Poly(3-hydroxybutyrate)/poly(ethylene glycol) scaffolds with different microstructure: the effect on growth of mesenchymal stem cells.

Development of biocompatible 3D scaffolds is one of the most important challenges in tissue engineering. In this study, we developed polymer scaffolds of different design and microstructure to study cell growth in them. To obtain scaffolds of various microstructure, e.g., size of pores, we used double- and one-stage leaching methods using porogens with selected size of crystals. A composite of poly(3-hydroxybutyrate) (PHB) with poly(ethylene glycol) (PEG) (PHB/PEG) was used as polymer biomaterial for scaffolds. The morphology of scaffolds was analyzed by scanning electron microscopy; the Young modulus of scaffolds was measured by rheometry. The ability to support growth of mesenchymal stem cells (MSCs) in scaffolds was studied using the XTT assay; the phenotype of MSC was preliminarily confirmed by flow cytometry and the activity of alkaline phosphatase and expression level of CD45 marker was studied to test possible MSC osteogenic differentiation. The obtained scaffolds had different microstructure: the scaffolds with uniform pore size of about 125 µm (normal pores) and 45 µm (small pores) and scaffolds with broadly distributed pores size from about 50-100 µm. It was shown that PHB/PEG scaffolds with uniform pores of normal size did not support MSCs growth probably due to their marked spontaneous osteogenic differentiation in these scaffolds, whereas PHB/PEG scaffolds with diverse pore size promoted stem cells growth that was not accompanied by pronounced differentiation. In scaffolds with small pores (about 45 µm), the growth of MSC was the lowest and cell growth suppression was only partially related to stem cells differentiation. Thus, apparently, the broadly distributed pore size of PHB/PEG scaffolds promoted MSC growth in them, whereas uniform size of scaffold pores stimulated MSC osteogenic differentiation.

Biosynthesis of poly(3-hydroxybutyrate) copolymers by Azotobacter chroococcum 7B: A precursor feeding strategy.

A precursor feeding strategy for effective biopolymer producer strain Azotobacter chroococcum 7B was used to synthesize various poly(3-hydroxybutyrate) (PHB) copolymers. We performed experiments on biosynthesis of PHB copolymers by A. chroococcum 7B using various precursors: sucrose as the primary carbon source, various carboxylic acids and ethylene glycol (EG) derivatives [diethylene glycol (DEG), triethylene glycol (TEG), poly(ethylene glycol) (PEG) 300, PEG 400, PEG 1000] as additional carbon sources. We analyzed strain growth parameters including biomass and polymer yields as well as molecular weight and monomer composition of produced copolymers. We demonstrated that A. chroococcum 7B was able to synthesize copolymers using carboxylic acids with the length less than linear 6C, including poly(3-hydroxybutyrate-co-3-hydroxy-4-methylvalerate) (PHB-4MHV) using Y-shaped 6C 3-methylvaleric acid as precursor as well as EG-containing copolymers: PHB-DEG, PHB-TEG, PHB-PEG, and PHB-HV-PEG copolymers using short-chain PEGs (with n ≤ 9) as precursors. It was shown that use of the additional carbon sources caused inhibition of cell growth, decrease in polymer yields, fall in polymer molecular weight, decrease in 3-hydroxyvalerate content in produced PHB-HV-PEG copolymer, and change in bacterial cells morphology that were depended on the nature of the precursors (carboxylic acids or EG derivatives) and the timing of its addition to the growth medium.

Biosynthesis of poly(3-hydroxybutyrateco-3-hydroxy-4-methylvalerate) by Strain Azotobacter chroococcum 7B.

Production of novel polyhydroxyalkanoates (PHAs), biodegradable polymers for biomedical applications, and biomaterials based on them is a promising trend in modern bioengineering. We studied the ability of an effective strain-producer Azotobacter chroococcum 7B to synthesize not only poly(3-hydroxybutyrate) homopolymer (PHB) and its main copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), but also a novel copolymer, poly(3-hydroxybutyrate-co-3-hydroxy-4-methylvalerate) (PHB4MV). For the biosynthesis of PHB copolymers, we used carboxylic acids as additional carbon sources and monomer precursors in the chain of synthesized copolymers. The main parameters of these polymers' biosynthesis were determined: strain-producer biomass yield, polymer yield, molecular weight and monomer composition of the synthesized polymers, as well as the morphology of A. chroococcum 7B bacterial cells. The physico-chemical properties of the polymers were studied using nuclear magnetic resonance spectroscopy (NMR), differential scanning calorimetry (DSC), contact angle test, and other methods. In vitro biocompatibility of the obtained polymers was investigated using stromal cells isolated from the bone marrow of rats with the XTT cell viability test. The synthesis of the novel copolymer PHB4MV and its chemical composition were demonstrated by NMR spectroscopy: the addition of 4-methylvaleric acid to the culture medium resulted in incorporation of 3-hydroxy-4-methylvalerate (3H4MV) monomers into the PHB polymer chain (0.6 mol%). Despite the low molar content of 3H4MV in the obtained copolymer, its physico-chemical properties were significantly different from those of the PHB homopolymer: it has lower crystallinity and a higher contact angle, i.e. the physico-chemical properties of the PHB4MV copolymer containing only 0.6 mol% of 3H4MV corresponded to a PHBV copolymer with a molar content ranging from 2.5% to 7.8%. In vitro biocompatibility of the obtained PHB4MV copolymer, measured in the XTT test, was not statistically different from the cell growth of PHB and PHBV polymers, which make its use possible in biomedical research and development.

