RESUMO
Porous alginate hydrogels possess many advantages as cell carriers. However, current pore generation methods require either complex or harsh fabrication processes, toxic components, or extra purification steps, limiting the feasibility and affecting the cellular survival and function. In this study, a simple and cell-friendly approach to generate highly porous cell-laden alginate hydrogels based on two-phase aqueous emulsions is reported. The pre-gel solutions, which contain two immiscible aqueous phases of alginate and caseinate, are crosslinked by calcium ions. The porous structure of the hydrogel construct is formed by subsequently removing the caseinate phase from the ion-crosslinked alginate hydrogel. Those porous alginate hydrogels possess heterogeneous pores around 100 µm and interconnected paths. Human white adipose progenitors (WAPs) encapsulated in these hydrogels self-organize into spheroids and show enhanced viability, proliferation, and adipogenic differentiation, compared to non-porous constructs. As a proof of concept, this porous alginate hydrogel platform is employed to prepare core-shell spheres for coculture of WAPs and colon cancer cells, with WAP clusters distributed around cancer cell aggregates, to investigate cellular crosstalk. This efficacious approach is believed to provide a robust and versatile platform for engineering porous-structured alginate hydrogels for applications as cell carriers and in disease modeling.
RESUMO
Alginate hydrogel beads are a common platform for generating 3D cell cultures in biomedical research. Simple methods for bead generation using a manual pipettor or syringe are low-throughput and produce beads showing high variability in size and shape. To address these challenges, we designed a 3D printed bead generator that uses an airflow to cleave beads from a stream of hydrogel solution. The performance of the proposed alginate bead generator was evaluated by changing the volume flow rates of alginate (QAlg) and air (QA), the diameter of device nozzle (d) and the concentration of alginate gel solution (C). We identified that the diameter of beads (D = 0.9 -2.8 mm) can be precisely controlled by changing QA and d. Also the bead generation frequency (f) can be tuned by changing QAlg. Finally, we demonstrated that viability and biological function (pericellular matrix deposition) of chondrocytes were not adversely affected by high f using this bead generator. Because 3D printing is becoming a more accessible technique, our unique design will allow greater access to average biomedical research laboratories, STEM education and industries in cost- and time-effective manner.
Assuntos
Alginatos , Técnicas de Cultura de Células , Hidrogéis , Impressão TridimensionalRESUMO
The architecture and morphogenetic properties of tissues are founded in the tissue-specific regulation of cell behaviors. In endochondral bones, the growth plate cartilage promotes bone elongation via regulated chondrocyte maturation within an ordered, three-dimensional cell array. A key event in the process that generates this cell array is the transformation of disordered resting chondrocytes into clonal columns of discoid proliferative cells aligned with the primary growth vector. Previous analysis showed that column-forming chondrocytes display planar cell divisions, and the resulting daughter cells rearrange by â¼90° to align with the lengthening column. However, these previous studies provided limited information about the mechanisms underlying this dynamic process. Here we present new mechanistic insights generated by application of a novel time-lapse confocal microscopy method along with immunofluorescence and electron microscopy. We show that, during cell division, daughter chondrocytes establish a cell-cell adhesion surface enriched in cadherins and ß-catenin. Rearrangement into columns occurs concomitant with expansion of this adhesion surface in a process more similar to cell spreading than to migration. Column formation requires cell-cell adhesion, as reducing cadherin binding via chelation of extracellular calcium inhibits chondrocyte rearrangement. Importantly, physical indicators of cell polarity, such as cell body alignment, are not prerequisites for oriented cell behavior. Our results support a model in which regulation of adhesive surface dynamics and cortical tension by extrinsic signaling modifies the thermodynamic landscape to promote organization of daughter cells in the context of the three-dimensional growth plate tissue.
