Thermal, mechanical and biodegradation studies of biofiller based poly-3-hydroxybutyrate biocomposites.

Biodegradable poly-3-hydroxybutyrate [P(3HB)] and natural fillers - clay, peat, and birch wood flour - were used to prepare powdered composites to form pellets and granules. Pellets were produced by cold pressing of polymer and filler powder whereas granules were produced from the powders wetted with ethanol. Characterization techniques like IR spectroscopy, differential scanning calorimetry, X-ray analysis, mechanical analysis and electron microscopy were employed to study the properties of the initial P(3HB) and fillers and the composites. Analysis of the IR spectra of the composites showed the absence of chemical bonds between the components, i.e. the composites were physical mixtures. Young's moduli of the pellets prepared from initial materials varied considerably, and the highest value was obtained for P(3HB) pellets (350 MPa). Studies of biodegradation of composite pellets and granules in the soil for 35 days showed that the residual mass of the pellets had decreased to 68% for P(3HB); 56.4% for P(3HB)/peat; 67% for P(3HB)/wood flour, and 64% for P(3HB)/clay; granules exhibited a similar mass loss, residual mass of the granules of P(3HB) was 68.4%, P(3HB)/peat 46.4%; P(3HB)/wood flour 77%, and P(3HB)/clay 74%. This shows the significance of the material as an eco-friendly composite without sacrificing its mechanical properties.

Biotechnological wound dressings based on bacterial cellulose and degradable copolymer P(3HB/4HB).

Hybrid wound dressings have been constructed using two biomaterials: bacterial cellulose (BC) and copolymer of 3-hydroxybutyric and 4-hydroxybutyric acids [P(3HB/4HB)] - a biodegradable polymer of microbial origin. Some of the experimental membranes were loaded with drugs promoting wound healing and epidermal cells differentiated from multipotent adipose-derived mesenchymal stem cells. A study has been carried out to investigate the structure and physical/mechanical properties of the membranes. The in vitro study showed that the most effective scaffolds for growing fibroblasts were composite BC/P(3HB/4HB) films loaded with actovegin. Two types of the experimental biotechnological wound dressings - BC/P(3HB/4HB)/actovegin and BC/P(3HB/4HB)/fibroblasts - were tested in vivo, on laboratory animals with model third-degree skin burns. Wound planimetry, histological examination, and biochemical and molecular methods of detecting factors of angiogenesis, inflammation, type I collagen, and keratin 10 and 14 were used to monitor wound healing. Experimental wound dressings promoted healing more effectively than VoskoPran - a commercial wound dressing.

[The study of effectiveness of polyhydroxyalkanoates based biodegradable stents in ureteropelvic segment pyeloplasty].

INTRODUCTION: Among urologic diseases, ureteropelvic segment stenosis with hydronephrosis is a common indication for instrumental or surgical correction. The restriction of urine flow with dilatation proximal to obstruction develops in 6.5-37% of cases at different times after the ureteral reconstruction. All this urges to develop and improve stents and search for effective ways to place stents and control their function. AIM: To investigate the effectiveness of polyhydroxyalkanoates based biodegradable stent compared with a commercial analogue in upper urinary tract drainage after ureteropelvic segment pyeloplasty. MATERIALS AND METHODS: Morphological and functional changes in the stented ureter were investigated in 45 male rabbits of "Soviet chinchilla" breed weighing 4550-5200 g that underwent stenting of ureteropelvic segment (UPS). The study used polymeric stents based on poly-3-hydroxybutyrate, poly-4-gidroksibutirotom P (3GB/4GB) and a mixture of poly-3-hydroxybutyrate with polycaprolactone II (3GB)/PCL with the inclusion of PCL 75%; the control material was polyurethane stents. Morphologic evaluation was conducted on ureteral fragments and UPSs in the area of the stent placement at 7, 14 and 28 days after operation. RESULTS: Throughout the experiment, excretory urography and spiral tomography in experimental groups showed no changes in the pelvicalyceal system after placing polymeric stents. The morphologic examination in the experimental group at day 28 after surgery revealed preserved longitudinal folding of ureteral mucosa and absence of muscle hypertrophy. Transitional epithelium had no signs of atrophy and desquamation, its mean thickness was 112.4+/-8.5 mm, whereas in the control group a productive inflammation resulting in sclerosis was found to develop. CONCLUSIONS: We conducted a comparative study of morphologic and functional changes in rabbit ureters after stenting using polyurethane stents (control group) and polymeric stents made of poly-3-hydroxybutyrate, poly-4-hydroxybutyrate II (3GB /4GB) and a mixture of poly-3-hydroxybutyrate with polycaprolactone II (3Gb)/PCL (experimental group). Despite the difference in physical and mechanical properties of biodegradable PGA-based stents, the tissue response to both types of stent was comparable. Ureteral wall preserved longitudinal folds, there was no muscle layer hypertrophy, and mucous membrane had smooth contours with a uniform thickness of the transitional epithelium, whereas in the control group a productive inflammation resulting in sclerosis was found to develop.

