Grazyna Ginalska

Characterization of the developed antimicrobial urological catheters

Antimicrobial urological catheters were developed by the mixed, covalent and non-covalent binding of sparfloxacin (SPA) to heparin (HP) film which was first deposited on the latex surface of biomaterial. The SPA-HP modified surface was characterized by SEM analysis and ATR-Fourier transform infrared spectroscopy. For the antimicrobial prevention, SPA as an antibiotic with a broad antimicrobial spectrum was chosen. Antimicrobial activity of antibiotic-modified catheter against Staphylococcus aureus, Staphylococcus epidermidis and Escherichia coli strains was assessed using various procedures. On the basis of the inhibition zone and diffusion assays the efficacy around the modified catheters was demonstrated. The test samples clearly showed an antibacterial activity against all tested bacterial stains for a least one month. Inhibition of the bacterial colonization on the modified catheter surface was proved by the biofilm test. Antimicrobial activity of SPA-treated catheter surface was also quantitatively evaluated according to standard method of ISO based on JIS. The R-values were found to be higher than 3.8. The performed research indicated that the immobilization of SPA on the catheter surface by means of the mixed-type bonds resulted in stable antibacterial protection of the urological catheters for a long time.

Covalent coating of hydroxyapatite by keratin stabilizes gentamicin release

A novel hybrid hydroxyapatite (HAP) matrix, covalently coated with rarely applied, hardly degradable keratin and effectively modified by gentamicin immobilized in mixed-type mode (via interactions of diverse strength), was created. This hybrid showed a remarkably high drug immobilization yield and the most sustainable antibiotic release among all tested composites. It was also able to inhibit bacterial growth, both in surrounding liquid and on matrix surface, much longer (for at least 121 days of experiment) than analogous gelatin-modified and nonmodified matrices. Gentamicin-keratin-coated-HAP granules were nontoxic to human osteoblasts and enabled their proliferation with a rate similar as noncoated HAP. Presence of keratin on HAP granules seemed to slightly enhance the osteoblast proliferation. The results indicate that newly created HAP hybrid with covalently immobilized keratin and gentamicin--nontoxic and osteoblast-friendly--is a promising biomaterial of significantly prolonged antibacterial activity.

A comparative analysis of phenotypic and genotypic methods for the determination of the biofilm-forming abilities of Staphylococcus epidermidis

The collection of 146 Staphylococcus epidermidis strains isolated from the nasopharynx of lung cancer patients has been studied for the ability of slime secretion and biofilm formation using the Congo red agar (CRA) test and the microtiter plate (MtP) method, respectively. The prevalence of the icaAD and the aap genes was also analyzed. Some isolates (35.6%) were biofilm positive by the MtP method, while 58.9% of isolates exhibited a slime-positive phenotype by the CRA test. The sensitivities of the CRA test evaluated using the MtP method as a gold standard of biofilm production were 73.1%, 97.3% and 13.3% for all the strains screened, ica-positive and ica-negative strains, respectively. The genotype ica(+)aap(+) was correlated with a strong biofilm-producer phenotype. Interestingly, some of the ica(-)aap(-) isolates could also form a biofilm. The correlation between the presence of icaAD genes and the biofilm-positive phenotype by the MtP method as well as slime production by the CRA test was statistically significant (P<0.0001). However, some S. epidermidis strains possess the potential ability of ica-independent biofilm formation; thus, further studies are needed to determine reliable, short-time criteria for an in vitro assessment of biofilm production by staphylococci.

Cooperation of fungal laccase and glucose 1-oxidase in transformation of Björkman lignin and some phenolic compounds

Summary A screening of wood-rotting basidiomycete fungi was conducted for glucose 1-oxidase (GOD) and laccase (LAC) production as well as for ligninolytic activity measured by a Rhemazol reaction. The results showed that genera rich in GOD are lignin degraders as well as effective producers of extracellular LAC. The fungi poor in GOD neither showed LAC, nor ligninolytic activity. The Björkman lignin and 3 phenolic compounds, hydroquinone and syringic and vanillic acids, were tested on the sequential activity of LAC and GOD. In the presence of LAC, quinoid intermediates formed from Björkman lignin and phenolic compounds were observed. The addition of GOD caused a diminution of the quinone level. During incubation of Björkman lignin with LAC and GOD depolymerization occurred, and in the experiments omitting GOD the quantities of low molecular products were markedly lower. Consequently, the consecutive ping-pong activity of LAC and GOD reduced the polymerization and improved the efficiency of depolymerization processes.

