Biomechanical Aspects in Bone Tumor Engineering.

In the past decades, anticancer drug development brought the field of tumor engineering to a new level by the need of robust test systems. Simulating tumor microenvironment in vitro remains a challenge, and osteosarcoma-the most common primary bone cancer-is no exception. The growing evidence points to the inevitable connection between biomechanical stimuli and tumor chemosensitivity and aggressiveness, thus making this component of the microenvironment a mandatory requirement to the developed models. In this review, we addressed the question: is the "in vivo - in vitro" gap in osteosarcoma engineering bridged from the perspective of biomechanical stimuli? The most notable biomechanical cues in the tumor cell microenvironment are observed and compared in the contexts of in vivo conditions and engineered three-dimensional in vitro models. Impact statement The importance of biomechanical stimuli in three-dimensional in vitro models for drug testing is becoming more pronounced nowadays. This review might assist in understanding the key players of the biophysical environment of primary bone cancer and the current state of bone tumor engineering from this perspective.

A 3D Biomimetic System for Testing Anticancer Drug Sensitivity.

3D cultures of cancer cells enable better mimicking of physiological conditions compared to traditional monolayer 2D cultures. Here we describe alginate scaffold-based model that can be used in both static and biomimetic conditions for studying drug sensitivity in cancer cells and multidrug resistance (MDR) mechanisms. This 3D culture model resembles in vivo conditions and provides relevant and reproducible results. It is easy to set up and allows for facile manipulation for downstream analyses. All these remarkable features make this 3D culture model a promising tool in drug discovery and cancer cell biology research.

Effects of poly(vinyl alcohol) blending with Ag/alginate solutions to form nanocomposite fibres for potential use as antibacterial wound dressings.

In this work, nanocomposite fibres and microfibres based on alginate and poly(vinyl alcohol) (PVA) with silver nanoparticles (AgNPs) were produced and characterized for potential application as antibacterial wound dressings. PVA/Ag/Na-alginate colloid solution was used for the preparation of the fibres by a simple extrusion technique followed by freezing-thawing cycles. UV-Visible spectroscopy confirmed successful preservation of AgNPs in fibres while Fourier transform infrared spectroscopy has shown a balanced combined effect on the Ca-alginate spatial arrangement with the addition of both AgNPs and PVA. The presence of PVA in fibres induced an increase in the swelling degree as compared with that of Ag/Ca-alginate fibres (approx. 28 versus approx. 14). Still, the initially produced PVA/Ca-alginate fibres were mechanically weaker than Ca-alginate fibres, but after drying and rehydration exhibited better mechanical properties. Also, the obtained fibres released AgNPs and/or silver ions at the concentration of approximately 2.6 µg cm-3 leading to bacteriostatic effects against Staphylococcus aureus and Escherichia coli. These results are relevant for practical utilization of the fibres, which could be stored and applied in the dry form with preserved mechanical stability, sorption capacity and antibacterial activity.

Development and Validation of a Long-Term 3D Glioblastoma Cell Culture in Alginate Microfibers as a Novel Bio-Mimicking Model System for Preclinical Drug Testing.

BACKGROUND: Various three-dimensional (3D) glioblastoma cell culture models have a limited duration of viability. Our aim was to develop a long-term 3D glioblastoma model, which is necessary for reliable drug response studies. METHODS: Human U87 glioblastoma cells were cultured in alginate microfibers for 28 days. Cell growth, viability, morphology, and aggregation in 3D culture were monitored by fluorescent and confocal microscopy upon calcein-AM/propidium iodide (CAM/PI) staining every seven days. The glioblastoma 3D model was validated using temozolomide (TMZ) treatments 3 days in a row with a recovery period. Cell viability by MTT and resistance-related gene expression (MGMT and ABCB1) by qPCR were assessed after 28 days. The same TMZ treatment schedule was applied in 2D U87 cell culture for comparison purposes. RESULTS: Within a long-term 3D model system in alginate fibers, U87 cells remained viable for up to 28 days. On day 7, cells formed visible aggregates oriented to the microfiber periphery. TMZ treatment reduced cell growth but increased drug resistance-related gene expression. The latter effect was more pronounced in 3D compared to 2D cell culture. CONCLUSION: Herein, we described a long-term glioblastoma 3D model system that could be particularly helpful for drug testing and treatment optimization.

Novel composite scaffolds based on alginate and Mg-doped calcium phosphate fillers: Enhanced hydroxyapatite formation under biomimetic conditions.

