Do Curdlan Hydrogels Improved with Bioactive Compounds from Hop Exhibit Beneficial Properties for Skin Wound Healing?

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.

Polymer microspheres modified with pyrazole derivatives as potential agents in anticancer therapy - Preliminary studies.

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.

Novel C60 Fullerenol-Gentamicin Conjugate-Physicochemical Characterization and Evaluation of Antibacterial and Cytotoxic Properties.

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.

Can Extracts from the Leaves and Fruits of the Cotoneaster Species Be Considered Promising Anti-Acne Agents?

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.

Highly Porous Fluorapatite/ß-1,3-Glucan Composite for Bone Tissue Regeneration: Characterization and In-Vitro Assessment of Biomedical Potential.

A novel fluorapatite/glucan composite ("FAP/glucan") was developed for the treatment of bone defects. Due to the presence of polysaccharide polymer (ß-1,3-glucan), the composite is highly flexible and thus very convenient for surgery. Its physicochemical and microstructural properties were evaluated using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), mercury intrusion, mechanical testing and compared with the reference material, which was a hydroxyapatite/glucan composite ("HAP/glucan") with hydroxyapatite granules (HAP) instead of FAP. It was found that FAP/glucan has a higher density and lower porosity than the reference material. The correlation between the Young's modulus and the compressive strength between the materials is different in a dry and wet state. Bioactivity assessment showed a lower ability to form apatite and lower uptake of apatite-forming ions from the simulated body fluid by FAP/glucan material in comparison to the reference material. Moreover, FAP/glucan was determined to be of optimal fluoride release capacity for osteoblasts growth requirements. The results of cell culture experiments showed that fluoride-containing biomaterial was non-toxic, enhanced the synthesis of osteocalcin and stimulated the adhesion of osteogenic cells.

The Chemical and Biological Properties of Nanohydroxyapatite Coatings with Antibacterial Nanometals, Obtained in the Electrophoretic Process on the Ti13Zr13Nb Alloy.

The risk of an early inflammation after implantation surgery of titanium implants has caused the development of different antimicrobial measures. The present research is aimed at characterizing the effects of nanosilver and nanocopper dispersed in the nanohydroxyapatite coatings, deposited on the Ti13Zr13Nb alloy, and on the chemical and biological properties of the coatings. The one-stage deposition process was performed by the electrophoretic method at different contents of nanomaterials in suspension. The surface topography of the coatings was examined with scanning electron microscopy. The wettability was expressed as the water contact angle. The corrosion behavior was characterized by the potentiodynamic technique. The release rate of copper and silver into the simulated body fluid was investigated by atomic absorption spectrometry. The antibacterial efficiency was evaluated as the survivability and adhesion of the bacteria and the growth of the biofilm. The cytotoxicity was assessed for osteoblasts. The results demonstrate that silver and copper increase the corrosion resistance and hydrophilicity. Both elements together effectively kill bacteria and inhibit biofilm growth but appear to be toxic for osteoblasts. The obtained results show that the nanohydroxyapatite coatings doped with nanosilver and nanocopper in a one-stage electrophoretic process can be valuable for antibacterial coatings.

Application of Raman Spectroscopic Imaging to Assess the Structural Changes at Cell-Scaffold Interface.

Raman spectroscopic imaging and mapping were applied to characterise three-compound ceramic composite biomaterial consisting of chitosan, ß-1,3-d-glucan (curdlan) and hydroxyapatite (HA) developed as a bone tissue engineering product (TEP). In this rapidly advancing domain of medical science, the urge for quick, reliable and specific method for products evaluation and tissue-implant interaction, in this case bone formation process, is constantly present. Two types of stem cells, adipose-derived stem cells (ADSCs) and bone marrow-derived stem cells (BMDSCs), were cultured on composite surface. Raman spectroscopic imaging provided advantageous information on molecular differences and spatial distribution of compounds within and between the cell-seeded and untreated samples at a microscopic level. With the use of this, it was possible to confirm composite biocompatibility and bioactivity in vitro. Deposition of HA and changes in its crystallinity along with protein adsorption proved new bone tissue formation in both mesenchymal stem cell samples, where the cells proliferated, differentiated and produced biomineralised extracellular matrix (ECM). The usefulness of spectroscopic Raman imaging was confirmed in tissue engineering in terms of both the organic and inorganic components considering composite-cells interaction.

