Growth of Mesenchymal Stem Cells on Poly(3-Hydroxybutyrate) Scaffolds Loaded with Simvastatin.

We studied the effect of porous composite scaffolds based on poly(3-hydroxybutyrate) (PHB) loaded with simvastatin on the growth and differentiation of mesenchymal stem cells. The scaffolds have a suitable microstructure (porosity and pore size) and physicochemical properties to support the growth of mesenchymal stem cells. Scaffold loading with simvastatin suppressed cell growth and increased alkaline phosphatase activity, which can attest to their osteoinductive properties.

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.

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.

[Sustained release of the antitumor drug paclitaxel from poly(3-hydroxybutyrate)-based microspheres].

Development of systems of medicines with sustained action on the basis of biodegradable polymers is a promising trend in modem pharmacology. Polyhydroxyalkanoates (POA) attract increasing attention due to their biodegradability and high biocompatibility, which make them suitable for development of novel drug dosage forms. We obtained microspheres on the basis of poly(3-hydroxybutyrate) (PHB) loaded with the antitumor drug paclitaxel. Morphology, drug release kinetics and effect on tumor cells in vitro of microspheres were studied. The data on the kinetics of drug release, biocompatibility and biological activity of the biopolymer microspheres in vitro showed that the studied system of prolonged drug release had lower toxicity and higher efficiency compared to the traditional dosage forms of paclitaxel.

[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.

[New poly-(3-hydroxybutyrate)-based systems for controlled release of dipyridamole and indomethacin].

New poly-(3-hydroxybutyrate)-based systems for controlled release of anti-inflammatory and antithrombogenic drugs have been studied. The release occurs via two mechanisms (diffusion and degradation) operating simultaneously. Dipyridamole and indomethacin diffusion processes determine the rate of the release at the early stages of the contact of the system with the environment (the first 6-8 days). The coefficient of the release diffusion of a drug depends on its nature, the thickness of the poly-(3-hydroxybutyrate) films containing the drug, the concentrations of dipyridamole and indomethacin, and the molecular weight of the poly-(3-hydroxybutyrate). The results obtained are critical for developing systems of release of diverse drugs, thus, enabling the attainment of the requisite physiological effects on tissues and organs of humans.

[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.