Optimization of the Composition of Mesoporous Polymer-Ceramic Nanocomposite Granules for Bone Regeneration.

Difficult-to-treat bone damage resulting from metabolic bone diseases, mechanical injuries, or tumor resection requires support in the form of biomaterials. The aim of this research was to optimize the concentration of individual components of polymer-ceramic nanocomposite granules (nanofilled polymer composites) for application in orthopedics and maxillofacial surgery to fill small bone defects and stimulate the regeneration process. Two types of granules were made using nanohydroxyapatite (nanoHA) and chitosan-based matrix (agarose/chitosan or curdlan/chitosan), which served as binder for ceramic nanopowder. Different concentrations of the components (nanoHA and curdlan), foaming agent (sodium bicarbonate-NaHCO3), and chitosan solvent (acetic acid-CH3COOH) were tested during the production process. Agarose and chitosan concentrations were fixed to be 5% w/v and 2% w/v, respectively, based on our previous research. Subsequently, the produced granules were subjected to cytotoxicity testing (indirect and direct contact methods), microhardness testing (Young's modulus evaluation), and microstructure analysis (porosity, specific surface area, and surface roughness) in order to identify the biomaterial with the most favorable properties. The results demonstrated only slight differences among the resultant granules with respect to their microstructural, mechanical, and biological properties. All variants of the biomaterials were non-toxic to a mouse preosteoblast cell line (MC3T3-E1), supported cell growth on their surface, had high porosity (46-51%), and showed relatively high specific surface area (25-33 m2/g) and Young's modulus values (2-10 GPa). Apart from biomaterials containing 8% w/v curdlan, all samples were predominantly characterized by mesoporosity. Nevertheless, materials with the greatest biomedical potential were obtained using 5% w/v agarose, 2% w/v chitosan, and 50% or 70% w/v nanoHA when the chitosan solvent/foaming agent ratio was equal to 2:2. In the case of the granules containing curdlan/chitosan matrix, the most optimal composition was as follows: 2% w/v chitosan, 4% w/v curdlan, and 30% w/v nanoHA. The obtained test results indicate that both manufactured types of granules are promising implantable biomaterials for filling small bone defects that can be used in maxillofacial surgery.

In Vitro Screening Studies on Eight Commercial Essential Oils-Derived Compounds to Identify Promising Natural Agents for the Prevention of Osteoporosis.

Over the years, essential oils (EOs) and their compounds have gained growing interest due to their anti-inflammatory, antimicrobial, antioxidant, and immunomodulatory properties. The aim of this study was to evaluate the effect of eight commercially available EO-derived compounds ((R)-(+)-limonene, (S)-(-)-limonene, sabinene, carvacrol, thymol, alpha-pinene (α-pinene), beta-pinene (ß-pinene), and cinnamaldehyde) on the bone formation process in vitro to select the most promising natural agents that could potentially be used in the prevention or treatment of osteoporosis. Within this study, evaluation of cytotoxicity, cell proliferation, and osteogenic differentiation was performed with the use of mouse primary calvarial preosteoblasts (MC3T3-E1). Moreover, extracellular matrix (ECM) mineralization was determined using MC3T3-E1 cells and dog adipose tissue-derived mesenchymal stem cells (ADSCs). The two highest non-toxic concentrations of each of the compounds were selected and used for testing other activities. The conducted study showed that cinnamaldehyde, thymol, and (R)-(+)-limonene significantly stimulated cell proliferation. In the case of cinnamaldehyde, the doubling time (DT) for MC3T3-E1 cells was significantly shortened to approx. 27 h compared to the control cells (DT = 38 h). In turn, cinnamaldehyde, carvacrol, (R)-(+)-limonene, (S)-(-)-limonene, sabinene, and α-pinene exhibited positive effects on either the synthesis of bone ECM or/and mineral deposition in ECM of the cells. Based on the conducted research, it can be assumed that cinnamaldehyde and (R)-(+)-limonene are the most promising among all tested EO-derived compounds and can be selected for further detailed research in order to confirm their biomedical potential in the chemoprevention or treatment of osteoporosis since they not only accelerated the proliferation of preosteoblasts, but also significantly enhanced osteocalcin (OC) synthesis by preosteoblasts (the OC level was approx. 1100-1200 ng/mg compared to approx. 650 ng/mg in control cells) and ECM calcification of both preosteoblasts and mesenchymal stem cells. Importantly, cinnamaldehyde treatment led to a three-fold increase in the mineral deposition in ADSCs, whereas (R)-(+)-limonene caused a two-fold increase in the ECM mineralization of both MC3T3-E1 cells and ADSCs.

