Phosphoglucose isomerase (EC 5.3.1.9; PGI) is an enzyme participating in the metabolism of the sugar and carrying the reversible isomerization of glucose-6-phosphate and D-fructose-6-phosphate. It plays key role in the process of the extracellular polysaccharides (EPS) synthesis in vivo – creating the bacterial biofilm. The complete understanding of PGI catalysis and inhibition mechanisms may results in discovery of new PGI inhibitors – antibacterial drugs. In this pilot study we described the procedure of partial purification of E. coli ATCC 25922 PGI from cytosolic fraction. The activity of enzymes was measured by the conversion of D-fructose-6P into D-glucose-6P. Two forms (isoenzymes I and II) of this enzyme were separated. Some properties of dominant form (isoenzyme I) of PGI were studied. The Km constant for D-fructose-6P was 1 mM. The enzyme had optimum activity in TRIS/HCl buffer, pH 7.65. Phosphoglucose isomerase had molecular weight 120 kDa. Our results indicated that amino acids - cysteine, tryptophan, tyrosine and lysine residues were to be presented in the active site of PGI from E. coli ATCC 25922. The lack of the activity inhibition of characterized enzyme by chelators as EDTA, 1,10-phenantroline and TRIS indicated that the PGI is not a metallprotein. A large similarity between properties of phosphoglucose isomerases isolated from E. coli ATCC 25922 and other E. coli strains such as K10 and ATCC 35218 was discovered.

The development of biomaterials for medical applications is an evolving subject in the area of human health care. Osteoconductive properties and biocompatibility of calcium phosphate ceramic materials such as hydroxyapatite (HAp) allow their use in skeletal tissue engineering. In order to overcome their limitations, such as brittleness, various composites have been developed so far. Combining different materials in composites generates better physiochemical properties of biomaterial. However, such a new mixture needs special attention in terms of its biocompatibility. Biomaterials used in bone repair, by nature of their in vivo applications, inevitably cause multi gene response of host organism. Gene microarray technology has been used successfully in bioengineering to characterize genes expression on mRNA level. The poor correlation between mRNA level and corresponding protein expression leads to the development of protein microarray. Direct screening of the proteins level changes in cells responding to different biomaterials stimulation, permits evaluation of their biocompatibility. Our study has been conducted with osteoblast cell line stimulated by hydroxyapatite and hydroxyapatite/plant-derived polymer composite. The gene expression changes have been analyzed on the protein level with the use of The Panorama Ab Microarray Cell Signaling kit. The microarray tool for cellular-signaling protein has been chosen for noting the development of changes in signaling pathways with the use of pathway-finder software. In our studies, cells stimulated by HAp and HAp/plant-derived polymer composite showed no differences in the level of protein expression in almost 90%. Interestingly, the p53-controlled signaling pathways have not been changed by hydroxyapatite composite. Further analysis should be performed to identify the role of up- or down-regulated proteins in apoptosis and cell cycle pathways crucial for bioengineering applications.

The aim of this work is to provide a comprehensive set of biological tests to assess the biomedical potential of novel osteochondral scaffolds with methods proposed to comply with the 3Rs principle, focusing here on a biphasic Curdlan-based osteochondral scaffold as a promising model biomaterial. In vitro experiments include the evaluation of cytotoxicity, mutagenicity, and genotoxicity referring to ISO standards, the assessment of the viability and proliferation of human chondrocytes and osteoblasts, and the estimation of inflammation after direct contact of biomaterials with human macrophages. Ex vivo experiments include assessments of the response of the surrounding osteochondral tissue after incubation with the implanted biomaterial. In vivo experiments involve an evaluation of the toxicity and regenerative potential of the biomaterial in zebrafish (larvae and adults) and in osteochondral defects in dogs (veterinary patients). The applied set of tests allows us to show that the Curdlan-based scaffold does not induce cytotoxicity (cell viability close to 100%), mutagenicity (the level of reversion is not 2× higher compared to the control), and genotoxicity (it does not exhibit any change in chromosomal aberration; the frequency of micronuclei, micronucleated binucleated cells, and cytokinesis-block proliferation index is comparable to the control; moreover, it does not cause the formation of comets in cells). This biomaterial also promotes the viability and proliferation of chondrocytes and osteoblasts (the OD values between the fourth and seventh day of incubation increase by approximately 1.6×). The Curdlan-based scaffold stimulates only a transient inflammatory response in vitro and ex vivo. This biomaterial does not cause Danio rerio larvae malformation and also enables proper regeneration of the caudal fin in adults. Finally, it supports the regeneration of an osteochondral defect in veterinary patients. Thus, this is a proposal to use alternative methods for biological assessment of osteochondral scaffolds as opposed to commonly used tests using large numbers of laboratory animals.