Assuntos
Cartilagem/citologia , Membrana Celular/metabolismo , Lâmina de Crescimento/citologia , Animais , Animais Recém-Nascidos , Cartilagem/crescimento & desenvolvimento , Cartilagem/metabolismo , Adesão Celular , Divisão Celular , Polaridade Celular , Forma Celular , Células Cultivadas , Condrócitos/citologia , Condrócitos/fisiologia , Feminino , Lâmina de Crescimento/crescimento & desenvolvimento , Lâmina de Crescimento/metabolismo , Masculino , Camundongos , Camundongos TransgênicosRESUMO
For tissues that develop throughout embryogenesis and into postnatal life, the generation of differentiated cells to promote tissue growth is at odds with the requirement to maintain the stem cell/progenitor cell population to preserve future growth potential. In the growth plate cartilage, this balance is achieved in part by establishing a proliferative phase that amplifies the number of progenitor cells prior to terminal differentiation into hypertrophic chondrocytes. Here, we show that endogenous calcium/calmodulin-dependent protein kinase II (CamkII, also known as Camk2) activity is upregulated prior to hypertrophy and that loss of CamkII function substantially blocks the transition from proliferation to hypertrophy. Wnt signaling and Pthrp-induced phosphatase activity negatively regulate CamkII activity. Release of this repression results in activation of multiple effector pathways, including Runx2- and ß-catenin-dependent pathways. We present an integrated model for the regulation of proliferation potential by CamkII activity that has important implications for studies of growth control and adult progenitor/stem cell populations.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Condrócitos/citologia , Condrócitos/enzimologia , Lâmina de Crescimento/embriologia , Lâmina de Crescimento/enzimologia , Animais , Animais Geneticamente Modificados , Sequência de Bases , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Crescimento Celular , Proliferação de Células , Embrião de Galinha , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Primers do DNA/genética , Receptores Frizzled/metabolismo , Lâmina de Crescimento/citologia , Camundongos , Camundongos Mutantes , Modelos Biológicos , Proteína Relacionada ao Hormônio Paratireóideo/metabolismo , Transdução de Sinais , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Adipose tissue is central to the regulation of energy balance. Two functionally different types of fat are present in mammals: white adipose tissue, the primary site of triglyceride storage, and brown adipose tissue, which is specialized in energy expenditure and can counteract obesity. Factors that specify the developmental fate and function of white and brown adipose tissue remain poorly understood. Here we demonstrate that whereas some members of the family of bone morphogenetic proteins (BMPs) support white adipocyte differentiation, BMP7 singularly promotes differentiation of brown preadipocytes even in the absence of the normally required hormonal induction cocktail. BMP7 activates a full program of brown adipogenesis including induction of early regulators of brown fat fate PRDM16 (PR-domain-containing 16; ref. 4) and PGC-1alpha (peroxisome proliferator-activated receptor-gamma (PPARgamma) coactivator-1alpha; ref. 5), increased expression of the brown-fat-defining marker uncoupling protein 1 (UCP1) and adipogenic transcription factors PPARgamma and CCAAT/enhancer-binding proteins (C/EBPs), and induction of mitochondrial biogenesis via p38 mitogen-activated protein (MAP) kinase-(also known as Mapk14) and PGC-1-dependent pathways. Moreover, BMP7 triggers commitment of mesenchymal progenitor cells to a brown adipocyte lineage, and implantation of these cells into nude mice results in development of adipose tissue containing mostly brown adipocytes. Bmp7 knockout embryos show a marked paucity of brown fat and an almost complete absence of UCP1. Adenoviral-mediated expression of BMP7 in mice results in a significant increase in brown, but not white, fat mass and leads to an increase in energy expenditure and a reduction in weight gain. These data reveal an important role of BMP7 in promoting brown adipocyte differentiation and thermogenesis in vivo and in vitro, and provide a potential new therapeutic approach for the treatment of obesity.
