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INTRODUCTION: 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. EXPERIMENTAL: The impregnated gauzes were prepared by a three-step process: pretreatment, impregnation, and gelling. Pretreatment involved passing cotton gauzes through a Ca(NO3)2 solution (5-15 wt.%), while impregnation was carried out using solutions containing alginate and glycerol (mass ratio 4:10) with a dynamic viscosity of 0.3 Pa s measured at a shear rate of 10 s-1 and at 25°C. Two different gelling solutions were used: one based on 2 wt.% Ca(NO3)2 and the second based on 2.3 wt.% Zn(NO3)2, both supplemented with glycerol (5-20 wt.%) and polyethylene glycol (10- 30 wt.%). All produced gauzes were dried at 40 °C overnight and the best impregnated gauze candidates were further characterized. Dressings produced following the same procedure, just without gauze, served as controls. RESULTS AND DISCUSSION: The impregnated gauzes were evaluated for the polymer layer thickness, sorption capacity, adhesion, mechanical properties and active component release. While polymer retention was consistent (87.6 ± 3.3 g/m²) regardless of the gelling agent (Ca(NO3)2 or Zn(NO3)2), differences were noted in polymer thickness and sorption capacity. Under Ca(NO3)2 gelling, the polymer thickness was 42.6 ± 7.7 µm, with a sorption capacity of 770%. In contrast, Zn(NO3)2 gelling resulted in a thickness of 77.1 ± 10.5 µm and a sorption capacity of 700%, indicating stronger hydrogel formation with Ca ions. Peel-off tests showed low adhesion force (1.91 ± 1.25 N), making the dressings painless upon removal. In mechanical tests, impregnated gauze exhibited superior strength compared to the control films, with nearly double the tearing force and higher elongation at breakage (approximately 23%). Zn-alginate dressings achieved complete release of Zn ions after 72 h, with 70-80 % release after 24 h, which is suitable for dressing changes every 1-3 days. CONCLUSIONS: The advantages of the enhanced dressings obtained as a result of this study include: i) high absorption capacity indicating capacity for moisture regulation and absorption of excess fluids in the wound, ii) expected painless removal of the dressing after the treatment, without leaving any residue of cotton threads in the wound, iii) the possibility of incorporating various active agents (e.g. antimicrobial, immune-stimulating, etc.) and their controlled release for faster and more efficient wound healing (as confirmed by the release of zinc ions from the developed enhanced dressings). During the process, a final formulation has been developed to ensure good flexibility and toughness of the impregnated dressings, making their cutting, and shaping according to the needs of the wound treatment very simple and efficient. [ABSTRACT FROM AUTHOR]

INTRODUCTION: Common clinical problems frequently place a significant stress on the clinical system, and the musculoskeletal system is particularly susceptible to aging and traumatic occurrences. New solutions are required to address significant unmet needs for patients who require bone-substitute implants to treat critical-size bone defects, including personalized solutions for better clinical outcomes, material advancements to ensure higher mechanical reliability without sacrificing bioactive and bioresorbable properties, and optimized manufacturing techniques for materials and products of high reliability and quality. The four-year ReBone Doctoral Network, funded by the Europe Horizon Marie Sklodowska programme, aims to train a new generation of researchers in an innovative and multidisciplinary optimization process to provide technologies for customized bone-substitute implants based on bioactive ceramics and cutting-edge additive manufacturing techniques, to address the health and societal burdens of trauma and bone diseases. MATERIALS and METHODS: A multidisciplinary network and training program have been planned in which ten doctoral candidates will jointly develop an innovative and integrated methodology to the design of personalized ceramic-based bone substitute implants. In order to achieve the purpose, a European network of partners and associated partners has been established encompassing diverse disciplines including biomechanics, clinics, materials engineering, mechano-biology, additive manufacturing technologies and mixed reality models for surgical planning simulations. Materials play a significant role in this project in terms of mechanical properties, bioactivity, biocompatibility, printing technology, and pertinent fidelity. Biologists, material engineers, bioengineers, and technology developers will collaborate to design and thoroughly characterize glass-ceramic based materials for the intended use. Four clinical cases of patients in the need of bone repair will be purposely selected with the aim to develop real-case scenarios of personalized design of bone-substitute implants. Clinical data will be used to create personalized multi-scale models of the implant at the organ level; concurrently, the design of device architecture, materials and parameters for manufacturing technology will be optimized to achieve improved implant outcome in terms of optimal mechanical performance in relationship to the shape and the anatomical location of the implant. RESULTS AND DISCUSSION: As a primary result, a consortium of nine European countries has been constituted and an up-to-date multidisciplinary training program has been set. Furthermore, ten interdisciplinary doctoral research projects have been drawn; ten Doctoral Candidates will undertake the above-mentioned research program in a multidisciplinary environment. Preliminary results achieved by the research institutes involved in the project in the area of ceramic materials development and characterization, additive manufacturing and biomechanics and a clinical research institute will constitute the solid background of the whole activity. CONCLUSIONS: The Europe Horizon Marie Sklodowska program funds the ReBone Doctoral Network, which addresses issues with bone-substitute implants for critical-size bone lesions. A variety of research fields will be addressed by the multidiscciplinary approach. Among these, technological and material development advancements are crucial and will serve as the foundation for the design of customized solutions. [ABSTRACT FROM AUTHOR]

