Nico Scharnagl

Design principles for polymers as substratum for adherent cells

The next generation of biomaterials for regenerative therapies requires the development of substances, which are able to influence and activate specific phenotype characteristics of cells and tissues. Research towards this aim has resulted in an increasing number of reports about material induced cellular functions and cell-cell interactions. In this context, polymeric materials, which are not intended to degrade can provide helpful in-vitro tools to gain more detailed knowledge about the cell-substrate crosstalk and the resulting cell specific effects. This review aims to consolidate current strategies to induce specific effects on adhesive cells which are related to defined characteristics of two-dimensional systems starting with the molecular dimension, following up with the nanostructure and ending with the surface microstructure. This includes approaches to induce direct or indirect biological responses towards cells by systematic changes in material properties such as hydrophilicity or elasticity. These properties are explained as a function of chemical composition such as the type and ratio of copolymers used for linear polymers, or the geometric arrangement of branching points for network polymer architectures. Surface topographical features are identified to strongly influence cell-substrate interactions and techniques are described to control the surface patterning of polymeric materials on the nano- or microscale. Finally we offer a strategy on how to develop complex and multifunctional materials, which might fulfill the requirements of cell and tissue adapted biomaterials for regenerative therapies.

Surface Functionalization of Poly(ether imide) Membranes with Linear, Methylated Oligoglycerols for Reducing Thrombogenicity

Materials for biomedical applications are often chosen for their bulk properties. Other requirements such as a hemocompatible surface shall be fulfilled by suitable chemical functionalization. Here we show, that linear, side-chain methylated oligoglycerols (OGMe) are more stable to oxidation than oligo(ethylene glycol) (OEG). Poly(ether imide) (PEI) membranes functionalized with OGMes perform at least as good as, and partially better than, OEG functionalized PEI membranes in view of protein resistance as well as thrombocyte adhesion and activation. Therefore, OGMes are highly potent surface functionalizing molecules for improving the hemocompatibility of polymers.

Behaviour of fibroblasts on water born acrylonitrile-based copolymers containing different cationic and anionic moieties.

The chemical composition of a substrate can influence the adhesion, viability and proliferation of cells seeded on the substrate. The aim of this work was to investigate the influence of different cationic or anionic moieties in acrylonitrile-based copolymers on the interaction with fibroblasts. A series of ten different types of acrylonitrile-based copolymers with a random sequence structure was prepared using a water born synthesis process to exclude potential residues of organic solvents. As charged comonomers cationic methacrylic acid-2-aminoethylester hydrochloride (AEMA), N-3-amino-propyl-methacrylamide hydrochloride (APMA) and anionic 2-methyl-2-propene-1-sulfonic acid sodium salt (NaMAS) were utilized. By application of a specific sintering procedure the copolymer materials were processed into transparent disks for conducting cell tests in direct contact. The copolymers were analyzed with respect to their composition and surface properties. Cytotoxicity tests of the polymer extracts, as well as of the disks were performed with L929 mouse fibroblasts. All copolymers showed no cytotoxic effects. Furthermore, for higher molar ratios of AEMA an increase in cell growth could be observed, which might be a hint that higher charge densities are favorable for the proliferation of L929 cells.

Adherence and viability of primary human keratinocytes and primary human dermal fibroblasts on acrylonitrile-based copolymers with different concentrations of positively charged functional groups

As shown in several studies, various properties of biomaterials such as stiffness, surface roughness, chemical composition or the amount of functional groups at the surface can influence adhesion, viability, proliferation and functionalities of cells. The aim of this work was to explore whether a cell-selective effect could be achieved for acrylonitrile-based copolymers containing different contents of positively charged functional groups, which were introduced by incorporation of methacrylic acid-2-aminoethylester hydrochloride (AEMA) units. The p(AN-co-AEMA) copolymers were synthesized by suspension polymerization in water and processed into disk shaped test specimen via a sintering process to ensure the absence of organic solvents in the copolymers. Copolymers with an AEMA content of 1.4, 1.6, and 4.4 mol-% were investigated according to their cell-selective capacity, which should support the adhesion, viability and proliferation of keratinocytes, while the adherence of fibroblasts should rather be disabled. The test samples were seeded with primary human keratinocytes and primary human dermal fibroblasts in mono- as well as in co-cultures. Tissue culture plate polystyrene (TCP) was used to control the physiologic growth of the cells. Density and viability of attached and non-adherent cells were analyzed by live/dead staining, lactate dehydrogenase (LDH) assay and flow cytometry with DAPI staining. For the assured discrimination of adherent cell types in coculture a keratin/vimentin-staining was performed. On copolymers with 4.4 mol-% AEMA adherent keratinocytes in monoculture and cocultured keratinocytes and fibroblasts showed a higher viability, a lower impairment of cell membranes and higher densities of viable cells compared to both other copolymers. For adherent fibroblasts these parameters did not differ between the copolymers and an increasing ratio of keratinocytes to fibroblasts in cocultures were found with increasing AEMA content. The results showed that keratinocytes and fibroblasts can be influenced by copolymers with different contents of positively charged functional groups. Since the tendency of a better adherence and viability of keratinocytes with increasing amounts of positively charged functional groups was shown, the potential enhancement by further increase of the amount of positively charged functional groups shall be tested in a future study.

