Epiregulin (EREG), a ligand of the epidermal growth factor receptor, has emerged as a potent modulator of mesenchymal stem cell (MSC) function, particularly in inflammatory conditions. Recent evidence indicates that EREG signaling significantly enhances the migration, chemotaxis, and lineage-specific differentiation of MSCs, overcoming critical barriers to cell survival and engraftment in hostile microenvironments. This review summarizes recent advances in the synergistic application of EREG and MSCs in various tissue engineering applications, such as bone, skin, liver, and intestinal regeneration. We discuss the context-dependent nature of EREG activity and emerging biomaterial-based delivery strategies. We also explore the underappreciated role of the gut microbiota as a systemic regulator of EREG expression and a dynamic biomarker of therapeutic efficacy. By integrating mechanistic insights with translational prospects, we outline how EREG-primed MSC therapies can be optimized for clinical implementation in complex regenerative settings.

Alginates are anionic unbranched plant and bacterial polysaccharides composed of mannuronic and guluronic acid residues. Alginates can form hydrogels under mild conditions in the presence of divalent cations (e.g., Ca2+). Because of their capacity to form gels, high biocompatibility, and relatively low cost, these polysaccharides are employed in pharmaceutical industry, medicine, food industry, cosmetology, and agriculture. Alginate oligomers produced by enzymatic cleavage of high-molecular-weight algal alginates are used as medicinal agents and dietary supplements. The global market for alginate-based products exceeds 1 billion. Alginates and their oligomers have attracted a special interest in biomedical sciences due to manifestation of various types of therapeutic activity. Across more than 50-year history of studies of alginates, over 60

The mini-review explores the potential use of alginates produced biotechnologically by bacteria for the development of various implantable biomaterials intended for bone and cartilage tissue regeneration in orthopedics: the recent studies on the use of algal alginate-based biomaterials in the form of hydrogels, scaffolds, and microparticles for medical applications are considered as a potential opportunity to use bacterial alginate for these applications, taking into account the advantages of biotechnological production of a polymer with desired properties. The methods of producing different alginate-based biomaterials, the manufacturing of implantable medical devices using them, and the surgical techniques for bone and cartilage tissue regeneration using these materials for orthopedic purposes are discussed.

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.

In this study, composite films and scaffolds of polyester poly(3-hydroxybutyrate) and polysaccharide chitosan obtained via a simple and reproducible blending method using acetic acid as a solvent were considered. The degradation process of the films was studied gravimetrically in a model biological medium in the presence of enzymes in vitro for 180 days. The kinetics of weight reduction depended on the amount of chitosan in the composition. The biocompatibility of the films was evaluated using the Alamar blue test and fluorescence microscopy. The materials were non-cytotoxic, and the addition of poly(3-hydroxybutyrate) to chitosan improved its matrix properties on mesenchymal stem cells. Then, the 3D composites were prepared by freeze-drying. Their structure (using SEM), rheological behavior, moisture absorption, and porosity were investigated. The addition of different amounts of chitosan allowed us to vary the chemical and biological properties of poly(3-hydroxybutyrate) materials and their degradation rate, which is extremely important in the development of biomedical poly(3-hydroxybutyrate) materials, especially implantable ones.

Physiological resorption of bone tissue after tooth extraction leads to a decrease in the volume of bone tissue available for implantation and makes it difficult to install dental implants. Preservation of the well after tooth extraction is the solution to this problem, with the choice of bone plastic material playing an important role. The development of an "ideal" bone plastic material with osteoinductive properties that promotes reparative bone regeneration remains an urgent task. The aim of the study was to evaluate the regeneration of bone tissue of the alveolar ridge during implantation of a new osteoinductive bone plastic material containing simvastatin into the wells of extracted teeth in sheep using microcomputer tomography. Materials and Methods. The study was conducted on 24 adult sheep with a total of 48 teeth removed. 12 wells were filled with material based on poly(3-hydroxybutyrate) (PHB) with simvastatin; 12 wells were filled with PHB-based material without simvastatin, 24 wells were used as a control. Micro-CT was used for comparative analysis of bone tissue formation between the test groups after 3 and 6 months. Results. The results of the study confirm the positive effect of simvastatin released from the PHB-based osteoplastic material on the volume of the formed bone tissue and the total bone volume in the defect area (BV/TV) and bone mineral density (BMD) 3 and 6 months after surgery. Conclusion. The study demonstrated that simvastatin, released from the PHB-based osteoplastic material, has an osteoinductive effect, promoting bone tissue regeneration in the wells left after tooth removal. Higher BV/TV and BMD values in the wells indicate better efficacy of the material in terms of regeneration support.

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.