In the present study, we synthesized hydroxyapatite (HAP) powders followed by the production of alginate based macroporous scaffolds with the aim to imitate the natural bone structure. HAP powders were synthesized by using a hydrothermal method, and after calcination, dominant phases in the powders, undoped and doped with Mg2+ were HAP and ß-tricalcium phosphate, respectively. Upon mixing with Na-alginate, followed by gelation and freeze-dying, highly macroporous composite scaffolds were obtained with open and connected pores and uniformly dispersed mineral phase as determined by scanning electron microscopy. Mechanical properties of the scaffolds were influenced by the composition of calcium phosphate fillers being improved as Ca2+ concentration increased while Mg2+ concentration decreased. HAP formation within all scaffolds was investigated in simulated body fluid (SBF) during 28 days under static conditions while the best candidate (Mg substituted HAP filler, precursor solution with [Ca + Mg]/P molar ratio of 1.52) was investigated under more physiological conditions in a biomimetic perfusion bioreactor. The continuous SBF flow (superficial velocity of 400 µm/s) induced the formation of abundant HAP crystals throughout the scaffolds leading to improved mechanical properties to some extent as compared to the initial scaffolds. These findings indicated potentials of novel biomimetic scaffolds for use in bone tissue engineering.

Evaluation of Nisin and LL-37 Antimicrobial Peptides as Tool to Preserve Articular Cartilage Healing in a Septic Environment.

Cartilage repair still represents a challenge for clinicians and only few effective therapies are nowadays available. In fact, surgery is limited by the tissue poor self-healing capacity while the autologous transplantation is often forsaken due to the poor in vitro expansion capacity of chondrocytes. Biomaterials science offers a unique alternative based on the replacement of the injured tissue with an artificial tissue-mimicking scaffold. However, the implantation surgical practices and the scaffold itself can be a source of bacterial infection that currently represents the first reason of implants failure due to the increasing antibiotics resistance of pathogens. So, alternative antibacterial tools to prevent infections and consequent device removal are urgently required. In this work, the role of Nisin and LL-37 peptides has been investigated as alternative to antibiotics to their antimicrobial performances for direct application at the surgical site or as doping chemicals for devices aimed at articular cartilage repair. First, peptides cytocompatibility was investigated toward human mesenchymal stem cells to determine safe concentrations; then, the broad-range antibacterial activity was verified toward the Gram-positive Staphylococcus aureus and Staphylococcus epidermidis as well as the Gram-negative Escherichia coli and Aggregatibacter actinomycetemcomitans pathogens. The peptides selective antibacterial activity was verified by a cells-bacteria co-culture assay, while chondrogenesis was assayed to exclude any interference within the differentiation route to simulate the tissue repair. In the next phase, the experiments were repeated by moving from the cell monolayer model to 3D cartilage-like spheroids to revisit the peptides activity in a more physiologically relevant environment model. Finally, the spheroid model was applied in a perfusion bioreactor to simulate an infection in the presence of circulating peptides within a physiological environment. Results suggested that 75 µg/ml Nisin can be considered as a very promising candidate since it was shown to be more cytocompatible and potent against the investigated bacteria than LL-37 in all the tested models.

Preclinical functional characterization methods of nanocomposite hydrogels containing silver nanoparticles for biomedical applications.

Nanocomposite hydrogels that contain silver nanoparticles (AgNPs) are especially attractive for various biomedical applications (e.g., antimicrobial wound dressings, coatings and soft tissue implants) due to strong antimicrobial activity of released silver nanoparticles and/or ions over prolonged times. However, all potential biomedical products have to be thoroughly specified fulfilling strict safety requirements. Characterization of nanocomposites is additionally complicated due to potential harmful effects of nanoparticles and accumulation in cells and tissues. This paper summarizes methods for preclinical characterization of hydrogel nanocomposites containing AgNPs with the particular attention on Ag/alginate hydrogels. Standard physicochemical characterization methods include transmission electron microscopy (TEM), scanning electron microscopy (SEM) and atomic force microscopy (AFM), UV-visible spectroscopy, and Fourier transform infrared spectroscopy (FTIR). Functional in vitro characterization relies on different methods for estimation of silver release, antimicrobial activity, and nanocomposite cytotoxicity. Here, we specially focus on utilization of 3D bioreactor systems that mimic native physiological environments with the aim to reliably predict nanocomposite behavior during implementation and so to decrease the need for animal experimentation. These systems were shown to provide more accurate and relevant data on silver release and cytotoxicity as compared to static systems such as 2D cell monolayer cultures. Finally, nanocomposites are evaluated in vivo in different animal models, which are in the case of wound dressings typically mice, rats, and pigs. The present review provides a basis for defining a strategy for comprehensive and efficient preclinical characterization of novel nanocomposites attractive not only for those containing AgNPs but also other metallic nanoparticles aimed for biomedical applications.Key points• A platform for devising comprehensive preclinical evaluation of nanocomposites. • Biomimetic bioreactors provide reliable functional nanocomposite evaluation. • Cells in 2D cultures are more sensitive to silver nanoparticles than in 3D cultures. • Biomimetic bioreactor 3D cell/tissue cultures can address the in vitro-in vivo gap.

