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Comprehensive research, particularly in evaluating drug efficacy, still heavily relies on the results obtained by the utilization of cell monolayers and animals. However, the inherent limitations of these models such as their physiological disparities from humans pose significant obstacles to acquiring reliable results thus impeding further scientific progression. To address this challenge, 3D in vitro cell culture models emerged as physiologically relevant models having the potential to enhance research and drug discovery. Our study aimed to develop a 3D in vitro cell culture model based on bone-like scaffolds in conjunction with a perfusion bioreactor (“3D Perfuse”, Innovation Center FTM, Belgrade, Serbia) for studying both physiological and pathological (i.e. tumors) bone conditions.

The slow advance in anticancer drug development can be attributed to the limitations of conventional models, predominantly monolayer cell (2D) cultures and animal models, which inadequately recapitulate the complex nature of human malignant tumors. Three-dimensional (3D) in vitro models are invaluable tools in drug screening; however, creating a universal model for all cancer types poses challenges due to the diverse nature of cancers. The aim of this work was to develop a single, versatile model using alginate microfibers to accommodate cultivation of various cancer cells.

Development of novel, effective, and safe anti-tumor drugs is still a slow and cumbersome process, which is often attributed to weaknesses of current preclinical assays and low correlation of the preclinical in vitro and in vivo data with the results obtained in clinical trials. Consequently, there is a clear need for development of more reliable in vitro three dimensional (3D) tumor models, which will capture key features of the in vivo tumor cell microenvironment and provide drug testing results relevant for human patients. The aim of the project “Biomimetic tumor engineering to enhance drug discovery – BioengineeredTumor” funded by the Science Fund of the Republic of Serbia is to develop 2 novel, simple and robust 3D models for cultures of carcinoma and osteosarcoma cells by applying systematic and integrated methodology to comprehensively define the key model components. In specific, the aim is to use different human and animal cancer cell lines in conjunction with alginate-based biomaterials as artificial extracellular matrices imitating tumor environments and to cultivate the obtained constructs in perfusion bioreactors providing enhanced transport of nutrients, gases and biochemical signals to the cells as well as adequate levels of hydrodynamic shear stresses. Thus, the strategic goal is to establish an adaptable platform suited to the use by scientists without technical expertise for long-term in vitro studies of cancer cells for applications in anti-cancer drug discovery and validation, development of personalized medical treatments, and cancer research.

Osteosarcoma (OS) is a highly aggressive primary malignant bone tumor that most commonly affects children, adolescents, and young adults. The standard treatment for OS consists of surgical resection and chemotherapy, whereas radiation therapy is recommended for the unresectable tumor. Due to its easy metastasis and recurrence, the 5-year overall survival rate is only 66.5 %. Thus, there is a critical need to recognize the molecular mechanisms underlying OS development and pathogenesis. Traditionally, two-dimensional (2D) cells are widely used in cancer biology and pre-clinical studies. However, 2D models are unable to mimic cell–cell and cell-extracellular matrix interactions which are crucial for adequate cellular function. Three-dimensional (3D) model systems are able to recapitulate key features of human cancer and are recognized as a promising platform for fundamental and translational research. In the present work, we established an osteosarcoma 3D model based on alginate microbeads and studied the effect of combined treatment with doxorubicin (Doxo), widely used chemotherapeutic, and quercetin (Quer), a plant pigment with anticancer properties, on OS model systems.

Traditional cotton wound dressings, like gauze and bandages, remain popular in wound care due to their affordability. However, they have drawbacks: adhering to wounds, risking tissue damage and low absorbance of secretions, requiring multiple layers, and causing discomfort. Modern alternatives, such as alginate hydrogel dressings, target these issues. Designed for moderate to intense exudate wounds, they enhance comfort and treatment effectiveness. Yet, their higher cost limits accessibility. Moreover, neither alginate nor cotton gauze offer bioactivity, while mechanical strength of alginate hydrogel may be inadequate. This work aims to develop enhanced gauzes to overcome these challenges, offering improved functionality at affordable costs and superior wound care.

Tehničko rešenje obuhvata formulaciju i tehnologiju dobijanja unapređenih gaza za tretman rana koje su porozne, ojačane tkaninom i ne lepe se za ranu, pri tome upijaju sekret rane i regulišu njenu vlažnost, dok kontrolisano mogu da otpuštaju različite aktivne komponente, kao što su npr. joni srebra ili cinka. Prema tome, tehničko rešenje pripada oblasti materijali i hemijske tehnologije.

