Millions of people worldwide suffer from musculoskeletal damage, thus using the largest proportion of rehabilitation services. The limited self-regenerative capacity of bone and cartilage tissues necessitates the development of functional biomaterials. Magnetoactive materials are a promising solution due to clinical safety and deep tissue penetration of magnetic fields (MFs) without attenuation and tissue heating. Herein, electrospun microfibrous scaffolds were developed based on piezoelectric poly(3-hydroxybutyrate) (PHB) and composite magnetic nanofillers [magnetite with graphene oxide (GO) or reduced GO]. The scaffolds' morphology, structure, mechanical properties, surface potential, and piezoelectric response were systematically investigated. Furthermore, a complex mechanism of enzymatic biodegradation of these scaffolds is proposed that involves (i) a release of polymer crystallites, (ii) crystallization of the amorphous phase, and (iii) dissolution of the amorphous phase. Incorporation of Fe3O4, Fe3O4-GO, or Fe3O4-rGO accelerated the biodegradation of PHB scaffolds owing to pores on the surface of composite fibers and the enlarged content of polymer amorphous phase in the composite scaffolds. Six-month biodegradation caused a reduction in surface potential (1.5-fold) and in a vertical piezoresponse (3.5-fold) of the Fe3O4-GO scaffold because of a decrease in the PHB β-phase content. In vitro assays in the absence of an MF showed a significantly more pronounced mesenchymal stem cell proliferation on composite magnetic scaffolds compared to the neat scaffold, whereas in an MF (68 mT, 0.67 Hz), cell proliferation was not statistically significantly different when all the studied scaffolds were compared. The PHB/Fe3O4-GO scaffold was implanted into femur bone defects in rats, resulting in successful bone repair after nonperiodic magnetic stimulation (200 mT, 0.04 Hz) owing to a synergetic influence of increased surface roughness, the presence of hydrophilic groups near the surface, and magnetoelectric and magnetomechanical effects of the material.

Peripheral nerve injury poses a threat to the mobility and sensitivity of a nerve, thereby leading to permanent function loss due to the low regenerative capacity of mature neurons. To date, the most widely clinically applied approach to bridging nerve injuries is autologous nerve grafting, which faces challenges such as donor site morbidity, donor shortages, and the necessity of a second surgery. An effective therapeutic strategy is urgently needed worldwide to overcome the current limitations. Herein, a magnetic nerve guidance conduit (NGC) based on biocompatible biodegradable poly(3-hydroxybutyrate) (PHB) and 8 wt % of magnetite nanoparticles modified by citric acid (Fe3O4-CA) was fabricated by electrospinning. The crystalline structure of NGCs was studied by X-ray diffraction, which indicated an enlarged beta-phase of PHB in the composite conduit compared to a pure PHB conduit. Tensile tests revealed greater ductility of PHB/Fe3O4-CA: the composite conduit has Young's modulus of 221 +/- 52 MPa and an elongation at break of 28.6 +/- 2.9%, comparable to clinical materials. Saturation magnetization (sigma(s)) of Fe3O4-CA and PHB/Fe3O4-CA is 61.88 +/- 0.29 and 7.44 +/- 0.07 emu/g, respectively. The water contact angle of the PHB/Fe3O4-CA conduit is lower as compared to pure PHB, while surface free energy (sigma) is significantly higher, which was attributed to higher surface roughness and an amorphous phase as well as possible PHB/Fe3O4-CA interface interactions. In vitro, the conduits supported the proliferation of rat mesenchymal stem cells (rMSCs) and SH-SY5Y cells in a low-frequency magnetic field (0.67 Hz, 68 mT). In vivo, the conduits were used to bridge damaged sciatic nerves in rats; pure PHB and composite PHB/Fe3O4-CA conduits did not cause acute inflammation and performed a barrier function, which promotes nerve regeneration. Thus, these conduits are promising as implants for the regeneration of peripheral nerves.

Учебно-методическое пособие. В учебно-методическом пособии представлены последние достижения в области разработки и биомедицинских исследований электро- и магнитоактивных биоматериалов: роль пьезоэлектрического эффекта и механотрансдукции для живых клеток и тканей, применение различных электро- и магнитоактивных биоматериалов в тканевой инженерии и регенеративной медицине, механизмы воздействия электрических и механических стимулов на рост клеток млекопитающих и адгезию бактерий на этих биоматериалах. В отдельном разделе представлены два задания для практикума по исследованию роста клеток млекопитающих и адгезии бактерий на биополимерных композитах с магнитными наночастицами под воздействием магнитного поля. Учебно-методическое пособие разработано для реализуемых основных профессиональных образовательных программ высшего образования по направлению подготовки «Биоинженерия и биотехнология» (программ специалитета и бакалавриата, магистратуры, реализуемые последовательно по схеме интегрированной подготовки). Материалы учебно-методического пособия использованы в учебных программах Биологического факультета Московского государственного университета имени М.В. Ломоносова «Теоретическая биоинженерия» (курс лекций) и «Биоинженерия биосовместимых полимеров» (практикум). Учебно-методическое пособие разработано в рамках проекта РНФ № 20-64-47008.