Bioactive Compounds Obtained from Polish "Marynka" Hop Variety Using Efficient Two-Step Supercritical Fluid Extraction and Comparison of Their Antibacterial, Cytotoxic, and Anti-Proliferative Activities In Vitro.

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, ß-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.

Design, synthesis and antimycobacterial activity of thiazolidine-2,4-dione-based thiosemicarbazone derivatives.

The two series of thiosemicarbazone derivatives with thiazolidine-2,4-dione (TZD) core were designed and synthesized. The antimycobacterial activity of the target compounds was tested against Mycobacterium tuberculosis H37Ra by broth microdilution method with resazurin as an indicator of the metabolic activity of mycobacteria. Conducted studies revealed antimycobacterial activity in the concentration range of 0.031-64 µg/ml for 31 synthesized derivatives with TZD core. The highest antimycobacterial activity (MIC = 0.031-0.125 µg/ml) was demonstrated for the new group of compounds: TZD-based hybrids with 4-unsubstituted thiosemicarbazone substituent. Furthermore, all the tested compounds within this group were characterized by low cytotoxicity. Among tested compounds, two compounds are the most promising potential antimycobacterial agents since they not only show very low MIC values, but also non-toxicity against Vero cells at tested concentration range. High effectiveness and safety of these synthesized compounds makes them promising candidates as antimycobacterial agents.

1,2,4-Triazolin-5-thione derivatives with anticancer activity as CK1γ kinase inhibitors.

The optimization and synthesis of new CK2 and CK1 inhibitors are the basis for the development of new therapeutic strategies for the treatment of cancer and neurodegenerative disorders associated with overexpression and abnormal functioning of these enzymes. Triazole derivatives appear to be especially interesting as potential kinase inhibitors. In this context we synthesized a series of 1,2,4-triazolin-5-thione derivatives as CK1γ kinase inhibitors. The antiproliferative activity of synthesized compounds was assessed against cancer cells: human lung adenocarcinoma (A549), human hepatoma (HepG2), and human breast adenocarcinoma (MCF-7). Compound 1 exhibited antiproliferative potency against A549 cancer cells and was characterized by a selective antiproliferative effect. Additionally, this compound has high apoptotic activity against A549, HepG2, MCF-7 cells and induced only slight amount of necrotic cells in these cell lines. In order to decipher the mechanism of anticancer activity of the studied compounds PASS software was used and these compounds were assayed for the inhibition of CK1γ and CK2α kinases. The reported series of 1,2,4-triazolin-5-thiones inhibits CK1γ and CK2α kinases in micromolar range. The most active compound shows activity against isoform γ3 which at concentration of 50 µM reduced the kinase activity by 69% while at 100 µM by 80%. CK2α was found to be less susceptible to the effects of the triazoles tested, as the reduction in kinase activity by 29% was observed for compound 15, and by 27% for compound 1 only at the concentration of 100 µM. The inhibition of CK1γ and CK2α kinases was rationalized using molecular docking.

Positive Effect of Cold Atmospheric Nitrogen Plasma on the Behavior of Mesenchymal Stem Cells Cultured on a Bone Scaffold Containing Iron Oxide-Loaded Silica Nanoparticles Catalyst.

Low-temperature atmospheric pressure plasma was demonstrated to have an ability to generate different reactive oxygen and nitrogen species (RONS), showing wide biological actions. Within this study, mesoporous silica nanoparticles (NPs) and FexOy/NPs catalysts were produced and embedded in the polysaccharide matrix of chitosan/curdlan/hydroxyapatite biomaterial. Then, basic physicochemical and structural characterization of the NPs and biomaterials was performed. The primary aim of this work was to evaluate the impact of the combined action of cold nitrogen plasma and the materials produced on proliferation and osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells (ADSCs), which were seeded onto the bone scaffolds containing NPs or FexOy/NPs catalysts. Incorporation of catalysts into the structure of the biomaterial was expected to enhance the formation of plasma-induced RONS, thereby improving stem cell behavior. The results obtained clearly demonstrated that short-time (16s) exposure of ADSCs to nitrogen plasma accelerated proliferation of cells grown on the biomaterial containing FexOy/NPs catalysts and increased osteocalcin production by the cells cultured on the scaffold containing pure NPs. Plasma activation of FexOy/NPs-loaded biomaterial resulted in the formation of appropriate amounts of oxygen-based reactive species that had positive impact on stem cell proliferation and at the same time did not negatively affect their osteogenic differentiation. Therefore, plasma-activated FexOy/NPs-loaded biomaterial is characterized by improved biocompatibility and has great clinical potential to be used in regenerative medicine applications to improve bone healing process.