[Development and preclinical studies of insulating membranes based on poly-3-hydroxybutyrate-co-3-hydroxyvalerate for guided bone regeneration].

Bone tissue damages are one of the dominant causes of temporary disability and developmental disability. Currently, there are some methods of guided bone regeneration employing different osteoplastic materials and insulation membranes used in surgery. In this study, we have developed a method of preparation of porous membranes from the biopolymer poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV), produced by a strain of Azotobacter chroococcum 7B. The biocompatibility of the porous membranes was investigated in vitro using mesenchymal stem cells (MSCs) and in vivo on laboratory animals. The cytotoxicity test showed the possibility of cell attachment on membrane and histological studies confirmed good insulating properties the material. The data obtained demonstrate the high biocompatibility and the potential application of insulating membranes based on PHBV in bone tissue engineering.

Culturing of Mouse Mesenchymal Stem Cells on Poly-3-Hydroxybutyrate Scaffolds.

We studied the possibility of long-term culturing of mouse mesenchymal stem cells on a porous scaffold made of biocompatible polymer poly-3-hydroxybutyrate. The cells remained viable for at least 2 months and passed more than 65 population doublings in culture. Culturing on the scaffold did not change surface phenotype of cells. 3D poly-3-hydroxybutyrate scaffolds are appropriate substrate for long-term culturing of mesenchymal stem cells.

[The effect of poly(3-hydroxybutyrate) modification by poly(ethylene glycol) on the viability of cells grown on the polymer films].

A biodegradable polymer of bacterial origin, poly(3-hydroxybutyrate) (PHB), is intensively studied as biomaterial for tissue engineering. However, factors determining its biocompatibility still require better understanding. To analyze the PHB films biocompatibility, the polymer material was modified by hydrophilic polymer, poly(ethylene glycol) 300 (PEG). The blends PHB/PEG with different PEG content (10, 20, 30 and 50%) were produced by subsequent incubation in water resulted in removal of 95% PEG. The surface roughness and hydrophilicity were studied by atomic force microscopy (AFM) and contact angle "water-polymer" measurement, respectively. The film biocompatibility on cell culture of COS-1 fibroblasts was studied in vitro. It was shown that both roughness and hydrophobicity are directly proportional to initial PEG content in the PHB/PEG blends. The growth rate of COS-1 fibroblasts on polymer films is determined by combination of two basic physicochemical properties of the polymer surface: the roughness and hydrophilicity. The optimal roughness requred for COS-1 cells growth is the average roughness more than 25 nm, whereas the limit values of the contact angle "water-polymer" that was responsible for relatively high cell viability were not found. These data indicate that the film surface roughness had the greatest effect on the cell growth, whereas the increase in the polymer surface hydrophilicity caused the additional positive effect on viability of attached cells. Thus, the modification of PHB polymer material by PEG resulted in the improved viability of cells cultivated on the polymer films in vitro. The obtained data can be used for development of such medical devices as surgeon patches and periodontal membranes.

[Prolonged release of chlorambucil and etoposide from poly-3-oxybutyrate-based microspheres].

Microspheres were obtained on the basis of poly(3-oxibutyrate) (POB) with the inclusion of the Chlorambucil and Etoposide cytostatic drugs in a polymer matrix, and the morphology, kinetics of drug release from microspheres, and the interaction between microspheres and tumor cells in vitro were studied. Data on the kinetics of drug release suggests that a prolonged release occurs by drug diffusion from the polymer matrix at the initial stage and at the expense of hydrolytic degradation of the polymer at a later stage. A study of the biocompatibility and biological activity of biopolymeric microspheres showed that chlorambucil operates actively and strongly inhibits the growth of cultured cells for a short time (24 h). Etoposide acts weaker (the percentage of cell growth suppression during 48 h does not exceed 50%), but subsequently it has a basis for the creation of new dosage forms with prolonged action of Etoposide and chlorambucil for cancer therapy.

[Biosynthesis of poly-3-hydroxybutyrate-3-hydroxyvalerate copolymer by Azotobacter chroococcum strain 7B].

