Creation of a Composite Bioactive Coating with Antibacterial Effect Promising for Bone Implantation.

When creating titanium-containing bone implants, the bioactive coatings that promote their rapid engraftment are important. The engraftment rate of titanium implants with bone tissue depends significantly on the modification of the implant surface. It is achieved by changing either the relief or the chemical composition of the surface layer, as well as a combination of these two factors. In this work, we studied the creation of composite coatings with a two-level (the micro- and nanolevel) hierarchy of the surface relief, which have bioactive and bactericidal properties, which are promising for bone implantation. Using the developed non-lithographic template electrochemical synthesis, a composite coating on titanium with a controlled surface structure was created based on an island-type TiO2 film, silver and hydroxyapatite (HAp). This TiO2/Ag/HAp composite coating has a developed surface relief at the micro- and nanolevels and has a significant cytological response and the ability to accelerate osteosynthesis, and also has an antibacterial effect. Thus, the developed biomaterial is suitable for production of dental and orthopedic implants with improved biomedical properties.

The Use of Mesenchymal Stem Cells in the Complex Treatment of Kidney Tuberculosis (Experimental Study).

In recent years, the application of mesenchymal stem cells (MSCs) has been recognized as a promising method for treatment of different diseases associated with inflammation and sclerosis, which include nephrotuberculosis. The aim of our study is to investigate the effectiveness of MSCs in the complex therapy of experimental rabbit kidney tuberculosis and to evaluate the effect of cell therapy on the reparative processes. Methods: To simulate kidney tuberculosis, a suspension of the standard strain Mycobacterium tuberculosis H37Rv (106 CFU) was used, which was injected into the cortical layer of the lower pole parenchyma of the left kidney under ultrasound control in rabbits. Anti-tuberculosis therapy (aTBT) was started on the 18th day after infection. MSCs (5 × 107 cells) were transplanted intravenously after the start of aTBT. Results: 2.5 months after infection, all animals showed renal failure. Conducted aTBT significantly reduced the level of albumin, ceruloplasmin, elastase and the severity of disorders in the proteinase/inhibitor system and increased the productive nature of inflammation. A month after MSC transplantation, the level of inflammatory reaction activity proteins decreased, the area of specific and destructive inflammation in kidneys decreased and the formation of mature connective tissue was noted, which indicates the reparative reaction activation.

Chromone-Containing Allylmorpholines Influence Ion Channels in Lipid Membranes via Dipole Potential and Packing Stress.

Herein, we report that chromone-containing allylmorpholines can affect ion channels formed by pore-forming antibiotics in model lipid membranes, which correlates with their ability to influence membrane boundary potential and lipid-packing stress. At 100 µg/mL, allylmorpholines 1, 6, 7, and 8 decrease the boundary potential of the bilayers composed of palmitoyloleoylphosphocholine (POPC) by about 100 mV. At the same time, the compounds do not affect the zeta-potential of POPC liposomes, but reduce the membrane dipole potential by 80-120 mV. The allylmorpholine-induced drop in the dipole potential produce 10-30% enhancement in the conductance of gramicidin A channels. Chromone-containing allylmorpholines also affect the thermotropic behavior of dipalmytoylphosphocholine (DPPC), abolishing the pretransition, lowering melting cooperativity, and turning the main phase transition peak into a multicomponent profile. Compounds 4, 6, 7, and 8 are able to decrease DPPC's melting temperature by about 0.5-1.9 °C. Moreover, derivative 7 is shown to increase the temperature of transition of palmitoyloleoylphosphoethanolamine from lamellar to inverted hexagonal phase. The effects on lipid-phase transitions are attributed to the changes in the spontaneous curvature stress. Alterations in lipid packing induced by allylmorpholines are believed to potentiate the pore-forming ability of amphotericin B and gramicidin A by several times.

Plant Alkaloids Inhibit Membrane Fusion Mediated by Calcium and Fragments of MERS-CoV and SARS-CoV/SARS-CoV-2 Fusion Peptides.

To rationalize the antiviral actions of plant alkaloids, the ability of 20 compounds to inhibit calcium-mediated fusion of lipid vesicles composed of phosphatidylglycerol and cholesterol was investigated using the calcein release assay and dynamic light scattering. Piperine, tabersonine, hordenine, lupinine, quinine, and 3-isobutyl-1-methylxanthine demonstrated the most potent effects (inhibition index greater than 50%). The introduction of phosphatidylcholine into the phosphatidylglycerol/cholesterol mixture led to significant changes in quinine, hordenine, and 3-isobutyl-1-methylxanthine efficiency. Comparison of the fusion inhibitory ability of the tested alkaloids, and the results of the measurements of alkaloid-induced alterations in the physical properties of model membranes indicated a potent relationship between a decrease in the cooperativity of the phase transition of lipids and the ability of alkaloids to prevent calcium-mediated vesicle fusion. In order to use this knowledge to combat the novel coronavirus pandemic, the ability of the most effective compounds to suppress membrane fusion induced by fragments of MERS-CoV and SARS-CoV/SARS-CoV-2 fusion peptides was studied using the calcein release assay and confocal fluorescence microscopy. Piperine was shown to inhibit vesicle fusion mediated by both coronavirus peptides. Moreover, piperine was shown to significantly reduce the titer of SARS-CoV2 progeny in vitro in Vero cells when used in non-toxic concentrations.