Calcium phosphate ceramic materials such as hydroxyapatite HAp (Ca10(PO4)6(OH)2) are used for skeletal tissue engineering because of their biocompatibility and osteoconductive properties. The interconnected porous structure, good mechanical properties, biocompatibility of biodegradable composite provide a suitable microenvironment to promote osteoblast proliferation and osteogenesis. Culturing of osteogenic cells on a porous HAp scaffold offers a new solution to bone grafting using autologous human mesenchymal stem cells (hMSC) from the patient. These scaffolds could be designed as a perfectly fit replacements to reconstruct the patient's skeleton. Before their use as bone replacement, HAp and its composites should be tested in vivo and in vitro. Microarray is a tool for gene expresion analysis of human osteoblasts in response to HAp biomaterials. By using bioactive ceramic materials that mimic the mineral composition of natural bones, the stimulation of a gene expression which is involved in the commitment of MPCs to osteoblasts was noted by microarray technology. Among up-regulated eleven genes were the genes encoding SOX9 (transcription factor SOX-9) and ERK (extracellular-signal-regulated protein kinases).

An attempt was made to develop a simple, rapid, specific, precise and accurate RP-HPLC method with combined fluorescence and spectrophotometric detection and with simple mobile phase for determination of sparfloxacin (SPA). The comparison of both detection methods showed no significant differences with reference to precision and accuracy. A method thus developed was applied for the quantification of sparfloxacin in pure substance. The method was validated according to the ICH guidelines in respect to linearity, limit of detection, precision, accuracy, robustness, and selectivity. The developed HPLC method was successfully applied for assay of SPA pharmaceutical formulation to check the applicability of the proposed methodology.

Some reports in the literature show the advantages of fluoride-containing apatite ceramics over hydroxyapatite (HAP), at least in some aspects. While HAP has been used extensively in the treatment of bone defects, fluoridated apatite has hardly been tested in vivo. In order to verify the biological properties of fluoride-doped apatite and to assess its therapeutic potential, we synthesized fluorapatite (FAP) and applied it as a filling in bone defects of experimental animals (rabbits). The treatment effects were evaluated on extracted bones after 3 and 6 months from implantation using peripheral quantitative computed tomography (pQCT), dual-energy X-ray absorptiometry (DXA), radiography (X-ray) and histological staining. The study proved the integration between FAP and the bone tissue, thus indicating its stimulating effect on new bone formation and mineralization. The results achieved after 3 months of treatment were difficult to interpret unequivocally and suggested the transient delay in FAP integration of bone in comparison with HAP. The reasons for this phenomenon are unclear. Most likely, these differences between FAP and HAP resulted mainly from the different porosities, densities and ionic reactivity of the ceramics, which in our opinion affected their solubility, integration and degree of bone tissue resorption. However, it was shown that 6 months after implantation, similar level of bone defect regeneration was achieved for both FAP and HAP. In this article, we present our hypothesis concerning the basis of this phenomenon.