Assuntos
Adipogenia , Tecido Adiposo Marrom/crescimento & desenvolvimento , Tecido Adiposo Marrom/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Metabolismo Energético , Fator de Crescimento Transformador beta/metabolismo , Células 3T3-L1 , Tecido Adiposo Branco/crescimento & desenvolvimento , Animais , Proteína Morfogenética Óssea 7 , Linhagem Celular , Metabolismo Energético/genética , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Mitocôndrias/fisiologia , Termogênese , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Spatial and temporal regulation of chondrocyte maturation in the growth plate drives growth of many bones. One essential event to generate the ordered cell array characterizing growth plate cartilage is the formation of chondrocyte columns in the proliferative zone via 90-degree rotation of daughter cells to align with the long axis of the bone. Previous studies have suggested crucial roles for cadherins and integrin ß1 in column formation. The purpose of this study was to determine the relative contributions of cadherin- and integrin-mediated cell adhesion in column formation. Here we present new mechanistic insights generated by application of live time-lapse confocal microscopy of cranial base explant cultures, robust genetic mouse models, and new quantitative methods to analyze cell behavior. We show that conditional deletion of either the cell-cell adhesion molecule Cdh2 or the cell-matrix adhesion molecule Itgb1 disrupts column formation. Compound mutants were used to determine a potential reciprocal regulatory interaction between the two adhesion surfaces and identified that defective chondrocyte rotation in a N-cadherin mutant was restored by a heterozygous loss of integrin ß1. Our results support a model for which integrin ß1, and not N-cadherin, drives chondrocyte rotation and for which N-cadherin is a potential negative regulator of integrin ß1 function.
Assuntos
Caderinas , Cartilagem , Lâmina de Crescimento , Integrina beta1 , Animais , Camundongos , Caderinas/metabolismo , Cartilagem/metabolismo , Adesão Celular/fisiologia , Lâmina de Crescimento/metabolismo , Integrina beta1/metabolismoRESUMO
Proteins that are localized to the cell surface via glycosylphosphatidylinositol (gpi) anchors have been proposed to regulate cell signaling and cell adhesion events involved in tissue patterning. Conditional deletion of Piga, which encodes the catalytic subunit of an essential enzyme in the gpi-biosynthetic pathway, in the lateral plate mesoderm results in normally patterned limbs that display chondrodysplasia. Analysis of mutant and mosaic Piga cartilage revealed two independent cell autonomous defects. First, loss of Piga function interferes with signal reception by chondrocytes as evidenced by delayed maturation. Second, the proliferative chondrocytes, although present, fail to flatten and arrange into columns. We present evidence that the abnormal organization of mutant proliferative chondrocytes results from errors in cell intercalation. Collectively, our data suggest that the distinct morphological features of the proliferative chondrocytes result from a convergent extension-like process that is regulated independently of chondrocyte maturation.
Assuntos
Condrócitos/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Lâmina de Crescimento/embriologia , Lâmina de Crescimento/metabolismo , Proteínas de Membrana/metabolismo , Animais , Padronização Corporal , Diferenciação Celular , Polaridade Celular , Proliferação de Células , Células Cultivadas , Condrócitos/citologia , Extremidades/embriologia , Lâmina de Crescimento/citologia , Proteínas de Membrana/genética , Camundongos , Mutação , Osteocondrodisplasias/embriologia , Osteocondrodisplasias/genética , Osteocondrodisplasias/metabolismoRESUMO
Conditional mutations and transcription-based reporters are important new tools for exploring the dynamic functions of biological pathways in vivo. While studying the role of the Wnt signaling pathway in cartilage, we observed that the ß-catenin-dependent reporter TOPGAL was expressed in chondrocytes in which ß-catenin was conditionally inactivated using a Col2a1::cre driver. Here we show that in these embryos recombination is complete and full-length ß-catenin protein is absent in chondrocytes. Although a null allele in this context, the recombined ß-catenin locus produces a stable transcript that encodes a truncated protein. The truncated protein alone fails to activate TOPFLASH, but strongly potentiates reporter activity in the presence of expressed ß-catenin or Tcf4. Together, these data show that each mouse model exhibits specific undesirable properties, findings that strongly suggest the need for specific standards to ensure proper validation of this new generation of genetic tools.
Assuntos
Via de Sinalização Wnt/fisiologia , Alelos , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Western Blotting , Células Cultivadas , Condrócitos/metabolismo , Imunofluorescência , Genótipo , Hibridização In Situ , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição 4 , Via de Sinalização Wnt/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMO
We highlight the force-sensing function of extracellular matrix and present a complementary mechanotransduction paradigm.