Influence of co-monomer ratio on the chemical properties and cytotoxicity of poly[acrylonitrile-co-(N-vinylpyrrolidone)] nanoparticles

Purpose A system of nanoparticles with varying hydrophilicities may include promising biomaterial candidates as they offer various cellular uptake properties and a range of drug encapsulation efficacies, which would be advantageous in regenerative therapies. Therefore, a model system of nanoparticles with varying hydrophilicities was synthesized and assessed for its candidacy as a biomaterial. Methods Here, acrylonitrile (AN) was copolymerized with N-vinylpyrrolidone (NVP) in a mini-emulsion to form a family of nanoparticles, thereby enabling the systematic variation of the copolymer hydrophilicity. The nanoparticles based on these copolymers were prepared and characterized using 1H-NMR, dynamic light scattering, differential scanning calorimetry, and thermal gravimetric analysis. Finally, the cytotoxicity of the nanoparticles was assessed by conducting indirect tests using L929 fibroblasts. Results The nanoparticles showed well controlled NVP/AN molar ratios as determined by 1H NMR, well defined diameters ranging from approximately 100 nm to 200 nm, and increasing glass transition temperatures with increasing molar NVP content. Finally, L929 fibroblasts only slightly changed their morphology upon incubation with material eluates. Conclusions Poly[acrylonitrile-co-(N-vinylpyrrolidone)] nanoparticles with varying amounts of NVP were shown to be a promising model system for further biological assessment.

Influence of a silicon (Si14)-based coating substrate for biomaterials on fibroblast growth and human C5a

Despite considerable efforts in biomaterial development there is still a lack on substrates for cardiovascular tissue engineering approaches which allow the establishment of a tight a functional endothelial layer on their surface to provide hemocompatibility. The study aimed to test the biocompatibility of a silicon (Si14)-based coating substrate (Supershine Medicare, Permanon) which was designed to resist temperatures from -40°C up to 300°C and which allows the use of established heat-inducing sterilization techniques respectively. By X-ray photoelectron spectroscopy it could be validated that this substrate is able to establish a 40-50 nm thick layer of silica, oxygen and carbon without including any further elements from the substrate on an exemplary selection of materials (silicone, soda-lime-silica glass, stainless steel). Analysis of the LDH-release, the cell activity/proliferation (MTS assay) and the cell phenotype after growing 3T3 cells with extracts of the coated materials did not indicate any signs of cytotoxicity. Additionally by measuring the C5a release after exposure of the coated materials with human serum it could be demonstrated, that the coating had no impact on the activation of the complement system. These results generally suggest the tested substrate as a promising candidate for the coating of materials which are aimed to be used in cardiovascular tissue engineering approaches.

In vitro evaluation of a nitinol based vein cuff for external valvuloplasty

This study shows first in vitro tests of a nitinol based vein cuff developed for external valvuloplasty. In contrary to currently existing vein cuffs the tested model enables minimal invasive implantation and also maintains its round pre-shaped profile at body temperature (37 degrees C). The examination of the cuff surface structure by scanning electron microscopy, profilometry and X-ray photoelectron spectroscopy after sterilization with ethylene oxide and before cyto-compatibility testing revealed a nearly smooth surface (mean square roughness Rq 66 +/- 33 nm) which was primarily composed of nickel, oxygen, titanium, carbon and silicon where nickel was the least fraction (Ni: 0.7%, Ti: 1.7%, Si: 15.8%, O: 29.5%, C: 52.3%) of the surface elements. Si and C are supposed to be contaminations caused by a final cuff polishing with silicon carbide at the end of the manufacturing process. To evaluate cyto-compatibility initial cell adherence and cell activity were assessed. The results showed good initial cell adherence of L929 fibroblast-like cells on the cuff surface already after 24 h. The results also revealed no inhibitory effects on the activity of these cells (MTS test) later on. The test setup developed to analyse functionality in a dynamic mode was shown to be suited at blood pressures up to 300 mmHg. The cuff successfully limited dilation of varicose veins (Vena saphena magna) at physiological blood pressures (< 120 mmHg) and also in cases of hypertonia (300 mmHg) to the diameter determined by the cuff (4.0 mm) over thecomplete testing period. This indicates that the clasp based cuff closure mechanism is suited to close the cuff under variable physiological and pathological blood pressure conditions. The cuff structure only allowed minimal adaptation on the inhomogenously dilating vein profile in the both peripheral cuff modules. Both peripheral modules followed the vessel dilation in correlation to the applied pressure. At pressures within the physiological range </= 120 mmHg) the variation of the lateral arch module diameter was only marginal, whereas at 300 mmHg pressure the peripheral modules followed vein dilation up to a diameter of 5.0 to 5.5 mm. The cuff also maintained the pre-shaped round profile in the central and peripheral modules during the pressure increase and the consecutive cuff expansion. The study showed that the first nitinol based vein cuff for external valvuloplasty was processed well enough by electropolishing and sterilization to allow culturing of L929 fibroblast-like cells on the cuff surface as a test of general biocompatibility. The cuff also proved to limit dilation of varicose veins at physiological and pathological blood pressures in vitro. Further tests with primary cells from the venous wall will follow to test the specific biocompatibility before tests in vivo can be envisaged.

