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There is increasing evidence that surface curvature at a near-cell-scale influences cell behaviour. Epithelial or endothelial cells lining small acinar or tubular body lumens, as those of the alveoli or blood vessels, experience such highly curved surfaces. In contrast, the most commonly used culture substrates for in vitro modelling of these human tissue barriers, ion track-etched membranes, offer only flat surfaces. Here, we propose a more realistic culture environment for alveolar cells based on biomimetically curved track-etched membranes, preserving the mainly spherical geometry of the cells' native microenvironment. The curved membranes were created by a combination of three-dimensional (3D) micro film (thermo)forming and ion track technology. We could successfully demonstrate the formation, the growth and a first characterization of confluent layers of lung epithelial cell lines and primary alveolar epithelial cells on membranes shaped into an array of hemispherical microwells. Besides their application in submerged culture, we could also demonstrate the compatibility of the bioinspired membranes for air-exposed culture. We observed a distinct cellular response to membrane curvature. Cells (or cell layers) on the curved membranes reveal significant differences compared to cells on flat membranes concerning membrane epithelialization, areal cell density of the formed epithelial layers, their cross-sectional morphology, and proliferation and apoptosis rates, and the same tight barrier function as on the flat membranes. The presented 3D membrane technology might pave the way for more predictive barrier in vitro models in future.
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Células Endoteliales , Alveolos Pulmonares , Estudios Transversales , Células Epiteliales , Humanos , MembranasRESUMEN
Microfluidic screening platforms offer new possibilities for performing in vitro cell-based assays with higher throughput and in a setting that has the potential to closely mimic the physiological microenvironment. Integrating functional biomaterials into such platforms is a promising approach to obtain a deeper insight into the interactions occurring at the cell-material interface. The success of such an approach is, however, largely dependent on the ability to miniaturize the biomaterials as well as on the choice of the assay used to study the cell-material interactions. In this work, we developed a microfluidic device, the main component of which is made of a widely used biocompatible polymer, polylactic acid (PLA). This device enabled cell culture under different fluidic regimes, including perfusion and diffusion. Through a combination of photolithography, two-photon polymerization and hot embossing, it was possible to microstructure the surface of the cell culture chamber of the device with highly defined geometrical features. Furthermore, using pyramids with different heights and wall microtopographies as an example, adhesion, morphology and distribution of human MG63 osteosarcoma cells were studied. The results showed that both the height of the topographical features and the microstructural properties of their walls affected cell spreading and distribution. This proof-of-concept study shows that the platform developed here is a useful tool for studying interactions between cells and clinically relevant biomaterials under controlled fluidic regimes.
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Materiales Biocompatibles , Adhesión Celular , Técnicas de Cultivo de Célula/instrumentación , Técnicas Analíticas Microfluídicas/instrumentación , Materiales Biocompatibles/metabolismo , Materiales Biocompatibles/farmacología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Línea Celular Tumoral , Diseño de Equipo , Humanos , Técnicas Analíticas Microfluídicas/métodosRESUMEN
Micro- and nano-topographies of scaffold surfaces play a pivotal role in tissue engineering applications, influencing cell behavior such as adhesion, orientation, alignment, morphology and proliferation. In this study, a novel microfabrication method based on the combination of soft-lithography and electrospinning for the production of micro-patterned electrospun scaffolds was proposed. Subsequently, a 3D screening device for electrospun meshes with different micro-topographies was designed, fabricated and biologically validated. Results indicated that the use of defined patterns could induce specific morphological variations in human mesenchymal stem cell cytoskeletal organization, which could be related to differential activity of signaling pathways. STATEMENT OF SIGNIFICANCE: We introduce a novel and time saving method to fabricate 3D micropatterns with controlled micro-architectures on electrospun meshes using a custom made collector and a PDMS mold with the desired topography. A possible application of this fabrication technique is represented by a 3D screening system for patterned electrospun meshes that allows the screening of different scaffold/electrospun parameters on cell activity. In addition, what we have developed in this study could be modularly applied to existing platforms. Considering the different patterned geometries, the cell morphological data indicated a change in the cytoskeletal organization with a close correspondence to the patterns, as shown by phenoplot and boxplot analysis, and might hint at the differential activity of cell signaling. The 3D screening system proposed in this study could be used to evaluate topographies favoring cell alignment, proliferation and functional performance, and has the potential to be upscaled for high-throughput.