Assuntos
Matriz Extracelular , Mecanotransdução Celular , Matriz Extracelular/fisiologia , Mecanotransdução Celular/fisiologiaRESUMO
In the past decade, cartilage tissue engineering has arisen as a promising therapeutic option for degenerative joint diseases, such as osteoarthritis, in the hope of restoring the structure and physiological functions. Hydrogels are promising biomaterials for developing engineered scaffolds for cartilage regeneration. However, hydrogel-delivered mesenchymal stem cells or chondrocytes could be exposed to elevated levels of reactive oxygen species (ROS) in the inflammatory microenvironment after being implanted into injured joints, which may affect their phenotype and normal functions and thereby hinder the regeneration efficacy. To attenuate ROS induced side effects, a multifunctional hydrogel with an innate anti-oxidative ability was produced in this study. The hydrogel was rapidly formed through a dynamic covalent bond between phenylboronic acid grafted hyaluronic acid (HA-PBA) and poly(vinyl alcohol) and was further stabilized through a secondary crosslinking between the acrylate moiety on HA-PBA and the free thiol group from thiolated gelatin. The hydrogel is cyto-compatible and injectable and can be used as a bioink for 3D bioprinting. The viscoelastic properties of the hydrogels could be modulated through the hydrogel precursor concentration. The presence of dynamic covalent linkages contributed to its shear-thinning property and thus good printability of the hydrogel, resulting in the fabrication of a porous grid construct and a meniscus like scaffold at high structural fidelity. The bioprinted hydrogel promoted cell adhesion and chondrogenic differentiation of encapsulated rabbit adipose derived mesenchymal stem cells. Meanwhile, the hydrogel supported robust deposition of extracellular matrix components, including glycosaminoglycans and type II collagen, by embedded mouse chondrocytesin vitro. Most importantly, the hydrogel could protect encapsulated chondrocytes from ROS induced downregulation of cartilage-specific anabolic genes (ACAN and COL2) and upregulation of a catabolic gene (MMP13) after incubation with H2O2. Furthermore, intra-articular injection of the hydrogel in mice revealed adequate stability and good biocompatibilityin vivo. These results demonstrate that this hydrogel can be used as a novel bioink for the generation of 3D bioprinted constructs with anti-ROS ability to potentially enhance cartilage tissue regeneration in a chronic inflammatory and elevated ROS microenvironment.
Assuntos
Bioimpressão , Engenharia Tecidual , Animais , Bioimpressão/métodos , Cartilagem , Condrócitos , Gelatina/farmacologia , Ácido Hialurônico/química , Hidrogéis/química , Hidrogéis/farmacologia , Peróxido de Hidrogênio , Camundongos , Coelhos , Espécies Reativas de Oxigênio/metabolismo , Engenharia Tecidual/métodos , Alicerces Teciduais/químicaRESUMO
The Ecdysoneless (Ecd) protein is required for cell-autonomous roles in development and oogenesis in Drosophila, but the function of its evolutionarily conserved mammalian orthologs is not clear. To study the cellular function of Ecd in mammalian cells, we generated Ecd(lox/lox) mouse embryonic fibroblast cells from Ecd floxed mouse embryos. Cre-mediated deletion of Ecd in Ecd(lox/lox) mouse embryonic fibroblasts led to a proliferative block due to a delay in G(1)-S cell cycle progression; this defect was reversed by the introduction of human Ecd. Loss of Ecd led to marked down-regulation of E2F target gene expression. Furthermore, Ecd directly bound to Rb at the pocket domain and competed with E2F for binding to hypophosphorylated Rb. Our results demonstrate that mammalian Ecd plays a role in cell cycle progression via the Rb-E2F pathway.
