Martin Parizek

Modulation of cell adhesion, proliferation and differentiation on materials designed for body implants.

The interaction of cells and tissues with artificial materials designed for applications in biotechnologies and in medicine is governed by the physical and chemical properties of the material surface. There is optimal cell adhesion to moderately hydrophilic and positively charged substrates, due to the adsorption of cell adhesion-mediating molecules (e.g. vitronectin, fibronectin) in an advantageous geometrical conformation, which makes specific sites on these molecules (e.g. specific amino acid sequences) accessible to cell adhesion receptors (e.g. integrins). Highly hydrophilic surfaces prevent the adsorption of proteins, or these molecules are bound very weakly. On highly hydrophobic materials, however, proteins are adsorbed in rigid and denatured forms, hampering cell adhesion. The wettability of the material surface, particularly in synthetic polymers, can be effectively regulated by physical treatments, e.g. by irradiation with ions, plasma or UV light. The irradiation-activated material surface can be functionalized by various biomolecules and nanoparticles, and this further enhances its attractiveness for cells and its effectiveness in regulating cell functions. Another important factor for cell-material interaction is surface roughness and surface topography. Nanostructured substrates (i.e. substrates with irregularities smaller than 100nm), are generally considered to be beneficial for cell adhesion and growth, while microstructured substrates behave more controversially (e.g. they can hamper cell spreading and proliferation but they enhance cell differentiation, particularly in osteogenic cells). A factor which has been relatively less investigated, but which is essential for cell-material interaction, is material deformability. Highly soft and deformable substrates cannot resist the tractional forces generated by cells during cell adhesion, and cells are not able to attach, spread and survive on such materials. Local variation in the physical and chemical properties of the material surface can be advantageously used for constructing patterned surfaces. Micropatterned surfaces enable regionally selective cell adhesion and directed growth, which can be utilized in tissue engineering, in constructing microarrays and in biosensorics. Nanopatterned surfaces are an effective tool for manipulating the type, number, spacing and distribution of ligands for cell adhesion receptors on the material surface. As a consequence, these surfaces are able to control the size, shape, distribution and maturity of focal adhesion plaques on cells, and thus cell adhesion, proliferation, differentiation and other cell functions.

Nanofibrous poly(lactide-co-glycolide) membranes loaded with diamond nanoparticles as promising substrates for bone tissue engineering.

Video abstract Video

Improved Adhesion, Growth and Maturation of Vascular Smooth Muscle Cells on Polyethylene Grafted with Bioactive Molecules and Carbon Particles

High-density polyethylene (PE) foils were modified by an Ar+ plasma discharge and subsequent grafting with biomolecules, namely glycine (Gly), polyethylene glycol (PEG), bovine serum albumin (BSA), colloidal carbon particles (C) or BSA and C (BSA + C). As revealed by atomic force microscopy (AFM), goniometry and Rutherford Backscattering Spectroscopy (RBS), the surface chemical structure and surface morphology of PE changed dramatically after plasma treatment. The contact angle decreased for the samples treated by plasma, mainly in relation to the formation of oxygen structures during plasma irradiation. A further decrease in the contact angle was obvious after glycine and PEG grafting. The increase in oxygen concentration after glycine and PEG grafting proved that the two molecules were chemically linked to the plasma-activated surface. Plasma treatment led to ablation of the PE surface layer, thus the surface morphology was changed and the surface roughness was increased. The materials were then seeded with vascular smooth muscle cells (VSMC) derived from rat aorta and incubated in a DMEM medium with fetal bovine serum. Generally, the cells adhered and grew better on modified rather than on unmodified PE samples. Immunofluorescence showed that focal adhesion plaques containing talin, vinculin and paxillin were most apparent in cells on PE grafted with PEG or BSA + C, and the fibres containing α-actin, β-actin or SM1 and SM2 myosins were thicker, more numerous and more brightly stained in the cells on all modified PE samples than on pristine PE. An enzyme-linked immunosorbent assay (ELISA) revealed increased concentrations of focal adhesion proteins talin and vinculin and also a cytoskeletal protein β-actin in cells on PE modified with BSA + C. A contractile protein α-actin was increased in cells on PE grafted with PEG or Gly. These results showed that PE activated with plasma and subsequently grafted with bioactive molecules and colloidal C particles, especially with PEG and BSA + C, promotes the adhesion, proliferation and phenotypic maturation of VSMC.

Injectable nanoparticle-loaded hydrogel system for local delivery of sodium alendronate.

Systemic administration of bisphosphonates, e.g. sodium alendronate (Aln) is characterized by extremely low bioavailability and high toxicity. To omit aforementioned drawbacks an injectable system for the intra-bone delivery of Aln based on Aln-loaded nanoparticles (NPs-Aln) suspended in a hydrogel matrix (gellan gum, GG) was developed. Aln was encapsulated in poly(lactide-co-glycolide) (PLGA 85:15) by solid-oil-water emulsification. Drug release tests showed that within 25 days all the encapsulated drug was released from NPs-Aln and the release rate was highest at the beginning and decreased with time. In contrast, by suspending NPs-Aln in a GG matrix, the release rate was significantly lower and more constant in time. The GG-NPs-Aln system was engineered to be easily injectable and was able to reassemble its structure after extrusion as shown by rheological measurements. Invitro studies showed that the GG-NPs-Aln was cytocompatible with MG-63 osteoblast-like cells and it inhibited RANKL-mediated osteoclastic differentiation of RAW 264.7 cells. The injectability, the sustained local delivery of small doses of Aln and the biological activity render the GG-NPs-Aln system promising for the local treatment of osteoporosis and other bone tissue disorders.

Perivascular sirolimus-delivery system.

