Dirk Hegemann

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.

Densification of functional plasma polymers by momentum transfer during film growth

Functional plasma polymers were deposited from pure ethylene discharges and with the addition of carbon dioxide or ammonia. The incorporation of oxygen and nitrogen-containing functional groups depends on the fragmentation in the gas phase as well as on the densification during film growth. While a minimum energy per deposited carbon atom is required for cross-linking, the densification and accompanying reduction of functional group incorporation was found to scale linearly with momentum transfer through ion bombardment during film growth.

Antibacterial burst-release from minimal Ag-containing plasma polymer coatings.

Biomaterials releasing silver (Ag) are of interest because of their ability to inhibit pathogenic bacteria including antibiotic-resistant strains. In order to investigate the potential of nanometre-thick Ag polymer (Ag/amino-hydrocarbon) nanocomposite plasma coatings, we studied a comprehensive range of factors such as the plasma deposition process and Ag cation release as well as the antibacterial and cytocompatible properties. The nanocomposite coatings released most bound Ag within the first day of immersion in water yielding an antibacterial burst. The release kinetics correlated with the inhibitory effects on the pathogens Pseudomonas aeruginosa or Staphylococcus aureus and on animal cells that were in contact with these coatings. We identified a unique range of Ag content that provided an effective antibacterial peak release, followed by cytocompatible conditions soon thereafter. The control of the in situ growth conditions for Ag nanoparticles in the polymer matrix offers the possibility to produce customized coatings that initially release sufficient quantities of Ag ions to produce a strong adjacent antibacterial effect, and at the same time exhibit a rapidly decaying Ag content to provide surface cytocompatibility within hours/days. This approach seems to be favourable with respect to implant surfaces and possible Ag-resistance/tolerance built-up.

Plasma-functionalized electrospun matrix for biograft development and cardiac function stabilization

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(ɛ-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).

Macroscopic investigation of reaction rates yielding plasma polymer deposition

Plasma polymers cover a broad range from organic to hard coatings. In order to investigate the influencing factors yielding plasma polymer deposition, a macroscopic relation between the deposition rate Rm and the energy input W/F is derived based on reaction rates following a quasi-Arrhenius approach. An activation barrier for a specific plasma chemical reaction pathway is thus assumed. As an important finding, it can be shown that the pre-factor in the quasi-Arrhenius equation is independent of the energy input for the generation of film-forming species and their flux towards a substrate. For a constant sticking probability, it follows that also the mass deposition rate solely varies with the exponential term of the quasi-Arrhenius equation. The influencing factors in plasma polymerization enclosed in the pre-factor are found to be (i) the reaction rate for the plasma chemical reaction pathway following Arrhenius behaviour, (ii) the power coupling to the plasma governed by the electron energy distribution function, (iii) the monomer gas flow rate, (iv) the deposition area, (v) the molecular mass of the film-forming species and (vi) their sticking probability, which also includes their transport to the substrate. Different plasma polymerization processes are discussed in order to examine the derived influencing factors.

Embroidered Electrode with Silver/Titanium Coating for Long-Term ECG Monitoring

For the long-time monitoring of electrocardiograms, electrodes must be skin-friendly and non-irritating, but in addition they must deliver leads without artifacts even if the skin is dry and the body is moving. Todays adhesive conducting gel electrodes are not suitable for such applications. We have developed an embroidered textile electrode from polyethylene terephthalate yarn which is plasma-coated with silver for electrical conductivity and with an ultra-thin titanium layer on top for passivation. Two of these electrodes are embedded into a breast belt. They are moisturized with a very low amount of water vapor from an integrated reservoir. The combination of silver, titanium and water vapor results in an excellent electrode chemistry. With this belt the long-time monitoring of electrocardiography (ECG) is possible at rest as well as when the patient is moving.

Suppression of Hydrophobic Recovery by Plasma Polymer Films with Vertical Chemical Gradients

Vertical chemical gradients extending over a few nanometers were explored. The gradients are based on plasma-polymerized oxygen-containing ethylene (ppOEt) films. Using plasma conditions with low CO2/C2H4 ratio and high energy input, cross-linked films were deposited as base layer, while increasing CO2 and lowering energy input resulted in less cross-linked yet highly functional films as applied as top layer. Aging studies indicate that, in particular, for very thin gradient structures, the cross-linked subsurface zone effectively hinders reorientation of the surface functional groups, thus restricting hydrophobic recovery and oxidation effects.

Composition and Stability of Plasma Polymer Films Exhibiting Vertical Chemical Gradients

Controlling the balance between stability and functional group density in grown plasma polymer films is the key to diverse applications such as drug release, tissue-engineered implants, filtration, contact lenses, microfluidics, electrodes, sensors, etc. Highly functional plasma polymer films typically show a limited stability in air or aqueous environments due to mechanisms like molecular reorganization, oxidation, and hydrolysis. Stabilization is achieved by enhancing cross-linking at the cost of the terminal functional groups such as -OH and -COOH, but also -NH2, etc. To overcome such limitations, a structural and chemical gradient was introduced perpendicular to the surface plane; this vertical gradient structure is composed of a highly cross-linked base layer, gradually changing into a more functional nanoscaled surface termination layer. This was achieved using CO2/C2H4 discharges with decreasing power input and increasing gas ratio during plasma polymer deposition. The aging behavior and stability of such oxygen-functional vertical gradient nanostructures were studied in air and in different aqueous environments (acidic pH 4, neutral pH ≈ 6.2, and basic pH 10). Complementary characterization methods were used, including angle-resolved X-ray photoelectron spectroscopy (ARXPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) as well as water contact angle (WCA) measurements. It was found that in air, the vertical gradient films are stabilized over a period of months. The same gradients also appear to be stable in neutral water over a period of at least 1 week. Changes in the oxygen depth profiles have been observed at pH 4 and pH 10 showing structural and chemical aging effects on different time scales. The use of vertical gradient plasma polymer nanofilms thus represents a novel approach providing enhanced stability, thus opening the possibility for new applications.

Plasma polymerization at different positions in an asymmetric ethylene discharge

The characteristics of plasma polymerization are investigated in an asymmetric, capacitively coupled plasma discharge. Here, the deposition in different plasma zones, i.e. on the driven electrode, within the plasma bulk and the plasma sheath as well as approximately at the plasma-sheath edge, is investigated. Principal expectations are perfectly met, though new interesting dependences of the obtained a-C : H coatings with respect to film properties and deposition rates are also found. That is, the deposition rates as measured on thin, small glass slides placed directly on the electrode are considerably higher than everywhere else in the plasma, yet only single-sided. In contrast, the deposition rates on the samples within the plasma are lowered depending on the exact placement, while a double-sided coating is obtained. Furthermore, film properties, such as the film density, are highly dependent on the sample placement in the plasma, which can even be higher under floating conditions. With simple physical arguments we are able to show the relations between the deposition rate and the energy input into the plasma as well as between the energy density during film growth and the film density itself.

General Protocol for the Culture of Cells on Plasma-Coated Electrospun Scaffolds

As opposed to culture on standard tissue-treated plastic, cell culture on three-dimensional scaffolds impedes additional challenges with respect to substrate preparation, cell seeding, culture maintenance, and analysis. We herewith present a general route for the culture of primary cells, differentiated cells, or stem cells on plasma-coated, electrospun scaffolds. We describe a method to prepare and fix the scaffolds in culture wells and discuss a convenient method for cell seeding and subsequent analysis by scanning electron microscopy or immunohistology.