O. A. Mogilnaya

Results of biomedical investigations of PHB and PHB/PHV fibers

Abstract The paper presents the results of biomedical investigations of sutures made of polyhydroxyalkanoates (PHAs) of two types (poly-3-hydroxybutyrate (PHB) and poly-3-hydroxybutyrate-co-poly-3-hydroxyvalerate (PHB/PHV)), synthesized by the bacteria Ralstonia eutropha B 5786 in the experiment on test animals in vivo, in comparison with silk and catgut sutures. PHB and PHB/PHV (PHBV) implants produced no adverse effect on physiological, biochemical and functional parameters of the animals during the post-surgery period. The tested PHA sutures featured the necessary strength throughout the healing period of the muscle-fascial cuts. The reaction of tissues to the implantation of PHB and PHB/PHV fibers fitted into the usual scheme characteristic of the wound process and of the reaction to a foreign-body invasion. This reaction and the reaction of tissues to silk had the similar nature and period of inflammation, but it was much less pronounced than the reaction to catgut. The tissue response to the implantation of PHAs consisted in a short-duration (up to 2 weeks) post-traumatic inflammation and the formation of a fibrous capsule less than 200xa0μm thick during weeks 4–8, which in 4–6 months was reduced to 40–60xa0μm in the course of reverse development. There were no adverse changes, such as suppurative inflammation, necrosis, calcification, and malignization of the cicatrice, at the site of implantation of PHA filaments, unlike in the cases with silk and catgut. In the case of PHA implantation there was a typical prolonged (throughout the post-surgery monitoring period) pronounced macrophagal stage with a large number of macrophages present. The macrophages were of the phagocytic type and multinucleate giant foreign cells, with a high activity of acid phosphomonoesterase that correlated with the activity of the enzyme in blood. Throughout the period of monitoring no differences in the tissue response to the implantation of the polymer filaments of two PHA types were recorded.

Biocompatibility of polyhydroxybutyrate microspheres: in vitro and in vivo evaluation

Microspheres have been prepared from the resorbable linear polyester of β-hydroxybutyric acid (polyhydroxybutyrate, PHB) by the solvent evaporation technique and investigated in vitro and in vivo. Biocompatibility of the microspheres has been proved in tests in the culture of mouse fibroblast cell line NIH 3T3 and in experiments on intramuscular implantation of the microspheres to Wistar rats for 3xa0months. Tissue response to the implantation of polymeric microspheres has been found to consist in a mild inflammatory reaction, pronounced macrophage infiltration that increases over time, involving mono- and poly-nuclear foreign body giant cells that resorb the polymeric matrix. No fibrous capsules were formed around polymeric microparticles; neither necrosis nor any other adverse morphological changes and tissue transformation in response to the implantation of the PHB microparticles were recorded. The results of the study suggest that polyhydroxybutyrate is a good candidate for fabricating prolonged-action drugs in the form of microparticles intended for intramuscular injection.

Tissue Response to Biodegradable Suture Threads Made of Polyhydroxyalkanoates

Physicomechanical characteristics of biodegradable suture threads, such as rigidity and elasticity, should meet a number or requirements. The rate of resorption of suture threads should correspond to the kinetics of tissue recovery. The products of destruction of the thread material should be easily eliminable from the implantation zone and harmless to the patientx92s health. They should not cause any adverse response of adjacent tissues and the body as a whole [1]. Polymers of hydroxy compounds of fatty acids (polyhydroxyalkanoates, PHA) are biocompatible and biodegradable materials of microbial origin. Polyhydroxyalkanoates hold much promise as materials for surgical elements. They can be used for tissue engineering and drug batching [25, 26, 28]. In distinction to polylactides and polyglycolides, PHA are thermoplastic and not susceptible to chemical hydrolysis. Thus, PHA are destroyed only gradually without losing their mechanical strength. Some basic properties of PHA can be adjusted by varying their composition [11]. Nonwoven fabrics, polymer films, suture threads, and other medical articles can be made of PHA [17, 18, 29]. Currently available data about the tissue response to PHA implants and the kinetics of PHA destruction in vivo are ambiguous. It was shown in [12] that the tissue response to PHA implants depends only slightly on the implant composition. On the other hand, PHA implants containing polyhydroxyvalerianate were shown to cause tissue inflammation [15]. The problem of in vivo destruction of PHA implants is also unsolved. According to [21, 27], PHA threads implanted into laboratory animals remained intact for up to 6-12 months. In other works [12, 14, 19] it was shown that the weight of a PHA implant decreased by 30-80%. The goal of this work was to study the tissue response to implanted PHA suture threads during the post-operative period and to compare PHA to conventional surgical materials (silk and catgut).

Tissue morphogenesis under the conditions of implantation of polyhydroxybutyrate, a biodegradable polymer.