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Técnicas de Cultivo de Célula/instrumentación , Diferenciación Celular , Citoesqueleto/metabolismo , Células Madre Mesenquimatosas , Andamios del Tejido/química , Adulto , Técnicas de Cultivo de Célula/métodos , Células Cultivadas , Humanos , Masculino , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , PorosidadRESUMEN
The amount of bone generated using current tissue engineering approaches is insufficient for many clinical applications. Previous in vitro studies suggest that culturing cells as 3D aggregates can enhance their osteogenic potential, but the effect on bone formation in vivo is unknown. Here, we use agarose wells to generate uniformly sized mesenchymal stromal cell (MSC) aggregates. When combined with calcium phosphate ceramic particles and a gel prepared from human platelet-rich plasma, we generated a tissue engineered construct which significantly improved in vivo bone forming capacity as compared to the conventional system of using single cells seeded directly on the ceramic surface. Histology demonstrated the reproducibility of this system, which was tested using cells from four different donors. In vitro studies established that MSC aggregation results in an up-regulation of osteogenic transcripts. And finally, the in vivo performance of the constructs was significantly diminished when unaggregated cells were used, indicating that cell aggregation is a potent trigger of in vivo bone formation by MSCs. Cell aggregation could thus be used to improve bone tissue engineering strategies.
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Células Madre Mesenquimatosas/citología , Osteogénesis , Anciano , Animales , Biomarcadores/metabolismo , Agregación Celular , Células Cultivadas , Femenino , Humanos , Implantes Experimentales , Masculino , Ratones SCID , Persona de Mediana Edad , Plasma Rico en Plaquetas/química , Implantación de Prótesis , Factores de Tiempo , Andamios del Tejido/químicaRESUMEN
A triphasic scaffold (TPS) for the regeneration of the bone-ligament interface was fabricated combining a 3D fiber deposited polycaprolactone structure and a polylactic co-glycolic acid electrospun. The scaffold presented a gradient of physical and mechanical properties which elicited different biological responses from human mesenchymal stem cells. Biological test were performed on the whole TPS and on scaffolds comprised of each single part of the TPS, considered as the controls. The TPS showed an increase of the metabolic activity with culturing time that seemed to be an average of the controls at each time point. The importance of differentiation media for bone and ligament regeneration was further investigated. Metabolic activity analysis on the different areas of the TPS showed a similar trend after 7 days in both differentiation media. Total alkaline phosphatase (ALP) activity analysis showed a statistically higher activity of the TPS in mineralization medium compared to the controls. A different glycosaminoglycans amount between the TPS and its controls was detected, displaying a similar trend with respect to ALP activity. Results clearly indicated that the integration of electrospinning and additive manufacturing represents a promising approach for the fabrication of scaffolds for the regeneration of tissue interfaces, such as the bone-to-ligament one, because it allows mimicking the structural environment combining different biomaterials at different scales.