Chronic wounds, among others, are mainly characterized by prolonged inflammation associated with the overproduction of reactive oxygen species and pro-inflammatory cytokines by immune cells. As a consequence, this phenomenon hinders or even precludes the regeneration process. It is known that biomaterials composed of biopolymers can significantly promote the process of wound healing and regeneration. The aim of this study was to establish whether curdlan-based biomaterials modified with hop compounds can be considered as promising candidates for the promotion of skin wound healing. The resultant biomaterials were subjected to an evaluation of their structural, physicochemical, and biological in vitro and in vivo properties. The conducted physicochemical analyses confirmed the incorporation of bioactive compounds (crude extract or xanthohumol) into the curdlan matrix. It was found that the curdlan-based biomaterials improved with low concentrations of hop compounds possessing satisfactory hydrophilicity, wettability, porosity, and absorption capacities. In vitro, tests showed that these biomaterials were non-cytotoxic, did not inhibit the proliferation of skin fibroblasts, and had the ability to inhibit the production of pro-inflammatory interleukin-6 by human macrophages stimulated with lipopolysaccharide. Moreover, in vivo studies showed that these biomaterials were biocompatible and could promote the regeneration process after injury (study on Danio rerio larvae model). Thus, it is worth emphasizing that this is the first paper demonstrating that a biomaterial based on a natural biopolymer (curdlan) improved with hop compounds may have biomedical potential, especially in the context of skin wound healing and regeneration.

The methods of fighting cancer are far from ideal, therefore it is necessary to search for innovative and effective drugs. In our work, we present pyrazole derivatives and their modifications with polymer microspheres as potential anticancer agents. Molecular and crystal structures of pyrazole derivatives were determined an X-ray analysis and characterized by theoretical calculations. Modifications of cross-linked polymer microspheres with pyrazole derivatives were made on the basis of divinylbenzene and glycidyl methacrylate. The in vitro antiproliferative activity of the pyrazole derivatives and their modified microspheres was assessed against a normal cell line, namely monkey epithelial renal cells (GMK) and cancer cell lines, such as human hepatocellular carcinoma cell line (HepG2), human breast adenocarcinoma cell line (MCF-7) as well as human lung adenocarcinoma cell line (A549), using the MTT assay. All the tested pyrazole derivatives and the polymer microspheres modified by them showed antiproliferative activity in vitro. Two of the modified substances showed the greatest ability to inhibit divisions of all cancer cells. In order to determine a potential target, molecular docking was performed. In silico studies carried out with the use of the human EphB1 receptor revealed that the analyzed compounds bound to the EphB1 binding site, and the compounds with the highest antiproliferative activity showed a better fit to the active site.

Guided tissue regeneration and guided bone regeneration membranes are some of the most common products used for bone regeneration in periodontal dentistry. The main disadvantage of commercially available membranes is their lack of bone cell stimulation and easy bacterial colonization. The aim of this work was to design and fabricate a new membrane construct composed of electrospun poly (D,L-lactic acid)/poly (lactic-co-glycolic acid) fibers sonocoated with layers of nanoparticles with specific properties, i.e., hydroxyapatite and bimetallic nanocomposite of zinc oxide–silver. Thus, within this study, four different variants of biomaterials were evaluated, namely: poly (D,L-lactic acid)/poly (lactic-co-glycolic acid) biomaterial, poly(D,L-lactic acid)/poly (lactic-co-glycolic acid)/nano hydroxyapatite biomaterial, poly (D,L-lactic acid)/poly (lactic-co-glycolic acid)/nano zinc oxide–silver biomaterial, and poly (D,L-lactic acid)/poly (lactic-co-glycolic acid)/nano hydroxyapatite/nano zinc oxide–silver biomaterial. First, it was demonstrated that the wettability of biomaterials—a prerequisite property important for ensuring desired biological response—was highly increased after the sonocoating process. Moreover, it was indicated that biomaterials composed of poly (D,L-lactic acid)/poly (lactic-co-glycolic acid) with or without a nano hydroxyapatite layer allowed proper osteoblast growth and proliferation, but did not have antibacterial properties. Addition of a nano zinc oxide–silver layer to the biomaterial inhibited growth of bacterial cells around the membrane, but at the same time induced very high cytotoxicity towards osteoblasts. Most importantly, enrichment of this biomaterial with a supplementary underlayer of nano hydroxyapatite allowed for the preservation of antibacterial properties and also a decrease in the cytotoxicity towards bone cells, associated with the presence of a nano zinc oxide–silver layer. Thus, the final structure of the composite poly (D,L-lactic acid)/poly (lactic-co-glycolic acid)/nano hydroxyapatite/nano zinc oxide–silver seems to be a promising construct for tissue engineering products, especially guided tissue regeneration/guided bone regeneration membranes. Nevertheless, additional research is needed in order to improve the developed construct, which will simultaneously protect the biomaterial from bacterial colonization and enhance the bone regeneration properties.

