ИСТИНА

Introduction The biodegradable and biocompatible polymers, polyhydroxyalkanoates (PHAs), are actively used in medicine for production of a wide range of medical devices and dosage formulations, as well as for the manufacture of scaffolds for tissue engineering. For tissue engineering, these polymers obtained by chemical synthesis (e.g. polylactides, polyglycolides, poly-ε-caprolactone, and their copolymers) are mainly used, but the interest in the bioengineering application of natural poly(3-hydroxyalkanoates) obtained biotechnologically is also growing. Actually, the chemically synthetic PHAs are biomimetic analogs of bacterial poly(3-hydroxybutyrate) (PHB) and its copolymers. Due to the favourable properties (high biocompatibility, the relatively low rate of biodegradation, and good mechanics) PHB is the most promising for bone tissue engineering and bone regeneration. However, the problem of the osteoinductive activity of PHB remains insufficiently studied. Thus, the purpose of our research is to analyze the possible osteoinductive activity of PHB. Experimental Methods PHB and its bioPEGylated copolymer (PHB-PEG) were produced by the original biotechnological technique using a highly productive producing strain Azotobacter chroococcum 7B [1]. The obtained copolymer PHB-PEG, the composite of PHB with PEG (PHB/PEG) and polylactide (PLA) were used as controls to PHB. The obtained polymeric biomaterials were used to prepare the 3D-scaffolds by various modifications of salt leaching method. The morphology of scaffolds was analyzed by the scanning electron microscopy and the wide-field light microscopy. The physicochemical properties of the produced scaffolds were studied by rheometry, differential scanning calorimetry, and water uptake test. The ability to support the growth of mesenchymal stem cells (MSCs) isolated from rat bone marrow and 3T3-NIH fibroblasts in scaffolds were studied using the XTT-assay. The osteogenic differentiation of MSCs in regular and osteogenic media was studied by Alkaline Phosphatase activity assay, Alizarin Red staining assay, and analysis of change in expression of MSCs phenotype markers (CD90, CD45, CD 11b/c, CD29) using flow cytometry, scanning electron and confocal microscopy [2]. The osteogenic activity of PHB scaffolds in vivo was studied on the noncritical 1.5 mm defect of famous bone and the 8 mm critical defect of parietal bone that were modeled in Wistar rats by surgery technique using a trephine С-reamer. The bone tissue regeneration after implantation of the scaffolds as bone substitutes was evaluated by histological methods and fluorescent microscopy [3]. To estimate the dynamics of neo-osteogenesis on 3 different stages the vital fluorescent labeling of the newly formed bone tissue was carried out using i.p. administration of doxycycline, tetracycline, and alizarine red C. All experiments were performed according to guidelines for ethical treatment of animals ISO 10993-2. Results and Discussion The produced PHB, PHB-PEG, PHB/PEG, and PLA scaffolds have a 3D porous structure: with various pore sizes (the micropores and macropores), mean porosity >90%, and interconnected pore system. The 3D-growth of MSCs and 3T3-NIH fibroblasts in the scaffolds were observed. The spontaneous osteogenic differentiation of MSCs in regular medium and the modulation of their osteogenic differentiation in the osteogenic medium on PHB and PHB/PEG scaffolds were demonstrated, whereas PHB-PEG and PLA scaffolds promote MSCs growth that was not accompanied by pronounced differentiation. The PHB, PHB-PEG, and PHB/PEG scaffolds stimulated the bone tissue regeneration after their implantation in the noncritical and critical bone defects as the bone substitutes. The osteogenic activity of PHB scaffolds depends on the physicochemical properties of the polymers, 3D- and surface microstructure of scaffolds, and also possibly the own biological activity of PHAs. But the reason and mechanisms of such biological activity of PHB remain unclear. We suggest here that the osteoinductive activity of PHB can be associated with its natural properties as a bacteria-origin biopolymer. This biopolymer is present in bacteria of mammalian microbiota, whereas endogenous PHB is containing in mammalian tissues. A series of studies indicate the possible regulating functions of PHB in bacteria of mammalian microbiota for their interaction with cells of host-organism [4]. The possible association of osteogenic properties of PHB with various biological functions of PHB in bacteria and eukaryotes, including in humans, are discussed. Conclusion Thus, we suggest the presence of own osteoinductive activity of PHB, which can be associated with its natural properties as a functional biopolymer in bacteria of mammalian microbiota.