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.

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.

In vitro and transdermal penetration of PHBV micro/nanoparticles.

The purpose of this study was to develop micro and nano sized drug carriers from poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and study the cell and skin penetration of these particles. PHBV micro/nanospheres were prepared by o/w emulsion method and were stained with a fluorescent dye, Nile Red. The particles were fractionated by centrifugation to produce different sized populations. Topography was studied by SEM and average particle size and its distribution were determined with particle sizer. Cell viability assay (MTT) was carried out using L929 fibroblastic cell line, and particle penetration into the cells were studied. Transdermal permeation of PHBV micro/nanospheres and tissue reaction were studied using a BALB/c mouse model. Skin response was evaluated histologically and amount of PHBV in skin was determined by gas chromatography-mass spectrometry. The average diameters of the PHBV micro/nanosphere batches were found to be 1.9 µm, 426 and 166 nm. Polydispersity indices showed that the size distribution of micro sized particles was broader than the smaller ones. In vitro studies showed that the cells had a normal growth trend. MTT showed no signs of particle toxicity. The 426 and 166 nm sized PHBV spheres were seen to penetrate the cell membrane. The histological sections revealed no adverse effects. In view of this data nano and micro sized PHBV particles appeared to have potential to serve as topical and transdermal drug delivery carriers for use on aged or damaged skin or in cases of skin diseases such as psoriasis, and may even be used in gene transfer to cells.

Study of the efficiency of doxorubicin deposited in microparticles from resorbable Bioplastotane™ on laboratory animals with Ehrlich's solid carcinoma.

Antitumor efficiency of an experimental form of an experimental form of anthracyclin antibiotic (doxorubicin), resorbable microparticles from Bioplastotane(TM), was studied on laboratory mice with transplanted Ehrlich's solid carcinoma. Use of the experimental form of the cytostatic in polymeric microparticles from resorbable Bioplastotane(TM)in animals with solid tumor led to inhibition of the cancerous process, comparable to that in response to intravenous free doxorubicin, but without negative effects on the blood system.

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.

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.

Tissue reaction to intramuscular injection of resorbable polymer microparticles.

Tissue reaction to implantation of polymeric microparticles from resorbable polymer (polyhydroxybutyrate) is characterized by slight inflammatory reaction and pronounced progressive macrophage infiltration with the presence of mono- and multinuclear foreign body giant cells resorbing the polymeric matrix. No fibrous capsules were formed around the polymeric microparticles; neither necrosis nor other adverse morphological changes and tissue transformation in response to implantation of the PHB microparticles were recorded. The results indicate good prospects of using polyhydroxybutyrate for the construction of long-acting dosage forms as microparticles for intramuscular injection.

A hybrid PHB-hydroxyapatite composite for biomedical application: production, in vitro and in vivo investigation.

Samples of a hybrid composite of polyhydroxybutyrate (PHB), a biodegradable polyester, and hydroxyapatite (HA), with different PHB/HA ratios, have been prepared using mechanical-physical method. Electron microscopy, X-ray structure analysis and differential thermal analysis have been used to investigate the structure and physicochemical properties of the composite, depending on the PHB/HA ratio. The properties of the surface of the HA-loaded composite are significantly different from those of the pure polymer. As the HA percentage in the composite increases, free interface energy, the cohesive force, i.e., the strength of the adhesive bond between the composite surface and the water phase, and surface wettability increase. The HA percentage of the composite does not influence its melting temperature, but affects the temperature for the onset of decomposition: as the HA content increases from 0 to 10% (w/w), Td decreases from 260 degrees C to 225 degrees C. The degree of crystallinity of PHB/HA increases from 77% to 89% with an increase in the HA fraction from 10% to 50%. Functional properties of the composites have been investigated in vitro and in vivo. The best parameters of growth and differentiation of murine marrow osteoblasts are registered on PHB/HA samples containing 10% and 20% HA. In ectopic bone formation assay it has been proven that the hybrid PHB/HA composites can function as scaffolds and that bone tissue develops on their surface and in pores.

Degradation of P(3HB) and P(3HB-co-3HV) in biological media.

