Electrospun magnetic composite poly-3-hydroxybutyrate/magnetite scaffolds for biomedical applications: composition, structure, magnetic properties, and biological performance

Magnetically responsive composite polymer scaffolds have good potential for a variety of biomedical applications. In this work, electrospun composite scaffolds made of polyhydroxybutyrate (PHB) and magnetite (Fe3O4) particles (MPs) were studied before and after degradation in either PBS or a lipase solution. MPs of different sizes with high saturation magnetization were synthesized by the coprecipitation method followed by coating with citric acid (CA). Nanosized MPs were prone to magnetite-maghemite phase transformation during scaffold fabrication, as revealed by Raman spectroscopy; however, for CA-functionalized nanoparticles, the main phase was found to be magnetite, with some traces of maghemite. Submicron MPs were resistant to the magnetite-maghemite phase transformation. MPs did not significantly affect the morphology and diameter of PHB fibers. The scaffolds containing CA-coated MPs lost 0.3 or 0.2% of mass in the lipase solution and PBS, respectively, whereas scaffolds doped with unmodified MPs showed no mass changes after 1 month of incubation in either medium. In all electrospun scaffolds, no alterations of the fiber morphology were observed. Possible mechanisms of the crystalline-lamellar-structure changes in hybrid PHB/Fe3O4 scaffolds during hydrolytic and enzymatic degradation are proposed. It was revealed that particle size and particle surface functionalization affect the mechanical properties of the hybrid scaffolds. The addition of unmodified MPs increased scaffolds' ultimate strength but reduced elongation at break after the biodegradation, whereas simultaneous increases in both parameters were observed for composite scaffolds doped with CA-coated MPs. The highest saturation magnetization-higher than that published in the literature-was registered for composite PHB scaffolds doped with submicron MPs. All PHB scaffolds proved to be biocompatible, and the ones doped with nanosized MPs yielded faster proliferation of rat mesenchymal stem cells. In addition, all electrospun scaffolds were able to support angiogenesis in vivo at 30 days after implantation in Wistar rats

Evaluation of eliciting activity of peptidil prolyl cys/trans isomerase from <em>Pseudonomas fluorescens</em> encapsulated in sodium alginate regarding plant resistance to viral and fungal pahogens

Use of chemical pesticides poses a threat for environment and human health, so green technologies of crop protection are of high demand. Some microbial proteins able to activate plant defense mechanisms and prevent the development of resistance in plant pathogens, may be good alternative to chemicals, but practical use of such elicitors is limited due to need to protect them against adverse environment prior their delivery to target receptors of plant cells. In this study we examined a possibility to encapsulate heat resistant FKBP-type peptidyl prolyl cis-trans isomerase (PPIase) from Pseudomonas fluorescens, which possesses a significant eliciting activity in relation to a range of plant pathogens, in sodium alginate microparticles and evaluated the stability of resulted complex under long-term UV irradiation and in the presence of proteinase K, as well as its eliciting activity in three different “plant-pathogen” models comparing to that of free PPIase. The obtained PPIase-containing microparticles consisted of 70% of sodium alginate, 20% of bovine serum albumin, and 10% of PPIase. In contrast to free PPIase, which lost its eliciting properties after 8-h UV treatment, encapsulated PPIase kept its eliciting ability unchanged; after being exposed to proteinase K, its eliciting ability twice exceeded that of free PPIase. Using “tobacco-TMV”, “tobacco-Alternaria longipes”, and “wheat-Stagonospora nodorum” model systems, we showed that encapsulation process did not influence on the eliciting activity of PPIase. In the case of the “wheat-S. nodorum” model system, we also observed a significant eliciting activity of alginate-albumin complex and almost doubled activity of encapsulated PPIase as compared to the free PPIase. As far as we know, this is the first observation of a synergistic interaction between alginate and other compound possessing any bioactive properties. The results of the study show some prospects for a PPIase use in agriculture

Competitive Biosynthesis of Bacterial Alginate Using <em>Azotobacter vinelandii</em> 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

The terpolymer produced by Azotobacter chroococcum 7B: effect of surface properties on cell attachment.

The copolymerization of poly(3-hydroxybutyrate) (PHB) is a promising trend in bioengineering to improve biomedical properties, e.g. biocompatibility, of this biodegradable polymer. We used strain Azotobacter chroococcum 7B, an effective producer of PHB, for biosynthesis of not only homopolymer and its main copolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-HV), but also novel terpolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate)-poly(ethylene glycol) (PHB-HV-PEG), using sucrose as the primary carbon source and valeric acid and poly(ethylene glycol) 300 (PEG 300) as additional carbon sources. The chemical structure of PHB-HV-PEG was confirmed by (1)H nuclear-magnetic resonance analysis. The physico-chemical properties (molecular weight, crystallinity, hydrophilicity, surface energy) of produced biopolymer, the protein adsorption to the terpolymer, and cell growth on biopolymer films were studied. Despite of low EG-monomers content in bacterial-origin PHB-HV-PEG polymer, the terpolymer demonstrated significant improvement in biocompatibility in vitro in contrast to PHB and PHB-HV polymers, which may be coupled with increased protein adsorption, hydrophilicity and surface roughness of PEG-containing copolymer

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 &deg;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

Poly(3-hydroxybutyrate) 3D-Scaffold–Conduit for Guided Tissue Sprouting

Scaffold biocompatibility remains an urgent problem in tissue engineering. An especially interesting problem is guided cell intergrowth and tissue sprouting using a porous scaffold with a special design. Two types of structures were obtained from poly(3-hydroxybutyrate) (PHB) using a salt leaching technique. In flat scaffolds (scaffold-1), one side was more porous (pore size 100–300 μm), while the other side was smoother (pore size 10–50 μm). Such scaffolds are suitable for the in vitro cultivation of rat mesenchymal stem cells and 3T3 fibroblasts, and, upon subcutaneous implantation to older rats, they cause moderate inflammation and the formation of a fibrous capsule. Scaffold-2s are homogeneous volumetric hard sponges (pore size 30–300 μm) with more structured pores. They were suitable for the in vitro culturing of 3T3 fibroblasts. Scaffold-2s were used to manufacture a conduit from the PHB/PHBV tube with scaffold-2 as a filler. The subcutaneous implantation of such conduits to older rats resulted in gradual soft connective tissue sprouting through the filler material of the scaffold-2 without any visible inflammatory processes. Thus, scaffold-2 can be used as a guide for connective tissue sprouting. The obtained data are advanced studies for reconstructive surgery and tissue engineering application for the elderly patients

AFM microphotography of film surface of produced PHAs.

<p>AFM microphotography of film surface of produced PHAs: (a) PHB, “rough” surface; (b) “smooth” surface; (c) PHB-HV, “smooth” surface; (d) PHB-HV-PEG, “smooth” surface.</p

Contact angles of different liquids on polymer films.

*<p>vs PHB, ^# vs PHB-HV, p<0.05.</p

Roughness of polymer films surfaces.

<p>Average roughness (R_a) and the root mean square roughness (R_q) of PHB, PHB-HV, and PHB-HV-PEG films surface.</p>*<p>vs PHB, ^# vs PHB-HV, p<0.05.</p