Porous 3D implants of degradable poly-3-hydroxybutyrate used to enhance regeneration of rat cranial defect.

The study describes preparation and testing of porous 3D implants of natural degradable polymer of 3-hydroxybutyric acid P(3HB) for regeneration of bone tissue defects. The ability of the P(3HB) implants to favor attachment and facilitate proliferation and directed differentiation of mesenchymal stem cells (MSCs) was studied in the culture of MSCs isolated from bone marrow and adipose tissue. Tissue-engineered hybrid systems (grafts) constructed using P(3HB) and P(3HB) in combination with osteoblasts were used in experiments on laboratory animals (n = 48) with bone defect model. The defect model (5 mm in diameter) was created in the rat parietal bone, and filling of the defect by the new bone tissue was monitored in the groups of animals with P(3HB) implants, with commercial material, and without implants (negative control). Computed tomography (CT) and histologic examination showed that after 120 days, in the group with the osteoblast-seeded P(3HB) implants, the defect was completely closed; in the group with the cell-free P(3HB) implants, the remaining defect was no more than 10% of the initial one (0.5 mm); in both the negative and positive controls, the size of the defect was about 1.0-1.2 mm. These results suggest that P(3HB) has good potential as osteoplastic material for reconstructive osteogenesis. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 566-577, 2017.

Surface wettability and energy effects on the biological performance of poly-3-hydroxybutyrate films treated with RF plasma.

The surface properties of poly-3-hydroxybutyrate (P3HB) membranes were modified using oxygen and an ammonia radio-frequency (RF, 13.56 MHz) plasma. The plasma treatment procedures used in the study only affected the surface properties, including surface topography, without inducing any significant changes in the crystalline structure of the polymer, with the exception being a power level of 250 W. The wettability of the modified P3HB surfaces was significantly increased after the plasma treatment, irrespective of the treatment procedure used. It was revealed that both surface chemistry and surface roughness changes caused by the plasma treatment affected surface wettability. A treatment-induced surface aging effect was observed and resulted in an increase in the water contact angle and a decrease in the surface free energy. However, the difference in the water contact angle between the polymers that had been treated for 4 weeks and the untreated polymer surfaces was still significant. A dependence between cell adhesion and proliferation and the polar component of the surface energy was revealed. The increase in the polar component after the ammonia plasma modification significantly increased cell adhesion and proliferation on biodegradable polymer surfaces compared to the untreated P3HB and the P3HB modified using an oxygen plasma.

Laser processing of polymer constructs from poly(3-hydroxybutyrate).