Effect of a carbonated HAP/β-glucan composite bone substitute on healing of drilled bone voids in the proximal tibial metaphysis of rabbits.

A novel elastic hydroxyapatite-based composite of high surgical handiness has been developed. Its potential application in orthopedics as a filler of bone defects has been studied. The biomaterial was composed of carbonated hydroxyapatite (CHAP) granules and polysaccharide polymer (β-1,3-glucan). Cylinders of 4mm in diameter and 6mm in length were implanted into bone cavities created in the proximal metaphysis of tibiae of 24 New Zealand white rabbits. 18 sham-operated animals were used as controls. After 1, 3 or 6 months, the rabbits were euthanized, the bones were harvested and subjected to analysis. Radiological images and histological sections revealed integration of implants with bone tissue with no signs of graft rejection. Peripheral quantitative computed tomography (pQCT) indicated the stimulating effect of the biomaterial on bone formation and mineralization. Densitometry (DXA) analysis suggested that biomineralization of bones was preceded by bioresorption and gradual disappearance of porous ceramic granules. The findings suggest that the CHAP-glucan composite material enables regeneration of bone tissue and could serve as a bone defect filler.

Application of β-1,3-glucan in production of ceramics-based elastic composite for bone repair

BackgroundUnsatisfactory surgical handiness is a commonly known disadvantage of implantable granular bioceramics. To overcome this problem, β-1,3-glucan, biotechnologically derived polysaccharide, has been proposed as a joining agent to combine granular ceramics into novel compact and elastic composite. Hydroxyapatite/glucan elastic material was processed and evaluated as a potential bone void filler.MethodologyThe procedure of composite formation was based on gelling properties of glucan. Its properties were studied using X-ray microtomography, SEM-EDS, FTIR spectroscopy, compression test and ultrasonic method. Sorption index was determined in phosphate buffered saline; bioactivity in simulated body fluid; sterility in growth broth and human blood plasma; implantation procedure in dog model.ResultsHAp/glucan composite is sterilizable, flexible and self-adapting to defect shape. It exhibits bioactivity, good surgical handiness, high sorption index and profitable mechanical properties, resembling those of spongy bone. Results of pilot clinical experiment on animal (dog) patients of a local clinic of animal surgery suggested good healing properties of the composite and its transformation into new bone tissue within critical-size defect.ConclusionsThe results obtained in this study confirm that flexible HAp/glucan composite has potential as a bone-substituting material. Promising results of pilot clinical experiment suggest that further in vivo experiments should be performed.

Biomedical potential of chitosan/HA and chitosan/β-1,3-glucan/HA biomaterials as scaffolds for bone regeneration — A comparative study

The aim of this work was to compare biomedical potential of chitosan/hydroxyapatite (chit/HA) and novel chitosan/β-1,3-glucan/hydroxyapatite (chit/glu/HA) materials as scaffolds for bone regeneration via characterization of their biocompatibility, porosity, mechanical properties, and water uptake behaviour. Biocompatibility of the scaffolds was assessed in direct-contact with the materials using normal human foetal osteoblast cell line. Cytotoxicity and osteoblast proliferation rate were evaluated. Porosity was assessed using computed microtomography analysis and mechanical properties were determined by compression testing. Obtained results demonstrated that chit/HA scaffold possessed significantly better mechanical properties (compressive strength: 1.23 MPa, Youngs modulus: 0.46 MPa) than chit/glu/HA material (compressive strength: 0.26 MPa, Youngs modulus: 0.25 MPa). However, addition of bacterial β-1,3-glucan to the chit/HA scaffold improved its flexibility and porosity. Moreover, chit/glu/HA scaffold revealed significantly higher water uptake capability (52.6% after 24h of soaking) compared to the chit/HA (30.7%) and thus can serve as a very good drug delivery carrier. Chit/glu/HA scaffold was also more favourable to osteoblast survival (near 100% viability after 24-h culture), proliferation, and spreading compared to the chit/HA (63% viability). The chit/glu/HA possesses better biomedical potential than chit/HA scaffold. Nevertheless, poor mechanical properties of the chit/glu/HA limit its application to non-load bearing implantation area.