Influence of the blood exposure time in dynamic hemocompatibility testing on coagulation and C5a activation

Within the hemocompatibility testing portfolio of medical devices a range of dynamic models were established in recent years. In contrast to the static hemocompatibility testing method the dynamic models allow considering the impact of hemorheological and hemodynamic blood characteristics on the hemocompatibility of medical devices. Unfortunately the EN DIN ISO 10993-4 for the biological evaluation of medical devices for interaction with blood gives no hints towards the period of time during which the medical devices should be exposed to the blood in these tests. To examine whether different exposure times impact the comparability of hemocompatibility test results low density polyethylene (LD-PE) tubes and nitinol stents were tested exemplarily in a closed loop model for changes of the fibrinogen content, the prothrombin time, the thrombin time, and the C5a activity after 30 and 90 min exposure to the blood. Low density polyethylene was used as negative control because it is one of the European reference materials for hemocompatibility testing. After 90 min blood exposure to the LD-PE tubing and the nitinol stents the prothrombin time was significantly longer and the fibrinogen content significantly lower (p < 0.05) than after 30 min. In contrast the thrombin time and the C5a were comparable after 30 and 90 min blood exposure time. These results might recommend to an initial 30 min exposure time, which is followed by a 90 min exposure time in cases of unclear results.

A double-layer patch design for local and controlled drug delivery as an intraoperative custom-made implant-coating technology

Background The specific biological need of patients frequently becomes obvious just in the intraoperative setting. We hypothesized that a double-layer patch approach that allowed rapid attachment to an implant surface would represent a potential solution for technically challenging intraoperative personalized local drug delivery. Methods Dexamethasone-loaded poly[(rac-lactide)-co-glycolide] (PLGA) microparticles were embedded within a polyvinyl alcohol (PVA) patch that was attached to metal implant surfaces by in situ polymerization of alkyl-2-cyanoacrylates (CAs). Hydroxyapatite (HA) nanoparticles were also embedded in the PVA patch. Results Very rapid dexamethasone-release profiles were observed from the PLGA microparticles / PVA patches. The incorporation of HA nanoparticles into the PVA enabled control of CA penetration within the patch, and improved significantly its attachment, while no interference with the drug release was observed. Conclusions Double-layered patches with 1 layer for drug delivery and 1 as gluing interface could represent a solution for safe and controlled local drug delivery from implant surfaces or other, even biological, materials. The technology platform presented here opens the opportunity for personalized medicine by allowing local administration of drugs with customized release based on an intraoperative application.

Local drug delivery by personalized, intraoperative custom-made implant coating

Local administration of drugs can enhance regeneration, prevent infection, or treat postsurgical pain. If used in conjunction with implants, coating strategies should allow the choice of a drug or combination of drugs, their doses, localization, and release due to intraoperative considerations. Current coating technologies lack the ability for personalized medicine strategies. Here, we describe a new intraoperative strategy for drug delivery that allows a personalized approach as local drug delivery by implant coating. A polyvinylalcohol (PVA) patch provides rapid attachment to implant surfaces by cyanoacrylate (CA) adhesives. The CA polymerization was initiated by water uptake of the patch due to exposure to a humid environment. The coating strength depended on the type of the CA, the time of external pressing load and humidification, the properties of the patch and the implant surface. The CA adhesive penetrated and polymerized within the patch without impeding the bioactivity of the embedded molecules or strongly altering the protein release pattern after attachment to the implant surface. The use of CA in combination with the PVA patch proved to be noncytotoxic in vitro. This technology platform opens the possibility for personalized medicine to locally administer drugs due to intraoperative requirements.