Anti-Acne Action of Peptides Isolated from Burdock Root-Preliminary Studies and Pilot Testing.

This work aimed to study the anti-bacterial, anti-biofilm and anti-oxidant potential effects of low molecular weight (LMW) peptides (Br-p) isolated from burdock (Arctium lappa L.) roots. We conducted a preliminary study to exclude or confirm the antibiotic activity of the LMW peptides fraction of this plant. Br-p were isolated using gel filtration and a 10 kDa cut-off membrane. The obtained peptides were identified by MALDI TOF/TOF. Antibacterial activity was tested against acne strains using diffusion tests, MIC and MBC. The fibroblast cytotoxicity of Br-p was tested, and the selectivity index (SI) value was determined. The fraction of 46 Br-p peptides isolated from burdock root with a molecular weight below 5000 Da and theoretic pI (isoelectric point) of 3.67-11.83 showed a narrow spectrum of activity against Gram-positive acne bacterial strains. One of the Br-p peptides assessed on MALDI RapidDeNovo was LRCDYGRFFASKSLYDPLKKRR cationic peptide. It was analogous to that contained in A. lappa protein, and theoretically it was matched as a peptide with antibiotic nature. Br-p did not show toxicity to fibroblasts in the tested concentration up to 10 mg/mL, obtaining CC50 10 mg/mL. The SI value for the tested Propionibacterium strains ranged from 160 to 320. Finally, an active dressing based on chitosan/alginate/genipin was prepared using freeze-drying. The formed dressing was evaluated for its anti-acne activity. To sum up: preliminary biological studies confirmed the anti-acne properties of the isolated peptide fraction from burdock root and pointed to the possibility of using it to create an active dressing on the skin.

New Application of 1,2,4-Triazole Derivatives as Antitubercular Agents. Structure, In Vitro Screening and Docking Studies.

A series of 1,2,4-triazole derivatives were synthesized and assigned as potential anti-tuberculosis substances. The molecular and crystal structures for the model compounds C1, C12, and C13 were determined using X-ray analysis. The X-ray investigation confirmed the synthesis pathway and the assumed molecular structures for analyzed 1,2,4-triazol-5-thione derivatives. The conformational preferences resulting from rotational degrees of freedom of the 1,2,4-triazole ring substituents were characterized. The lipophilicity (logP) and electronic parameters as the energy of frontier orbitals, dipole moments, NBO net charge distribution on the atoms, and electrostatic potential distribution for all structures were calculated at AM1 and DFT/B3LYP/6-311++G(d,p) level. The in vitro test was done against M. tuberculosis H37Ra, M. phlei, M. smegmatis, and M. timereck. The obtained results clearly confirmed the antituberculosis potential of compound C4, which turned out to be the most active against Mycobacterium H37Ra (MIC = 0.976 µg/mL), Mycobaterium pheli (MIC = 7.81 µg/mL) and Mycobacerium timereck (62.6 µg/mL). Satisfactory results were obtained with compounds C8, C11, C14 versus Myc. H37Ra, Myc. pheli, Myc. timereck (MIC = 31.25-62.5 µg/mL). The molecular docking studies were carried out for all investigated compounds using the Mycobacterium tuberculosis cytochrome P450 CYP121 enzyme as molecular a target connected with antimycobacterial activity.

Synthesis, In Vitro Screening and Docking Studies of New Thiosemicarbazide Derivatives as Antitubercular Agents.

A series of thiosemicarbazide derivatives was designed and synthesized by reaction of carboxylic acid hydrazide with isothiocyanates. The molecular structures of the investigated thiosemicarbazides were confirmed and characterized by spectroscopic analysis. The conformational preference of carbonylthiosemicarbazide chain and intra- and intermolecular interactions in the crystalline state were characterized using X-ray analysis. The antituberculosis activity of the target compounds were tested in vitro against four Mycobacterium strains: M. H37Ra, M. phlei, M. smegmatis, M. timereck. The most active compounds were those with 2-pyridine ring. They exhibited lower minimal inhibitory concentration (MIC) values in the range 7.81⁻31.25 µg/mL in comparison to the other isomers. Compound 5 had activity against M. smegmatis at a concentration of 7.81 µg/mL whereas compound 2 had activity against all tested strains at a concentration of 15.625 µg/mL. The molecular docking studies were performed for investigated compounds using the Mycobacterium tuberculosis glutamine synthetase MtGS as their molecular target.