Autologous vein grafts are often used for treating damaged vessels, e.g. arteriovenous fistulas or arterial bypass conduits. Veins have a different histological structure from arteries, which often leads to intimal hyperplasia and graft restenosis. The aim of this study was to develop a perivascular sirolimus-delivery system that would release the antiproliferative drug sirolimus in a controlled manner. Polyester Mesh I was coated with purasorb, i.e. a copolymer of L-lactide and ɛ-caprolactone, with dissolved sirolimus; Mesh II was coated with two copolymer layers; the layer with dissolved sirolimus was overlaid with pure purasorb. This arrangement allowed sirolimus to be released for 6 and 4 weeks, for Mesh I and Mesh II, respectively. Mesh II released sirolimus more homogeneously, without the initial burst effect during the first week. However, the cumulative release curve was steeper at later time points than the curve for Mesh I. Both meshes inhibited proliferation of rat vascular smooth muscle cells during 14-day culture in vitro and preserved excellent cell viability. Newly developed sirolimus-releasing perivascular meshes are promising devices for preventing autologous graft restenosis.

Adhesion, Growth, and Maturation of Vascular Smooth Muscle Cells on Low-Density Polyethylene Grafted with Bioactive Substances

The attractiveness of synthetic polymers for cell colonization can be affected by physical, chemical, and biological modification of the polymer surface. In this study, low-density polyethylene (LDPE) was treated by an Ar+ plasma discharge and then grafted with biologically active substances, namely, glycine (Gly), polyethylene glycol (PEG), bovine serum albumin (BSA), colloidal carbon particles (C), or BSA+C. All modifications increased the oxygen content, the wettability, and the surface free energy of the materials compared to the pristine LDPE, but these changes were most pronounced in LDPE with Gly or PEG, where all the three values were higher than in the only plasma-treated samples. When seeded with vascular smooth muscle cells (VSMCs), the Gly- or PEG-grafted samples increased mainly the spreading and concentration of focal adhesion proteins talin and vinculin in these cells. LDPE grafted with BSA or BSA+C showed a similar oxygen content and similar wettability, as the samples only treated with plasma, but the nano- and submicron-scale irregularities on their surface were more pronounced and of a different shape. These samples promoted predominantly the growth, the formation of a confluent layer, and phenotypic maturation of VSMC, demonstrated by higher concentrations of contractile proteins alpha-actin and SM1 and SM2 myosins. Thus, the behavior of VSMC on LDPE can be regulated by the type of bioactive substances that are grafted.

Multistage-targeted pH-responsive polymer conjugate of Auger electron emitter: optimized design and in vivo activity.

Auger electrons-emitting radioisotopes (such as iodine-125) are a potentially effective cancer treatment. They are extremely biologically effective, but only within a short range (nanometers). Their use as an effective cancer therapy requires that they will be transported within close proximity of DNA by an intercalator, where they induce double-strand breaks leading to cell death. This type of therapy may be even more beneficial when associated with drug delivery systems. In this report, we describe an optimized triple-targeted polymer delivery system for the intercalator ellipticine, which contains radioisotope iodine-125 with high specific radioactivity (63.2 GBq/mg). This compound is linked to an N-(2-hydroxypropyl)methacrylamide copolymer via an optimized acid-sensitive hydrazone linker. The system is stable at pH 7.4 (representing the pH of blood plasma), and the radioiodine-containing biologically active intercalator is released upon a decrease in pH (44% of the intercalator is released after 24h of incubation in pH 5.0 buffer, which mimics the pH in late endosomes). The active compound is a potent intercalator, as shown with direct titration with a DNA solution, and readily penetrates into cell nuclei, as observed by confocal microscopy. Its polymer conjugate is internalized into endosomes and releases the radioactive intercalator, which accumulates in the cell nuclei. In vivo experiments on mice with 4T1 murine breast cancer resulted in a statistically significant increase in the survival of mice treated with the polymer radioconjugate. The free radiolabeled intercalator was also shown to be effective, but it was less potent than the polymer conjugate.

Porous Heat-Treated Polyacrylonitrile Scaffolds for Bone Tissue Engineering

Heat-treated polyacrylonitrile (HT-PAN), also referred to as black orlon (BO), is a promising carbon-based material used for applications in tissue engineering and regenerative medicine. To the best of our knowledge, no such complex bone morphology-mimicking three-dimensional (3D) BO structure has been reported to date. We report that BO can be easily made into 3D cryogel scaffolds with porous structures, using succinonitrile as a porogen. The cryogels possess a porous morphology, similar to bone tissue. The prepared scaffolds showed strong osteoconductive activity, providing excellent support for the adhesion, proliferation, and mitochondrial activity of human bone-derived cells. This effect was more apparent in scaffolds prepared from a matrix with a higher content of PAN (i.e., 10% rather than 5%). The scaffolds with 10% of PAN also showed enhanced mechanical properties, as revealed by higher compressive modulus and higher compressive strength. Therefore, these scaffolds have a robust potential for use in bone tissue engineering.

Modified glycogen as construction material for functional biomimetic microfibers

We describe a conceptually new, microfibrous, biodegradable functional material prepared from a modified storage polysaccharide also present in humans (glycogen) showing strong potential as direct-contact dressing/interface material for wound healing. Double bonds were introduced into glycogen via allylation and were further exploited for crosslinking of the microfibers. Triple bonds were introduced by propargylation and served for further click functionalization of the microfibers with bioactive peptide. A simple solvent-free method allowing the preparation of thick layers was used to produce microfibers (diameter ca 2μm) from allylated and/or propargylated glycogen. Crosslinking of the samples was performed by microtron beta-irradiation, and the irradiation dose was optimized to 2kGy. The results from biological testing showed that these highly porous, hydrophilic, readily functionalizable materials were completely nontoxic to cells growing in their presence. The fibers were gradually degraded in the presence of cells.