CO2 laser radiation was used to process poly(3-hydroxybutyrate) constructs - films and 3D pressed plates. Laser processing increased the biocompatibility of unperforated films treated with moderate uniform radiation, as estimated by the number and degree of adhesion of NIH 3T3 mouse fibroblast cells. The biocompatibility of perforated films modified in the pulsed mode did not change significantly. At the same time, pulsed laser processing of the 3D plates produced perforated scaffolds with improved mechanical properties and high biocompatibility with bone marrow-derived multipotent, mesenchymal stem cells, which show great promise for bone regeneration.

The use of polymeric microcarriers loaded with anti-inflammatory substances in the therapy of experimental skin wounds.

We studied the effects of anti-inflammatory substances incorporated in polymeric microparticles made of degradable natural polyhydroxyalkanoate polyesters on experimental skin wounds caused by chemical burns in laboratory animals. Treatment with encapsulated forms of anti-inflammatory substances (applied in gel) accelerated wound healing in comparison with routine therapy (estimated by area of burn wound, wound healing activity, number of acanthotic cells, and number of hair and sebaceous follicles). The results showed the perspectives of usage of developed form of substances (degradable polymeric microparticles) for treatment of skin defects.

[Study of electrokinetic potential of drug micro-carriers prepared from resorbable polymers bioplastotan].

The study investigates electrokinetic potential of microparticles prepared from biodegradable poly-3-hydroxybutyrate depending upon the method of preparation employed and taking into account the size of particles maintained in liquid media. The ζ-potential of microparticles. prepared from emulsion by solvent evaporation method was -20 mV; the ζ-potential of microparticles prepared by spray drying was reduced to -95 mV. The value of ζ-potential was influenced by drug loading into microparticles; the drug-loaded microparticles maintained in balanced phosphate buffer for 30 days had higher physical stability than those without drug loading.

Microparticles prepared from biodegradable polyhydroxyalkanoates as matrix for encapsulation of cytostatic drug.

Microparticles made from degradable polyhydroxyalkanoates of different chemical compositions a homopolymer of 3-hydroxybutyric acid, copolymers of 3-hydroxybutyric and 4-hydroxybutyric acids (P3HB/4HB), 3-hydroxybutyric and 3-hydroxyvaleric acids (P3HB/3HV), 3-hydroxybutyric and 3-hydroxyhexanoic acids (P3HB/3HHx) were prepared using the solvent evaporation technique, from double emulsions. The study addresses the influence of the chemical compositions on the size and ξ-potential of microparticles. P3HB microparticles loaded with doxorubicin have been prepared and investigated. Their average diameter and ξ-potential have been found to be dependent upon the level of loading (1, 5, and 10 % of the polymer mass). Investigation of the in vitro drug release behavior showed that the total drug released from the microparticle into the medium increased with mass concentration of the drug. In this study mouse fibroblast NIH 3T3 cells were cultivated on PHA microparticles, and results of using fluorescent DAPI DNA stain, and MTT assay showed that microparticles prepared from PHAs of different chemical compositions did not exhibit cytotoxicity to cells cultured on them and proved to be highly biocompatible. Cell attachment and proliferation on PHA microparticles were similar to those on polystyrene. The cytostatic drug encapsulated in P3HB/3HV microparticles has been proven to be effective against HeLa tumor cells.

[Biodegradation of polyhydroxyalkanoates by soil microbiocoenoses of different structures and detection of microorganisms-destructors].

Biodegradation of microbial linear polymers of hydroxyalkanoic acids (polyhydroxyalkanoates, PHAs) by soil microbiocoenoses of different structures has been studied during two field seasons in different weather conditions. This process was shown to be influenced by the polymer chemical composition, temperature, humidity, and the microbial soil component. The PHA degradation was accompanied by a decrease in the polymer molecular weight and an increase in the degree of crystallinity, indicating the preferential destruction of the amorphous phase compared to the crystalline one. The quantity of the true PHA destructors developing at the surface of the polymer samples was lower than the quantity of accompanying bacteria. The dominant PHA destructors under the test conditions were identified as bacteria of the genera Variovorax, Stenotrophomonas, Acinetobacter, Pseudomonas, Bacillus, and Xanthomonas and as micromycetes from Penicillium, Paecilomyces, Acremonium, Verticillium. and Zygosporium.