The ability of Azotobacter chroococcum strain 7B, producer of polyhydroxybutyrate (PHB), to synthesize its copolymer poly-3-hydroxybutyrate-3-hydroxyvalerate (PHB-HV) was studied. It was demonstrated, for the first time, that A. chroococcum strain 7B was able to synthesize PHB-HV with various molar rates of HV in the polymer chain when cultivated on medium with sucrose and carboxylic acids as precursors of HV elements in the PHB chain, namely, valeric (13.1-21.6 mol %), propanoic (3.1 mol %), and hexanoic (2.1 mol %) acids. Qualitative and functional differences between PHB and PHB-HV were demonstrated by example of the release kinetic of methyl red from films made of synthesized polymers. Maximal HV incorporation into the polymer chain (28.8 mol %) was recorded when the nutrient medium was supplemented with 0.1% peptone on the background of 20 mM valerate. These results suggest that that the studied strain can be regarded as a potential producer of not only PHB but also PHB-HV.

[A comparative study of biodegradation kinetics of biopolymer systems based on poly(3-hydroxybutyrate)].

The aim of this study was to evaluate and to compare of long-term kinetics curves of biodegradation of poly(3-hydroxybutyrate) (PHB), its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate), and PHB/polylactic acid blend. The total weight loss and the change of average viscosity molecular weight were used as an index of biodegradation degree. The rate of biodegradation was analyzed in vitro in presence oflipase and in vivo when the films were implanted in animal tissues. The morphology of PHB films surface was studied by atomic force microscopy technique. It was shown that biodegradation of PHB is occurred by means of as polymer hydrolysis, and as its enzymatic biodegradation. The obtained data can be used for development of medical devices on the base of PHB.

[Effect of growth conditions on the molecular weight of poly-3-hydroxybutyrate produced by Azotobacter chroococcum 7B].

It has been shown that poly-3-hydroxybutyrate (PHB) of predetermined molecular weight can be obtained by varying the growth conditions of the producer strain, Azotobacter chroococcum 7B: pH, temperature, aeration, presence of sodium acetate as an additional carbon source, or growth on crude complex carbon sources (molasses, vinasse, or starch). High-molecular-weight polymer can be obtained at pH 7.0, optimal for the culture (1485 kDa), temperature 30-37 degrees C (1600-1450 kDa, respectively), and low aeration (2215 kDa). The following factors decrease PHB MW: pH deviation to the acidic (pH 6.0, 476 kDa) or alkaline (pH 8.0, 354 kDa) range or lower temperature (20 degrees C, 897 kDa). Introduction of additional carbon source (sodium acetate) at concentrations in the medium varying from 0 to 5 g/l provides an original method of production of PHB with predetermined MW in a wide range, from 270 to 1515 kDa, with high PHB content in the cell.

Aerobic and anaerobic microbial degradation of poly-beta-hydroxybutyrate produced by Azotobacter chroococcum.

Food industry wastewater served as a carbon source for the synthesis of poly-beta-hydroxybutyrate (PHB) by Azotobacter chroococcum. The content of polymer in bacterial cells grown on the raw materials reached 75%. PHB films were degraded under aerobic, microaerobic, and anaerobic conditions in the presence and absence of nitrate by microbial populations of soil, sludges from anaerobic and nitrifying/denitrifying reactors, and sediment from a sludge deposit site. Changes in molecular mass, crystallinity, and mechanical properties of PHB were studied. Anaerobic degradation was accompanied by acetate formation, which was the main intermediate utilized by denitrifying bacteria or methanogenic archaea. On a decrease in temperature from 20 to 5 degrees C in the presence of nitrate, the rate of PHB degradation was 7.3 times lower. Under anaerobic conditions and in the absence of nitrate, no PHB degradation was observed, even at 11 degrees C. The enrichment cultures of denitrifying bacteria obtained from soil and anaerobic sludge degraded PHB films for a short time (3-7 d). The dominant species in the enrichment culture from soil were Pseudomonas fluorescens and Pseudomonas stutzeri. The rate of PHB degradation by the enrichment cultures depended on the polymer molecular weight, which reduced with time during biodegradation.

[Reduction of nitrates by cultures of Azotobacter indicum and Azotobacter chroococcum ]. / Vosstanovlenie nitratov kul'turami Azotobacter indicum i Azotobacter chroococcum.

The capacity for denitrification was studied in Azotobacter bacteria, which are free-living nitrogen-fixing obligatory aerobes. Data on the nitrate reduction to nitrites and nitric oxide by A. indicum under anaerobic conditions were obtained for the first time for genus Azotobacter.

[The biodegradation of poly-beta-hydroxybutyrate (PHB) by a model soil community: the effect of cultivation conditions on the degradation rate and the physicochemical characteristics of PHB]. / Biodegradatsiia poli-beta-gidroksibutirata v model'nykh usloviiakh pochvennogo soobshchestva: vliianie uslovii sredy na skorost' protsessa i fisiko-mekhanicheskie kharakteristiki polimera.

The biodegradation of films made of poly-beta-hydroxybutyrate (PHB) with a molecular mass of 1500 kDa was studied using a model soil community in the presence and absence of nitrate and at different concentrations of oxygen in the gas phase. The biodegradation of PHB was investigated with respect to changes in its molecular mass, crystallinity, and some mechanical properties.