Vapor-Phase-Deposited Ag/Ir and Ag/Au Film Heterostructures for Implant Materials: Cytotoxic, Antibacterial and Histological Studies.

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.

Preservation of Mechanical and Morphological Properties of Porcine Cardiac Outflow Vessels after Decellularization and Wet Storage.

Widely used storage methods, including freezing or chemical modification, preserve the sterility of biological tissues but degrade the mechanical properties of materials used to make heart valve prostheses. Therefore, wet storage remains the most optimal option for biomaterials. Three biocidal solutions (an antibiotic mixture, an octanediol-phenoxyethanol complex solution, and a glycerol-ethanol mixture) were studied for the storage of native and decellularized porcine aorta and pulmonary trunk. Subsequent mechanical testing and microstructural analysis showed a slight increase in the tensile strength of native and decellularized aorta in the longitudinal direction. Pulmonary trunk elongation increased 1.3-1.6 times in the longitudinal direction after decellularization only. The microstructures of the tested specimens showed no differences before and after wet storage. Thus, two months of wet storage of native and decellularized porcine aorta and pulmonary trunks does not significantly affect the strength and elastic properties of the material. The wet storage protocol using alcohol solutions of glycerol or octanediol-phenoxyethanol mixture may be intended for further fabrication of extracellular matrix for tissue-engineered biological heart valve prostheses.

Functional Activity of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes on a Mouse Renal Subcapsular Xenograft Model.

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.

Cellular electrophysiological effects of botulinum toxin A on neonatal rat cardiomyocytes and on cardiomyocytes derived from human-induced pluripotent stem cells.

Botulinum toxin A is a well-known neurotransmitter inhibitor with a wide range of applications in modern medicine. Recently, botulinum toxin A preparations have been used in clinical trials to suppress cardiac arrhythmias, especially in the postoperative period. Its antiarrhythmic action is associated with inhibition of the nervous system of the heart, but its direct effect on heart tissue remains unclear. Accordingly, we investigate the effect of botulinum toxin A on isolated cardiac cells and on layers of cardiac cells capable of conducting excitation. Cardiomyocytes of neonatal rat pups and human cardiomyocytes obtained through cell reprogramming were used. A patch-clamp study showed that botulinum toxin A inhibited fast sodium currents and L-type calcium currents in a dose-dependent manner, with no apparent effect on potassium currents. Optical mapping showed that in the presence of botulinum toxin A, the propagation of the excitation wave in the layer of cardiac cells slows down sharply, conduction at high concentrations becomes chaotic, but reentry waves do not form. The combination of botulinum toxin A with a preparation of chitosan showed a stronger inhibitory effect by an order of magnitude. Further, the inhibitory effect of botulinum toxin A is not permanent and disappears after 12 days of cell culture in a botulinum toxin A-free medium. The main conclusion of the work is that the antiarrhythmic effect of botulinum toxin A found in clinical studies is associated not only with depression of the nervous system but also with a direct effect on heart tissue. Moreover, the combination of botulinum toxin A and chitosan reduces the effective dose of botulinum toxin A.

Biological Studies of New Implant Materials Based on Carbon and Polymer Carriers with Film Heterostructures Containing Noble Metals.

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.

Do Human iPSC-Derived Cardiomyocytes Cultured on PLA Scaffolds Induce Expression of CD28/CTLA-4 by T Lymphocytes?

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.

Noble Metals for Modern Implant Materials: MOCVD of Film Structures and Cytotoxical, Antibacterial, and Histological Studies.

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.

Botulinum Toxin-Chitosan Nanoparticles Prevent Arrhythmia in Experimental Rat Models.

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.

Vascular Stents Coated with Electrospun Drug-Eluting Material: Functioning in Rabbit Iliac Artery.

A stenting procedure aimed at blood flow restoration in stenosed arteries significantly improves the efficiency of vascular surgery. However, the current challenge is to prevent neointimal growth, which reduces the vessel lumen, in the stented segments in the long run. We tested in vivo drug-eluting coating applied by electrospinning to metal vascular stents to inhibit the overgrowth of neointimal cells via both the drug release and mechanical support of the vascular wall. The blend of polycaprolactone with human serum albumin and paclitaxel was used for stent coating by electrospinning. The drug-eluting stents (DESs) were placed using a balloon catheter to the rabbit common iliac artery for 1, 3, and 6 months. The blood flow rate was ultrasonically determined in vivo. After explantation, the stented arterial segment was visually and histologically examined. Any undesirable biological responses (rejection or hemodynamically significant stenosis) were unobservable in the experimental groups. DESs were less traumatic and induced weaker neointimal growth; over six months, the blood flow increased by 37% versus bare-metal stents, where it increased by at least double the rate. Thus, electrospun-coated DESs demonstrate considerable advantages over the bare-metal variants.