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Regeneración Ósea/fisiología , Huesos/fisiología , Regeneración Tisular Dirigida/instrumentación , Ligamentos/fisiología , Células Madre Mesenquimatosas/citología , Andamios del Tejido , Huesos/citología , Células Cultivadas , Diseño de Equipo , Análisis de Falla de Equipo , Humanos , Ácido Láctico/química , Ligamentos/citología , Masculino , Ensayo de Materiales , Células Madre Mesenquimatosas/fisiología , Transición de Fase , Poliésteres , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Impresión Tridimensional , Estrés Mecánico , Resistencia a la Tracción , Ingeniería de Tejidos/instrumentación , Adulto JovenRESUMEN
3D organoids using stem cells to study development and disease are now widespread. These models are powerful to mimic in vivo situations but are currently associated with high variability and low throughput. For biomedical research, platforms are thus necessary to increase reproducibility and allow high-throughput screens (HTS). Here, we introduce a microwell platform, integrated in standard culture plates, for functional HTS. Using micro-thermoforming, we form round-bottom microwell arrays from optically clear cyclic olefin polymer films, and assemble them with bottom-less 96-well plates. We show that embryonic stem cells aggregate faster and more reproducibly (centricity, circularity) as compared to a state-of-the-art microwell array. We then run a screen of a chemical library to direct differentiation into primitive endoderm (PrE) and, using on-chip high content imaging (HCI), we identify molecules, including regulators of the cAMP pathway, regulating tissue size, morphology and PrE gene activity. We propose that this platform will benefit to the systematic study of organogenesis in vitro.
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Evaluación Preclínica de Medicamentos/métodos , Cuerpos Embrioides/citología , Cuerpos Embrioides/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento/métodos , Microtecnología/métodos , Temperatura , Animales , Agregación Celular/efectos de los fármacos , Línea Celular , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Activación Enzimática/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Cinética , Ratones , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Reproducibilidad de los ResultadosRESUMEN
In regenerative medicine studies, cell seeding efficiency is not only optimized by changing the chemistry of the biomaterials used as cell culture substrates, but also by altering scaffold geometry, culture and seeding conditions. In this study, the importance of seeding parameters, such as initial cell number, seeding volume, seeding concentration and seeding condition is shown. Human mesenchymal stem cells (hMSCs) were seeded into cylindrically shaped 4 × 3 mm polymeric scaffolds, fabricated by fused deposition modelling. The initial cell number ranged from 5 × 10(4) to 8 × 10(5) cells, in volumes varying from 50 µl to 400 µl. To study the effect of seeding conditions, a dynamic system, by means of an agitation plate, was compared with static culture for both scaffolds placed in a well plate or in a confined agarose moulded well. Cell seeding efficiency decreased when seeded with high initial cell numbers, whereas 2 × 10(5) cells seemed to be an optimal initial cell number in the scaffolds used here. The influence of seeding volume was shown to be dependent on the initial cell number used. By optimizing seeding parameters for each specific culture system, a more efficient use of donor cells can be achieved. Copyright © 2013 John Wiley & Sons, Ltd.
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Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Andamios del Tejido/química , Anciano , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Although bone marrow stromal cells (BMSCs) appear promising for cartilage repair, current clinical results are suboptimal and the success of BMSC-based therapies relies on a number of methodological improvements, among which is better understanding and control of their differentiation pathways. We investigated here the role of the cellular environment (paracrine vs juxtacrine signalling) in the chondrogenic differentiation of BMSCs. Bovine BMSCs were encapsulated in alginate beads, as dispersed cells or as small micro-aggregates, to create different paracrine and juxtacrine signalling conditions. BMSCs were then cultured for 21 days with TGFß3 added for 0, 7 or 21 days. Chondrogenic differentiation was assessed at the gene (type II and X collagens, aggrecan, TGFß, sp7) and matrix (biochemical assays and histology) levels. The results showed that micro-aggregates had no beneficial effects over dispersed cells: matrix production was similar, whereas chondrogenic marker gene expression was lower for the micro-aggregates, under all TGFß conditions tested. This weakened chondrogenic differentiation might be explained by a different cytoskeleton organization at day 0 in the micro-aggregates. Copyright © 2014 John Wiley & Sons, Ltd.
