RESUMEN
Elastomers have been extensively exploited to study cell physiology in fields such as mechanobiology, however, their intrinsic high hydrophobicity renders their surfaces incompatible for prolonged cell adhesion and proliferation. Electrospun fiber networks on the other side provide a promising environment for enhanced cell adhesion and growth due to their architecture closely mimicking the structure of the extracellular matrix present within tissues of the human body. Here, we explored the stable integration of electrospun fibers onto the surfaces of elastomeric materials to promote cytocompatibility of these composites. Elastomers based on room temperature vulcanizing silicone (RTV), polydimethylsiloxane (PDMS) as well as functionalized PDMS-based materials were chosen as wafer substrates for attachment of poly(vinylidene fluoride-co-hexafluoropropylene) (PVDFhfp) fibers, a well-known antithrombotic polymer. Electrospinning the fibers onto uncured interfaces acted as bonding agents on the wafers, enabling penetration and formation of a stable bond between the fibers surfaces and the elastomers after curing the interface. Dimensional analysis revealed a relationship between peeling force, intrusion depth and the elastic modulus of the wafers. A design parameter Πα was extrapolated to be used as a predictive tool of the peeling force when intrusion depth of PVDFhfp fibers and elastic modulus of the wafers are known. Cultivating fibroblasts on these hybrid membranes showed cell attachment and growth over 7â¯days regardless of the composition of the substrate, confirming high cytocompatibility for all composite materials. The presented approach opens avenues to establish nanofiber morphologies as a novel, stable surface texturing tool for tissue engineering, cell biology, medical devices and textiles.
Asunto(s)
Biomimética/métodos , Nanofibras/química , Adhesión Celular/fisiología , Células Cultivadas , Dimetilpolisiloxanos/química , Humanos , Microscopía Electrónica de Rastreo , Nanoestructuras/química , Nanoestructuras/ultraestructura , Ingeniería de Tejidos/métodos , Andamios del Tejido/químicaRESUMEN
OBJECTIVE: Lumican (LUM) is a major extracellular matrix glycoprotein in adult articular cartilage and its expression is known to be upregulated upon cartilage degeneration. LUM is associated with the pathogen-associated molecular pattern (PAMP) activation of the TLR4 signalling cascade, with TLR4 being highly associated with inflammation in rheumatic diseases. However, the main role of the LUM structural molecule in osteoarthritis (OA) remains elusive. The aim of this study was, therefore, to understand the role of LUM during TLR4-mediated activation in OA. METHODS: After measuring LUM levels in synovial fluid (SF) of OA patients and lipopolysaccharide (LPS)-induced TLR4 activation, the role of LUM in the expression of pro-inflammatory molecules and cartilage degradation was assessed in vitro and ex vivo in a cartilage explant model. Primary macrophage activation and polarization were studied upon LUM co-stimulation with LPS. RESULTS: We demonstrate that LUM is not only significantly upregulated in SF from OA patients compared to healthy controls, but also that LUM increases lipopolysaccharide (LPS)-induced TLR4 activation. Furthermore, we show that a pathophysiological level of LUM augments the LPS-induced TLR4 activation and expression of downstream pro-inflammatory molecules, resulting in extensive cartilage degradation. LUM co-stimulation with LPS also provided a pro-inflammatory stimulus, upregulating primary macrophage activation and polarization towards the M1-like phenotype. CONCLUSIONS: These findings strongly support the role of LUM as a mediator of PAMP-induced TLR4 activation of inflammation, cartilage degradation, and macrophage polarization in the OA joint and potentially other rheumatic diseases.