INTRODUCTION: Musculoskeletal diseases are a major burden on individuals, healthcare and welfare systems. Treatment of musculoskeletal disorders is currently based either on prosthetic or regenerative surgical procedures, often involving medical device implantation. In both cases, individual tissue healing and regeneration, together with the appropriateness of the implanted device, markedly affect the outcome. A great improvement could be achieved by precision medicine, specifically designed on patient’s individual characteristics. This implies combining the personalized clinical approach with individual ‘omic’ characterization and proper choice of medical device. The concept is “To take care with care”. This is the meaning of the Italian word “premurosa” and the ultimate goal of the Innovative Training Network PREMUROSA project, aimed to train a new generation of scientists with an integrated vision of the whole value chain in musculoskeletal regeneration technologies and able to boost the necessary innovations to achieve precision principles in developing innovative devices and optimized clinical applications. MATERIALS and METHODS This aim have been achieved by a “triple i” (interdisciplinary, intersectoral, international) approach of thirteen Early Scientific Researchers (ESRs), who have had benefit from an excellent scientific environment, up-date technologies, and supervision by international leaders in the field. RESULTS AND DISCUSSION: ESRs have investigated the links between physico-chemical properties of metallic (titanium and its alloys and ceramic (bioactive glasses and glass-ceramics) materials, including chemistry (chemical composition, surface functionalization), surface morphology (topography, roughness) and hierarchical porosity (at the macro-, meso-nanoscale), and cells (pro- and eukaryotic) and tissues functionality and they studied the impact of surface chemistry, charge and topography on model protein adsorptions and conformation, stem cells fate and extracellular vesicles release and composition. They clarify the role of the extracellular matrix composition as well as the role of the vascular, nervous, and immune system on musculoskeletal tissue regeneration and they developed ad hoc technologies to test safety and efficacy of biomaterials including bioreactors, cellular and computational methods. In addition, they learnt to integrate academic and industrial aspects and they sharpen their experimental and complementary skills in a well-designed and diversified and unprecedented training program. More than 30 open access papers have been published on high impacted journal and posted on Zenodo.org, and more than 70 conference communications have been released. CONCLUSIONS: PREMUROSA has contributed to develop personalized tools for the rational and appropriate application of the musculoskeletal regeneration technologies and to clarify the interplay between tissues, cells, and materials in view of regeneration technologies optimization. [ABSTRACT FROM AUTHOR]

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. The project is structured based on 3 concept points: (i) development of a longer-term in vitro 3D tumor model relies on the use of biomimetic scaffolds and bioreactors providing adequate biochemical and physical signals, (ii) bottom-up approach starting form single well-defined components can yield controlled, reproducible and physiologically relevant 3D tumor models, and (iii) simple in vitro 3D tumor models capturing some of the key features of the tumor environment in vivo can present a useful and expandable platform for reliable anti-cancer drug testing and cancer research. The planned methodology is designed accordingly so that the project will comprise 3 phases: I) development of 3D tumor models, II) validation of the models, and III) utilization of the optimized 3D tumor models in short-term (up to 7 days) and longer-term (up to 28 days) studies of the effects of standard anti-cancer drugs (e.g. cisplatin, doxorubicin, 5- fluorouracil, or paclitaxel) on the cultured cells. In addition, the second phase of the project will include development or adaptation of analytical methods for comprehensive characterization of the cells in 3D cultures as well as mathematical modelling in order to assess the effects of culture conditions on cell viability, morphology, apoptosis and cytokine profiles. Such a systematic experimental and analytical approach will provide significant insights in cancer cell biology regarding 3D environment and guidelines for further optimization of 3D tumor models in general. Overall, the project BioengineeredTumor is addressing an urgent clinical problem, aiming to provide important fundamental insights in cancer cell biology as well as usable products and methods for advancements in pharmaceutical and healthcare sectors. [ABSTRACT FROM AUTHOR]