The number of bone fractures and cracks requiring surgical interventions increases every year; hence, there is a huge need to develop new potential bone scaffolds for bone regeneration. The goal of this study was to gain knowledge about the basic properties of novel curdlan/whey protein isolate/hydroxyapatite biomaterials in the context of their use in bone tissue engineering. The purpose of this research was also to determine whether the concentration of whey protein isolate in scaffolds has an influence on their properties. Thus, two biomaterials differing in the concentration of whey protein isolate (i.e., 25 wt.% and 35 wt.%; hereafter called Cur_WPI25_HAp and Cur_WPI35_HAp, respectively) were fabricated and subjected to evaluation of porosity, mechanical properties, swelling ability, protein release capacity, enzymatic biodegradability, bioactivity, and cytocompatibility towards osteoblasts in vitro. It was found that both biomaterials fulfilled a number of requirements for bone scaffolds, as they demonstrated limited swelling and the ability to undergo controllable enzymatic biodegradation, to form apatite layers on their surfaces and to support the viability, growth, proliferation, and differentiation of osteoblasts. On the other hand, the biomaterials were characterized by low open porosity, which may hinder the penetration of cells though their structure. Moreover, they had low mechanical properties compared to natural bone, which limits their use to filling of bone defects in non-load bearing implantation areas, e.g., in the craniofacial area, but then they will be additionally supported by application of mechanically strong materials such as titanium plates. Thus, this preliminary in vitro research indicates that biomaterials composed of curdlan, whey protein isolate, and hydroxyapatite seem promising for bone tissue engineering applications, but their porosity and mechanical properties should be improved. This will be the subject of our further work.

This study aimed to evaluate the phenolic profile and biological activity of the extracts from the leaves and fruits of Cotoneaster nebrodensis and Cotoneaster roseus. Considering that miscellaneous species of Cotoneaster are thought to be healing in traditional Asian medicine, we assumed that this uninvestigated species may reveal significant therapeutic properties. Here, we report the simultaneous assessment of chemical composition as well as biological activities (antioxidant, anti-inflammatory, antibacterial, and cytotoxic properties) of tested species. Complementary LC-MS analysis revealed that polyphenols (especially flavonoids and proanthocyanidins) are the overriding phytochemicals with the greatest significance in tested biological activities. In vitro chemical tests considering biological activities revealed that obtained results showed different values depending on concentration, extraction solvent as well as phenolic content. Biological assays demonstrated that the investigated extracts possessed antibacterial properties and were not cytotoxic toward normal skin fibroblasts. Given the obtained results, we concluded that knowledge of the chemical composition and biological activities of investigated species are important to achieve a better understanding of the utilization of these plants in traditional medicine and be useful for further research in their application to treat various diseases, such as skin disorders.