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Células de la Médula Ósea/metabolismo , Microambiente Celular , Condrogénesis , Animales , Antígenos de Diferenciación/biosíntesis , Células de la Médula Ósea/citología , Bovinos , Agregación Celular , Diferenciación Celular , Células Cultivadas , Regulación de la Expresión Génica , Células del Estroma/citología , Células del Estroma/metabolismoRESUMEN
An increasing demand exists for biomaterials that are able to actively participate in the process of repair and regeneration of damaged or diseased organs and tissues. Patterning of surfaces of biomaterials with distinct chemical or physical cues is an attractive way to obtain spatial control over their interactions with the biological system. In the current study, micromoulding in capillaries method was used to pattern silicon substrates with bioinert yttria-stabilised zirconia or with bioactive calcium phosphate ceramics, both widely used biomaterials in orthopaedics and dentistry. Micrometer-scale patterns consisted of parallel lines with varying width and spacing. Both ceramics were successfully deposited on the substrate in a pattern defined by the mould. While the yttria-stabilised zirconia pattern was highly homogenous and smooth (Rq = 5.5 nm), the calcium phosphate pattern, consisting of dicalcium phosphate anhydrous before, and of ß-tricalcium phosphate after annealing, exhibited a less homogenous morphology and higher roughness (Rq = 893 nm). Both materials allowed attachment and proliferation of the MG-63 osteosarcoma cell line, independent of the pattern used. While a preferential orientation of cells in the direction of the pattern lines was observed for all patterns, this effect was more pronounced on the lines with a width of up to 20 µm on both yttria-stabilised zirconia and calcium phosphate ceramics, as compared to wider patterns. Furthermore, the cells retained an elongated morphology for a longer period of time on narrow patterns. Micromoulding in capillaries appeared to be a suitable method to pattern both types of ceramics, however further optimisation is needed to improve homogeneity and obtain better control over the chemical phase and crystalline structure of calcium phosphate patterns.
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Many studies have shown the influence of soluble factors and material properties on the differentiation capacity of mesenchymal stromal cells (MSCs) cultured as monolayers. These types of two-dimensional (2D) studies can be used as simplified models to understand cell processes related to stem cell sensing and mechano-transduction in a three-dimensional (3D) context. For several other mechanisms such as cell-cell signaling, cell proliferation and cell morphology, it is well-known that cells behave differently on a planar surface compared to cells in 3D environments. In classical tissue engineering approaches, a combination of cells, 3D scaffolds and soluble factors are considered as the key ingredients for the generation of mechanically stable 3D tissue constructs. However, when MSCs are used for tissue engineering strategies, little is known about the maintenance of their differentiation potential in 3D scaffolds after the removal of differentiation soluble factors. In this study, the differentiation potential of human MSCs (hMSCs) into the chondrogenic and osteogenic lineages on two distinct 3D scaffolds, additive manufactured electrospun scaffolds, was assessed and compared to conventional 2D culture. Human MSCs cultured in the presence of soluble factors in 3D showed to differentiate to the same extent as hMSCs cultured as 2D monolayers or as scaffold-free pellets, indicating that the two scaffolds do not play a consistent role in the differentiation process. In the case of phenotypic changes, the achieved differentiated phenotype was not maintained after the removal of soluble factors, suggesting that the plasticity of hMSCs is retained in 3D cell culture systems. This finding can have implications for future tissue engineering approaches in which the validation of hMSC differentiation on 3D scaffolds will not be sufficient to ensure the maintenance of the functionality of the cells in the absence of appropriate differentiation signals.