The study involved the fabrication of films with different roughness and scaffolds made of poly-3-hydroxybutyrate using various methods. Chaotic and oriented scaffolds with varying fiber thickness were obtained through the electrospinning method, depending on the polymer concentration and electrospinning parameters. Films with different surface roughness were obtained using spin coating and self-assembly methods. It was demonstrated that the varying microstructure of the surface does not affect the growth of mesenchymal stem cells over the course of 1 week; however, it does influence the morphology of the adhered cells.

Scaffold biocompatibility remains an urgent problem in tissue engineering. An especially interesting problem is guided cell intergrowth and tissue sprouting using a porous scaffold with a special design. Two types of structures were obtained from poly(3-hydroxybutyrate) (PHB) using a salt leaching technique. In flat scaffolds (scaffold-1), one side was more porous (pore size 100–300 μm), while the other side was smoother (pore size 10–50 μm). Such scaffolds are suitable for the in vitro cultivation of rat mesenchymal stem cells and 3T3 fibroblasts, and, upon subcutaneous implantation to older rats, they cause moderate inflammation and the formation of a fibrous capsule. Scaffold-2s are homogeneous volumetric hard sponges (pore size 30–300 μm) with more structured pores. They were suitable for the in vitro culturing of 3T3 fibroblasts. Scaffold-2s were used to manufacture a conduit from the PHB/PHBV tube with scaffold-2 as a filler. The subcutaneous implantation of such conduits to older rats resulted in gradual soft connective tissue sprouting through the filler material of the scaffold-2 without any visible inflammatory processes. Thus, scaffold-2 can be used as a guide for connective tissue sprouting. The obtained data are advanced studies for reconstructive surgery and tissue engineering application for the elderly patients.

Magnetically responsive composite polymer scaffolds have good potential for a variety of biomedical applications. In this work, electrospun composite scaffolds made of polyhydroxybutyrate (PHB) and magnetite (Fe3O4) particles (MPs) were studied before and after degradation in either PBS or a lipase solution. MPs of different sizes with high saturation magnetization were synthesized by the coprecipitation method followed by coating with citric acid (CA). Nanosized MPs were prone to magnetite-maghemite phase transformation during scaffold fabrication, as revealed by Raman spectroscopy; however, for CA-functionalized nanoparticles, the main phase was found to be magnetite, with some traces of maghemite. Submicron MPs were resistant to the magnetite-maghemite phase transformation. MPs did not significantly affect the morphology and diameter of PHB fibers. The scaffolds containing CA-coated MPs lost 0.3 or 0.2% of mass in the lipase solution and PBS, respectively, whereas scaffolds doped with unmodified MPs showed no mass changes after 1 month of incubation in either medium. In all electrospun scaffolds, no alterations of the fiber morphology were observed. Possible mechanisms of the crystalline-lamellar-structure changes in hybrid PHB/Fe3O4 scaffolds during hydrolytic and enzymatic degradation are proposed. It was revealed that particle size and particle surface functionalization affect the mechanical properties of the hybrid scaffolds. The addition of unmodified MPs increased scaffolds' ultimate strength but reduced elongation at break after the biodegradation, whereas simultaneous increases in both parameters were observed for composite scaffolds doped with CA-coated MPs. The highest saturation magnetization─higher than that published in the literature─was registered for composite PHB scaffolds doped with submicron MPs. All PHB scaffolds proved to be biocompatible, and the ones doped with nanosized MPs yielded faster proliferation of rat mesenchymal stem cells. In addition, all electrospun scaffolds were able to support angiogenesis in vivo at 30 days after implantation in Wistar rats.

The development of orthodontics and orthopedics requires new materials for guided bone regeneration. These materials must meet certain requirements for affinity with surrounding tissues and the ability to induce osteogenesis. Poly-3(hydroxybutyrate) (PHB) is a biocompatible and biodegradable material, actively used in different biomedical applications. Recent studies have shown piezoelectric properties of PHB. Since electrical signals have a huge impact on differentiation and cell growth, novel materials with piezoelectric properties could be effective tool for tissue regeneration. To enhance this effect in PHB polymer was combined with magnetic nanoparticles (MNs). The main component of MNs was magnetite (Fe3O4) synthesizing under different conditions using the magnetic separation method. Assessment of toxicity was obtained with composite films on mesenchymal stem cells (MSCs). Our work demonstrates that composites PHB/MNs have a potential to be used as a smart new material for bone tissue engineering applications.