Novel nano-composite hydrogels with honey effective against multi-resistant clinical strains of Acinetobacter baumannii and Pseudomonas aeruginosa.

Novel alginate hydrogels with silver nanoparticles (AgNPs) and honey components were produced with the aim to target multidrug-resistant bacterial strains causing nosocomial wound infections. AgNP synthesis was optimized in highly concentrated honey solutions so that a 5-month stable, colloid solution with 50% of honey and ~ 8 nm AgNPs at neutral pH was obtained. The colloid solution was further used to produce nano-composite Ag/alginate hydrogels in different forms (microbeads, microfibers and discs) that retained all AgNPs and high fractions of honey components (40-60%) as determined by the phenol-sulfuric acid and Folin-Ciocalteu methods. The hydrogels were characterized by UV-Vis spectroscopy and Fourier-transform infrared-attenuated total reflectance spectroscopy while the antibacterial activity was investigated against a broad spectrum of Gram-negative and Gram-positive bacteria, including 13 multi-resistant clinical strains of Acinetobacter baumannii, one clinical strain of Pseudomonas aeruginosa and one clinical strain of Staphylococcus aureus. At the total released silver concentration of ~ 9 µg/ml, the hydrogels exhibited strong bactericidal activity against standard and most of the investigated multi-resistant hospital strains with the exemption of 3 clinical strains of A. baumannii in which antibacterial effects were absent. These results reveal the need for further in-depth studies of bacterial resistance mechanisms and, in the same time, potentials of the novel Ag/alginate hydrogels with honey components to combat wound infections and enhance healing as non-sticky, antibacterial, and bioactive dressings.

Multifunctional ternary composite films based on PLA and Ag/alginate microbeads: Physical characterization and silver release kinetics.

Novel multifunctional composite poly(lactic acid) (PLA) films with alginate microbeads containing silver nanoparticles (AgNPs) were developed for potential antimicrobial food packaging applications. AgNPs, 10-20 nm in size, were synthesized in a Na-alginate solution by a hydrothermal method yielding a sterile, pH neutral colloid solution of low viscosity that was electrostatically extruded to produce Ag/alginate microbeads (190 µm in size) with retained AgNPs. Dried microbeads were uniformly dispersed in PLA films with retained AgNPs as confirmed by UV-Vis spectroscopy and scanning electron microscopy. The films were characterized regarding thermal and mechanical properties as well as silver release in different food simulants. Results show that PLA matrix served as a diffusion barrier so that the released silver concentration in water after 10 days was within the prescribed limit of 0.05 mg kg-1 while the films induced inhibitory effects against Staphylococcus aureus in the agar diffusion test.

Functional bioreactor characterization to assess potentials of nanocomposites based on different alginate types and silver nanoparticles for use as cartilage tissue implants.

In this work, functional characterization of biomaterials concerning potential application as articular cartilage implants was performed by using a biomimetic bioreactor with dynamic compression in the physiological regime (10% strain, 0.84 Hz frequency, 1 h on/1 h off). Specifically, two alginate types with low (LG) and high (HG) guluronic/mannuronic residue ratios with electrochemically synthesized silver nanoparticles (AgNPs) were evaluated. HG Ag/alginate hydrogels were clearly indicated as potential candidates due to better initial mechanical properties as compared to LG hydrogels (dynamic compression modulus of ~60 vs. ~40 kPa) as well as the mechanical stability displayed during 7 days of dynamic compression. Cytotoxicity studies in 3D bovine cartilage explant cultures under dynamic compression have shown negligible effects as compared to standard 2D monolayers of bovine chondrocytes where moderate cytotoxicity was observed. Finally, experimental and mathematical modeling studies revealed different mechanisms of AgNP release under physiological-like bioreactor conditions as compared to static conditions. Overall, the results clearly demonstrate bioreactor advantages in characterization and selection of candidate biomaterials as well as potentials to bridge the in vitro-in vivo gap. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 755-768, 2019.