Assuntos
Proteínas de Transporte/fisiologia , Ciclo Celular/fisiologia , Proliferação de Células , Fibroblastos/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F1/metabolismo , Feminino , Fibroblastos/citologia , Fase G1/fisiologia , Expressão Gênica , Humanos , Immunoblotting , Imunoprecipitação , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fosforilação , Ligação Proteica , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Fase S/fisiologia , Fatores de TempoRESUMO
Marfan syndrome (MFS) is a highly variable genetic connective tissue disorder caused by mutations in the calcium binding extracellular matrix glycoprotein fibrillin-1. Patients with the most severe form of MFS (neonatal MFS; nMFS) tend to have mutations that cluster in an internal region of fibrillin-1 called the neonatal region. This region is predominantly composed of eight calcium-binding epidermal growth factor-like (cbEGF) domains, each of which binds one calcium ion and is stabilized by three highly conserved disulfide bonds. Crucially, calcium plays a fundamental role in stabilizing cbEGF domains. Perturbed calcium binding caused by cbEGF domain mutations is thus thought to be a central driver of MFS pathophysiology. Using steered molecular dynamics (SMD) simulations, we demonstrate that cbEGF domain calcium binding decreases under mechanical stress (i.e. cbEGF domains are mechanosensitive). We further demonstrate the disulfide bonds in cbEGF domains uniquely orchestrate protein unfolding by showing that MFS disulfide bond mutations markedly disrupt normal mechanosensitive calcium binding dynamics. These results point to a potential mechanosensitive mechanism for fibrillin-1 in regulating extracellular transforming growth factor beta (TGFB) bioavailability and microfibril integrity. Such mechanosensitive "smart" features may represent novel mechanisms for mechanical hemostasis regulation in extracellular matrix that are pathologically activated in MFS.
Assuntos
Cálcio/metabolismo , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Fibrilina-1/genética , Fibrilina-1/metabolismo , Síndrome de Marfan/genética , Mecanotransdução Celular/genética , Mecanotransdução Celular/fisiologia , Simulação de Dinâmica Molecular , Mutação , Domínios Proteicos , Disponibilidade Biológica , Cálcio/fisiologia , Dissulfetos/metabolismo , Matriz Extracelular/metabolismo , Humanos , Recém-Nascido , Microfibrilas/metabolismo , Ligação Proteica/genética , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth plate cartilage architecture and results in growth modulation of long bones of children. To determine the role of compressive stress in bone growth, we created a microfluidic device actuated by pneumatic pressure, to dynamically (or statically) compress growth plate chondrocytes embedded in alginate hydrogel cylinders. In this article, we describe detailed methods for fabricating and characterizing this device. The advantages of our protocol are: 1) Five different magnitudes of compressive stress can be generated on five technical replicates in a single platform, 2) It is easy to visualize cell morphology via a conventional light microscope, 3) Cells can be rapidly isolated from the device after compression to facilitate downstream assays, and 4) The platform can be applied to study mechanobiology of any cell type that can grow in hydrogels.
Assuntos
Condrócitos/citologia , Dispositivos Lab-On-A-Chip , Microfluídica , Estresse Mecânico , Alginatos , Animais , Desenvolvimento Ósseo , Cartilagem , Técnicas de Cultura de Células , Força Compressiva , Desenho de Equipamento , Lâmina de Crescimento , Humanos , Hidrogéis/metabolismo , PressãoRESUMO
Hyaline cartilage is a specialized type of connective tissue that lines many moveable joints (articular cartilage) and contributes to bone growth (growth plate cartilage). Hyaline cartilage is composed of a single cell type, the chondrocyte, which produces a unique hydrated matrix to resist compressive stress. Although compressive stress has profound effects on transcriptional networks and matrix biosynthesis in chondrocytes, mechanistic relationships between strain, signal transduction, cell metabolism, and matrix production remain superficial. Here, we describe development and validation of a polydimethylsiloxane (PDMS)-based pneumatic microfluidic cell compression device which generates multiple compression conditions in a single platform. The device contained an array of PDMS balloons of different sizes which were actuated by pressurized air, and the balloons compressed chondrocytes cells in alginate hydrogel constructs. Our characterization and testing of the device showed that the developed platform could compress chondrocytes with various magnitudes simultaneously with negligible effect on cell viability. Also, the device is compatible with live cell imaging to probe early effects of compressive stress, and it can be rapidly dismantled to facilitate molecular studies of compressive stress on transcriptional networks. Therefore, the proposed device will enhance the productivity of chondrocyte mechanobiology studies, and it can be applied to study mechanobiology of other cell types.
