This study presents a novel method to enhance the biocompatibility of decellularized porcine aortic segments while preserving their mechanical properties and histological structure. Detergent-decellularized aortic segments were treated with modified globular chitosan (Novochizol™) at varying concentrations (0.5%, 1%, 2%, and 3%) by sonication and subsequently subjected to mechanical testing. To further improve cell infiltration, blind-ended laser channels were created within the decellularized segments. The modified grafts were then seeded with porcine vascular interstitial cells in vitro for 7 days or implanted into the thoracic aorta of minipigs for 30 days. Histological analysis was performed at each stage of the study. Impregnation with Novochizol™ significantly increased the specific strength (from 0.97 ± 0.19 MPa to 4.99 ± 2.43 MPa) and Young’s modulus (from 0.73 ± 0.06 MPa to 14.66 ± 7.14 MPa) of the decellularized aortic segments. Histological examination confirmed the preservation of the connective tissue matrix’s morphological structure. Optimal modification conditions were identified as a 30 min sonication in a 1% Novochizol™ solution at 25 °C. A 35 ms continuous laser treatment was sufficient to create a 1 mm deep blind-ended channel, thereby promoting the seeding of vascular interstitial cells within the acellular graft, as confirmed by implantation in minipigs.

Polymer heart valves are a promising alternative to bioprostheses, the use of which is limited by the risks of calcific deterioration of devitalized preserved animal tissues. This is especially relevant in connection with the increasingly widespread use of transcatheter valves. Advances in modern organic chemistry provide a wide range of polymers that can replace biological material in the production of valve prostheses. In this work, the main properties of REPEREN® polymer film, synthesized from methacrylic oligomers reinforced with ultra-thin (50 µm) polyamide fibers, are studied. The film structure was studied using scanning electron microscopy (SEM) and atomic force microscopy (AFM). The hydrophilicity and cytocompatibility with EA.hy926 endothelial cells were assessed, and a hemocompatibility evaluation was carried out by studying the platelet aggregation and adhesion upon contact of the REPEREN® with blood. The mechanical behavior and biocompatibility (subcutaneous implantation in rats for up to 90 days, followed by a histological examination) were studied in comparison with a bovine pericardium (BP) cross-linked with an ethylene glycol diglycidyl ether (DE). The results showed that REPEREN® films have two surfaces with a different relief, smooth and rough. The rough surface is more hydrophilic, hemo- and cytocompatible. Compared with the DE-BP, REPEREN® has a higher ultimate tensile stress and better biocompatibility when implanted subcutaneously in rats. The key properties of REPEREN® showed its potential for the development of a polymeric heart valve. Further studies should be devoted to assessing the durability of REPEREN® valves and evaluating their function during orthotopic implantation in large animals.

Cell-based therapies using corneal stromal stem cells (CSSC), corneal keratocytes, or a combination of both suppress corneal scarring. The number of quiescent keratocytes in the cornea is small; it is difficult to expand them in vitro in quantities suitable for transplantation. This study examined the therapeutic effect of corneal fibroblasts reversed into keratocytes (rCF) in a mouse model of mechanical corneal injury. The therapeutic effect of rCF was studied in vivo (slit lamp, optical coherence tomography) and ex vivo (transmission electron microscopy and immunofluorescence staining). Injection of rCF into the injured cornea was accompanied by recovery of corneal thickness, improvement of corneal transparency, reduction of type III collagen in the stroma, absence of myofibroblasts, and the improvement in the structural organization of collagen fibers. TEM results showed that 2 months after intrastromal injection of cells, there was a decrease in the fibril density and an increase in the fibril diameter and the average distance between collagen fibrils. The fibrils were well ordered and maintained the short-range order and the number of nearest-neighbor fibrils, although the averaged distance between them increased. Our results demonstrated that the cell therapy of rCF from ReLEx SMILe lenticules promotes the recovery of transparent corneal stroma after injury.