The biodegradability of oriented fibers made of polyhydroxybutyrate (P(3HB)) and its co-polymer with beta-hydroxyvalerate (P(3HB-co-3HV)) was investigated in buffer solutions and in biological media in vitro and in vivo. The fibers of both polymer types demonstrated resistance to hydrolytic degradation in buffer solutions at 38 degrees C and pH from 4.5 to 7.0 (for up to 180 days). It has been found that the biodegradation of the fibers in vitro in blood and serum and in vivo is accompanied by weight losses and minor changes in the microstructure with no significant losses in the tensile strength over a long time (up to 180 days). The biodegradation rate of the less crystalline co-polymer P(3HB-co-3HV) fibers was 1.4-2.0-times higher than that of the homopolymer P(3HB). It has also been shown that the degradation of the fibers in vivo is influenced both by tissue fluid enzymes and cells (macrophages and foreign-body giant cells). The fibers were eroded on the surface only with no gross defects and no dramatic effects on their mechanical performance.

A comparative investigation of biodegradable polyhydroxyalkanoate films as matrices for in vitro cell cultures.

The paper describes the production and investigation of flexible films made of high-purity polyhydroxyalkanoates (PHAs)--polyhydroxybutyrate [poly-(3HB)] and poly-3-hydroxybutyrate-co-poly-3-hydroxyvalerate [poly(3HB-co-3HV)], containing 4-30 mol % hydroxyvalerate. Poly(3HB-co-3HV) films have a more porous structure than poly-(3HB) films, which are more compact, but their surface properties, such as wettability and surface and interface energies, are the same. Sterilisation of the PHA films by conventional methods (heat treatment and gamma-irradiation) did not impair their strength. Cells cultured on PHA films exhibited high levels of cell adhesion. Cell morphology, protein synthesis and DNA synthesis were estimated by extent of 3H-thymidine incorporation into the animal cell cultures of various origins (fibroblasts, endothelium cells, and isolated hepatocytes) in direct contact with PHAs. The investigation showed that this material can be used to make matrices for in vitro proliferous cells. The investigated properties of poly-(3HB) and poly(3HB-co-3HV) films proved to be fundamentally similar.

Tissue response to the implantation of biodegradable polyhydroxyalkanoate sutures.

Polyhydroxyalkanoate (PHA) sutures were implanted to test animals intramuscularly, and tissue reaction was investigated and compared with the reaction to silk and catgut. Tested monofilament sutures made of PHAs of two types--polyhydroxybutyrate (PHB) and a copolymer of hydroxybutyrate and hydroxyvalerate (PHV)--featured the strength necessary for the healing of muscle-fascial wounds. The reaction of tissues to polymeric implants was similar to their reaction to silk and was less pronounced than the reaction to catgut; it was expressed in a transient post-traumatic inflammation (up to four weeks) and the formation of a fibrous capsule less than 200 microm thick, which became as thin as 40-60 microm after 16 weeks, in the course of reverse development. Macrophages and foreign-body giant cells with a high activity of acid phosphatase were actively involved in this process. PHB and PHB/PHV sutures implanted intramuscularly for an extended period (up to one year) did not cause any acute vascular reaction at the site of implantation or any adverse events, such as suppurative inflammation, necrosis, calcification of the fibrous capsule or malignant tumor formation. No statistically significant differences were revealed in the tissue response to polymer sutures of the two types. Capsules around silk and catgut sutures did not become significantly thinner.

Production of purified polyhydroxyalkanoates (PHAs) for applications in contact with blood.

Samples of olyhydroxyalkanoates (PHAs), polyhydroxybutyrate (PHB) and copolymers poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) with 4 and 18 mol% hydroxyvalerate, synthesized by the bacteria Ralstonia eutropha B5786, were investigated. PHA films in contact with blood did not activate the hemostasis system at the level of cell response, but they did activate the coagulation system and the complement reaction. To detect biologically-active components in the PHAs, a detailed analysis of the composition of the polymers was conducted. Gas chromatography-mass spectrometry revealed long-chain fatty acids (FAs) in the tested PHAs. Their total concentration in the polymer ranged from tenths of mol% to 2-3 mol%, depending on the purification method. C16:0 constituted the largest proportion, up to 70%. Of the long-chain hydroxy acids, only beta-OH-C14:0 was detected and it did not exceed 0.06 mol%. The analysis of the hemocompatibility properties of the PHAs purified by a specialized procedure, including the quantitative and morphological estimation of platelets adherent to the surface of polymer films, the plasma recalcification time and complement activation studies, indicated that PHB and PHBV can be used in contact with blood. It has been found out that the lipopolysaccharides of bacteria producing PHAs, which contain mostly long-chain hydroxy acids, can be the factor activating the hemostasis systems. Thus, the technology of PHA purification must satisfy rather stringent specific requirements.