This study aimed to develop, characterize, and evaluate antibacterial and cytotoxic properties of novel fullerene derivative composed of C60 fullerenol and standard aminoglycoside antibiotic–gentamicin (C60 fullerenol-gentamicin conjugate). The successful introduction of gentamicin to fullerenol was confirmed by X-ray photoelectron spectroscopy which together with thermogravimetric and spectroscopic analysis revealing the formula of the composition as C60(OH)12(GLYMO)11(Gentamicin)0.8. The dynamic light scattering (DLS) revealed that conjugate possessed ability to form agglomerates in water (size around 115 nm), while Zeta potential measurements demonstrated that such agglomerates possessed neutral character. In vitro biological assays indicated that obtained C60 fullerenol-gentamicin conjugate possessed the same antibacterial activity as standard gentamicin against Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Escherichia coli, which proves that combination of fullerenol with gentamicin does not cause the loss of antibacterial activity of antibiotic. Moreover, cytotoxicity assessment demonstrated that obtained fullerenol-gentamicin derivative did not decrease viability of normal human fibroblasts (model eukaryotic cells) compared to control fibroblasts. Thus, taking into account all of the results, it can be stated that this research presents effective method to fabricate C60 fullerenol-gentamicin conjugate and proves that such derivative possesses desired antibacterial properties without unfavorable cytotoxic effects towards eukaryotic cells in vitro. These promising preliminary results indicate that obtained C60 fullerenol-gentamicin conjugate could have biomedical potential. It may be presumed that obtained fullerenol may be used as an effective carrier for antibiotic, and developed fullerenol-gentamicin conjugate may be apply locally (i.e., at the wound site). Moreover, in future we will evaluate possibility of its applications in inter alia tissue engineering, namely as a component of wound dressings and implantable biomaterials.

The purpose of this pilot study was to establish whether a novel freeze-dried curdlan/whey protein isolate-based biomaterial may be taken into consideration as a potential scaffold for matrix-associated autologous chondrocyte transplantation. For this reason, this biomaterial was initially characterized by the visualization of its micro- and macrostructures as well as evaluation of its mechanical stability, and its ability to undergo enzymatic degradation in vitro. Subsequently, the cytocompatibility of the biomaterial towards human chondrocytes (isolated from an orthopaedic patient) was assessed. It was demonstrated that the novel freeze-dried curdlan/whey protein isolate-based biomaterial possessed a porous structure and a Young's modulus close to those of the superficial and middle zones of cartilage. It also exhibited controllable degradability in collagenase II solution over nine weeks. Most importantly, this biomaterial supported the viability and proliferation of human chondrocytes, which maintained their characteristic phenotype. Moreover, quantitative reverse transcription PCR analysis and confocal microscope observations revealed that the biomaterial may protect chondrocytes from dedifferentiation towards fibroblast-like cells during 12-day culture. Thus, in conclusion, this pilot study demonstrated that novel freeze-dried curdlan/whey protein isolate-based biomaterial may be considered as a potential scaffold for matrix-associated autologous chondrocyte transplantation.

Iron oxide on mesoporous silica gave a synergy with plasma jet for HO˙ radical production at neutral pH.

This paper presents the properties of the wood-resin composites. For improving their antibacterial character, silver nanoparticles were incorporated into their structures. The properties of the obtained materials were analyzed in vitro for their anti-biofilm potency in contact with aerobic Gram-positive Staphylococcus aureus and Staphylococcus epidermidis; and aerobic Gram-negative Escherichia coli and Pseudomonas aeruginosa. These pathogens are responsible for various infections, including those associated with healthcare. The effect of silver nanoparticles incorporation on mechanical and thermomechanical properties as well as gloss were investigated for the samples of composites before and after accelerating aging tests. The results show that bacteria can colonize in various wrinkles and cracks on the composites with wood flour but also the surface of the cross-linked unsaturated polyester resin. The addition of nanosilver causes the death of bacteria. It also positively influences mechanical and thermomechanical properties as well as gloss of the resin.