Introduction: The NT2/D1 embryonal carcinoma cell line represents a well-established in vitro model of human neurogenesis. It’s widely used for studying neurodevelopmental processes, neurotoxicity, and neurodegenerative disorders. The utilization of alginate fibers as a 3D cell culture system offers a biocompatible and structurally supportive environment for neural differentiation and maturation of cells, making it a suitable tool for investigating neurodevelopmental processes. Methods: In thisstudy, we evaluated the alginate microfibers as a 3D modelsystem for in vitro neural differentiation of NT2/D1 cells.We described the immobilization of NT2/D1 cellsin alginate microfibers and the effect of propagation in this 3D model on morphological features, viability, and proliferation of immobilized cells. We also assessed the RA-induced initiation of neural differentiation of NT2/D1 cellsin alginate microfibers by comparison with the initiation of neural differentiation in adherent 2D cell culture. Results: Our results showed that immobilized NT2/D1 acquired morphological features characteristic of cells propagated in 3D model systems and retain viability, proliferative capacity, and ability to attach to adherent surfaces. In addition, immobilized NT2/D1 cells preserved neural differentiation capacity. Upon RA induction we detected a marked decrease in the expression of specific pluripotency-maintaining markers, SOX2, OCT4, and NANOG. Consecutively, the expression of early neural markers, SOX3, PAX6, and miR219 was significantly increased. Conclusion: Neural differentiation of NT2/D1 cellsimmobilized within alginate fibersrepresents a highly promising 3D modelsystem forstudying human neurogenesis and offers a valuable platform forscreening the effect of drugs and bioactive compounds on human neural differentiation.

Development of novel biomaterials for potential biomedical applications requires comprehensive preclinical studies. Traditional methods for biomaterial evaluation are based on in vitro testing routinely performed in monolayer cell cultures followed by in vivo animal studies. However, these methods have numerous limitations. Although studies in cell monolayers allow rapid evaluation of biomaterials by standardized protocols, quantitative and comparable results, cell metabolism and morphology is changed in the 2-dimensional environment often leading to unreliable results. On other hand, animal studies are complex, time-consuming, expensive and raise ethical concerns. One of the approaches to address this problem and obtain reliable results in a more efficient way is utilization of biomimetic bioreactors. These bioreactors are primarily developed as an essential component in tissue engineering mimicking physiological in vivo conditions in particular tissue or organ by providing all necessary biochemical (e.g. pH, nutrients, gases, growth factors) and biophysical signals (e.g., shear stress, hydrostatic pressure, mechanical strains) for cell differentiation and metabolic activity. Some examples include perfusion bioreactors, bioreactors with shear stresses and/or dynamic compression, and bioreactor with stretch and shear stresses imitating conditions in vascularized tissues, articular cartilage, and vascular grafts, respectively. Such physiologically relevant, while strictly controlled environment is also beneficial for biomaterial assessment, investigation of cell-biomaterial interactions, and prediction of biomaterial behaviour upon application. The present review provides readers with up-to-date studies and results regarding utilization of biomimetic bioreactors as tools for comprehensive and efficient evaluation of novel biomaterials, such as determination of mechanical characteristics, release of bioactive substances, cell-biomaterial interactions and cytotoxicity.

Biomaterials engineering is one of the strongest research areas at the Faculty of Technology and Metallurgy (FTM), University of Belgrade including development of novel biomaterials for orthopaedic and dental applications. Especially, synthesis and physico-chemical characterization methods are well established resulting in variety of novel inorganic, polymer and composite biomaterials produced in different forms with controlled compositions and structures. Still, expertise in further advancement of the research results towards utilization in medical devices is lacking. The ExcellMater Horizon 2020 project aims to further advance the scientific and technological capacity of the Faculty in this area by twinning the knowledge from the three European partners, internationally leading institutions, with recognized expertise in biological characterization methods, intellectual property protection and utilization, evaluation of health technologies and regulatory aspects of medical devices. The aim of the project is also to increase visibility of scientific research and enhance collaboration of engineers at FTM with life scientists, medical doctors and pharmacists as well as with industry. Here, we will present some of the novel bioceramic and composite biomaterials developed at FTM aimed for potential applications in orthopaedics, dentistry, bone tissue engineering, and cancer research.