Using gas-phase deposition (Physical Vapor Deposition (PVD) and Metal Organic Chemical Vapor Deposition (MOCVD)) methods, modern implant samples (Ti alloy and CFR-PEEK polymer, 30% carbon fiber) were functionalized with film heterostructures consisting of an iridium or gold sublayer, on the surface of which an antibacterial component (silver) was deposited: Ag/Ir(Au)/Ti(CFR-PEEK). The biocidal effect of the heterostructures was investigated, the effect of the surface relief of the carrier and the metal sublayer on antibacterial activity was established, and the dynamics of silver dissolution was evaluated. It has been shown that the activity of Ag/Ir heterostructures was due to high Ag+ release rates, which led to rapid (2–4 h) inhibition of P. aeruginosa growth. In the case of Ag/Au type heterostructures, the inhibition of the growth of P. aeruginosa and S. aureus occurred more slowly (from 6 h), and the antibacterial activity appeared to be due to the contribution of two agents (Ag+ and Au+ ions). It was found, according to the in vitro cytotoxicity study, that heterostructures did not exhibit toxic effects (cell viability > 95–98%). An in vivo biocompatibility assessment based on the results of a morphohistological study showed that after implantation for a period of 30 days, the samples were characterized by the presence of a thin fibrous capsule without volume thickening and signs of inflammation.

This study presents a novel method to enhance the biocompatibility of decellularized porcine aortic segments while preserving their mechanical properties and histological structure. Detergent-decellularized aortic segments were treated with modified globular chitosan (Novochizol™) at varying concentrations (0.5%, 1%, 2%, 3%) by sonication and subsequently subjected to mechanical testing. To further improve cell infiltration, blind-ended laser channels were created within the decellularized segments. The modified grafts were then seeded with porcine vascular interstitial cells in vitro for 7 days or implanted into the thoracic aorta of minipigs for 30 days. Histological analysis was performed at each stage of the study. Impregnation with Novochizol™ significantly increased the specific strength (from 0.97 ± 0.19 MPa to 4.99 ± 2.43 MPa) and Young's modulus (from 0.73 ± 0.06 MPa to 14.66 ± 7.14 MPa) of the decellularized aortic segments. Histological examination confirmed the preservation of the connective tissue matrix's morphological structure. Optimal modification conditions were identified as a 30-minute sonication in a 1% Novochizol™ solution at 25°C. A 35-ms continuous laser treatment was sufficient to create a 1 mm deep blind-ended channel, thereby promoting the seeding of vascular interstitial cells within the acellular graft, as confirmed by implantation in minipigs.

In this work, approaches to the formation of multifunctional film heterostructures based on noble metals for the modification of the surface of implant materials (titanium alloy TiAl6V4 and carbon-fiber-reinforced polyetheretherketone CFR-PEEK) are developed. Such heterostructures consist of continuous layers of platinum (Pt) or iridium (Ir) and antibacterial components on their surface, namely silver (nanoparticles or discontinuous films) and gold (nanoparticles). Chemical or physical gas-phase deposition methods were used for their preparation. The influence of the concentration and form of the antibacterial component on the antibacterial activity and in vivo biocompatibility of the film structures was evaluated for the first time. Differences in the dynamics of silver dissolution depending on Ag concentration in the sample and the type of bottom surface (the noble metal layer = Ir, Pt or TiAl6V4) surfaces allowed us to better understand the nature of the antibacterial action against Staphylococcus aureus and Pseudomonas aeruginosa (S. aureus and P. aeruginosa) of Ag/M heterostructures. From in vivo histological studies using rats, the best biocompatibility was shown by the Ag/M heterostructure with a prolonged release of the low fraction of antibacterial component (Ag).

In the treatment of coronary heart disease, the most promising approach for replacing lost contractile elements involves obtaining cardiomyocytes through cardiac differentiation of pluripotent cells. The objective of this study is to develop a technology for creating a functional layer of cardiomyocytes derived from iPSCs, capable of generating rhythmic activity and synchronous contractions. To expedite the maturation of cardiomyocytes, a renal subcapsular transplantation model was employed in SCID mice. Following explantation, the formation of the cardiomyocyte contractile apparatus was assessed using fluorescence and electron microscopy, while the cytoplasmic oscillation of calcium ions was evaluated through visualization using the fluorescent calcium binding dye Fluo-8. The results demonstrate that transplanted human iPSC-derived cardiomyocyte cell layers, placed under the fibrous capsules of SCID mouse kidneys (for up to 6 weeks), initiate the development of an organized contractile apparatus and retain functional activity along with the ability to generate calcium ion oscillations even after removal from the body.

