Electrospinning Auricular Shaped Scaffolds for Tissue Engineering

Poly(ɛ)caprolactone scaffolds have been electrospun directly into an auricular shaped conductive mould. Bovine chondrocytes were harvested from articular cartilage and seeded onto 16 of the produced scaffolds, which received either an ethanol (group A) or a plasma treatment (group B) for sterilisation before seeding. The seeded scaffolds were cultured for 3 weeks in vitro and analysed with regard to total DNA and GAG content as well as the expression of AGG, COL1, COL2, MMP3 and MMP13. Rapid cell proliferation and GAG accumulation was observed until week 2. However, total DNA and GAG content decreased again in week 3. qPCR data shows a slight increase in the expression of anabolic genes and a slight decrease for the catabolic genes, with a significant difference between the groups A and B only for COL2 and MMP13.

Direct electrospinning of 3D auricle-shaped scaffolds for tissue engineering applications.

Thirty-two poly(ε)caprolactone (PCL) scaffolds have been produced by electrospinning directly into an auricle-shaped mould and seeded with articular chondrocytes harvested from bovine ankle joints. After seeding, the auricle shaped constructs were cultured in vitro and analysed at days 1, 7, 14 and 21 for regional differences in total DNA, glycosaminoglycan (GAG) and collagen (COL) content as well as the expression of aggrecan (AGG), collagen type I and type II (COL1/2) and matrix metalloproteinase 3 and 13 (MMP3/13). Stress-relaxation indentation testing was performed to investigate regional mechanical properties of the electrospun constructs. Electrospinning into a conductive mould yielded stable 3D constructs both initially and for the whole in vitro culture period, with an equilibrium modulus in the MPa range. Rapid cell proliferation and COL accumulation was observed until week 3. Quantitative real time PCR analysis showed an initial increase in AGG, no change in COL2, a persistent increase in COL1, and only a slight decrease initially for MMP3. Electrospinning of fibrous scaffolds directly into an auricle-shape represents a promising option for auricular tissue engineering, as it can reduce the steps needed to achieve an implantable structure.

Fine-tuning of substrate architecture and surface chemistry promotes muscle tissue development

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.

Anisotropically oriented electrospun matrices with an imprinted periodic micropattern: a new scaffold for engineered muscle constructs

Engineered muscle constructs provide a promising perspective on the regeneration or substitution of irreversibly damaged skeletal muscle. However, the highly ordered structure of native muscle tissue necessitates special consideration during scaffold development. Multiple approaches to the design of anisotropically structured substrates with grooved micropatterns or parallel-aligned fibres have previously been undertaken. In this study we report the guidance effect of a scaffold that combines both approaches, oriented fibres and a grooved topography. By electrospinning onto a topographically structured collector, matrices of parallel-oriented poly(ε-caprolactone) fibres with an imprinted wavy topography of 90 µm periodicity were produced. Matrices of randomly oriented fibres or parallel-oriented fibres without micropatterns served as controls. As previously shown, un-patterned, parallel-oriented substrates induced myotube orientation that is parallel to fibre direction. Interestingly, pattern addition induced an orientation of myotubes at an angle of 24° (statistical median) relative to fibre orientation. Myotube length was significantly increased on aligned micropatterned substrates in comparison to that on aligned substrates without pattern (436 ± 245 µm versus 365 ± 212 µm; p < 0.05). We report an innovative, yet simple, design to produce micropatterned electrospun scaffolds that induce an unexpected myotube orientation and an increase in myotube length.

Covalent immobilisation of VEGF on plasma-coated electrospun scaffolds for tissue engineering applications.

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(ɛ-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.