A multidisciplinary approach based on experiments and mathematical modeling was used in biomimetic system development for three-dimensional (3D) cultures of cancer cells. Specifically, two cancer cell lines, human embryonic teratocarcinoma NT2/D1 and rat glioma C6, were immobilized in alginate microbeads and microfibers, respectively, and cultured under static and flow conditions in perfusion bioreactors. At the same time, chemical engineering methods were applied to explain the obtained results. The superficial medium velocity of 80 mu m s(-1) induced lower viability of NT2/D1 cells in superficial microbead zones, implying adverse effects of fluid shear stresses estimated as similar to 67 mPa. On the contrary, similar velocity (100 mu m s(-1)) enhanced the proliferation of C6 glioma cells within microfibers compared to static controls. An additional study of silver release from nanocomposite Ag/honey/alginate microfibers under perfusion indicated that the medium partially flows through the hydrogel (interstitial velocity of similar to 10 nm s(-1)). Thus, a diffusion-advection-reaction model described the mass transport to immobilized cells within microfibers. Substances with diffusion coefficients of similar to 10(-)(19)-10(-11) m(2) s(-)(1) are sufficiently supplied by diffusion only, while those with significantly lower diffusivities (similar to 10(-1)(9) m(2) s(-1)) require additional convective transport. The present study demonstrates the selection and contribution of chemical engineering methods in tumor model system development.

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 beta-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 mu m/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.

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.

The exibits presents a range of new multifunctional biomaterials for potential medical application. Biocompatible composites based on hydrogels of alginate, polyvinyl alcohol, and chitosan with incorporated bioactive agents such as silver nanoparticles, honey and activated charcoal particles with adsorbed povidone-iodine provide efficient release of the immoblized agents and antibacterial activity, which is attractive for applocation in wound dressing. Bioactive macroporous composites based on polzmer hzdrogels with bioceramic particles as well as metal foundations with multicomponent coatings are attractive as bone tissue implants. Finallz, dental inserts were developed for dentin replacement. In addition, the exibit will include a bioreactor system for cultivation of cancer cells in alginate hydrogels in 3-dimensionalenvironment, aimed to provide more reliable antitumor drug testing and development of personalized medical therapies.

Cancer is the second leading cause of death globally, making the search for its cure one of the most important challenges of the 21st century. With ethical questions regarding animal testing and inconsistency of results of cancer drug testing in standard two-dimensional (2D) monolayer cell cultures with the results in vivo, there is a pressing need for better in vitro models of human cancers that will provide more relevant systems for cancer drug screening. Three-dimensional (3D) in vitro systems based on natural polymers with immobilized cancer cells that mimic cancerous tissue and bioreactors that provide relevant chemical and physical signals could close the gap between 2D in vitro and in vivo cancer models. The aim of this study was to optimize culture conditions for the rat glioma cell line C6 immobilized in alginate microfibers in perfusion bioreactors in terms of cell density and perfusion rate. In this study we investigated following sets of parameters: perfusion rate of 0.12, 0.25 and 0.30 ml min-1 coupled with the cell density of 4∙106 cells ml-1 , and perfusion rate of 0.30 ml min-1 coupled with the cell density of 8∙106 cells ml-1 . Microfiber cultures under static conditions in Petri dishes served as controls. The results have shown that the perfusion rate of 0.30 ml min-1 in combination with the cell density of 8∙106 cells ml-1 yields higher cell viability and proliferation compared to the control static culture. These results indicate the importance of culture medium perfusion in the bioreactor for improved mass transfer of nutrients and oxygen to alginate microfibers so that the investigated system shows potentials for use as a model system in cancer research.

INTRODUCTION: Alginate dressings are widely used in the treatment of exuding wounds29. The enzymatically synthesized lignin model compound dehydrogenate polymer (DHP) from coniferyl alcohol by the enzyme peroxidase, is the best lignin substitute used in various experiments30. In our previous work, we have shown that synthesized DHP has antibacterial and antibiofilm properties, and in combination with alginate has good potential for wound treatment31. OBJECTIVES: The objective of this paper was to study the sustained release of DHP from low and medium viscosity alginate beads.

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.

