The treatment of chronic wounds is still a meaningful challenge to physicians. The aim of this work was to produce vitamin C-enriched chitosan/agarose (CHN/A) film that could serve as potential artificial skin substitute for chronic wound treatment. The biomaterial was fabricated by a newly developed and simplified method via mixing acidic chitosan solution with alkaline agarose solution that allowed to obtain slightly acidic pH (5.97) of the resultant material, which is known to support skin regeneration. Vitamin C was immobilized within the matrix of the film by entrapment method during production process. Produced films (CHN/A and CHN/A + vit C) were subjected to comprehensive evaluation of cellular response with the use of human skin fibroblasts, epidermal keratinocytes, and macrophages. It was demonstrated that novel biomaterials support adhesion and growth of human skin fibroblasts and keratinocytes, have ability to slightly reduce transforming growth factor-beta 1 (TGF-β1) (known to be present at augmented levels in the epidermis of chronic wounds), and increase platelet-derived growth factor-BB (PDGF-BB) secretion by the cells. Nevertheless, addition of vitamin C to the biomaterial formulation does not significantly improve its biological properties due to burst vitamin release profile. Obtained results clearly demonstrated that produced CHN/A film has great potential to be used as cellular dermal, epidermal, or dermo-epidermal graft pre-seeded with human skin cells for chronic wound treatment.

There are many modern wound dressings that have promising properties for repairing skin damage. However, due to various types of wounds and the problems they cause, there is still a great demand for new, effective healing strategies. The aim of this study was to create superabsorbent wound dressing made of marine-derived polysaccharides (agarose and chitosan) using the freeze-drying method. The secondary goal was its comprehensive evaluation for potential use as an external superabsorbent bandage for wounds with high exudation. Due to the well-known positive effect of ascorbic acid (vitamin C) on the healing process, biomaterial enriched with vitamin C was prepared and compared to the variant without the addition of ascorbic acid. It was shown that the produced foam-like wound dressing had a very porous structure, which was characterized by hydrophilicity, allowing a large amount of human fluids to be absorbed. According to in vitro tests on human fibroblasts, biomaterial was nontoxic and supportive to cell proliferation. Vitamin C-enriched dressing also had the ability to significantly reduce matrix metalloproteinase-2 production and to promote platelet-derived growth factor-BB synthesis by fibroblasts, which is desired during chronic wound treatment. The material has features of the eco-friendly wound care product since it was made of naturally-derived polysaccharides and was proved to be biodegradable. Importantly, despite degradable character, it was stable in the chronic and infected wound microenvironment, maintaining high integrity after 8-week incubation in the enzymatic solutions containing lysozyme and collagenases. The obtained results clearly showed that developed biomaterial possesses all necessary features of the external dressing for the management of exudate from both acute and chronic non-healing wounds.

Silver salts and azole derivatives are well known for their antimicrobial properties. Recent evidence has demonstrated also their cytotoxic and genotoxic potential toward both normal and cancer cells. Still, little is known about the action of complexes of azoles with silver(I) salts. Thus, the goal of the study was to compare the chemical, cytotoxic and antimicrobial properties of metronidazole complexes with silver(I) nitrate and silver(I) sulfate to metronidazole and pure silver(I) salts. We synthetized a novel complex, [Ag(MTZ)₂]₂SO₄, and confirmed its chemical structure and properties using ¹H and ¹³C NMR spectroscopy and X-Ray, IR and elemental analysis. To establish the stability of complexes [Ag(MTZ)₂NO₃] and [Ag(MTZ)₂]₂SO₄, they were exposed to daylight and UV-A rays and were visually assessed. Their cytotoxicity toward human cancer cells (HepG2, Caco-2) and mice normal fibroblasts (Balb/c 3T3 clone A31) was determined by MTT, NRU, TPC and LDH assays. The micro-dilution broth method was used to evaluate their antimicrobial properties against Gram-positive and Gram-negative bacteria. A biofilm eradication study was also performed using the crystal violet method and confocal laser scanning microscopy. The photo-stability of the complexes was higher than silver(I) salts. In human cancer cells, [Ag(MTZ)₂]₂SO₄ was more cytotoxic than Ag₂SO₄ and, in turn, AgNO₃ was more cytotoxic than [Ag(MTZ)₂NO₃]. For Balb/c 3T3 cells, Ag₂SO₄ was more cytotoxic than [Ag(MTZ)₂]₂SO₄, while the cytotoxicity of AgNO₃ and [Ag(MTZ)₂NO₃] was similar. Metronidazole in the tested concentration range was non-cytotoxic for both normal and cancer cells. The complexes showed increased bioactivity against aerobic and facultative anaerobic bacteria when compared to metronidazole. For the majority of the tested bacterial strains, the silver(I) salts and complexes showed a higher antibacterial activity than MTZ; however, some bacterial strains presented the reverse effect. Our results showed that silver(I) complexes present higher photo-stability, cytotoxicity and antimicrobial activity in comparison to MTZ and, to a certain extent, to silver(I) salts.

