Anna Belcarz

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.

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.

Biphasic mode of antibacterial action of aminoglycoside antibiotics-loaded elastic hydroxyapatite–glucan composite

Following the quest for new composite materials for bone tissue engineering, a novel elastic hydroxyapatite-glucan composite loaded with two aminoglycoside antibiotics was prepared. The porosity of the composite and the drug release profiles in closed-loop and semi-open systems were tested. The antibacterial activity of the drug was estimated against two Gram-positive and two Gram-negative bacterial strains causing orthopedic infections. It was found that the loaded antibiotic acted in a biphasic mode. The majority of the drug was released within 48-119 h in a pore-dependent manner and inhibited the bacterial growth in the culture medium. However, a small residual amount of the drug was bound to the composite microstructure via ionic interactions and acted as a short-lived barrier against bacterial adhesion to the composite, although the surrounding medium was no longer protected against bacterial infection. Sub-inhibitory concentrations of the released drug were observed in the medium only during the last two days of the experiment (minimized risk of occurrence of drug-resistant strains). Thus the novel drug-loaded elastic hydroxyapatite-glucan composite, demonstrating a biphasic mode of antibacterial action, may be recommended for antibiotic prophylaxis in bone substitute implantation, with less emphasis on the treatment of bone infections.

TiO2 nanotube composite layers as delivery system for ZnO and Ag nanoparticles — An unexpected overdose effect decreasing their antibacterial efficacy

Enhancement of biocompatibility and antibacterial properties of implant materials is potentially beneficial for their practical value. Therefore, the use of metallic and metallic oxide nanoparticles as antimicrobial coatings components which induce minimized antibacterial resistance receives currently particular attention. In this work, TiO2 nanotubes layers loaded with ZnO and Ag nanoparticles were designed for biomedical coatings and delivery systems and evaluated for antimicrobial activity. TiO2 nanotubes themselves exhibited considerable and diameter-dependent antibacterial activity against planktonic Staphylococcus epidermidis cells but favored bacterial adhesion. Loading of nanotubes with moderate amount of ZnO nanoparticles significantly diminished S. epidermidis cell adhesion and viability just after 1.5h contact with modified surfaces. However, an increase of loaded ZnO amount unexpectedly altered the structure of nanoparticle-nanolayer, caused partial closure of nanotube interior and significantly reduced ZnO solubility and antibacterial efficacy. Co-deposition of Ag nanoparticles enhanced the antibacterial properties of synthesized coatings. However, the increase of ZnO quantity on Ag nanoparticles co-deposited surfaces favored the adhesion of bacterial cells. Thus, ZnO/Ag/TiO2 nanotube composite layers may be promising delivery systems for combating post-operative infections in hard tissue replacement procedures. However, the amount of loaded antibacterial agents must be carefully balanced to avoid the overdose and reduced efficacy.

Do Ca2+-adsorbing ceramics reduce the release of calcium ions from gypsum-based biomaterials?

Bone implantable materials based on calcium sulfate dihydrate dissolve quickly in tissue liquids and release calcium ions at very high levels. This phenomenon induces temporary toxicity for osteoblasts, may cause local inflammation and delay the healing process. Reduction in the calcium ion release rate by gypsum could be therefore beneficial for the healing of gypsum-filled bone defects. The aim of this study concerned the potential use of calcium phosphate ceramics of various porosities for the reduction of high Ca(2+) ion release from gypsum-based materials. Highly porous ceramics failed to reduce the level of Ca(2+) ions released to the medium in a continuous flow system. However, it succeeded to shorten the period of high calcium level. It was not the phase composition but the high porosity of ceramics that was found crucial for both the shortening of the Ca(2+) release-related toxicity period and intensification of apatite deposition on the composite. Nonporous ceramics was completely ineffective for this purpose and did not show any ability to absorb calcium ions at a significant level. Moreover, according to our observations, complex studies imitating in vivo systems, rather than standard tests, are essential for the proper evaluation of implantable biomaterials.

Do Ca2+-chelating polysaccharides reduce calcium ion release from gypsum-based biomaterials?

BackgroundCalcium sulphate, a widely used bone filler, may negatively affect human osteoblasts due to release of high quantities of calcium ions. To reduce this effect, an attempt was made to enrich calcium sulphate with Ca2+-chelating plant and rhizobial exopolysaccharides (EPS).MethodologyIncubation of polysaccharide-enriched calcium sulphate composites was performed in DMEM/F12 medium. Ca2+ (and Mg2+ and Pi) levels were estimated using standardised, spectrophotometry-based kits. Composite surface morphology was tested using SEM technique.ResultsRhizobial EPS was found slightly less effective at Ca2+ chelation than sodium alginate. Both polysaccharides may be used as gypsum supplements in the form of setting liquids (0.3% total mass), but only sodium alginate may be used as a powder (up to 5% total mass of the composite). Polysaccharide-triggered reduction of Ca2+ release reached the level of 50% during the first 2.5 h of incubation, then decreased significantly.ConclusionsBoth tested polysaccharides possess calcium-chelating properties. However, although alginate caused a reduction in Ca2+ levels in the media incubated with the gypsum samples, the reduction was too short lived to provide a long-term effect. Further modification of the composite content using calcium-deficient hydroxyapatite and low-molecular weight rhizobial EPS with higher solubility could bring more satisfactory results.