Electrospun polyurethane-based vascular grafts: physicochemical properties and functioning in vivo.

General physicochemical properties of the vascular grafts (VGs) produced from the solutions of Tecoflex (Tec) with gelatin (GL) and bivalirudin (BV) by electrospinning are studied. The electrospun VGs of Tec-GL-BV and expanded polytetrafluoroethylene (e-PTFE) implanted in the abdominal aorta of 36 Wistar rats have been observed over different time intervals up to 24 weeks. A comparison shows that 94.5% of the Tec-GL-BV VGs and only 66.6% of e-PTFE VGs (р = 0.0438) are free of occlusions after a 6 month implantation. At the intermediate observation points, Tec-GL-BV VGs demonstrate severe neovascularization of the VG neoadventitial layer as compared with e-PTFE grafts. A histological examination demonstrates a small thickness of the neointima layer and a low level of calcification in Tec-GL-BV VGs as compared with the control grafts. Thus, polyurethane-based protein-enriched VGs have certain advantages over e-PTFE VGs, suggesting their utility in clinical studies.

Long-term suppression of atrial fibrillation by botulinum toxin injection into epicardial fat pads in patients undergoing cardiac surgery: Three-year follow-up of a randomized study.

BACKGROUND: Botulinum toxin (BTX) injections into epicardial fat pads in patients undergoing coronary artery bypass grafting (CABG) has resulted in suppression of atrial fibrillation (AF) during the early postoperative period through 1-year of follow-up in a pilot program. OBJECTIVE: The purpose of this study was to report 3-year AF patterns by the use of implantable cardiac monitors (ICMs). METHODS: Sixty patients with a history of paroxysmal AF and indications for CABG were randomized 1:1 to either BTX or placebo injections into 4 posterior epicardial fat pads. All patients received an ICM with regular follow-up for 3 years after surgery. The primary end point of the extended follow-up period was incidence of any atrial tachyarrhythmia after 30 days of procedure until 36 months on no antiarrhythmic drugs. The secondary end points included clinical events and AF burden. RESULTS: At the end of 36 months, the incidence of any atrial tachyarrhythmia was 23.3% in the BTX group vs 50% in the placebo group (hazard ratio 0.36; 95% confidence interval 0.14-0.88; P = .02). AF burden at 12, 24, and 36 months was significantly lower in the BTX group than in the placebo group: 0.22% vs 1.88% (P = .003), 1.6% vs 9.5% (P < .001), and 1.3% vs 6.9% (P = .007), respectively. In the BTX group, 2 patients (7%) were hospitalized during follow-up compared with 10 (33%) in the placebo group (P = .02). CONCLUSION: Injection of BTX into epicardial fat pads in patients undergoing CABG resulted in a sustained and substantial reduction in atrial tachyarrhythmia incidence and burden during 3-year follow-up, accompanied by reduction in hospitalizations.

Globular chitosan prolongs the effective duration time and decreases the acute toxicity of botulinum neurotoxin after intramuscular injection in rats.

Botulinum neurotoxin (BoNT) is used for an increasing number of neurological and non-neurological indications and disorders. Since the duration of action of this neurotoxin is limited, the goal of the work was to improve the pharmacological time course of BoNT. We explored the effect of several polysaccharides on the duration of action of BoNT/A1 in rat electromyography. The formulation of BoNT/A1 containing globular chitosan increased the threshold stimulation intensity almost 2 times in 30 days after injection if compared with the baseline threshold. However, conventional linear chitosan, heparin and hyaluronic acid did not have such an effect. In addition, we compared the effectiveness of different doses of BoNT/A1 (25, 50, 75, and 100 U) with globular chitosan and compared the acute toxicity of this formulation with that of BoNT/A1 in physiological saline after intramuscular injection. The results demonstrated that the dose 25 U of BoNT/A1 with globular chitosan was both effective and safe for animals after intramuscular injection. The assessed median lethal dose (LD50) for intramuscular injection in rats was 1.4 times higher for a combination of BoNT/A1 with globular chitosan than that for a solution of BoNT/A1 in physiological saline. Thus, the results of our study have provided evidence that intramuscular injection of the formulation of BoNT/A1 (25 U) containing globular chitosan in rats is safe and significantly prolongs the effective duration time of BoNT/A1.

Globular chitosan treatment of bovine jugular veins: evidence of anticalcification efficacy in the subcutaneous rat model.