Given the health-beneficial properties of compounds from hop, there is still a growing trend towards developing successful extraction methods with the highest yield and also receiving the products with high added value. The aim of this study was to develop efficient extraction method for isolation of bioactive compounds from the Polish "Marynka" hop variety. The modified two-step supercritical fluid extraction allowed to obtain two hop samples, namely crude extract (E1), composed of α-acids, β-acids, and terpene derivatives, as well as pure xanthohumol with higher yield than that of other available methods. The post-extraction residues (R1) were re-extracted in order to obtain extract E2 enriched in xanthohumol. Then, both samples were subjected to investigation of their antibacterial (anti-acne, anti-caries), cytotoxic, and anti-proliferative activities in vitro. It was demonstrated that extract (E1) possessed more beneficial biological properties than xanthohumol. It exhibited not only better antibacterial activity against Gram-positive bacteria strains (MIC, MBC) but also possessed a higher synergistic effect with commercial antibiotics when compared to xanthohumol. Moreover, cell culture experiments revealed that crude extract neither inhibited viability nor divisions of normal skin fibroblasts as strongly as xanthohumol. In turn, calculated selectivity indexes showed that the crude extract had from slightly to significantly better selective anti-proliferative activity towards cancer cells in comparison with xanthohumol.

In the present study structural characteristics and physicochemical properties of tri-component biomaterial (consisting of chitosan, beta-1,3-glucan and hydroxyapatite) seeded with mesenchymal stem cells were investigated with the use of diffuse reflectance infrared Fourier transform spectroscopy (DRIFT), X-ray photoelectron spectroscopy (XPS), and scanning electron microscopy (SEM). In this study we use non-conventional approach of DRIFT spectroscopy for investigating biomaterial changes under simulated physiological conditions. Particular cell-induced changes were intended to be properly evaluated with analytical methods. Abovementioned techniques allowed to precisely assess the changes on the surface of the biomaterial caused by two kinds of stem cells (ADSCs - Adipose tissue-Derived Stem Cells and BMDSCs - Bone Marrow-Derived Stem Cells) cultured directly on the surface of bioceramic-based biomaterial. The bioactivity and biocompatibility of designed bone biomaterial were demonstrated and hence it seems to be a promising scaffold used in tissue engineering. Designed chitosan, beta-1,3-glucan, and hydroxyapatite biomaterial was proven to be non-toxic, surgically handy with cellular compatibility. The obtained results are interesting and promising in terms of spectroscopic methods suitability for qualitative assessment of material-cell interactions. (C) 2021 Elsevier B.V. All rights reserved.

The aim of this work was to establish whether novel curdlan-based hydrogels enriched with Ca2+ ions may be considered as potential candidates for dressings, for the acceleration of skin wound healing. Firstly, biomaterials were allocated for evaluation of structural and mechanical properties. Subsequently, the ability of hydrogels to absorb simulated wound fluid and water vapor permeability, as well their capacity to release calcium ions, was evaluated. The biocompatibility of biomaterials was assessed using normal human skin fibroblasts. Importantly, the main features of the obtained curdlan-based hydrogels were compared with those of KALTOSTAT® (a commercial calcium sodium alginate wound dressing). The obtained results showed that curdlan-based biomaterials possessed a mesoporous structure (pore diameter ranged from 14–48 nm) and exhibited a good ability to absorb simulated wound fluid (swelling ratio close to 974–1229%). Moreover, in a wet state, they enabled proper water vapor transmission rate (>2000 g/m2/day), thanks to their hydrogel structure. Finally, it was found that biomaterial composed of 11 wt.% of curdlan (Cur_11%) possessed the most desirable biological properties in vitro. It released a beneficial amount of calcium ions to the aqueous environment (approximately 6.12 mM), which significantly enhanced fibroblast viability and proliferation. Taking into account the beneficial properties of Cur_11% biomaterial, it seems justified to subject it to more advanced cell culture experiments in vitro and to in vivo studies in order to determine its precise influence on skin wound healing.