Fibroblasts isolated and expanded from ReLEx SMILE lenticules can be a source of human keratocytes. Since corneal keratocytes are quiescent cells, it is difficult to expand them in vitro in suitable numbers for clinical and experimental use. In the present study, this problem was solved by isolating and growing corneal fibroblasts (CFs) with a high proliferative potential and their reversion to keratocytes in a selective serum-free medium. Fibroblasts reversed into keratocytes (rCFs) had a dendritic morphology and ultrastructural signs of activation of protein synthesis and metabolism. The cultivation of CFs in a medium with 10% FCS and their reversion into keratocytes was not accompanied by the induction of myofibroblasts. After reversion, the cells spontaneously formed spheroids and expressed keratocan and lumican markers, but not mesenchymal ones. The rCFs had low proliferative and migratory activity, and their conditioned medium contained a low level of VEGF. CF reversion was not accompanied by a change with the levels of IGF-1, TNF-alpha, SDF-1a, and sICAM-1. In the present study, it has been demonstrated that fibroblasts from ReLEx SMILE lenticules reverse into keratocytes in serum-free KGM, maintaining the morphology and functional properties of primary keratocytes. These keratocytes have a potential for tissue engineering and cell therapy of various corneal pathologies.

Improved methods for obtaining connective tissue structures in cardiac surgery are actively developed over the world. An important aspect of this research pertains to long-term wet storage of cardiovascular biomaterial that optimally preserves the initial morphological characteristics. Native and decellularized porcine aortas were stored for 50 days in several biocidal solutions (complex alcohol solution, a mixture of ethanol and glycerol, a mixture of antibiotics), and subsequently implanted in animals and studied histologically. The aortic specimens were implanted subcutaneously into 15 adult WAG rats. The samples were retrieved after 3 months and evaluated for calcium content using a quantitative spectroanalytical procedure. Aortic specimens were also orthotopically implanted into the vascular bed of 3 minipigs, harvested after 6 months, and histologically evaluated. Atomic absorption spectroscopy showed that decellularized materials accumulated 4.8–9.2 times less calcium, depending on the storage solution used. Morphological and morphometrical analysis of orthotopically implanted aortic fragments in minipigs showed better preservation of decellularized material that had been stored in a complex alcohol solution and mixture of antibiotics. Functional and histological evidence was obtained demonstrating long-term storage without freezing of o aortic specimens without alteration of their biophysical properties and structure. The developed technique will be used in the future for the fabrication of vascular prostheses and heart valves.

Recently, film materials based on the combination of noble metals have showed promising results for surface modification of medical implants, allowing both to improve biocompatibility and to acquire the increased antibacterial effect. An important challenge here is to combine the developed coating morphology, which is favorable for biological response, with a high protective function, which, on the contrary, requires a compact coating microstructure. In this work, we aimed to solve this problem with respect to the TiNi implant material. We have tested two types of compact thin sublayers: Iridium (Ir’), formed by metal-organic chemical vapor deposition (MOCVD), and gold (Au), formed by physical vapor deposition (PVD). Subsequently these sublayers were coated with a developed-columnar-iridium (Ir) by MOCVD. Features of the microstructure, chemical and phase composition of all these film materials were studied using powder X-ray diffraction (XRD), scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). The changes in the characteristics of TiNi martensitic transformation due to MOCVD experiments were also studied by differential scanning calorimetry (DSC). The biocompatibility of Ir’/TiNi, Au/TiNi, Ir/Ir’/TiNi, Ir/Au/TiNi samples was assessed by cytoxicity testing (Man-1 cells) and measuring of nickel content in the biological extracts. The application of both sublayers effectively reduces the release of nickel, which was previously shown for Ir/TiNi samples. This prevents the toxic effect. Note that the Ir’ sublayer better protects against nickel release, while the Au sublayer promotes cell proliferation.