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Células Madre Mesenquimatosas/citología , Fosfatasa Alcalina/metabolismo , Técnicas de Cultivo de Célula , Desdiferenciación Celular , Diferenciación Celular , Condrogénesis , Matriz Extracelular/metabolismo , Humanos , Mecanotransducción Celular , Células Madre Mesenquimatosas/fisiología , Microscopía Electrónica de Rastreo , Osteogénesis , Fenotipo , Polímeros/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ingeniería de Tejidos , Andamios del Tejido/químicaRESUMEN
In clinical islet transplantation, allogeneic islets of Langerhans are transplanted into the portal vein of patients with type 1 diabetes, enabling the restoration of normoglycemia. After intra-hepatic transplantation several factors are involved in the decay in islet mass and function mainly caused by an immediate blood mediated inflammatory response, lack of vascularization, and allo- and autoimmunity. Bioengineered scaffolds can potentially provide an alternative extra-hepatic transplantation site for islets by improving nutrient diffusion and blood supply to the scaffold. This would ultimately result in enhanced islet viability and functionality compared to conventional intra portal transplantation. In this regard, the biomaterial choice, the three-dimensional (3D) shape and scaffold porosity are key parameters for an optimal construct design and, ultimately, transplantation outcome. We used 3D bioplotting for the fabrication of a 3D alginate-based porous scaffold as an extra-hepatic islet delivery system. In 3D-plotted alginate scaffolds the surface to volume ratio, and thus oxygen and nutrient transport, is increased compared to conventional bulk hydrogels. Several alginate mixtures have been tested for INS1E ß-cell viability. Alginate/gelatin mixtures resulted in high plotting performances, and satisfactory handling properties. INS1E ß-cells, human and mouse islets were successfully embedded in 3D-plotted constructs without affecting their morphology and viability, while preventing their aggregation. 3D plotted scaffolds could help in creating an alternative extra-hepatic transplantation site. In contrast to microcapsule embedding, in 3D plotted scaffold islets are confined in one location and blood vessels can grow into the pores of the construct, in closer contact to the embedded tissue. Once revascularization has occurred, the functionality is fully restored upon degradation of the scaffold.
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Hidrogel de Polietilenoglicol-Dimetacrilato/química , Trasplante de Islotes Pancreáticos , Andamios del Tejido , Alginatos/química , Animales , Cápsulas/química , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Gelatina/química , Glucosa/metabolismo , Glucosa/farmacología , Ácido Glucurónico/química , Ácidos Hexurónicos/química , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato/toxicidad , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/citología , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Ratones , Ratones Transgénicos , Microscopía Fluorescente , Porosidad , RatasRESUMEN
The main objective of this study was to evaluate the effectiveness of a mesenchymal stem cell (MSC)-seeded polyethylene-oxide-terephthalate/polybutylene-terephthalate (PEOT/PBT) scaffold for cartilage tissue repair in an osteochondral defect using a rabbit model. Material characterisation using scanning electron microscopy indicated that the scaffold had a 3D architecture characteristic of the additive manufacturing fabrication method, with a strut diameter of 296 ± 52 µm and a pore size of 512 ± 22 µm × 476 ± 25 µm × 180 ± 30 µm. In vitro optimisation revealed that the scaffold did not generate an adverse cell response, optimal cell loading conditions were achieved using 50 µg/ml fibronectin and a cell seeding density of 25 × 10(6) cells/ml and glycosaminoglycan (GAG) accumulation after 28 days culture in the presence of TGFß3 indicated positive chondrogenesis. Cell-seeded scaffolds were implanted in osteochondral defects for 12 weeks, with cell-free scaffolds and empty defects employed as controls. On examination of toluidine blue staining for chondrogenesis and GAG accumulation, both the empty defect and the cell-seeded scaffold appeared to promote repair. However, the empty defect and the cell-free scaffold stained positive for collagen type I or fibrocartilage, while the cell-seeded scaffold stained positive for collagen type II indicative of hyaline cartilage and was statistically better than the cell-free scaffold in the blinded histological evaluation. In summary, MSCs in combination with a 3D PEOT/PBT scaffold created a reparative environment for cartilage repair.