Poly(levodopa)-Functionalized Polysaccharide Hydrogel Enriched in Fe3O4 Particles for Multiple-Purpose Biomedical Applications.

In recent years, there has been a significant increase in interest in the use of curdlan, a naturally derived polymer, for medical applications. However, it is relatively inactive, and additives increasing its biomedical potential are required; for example, antibacterial compounds, magnetic particles, or hemostatic agents. The stability of such complex constructs may be increased by additional functional networks, for instance, polycatecholamines. The article presents the production and characterization of functional hydrogels based on curdlan enriched with Fe3O4 nanoparticles (NPs) or Fe3O4-based heterostructures and poly(L-DOPA) (PLD). Some of the prepared modified hydrogels were nontoxic, relatively hemocompatible, and showed high antibacterial potential and the ability to convert energy with heat generation. Therefore, the proposed hydrogels may have potential applications in temperature-controlled regenerative processes as well as in oncology therapies as a matrix of increased functionality for multiple medical purposes. The presence of PLD in the curdlan hydrogel network reduced the release of the NPs but slightly increased the hydrogel's hemolytic properties. This should be taken into account during the selection of the final hydrogel application.

Wound Dressing Modifications for Accelerated Healing of Infected Wounds.

Infections that occur during wound healing involve the most frequent complications in the field of wound care which not only inhibit the whole process but also lead to non-healing wound formation. The diversity of the skin microbiota and the wound microenvironment can favor the occurrence of skin infections, contributing to an increased level of morbidity and even mortality. As a consequence, immediate effective treatment is required to prevent such pathological conditions. Antimicrobial agents loaded into wound dressings have turned out to be a great option to reduce wound colonization and improve the healing process. In this review paper, the influence of bacterial infections on the wound-healing phases and promising modifications of dressing materials for accelerated healing of infected wounds are discussed. The review paper mainly focuses on the novel findings on the use of antibiotics, nanoparticles, cationic organic agents, and plant-derived natural compounds (essential oils and their components, polyphenols, and curcumin) to develop antimicrobial wound dressings. The review article was prepared on the basis of scientific contributions retrieved from the PubMed database (supported with Google Scholar searching) over the last 5 years.

Biocompatible curdlan-based biomaterials loaded with gentamicin and Zn-doped nano-hydroxyapatite as promising dressing materials for the treatment of infected wounds and prevention of surgical site infections.

A topical application of antibiotic-loaded wound dressings is recommended only for chronically infected wounds with poor vascularization. Thus, more often dressing materials loaded with antibacterial metal ions are produced. In turn, gentamicin sponges are commonly used to prevent surgical site infections. The aim of this study was to produce curdlan-based biomaterials enriched with gentamicin and zinc (Zn)-doped nano-hydroxyapatite to prevent wound and surgical site infections. Developed biomaterials were subjected to basic microstructural characterization, cytotoxicity test against human skin fibroblasts (BJ cell line), and comprehensive microbiological experiments using Staphylococcus aureus and Pseudomonas aeruginosa strains. To evaluate the in vivo healing capacity of the developed biomaterials, severely infected chronic wound in a veterinary patient was treated with the use of gentamicin-loaded dressing. Fabricated biomaterials were characterized by a highly porous microstructure with high plasma absorption capacity (approx. 7 mL/g for Zn-loaded biomaterial and 13 mL/g for gentamicin-enriched dressing) and optimal water vapor transmission rate (approx. 1700 g/m2/day). Due to the presence of bioceramics, material containing Zn showed slightly higher compressive strength (0.37 MPa) and Young's modulus (3.33 MPa) values compared to gentamicin-loaded biomaterial (0.12 MPa and 1.29 MPa, respectively). Gentamicin-enriched biomaterial showed burst release of the drug within the first 5 h, while, the zinc-loaded biomaterial exhibited a constant gradual release of the zinc ions. Conducted assays showed that developed biomaterials were non-toxic against human skin fibroblasts (cell viability in the range of 71-95 %) and revealed strong bactericidal activity (99.9 % reduction in the number of viable bacterial CFUs in direct contact test) against S. aureus. In the case of P. aeruginosa, only gentamicin-loaded biomaterial exhibited bactericidal effect. Additionally, biomaterials had the ability to uptake, lock in, and kill bacteria within their gel structure, enabling the cleansing of the wound bed at every dressing change. Finally, the treatment of severely infected wound in veterinary patient confirmed the effectiveness of gentamicin-loaded biomaterial. Biomaterial enriched with gentamicin possesses great potential to be used as a dressing material or sponge for the treatment of chronically infected wounds and surgical site infections. In turn, the zinc-loaded biomaterial may be used as a wound dressing to reduce and prevent microbial contamination.