Assuntos
Condrócitos/citologia , Dispositivos Lab-On-A-Chip , Fenômenos Mecânicos , Fenômenos Biomecânicos , Sobrevivência Celular , Fatores de TempoRESUMO
Defining the final size and geometry of engineered tissues through precise control of the scalar and vector components of tissue growth is a necessary benchmark for regenerative medicine, but it has proved to be a significant challenge for tissue engineers. The growth plate cartilage that promotes elongation of the long bones is a good model system for studying morphogenetic mechanisms because cartilage is composed of a single cell type, the chondrocyte; chondrocytes are readily maintained in culture; and growth trajectory is predominately in a single vector. In this cartilage, growth is generated via a differentiation program that is spatially and temporally regulated by an interconnected network composed of long- and short-range signaling mechanisms that together result in the formation of functionally distinct cellular zones. To facilitate investigation of the mechanisms underlying anisotropic growth, we developed an in vitro model of the growth plate cartilage by using neonatal mouse growth plate chondrocytes encapsulated in alginate hydrogel beads. In bead cultures, encapsulated chondrocytes showed high viability, cartilage matrix deposition, low levels of chondrocyte hypertrophy, and a progressive increase in cell proliferation over 7 days in culture. Exogenous factors were used to test functionality of the parathyroid-related protein-Indian hedgehog (PTHrP-IHH) signaling interaction, which is a crucial feedback loop for regulation of growth. Consistent with in vivo observations, exogenous PTHrP stimulated cell proliferation and inhibited hypertrophy, whereas IHH signaling stimulated chondrocyte hypertrophy. Importantly, the treatment of alginate bead cultures with IHH or thyroxine resulted in formation of a discrete domain of hypertrophic cells that mimics tissue architecture of native growth plate cartilage. Together, these studies are the first demonstration of a tunable in vitro system to model the signaling network interactions that are required to induce zonal architecture in growth plate chondrocytes, which could also potentially be used to grow cartilage cultures of specific geometries to meet personalized patient needs.
Assuntos
Alginatos/química , Cartilagem/citologia , Lâmina de Crescimento/citologia , Alicerces Teciduais/química , Animais , Diferenciação Celular , Células Cultivadas , Citometria de Fluxo , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Camundongos , Transdução de SinaisAssuntos
Extremidades/embriologia , Monoéster Fosfórico Hidrolases/metabolismo , Animais , Embrião de Galinha , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Fator 8 de Crescimento de Fibroblasto , Fatores de Crescimento de Fibroblastos/metabolismo , Camundongos , Modelos Biológicos , Morfogênese , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Peixe-Zebra/embriologiaRESUMO
Common anaesthetic and analgesic agents used during pregnancy in mice have been observed to cause fetal growth restriction. We investigated the impact of therapeutic doses of three anaesthetics (ketamine/xylazine, isoflurane, and tribromoethanol) and two analgesics (buprenorphine and meloxicam) on fetal and placental growth. Pregnant mice were treated with one of these agents at fertilization (E0), attachment (E4), beginning of organogenesis (E6), end of organogenesis (E12), or during the logarithmic growth phase (E15), or they were placed into an untreated control group. At term (E18), fetal and placental growth were evaluated, morphological analyses were performed, and skeletal measurements were conducted. Fetal growth was reduced significantly (P < 0.01) by ketamine/xylazine treatment at E0, E4, E12, or E15, by isoflurane administered at E0 or E6, and by tribromoethanol administered at E6 or E12. Two-day treatment with buprenorphine beginning at E4 or E6, or with meloxicam at E0 also significantly reduced fetal growth (P < 0.01). Neither placental growth nor litter size was significantly affected by any of these agents. The occurrence of microphthalmia was nearly eight-fold higher (P < 0.05) in response to buprenorphine administration at E6 compared with controls. The length of the humerus was reduced at most gestation times in response to each of these agents and was correlated (P < 0.01) with fetal weight for ketamine/xylazine, tribromoethanol, and meloxicam. These data reveal patterns of acceptable and detrimental anaesthetic and analgesic use during fetal development and have refined our capability to provide recommendations for the use of these agents during pregnancy in the mouse.