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Cartílago/lesiones , Cartílago/metabolismo , Condrogénesis , Células Madre Mesenquimatosas/metabolismo , Poliésteres , Polietilenglicoles , Andamios del Tejido , Animales , Cartílago/inervación , Células Madre Mesenquimatosas/patología , ConejosRESUMEN
In the field of tissue engineering, mechano-regulation theories have been applied to help predict tissue development in tissue engineering scaffolds in the past. For this, finite element models (FEMs) were used to predict the distribution of strains within a scaffold. However, the strains reported in these studies are volumetric strains of the material or strains developed in the extracellular matrix occupying the pore space. The initial phase of cell attachment and growth on the biomaterial surface has thus far been neglected. In this study, we present a model that determines the magnitude of biomechanical signals on the biomaterial surface, enabling us to predict cell differentiation stimulus values at this initial stage. Results showed that magnitudes of the 2D strain--termed surface strain--were lower when compared to the 3D volumetric strain or the conventional octahedral shear strain as used in current mechano-regulation theories. Results of both µCT and CAD derived FEMs from the same scaffold were compared. Strain and fluid shear stress distributions, and subsequently the cell differentiation stimulus, were highly dependent on the pore shape. CAD models were not able to capture the distributions seen in the µCT FEM. The calculated mechanical stimuli could be combined with current mechanobiological models resulting in a tool to predict cell differentiation in the initial phase of tissue engineering. Although experimental data is still necessary to properly link mechanical signals to cell behavior in this specific setting, this model is an important step towards optimizing scaffold architecture and/or stimulation regimes.
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Fenómenos Químicos , Fenómenos Mecánicos , Andamios del Tejido , Adhesión Celular , Diferenciación Celular , Proliferación Celular , Modelos Biológicos , ReologíaRESUMEN
A material-based bottom-up approach is proposed towards an assembly of cells and engineered micro-objects at the macroscale. We show how shape, size and wettability of engineered micro-objects play an important role in the behavior of cells on these objects. This approach can, among other applications, be used as a tool to engineer complex 3D tissues of clinically relevant size.
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Microtecnología/métodos , Ingeniería de Tejidos/métodos , Andamios del Tejido , Animales , Agregación Celular , Línea Celular , Supervivencia Celular , Humanos , Células Madre Mesenquimatosas/citología , RatonesRESUMEN
The rapidly developing field of tissue engineering produces living substitutes that restore, maintain or improve the function of tissues or organs. In contrast to standard therapies, the engineered products become integrated within the patient, affording a potentially permanent and specific cure of the disease, injury or impairment. Despite the great progress in the field, development of clinically relevantly sized tissues with complex architecture remains a great challenge. This is mostly due to limitations of nutrient and oxygen delivery to the cells and limited availability of scaffolds that can mimic the complex tissue architecture. This study presents the development of a multilayer tissue construct by rolling pre-seeded electrospun sheets [(prepared from poly (l-lactic acid) (PLLA) seeded with C2C12 pre-myoblast cells)] around a porous multibore hollow fibre (HF) membrane and its testing using a bioreactor. Important elements of this study are: 1) the medium permeating through the porous walls of multibore HF acts as an additional source of nutrients and oxygen to the cells, which exerts low shear stress (controllable by trans membrane pressure); 2) application of dynamic perfusion through the HF lumen and around the 3D construct to achieve high cell proliferation and homogenous cell distribution across the layers, and 3) cell migration occurs within the multilayer construct (shown using pre-labeled C2C12 cells), illustrating the potential of using this concept for developing thick and more complex tissues.
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Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Animales , Reactores Biológicos , Técnicas de Cultivo de Célula , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Ácido Láctico/farmacología , Membranas Artificiales , Ratones , Poliésteres , Polímeros/farmacología , Coloración y Etiquetado , AguaRESUMEN
OBJECTIVE: Previous studies indicated a difference in crosstalk between canonical WNT pathway and nuclear factor-κB (NFκB) signaling in human and animal chondrocytes. To assess whether the differences found were dependent on cell types used, we tested the effect of WNT modulation on NFκB signaling in human primary articular chondrocytes in comparison with the immortalized human costal chondrocyte cell line C20/A4. DESIGN: We used gene expression analysis to study the effect of WNT modulation on IL1ß-induced matrix metalloproteinase (MMP) expression as well as on WNT and NFκB target gene expression. In addition, we tested the involvement of RelA and TCF4 on activation of the WNT and NFκB pathway by TCF/LEF and NFκB reporter experiments, respectively. RESULTS: We found an inhibitory effect of both induction and inhibition of WNT signaling on IL1ß-induced MMP mRNA expression in primary chondrocytes, whereas WNT modulation did not affect MMP expression in C20/A4 cells. Furthermore, TCF/LEF and NFκB reporter activation and WNT and NFκB target gene expression were regulated differentially by TCF4 and RelA in a cell type-dependent manner. Additionally, we found significantly higher mRNA and protein expression of TCF4 and RelA in C20/A4 cells in comparison with primary chondrocytes. CONCLUSIONS: We conclude that WNT modulation of NFκB is, at least in part, cell type dependent and that the observed differences are likely because of impaired sensitivity of the NFκB pathway in C20/A4 cells to modulations in WNT signaling. This might be caused by higher basal levels of TCF4 and RelA in C20/A4 cells compared to primary chondrocytes.