Intervertebral cages made of Ti6Al4V alloy show excellent osteoconductivity, but also higher stiffness, compared to commonly used polyether-ether-ketone (PEEK) materials, that may lead to a stress-shielding effect and implant subsidence. In this study, a metallic intervertebral fusion cage, with improved mechanical behavior, was manufactured by the introduction of a three-dimensional (3D) mesh structure to Ti6Al4V material, using an additive manufacturing method. Then, the mechanical and biological properties of the following were compared: (1) PEEK, with a solid structure, (2) 3D-printed Ti6Al4V, with a solid structure, and (3) 3D-printed Ti6Al4V, with a mesh structure. A load-induced subsidence test demonstrated that the 3D-printed mesh Ti6Al4V cage had significantly lower tendency (by 15%) to subside compared to the PEEK implant. Biological assessment of the samples proved that all tested materials were biocompatible. However, both titanium samples (solid and mesh) were characterized by significantly higher bioactivity, osteoconductivity, and mineralization ability, compared to PEEK. Moreover, osteoblasts revealed stronger adhesion to the surface of the Ti6Al4V samples compared to PEEK material. Thus, it was clearly shown that the 3D-printed mesh Ti6Al4V cage possesses all the features for optimal spinal implant, since it carries low risk of implant subsidence and provides good osseointegration at the bone-implant interface.

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 Fe_xO_y/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 Fe_xO_y/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 Fe_xO_y/NPs catalysts and increased osteocalcin production by the cells cultured on the scaffold containing pure NPs. Plasma activation of Fe_xO_y/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 Fe_xO_y/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.

Bioactive dressings are usually produced using natural or synthetic polymers. Recently, special attention has been paid to β-glucans that act as immunomodulators and have pro-healing properties. The aim of this research was to use β-1,3-glucan (curdlan) as a base for the production of bioactive dressing materials (curdlan/agarose and curdlan/chitosan) that were additionally enriched with vitamin C and/or hydrocortisone to improve healing of chronic and burn wounds. The secondary goal of the study was to compressively evaluate biological properties of the biomaterials. In this work, it was shown that vitamin C/hydrocortisone-enriched biomaterials exhibited faster vitamin C release profile than hydrocortisone. Consecutive release of the drugs is a desired phenomenon since it protects wounds against accumulation of high and toxic concentrations of the bioactive molecules. Moreover, biomaterials showed gradual release of low doses of the hydrocortisone, which is beneficial during management of burn wounds with hypergranulation tissue. Among all tested variants of biomaterials, dressing materials enriched with hydrocortisone and a mixture of vitamin C/hydrocortisone showed the best therapeutic potential since they had the ability to significantly reduce MMP-2 synthesis by macrophages and increase TGF-β1 release by skin cells. Moreover, materials containing hydrocortisone and its blend with vitamin C stimulated type I collagen deposition by fibroblasts and positively affected their migration and proliferation. Results of the experiments clearly showed that the developed biomaterials enriched with bioactive agents may be promising dressings for the management of non-healing chronic and burn wounds.