[Synthesis of 3-hydroxybutyrate-CO-4-hydroxybutyrate copolymers by hydrogen-oxidizing bacteria].

Synthesis of 3- and 4-hydroxybutyrate copolymer (3HB-CO-4HB), the most promising member of the biodegradable polyhydroxyalcanoate (PHA) family, has been studied. Cultivation conditions of naturally occurring strains of hydrogen-oxidizing bacteria Ralstonia eutropha B5786 and Cupriavidus eutrophus B10646 have been optimized to ensure efficient synthesis of the 3HB-CO-4HB copolymer. A set of highly pure samples of the 3HB-CO-4HB copolymer with 4HB content varying from 8.7 to 24.3 mol % has been obtained. Incorporation of 4-HB into the copolymer was shown to cause a more pronounced decrease in polymer crystallinity than the incorporation of 3-hydroxyvalerate or 3-hydroxyhexanoate; samples with a degree of crystallinity below 30% have been obtained. The weight average molecular mass of the 3HB-CO-4HB copolymers was shown to be independent on the monomer ratio and to vary broadly (from 540 to 1110 kDa).

[Modification of the surface of bioresorbed polymer matrices by laser treatment].

The effect of laser irradiation on the properties of the surface of film matrices obtained from the bioresorbed polymer polyhydroxybuturate has been studied. To determine the spectral region of the polymer optimal for the effective action of radiation on electron molecular bonds, theoretical investigations have been performed, which have shown that, for modifying the surface of matrices obtained from polyhydroxybuturate, it is expedient to use a vacuum laser at a wavelength of 160 nm. Using the laser regime of irradiation of matrices at a radiation power from 3.0 to 30 Wt, a series of films with modified surface, from roughnesses to perforations, have been obtained. The microstructure and properties of the film surface depending on the mode of irradiation have been examined, and conditions have been found under which the contact marginal angles of moistening of films with water can be decreased to 50 degrees (compared with 76-80 degrees in starting products). Thus, the conditions of laser treatment of polyhydroxybuturate matrices have been theoretically substantiated and experimentally realized that provide a beneficial effect on the properties of the surface without destroying the structure of the material.

[Cloning and molecular organization of the polyhydroxyalkanoic acid synthase gene (phaC) of Ralstonia eutropha strain B5786].

Class I polyhydroxyalkanoic acid (PHA) synthase gene (phaC) of Ralstonia eutropha strain B5786 was cloned and characterized. R. eutropha B5786 features the ability to synthesize multicomponent PHAs with short- and medium-chain-length monomers from simple carbohydrate substrate. A correlation was made between the molecular structure of PHA synthase and substrate specificity and the ability of strain-producers to accumulate PHAs of this or that structure. A strong similarity of PHA synthase of R. eutropha strain B5786 with PHA synthase of R. eutropha strain H16, which, as opposed to strain B5786, enables to incorporate medium chain length PHAs if hexanoate is used as carbon source, exhibited 99%. A correlation between the structure of PHA synthase of B5786 strain with synthases of microorganisms which synthesize short and medium chain length PHAs similarly to B5786 strain, showed an identity level from 26 to 41% (homology with synthase of Rhodospirillum rubrum makes 41%, Ectothiorhodospira shaposhnikovii makes 26%, Aeromonas punctata makes 40%, Thiococcus pfennigii makes 28%, Rhodococcus ruber makes 38%, and with PhaCl and PhaC2 synthases of Pseudomonas sp. 61-3 makes 34 and 37%, respectively). This allows for speaking about the absence of a direct connection between the molecular organization of PHA synthases and their functional abilities, namely, the ability to synthesize PHAs of a particular composition.