Functionalization of magnetite (Fe3O4) nanoparticles with reduced graphene oxide (rGO) with the preserved magnetic properties of the former presents great potential for applying the Fe3O4/rGO biocomposite in various biomedical applications, such as magnetic resonance imaging, as a therapeutic component in initiating tumour cell death in magnetic and photon ablation therapy, and as an effective carrier for drug delivery. In this study, magnetite nanoparticles (MNPs) with a high saturation magnetization were synthesized by co-precipitation under various conditions, followed by covalent functionalization with citric acid (CA) and subsequent attachment to rGO sheets by physical adsorption. Extensive characterization revealed increasing phase purity with a subsequent decrease in the crystallite size and average size of the MNPs synthesized in an inert atmosphere compared to ambient conditions. Meanwhile, further functionalization of the MNPs with CA by covalent binding does not affect the MNP structure and size, but decreases their agglomeration. To study the magnetic properties of the MNPs and the Fe3O4/rGO composite, magnetization curves were obtained with a vibrating sample magnetometer at a pulsed magnetic field of up to 6.5 kOe. The largest values of saturation magnetization are revealed for the samples synthesized without the addition of CA in an inert atmosphere (σs=80.27 emu/g). The addition of CA to the synthesized MNPs and Fe3O4/rGO composites reduced agglomeration, with σs values in the range from 64.10 to 60.97 emu/g. In vitro biological experiments revealed the MNP concentrations that did not cause any toxic effects on cells for use as magnetic fillers to investigate the strain-mediated effects of hybrid polymer composites on cellular behaviour due to external magnetic field exposure in the next stages of research.

We studied the effect of porous composite scaffolds based on poly(3-hydroxybutyrate) (PHB) loaded with simvastatin on the growth and differentiation of mesenchymal stem cells. The scaffolds have a suitable microstructure (porosity and pore size) and physicochemical properties to support the growth of mesenchymal stem cells. Scaffold loading with simvastatin suppressed cell growth and increased alkaline phosphatase activity, which can attest to their osteoinductive properties.

A critical-sized calvarial defect in rats is employed to reveal the osteoinductive properties of biomaterials. In this study, we investigate the osteogenic efficiency of hybrid scaffolds based on composites of a biodegradable and biocompatible polymer, poly(3-hydroxybutyrate) (PHB) with hydroxyapatite (HA) filled with alginate (ALG) hydrogel containing mesenchymal stem cells (MSCs) on the regeneration of the critical-sized radial defect of the parietal bone in rats. The scaffolds based on PHB and PHB/HA with desired shapes were prepared by two-stage salt leaching technique using a mold obtained by three-dimensional printing. To obtain PHB/HA/ALG/MSC scaffolds seeded with MSCs, the scaffolds were filled with ALG hydrogel containing MSCs; acellular PHB/ALG and PHB/ALG filled with empty ALG hydrogel were prepared for comparison. The produced scaffolds have high porosity and irregular interconnected pore structure. PHB/HA scaffolds supported MSC growth and induced cell osteogenic differentiation in a regular medium in vitro that was manifested by an increase in ALP activity and expression of the CD45 phenotype marker. The data of computed tomography and histological studies showed 94% and 92%, respectively, regeneration of critical-sized calvarial bone defect in vivo at 28th day after implantation of MSC-seeded PHB/HA/ALG/MSC scaffolds with 3.6 times higher formation of the main amount of bone tissue at 22-28 days in comparison with acellular PHB/HA/ALG scaffolds that was shown at the first time by fluorescent microscopy using the original technique of intraperitoneal administration of fluorescent dyes to living postoperative rats. The obtained in vivo results can be associated with the MSC-friendly microstructure and in vitro osteogenic properties of PHB/HA base-scaffolds. Thus, the obtained data demonstrate the potential of MSCs encapsulated in the bioactive biopolymer/mineral/hydrogel scaffold to improve the bone regeneration process in critical-sized bone defects.

Изготовлены пористые матриксы для тканевой инженерии на основе поли-3-оксибутирата (ПОБ) и его сополимера с полиэтиленгликолем (ПОБ-ПЭГ), который был получен по технологии биоПЭГилирования. Исследована морфология полученных матриксов и адсорбция на них модельного белка, бычьего сывороточного альбумина (БСА). Показано, что матриксы на основе биоПЭГили­рованного ПОБ сорбировали больше БСА, а доля необратимо сорбированного белка на них значительно меньше (33%), чем на матриксах из гомополимера ПОБ (47%). Влияние адсорбции белка на биосовместимость матриксов in vitro было проверено путем культивирования на них фибробластов линии COS-1. Показано, что матриксы на основе ПОБ-ПЭГ в большей степени поддерживали рост клеток на них по сравнению с матриксами на основе ПОБ. Таким образом, полученные полимерные матриксы на основе биоПЭГилированного ПОБ могут быть более перспективны для использования в инженерии мягких тканей.

