regulates the expression of a wide array of genes encoding antioxidant proteins, thiol molecules and their generating enzymes, detoxifying enzymes, and stress response proteins, all of which can counteract inflammatory and oxidative damages. There is considerable experimental evidence suggesting that NRF2 signaling plays a protective role in renal injuries that are caused by various pathologic conditions. In addition, impaired NRF2 activity and consequent target gene repression have been observed in CKD animals. Therefore, a pharmacological intervention activating NRF2 signaling can be beneficial in protecting against kidney dysfunction in CKD. This review article provides an overview of the role of NRF2 in experimental CKD models and describes current findings on the renoprotective effects of naturally occurring NRF2 activators, including sulforaphane, resveratrol, curcumin, and cinnamic aldehyde. These experimental results, coupled with recent clinical experiences with a synthetic triterpenoid, bardoxolone methyl, have brought a light of hope for ameliorating CKD progression by preventing oxidative stress and maintaining cellular redox homeostasis.
Asunto(s)
Antioxidantes/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Oxidación-Reducción , Insuficiencia Renal Crónica/genética , Animales , Humanos , Inflamación/tratamiento farmacológico , Inflamación/genética , Inflamación/patología , Masculino , Factor 2 Relacionado con NF-E2/genética , Estrés Oxidativo/genética , Insuficiencia Renal Crónica/tratamiento farmacológico , Insuficiencia Renal Crónica/metabolismo , Transducción de Señal/genéticaRESUMEN
The time-varying electromagnetic field (EMF) has been widely studied as one of the exogenous stimulation methods for improving bone healing. Our previous study showed that osteogenic differentiation of adipose-derived stem cells was accelerated by a 45-Hz EMF, whereas a 7.5-Hz EMF inhibited osteogenic marker expression. Accordingly, we hypothesized that each negative and positive condition for the osteogenic differentiation could inversely influence osteoclast formation and differentiation. Here, we demonstrated that osteoclast formation, differentiation, and activity can be regulated by altering the frequency of the electromagnetic stimulation, such as 7.5 (negative for osteogenic differentiation) and 45 Hz (positive for osteogenic differentiation). A 45 Hz EMF inhibited osteoclast formation whereas a 7.5-Hz EMF induced differentiation and activity. Osteoclastogenic markers, such as NFATc1, TRAP, CTSK, MMP9, and DC-STAMP were highly expressed under the 7.5-Hz EMF, while they were decreased at 45 Hz. We found that the 7.5-Hz EMF directly regulated osteoclast differentiation through ERK and p38 MAPK activation, whereas the EMF at 45 Hz suppressed RANKL-induced phosphorylation of IκB. Additionally, actin ring formation with tubules and bone resorptive activity were enhanced at 7.5 Hz through increased integrin ß3 expression. However, these were inhibited at 45 Hz. Although many questions remain unanswered, our study indicates that osteoclast formation and differentiation were controllable using physical tools, such as an EMF. It will now be of great interest to study the ill-defined correlation between electromagnetic conditions and osteoclast activities, which eventually could lead to determining the therapeutic characteristics of an EMF that will treat bone-related diseases.
Asunto(s)
Campos Electromagnéticos , Osteoclastos/citología , Actinas/metabolismo , Animales , Biomarcadores/metabolismo , Resorción Ósea/patología , Diferenciación Celular/efectos de los fármacos , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones Endogámicos C57BL , Osteoclastos/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Ligando RANK/farmacología , Reproducibilidad de los ResultadosRESUMEN
Pretreatment using various types of biophysical stimuli could provide appropriate potential to cells during construction of the engineered tissue in vitro. We hypothesized that multiple combinations of these biophysical stimuli could enhance osteogenic differentiation in vitro and bone formation in vivo. Cyclic strain, an electromagnetic field, and ultrasound were selected and combined as effective stimuli for osteogenic differentiation using a developed bioreactor. Here we report the experimental evaluation of the osteogenic effects of various combinations of three different biophysical stimuli in vitro and in vivo using human adipose-derived stem cells (ASCs). Osteogenic differentiation of ASCs was accelerated by multiple-combination biophysical stimulation in vitro. However, both single stimulation and double-combination stimulation were sufficient to accelerate bone regeneration in vivo, while the osteogenic marker expression of those groups was not as high as that of triple-combination stimulation in vitro. We inferred from these data that ASCs appropriately differentiated into the osteogenic lineage by biophysical stimulation could be a better option for accelerating bone formation in vivo than relatively undifferentiated or completely differentiated ASCs. Although many questions remain about the mechanisms of combined effects of various biophysical stimuli, this approach could be a more powerful tool for bone tissue regeneration.
Asunto(s)
Fenómenos Biofísicos , Regeneración Ósea , Animales , Reactores Biológicos , Regeneración Ósea/efectos de los fármacos , Fosfatos de Calcio/farmacología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Supervivencia Celular/efectos de los fármacos , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inmunohistoquímica , Ácido Láctico/farmacología , Laminas/metabolismo , Masculino , Ratones Endogámicos BALB C , Osteogénesis/efectos de los fármacos , Poliésteres/farmacología , Ácido Poliglicólico/farmacología , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Estrés Fisiológico/efectos de los fármacosRESUMEN
Cell-based drug delivery systems (DDSs) have been increasingly exploited because cells can be utilized as a continuous drug delivering system to produce therapeutic molecules over a more extended period compared to the simple drug carriers. Although hydrogels have many advantages for this application, their mechanical properties are generally not desirable to structurally protect implanted cells. Here, we present a three-dimensional (3D) hybrid scaffold with a combination of a 3D framework and a hydrogel to enhance the mechanical properties without chemically altering the transport properties of the hydrogel. Based on the 3D Ormocomp scaffold (framework) fabricated by projection-based microstereolithography with defined parameters, we developed a 3D hybrid scaffold by injection of the mixture of cells and the alginate gel into the internal space of the framework. This hybrid scaffold showed the improved mechanical strength and the framework in the scaffold played the role of an adhesion site for the encapsulated cells during the culture period. Additionally, we confirmed its protection of exogenous human cells from acute immune rejection in a mouse model. Eventually, we demonstrated the feasibility of applying this hybrid scaffold to the treatment of Parkinson's disease as a cell-based DDS. Dopamine released from the 3D hybrid scaffolds encapsulating dopamine-secreting cells for 8weeks suggested its clinical applicability. Further study on its long-term efficacy is necessary for the clinical applicability of this 3D hybrid scaffold for the treatment of Parkinson's disease.