Synthesis and in vitro antiproliferative and antibacterial activity of new thiazolidine-2,4-dione derivatives.

In our present research, we synthesised new thiazolidine-2,4-diones (12-28). All the newly synthesised compounds were evaluated for antiproliferative and antibacterial activity. Antiproliferative evaluation was carried out using normal human skin fibroblasts and tumour cell lines: A549, HepG2, and MCF-7. The IC50 values were determined for tested compounds revealing antiproliferative activity. Moreover, safety index (SI) was calculated. Among all tested derivatives, the compound 18 revealed the highest antiproliferative activity against human lung, breast, and liver cancer cells. More importantly, the derivative 18 showed meaningfully lower IC50 values when compared to the reference substance, irinotecan, and relatively high SI values. Moreover, newly synthesised compounds were screened for the bacteria growth inhibition in vitro. According to our screening results, most active compound was the derivative 18 against Gram-positive bacteria. Therefore, it may be implied that the novel compound 18 appears to be a very promising agent for anticancer treatment.

Nigella damascena L. Essential Oil-A Valuable Source of ß-Elemene for Antimicrobial Testing.

The most commonly used plant source of ß-elemene is Curcuma wenyujin Y. H. Chen & C. Ling (syn. of Curcuma aromatic Salisb.) with its content in supercritical CO2 extract up to 27.83%. However, the other rich source of this compound is Nigella damascena L. essential oil, in which ß-elemene accounts for 47%. In this work, the effective protocol for preparative isolation of ß-elemene from a new source-N. damascena essential oil-using high performance counter-current chromatography HPCCC was elaborated. Furthermore, since sesquiterpens are known as potent antimicrobials, the need for finding new agents designed to combat multi-drug resistant strains was addressed and the purified target compound and the essential oil were tested for its activity against a panel of Gram-positive and Gram-negative bacteria, fungi, and mycobacterial strains. The application of the mixture of petroleum ether, acetonitrile, and acetone in the ratio 2:1.5:0.5 (v/v) in the reversed phase mode yielded ß-elemene with high purity in 70 min. The results obtained for antimicrobial assay clearly indicated that N. damascena essential oil and isolated ß-elemene exert action against Mycobacterium tuberculosis strain H37Ra.

Antimycobacterial Activity of Cinnamaldehyde in a Mycobacterium tuberculosis(H37Ra) Model.

The purpose of the study was to evaluate the antimycobacterial activity and the possible action mode of cinnamon bark essential oil and its main constituent-cinnamaldehyde-against the Mycobacterium tuberculosis ATCC 25177 strain. Cinnamaldehyde was proved to be the main bioactive compound responsible for mycobacterial growth inhibition and bactericidal effects. The antimycobacterial activity of cinnamaldehyde was found to be comparable with that of ethambutol, one of the first-line anti-TB antibiotics. The selectivity index determined using cell culture studies in vitro showed a high biological potential of cinnamaldehyde. In M. tuberculosis cells exposed to cinnamaldehyde the cell membrane stress sensing and envelope preserving system are activated. Overexpression of clgR gene indicates a threat to the stability of the cell membrane and suggests a possible mechanism of action. No synergism was detected with the basic set of antibiotics used in tuberculosis treatment: ethambutol, isoniazid, streptomycin, rifampicin, and ciprofloxacin.

The Effect of Combining Natural Terpenes and Antituberculous Agents against Reference and Clinical Mycobacterium tuberculosis Strains.