Heart failure is a leading cause of death in patients who have suffered a myocardial infarction. Despite the timely use of modern reperfusion therapies such as thrombolysis, surgical revascularization and balloon angioplasty, they are sometimes unable to prevent the development of significant areas of myocardial damage and subsequent heart failure. Research efforts have focused on developing strategies to improve the functional status of myocardial injury areas. Consequently, the restoration of cardiac function using cell therapy is an exciting prospect. This review describes the characteristics of various cell types relevant to cellular cardiomyoplasty and presents findings from experimental and clinical studies investigating cell therapy for coronary heart disease. Cell delivery methods, optimal dosage and potential treatment mechanisms are discussed.

Many research groups have developed various types of tissue-engineered cardiac constructs. However, the immunological properties of such artificial tissues are not yet fully understood. Previously, we developed microfiber scaffolds carrying human iPSC-derived cardiomyocytes (hiPSC-CM). In this work, we evaluated the ability of these tissue-engineered constructs to activate the expression of CD28 and CTLA-4 proteins on T lymphocytes, which are early markers of the immune response. For this purpose, electrospun PLA microfiber scaffolds were seeded with hiPSC-CM and cultured for 2 weeks. Allogeneic mononuclear cells were then co-cultured for 48 h with three groups of samples: bare scaffolds, pure cardiomyocyte culture and tissue-engineered constructs, followed by analysis of CD28/CTLA-4 expression on T lymphocytes using flow cytometry. PLA scaffolds and concanavalin A stimulation (positive control) statistically significantly increased CD28 expression on CD4+ T cells (up to 61.3% and 66.3%) CD8+ T cells (up to 17.8% and 21.7%). CD28/CTLA-4 expression was not increased when T lymphocytes were co-cultured with cardiac tissue-engineered constructs and iPSC-CM monolayers. Thus, iPSC-CM in monolayers and on PLA microfiber scaffolds did not induce T cell activation, which suggests that such cardiac constructs would not be a cause of rejection after implantation.

Titanium oxynitride coatings deposited by reactive magnetron sputtering improve biocompatibility of vascular stents by increasing NO production, viability, and adhesion of EA.hy926 cells. Thus, the application of titanium oxynitride coatings is a promising strategy for increasing the biocompatibility of nitinol stents.

This paper presents pioneering results on the evaluation of noble metal film hetero-structures to improve some functional characteristics of carbon-based implant materials: carbon-composite material (CCM) and carbon-fiber-reinforced polyetheretherketone (CFR-PEEK). Metal-organic chemical vapor deposition (MOCVD) was successfully applied to the deposition of Ir, Pt, and PtIr films on these carriers. A noble metal layer as thin as 1 µm provided clear X-ray imaging of 1–2.5 mm thick CFR-PEEK samples. The coated and pristine CCM and CFR-PEEK samples were further surface-modified with Au and Ag nanoparticles (NPs) through MOCVD and physical vapor deposition (PVD) processes, respectively. The composition and microstructural features, the NPs sizes, and surface concentrations were determined. In vitro biological studies included tests for cytotoxicity and antibacterial properties. A series of samples were selected for subcutaneous implantation in rats (up to 3 months) and histological studies. The bimetallic PtIr-based heterostructures showed no cytotoxicity in vitro, but were less biocompatible due to a dense two-layered fibrous capsule. AuNP heterostructures on CFR-PEEK promoted cell proliferation in vitro and exhibited a strong inhibition of bacterial growth (p < 0.05) and high in vitro biocompatibility, especially Au/Ir structures. AgNP heterostructures showed a more pronounced antibacterial effect, while their in vivo biocompatibility was better than that of the pristine CFR-PEEK, but worse than that of AuNP heterostructures.