Chitosan/β-1,3-glucan/calcium phosphate ceramics composites—Novel cell scaffolds for bone tissue engineering application

Bone tissue engineering put emphasis on fabrication three-dimensional biodegradable porous scaffolds that possess ability to enhance adhesion, proliferation and differentiation of osteoblast cells, therefore supporting bone regeneration and functional bone tissue formation. The aim of this work was to fabricate novel tri-component scaffolds composed of chitosan, β-1,3-glucan, and bioceramics and to evaluate their basic structural, mechanical, and biological properties. It should be noted that we are the first who describe fabrication and characterization of tri-component composites containing β-1,3-glucan. Microstructure of novel composites was visualized by computed tomography scanning and SEM. Compressive strength and Youngs modulus of the composites were evaluated by compression testing. The biocompatibility was assessed in vitro by cytotoxicity, cell attachment and cell proliferation tests using human foetal osteoblast cell line. Our results demonstrated that novel composites possess good compressive strength as the effect of polysaccharide components of scaffolds, are very elastic, are non-toxic, favourable to cell adhesion and promote cell proliferation. However, novel biomaterials revealed relatively low Youngs modulus values. Thus, we infer that fabricated novel composites are promising materials for bone tissue engineering application as cell scaffolds to fill small bone losses rather than as massive bone fillers exposed to mechanical load.

Production of exopolysaccharide by Rhizobium leguminosarum bv. trifolii and its role in bacterial attachment and surface properties

Background and aimsThe acidic exopolysaccharide (EPS) produced by Rhizobium leguminosarum bv. trifolii is required for the establishment of effective symbiosis with compatible host plants (Trifolium spp.). In the rhizobium-legume interaction, early stages of root infection and nodule development have been well studied from a genetic standpoint. However, factors important for colonization of several surfaces by rhizobia, including soil particles and roots, have not yet been thoroughly investigated. The aim of this study was establishing of environmental factors affecting production of EPS by R. leguminosarum bv. trifolii strain 24.2 and the role of this polysaccharide in bacterial surface properties and attachment ability.MethodsBesides the wild-type strain, its derivatives differing in the level of EPS produced were used to these analyses. The ability of attachment to abiotic and biotic surfaces of these strains were established using CFU counting experiments. Three-dimensional structure and other parameters of biofilms formed were characterized in confocal laser scanning microscopy. Electrokinetic (zeta) potential of rhizobial cells were determined using Laser Doppler Velocimetry.ResultsIt was evidenced that the ability of R. leguminosarum bv. trifolii to produce EPS significantly affected bacterial attachment and biofilm formation on both abiotic and biotic surfaces. In addition, the presence of this polysaccharide influenced the zeta potential of rhizobial cells. Mutant strains having a mutation in genes involved in EPS synthesis were significantly impaired in attachment, whereas strains overproducing this polysaccharide showed higher adhesion efficiency to all of the tested materials. EPS facilitated attachment of bacterial cells to the tested surfaces most probably due to hydrophobic interactions and heterogeneity of the envelope surface.ConclusionsEPS produced by R. leguminosarum bv. trifolii plays a significant role in attachment and biofilm formation to both abiotic and biotic surfaces as well as bacterial surface properties.

Prevention of biofilm formation on urinary catheters: Comparison of the sparfloxacin-treated long-term antimicrobial catheters with silver-coated ones†

Urinary catheters are widely used for hospitalized patients and are often associated with high risk of urinary tract infection. The agar and broth diffusion tests, visual TTC (triphenyltetrazolium chloride) method, and confocal scanning laser microscopic (CSLM) observations have shown highly satisfactory antimicrobial and antibiofilm activity of the novel sparfoxacin (SPA)-treated urinary catheters compared with the controversial effectiveness of silver(Ag)-coated catheters against a background of untreated catheters used as controls. SPA-treated catheters were significantly less likely to become colonized (less than 0.01%; inner and outer surfaces against Escherichia coli and Staphylococcus aureus) than both silver-coated (from 0.01% to 39.3 %; outer surface against E. coli and inner surface against S. aureus, resp.) and untreated catheters (from 88.43% to 99.72%; outer and inner surfaces, resp., against S. aureus), and maintained their broad spectrum of antimicrobial and antibiofilm activity during storage for at least 6 months.