Ayşe Karakeçili

Comparison of bacterial and tissue cell initial adhesion on hydrophilic/hydrophobic biomaterials

In this study, interactions of widely-used polymeric biomaterials, i.e. poly(hydroxyethyl methacrylate) (PHEMA) and its copolymer with dimethylaminoethyl methacrylate (PHEMA-20% DMAEMA), polyurethane (PU), polypropylene (PP), poly(vinyl chloride) (PVC), and poly(lactideglycolide) (PLGA), with three pathogenic bacteria and one nonpathogen were investigated comparatively with the adhesion of two tissue cells in different morphologies, i.e. fibroblast-like baby hamster kidney (BHK 21) cells and epithelial Madine Darby kidney (MDBK) cells. Biomaterials were prepared in the membrane form by bulk polymerization or solvent casting. Surface characterization studies showed that these polymers have different surface free energies in the range of 26.9-63.1 ergcm-2 and they have smooth surfaces. The bacteria used were; Escherichia coli ATCC 25922, Staphylococcus epidermidis ATCC 12228, Staphylococcus aureus, and Lactobacillus acidophilus B-13. Initial adhesion of bacteria to the polymeric surfaces was examined under static conditions and in a laminar flow cell. The adhesion behaviour of S.aureus and S.epidermidis was found independent of the polymeric surface hydrophobicity. However, the percentage of attached E. coli decreased when increasing the surface free energy of the polymer, while L. acidophilus showed just the opposite behaviour. The comparative results indicated that the adhesion of BHK and MDBK cell was lowest on the most hydrophilic PHEMA surface and highest on the most hydrophobic PP surface. In contrast to the case of bacterial adhesion, no relationship was found between polymer hydrophobicity and mammalian cell adherence.

Uses of thermoresponsive and RGD/insulin-modified poly(vinyl ether)-based hydrogels in cell cultures.

Thermoresponsive hydrogels were synthesized by radiation copolymerization of ethylene glycol vinyl ether (1) and butyl vinyl ether (2) in the presence of cross-linking agent diethylene glycol divinyl ether. The comonomer ratio (monomer 1/monomer 2) and the cross-linker concentration were kept constant at 60 :40 (mole percentage in the monomeric mixture) and 4% (mole basis), respectively. The hydrogels showed a volume-phase transition in the temperature range 10-25 ° C and their swelling behaviour was reversible. The gels were modified by a cell adhesion factor, the RGD sequence of fibronectin, and a cell growth factor, insulin. However, they lost their thermoresponsive character after modification. The use of the gels in cell culture was investigated without using a proteolytic enzyme or serum. Cell culture studies realized by human skin fibroblasts (HS An1) showed that the cells can attach and proliferate on the surface of a thermoresponsive polymer. 80% of the cultured cells were readily detached from the polymer surface by lowering the incubation temperature from 37 ° C to 10 ° C for 30 min. In the studies carried out with RGD or insulin-modified hydrogels in serum-free cultures, higher values of cell proliferation (9 × 105 cells/ml) were obtained on the insulin-modified hydrogels, whereas higher values of cell attachment were obtained on the RGD-immobilized surfaces.

Osteogenic activities of MC3T3-E1 cells on heparin-immobilized poly(caprolactone) membranes

The aim of this study was to investigate the effects of heparin on the activity of osteoblast-like cells seeded on poly(caprolactone) (PCL) membranes. The membranes were prepared by solvent-casting technique in ~150 µm thickness. Then they were treated with 1,6-hexanediamine solution and functionalized with covalently bound heparin. The morphology, proliferation, and differentiation of MC3T3-E1 preosteoblasts on these membranes were investigated in vitro. The heparin functionalized PCL membranes, compared to non-functionalized membranes, significantly stimulated osteoblast proliferation. The Scanning electron microscope images confirmed the stimulative effect of covalently bound heparin on the osteoblast-like cell proliferation. The alkaline phosphatase and osteocalcin levels for cells proliferated on heparin containing PCL membranes were higher than that of nonfunctionalized membranes.

Preparation of bioactive and antimicrobial PLGA membranes by magainin II/EGF functionalization

Development of dual functional materials that are capable of both reducing bacterial interaction and encouraging host tissue integration has gained importance in design of biomaterials. In this study, we prepared a bilayer poly (lactide co-glycolide) fibrous membrane with antibacterial and bioactive properties by electrospinning. The antibacterial layer was produced by covalent immobilization of antimicrobial peptide, Magainin II. The bioactive layer incorporating epidermal growth factor (EGF) molecules was subsequently electrospun on the antibacterial layer. The membranes were characterized by X-ray photoelectron spectroscopy, scanning electron microscopy and fluorescence microscopy. EGF release was detected by enzyme-linked immunosorbent assay. The antibacterial activity was tested against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The ability to support tissue cell integration was detected by using L-929 mouse fibroblasts. The dual functional membranes established enhanced antibacterial properties and increased tissue cell compatibility. This combined approach suggests a promising strategy for wound dressings, vascular grafts and dental membranes as well as catheters and fixation devices.