Polyhydroxybutyrate (PHB), a polymer of hydroxybutyric acid, is a biocompatible polyester of natural origin. Medical applications of this polymer as a material for tissue engineering, various surgical elements, and dosed supply of pharmacological substances are extensively studied worldwide [1–3]. In addition to reliable fixation of surgical suture, implanted biodegradable sutural material should also undergo gradual degradation. The rate of biodegradation of the suture should be adequate to the kinetics of tissue recovery. The products of degradation of a biodegradable sutural material should be absolutely harmless for human organism, easily eliminated from the zone of implantation, and cause no negative reactions in the surrounding tissues or the human body as a whole [4]. The goal of this work was to analyze the postoperative reaction of biological tissues in vivo induced by implanted PHB surgical sutural threads. The sutural threads used in our experiments were made of PHB synthesized by the bacteria Ralstonia eutropha B5786. The technology of manufacture of the PHB surgical sutural threads was developed at the Institute of Biophysics, Siberian Division, Russian Academy of Sciences. Conventional surgical materials (silk and catgut) were used as control. Multifiber sutural threads were manufactured using the gel-technology described in [5]. Polyhydroxybutyrate was synthesized at the Institute of Biophysics, Siberian Division, Russian Academy of Sciences, as described in [6]. The experimental procedure was descried in [7]. Sexually mature Wistar female rats with an initial body weight of 180–200 g were used. When the animals had been anesthetized by ether inhalation, a 2-cm-long longitudinal incision was made under aseptic conditions through the skin and muscles of the right femur. The muscle incisions in experimental animals were sutured with three PHB threads (the total suture length was 3.0–3.5 cm). The muscle incisions in two control groups were sutured with silk and catgut. Skin incisions were sutured with silk. Animals were euthanized by overdose of ether inhalation 1, 2, 4, 8, 16, or 24 weeks after the surgery, and fragments of tissues surrounding the implants were assayed.

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.

Growth and Bioluminescence of Luminous Bacteria under the Action of Aflatoxin B1 before and after Its Treatment with Nanodiamonds

The effect of aflatoxin B1 on growth and luminescence of marine luminous bacteria P. phosphoreum and recombinant E. coli Z905 cells was investigated. The bidirectional effect of aflatoxin B1 on the studied bacterial species was detected—an inhibition of luminescence in P. phosphoreum and its stimulation in E. coli. It was shown that aflatoxin B1 influences the cell luminescence in the freshly grown cultures and bacteria restored after lyophilization. It was detected that the effect of aflatoxin B1 was graded after interaction with the modified nanodiamond (MND) of detonation synthesis. After mycotoxin’s treatment with MND, it does not cause significant changes in bacterial luminescence. The possibilities for the use of P. phosphoreum and E. coli bacteria in the bioluminescent monitoring of aflatoxin B1 and the use of MND for mycotoxin deactivation are discussed.

Creation of Bifunctional Indicating Complex Based on Nanodiamonds and Extracellular Oxidases of Luminous Fungus Neonothopanus nambi

A bifunctional indicating complex was created by immobilization of extracellular oxidases (glucose oxidase and peroxidases) of luminous fungus Neonothopanus nambi onto modified nanodiamonds (MNDs) synthesized by detonation. It was found that the enzymes firmly adsorb onto MND particles and exhibit their catalytic activity. Model in vitro experiments showed that the created MND–enzymes complex is suitable for repeated use for analyte (glucose and phenol) testing and retains its activity after storage at 4°C in deionized water for 1 month. The data obtained offer the prospects for developing a new class of reusable multifunctional indicating and diagnostic test systems on the basis of MNDs and higher fungal enzymes for medical and ecological analytics.

Extracellular Peroxidase Activity and Light Emission of the Mycelium of the Basidiomycete Neonothopanus nambi in the Presence of β-Glucosidase

A comparative evaluation of the level of extracellular peroxidase activity and light-emission intensity of the mycelium of the luminescent basidiomycete Neonothopanus nambi in the presence of β-glucosidase was performed. The enzyme activity damages the hyphae of the fungus leading to osmotic imbalance, partial degradation of the mycelium, and release of extracellular peroxidases into the incubation medium. The presence of β-glucosidase reduces the time necessary to reach the maximum luminescence. Putative biochemical mechanisms that underlie the stimulation of reactive oxygen species formation (first and foremost, of hydrogen peroxide) in the N. nambi mycelium in the presence of β-glucosidase are proposed.

Nanodiamonds as an effective adsorbent for immobilization of extracellular peroxidases from luminous fungus Neonothopanus nambi to construct a phenol detection system

Abstract Modified nanodiamonds (MNDs) produced by detonation synthesis can be used as an effective adsorbent to immobilize extracellular peroxidases of the luminous basidiomycete Neonothopanus nambi. The enzymes are firmly immobilized on MND particles and exhibit catalytic activity. The indicator system (the MND–enzyme complex) reused many times retains its ability to catalyze reaction of co-oxidation of phenol and 4-aminoantipirine in the presence of hydrogen peroxide and remains functionally active during long-term storage (for 1u2009month or longer) in aqueous suspensions at 4u2009°C. MNDs and enzymes of higher fungi can be effectively used to construct new reusable indicator systems for analytical applications such as monitoring contamination of aquatic environments by phenolic compounds.