Assuntos
Analgésicos/metabolismo , Anestésicos/metabolismo , Desenvolvimento Fetal/efeitos dos fármacos , Camundongos/embriologia , Camundongos/metabolismo , Analgésicos/administração & dosagem , Anestésicos/administração & dosagem , Animais , Feminino , Peso Fetal/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL/embriologia , Camundongos Endogâmicos C57BL/metabolismo , Placenta/efeitos dos fármacos , Placentação , Gravidez , Fatores de TempoRESUMO
Despite extensive genetic analysis of the dynamic multi-phase process that transforms a small population of lateral plate mesoderm into the mature limb skeleton, the mechanisms by which signaling pathways regulate cellular behaviors to generate morphogenetic forces are not known. Recently, a series of papers have offered the intriguing possibility that regulated cell polarity fine-tunes the morphogenetic process via orienting cell axes, division planes and cell movements. Wnt5a-mediated non-canonical signaling, which may include planar cell polarity, has emerged as a common thread in the otherwise distinct signaling networks that regulate morphogenesis in each phase of limb development. These findings position the limb as a key model to elucidate how global tissue patterning pathways direct local differences in cell behavior that, in turn, generate growth and form.
Assuntos
Polaridade Celular , Animais , Desenvolvimento Ósseo , Osso e Ossos/embriologia , Osso e Ossos/metabolismo , Extremidades/embriologia , Extremidades/crescimento & desenvolvimento , Humanos , Morfogênese , Transdução de Sinais , Proteínas Wnt/metabolismoRESUMO
Immunofluorescence detection of proteins in growth plate cartilage is often unsuccessful because of innate autofluorescence, fixative-induced fluorescence, and dense cartilage matrix, which can inhibit antibody penetration. To overcome these limitations, the authors have tested various chemical pretreatments, including the autofluorescence quencher sodium borohydride, the antigen retrieval method of boiling sodium citrate, sugar-degrading enzymes (hyaluronidase, heparinase, and chondroitinase), and the proteolytic enzyme protease XXIV. Here the authors show that, in most cases, background fluorescence in cartilage is the primary obstacle to high-quality imaging. Blocking intrinsic fluorescence of the specimen in combination with specific pretreatments allows visualization using antibodies that previously did not generate a robust signal in the growth plate. Each antibody requires a specific combination of chemical pretreatments that must be empirically determined to achieve optimal staining levels.
Assuntos
Lâmina de Crescimento/metabolismo , Animais , Animais Recém-Nascidos , Boroidretos , Condroitinases e Condroitina Liases , Citratos , Imunofluorescência , Heparina Liase , Técnicas de Preparação Histocitológica/métodos , Hialuronoglucosaminidase , Camundongos , Sensibilidade e Especificidade , Citrato de Sódio , SubtilisinaRESUMO
Bone growth is driven by cell proliferation and the subsequent hypertrophy of chondrocytes arranged in columns of discoid cells that resemble stacks of coins. However, the molecular mechanisms that direct column formation and the importance of columnar organization to bone morphogenesis are not known. Here, we show in chick that discoid proliferative chondrocytes orient the division plane to generate daughter cells that are initially displaced laterally and then intercalate into the column. Downregulation of frizzled (Fzd) signaling alters the dimensions of long bones and produces cell-autonomous changes in proliferative chondrocyte organization characterized by arbitrary division planes and altered cell stacking. These defects are phenocopied by disruption of noncanonical effector pathways but not by inhibitors of canonical Fzd signaling. These findings demonstrate that the regulation of cell polarity and cell arrangement by noncanonical Fzd signaling plays important roles in generating the unique morphological characteristics that shape individual cartilage elements.