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An important tenet in designing scaffolds for regenerative medicine consists in mimicking the dynamic mechanical properties of the tissues to be replaced to facilitate patient rehabilitation and restore daily activities. In addition, it is important to determine the contribution of the forming tissue to the mechanical properties of the scaffold during culture to optimize the pore network architecture. Depending on the biomaterial and scaffold fabrication technology, matching the scaffolds mechanical properties to articular cartilage can compromise the porosity, which hampers tissue formation. Here, we show that scaffolds with controlled and interconnected pore volume and matching articular cartilage dynamic mechanical properties, are indeed effective to support tissue regeneration by co-cultured primary and expanded chondrocyte (1:4). Cells were cultured on scaffolds in vitro for 4 weeks. A higher amount of cartilage specific matrix (ECM) was formed on mechanically matching (M) scaffolds after 28 days. A less protein adhesive composition supported chondrocytes rounded morphology, which contributed to cartilaginous differentiation. Interestingly, the dynamic stiffness of matching constructs remained approximately at the same value after culture, suggesting a comparable kinetics of tissue formation and scaffold degradation. Cartilage regeneration in matching scaffolds was confirmed subcutaneously in vivo. These results imply that mechanically matching scaffolds with appropriate physico-chemical properties support chondrocyte differentiation.
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Cartílago/fisiología , Fenómenos Químicos , Regeneración/fisiología , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Animales , Bovinos , ADN/metabolismo , Matriz Extracelular/metabolismo , Glicosaminoglicanos/metabolismo , Ensayo de Materiales , Ratones , Ratones Desnudos , Microscopía Electrónica de Rastreo , Tejido Subcutáneo/metabolismoRESUMEN
OBJECTIVE: When primary chondrocytes are cultured in monolayer, they undergo dedifferentiation during which they lose their phenotype and their capacity to form cartilage. Dedifferentiation is an obstacle for cell therapy for cartilage degeneration. In this study, we aimed to systemically evaluate the changes in gene expression during dedifferentiation of human articular chondrocytes to identify underlying mechanisms. METHODS: RNA was isolated from monolayer-cultured primary human articular chondrocytes at serial passages. Gene expression was analyzed by microarray. Based on the microarray analysis, relevant genes and pathways were identified. Their functions in chondrocyte dedifferentiation were further investigated. RESULTS: In vitro expanded human chondrocytes showed progressive changes in gene expression. Strikingly, an overall decrease in total gene expression was detected, which was both gradual and cumulative. DNA methylation was in part responsible for the expression downregulation of a number of genes. Genes involved in many pathways such as the extracellular-signal-regulated kinase (ERK) and Bone morphogenetic protein (BMP) pathways exhibited significant changes in expression. Inhibition of ERK pathway did not show dramatic effects in counteracting dedifferentiation process. BMP-2 was able to decelerate the dedifferentiation and reinforce the maintenance of chondrocyte phenotype in monolayer culture. CONCLUSION: Our study not only improves our knowledge of the intricate signaling network regulating maintenance of chondrocyte phenotype, but also contributes to improved chondrocyte expansion and chondrogenic performance for cell therapy.