The ability of materials to adhere bacteria on their surface is one of the most important aspects of their development and application in bioengineering. In this work, the effect of the properties of films and electrospun scaffolds made of composite materials based on biosynthetic poly(3-hydroxybutyrate) (PHB) with the addition of magnetite nanoparticles (MNP) and their complex with graphene oxide (MNP/GO) on the adhesion of E. coli and L. fermentum under the influence of a low-frequency magnetic field and without it was investigated. The physicochemical properties (crystallinity; surface hydrophilicity) of the materials were investigated by X-ray structural analysis, differential scanning calorimetry and “drop deposition” methods, and their surface topography was studied by scanning electron and atomic force microscopy. Crystal violet staining made it possible to reveal differences in the surface charge value and to study the adhesion of bacteria to it. It was shown that the differences in physicochemical properties of materials and the manifestation of magnetoactive properties of materials have a multidirectional effect on the adhesion of model microorganisms. Compared to pure PHB, the adhesion of E. coli to PHB-MNP/GO, and for L. fermentum to both composite materials, was higher. In the magnetic field, the adhesion of E. coli increased markedly compared to PHB-MNP/GO, whereas the effect on the adhesion of L. fermentum was reversed and was only evident in samples with PHB-MNP. Thus, the resultant factors enhancing and impairing the substrate binding of Gram-negative E. coli and Gram-positive L. fermentum turned out to be multidirectional, as they probably have different sensitivity to them. The results obtained will allow for the development of materials with externally controlled adhesion of bacteria to them for biotechnology and medicine.

The development of biopolymer scaffolds for intestine regeneration is one of the most actively developing areas in tissue engineering. However, intestinal regenerative processes after scaffold implantation depend on the activity of the intestinal microbial community that is in close symbiosis with intestinal epithelial cells. In this work, we study the impact of different scaffolds based on biocompatible poly(3-hydroxybutyrate) (PHB) and alginate (ALG) as well as PHB/ALG scaffolds seeded with probiotic bacteria on the composition of gut microbiota of Wistar rats. Implantation of PHB/ALG scaffolds on the large-intestine wall to close its injury showed that alpha diversity of the gut microbiota was not reduced in rats implanted with different PHB/ALG scaffolds except for the PHB/ALG scaffolds with the inclusion of Lactobacillus spheres (PHB/ALG-L). The composition of the gut microbiota of rats implanted with PHB/ALG scaffolds with probiotic bacteria or in simultaneous use of an antimicrobial agent (PHB/ALG-AB) differed significantly from other experimental groups. All rats with implanted scaffolds demonstrated shifts in the composition of the gut microbiota by individual operational taxonomic units. The PHB/ALG-AB construct led to increased abundance of butyrate-producing bacteria: Ileibacterium sp. dominated in rats with implanted PHB/ALG-L and Lactobacillus sp. and Bifidobacterium sp. dominated in the control group. In addition, the PHB/ALG scaffolds had a favourable effect on the growth of commensal bacteria. Thus, the effect of implantation of the PHB/ALG scaffold compared to other scaffolds on the composition of the gut microbiota was closest to the control variant, which may demonstrate the biocompatibility of this device with the microbiota.

Poly(3-hydroxybutyrate) and chitosan are among the most widely used polymers for biomedical applications due to their biocompatibility, renewability and low toxicity. The creation of composite materials based on biopolymers belonging to different classes makes it possible to overcome the disadvantages of each of the components and to obtain a material with specific properties. Solving this problem is associated with difficulties in the selection of conditions and solvents for obtaining the composite material. In our study, acetic acid was used as a common solvent for hydrophobic poly(3-hydroxybutyrate) and chitosan. Mechanical, thermal, physicochemical and surface properties of the composites and homopolymers were investigated. The composite films had less crystallinity and hydrophobicity than poly(3-hydroxybutyrate), and the addition of chitosan caused an increase in moisture absorption, a decrease in contact angle and changes in mechanical properties of the poly(3-hydroxybutyrate). The inclusion of varying amounts of chitosan controlled the properties of the composite, which will be important in the future for its specific biomedical applications.

The possibility of fabrication of microstructured poly-3-hydroxybutyrate films by self-assembly water microdroplets technique, using artificial templates and polymer inverse emulsions has been studied. It has been established that self-assembly water microdroplets technique allows forming ordered microstructures of poly-3-hydroxybutyrate with a hexagonal arrangement of cells with an adjustable diameter from 1 to 4 цт. It has been shown that application of inverse emulsions of poly-3-hydroxybutyrate allows us to fabricate porous films with a pore size in the range from 0.4 to 3 ^m, while the structure of the films and the pore size can be controlled by changing the polymer concentration in the dispersion medium and the volume ratio of the phases. Using spin-coating technique and artificial templates, it is possible to obtain poly-3-hydroxybutyrate microstructured replicas, which are characterized by a high degree of uniformity and the absence of defective areas. It has been shown that the formed microstructured poly-3-hydroxybutyrate films with controlled surface topography are promising for use as scaffolds for stem cells.