Vascular xenografts are widely used in cardiovascular surgery as an alternative to autologous vessels and vascular allografts. Calcification is one of the main drawbacks of vascular grafts, especially among young patients and children. Among different anticalcification approaches, chitosan emerges as a highly promising candidate due to its versatility, natural origin, and biocompatibility. We investigated the anticalcification efficacy of globular chitosan ("Chitozol") as it demonstrated the improved rate of water solubility as compared with conventional linear macromolecules of chitosan. In addition, we supposed that compact globular form of "Chitozol" molecules could provide effective penetration of extracellular matrix of bovine jugular veins (BJVs). Our results revealed that "Chitozol" treatment mitigated calcification in the experimental groups as compared to the control groups (without any treatment, conventional treatment with glutaraldehyde, and commercially available Contegra conduit). Different concentrations of "Chitozol" (0.3% and 3%), as well as different incubation times (15 and 30min), were equally effective in the prevention of calcification. In addition, "Chitozol" treatment with decellularization of BJVs demonstrated slightly improved stress-strain properties of unimplanted samples. Thus, the filling of fresh BJV with globular chitosan is proposed as a promising emerging treatment for the mitigation of calcific degeneration in BJVs xenografts.

Comprehensive study of hexarhenium cluster complex Na4[{Re6Te8}(CN)6] - In terms of a new promising luminescent and X-ray contrast agent.

Octahedral rhenium cluster complexes may have considerable potential as therapeutic and diagnostic drugs due to their luminescent and X-ray contrast properties, as well as their ability to generate singlet oxygen upon photoirradiation. However, their potential biological effects and toxicity in vitro and in vivo are rather far from being understood. Thus, the aim of our research was to study cytotoxicity, intracellular localization and cellular uptake/elimination kinetics in vitro, biodistribution and acute intravenous toxicity in vivo of a complex Na4[{Re6Te8}(CN)6] as the promising compound for biomedical application. The results have demonstrated that the complex penetrates through cell membranes with the maximum accumulation in cells in 24h of incubation and have low toxic effects in vitro and in vivo. The median lethal dose (LD50) of intravenously administrated Na4[{Re6Te8}(CN)6] is equal to 1082±83mg/kg. These findings will be useful for future development of cluster-based agents for different biomedical applications.

Long-Term Suppression of Atrial Fibrillation by Botulinum Toxin Injection Into Epicardial Fat Pads in Patients Undergoing Cardiac Surgery: One-Year Follow-Up of a Randomized Pilot Study.

BACKGROUND: Animal models suggest that the neurotransmitter inhibitor, botulinum toxin, when injected into the epicardial fat pads can suppress atrial fibrillation inducibility. The aim of this prospective randomized double-blind study was to compare the efficacy and safety of botulinum toxin injection into epicardial fat pads for preventing atrial tachyarrhythmias. METHODS AND RESULTS: Patients with history of paroxysmal atrial fibrillation and indication for coronary artery bypass graft surgery were randomized to botulinum toxin (Xeomin, Merz, Germany; 50 U/1 mL at each fat pad; n=30) or placebo (0.9% normal saline, 1 mL at each fat pad; n=30) injection into epicardial fat pads during surgery. Patients were followed for 1 year to assess maintenance of sinus rhythm using an implantable loop recorder. All patients in both groups had successful epicardial fat pad injections without complications. The incidence of early postoperative atrial fibrillation within 30 days after coronary artery bypass graft was 2 of 30 patients (7%) in the botulinum toxin group and 9 of 30 patients (30%) in the placebo group (P=0.024). Between 30 days and up to the 12-month follow-up examination, 7 of the 30 patients in the placebo group (27%) and none of the 30 patients in the botulinum toxin group (0%) had recurrent atrial fibrillation (P=0.002). There were no complications observed during the 1-year follow-up. CONCLUSIONS: Botulinum toxin injection into epicardial fat pads during coronary artery bypass graft provided substantial atrial tachyarrhythmia suppression both early as well as during 1-year follow-up, without any serious adverse events. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01842529.

Rapid Recovery from Chronic PRCA by MSC Infusion in Patient after Major ABO-Mismatched alloSCT.

Pure red cell aplasia (PRCA) is a rare complication in recipients of allogenic stem cell from ABO incompatible donors. It is characterized by reticulocytopenia and by an absence of red cell cell precursors in the bone marrow. Despite close isohemagglutinins monitoring and standard immunosupressive treatment in these patients prolong PRCA are still associated with severe transfusion dependence. We report the case of a 31 yr old male patient who underwent HLA-matched ABO-mismatched allo-SCT and developed resistance PRCA despite conventional immunosupressive therapy and prophylaxis cotrasplantation of bone marrow derived MSC at day 0. He responded dramatically to therapy with adipose tissue derived mesenchymal stem cells from HSC donors and continued to be transfusion-independent and AML-disease free. This method of the PRCA therapy of deserves further investigation.