INTRODUCTION: 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. EXPERIMENTAL: Two cancer cell types were used: osteosarcoma (human HOS and U2OS, and murine K7M2-wt cell lines) and non-small cell lung carcinoma (NCI-H460 human cell line). Cells were suspended (4×106 cells cm-3 ) in alginate solution (2 or 2.8 wt.%) or in a solution containing 2 wt. % alginate and 2 wt. % commercial hydroxyapatite (HAP) powder. To obtain microfibers, the suspensions were manually extruded through a 25 or 26-gauge needle into the gelling bath containing 0.18 M Ca2+ or 0.045 M Ba2+. The obtained microfibers were washed and transferred into culture flasks and then cultured up to 21 days. The 3D cultures were validated in anticancer drug testing: 3 cm of microfibers per well in a 96-well plate were treated with 0.25-20 µM doxorubicin (K7M2-wt) or 0.5-50 µM cisplatin (NCI-H460). Treated cells in monolayer served as a control. The viability and distribution of the cells were examined using live/dead assay and histology (H&E staining). The half-maximal inhibitory concentration (IC50) was determined by the MTT assay. RESULTS AND DISCUSSION: The obtained results of osteosarcoma cells immobilized in Ca-alginate microfibers with and without HAP, and lung cancer cells immobilized in Ba-alginate microfibers have shown that the microfibers supported cell viability, metabolic activity, and formation of cellular aggregates. The results of anticancer drug testing have shown that IC50 values for K7M2-wt cells immobilized in alginate microfibers with and without HAP, as well as for the 3D cultures of NCI-H460 cells were up to ~10-fold higher than the IC50 values of 2D cultures. These results align with the observed higher resistance to anticancer drugs in patients compared to traditional preclinical models. CONCLUSIONS: These findings demonstrate the potentials of the developed 3D model for more reliable anticancer drug screening and enhancement of the preclinical platforms for drug discovery. [ABSTRACT FROM AUTHOR]

INTRODUCTION: 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. EXPERIMENTAL: In our research, human permanent cell lines derived from osteosarcoma, SAOS-2 (ATCC) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS (fetal bovine serum) and 1 % AA (antibiotic-antimycotic) at 37°C with 10 % CO2. Cells were treated with doxorubicin (Ebewe), quercetin (Sigma), and their combination. The cells were immobilized in 1.5 wt.% alginate in the form of microbeads by manual extrusion followed by cultivation up to 21 days. Cell viability was determined using the MTT test, and viability rates were compared using Student's t-test with Graphpad Prism software. The experiment was performed in at least 3 technical replicates. RESULTS AND DISCUSSION: Cells were successfully immobilized in alginate microbeads(diameter: 1230 μm) and their viability significantly increased during the cultivation up to 21 days. Literature data have shown that quercetin could enhance chemotherapeutic effect of doxorubicin on cancer cells. Therefore, osteosarcoma cells were treated with both Doxo and Quer. Experimental results have shown that the combination of 1 µg/ml Doxo and 5 µM Quer significantly decreased the viability of SAOS-2 cells cultured in 2D conditions compared to cellstreated with 1 µg/ml Doxo. On the other hand, viability of the cells cultured in 3D conditionstreated with the same combination of Quer and Doxo did not show any statistically significant effect on cell viability. We can hypothesize that microenvironment-based mechanisms modulate doxorubicin sensitivity and increase resistance to treatment of osteosarcoma cells cultured in 3D conditions. CONCLUSION: Collectively, quercetin sensitized osteosarcoma cells to doxorubicin in 2D model. However, in an invivo like 3D model system, the effect on of the combined treatment was not observed. Further research is needed to investigate the possible role of quercetin in tumor treatment. [ABSTRACT FROM AUTHOR]