The results of comprehensive research on the thermal behavior and molecular and crystalline structures of poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-HV) films of different thicknesses, their molecular weights (M w ) and 3-hydroxyvalerate (3-HV) contents are reported. Increasing film thickness from 30 to 100 µm resulted in an isotropic crystal orientation, reducing the crystallite size of the orthorhombic α-phase in the b direction from 22 to 17 nm and increasing the degree of crystallinity of the PHB films without affecting their thermal behavior. Furthermore, despite resulting in the same degree of crystallinity and roughness, an ~8-fold decrease in PHB M w from 803 kDa to 102 kDa resulted in a decreased number of piezoactive domains. The addition of 5.9% 3-HV resulted in anisotropy in the PHB crystalline structure and increased D (020 ) from 19 nm to 24 nm. Additionally, a further increase in the 3-HV content to 17.5% in the PHB-HV films led to a decrease in the melting temperature and a decrease in the degree of crystallinity from 57% to 23%, which resulted in the absence of local piezoresponse. Notably, the decrease in the M w of PHB-HV (~17%) from 1177 kDa to 756 kDa resulted in an increase in the degree of crystallinity from 23% to 32%. Moreover, the PHB-HV films became smoother with increasing 3-HV content.

Various variants of poly(3-hydroxybutyrate) (PHB) were synthesized by varying three factors (sucrose concentration, phosphate concentration, and aeration level) while growing the producer Azotobacter vinelandii 12 using full-factorial design. Bacteria grown at elevated sucrose concentration, low phosphate concentration, and high aeration level (C+/P−/O+) showed the maximum PHB yield (0.49 g/L). Molecular weights (MW) of PHB samples obtained under diverse conditions differed by more than 30 times (from 49 to 1698 kDa). Low molecular weight PHBs were observed at low sucrose levels and high aeration (C–/O+). All other PHB samples had MW over 1200 kDa. The crystallinity of all PHB samples was determined by differential scanning calorimetry and was within the range of 62–68%. These results show that, with optimization, it will be possible to synthesize PHBs with different physicochemical properties for a wide range of biomedical problems, including tissue engineering.

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.

This study investigated the effect of various cultivation conditions (sucrose/phosphate concentrations, aeration level) on alginate biosynthesis using the bacterial producing strain Azotobacter vinelandii 12 by the full factorial design (FFD) method and physicochemical properties (e.g., rheological properties) of the produced bacterial alginate. We demonstrated experimentally the applicability of bacterial alginate for tissue engineering (the cytotoxicity testing using mesenchymal stem cells (MSCs)). The isolated synthesis of high molecular weight (Mw) capsular alginate with a high level of acetylation (25%) was achieved by FFD method under a low sucrose concentration, an increased phosphate concentration, and a high aeration level. Testing the viscoelastic properties and cytotoxicity showed that bacterial alginate with a maximal Mw (574 kDa) formed the densest hydrogels (which demonstrated relatively low cytotoxicity for MSCs in contrast to bacterial alginate with low Mw). The obtained data have shown promising prospects in controlled biosynthesis of bacterial alginate with different physicochemical characteristics for various biomedical applications including tissue engineering.

Polymer films are commonly used in different biotechnological applications. The effect of polymer film surface on its efficiency is of interest to study. In this article, porous polymer films (PHB and PHBHV) were obtained under different conditions using the honeycomb method—when water droplets concentrate on liquid polymer solution, while polymer dries thereby modifying the surface of polymer film into a regular structure. Pores on the surface of polymer films were characterized by mathematical processing—areas of pores, circularity, and aspect ratio were assessed. The data processing showed that by variation of the type of polymers and their concentration it is possible to influence the structure of the polymer film surface. This provides an opportunity to further study the effect of surface roughness in various applications.

Surface morphology affects cell attachment and proliferation. In this research, different films made of biodegradable polymers, poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-co-HV), containing different molecular weights, with microstructured surfaces were investigated. Two methods were used to obtain patterned films—water-assisted self-assembly (“breath figure”) and spin-coating techniques. The water-assisted technique made it possible to obtain porous films with a self-assembled pore structure, which is dependent on the monomer composition of a polymer along with its molecular weight and the technique parameters (distance from the nozzle, volume, and polymer concentration in working solution). Their pore morphologies were evaluated and their hydrophobicity was examined. Mesenchymal stem cells (MSCs) isolated from bone marrow were cultivated on a porous film surface. MSCs’ attachment differed markedly depending on surface morphology. On strip-formed stamp films, MSCs elongated along the structure, however, they interacted with a larger area of film surface. The honeycomb films and column type films did not set the direction of extrusion, but cell flattening depended on structure topography. Thus, stem cells can “feel” the various surface morphologies of self-assembled honeycomb films and change their behavior depending on it.