Alginate Gel Encapsulated with Enzybiotics Cocktail Is Effective against Multispecies Biofilms.

The development of new and effective antibacterials for pharmaceutical or cosmetic skin care that have a low potential for the emergence and expansion of bacterial resistance is of high demand in scientific and applied research. Great hopes are placed on alternative agents such as bactericidal peptidoglycan hydrolases, depolymerases, etc. Enzybiotic-based preparations are being studied for the treatment of various infections and, among others, can be used as topical formulations and dressings with protein-polysaccharide complexes. Here, we investigate the antibiofilm properties of a novel enzybiotic cocktail of phage endolysin LysSi3 and bacteriocin lysostaphin, formulated in the alginate gel matrix and its ability to control the opportunistic skin-colonizing bacteria Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae, as well as mixed-species biofilms. Our results propose that the application of SiL-gel affects different components of biofilm extracellular polymeric substances, disrupts the matrix, and eliminates the bacteria embedded in it. This composition is highly effective against biofilms composed of Gram-negative and Gram-positive species and does not possess significant cytotoxic effects. Our data form the basis for the development of antibacterial skin care products with a gentle but effective mode of action.

Honeycomb-Structured Porous Films from Poly(3-hydroxybutyrate) and Poly(3-hydroxybutyrate-co-3-hydroxyvalerate): Physicochemical Characterization and Mesenchymal Stem Cells Behavior.

Surface morphology affects cell attachment and proliferation. In this research, different films made of biodegradable polymers, poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-co-HV), containing different molecular weights, with microstructured surfaces were investigated. Two methods were used to obtain patterned films-water-assisted self-assembly ("breath figure") and spin-coating techniques. The water-assisted technique made it possible to obtain porous films with a self-assembled pore structure, which is dependent on the monomer composition of a polymer along with its molecular weight and the technique parameters (distance from the nozzle, volume, and polymer concentration in working solution). Their pore morphologies were evaluated and their hydrophobicity was examined. Mesenchymal stem cells (MSCs) isolated from bone marrow were cultivated on a porous film surface. MSCs' attachment differed markedly depending on surface morphology. On strip-formed stamp films, MSCs elongated along the structure, however, they interacted with a larger area of film surface. The honeycomb films and column type films did not set the direction of extrusion, but cell flattening depended on structure topography. Thus, stem cells can "feel" the various surface morphologies of self-assembled honeycomb films and change their behavior depending on it.

Competitive Biosynthesis of Bacterial Alginate Using Azotobacter vinelandii 12 for Tissue Engineering Applications.

This study investigated the effect of various cultivation conditions (sucrose/phosphate concentrations, aeration level) on alginate biosynthesis using the bacterial producing strain Azotobacter vinelandii 12 by the full factorial design (FFD) method and physicochemical properties (e.g., rheological properties) of the produced bacterial alginate. We demonstrated experimentally the applicability of bacterial alginate for tissue engineering (the cytotoxicity testing using mesenchymal stem cells (MSCs)). The isolated synthesis of high molecular weight (Mw) capsular alginate with a high level of acetylation (25%) was achieved by FFD method under a low sucrose concentration, an increased phosphate concentration, and a high aeration level. Testing the viscoelastic properties and cytotoxicity showed that bacterial alginate with a maximal Mw (574 kDa) formed the densest hydrogels (which demonstrated relatively low cytotoxicity for MSCs in contrast to bacterial alginate with low Mw). The obtained data have shown promising prospects in controlled biosynthesis of bacterial alginate with different physicochemical characteristics for various biomedical applications including tissue engineering.

Poly(3-hydroxybutyrate)/hydroxyapatite/alginate scaffolds seeded with mesenchymal stem cells enhance the regeneration of critical-sized bone defect.

A critical-sized calvarial defect in rats is employed to reveal the osteoinductive properties of biomaterials. In this study, we investigate the osteogenic efficiency of hybrid scaffolds based on composites of a biodegradable and biocompatible polymer, poly(3-hydroxybutyrate) (PHB) with hydroxyapatite (HA) filled with alginate (ALG) hydrogel containing mesenchymal stem cells (MSCs) on the regeneration of the critical-sized radial defect of the parietal bone in rats. The scaffolds based on PHB and PHB/HA with desired shapes were prepared by two-stage salt leaching technique using a mold obtained by three-dimensional printing. To obtain PHB/HA/ALG/MSC scaffolds seeded with MSCs, the scaffolds were filled with ALG hydrogel containing MSCs; acellular PHB/ALG and PHB/ALG filled with empty ALG hydrogel were prepared for comparison. The produced scaffolds have high porosity and irregular interconnected pore structure. PHB/HA scaffolds supported MSC growth and induced cell osteogenic differentiation in a regular medium in vitro that was manifested by an increase in ALP activity and expression of the CD45 phenotype marker. The data of computed tomography and histological studies showed 94% and 92%, respectively, regeneration of critical-sized calvarial bone defect in vivo at 28th day after implantation of MSC-seeded PHB/HA/ALG/MSC scaffolds with 3.6 times higher formation of the main amount of bone tissue at 22-28 days in comparison with acellular PHB/HA/ALG scaffolds that was shown at the first time by fluorescent microscopy using the original technique of intraperitoneal administration of fluorescent dyes to living postoperative rats. The obtained in vivo results can be associated with the MSC-friendly microstructure and in vitro osteogenic properties of PHB/HA base-scaffolds. Thus, the obtained data demonstrate the potential of MSCs encapsulated in the bioactive biopolymer/mineral/hydrogel scaffold to improve the bone regeneration process in critical-sized bone defects.

The Growth of 3T3 Fibroblasts on PHB, PLA and PHB/PLA Blend Films at Different Stages of Their Biodegradation In Vitro.

Over the past century there was a significant development and extensive application of biodegradable and biocompatible polymers for their biomedical applications. This research investigates the dynamic change in properties of biodegradable polymers: poly(3-hydroxybutyrate (PHB), poly-l-lactide (PLA), and their 50:50 blend (PHB/PLA)) during their hydrolytic non-enzymatic (in phosphate buffered saline (PBS), at pH = 7.4, 37 °C) and enzymatic degradation (in PBS supplemented with 0.25 mg/mL pancreatic lipase). 3T3 fibroblast proliferation on the polymer films experiencing different degradation durations was also studied. Enzymatic degradation significantly accelerated the degradation rate of polymers compared to non-enzymatic hydrolytic degradation, whereas the seeding of 3T3 cells on the polymer films accelerated only the PLA molecular weight loss. Surprisingly, the immiscible nature of PHB/PLA blend (showed by differential scanning calorimetry) led to a slower and more uniform enzymatic degradation in comparison with pure polymers, PHB and PLA, which displayed a two-stage degradation process. PHB/PLA blend also displayed relatively stable cell viability on films upon exposure to degradation of different durations, which was associated with the uneven distribution of cells on polymer films. Thus, the obtained data are of great benefit for designing biodegradable scaffolds based on polymer blends for tissue engineering.