Achieving high antimicrobial activity: Composite alginate hydrogel beads releasing activated charcoal with an immobilized active agent.

New composites based on Ca-alginate hydrogels were produced that release activated charcoal (AC) particles with adsorbed povidone iodine (PVP-I) as a model antimicrobial substance in a physiological-like environment. Composite beads with different alginate (0.5-1.5%w/w) and AC (1-20%w/w) concentrations were analyzed by FE-SEM and characterized regarding textural parameters, swelling, and AC release kinetics. PVP-I was easily adsorbed onto AC particles within the optimized beads (0.5%w/w alginate, 20%w/w AC) as indicated by UV-vis spectroscopy, EDX and FT-IR analyses. The obtained beads have shown strong bactericidal effects against two standard bacterial strains (Staphylococcus aureus and Pseudomonas aeruginosa) and clinical multi-resistant wound isolates (MRSA, Escherichia coli, Pseudomonas aeruginosa, Еnterococcus faecalis and Proteus mirabilis) and, at the same time, exhibited negligible PVP-I desorption in physiological saline solution. Thus, the obtained composites could provide utilization of potent antiseptics such as iodine, in wound dressings, without the concern of systemic absorption.

Comparative in vivo evaluation of novel formulations based on alginate and silver nanoparticles for wound treatments.

In the present study, possibilities for using novel nanocomposites based on alginate and silver nanoparticles for wound treatment were investigated in a second-degree thermal burn model in Wistar rats. Silver nanoparticles (AgNPs) were electrochemically synthesized in alginate solutions that were further utilized to obtain the Ag/alginate solution and microfibers for subsequent in vivo studies. Daily applications of the Ag/alginate colloid solution, containing AgNPs, alginate and ascorbic acid (G3), wet Ag/alginate microfibers containing AgNPs (G5) and dry Ag/alginate microfibers containing AgNPs (G6) were compared to treatments with a commercial cream containing silver sulfadiazine (G2) and a commercial Ca-alginate wound dressing containing silver ions (G4), as well as to the untreated controls (G1). Results of the in vivo study have shown faster healing in treated wounds, which completely healed on day 19 (G4, G5 and G6) and 21 (G2 and G3) after the thermal injury, while the period for complete reepitelization of untreated wounds (G1) was 25 days. The macroscopic analysis has shown that scabs fell off between day 10 and 12 after the thermal injury induction in treated groups, whereas between day 15 and 16 in the control group. These macroscopic findings were supported by the results of histopathological analyses, which have shown enhanced granulation and reepithelization, reduced inflammation and improved organization of the extracellular matrix in treated groups without adverse effects. Among the treated groups, dressings based on Ca-alginate (G4-G6) induced enhanced healing as compared to the other two groups (G2, G3), which could be attributed to additional stimuli of released Ca2+. The obtained results indicated potentials of novel nanocomposites based on alginate and AgNPs for therapeutic applications in wound treatments.

Bioreactors with hydrostatic pressures imitating physiological environments in intervertebral discs.

Intervertebral discs are normally exposed to a variety of loads and stresses but hydrostatic pressure (HP) could be the main biosignal for chondrogenic cell differentiation and maintenance of this tissue. Although there are simple approaches to intermittently expose cell cultures to HP in separate material testing devices, utilization of biomimetic bioreactors aiming to provide in vitro conditions mimicking those found in vivo, attracts special attention. However, design of such bioreactors is complex due to the requirement of high HP magnitudes (up to 3 MPa) applied in different regimes mimicking pressures arising in intervertebral disc during normal daily activities. Furthermore, efficient mass transfer has to be facilitated to cells within 3D scaffolds, and the engineering challenges include avoidance or removal of gas bubbles in the culture medium before pressurization as well as selection of appropriate, biocompatible construction materials and maintenance of sterility during cultivation. Here, we review approaches to induce HP in 2D and 3D cell cultures categorized into 5 groups: (I) discontinuous systems with direct pressurization of the cultivation medium by a piston, (II) discontinuous systems with indirect pressurization by a compression fluid, (III) continuous systems with direct pressurization of the cultivation medium, static culture, (IV) continuous systems with culture perfusion, and (V) systems applying HP in conjunction with other physical signals. Although the complexity is increasing as additional features are added to the systems, the need to understand HP effects on cells and tissues in a physiologically relevant, yet precisely controlled, environment together with current technological advancements are leading towards innovative bioreactor solutions.