Antibacterial activity on electrospun poly(lactide-co-glycolide) based membranes via Magainin II grafting

An antimicrobial peptide (AMP), Magainin II (Mag II) was covalently immobilized on poly(lactide-co-glycolide) (PLGA) and PLGA/gelatin electrospun fibrous membranes. The surface immobilization was characterized by X-ray Photoelectron Spectroscopy (XPS). Scanning Electron Microscopy (SEM) and Atomic Force Microscopy studies showed that the surface morphology of the fibers at micron scale was not affected by the immobilization process. The antibacterial activity of the bound Mag II was tested against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. Bacterial adhesion tests, SEM and confocal analyses revealed that the attachment and survival of bacteria were inhibited on Mag II functionalized membranes. AMP immobilization strategy was introduced as a new perspective for the modulation of antibacterial properties on PLGA based materials prepared by electrospinning.

Chitosan and polycaprolactone membranes patterned via electrospinning: Effect of underlying chemistry and pattern characteristics on epithelial/fibroblastic cell behavior

Electrospinning was used as an effective route to pattern chitosan (CS) and polycaprolactone (PCL) membranes with submicron fibers having different chemical structure (PCL or PCL/collagen) and physical characteristics (size: between ≈200 and 550 nm; randomly oriented or aligned form). While the PCL fibers with diameters in the same range (≈200 nm) were patterned on both of CS and PCL membranes to evaluate the influence of the underlying membrane chemistry, only CS membranes were patterned with PCL fibers having different sizes simply by changing the electrospinning conditions to investigate the effects of pattern characteristics. Furthermore, collagen was added to the PCL fiber structure to change the chemical composition of the fibers in a cell-attractive way. Two cell lines with different morphologies, fibroblastic MC3T3-E1 preosteoblasts and epithelial Madine Darby Bovine Kidney (MDBK) cells, were cultured on the patterned membranes. The observation of cellular behavior in terms of cell morphology and F-actin synthesis was realized by scanning electron microscopy and confocal microscopy analysis during the first 12 h of culture period. The viability of cells was controlled by MTT assay through 96 h of cell culture. The cell culture studies indicated that the leading aspect for the morphology change on patterned membranes was the fiber orientation. The aligned topography controlled the morphology of cells both on CS and PCL membranes. In the presence of collagen in the fiber structure, F-actin filament synthesis increased for MC3T3-E1 and MDBK cell lines.

Effect of emulsification-diffusion parameters on the formation of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) particles

The aim of this work was to evaluate the effect of various production parameters on the formation and particle size of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) PHBV particles prepared by the emulsification-diffusion technique. The increase in homogenization time and speed caused a decrease in particle size. No particle formation was observed below 2% (w/v) PHBV in the organic phase. Smaller particle size and narrower size distribution were observed when polyvinyl alcohol (PVA) was used as a stabilizer, when compared to didodecyldimethylammonium bromide. Submicron particles of 531 ± 150 nm size were obtained with 2% (w/v) PVA at 17 500 rpm and 15 min homogenization conditions with dichloromethane as the organic solvent.

Synthesis and characterization of Fe3O4-MPTMS-PLGA nanocomposites for anticancer drug loading and release studies

Abstract Magnetic nanocomposites (Fe3O4-MPTMS-PLGA) were synthesized by single oil emulsion method and characterized by transmission electron microscopy (TEM), X-Ray diffraction (XRD), and vibrating sample magnetometer (VSM). Particle size of nanocomposites was between 117 nm and 246 nm. High performance liquid chromatography (HPLC) was used to investigate drug loading (paclitaxel, PTX) and release from Fe3O4-MPTMS-PLGA-PTX nanocomposites. The percentages of drug loading and encapsulation efficiency onto nanocomposites were found as 7.35 and 68.58, respectively. Cytotoxities of free anticancer drug and anticancer drug-loaded nanocomposites were determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. In vitro cell culture studies indicated that Fe3O4-MPTMS-PLGA-PTX had significant toxicity on MG-63 cancer cells.

Designing double-layered nanofibrous membranes as a wound dressing material

New generation wound dressings require the criteria that both bioactive and conventional wound dressing materials can recompense the fundamental properties like defense of wound from microbial invasion, dehydration during the wound care duration and mimic the healing process. In this study, functional double-layered nanofibrous composite membranes were fabricated via electrospinning method. The matrices consist of a sheet of ampicillin loaded poly(2-hydroxylethyl methacrylate/polyacrylic acid (pHEMA/pAA) nanofibers on the upper side (first layer: pH sensitive antibacterial barrier) and a sheet of poly(ε-caprolactone) (PCL)/gelatin nanofibers (second layer: bioactive part). Ampicillin was successfully incorporated to double-layered matrices which greatly changed the mechanical properties, biodegradability and water uptake ratios (up to 4 fold higher values). The success of the antimicrobial activity of ampicillin on Staphylococcus aureus and Escherichia coli was indicated by the inhibition zone test. pH sensitivity was confirmed by the swelling and ampicillin release studies by shifting pH value to basic environment. Thus, double-layered pHEMA-pAA nanofibers suggest as a potential wound dressing material for its pH sensitive drug delivery ability and its bioactive part.