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Novel approaches, combining technology, biomaterial design, and cutting-edge cell culture, have been increasingly considered to advance the field of tissue engineering and regenerative medicine. Within this context, acoustic manipulation to remotely control spatial cellular organization within a carrier matrix has arisen as a particularly promising method during the last decade. Acoustic or sound-induced manipulation takes advantage of hydrodynamic forces exerted on systems of particles within a liquid medium by standing waves. Inorganic or organic particles, cells, or organoids assemble within the nodes of the standing wave, creating distinct patterns in response to the applied frequency and amplitude. Acoustic manipulation has advanced from micro- or nanoparticle arrangement in 2D to the assembly of multiple cell types or organoids into highly complex in vitro tissues. In this review, we discuss the past research achievements in the field of acoustic manipulation with particular emphasis on biomedical application. We survey microfluidic, open chamber, and high throughput devices for their applicability to arrange non-living and living units in buffer or hydrogels. We also investigate the challenges arising from different methods, and their prospects to gain a deeper understanding of in vitro tissue formation and application in the field of biomedical engineering.
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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.
Assuntos
Células Imobilizadas , Técnicas Eletroquímicas/métodos , Técnicas Analíticas Microfluídicas , Engenharia Tecidual , Alicerces Teciduais/química , Animais , Linhagem Celular , Células Imobilizadas/citologia , Células Imobilizadas/metabolismo , Camundongos , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Engenharia Tecidual/instrumentação , Engenharia Tecidual/métodosRESUMO
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.
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Materiais Biocompatíveis/química , Vasos Sanguíneos/fisiologia , Animais , Materiais Biocompatíveis/farmacologia , Prótese Vascular , Bombyx/metabolismo , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Fibroínas/química , Células Endoteliais da Veia Umbilical Humana , Humanos , Microscopia Confocal , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Poliésteres/química , Seda/química , Espectroscopia de Infravermelho com Transformada de Fourier , Engenharia Tecidual , Alicerces Teciduais/química , Difração de Raios XRESUMO
Conjugated polymers have been proposed as promising materials for scaffolds in tissue engineering applications. The restricted processability and biodegradability of conjugated polymers limit their use for biomedical applications however. Here we synthesised a block-co-polymer of aniline tetramer and PCL (AT-PCL), and processed it into fibrous non-woven scaffolds by electrospinning. We showed that fibronectin (Fn) adhesion was dependant on the AT-PCL oxidative state, with a reduced Fn unfolding length on doped membranes. Furthermore, we demonstrated the cytocompatibility and potential of these membranes to support the growth and osteogenic differentiation of MC3T3-E1 over 21 days.
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Recent findings in the field of biomaterials and tissue engineering provide evidence that surface immobilised growth factors display enhanced stability and induce prolonged function. Cell response can be regulated by material properties and at the site of interest. To this end, we developed scaffolds with covalently bound vascular endothelial growth factor (VEGF) and evaluated their mitogenic effect on endothelial cells in vitro. Nano- (254±133 nm) or micro-fibrous (4.0±0.4 µm) poly(É-caprolactone) (PCL) non-wovens were produced by electrospinning and coated in a radio frequency (RF) plasma process to induce an oxygen functional hydrocarbon layer. Implemented carboxylic acid groups were converted into amine-reactive esters and covalently coupled to VEGF by forming stable amide bonds (standard EDC/NHS chemistry). Substrates were analysed by X-ray photoelectron spectroscopy (XPS), enzyme-linked immuno-assays (ELISA) and immunohistochemistry (anti-VEGF antibody and VEGF-R2 binding). Depending on the reaction conditions, immobilised VEGF was present at 127±47 ng to 941±199 ng per substrate (6mm diameter; concentrations of 4.5 ng mm(-2) or 33.3 ng mm(-2), respectively). Immunohistochemistry provided evidence for biological integrity of immobilised VEGF. Endothelial cell number of primary endothelial cells or immortalised endothelial cells were significantly enhanced on VEGF-functionalised scaffolds compared to native PCL scaffolds. This indicates a sustained activity of immobilised VEGF over a culture period of nine days. We present a versatile method for the fabrication of growth factor-loaded scaffolds at specific concentrations.
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Poliésteres/química , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Fator A de Crescimento do Endotélio Vascular/química , Plasma/químicaRESUMO
Cardiac tissue engineering approaches can deliver large numbers of cells to the damaged myocardium and have thus increasingly been considered as a possible curative treatment to counteract the high prevalence of progressive heart failure after myocardial infarction (MI). Optimal scaffold architecture and mechanical and chemical properties, as well as immune- and bio-compatibility, need to be addressed. We demonstrated that radio-frequency plasma surface functionalized electrospun poly(É-caprolactone) (PCL) fibres provide a suitable matrix for bone-marrow-derived mesenchymal stem cell (MSC) cardiac implantation. Using a rat model of chronic MI, we showed that MSC-seeded plasma-coated PCL grafts stabilized cardiac function and attenuated dilatation. Significant relative decreases of 13% of the ejection fraction (EF) and 15% of the fractional shortening (FS) were observed in sham treated animals; respective decreases of 20% and 25% were measured 4 weeks after acellular patch implantation, whereas a steadied function was observed 4 weeks after MSC-patch implantation (relative decreases of 6% for both EF and FS).
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Testes de Função Cardíaca , Engenharia Tecidual , Animais , Modelos Animais de Doenças , Masculino , Infarto do Miocárdio/complicações , Infarto do Miocárdio/fisiopatologia , Ratos , Ratos Endogâmicos Lew , Alicerces TeciduaisRESUMO
Tissue engineering has been increasingly brought to the scientific spotlight in response to the tremendous demand for regeneration, restoration or substitution of skeletal or cardiac muscle after traumatic injury, tumour ablation or myocardial infarction. In vitro generation of a highly organized and contractile muscle tissue, however, crucially depends on an appropriate design of the cell culture substrate. The present work evaluated the impact of substrate properties, in particular morphology, chemical surface composition and mechanical properties, on muscle cell fate. To this end, aligned and randomly oriented micron (3.3±0.8 µm) or nano (237±98 nm) scaled fibrous poly(ε-caprolactone) non-wovens were processed by electrospinning. A nanometer-thick oxygen functional hydrocarbon coating was deposited by a radio frequency plasma process. C2C12 muscle cells were grown on pure and as-functionalized substrates and analysed for viability, proliferation, spatial orientation, differentiation and contractility. Cell orientation has been shown to depend strongly on substrate architecture, being most pronounced on micron-scaled parallel-oriented fibres. Oxygen functional hydrocarbons, representing stable, non-immunogenic surface groups, were identified as strong triggers for myotube differentiation. Accordingly, the highest myotube density (28±15% of total substrate area), sarcomeric striation and contractility were found on plasma-coated substrates. The current study highlights the manifold material characteristics to be addressed during the substrate design process and provides insight into processes to improve bio-interfaces.