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Cartílago Articular/citología , Condrocitos/citología , Anciano , Proteínas Morfogenéticas Óseas/fisiología , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Células Cultivadas , Metilación de ADN , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/fisiología , Humanos , Sistema de Señalización de MAP Quinasas/fisiología , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos/métodosRESUMEN
Upon contact with a biomaterial, cells and surrounding tissues respond in a manner dictated by the physicochemical and mechanical properties of the material. Traditionally, cellular responses are monitored using invasive analytical methods that report the expression of genes or proteins. These analytical methods involve assessing commonly used markers for a predefined readout, masking the actual situation induced in the cells. Hence, a broader expression profile of the cellular response should be envisioned, which technically limits up scaling to higher throughput systems. However, it is increasingly recognized that morphometric readouts, obtained non-invasively, are related to gene expression patterns. Here, we introduced distinct surface roughness to three PLA surfaces, by exposure to oxygen plasma of different duration times. The response of mesenchymal stromal cells was compared to smooth untreated PLA surfaces without the addition of differentiation agents. Morphological and genome wide expression profiles revealed underlying cellular changes which was hidden for the commonly used gene markers for osteo-, chondro- and adipogenesis. Using 3 morphometric parameters, obtained by high content imaging, we were able to build a classifier and discriminate between oxygen plasma-induced modified sheets and non-modified PLA sheets where evaluating classical candidates missed this effect. This approach shows the feasibility to use noninvasive morphometric data in high-throughput systems to screen biomaterial surfaces indicating the underlying genetic biomaterial-induced changes.
Asunto(s)
Materiales Biocompatibles/química , Perfilación de la Expresión Génica/métodos , Ácido Láctico/química , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/fisiología , Imagen Molecular/métodos , Polímeros/química , Proteoma/metabolismo , Adhesión Celular/fisiología , Células Cultivadas , Estudios de Factibilidad , Humanos , Ensayo de Materiales , Poliésteres , Propiedades de SuperficieRESUMEN
Plasma treatment is a method to modify the physicochemical properties of biomaterials, which consequently may affect interactions with cells. Based on the rationale that physical cues on the surface of culture substrates and implants, such as surface roughness, have proven to alter cell behaviour, we used electrospinning to fabricate fibrous three-dimensional scaffolds made of a poly (ethylene oxide terephthalate)/poly (butylene terephthalate) copolymer to mimic the physical microenvironment of extracellular matrix and applied radio-frequency oxygen plasma treatment to create nanoscale roughness. Scanning electron microscopy (SEM) analysis revealed a fibre diameter of 5.49 ± 0.96 µm for as-spun meshes. Atomic force microscopy (AFM) measurements determined an exponential increase of surface roughness with plasma treatment time. An increase in hydrophilicity after plasma treatment was observed, which was associated with higher oxygen content in plasma treated scaffolds compared to untreated ones. A more pronounced adsorption of bovine serum albumin occurred on scaffolds treated with plasma for 15 and 30 min compared to untreated fibres. Clinically relevant human mesenchymal stromal cells (hMSCs) were cultured on untreated, 15 and 30 min treated scaffolds. SEM analysis confirmed cell attachment and a pronounced spindle-like morphology on all scaffolds. No significant differences were observed between different scaffolds regarding the amount of DNA, metabolic activity and alkaline phosphatase (ALP) activity after 7 days of culture. The amount of ALP positive cells increased between 7 and 21 days of culture on both untreated and 30 min treated meshes. In addition, ALP staining of cells on plasma treated meshes appeared more pronounced than on untreated meshes after 21 days of culture. Quantitative polymerase chain reaction showed significant upregulation of bone sialoprotein and osteonectin expression on oxygen plasma treated fibres compared to untreated fibres in basic culture medium after 7 days of culture, while no differences were observed in the expression of other osteogenic markers. At 21 days, no osteocalcin protein could be detected by ELISA at any of the substrates. In conclusion, this study shows that oxygen plasma treatment can successfully be applied to modify the nanoscale surface properties of polymeric electrospun fibre meshes, which in turn may positively affect osteogenic differentiation of hMSCs.