Poly-3-hydroxybutyrate/chitosan composite films and nonwoven mats.

Composite films and nonwoven mats of the poly-3-hydroxybutyrate and chitosan natural polymers were prepared and investigated. DSC and FTIR techniques were used to confirm that P(3HB) blending with chitosan resulted in a decrease in P(3HB) crystallinity to 47% and 62% in the films and nonwoven mats, respectively. Scanning electron microscopy showed that addition of chitosan induced changes in the surface morphology of the composite films and a reduction in the diameter of ultrafine fibers in the nonwoven mats from 800 nm to 460 nm. The values of water contact angle for films (53°) and nonwoven mats (50.6°) suggested that chitosan enhanced hydrophilic properties and moisture absorption capacity of the composite materials. On the other hand, P(3HB) showed its reinforcing ability and improved the physical/mechanical properties of chitosan. The work included studies of in vitro biodegradation of the composite specimens and their ability to maintain cell growth and attachment in NIH 3T3 fibroblast culture.

[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.

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.

Population dynamics of transgenic strain Escherichia coli Z905/pPHL7 in freshwater and saline lake water microcosms with differing microbial community structures.

Populations of Escherichia coli Z905/pPHL7, a transgenic microorganism, were heterogenic in the expression of plasmid genes when adapting to the conditions of water microcosms of various mineralization levels and structure of microbial community. This TM has formed two subpopulations (ampicillin-resistant and ampicillin-sensitive) in every microcosm. Irrespective of mineralization level of a microcosm, when E. coli Z905/pPHL7 alone was introduced, the ampicillin-resistant subpopulation prevailed, while introduction of the TM together with indigenous bacteria led to the dominance of the ampicillin-sensitive subpopulation. A high level of lux gene expression maintained longer in the freshwater microcosms than in sterile saline lake water microcosms. A horizontal gene transfer has been revealed between the jointly introduced TM and Micrococcus sp. 9/pSH1 in microcosms with the Lake Shira sterile water.

Survival and alteration of the plasmid-containing microorganism Escherichia coli Z905/pPHL7 introduced into manmade closed aquatic microcosms.

It has been demonstrated that the transgenic microorganism Escherichia coli Z905/pPHL7 (AprLux+) can exist for a long time at an elevated concentration of mineral salts. The microorganism was introduced into microcosms with sterile brackish water (salinity variable from 21 to 22 g l-1) taken from Lake Shira (Khakasia, Russia). The survival of the microorganism was estimated both by measuring the growth of the colonies on solid nutrient media and by the bioluminescence exhibited by the transgenic strain in samples from the microcosms and in the enrichment culture with the added selective factor-ampicillin (50 micrograms/ml). In the enrichment culture, the bioluminescent signal was registered through the 160-day experiment. It has been shown that in the closed microcosms with brackish water the E. coli strain becomes heterogeneous in its ampicillin resistance. The populations of the transgenic strain were mainly represented by isolates able to persist in the medium containing 50 micrograms/ml, but there were also the cells (about 10%) with the threshold of ampicillin resistance not more than 0.05 micrograms/ml. Thus, it was shown that in the microcosms with brackish water and in the absence of the selective factor the transgenic strain survives and retails the recombinant plasmid.

Experimental evaluation of the processes resulting from the introduction of the transgenic microorganism Escherichia coli Z905/pPHL7 (lux+) into aquatic microcosms.

The processes resulting from the introduction of the tranagenic microorganism (TM) E. coli Z905/pPHL7 into aquatic microcosms have been modeled experimentally. It has been shown that the TM E. coli is able to adapt to a long co-existence with indigenous heterotrophic microflora in variously structured microcosms. In more complex microcosms the numerical dynamics of the introduced E. coli Z905/pPHL7 population is more stable. In the TM populations staying in the microcosms for a prolonged time, changes are recorded in the phenotypic expression of plasmid genes (ampicillin resistance and the luminescence level) and chromosome genes (morphological and physiological traits). However, in our study microcosms, the recombinant plasmid persisted in the TM cells for 6 years after the introduction, and as the population adapts to the conditions of the microcosms, the efficiency of the cloned gene expression in the cells is restored. In the microcosms with high microalgal counts (10(7) cells/ml), cells with a high threshold of sensitivity to ampicillin dominate in the population of the TM E. coli Z905/pPHL7.

Expression of lux-genes as an indicator of metabolic activity of cells in model ecosystem studies.

Quick response to different impacts and easy measurement make the luminescent systems of luminous bacteria an object convenient for application in various fields. Cloning of gene luminescence in different organisms is currently used to study both the survival of microbial cells and the effect of different factors on their metabolic activity, including the environment. A primary test-object in estimating bacteriological contamination of water bodies, Escherichia coli, can be conveniently used as an indicator of bactericidal properties of aquatic ecosystems. The application of Escherichia coli Z905/pPHL7 (lux+) as a marker microorganism can facilitate monitoring the microbiological status of closed biocenoses, including systems with higher organisms. The investigation of various parameters of microecosystems (carbon nutrition type, concentrations of inorganic ions and toxic compounds) shows that the recombinant strain E. coli Z905/pPHL7 can be effectively used as a marker.

Population dynamics of transgenic microorganisms in the different microecosystem conditions.

The role of key environmental factors in adaptation of spore-forming and non-spore-forming transgenic microorganisms (TM) have been studied in model ecosystems. Model TM Escherichia coli Z905 (bearing plasmid genes of bacterial luminescence Ap (r) Lux+) has been found to have a higher adaptation potential than TM Bacillus subtilis 2335/105 (bearing genes of human alpha 2-interferon Km (r) Inf+), planned for employment as a living vaccine under varying environmental conditions. Effects of abiotic factors on migration of natural and recombinant plasmids between microorganisms under model ecosystem conditions has been estimated. The transgenic microorganisms with low copy number survived better under introduction conditions in the microcosms studied. This trend has been shown to be independent of the microcosm type and its complexity. Grant numbers: 99-04-96017, 25, 00-07-9011.

Experimental microcosms as models of natural ecosystems for monitoring survival of genetically modified microorganism.

An experimental approach for investigation of genetically modified microorganisms (GMMO) introduced into model ecosystems to evaluate potential risk of propagation of recombinant plasmids in surrounding medium has been developed. The object of modeling was Escherichia coli Z905 strain with a recombinant plasmid with bacterial luminescence genes, which was introduced into water microcosms of different structure. The approach involves comprehensive investigation of GMMO at four hierarchical levels: molecular (retaining the structure of the plasmid and expression of cloned genes); cellular (variation of metabolic activity); population (competitive power and metabolic interactions of GMMO with indigenous microflora, migration of recombinant and natural plasmids); ecosystem (effect of GMMO and cloned genes on ecosystem parameters). The experimental evidence and theoretical estimates are intended to form grounds to develop a basic version of an ecological certificate for different GMMO variants.