Fractionation of Lycopodiaceae Alkaloids and Evaluation of Their Anticholinesterase and Cytotoxic Activities.

In view of the abundant evidence that Lycopodiaceae alkaloids, including the well-known huperzine A (HupA), are among the potent acetylcholinesterase (AChE) inhibitors, an attempt was made to search for new compounds responsible for this property. For this purpose, three plant species belonging to the Lycopodiaceae family, commonly found in the Euro-Asia region, were subjected to the isolation of bioactive compounds, their identification and subsequent evaluation of their anticholinesterase and cytotoxic activities. Methanolic extracts of two Lycopodium and one Hupezia species were obtained via optimized pressurized liquid extraction (PLE) and then pre-purified using innovative gradient vacuum liquid chromatography (gVLC). For the first time, three sorbents of different porosity packed in polypropylene cartridges and mobile phase systems of different polarity were used to elute the target compounds. This technique proved to be a rapid tool for the obtainment of alkaloid fractions and allowed one to select the appropriate process conditions to yield potent AChE inhibitors in each of the species studied. More than 100 collected fractions were analyzed via HPLC/ESI-QTOF-MS, which enabled one to detect more than 50 compounds, including several new ones previously unreported. Some of them were present in high purity fractions (60-90% of the established purity). TLC bioautography assays proved that the analyzed species are rich sources of AChE inhibitors, but H. selago showed the highest anti-AChE activity. Additionally, the modified silanized silica gel sorbent used allowed one to isolate L. clavatum alkaloids more efficiently using an aqueous reversed-phase solvent system. Furthermore, the tested extracts from the three plant extracts were found to be safe, as they did not exhibit cytotoxicity to skin fibroblasts.

Superabsorbent curdlan-based foam dressings with typical hydrocolloids properties for highly exuding wound management.

Effective management of chronic wounds with excessive exudate may be challenging for medical doctors. Over the years, there has been an increasing interest in the engineering of biomaterials, focusing on the development of polymer-based wound dressings to accelerate the healing of exuding wounds. The aim of this study was to use curdlan, which is known to support wound healing, as a base for the production of superabsorbent hybrid biomaterials (curdlan/agarose and curdlan/chitosan) with the intended use as wound dressings for highly exuding wound management. To evaluate the biomedical potential of the fabricated curdlan-based biomaterials, they were subjected to a comprehensive assessment of their microstructural, physicochemical, and biological properties. The obtained results showed that foam-like biomaterials with highly porous structure (66-77%) transform into soft gel after contact with the wound fluid, acting as typical hydrocolloid dressings. Novel biomaterials have the superabsorbent ability (1 g of the biomaterial absorbs approx. 15 ml of exudate) with horizontal wicking direction while keeping dry edges, and show water vapor transmission rate of approx. 1700-1800 g/m2/day which is recommended for optimal wound healing. Moreover, they are stable in the presence of collagenases, but prone to biodegradation in lysozyme solution (simulated infected wound environment). Importantly, the developed biomaterials are non-toxic and their surface hinders fibroblast attachment, which is essential during dressing changes to avoid damage to newly formed tissues in the wound bed. All mentioned features make the developed biomaterials promising candidates to be used as the wound dressings for the management of chronic wounds with moderate to high exudate.

Cellular Response to Vitamin C-Enriched Chitosan/Agarose Film with Potential Application as Artificial Skin Substitute for Chronic Wound Treatment.

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-ß1) (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.