Development of biocompatible 3D scaffolds is one of the most important challenges in tissue engineering. In this study, we developed polymer scaffolds of different design and microstructure to study cell growth in them. To obtain scaffolds of various microstructure, e.g., size of pores, we used double- and one-stage leaching methods using porogens with selected size of crystals. A composite of poly(3-hydroxybutyrate) (PHB) with poly(ethylene glycol) (PEG) (PHB/PEG) was used as polymer biomaterial for scaffolds. The morphology of scaffolds was analyzed by scanning electron microscopy; the Young modulus of scaffolds was measured by rheometry. The ability to support growth of mesenchymal stem cells (MSCs) in scaffolds was studied using the XTT assay; the phenotype of MSC was preliminarily confirmed by flow cytometry and the activity of alkaline phosphatase and expression level of CD45 marker was studied to test possible MSC osteogenic differentiation. The obtained scaffolds had different microstructure: the scaffolds with uniform pore size of about 125 µm (normal pores) and 45 µm (small pores) and scaffolds with broadly distributed pores size from about 50–100 µm. It was shown that PHB/PEG scaffolds with uniform pores of normal size did not support MSCs growth probably due to their marked spontaneous osteogenic differentiation in these scaffolds, whereas PHB/PEG scaffolds with diverse pore size promoted stem cells growth that was not accompanied by pronounced differentiation. In scaffolds with small pores (about 45 µm), the growth of MSC was the lowest and cell growth suppression was only partially related to stem cells differentiation. Thus, apparently, the broadly distributed pore size of PHB/PEG scaffolds promoted MSC growth in them, whereas uniform size of scaffold pores stimulated MSC osteogenic differentiation.

Porous scaffolds for tissue engineering have been prepared from poly(3-hydroxybutyrate) (PHB) and a copolymer of poly(3-hydroxybutyrate) and polyethylene glycol (PHB-PEG) produced by bioPEGylation. The morphology of the scaffolds and their capacity for adsorption of the model protein bovine serum albumin (BSA) have been studied. Scaffolds produced from bioPEGylated PHB adsorbed more BSA, whereas the share of protein irreversibly adsorbed on these scaffolds was significantly lower (33%) than in the case of PHB homopolymer-based scaffolds (47%). The effect of protein adsorption on scaffold biocompatibility in vitro was tested in an experiment that involved the cultivation of fibroblasts (line COS-1) on the scaffolds. PHB-PEG scaffolds had a higher capacity for supporting cell growth than PHB-based scaffolds. Thus, the bioPEGylated PHB-based polymer scaffolds developed in the present study have considerable potential for use in soft tissue engineering.

В работе изучается влияние полиэлектролитных слоев на сорбцию белка на примере пероксидазы хрена (ПХ) в стеклянных мультикапиллярах для разработки способа контроля селективной сорбции. Показано, что более эффективная сорбция ПХ наблюдается для бислоя полиэтиленимин (ПЕИ) / полистиролсульфонат (ПСС). Такой эффект объяснен тем, что поликатион ПЕИ модифицирует стеклянную поверхность, а полианион ПСС создает слой для эффективной сорбции ПХ.

Background: Poly(hydroxyalkanoates) (PHA) have recently attracted increasing attention due to their biodegradability and high biocompatibility, which makes them suitable for the development of new prolong drug formulations.Objective: This study was conducted to develop new prolong paclitaxel (PTX) formulation based on poly(3-hydroxybutyrate) (PHB) microparticles.Method: PHB microparticles loaded with antitumor cytostatic drug PTX were obtained by spray-drying method using Nano Spray Dryer B-90. The PTX release kinetics in vitro from PHB microparticles and their cytotoxity on murine hepatoma cell line MH-22a were studied. Microparticles antitumor activity in vivo was studied using intraperitoneally (i.p.) transplanted tumor models: murine Lewis lung carcinoma and xenografts of human breast cancer RMG1.Results: Uniform PTX release from PHB-microparticles during 2 months was observed. PTX-loaded PHB microparticles have demonstrated a significant antitumor activity versus pure drug both in vitro in murine hepatoma cells and in vivo when administered i.p. to mice with murine Lewis lung carcinoma and xenografts of human breast cancer RMG1.Conclusion: The developed technique of PTX sustained delivery from PHB-microparticles has therapeutic potential as prolong anticancer drug formulation.

occurs or not during supercontraction. Furthermore, although it has already been established that supercontraction induces a disorientation of the molecular units, this effect has not been quantified yet. Therefore, we will investigate and quantify, for different species, the conformational and orientational variations of the silk proteins induced by supercontraction. To achieve our goals, we will use Raman spectromicroscopy, a technique that as long been proven to be a useful tool to probe silk. Moreover, the effects of drawing speed on the magnitude of supercontraction will also be examined.