Cilj istraživanja u ovoj doktorskoj disertaciji je bila primena biomimičnih bioreaktora za sveobuhvatnu i fiziološki relevantnu karakterizaciju novih bioaktivnih biomaterijala za potencijalnu primenu u inženjerstvu tkiva, a time i razvoj novih metodologija za pouzdanu evaluaciju biomaterijala. Ispitani su nanokompozitni hidrogelovi alginata sa nanočesticama srebra za inženjerstvo tkiva artikularne hrskavice i hidrogelovi gelanske gume sa nanočesticama bioaktivnog stakla za inženjerstvo tkiva kosti i osteohondralnog tkiva primenom dva biomimična bioreaktora: protočnog i bioreaktora sa dinamičkom kompresijom. Poseban cilj ove disertacije je bio i razvoj novog bioreaktora za inženjerstvo tkiva intervertebralnog diska. Ispitivanja nanokompozitnih Ag/alginatih hidrogelova baziranih na dve vrste alginata pokazala su da polazni sastav alginata utiče na veličinu i kinetiku otpuštanja nanočestica srebra i mehaničke karakteristike hidrogelova u fiziološki relevantnim bioreaktorskim uslovima, na osnovu kojih je određena njihova potencijalna primena. Kinetika otpuštanja srebra je uspešno modelovana prenosom mase difuzijom i konvekcijom. Makroporozni hidrogelovi na bazi gelanske gume i bioaktivnog stakla sa otvorenim porama su dobijeni unapređenom procedurom, a u fiziološki relevantnim uslovima u protočnom bioreaktoru došlo je do značajnog formiranja kalcijum fosfata i poboljšanja mehaničkih karakteristika. Razvijena je i jednostavna metoda za dobijanje dvofaznih osteohondralnih implantata sa dobrom integracijom između slojeva. Rezultati dobijeni u ovoj doktorskoj disertaciji su potvrdili značaj i široki potencijal primene biomimičnih bioreaktora i sprovedene metodologije u više naučnih oblasti, od razvoja novih biomaterijala i inženjerstva tkiva do nanotoksikologije i biologije ćelija., The aim of this doctoral dissertation was application of biomimetic bioreactors for comprehensive and physiologically relevant characterization of novel, bioactive biomaterials intended for tissue engineering (TE), and, consequently development of new methodologies for reliable biomaterial evaluation, as well. The focus was on nanocomposite alginate hydrogels with silver nanoparticles (AgNPs) for articular cartilage TE and gellan gum hydrogels with bioactive glass nanoparticles (GG-BAG) for bone and osteochondral TE, while two biomimetic bioreactors were applied: perfusion and a bioreactor with dynamic compression. The specific goal of this dissertation was also development of a novel bioreactor for intervertebral disc TE. Investigation of nanocomposite Ag/alginate hydrogels based on two alginate types has shown that the initial alginate composition affects the size and release kinetics of AgNPs, as well as hydrogel mechanical properties under physiologically relevant bioreactor conditions, which indicated the potential use for each alginate hydrogel type. The silver release kinetics was successfully modeled by mass transfer by diffusion and advection-diffusion. Macroporous GG-BAG hydrogels with open pores were obtained by a novel procedure. Physiologically relevant conditions in perfusion bioreactors enhanced calcium phosphate formation and improved hydrogel mechanical properties. Also, a simple method was developed for obtaining biphasic osteochondral implants with good integration between layers. The obtained results indicate the significance and vast potentials of biomimetic bioreactors and methodologies developed in this doctoral dissertation for applications in different scientific fields from biomaterials science and TE to nanotoxicology and cell biology.

Development of drugs is a complex, time- and cost-consuming process due to the lack of standardized and reliable characterization techniques and models. Traditionally, drug screening is based on in vitro analysis using two-dimensional (2D) cell cultures followed by in vivo animal testing. Unfortunately, application of the obtained results to humans in about 90 % of cases fails. Therefore, it is important to develop and improve cell-based systems that can mimic the in vivo-like conditions to provide more reliable results. In this paper, we present development and validation of a novel, user-friendly perfusion bioreactor system for single use aimed for cancer research, drug screening, anti-cancer drug response studies, biomaterial characterization, and tissue engineering. Simple design of the perfusion bioreactor provides direct medium flow at physiological velocities (100-250 mu m s(-1)) through samples of different sizes and shapes. Biocompatibility of the bioreactor was confirmed in short term cultivation studies of cervical carcinoma SiHa cells immobilized in alginate microfibers under continuous medium flow. The results have shown preserved cell viability indicating that the perfusion bioreactor in conjunction with alginate hydrogels as cell carriers could be potentially used as a tool for controlled anti-cancer drug screening in a 3D environment.