Dissolution of copper mineral phases in biological fluids and the controlled release of copper ions from mineralized alginate hydrogels.

Here we investigate the dissolution behaviour of copper minerals contained within biocompatible alginate hydrogels. Copper has a number of biological effects and has most recently been evaluated as an alternative to expensive and controversial growth factors for applications in tissue engineering. Precise control and sustained release of copper ions are important due to a narrow therapeutic window of this potentially toxic ion, and alginate would appear to be a good material of choice for this purpose. We found that aqueously insoluble copper minerals could be precipitated during gelling within or mixed into alginate hydrogels in the form of microbeads prior to gelling to serve as depots of copper. These minerals were found to be soluble in a variety of biological fluids relevant to in vitro and in vivo investigations, and the alginate carrier served as a barrier to diffusion of these ions and therefore offered control over the rate and duration of release (Cu(2+) release rates observed between 10-750 µMol g(-1) h(-1) and duration for up to 32 d). Copper mineral and copper mineralized alginate microbeads were characterized using powder x-ray diffraction, FTIR, thermogravimetric analysis and scanning electron microscopy. Dissolution kinetics were studied based on measurements of copper ion concentrations using colourimetric methods. In addition we characterized the complexes formed between released copper ions and biological fluids by electron paramagnetic spectroscopy which offers an insight into the behaviour of these materials in the body.

A comprehensive approach to in vitro functional evaluation of Ag/alginate nanocomposite hydrogels.

In this work, we present a comprehensive approach to evaluation of alginate microbeads with included silver nanoparticles (AgNPs) at the concentration range of 0.3-5mM for potential biomedical use by combining cytotoxicity, antibacterial activity, and silver release studies. The microbeads were investigated regarding drying and rehydration showing retention of ∼ 80-85% of the initial nanoparticles as determined by UV-vis and SEM analyses. Both wet and dry microbeads were shown to release AgNPs and/or ions inducing similar growth delays of Staphylococcus aureus and Escherichia coli at the total released silver concentrations of ∼ 10 µg/ml. On the other hand, these concentrations were highly toxic for bovine chondrocytes in conventional monolayer cultures while nontoxic when cultured in alginate microbeads under biomimetic conditions in 3D perfusion bioreactors. The applied approach outlined directions for further optimization studies demonstrating Ag/alginate microbeads as potentially attractive components of soft tissue implants as well as antimicrobial wound dressings.

Bioreactor validation and biocompatibility of Ag/poly(N-vinyl-2-pyrrolidone) hydrogel nanocomposites.

Silver/poly(N-vinyl-2-pyrrolidone) (Ag/PVP) nanocomposites containing Ag nanoparticles at different concentrations were synthesized using γ-irradiation. Cytotoxicity of the obtained nanocomposites was determined by MTT assay in monolayer cultures of normal human immunocompetent peripheral blood mononuclear cells (PBMC) that were either non-stimulated or stimulated to proliferate by mitogen phytohemagglutinin (PHA), as well as in human cervix adenocarcinoma cell (HeLa) cultures. Silver release kinetics and mechanical properties of nanocomposites were investigated under bioreactor conditions in the simulated body fluid (SBF) at 37°C. The release of silver was monitored under static conditions, and in two types of bioreactors: perfusion bioreactors and a bioreactor with dynamic compression coupled with SBF perfusion simulating in vivo conditions in articular cartilage. Ag/PVP nanocomposites exhibited slight cytotoxic effects against PBMC at the estimated concentration of 0.4 µmol dm(-3), with negligible variations observed amongst different cell cultures investigated. Studies of the silver release kinetics indicated internal diffusion as the rate limiting step, determined by statistically comparable results obtained at all investigated conditions. However, silver release rate was slightly higher in the bioreactor with dynamic compression coupled with SBF perfusion as compared to the other two systems indicating the influence of dynamic compression. Modelling of silver release kinetics revealed potentials for optimization of Ag/PVP nanocomposites for particular applications as wound dressings or soft tissue implants.

Novel kinetic model of the removal of divalent heavy metal ions from aqueous solutions by natural clinoptilolite.

