Background: Cytotoxicity testing is a primary method to establish the safety of biomaterials, e.g., biocomposites. Biomaterials involve a wide range of medical materials, which are usually solid materials and are used in bone regeneration, cardiology, or dermatology. Current advancements in science and technology provide several standard cytotoxicity testing methods that are sufficiently sensitive to detect various levels of cellular toxicity, i.e., from low to high. The aim was to compare the direct and indirect methodology described in the ISO guidelines UNE-EN ISO 10993-5:2009 Part 5. Methods: Cell proliferation was measured using WST-1 assay, and cytotoxicity was measured using LDH test kit. Results: The results indicate that the molecular surface of biomaterials have impact on the cytotoxicity and proliferation profile. Based on these results, we confirm that the indirect method does not provide a clear picture of the cell condition after the exposure to the surface, and moreover, cannot provide complete results about the effects of the material. Conclusions: Comparison of both methods shows that it is pivotal to investigate biomaterials at the very early stages using both indirect and direct methods to access the influence of the released toxins and surface of the material on the cell condition.

Chitosan is one of the most commonly employed natural polymers for biomedical applications. However, in order to obtain stable chitosan biomaterials with appropriate strength properties, it is necessary to subject it to crosslinking or stabilization. Composites based on chitosan and bioglass were prepared using the lyophilization method. In the experimental design, six different methods were used to obtain stable, porous chitosan/bioglass biocomposite materials. This study compared the crosslinking/stabilization of chitosan/bioglass composites with ethanol, thermal dehydration, sodium tripolyphosphate, vanillin, genipin, and sodium β-glycerophosphate. The physicochemical, mechanical, and biological properties of the obtained materials were compared. The results showed that all the selected crosslinking methods allow the production of stable, non-cytotoxic porous composites of chitosan/bioglass. The composite with genipin stood out with the best of the compared properties, taking into account biological and mechanical characteristics. The composite stabilized with ethanol is distinct in terms of its thermal properties and swelling stability, and it also promotes cell proliferation. Regarding the specific surface area, the highest value exposes the composite stabilized by the thermal dehydration method.

Background: Immunotherapy is emerging as a powerful treatment approach for several types of cancers. Modulating the immune system to specifically target cancer cells while sparing healthy cells, is a very promising approach for safer therapies and increased survival of cancer patients. Tumour-associated antigens are favorable targets for cancer immunotherapy, as they are exclusively expressed by the cancer cells, minimizing the risk of an autoimmune reaction. The ability to initiate the activation of the immune system can be achieved by virus-like particles (VLPs) which are safe and potent delivery tools. VLP‐based vaccines have evolved dramatically over the last few decades and showed great potential in preventing infectious diseases. Immunogenic potency of engineered VLPs as a platform for the development of effective therapeutic cancer vaccines has been studied extensively. This study involves recombinant VLPs presenting multiple copies of tumour-specific mucin 1 (MUC1) epitope as a potentially powerful tool for future immunotherapy. Results: In this report VLPs based on the structural protein of Norovirus (NoV VP1) were genetically modified to present multiple copies of tumour-specific MUC1 epitope on their surface. Chimeric MUC1 particles were produced in the eukaryotic Leishmania tarentolae expression system and used in combination with squalene oil-in-water emulsion MF59 adjuvant to immunize BALB/c mice. Sera from vaccinated mice demonstrated high titers of IgG and IgM antibodies which were specifically recognizing MUC1 antigen. Conclusions: The obtained results show that immunization with recombinant chimeric NoV VP1- MUC1 VLPs result in high titers of MUC1 specific IgG antibodies and show great therapeutic potential as a platform to present tumour-associated antigens. Graphical

Background: Noroviruses are a major cause of epidemic and sporadic acute non-bacterial gastroenteritis worldwide. Unfortunately, the development of an effective norovirus vaccine has proven difficult and no prophylactic vaccine is currently available. Further research on norovirus vaccine development should be considered an absolute priority and novel vaccine candidates are needed. One of the recent approaches in safe vaccine development is the use of virus-like particles (VLPs). VLP-based vaccines show great immunogenic potential as they mimic the morphology and structure of viral particles without the presence of the virus genome. Results: This study is the first report showing successful production of norovirus VLPs in the protozoan Leishmania tarentolae (L. tarentolae) expression system. Protozoan derived vaccine candidate is highly immunogenic and able to not only induce a strong immune response (antibody titer reached 10⁴) but also stimulate the production of neutralizing antibodies confirmed by receptor blocking assay. Antibody titers able to reduce VLP binding to the receptor by > 50% (BT₅₀) were observed for 1:5–1:320 serum dilutions. Conclusions: Norovirus VLPs produced in L. tarentolae could be relevant for the development of the norovirus vaccine.

Assessing the toxicity of new biomaterials dedicated to bone regeneration can be difficult. Many reports focus only on a single toxicity parameter, which may be insufficient for a detailed evaluation of the new material. Moreover, published data frequently do not include control cells exposed to the environment without composite or its extract. Here we present the results of two assays used in the toxicological assessment of materials’ extracts (the integrity of the cellular membrane and the mitochondrial activity/proliferation), and the influence of different types of controls used on the obtained results. Results obtained in the cellular membrane integrity assay showed a lack of toxic effects of all tested extracts, and no statistical differences between them were present. Control cells, cells incubated with chitosan extract or chitosan-bioglass extract were used as a reference in proliferation calculations to highlight the impact of controls used on the result of the experiment. The use of different baseline controls caused variability between obtained proliferation results, and influenced the outcome of statistical analysis. Our findings confirm the thesis that the type of control used in an experiment can change the final results, and it may affect the toxicological assessment of biomaterial.

Purpose: The purpose of this study was to design and synthesize the ug46 peptide, incorporate its fibrils into composite materials, and evaluate its structural and antimicrobial properties. Another objective was to utilize spectroscopy and molecular simulation, enhanced by Machine Vision methods, to monitor the aggregation process of the ug46 peptide and assess its potential as a scaffold for an antimicrobial peptide. Methods: The structural analysis of the ug46 peptide reveals its dynamic conformational changes. Initially, the peptide exhibits a disordered structure with minimal α-helix content, but as incubation progresses, it aggregates into fibrils rich in β-sheets. This transformation was validated by CD and ThT assays, which showed decreased molar ellipticity and an increase in ThT fluorescence. Results: Laser-induced fluorescence and molecular dynamics simulations further revealed the transition from a compact native state to extended “worm-like” filament structures, influenced by peptide concentration and temperature. TEM and AFM confirmed these changes, showing the evolution of protofibrils into mature fibrils with characteristic twists. When incorporated into chitosan- bioglass composites, these fibrils significantly enhanced antimicrobial activity against pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa. Conclusion: Overall, ug46 peptide fibrils show promise as a multifunctional scaffold with structural and antimicrobial benefits in composite biomaterials.