Behavior of new hydroxyapatite/glucan composite in human serum

Biomaterials for bone tissue regeneration, including polymer-based composites, are typically evaluated in vitro prior to the clinical trials. However, such composites tested in vivo may behave different due to the specific body conditions. For example, some composites implanted into the tissue acidified due to transient postoperative inflammation may unexpectedly swell which delays the wound healing. Such massive swelling in acidic medium was previously observed for new elastic hydroxyapatite (HAp)/β-glucan biomaterial. However, in further clinical cases concerning the composite implantation in patients without significant inflammation indicators, no side effects were observed. Therefore, it was reasonable to test the effect of human serum of neutral pH (typical for noninflamed tissues) on the composite parameters, in particular volume changes. Thus, this article shows the characterization of physicochemical parameters of the composite after incubation (5 days) in human serum of neutral pH by means of weight and volume measurement, scanning electron microscopy, X-ray diffraction, Fourier-transform infrared spectroscopy, microcomputed tomography, mercury intrusion, and biochemical techniques. Results showed that human serum collected from healthy people caused no uncontrolled changes in weight and volume, porosity and mechanical properties of the composite. Therefore, this suggests the lack of volume change-related side effects of HAp/glucan composite in bone defects treatment if postoperative inflammation is prevented.

Do new N-substituted 3-amino-4-phenyl-5-oxo-pyrazolinecarboxamide derivatives exhibit antitubercular potential?

&NA; As a continuation of previous tests concerning new N‐substituted 3‐amino‐4‐phenyl‐5‐oxo‐pyrazolinecarboxamide derivatives (R3, R4 and R8) of notable antibacterial activity, their antitubercular potential against different mycobacterial strains was estimated. Tests performed on virulent (reference and clinical) strains of Mycobacterium bovis and Mycobacterium tuberculosis revealed the highest therapeutic potential of R8 derivative: MIC within the range 7.8–15.6 &mgr;g/ml and TI (therapeutic index) within the range 46.5–93. Moreover, the synergistic interaction was found between R3, R4 and R8 derivatives and rifampicin, one of the front‐line antitubercular drugs. R8/rifampicin mixture in concentrations effective in inhibition of Mycobacterium tuberculosis strain was non‐cytotoxic against GMK cells, displaying cell viability approximately 88–97% when compared to control. Molecular docking study enabled to conclude that enoyl acyl carrier protein reductase (InhA) can be considered as a potential molecular target of tested pyrazole derivatives. Although further modifications of chemical structure of the investigated pyrazole derivatives is required, in order to increase their antitubercular efficacy and therapeutic safety, these compounds, in particular R8 compound, can be promising for the treatment of human and bovine tuberculosis. Graphical abstract Figure. No caption available.

Metal TiO2 Nanotube Layers for the Treatment of Dental Implant Infections

Titanium oxide nanotube layers with silver and zinc nanoparticles are attracting increasing attention in the design of bone and dental implants due to their antimicrobial potential and their ability to control host cell adhesion, growth, and differentiation. However, recent reports indicate that the etiology of dental infections is more complex than has been previously considered. Therefore, the antimicrobial potential of dental implants should be evaluated against at least several different microorganisms cooperating in human mouth colonization. In this study, Ag and Zn nanoparticles incorporated into titanium oxide nanotubular layers were studied with regard to how they affect Candida albicans, Candida parapsilosis, and Streptococcus mutans. Layers of titanium oxide nanotubes with an average diameter of 110 nm were fabricated by electrochemical anodization, annealed at 650 °C, and modified with approx. 5 wt % Ag or Zn nanoparticles. The surfaces were examined with the scanning electron microscopy-energy dispersive X-ray analysis, scanning transmission electron microscopy, and X-ray photoelectron spectroscopy techniques and subjected to evaluation of microbial-killing and microbial adhesion-inhibiting potency. In a 1.5 h long adhesion test, the samples were found more effective toward yeast strains than toward S. mutans. In a release-killing test, the microorganisms were almost completely eliminated by the samples, either within 3 h of contact (for S. mutans) or 24 h of contact (for both yeast strains). Although further improvement is advisable, it seems that Ag and Zn nanoparticles incorporated into TiO2 nanotubular surfaces provide a powerful tool for reducing the incidence of bone implant infections. Their high bidirectional activity (against both Candida species and S. mutans) makes the layers tested particularly promising for the design of dental implants.