Background: On account of emergence of multi- and extensively drug-resistant Mycobacterium tuberculosis (Mtb) strains, combinations of drugs with natural compounds were tested to search for antibiotic activity enhancers. In this work we studied terpenes (α-pinene, bisabolol, ß-elemene, (R)-limonene, (S)-limonene, myrcene, sabinene), which are the main constituents of essential oil obtained from Mutellina purpurea L., a plant with described antitubercular activity, to investigate their interactions with antibiotics against reference Mtb strains and multidrug-resistant clinical isolates. Methods: The serial dilution method was used to evaluate the minimal inhibitory concentration (MIC) of tested compounds, while the fractional inhibitory concentration index (FICI) was calculated for characterization of interactions. Moreover, IC50 values of tested compounds were determined using monkey kidney epithelial cell line (GMK). Results: The combinations of all studied terpenes with ethambutol or rifampicin resulted in a synergistic interaction. Bisabolol and (R)-limonene decreased the MIC for rifampicin at least two-fold for all tested strains, however no synergistic action was observed against virulent strains. The tested terpenes showed slight (bisabolol) or no cytotoxic effect against normal eukaryotic cells in vitro. Conclusions: The obtained enhanced activity (FICI < 0.5) of ethambutol and rifampicin against H37Ra strain under the influence of the studied terpenes may be correlated to the capability of essential oil constituents to modify bacterial resistance mechanisms in general. The observed differences in avirulent and virulent bacteria susceptibility to terpenes tested separately and in combinations with antibiotics can be correlated with the differences in the cell wall structure between H37Ra mutant and all virulent strains.

New approach in evaluation of ceramic-polymer composite bioactivity and biocompatibility.

Regeneration of bone defects was promoted by a novel ß-glucan/carbonate hydroxyapatite composite and characterized by Raman spectroscopy, microCT and electron microscopy. The elastic biomaterial with an apatite-forming ability was developed for bone tissue engineering and implanted into the critical-size defects of rabbits' tibiae. The bone repair process was analyzed on non-decalcified bone/implant sections during a 6-month regeneration period. Using spectroscopic methods, we were able to determine the presence of amides, lipids and assign the areas of newly formed bone tissue. Raman spectroscopy was also used to assess the chemical changes in the composite before and after the implantation process. SEM analyses showed the mineralization degree in the defect area and that the gap size decreased significantly. Microscopic images revealed that the implant debris were interconnected to the poorly mineralized inner side of a new bone tissue. Our study demonstrated that the composite may serve as a biocompatible background for collagen ingrowth and exhibits the advantages of applying Raman spectroscopy, SEM and microCT in studying these samples.

A simple and effective protocol for fast isolation of human Tenon's fibroblasts from a single trabeculectomy biopsy - a comparison of cell behaviour in different culture media.

BACKGROUND: Human Tenon's fibroblasts (HTFs) play a crucial role in wound healing. They cause postoperative scarring of the filtering bleb and are thus responsible for trabeculectomy failure. This study aimed to find an effective and fast protocol for HTF isolation from trabeculectomy biopsies. The protocol was compared with the commonly recommended HTF isolation procedure, which uses Dulbecco's modified Eagle's medium (DMEM). We used Eagle's minimum essential medium (EMEM) enriched with fibroblast growth factor (FGF), which selectively promoted the proliferation of HTF cells. A secondary goal was to compare HTF morphology, metabolism and growth during parallel cultivation of the isolated cells in FGF-enriched EMEM and DMEM. RESULTS: Standard procedures for HTF isolation from tissue biopsies require a 20- to 30-day culture of the explants to obtain the first monolayer. Our protocol yielded the first monolayer after approx. 15 days. More importantly, the majority of the cells were fibroblasts with only individual epithelium-derived cells present. Using FGF-enriched EMEM allowed 1.3 × 106 vimentin-positive fibroblasts to be obtained from a single biopsy within approx. 25 days. Using DMEM resulted in isolation failure and required exchange to FGF-enriched medium to recover the fibroblast culture. HTFs maintained in FGF-enriched EMEM also showed faster proliferation and a different type I collagen production ability compared to HTFs cultured in DMEM. Thus, FGF-enriched EMEM is recommended for fast propagation of HTFs unless the aim of the study is to assess the effect of a tested agent on proliferation ability or type I collagen production. CONCLUSIONS: Our fast protocol for HTF isolation allows easy setup of cell banks by researchers under laboratory conditions and could be very useful during testing of novel ophthalmologic anti-fibrotic agents in vitro. Molecular analysis of HTFs isolated from patients with known treatment histories may provide valuable information on the effects of some medications taken before glaucoma surgery on the subsequent wound-healing process and potential for trabeculectomy failure.