[Characteristics of proteins synthesized by hydrogen-oxidizing microorganisms].

The study was conducted to determine the biological value of proteins synthesized by hydrogen-oxidizing microorganisms--the hydrogen bacteria Alcaligenes eutrophus Z1 and Ralstonia eutropha B5786 and the CO-resistant strain of carboxydobacterium Seliberia carboxydohydrogena Z1062. Based on a number of significant parameters characterizing the biological value of a product, the proteins of hydrogen-oxidizing microorganisms have been found to occupy an intermediate position between traditional animal and plant proteins. The high total protein in biomass of these microorganisms, their complete amino acid content, and availability to proteolytic enzymes allow for us to consider these microorganisms as potential protein producers.

[Microbial polymers as a degradable carrier for pesticide delivery].

The possibility of use of polyhydroxyalkanoates (PHAs), biodegradable microbial polyesters, as a carrier for pesticides (alpha-hexachlorocyclohexane and lindane) for targeted and controlled delivery of these compounds to soil was investigated. The kinetics of polymer degradation and the dynamics of pesticide release from the extended-release formulations was studied. It is shown that pesticides embedded in a degradable polymer (PHA) carrier are released gradually and slowly, without surges, as the polymer is degraded by the soil microflora. The microbial soil component actively responded to the addition of the polymer as an additional nutrient substrate: the latter was degraded and then utilized. The rate of the pesticide release to the soil can be regulated by varying the polymer-pesticide ratio.

Distribution and resorption of polymeric microparticles in visceral organs of laboratory animals after intravenous injection.

Microparticles obtained by using (14)C-labeled resorbable hydroxyaminobutyric acid polymer were injected into the caudal vein of laboratory animals without negative aftereffects for their growth and development and without changes in the macro- and microstructure of organs and tissues. The distribution of microparticles in the viscera and the dynamics of accumulation of carbon-containing polymer degradation products in the viscera were studied. The main targets for the particles are liver tissues, as well as renal and splenic tissues. The polymeric matrix of the microparticles is most actively destroyed in the spleen and liver. The presence of high-molecular-weight polymeric matrix in organs indicates the integrity of microparticles and the possibility of long-term (up to 12 weeks) functioning of polymeric particles in vivo.

Evaluation of antitumor activity of rubomycin deposited in absorbable polymeric microparticles.

An experimental dosage form of rubomycin is developed: the drug is incorporated in absorbable polymeric (polyhydroxybutyrate) matrix in the form of microparticles. Antitumor efficiency of this rubomycin dosage form was studied in laboratory mice with transplanted Ehrlich ascitic carcinoma. Rubomycin deposited in polymeric microparticles exhibited pronounced antitumor activity, inhibited the proliferative activity of Ehrlich ascitic carcinoma, and improved survival of mice with tumors. This dosage form of the drug can be used for local injections.

[Synthesis of reserve polyhydroxyalkanoates by luminescent bacteria].

The ability of marine luminescent bacteria to synthesize polyesters of hydroxycarboxylic acids (polyhydroxyalkanoates, PHA) as reserve macromolecules was studied. Twenty strains from the collection of the luminescent bacteria CCIBSO (WDSM839) of the Institute of Biophysics, Siberian Branch, Russian Academy of Sciences, assigned to different taxa (Photobacterium leiognathi, Ph. phosphoreum, Vibrio harveyi, and V. fischeri) were analyzed. The most productive strains were identified, and the conditions ensuring high polymer yields in batch culture (40-70% of the cell dry mass weight) were determined. The capacity of synthesizing two- and three-component polymers containing hydroxybutyric acid as the main monomer and hydroxyvaleric and hydroxyhexanoic acids was revealed in Ph. leiognathi and V. harveyi strains. The results allow luminescent microorganisms to be regarded as new producers of multicomponent polyhydroxyalkanoates.