Removal of heavy metal ions from aqueous solutions using zeolites is widely described by pseudo-second order kinetics although this model may not be valid under all conditions. In this work, we have extended approaches used for derivation of this model in order to develop a novel kinetic model that is related to the ion exchange mechanism underlying sorption of metal ions in zeolites. The novel model assumed two reversible steps, i.e. release of sodium ions from the zeolite lattice followed by bonding of the metal ion. The model was applied to experimental results of Cu(II) sorption by natural clinoptilolite-rich zeolitic tuff at different initial concentrations and temperatures and then validated by predictions of ion exchange kinetics of other divalent heavy metal ions (i.e. Mn(II), Zn(II) and Pb(II)). Model predictions were in excellent agreements with experimental data for all investigated systems. In regard to the proposed mechanism, modeling results implied that the sodium ion release rate was constant for all investigated metals while the overall rate was mainly determined by the rate of heavy metal ion bonding to the lattice. In addition, prediction capabilities of the novel model were demonstrated requiring one experimentally determined parameter, only.

Novel alginate based nanocomposite hydrogels with incorporated silver nanoparticles.

Alginate colloid solution containing electrochemically synthesized silver nanoparticles (AgNPs) was investigated regarding the nanoparticle stabilization and possibilities for production of alginate based nanocomposite hydrogels in different forms. AgNPs were shown to continue to grow in alginate solutions for additional 3 days after the synthesis by aggregative mechanism and Ostwald ripening. Thereafter, the colloid solution remains stable for 30 days and could be used alone or in mixtures with aqueous solutions of poly(vinyl alcohol) (PVA) and poly(N-vinyl-2-pyrrolidone) (PVP) while preserving AgNPs as verified by UV-Vis spectroscopy studies. We have optimized techniques for production of Ag/alginate microbeads and Ag/alginate/PVA beads, which were shown to efficiently release AgNPs decreasing the Escherichia coli concentration in suspensions for 99.9% over 24 h. Furthermore, Ag/hydrogel discs based on alginate, PVA and PVP were produced by freezing-thawing technique allowing adjustments of hydrogel composition and mechanical properties as demonstrated in compression studies performed in a biomimetic bioreactor.

Evaluation of alginate hydrogels under in vivo-like bioreactor conditions for cartilage tissue engineering.

Alginate hydrogels in forms of discs and packed beds of microbeads (~800 µm) were tested in a novel bioreactor at 10% strain using two regimes: at a loading rate of 337.5 µm/s and at sequential increments of 50 µm displacement every 30 min. Compressive strength increased with the increase in alginate concentration (1.5 vs. 2% w/w) and the content of guluronic residues (38.5 vs. 67%). Packed beds of microbeads exhibited significantly higher (~1.5-3.4 fold) compression moduli than the respective discs indicating the effects of gel form and entrapped water. Short-term cultivation of microbeads with immobilized bovine calf chondrocytes (1.5% w/w, 33 × 10(6) cells/ml) under biomimetic conditions (dynamic compression: 1 h on/1 h off, 0.42 Hz, 10% strain) resulted in cell proliferation and bed compaction, so that the compression modulus slightly increased. Thus, the novel bioreactor demonstrated advantages in evaluation of biomaterial properties and cell-biomaterial interactions under in vivo-like settings.

Application of Electrostatic Extrusion - Flavour Encapsulation and Controlled Release.

The subject of this study was the development of flavour alginate formulationsaimed for thermally processed foods. Ethyl vanilline was used as the model flavourcompound. Electrostatic extrusion was applied for the encapsulation of ethyl vanilline inalginate gel microbeads. The obtained microbeads with approx. 10 % w/w of ethylvanilline encapsulated in about 2 % w/w alginate were uniformly sized spheres of about450 µm. Chemical characterization by H-NMR spectroscopy revealed that the alginateused in this study had a high content (67 %) of guluronic residues and was rich in GG diadblocks (FGG = 55%) and thus presented a high-quality immobilisation matrix. The thermalbehaviour of alginate beads encapsulating ethyl vanilline was investigated bythermogravimetric (TG) and differential scanning calorimetry measurements (TG-DSC)under heating conditions which mimicked usual food processing to provide informationabout thermal decomposition of alginate matrix and kinetics of aroma release. Two wellresolved weight losses were observed. The first one was in the 50-150 °C temperaturerange with the maximum at approx. 112 °C, corresponding to the dehydration of thepolymer network. The second loss in the 220-325 °C temperature range, with a maximumat ~ 247 °C corresponded to the release of vanilline. The obtained results indicate that up to230 °C most of the vanilline remained intacta, while prolonged heating at elevatedtemperatures led to the entire loss of the aroma compound.