INTRODUCTION: 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. EXPERIMENTAL: Bone-like scaffolds were obtained by cross-linking the mixture of Na-alginate solution (2 wt.%) and hydroxyapatite (2 wt.%) with calcium ions followed by slow freezing and lyophilization. Scaffold porosity and pore sizes were determined by using optical microscopy. To model osteosarcoma tumor, scaffolds were seeded with murine K7M2-wt osteosarcoma cells, whereas for mimicking bone physiological conditions either human bone marrow-derived mesenchymal stem cell line (hBMSCs) or primary mesenchymal stem cells were used. Each cell type was cultivated for 7 days in a perfusion bioreactor with medium flow rate of 0.27 cm³ /min, corresponding to the medium superficial velocity of 40 μm/s. Static cell cultures served as controls. Cell behavior was assessed by cell metabolic activity assays (MTT or resazurin), histological and immunocytochemical analysis, phalloidin/DAPI staining, and gene expression analysis (qPCR). Shear stresses were calculated from histological sections using a cylindrical pore model. RESULTS AND DISCUSSION: Obtained scaffolds had an initial porosity of 60 % and contained a variety of pore sizes with a predominant presence of macropores, mimicking in that manner trabecular bone structure. All cell types adhered to the scaffolds indicated by cell seeding efficiency exceeding 80 %. In perfusion culture, osteosarcoma cells exhibited characteristics corresponding to in vivo tumor cell behavior: high cell metabolic activity, spontaneous assembly into compact spheroid-like structures, secretion of extracellular matrix and expression of pluripotency-associated genes. Furthermore, immunocytochemical staining revealed an increased presence of α-tubulin as compared to the control. Regarding the healthy bone model, both types of mesenchymal stem cells retained their intrinsic cellular shape and selforganized into aligned structures which was strongly induced by perfusion conditions. The overall positive influence of perfusion conditions could be attributed not only to improved mass transport but also to adequate values of hydrodynamic shear stresses calculated to be up to 5 mPa. CONCLUSIONS: Our 3D in vitro model can support cultures of different bone cell types and shows potential for further adjustment and utilization in the fields of tumor and tissue engineering. [ABSTRACT FROM AUTHOR]

INTRODUCTION: Millions of patients are still awaiting new therapies as the traditional models rely on monolayer cell (2D) cultures and in vivo studies, which have numerous limitations resulting in misleading conclusions. Consequently, there is a burning need for the development of alternative 3D models able to accurately mimic the complexity of human diseases. This research aim is to create microenvironments based on biomimetic bioreactors and alginate hydrogels as cell carriers for reliable disease research and drug screening. EXPERIMENTAL: Alginate cell carriers in forms of microfibers and microbeads with immobilized different cells were obtained by extrusion techniques, while macroporous cell carriers for imitating bone tissue based on alginate or gellan gum and bioactive inorganic particles (hydroxyapatite, ß-tricalcium phosphate, bioactive glass) were prepared by a simple controlled gelation and freeze-drying method followed by manual seeding of cells onto the partially rehydrated scaffolds. The obtained carriers with cells were cultivated in perfusion bioreactors (“3D Perfuse”, Innovation Center of the Faculty of Technology and Metallurgy, Belgrade, Serbia) under continuous medium flow (superficial velocity: 15-100 µm s-1 ) for up to 7 days. To evaluate these cell carriers for drug screening, microfibers with different cancer cells were treated with cisplatin or doxorubicin, while 2D cultures served as control. The cells were assessed regarding the metabolic activity (viability) by MTT, morphology, and distribution within carriers by scanning electron microscopy and histology (H&E stain). RESULTS AND DISCUSSION: Cell immobilization in alginate microbeads (diameter: 300 µm) and microfibers (diameter in the range 300-500 µm) resulted in uniform distribution, while the macroporous scaffolds with open and connected pores (porosity: ~60 %) provided cell adherence as individual cells and in aggregates (seeding efficiency: above 80 %).The majority of cells stayed viable and metabolically active, while retrieved cells from alginate carriers retained their morphology, viability, and ability to proliferate under 2D conditions. Alginate carriers with cervical carcinoma, glioblastoma, and osteosarcoma cells were further cultivated in perfusion bioreactors. After cultivation under biomimetic conditions the cells retained viability and proliferative capacity, spontaneously formed spheroid-like structures, and exhibited higher metabolic activity as compared to static controls. The obtained results imply the positive effects of medium flow on cells due to providing efficient mass transport and controlled levels of hydrodynamic shear stresses. Evaluation of these models for drug screening has shown that the immobilized different cancer cells exhibited up to 10-fold higher half-maximal inhibitory concentration than the cells in 2D cultures. CONCLUSIONS: The overall results have shown potentials of the applied approach based on 3D models comprising biomimetic bioreactors and alginate-based scaffolds for disease research and drug screening. [ABSTRACT FROM AUTHOR]