Objective: to determine the optimal method for long-term wet storage of donor material (50 days after collection), with maximum ability to preserve the original mechanical characteristics.Materials and methods. Porcine aortic wall fragments were used as objects of study. Half of the original material underwent detergent-based decellularization. The entire material (native and processed) was placed for 50 days in biocidal solutions: complex alcohol solution; ethanol and glycerol mixture; antibiotics mixture. Then the tests for mechanical strength of native and decellularized samples were carried out by the method of uniaxial longitudinal and circumferential stress.Results. Storage of native material in all media resulted in a significant increase in tensile strength. In the «complex alcohol solution», «ethanol and glycerol mixture», and «antibiotic mixture» group, tensile strength increased by 1.38-, 1.72- and 1.62-fold compared to the native control in circumferential tension. Also, in the «complex alcohol solution» group, the decellularized material was 1.57-fold stronger than the native in circumferential tension. In the «antibiotic mixture» group, the decellularized material was 1.33-fold less strong than the native in longitudinal tension. According to elongation to rupture data, significantly greater plasticity was noted in the «ethanol-glycerol» storage group for the decellularized aortic wall compared to the control group (1.5-fold). Young’s modulus did not reliably differ from those of control in all experimental groups regardless of the stress direction. Notably, decellularized specimens clearly tended to be stiffer under circumferential stress.Conclusion. Detergent-based decellularization of the porcine aortic wall and subsequent storage of these samples in our chosen experimental solutions for 50 days does not significantly affect the elastic properties of the material. Our proposed treatment methods partially increase the stiffness of the material after storage in alcohol-containing solutions.

This work is aimed at developing the modification of the surface of medical implants with film materials based on noble metals in order to improve their biological characteristics. Gas-phase transportation methods were proposed to obtain such materials. To determine the effect of the material of the bottom layer of heterometallic structures, Ir, Pt, and PtIr coatings with a thickness of 1.4–1.5 μm were deposited by metal–organic chemical vapor deposition (MOCVD) on Ti6Al4V alloy discs. Two types of antibacterial components, namely, gold nanoparticles (AuNPs) and discontinuous Ag coatings, were deposited on the surface of these coatings. AuNPs (11–14 nm) were deposited by a pulsed MOCVD method, while Ag films (35–40 nm in thickness) were obtained by physical vapor deposition (PVD). The cytotoxic (24 h and 48 h, toward peripheral blood mononuclear cells (PBMCs)) and antibacterial (24 h) properties of monophase (Ag, Ir, Pt, and PtIr) and heterophase (Ag/Pt, Ag/Ir, Ag/PtIr, Au/Pt, Au/Ir, and Au/PtIr) film materials deposited on Ti-alloy samples were studied in vitro and compared with those of uncoated Ti-alloy samples. Studies of the cytokine production by PBMCs in response to incubation of the samples for 24 and 48 h and histological studies at 1 and 3 months after subcutaneous implantation in rats were also performed. Despite the comparable thickness of the fibrous capsule after 3 months, a faster completion of the active phase of encapsulation was observed for the coated implants compared to the Ti alloy analogs. For the Ag-containing samples, growth inhibition of S. epidermidis, S. aureus, Str. pyogenes, P. aeruginosa, and Ent. faecium was observed.

Noble metals such as Ir, Pt, Au are promising as coatings for metal medical implants to improve biocompatibility and corrosion resistance. Moreover, these coatings can be used as a basis for the further formation of bimetallic hetero-structures with enhanced antibacterial properties. In this work, we develop an approach to obtain such coatings by metal-organic chemical vapor deposition (MOCVD). We have been focused on the formation of Ir coating with developed morphology and subsequent discrete Au coating onto the titanium nickelide (TiNi) implant material. Iridium was deposited in an oxidizing atmosphere from the volatile precursor [Ir(cod)(acac)] (cod = cyclooctadiene-1,5, acac = acetylacetonate-anion). The effects of the deposition temperature (290–350 °C) and amount of introduced oxygen on the composition (Ir, Ir + IrO2) and microstructure of the samples were studied. Hetero-metallic Au/Ir coatings were obtained using [(CH3)2Au(thd)] precursor (thd = dpm = dipivaloylmethanate-anion) at a deposition temperature of 240 °C in the presence of oxygen. To assess the biocompatibility, the toxicity of Ir/TiNi, Au/Ir/TiNi, and uncoated TiNi in relation to human embryonic stem cell line Man-1 was examined after 1, 3, and 5 days of incubation. The results obtained were explained based on the coating microstructures.