Asunto(s)
Cartílago/metabolismo , Lumican/fisiología , Macrófagos/fisiología , Osteoartritis/metabolismo , Receptor Toll-Like 4/fisiología , Adulto , Anciano , Anciano de 80 o más Años , Diferenciación Celular , Condrocitos/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Lumican/análisis , Lumican/metabolismo , Activación de Macrófagos , Masculino , Persona de Mediana Edad , FN-kappa B/metabolismo , Líquido Sinovial/química , Líquido Sinovial/metabolismo , Receptor Toll-Like 4/metabolismo , Regulación hacia ArribaRESUMEN
The fabrication of functional 3D tissues is a major goal in tissue engineering. While electrospinning is a promising technique to manufacture a structure mimicking the extracellular matrix, cell infiltration into electrospun scaffolds remains challenging. The robust and in situ delivery of cells into such biomimetic scaffolds would potentially enable the design of tissue engineered constructs with spatial control over cellular distribution but often solvents employed in the spinning process are problematic due to their high cytotoxicity. Herein, microfluidic cell encapsulation is used to establish a temporary protection vehicle for the in situ delivery of cells for the development of a fibrous, cell-laden hybrid biograft. Therefore a layer-by-layer process is used by alternating fiber electrospinning and cell spraying procedures. Both encapsulation and subsequent electrospraying of capsules has no negative effect on the viability and myogenic differentiation of murine myoblast cells. Propidium iodide positive stained cells were analyzed to quantify the amount of dead cells and the presence of myosin heavy chain positive cells after the processes was shown. Furthermore, encapsulation successfully protects cells from cytotoxic solvents (such as dimethylformamide) during in situ delivery of the cells into electrospun poly(vinylidene fluoride-co-hexafluoropropylene) scaffolds. The resulting cell-populated biografts demonstrate the clear potential of this approach in the creation of viable tissue engineering constructs. STATEMENT OF SIGNIFICANCE: Infiltration of cells and their controlled spatial distribution within fibrous electrospun membranes is a challenging task but allows for the development of functional highly organized 3D hybrid tissues. Combining polymer electrospinning and cell electrospraying in a layer-by-layer approach is expected to overcome current limitations of reduced cell infiltration after traditional static seeding. However, organic solvents, used during the electrospinning process, impede often major issues due to their high cytotoxicity. Utilizing microfluidic encapsulation as a mean to embed cells within a protective polymer casing enables the controlled deposition of viable cells without interfering with the cellular phenotype. The presented techniques allow for novel cell manipulation approaches being significant for enhanced 3D tissue engineering based on its versatility in terms of material and cell selection.
Asunto(s)
Células Inmovilizadas , Técnicas Electroquímicas/métodos , Técnicas Analíticas Microfluídicas , Ingeniería de Tejidos , Andamios del Tejido/química , Animales , Línea Celular , Células Inmovilizadas/citología , Células Inmovilizadas/metabolismo , Ratones , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Ingeniería de Tejidos/instrumentación , Ingeniería de Tejidos/métodosRESUMEN
Engineering a small diameter vascular graft with mechanical and biological properties comparable to living tissues remains challenging. Often, current devices lead to thrombosis and unsatisfactory long-term patency as a result of poor blood compatibility and a mismatch between the mechanical properties of the living tissue and the implanted biomaterial. Addressing all these requirements is essential to produce scaffolds able to survive throughout the life of the patient. For this purpose, we fabricated a novel three-layered vascular graft by combining electrospinning and braiding. Mirroring the structure of human blood vessels, the proposed device is composed of three layers: the intima, the media, and the adventitia. The intima and media layers were obtained by sequentially electrospinning silk fibroin (SF) and poly(L-lactide-co-ε-caprolactone), with ratios selected to match the mechanical properties of the native tissue. For the outer layer, the adventitia, SF yarns were braided on top of the electrospun tubes to create a structure able to withstand high pressures. Compliance, Young's modulus and deformability of the obtained scaffold were similar to that of human blood vessels. Additionally, cytocompatibility of the two layers, media and intima, was assessed in vitro by analysing cell metabolic activity and proliferation of endothelial cells and smooth muscle cells, respectively. Furthermore, heparin functionalization of the scaffolds led to improved anticoagulant properties upon incubation in whole blood. The obtained results indicate a potential application of the herewith designed three-layered construct as a vascular graft for small diameter blood vessel engineering.
Asunto(s)
Materiales Biocompatibles/química , Vasos Sanguíneos/fisiología , Animales , Materiales Biocompatibles/farmacología , Prótesis Vascular , Bombyx/metabolismo , Adhesión Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Fibroínas/química , Células Endoteliales de la Vena Umbilical Humana , Humanos , Microscopía Confocal , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Poliésteres/química , Seda/química , Espectroscopía Infrarroja por Transformada de Fourier , Ingeniería de Tejidos , Andamios del Tejido/química , Difracción de Rayos XRESUMEN
Cell shape and regulation of biological processes such as proliferation and differentiation are to a large degree connected. Investigation of the possible relationship between cell shape and function is therefore important for developing new material concepts for medical applications as well as developing novel cell based sensors. Cell spreading requires a firm contact with the underlying substrate, with focal contacts (FC) being the primary sites of adhesion. They consist of a large number of clustered transmembrane proteins (integrins). FC integrins connect the cell cytoskeleton with the cell substratum. It has been demonstrated that cell spreading increases osteoblast differentiation in pre-osteoblastic progenitors. The gradual process of osteogenesis can be followed by different proteins being expressed at various time points, comprising early (e.g., bone-specific alkaline phosphatase (bALP)) and late genes (e.g., osteocalcin (OC)). In the present study we have used immunohistochemistry and RT-PCR to determine osteogenic differentiation of human bone cells (HBC). For online monitoring, fluorescently-tagged actin and vinculin were used for transfection of HBCs. Transfection of HBCs with an OC promoter gene construct allowed us to online monitor the gradual process of osteogenesis. We found distinct changes in cell architecture upon osteogenic differentiation thus providing evidence for the connection between cell shape and functional state.