Titanium oxynitride (TiOxNy) coatings increase the bio- and hemocompatibility of stainless steel and Co-Cr vascular stents, possibly by directly releasing NO. Herein, to examine the applicability of this approach to nitinol (NiTi) devices, we coated NiTi samples with TiOx or TiOxNy by reactive magnetron sputtering using different reaction gas compositions and bias voltages. One side of the samples was preliminarily abraded, while the other remained in its original state. Samples morphology, composition and structure were studied by atomic force microscopy (AFM), X-ray diffraction (XRD) and field emission scanning electron microscopy (FE SEM). We investigated how various types of TiOx and TiOxNy coatings interact with EA.hy926 endothelial cells. None of the tested samples exhibited cytotoxicity, and an N2:O2 ratio of 3:1 regardless of the bias voltage was found to be optimal for NO production and cell adhesion, spreading, and viability, which was ascribed to a cumulative positive effect of the composition and nanoroughness of TiOxNy coatings formed under these conditions. Thus, the deposition of TiOxNy coatings was concluded to be a promising strategy of increasing the biocompatibility of NiTi stents.

Aim.To evaluate the effects of long-term wet treatment of porcine aortic wall fragments with biocidal solutions (glutaraldehyde, antibiotics, alcohol-glycerin mixture, complex alcohol solution (CAS)) on the severity of degradation of samples’ connective matrix and their long-term ability to accumulate calcium during subcutaneous implantation in laboratory rats (1 and 3 months).Methods.Histological analysis of freshly collected material was performed after 50 days of treatment with various biocidal solutions and after subcutaneous implantation of samples in small laboratory animals at 1 and 3 months. Calcium was measured with the atomic absorption spectroscopy after 3 months of subcutaneous implantation.Results.Histological analysis reported that connective matrix of samples treated with glutaraldehyde, antibiotics, and CAS was similar to the native one (with the exception of partial loss of cellular components in samples). After 1 month of implantation, a moderate degradation of connective tissue was found. The treatment with the alcohol-glycerin mixture was associated with the most severe degradation. After 3 months of implantation, glutaraldehyde-treated samples had the most preserved connective matrix, while alcohol-glycerin-treated samples demonstrated severe connective matrix injury. However, severe calcific deposits were found in samples treated with glutaraldehyde, whereas mild ones were detected in samples treated with alcohol-glycerin. Atomic absorption spectroscopy reported that calcific deposits in Cas- and alcohol-glycerin-treated samples were 2.3 and 1.8 times lower than those in glutaraldehyde-treated samples. The calcium content in samples treated with antibiotics did not differ significantly from those in glutaraldehyde-treated samplesConclusion.The comprehensive analysis of the effects of various treatment media on the stability of connective matrix to subsequent degradation and its ability to accumulate calcium found that CAS was the most preferred medium for long-term wet treatment of xenoprosthetic vascular tissue.

Several experimental studies have recently demonstrated that temporary autonomic block using botulinum toxin (BoNT/A1) might be a novel option for the treatment of atrial fibrillation. However, the assessment of antiarrhythmic properties of BoNT has so far been limited, relying exclusively on vagal stimulation and rapid atrial pacing models. The present study examined the antiarrhythmic effect of specially formulated BoNT/A1-chitosan nanoparticles (BTN) in calcium chloride-, barium chloride- and electrically induced arrhythmia rat models. BTN enhanced the effect of BoNT/A1. Subepicardial injection of BTN resulted in a significant antiarrhythmic effect in investigated rat models. BTN formulation antagonizes arrhythmia induced by the activation of Ca, K and Na channels.