Proteomic Profiling of the Human Fetal Multipotent Mesenchymal Stromal Cells Secretome.

Secretome of multipotent mesenchymal stromal cells (MSCs) is actively used in biomedical applications such as alveolar bone regeneration, treatment of cardiovascular disease, and neurodegenerative disorders. Nevertheless, hMSCs have low proliferative potential and production of the industrial quantity of their secretome might be challenging. Human fetal multipotent mesenchymal stromal cells (FetMSCs) isolated from early human embryo bone marrow are easy to expand and might be a potential source for pharmaceutical substances production based on their secretome. However, the secretome of FetMSCs was not previously analyzed. Here, we describe the secretome of FetMSCs using LC-MALDI shotgun proteomics. We identified 236 proteins. Functional annotation of the identified proteins revealed their involvement in angiogenesis, ossification, regulation of apoptosis, and immune response processes, which made it promising for biomedical applications. The proteins identified in the FetMSCs secretome are involved in the same biological processes as proteins from previously described adult hMSCs secretomes. Nevertheless, many of the common hMSCs secretome components (such as VEGF, FGF, Wnt and TGF-ß) have not been identified in the FetMSCs secretome.

Urethroplasty with a bilayered poly-D,L-lactide-co-ε-caprolactone scaffold seeded with allogenic mesenchymal stem cells.

Reconstructive surgery for urethral defects employing tissue-engineered scaffolds represents an alternative treatment for urethroplasty. The aim of this study was to compare the therapeutic efficacy of the bilayer poly-D,L-lactide/poly-ε-caprolactone (PL-PC) scaffold seeded with allogenic mesenchymal stem cells (MSCs) for urethra reconstruction in a rabbit model with conventional urethroplasty employing an autologous buccal mucosa graft (BG). The inner layer of the scaffold based on poly-D,L-lactic acid (PL) was seeded with MSCs, while the outer layer, prepared from poly-ε-caprolactone, protected the surrounding tissues from urine. To track the MSCs in vivo, the latter were labeled with superparamagnetic iron oxide nanoparticles. In rabbits, a dorsal penile defect was reconstructed employing a BG or a PL-PC graft seeded with nanoparticle-labeled MSCs. In the 12-week follow-up period, no complications were detected. Subsequent histological analysis demonstrated biointegration of the PL-PC graft with surrounding urethral tissues. Less fibrosis and inflammatory cell infiltration were observed in the experimental group as compared with the BG group. Nanoparticle-labeled MSCs were detected in the urothelium and muscular layer, co-localizing with the urothelium cytokeratin marker AE1/AE3, indicating the possibility of MSC differentiation into neo-urothelium. Our results suggest that a bilayer MSCs-seeded scaffold could be efficiently employed for urethroplasty.

Evaluation of the temporary effect of physical vapor deposition silver coating on resistance to infection in transdermal skin and bone integrated pylon with deep porosity.

Periprosthetic infection via skin-implant interface is a leading cause of failures and revisions in direct skeletal attachment of limb prostheses. Implants with deep porosity fabricated with skin and bone integrated pylons (SBIP) technology allow for skin ingrowth through the implant's structure creating natural barrier against infection. However, until the skin cells remodel in all pores of the implant, additional care is required to prevent from entering bacteria to the still nonoccupied pores. Temporary silver coating was evaluated in this work as a means to provide protection from infection immediately after implantation followed by dissolution of silver layer in few weeks. A sputtering coating with 1 µm thickness was selected to be sufficient for fighting infection until the deep ingrowth of skin in the porous structure of the pylon is completed. In vitro study showed less bacterial (Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa) growth on silver coated tablets compared to the control group. Analysis of cellular density of MG-63 cells, fibroblasts, and mesenchymal stem cells (MSCs) showed that silver coating did not inhibit the cell growth on the implants and did not affect cellular functional activity. The in vivo study did not show any postoperative complications during the 6-month observation period in the model of above-knee amputation in rabbits when SBIP implants, either silver-coated or untreated were inserted into the bone residuum. Three-phase scintigraphy demonstrated angiogenesis in the pores of the pylons. The findings suggest that a silver coating with well-chosen specifications can increase the safety of porous implants for direct skeletal attachment. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 169-177, 2019.

Improved osseointegration properties of hierarchical microtopographic/nanotopographic coatings fabricated on titanium implants.

BACKGROUND: Titanium (Ti) implants are extensively used in reconstructive surgery and orthopedics. However, the intrinsic inertness of untreated Ti implants usually results in insufficient osseointegration. In order to improve the osteoconductivity properties of the implants, they are coated with hierarchical microtopographic/nanotopographic coatings employing the method of molecular layering of atomic layer deposition (ML-ALD). RESULTS: The analysis of the fabricated nanostructured relief employing scanning electron microscopy, atomic force microscopy, and electron spectroscopy for chemical analysis clearly demonstrated the formation of the nanotopographic (<100 nm) and microtopographic (0.1-0.5 µm) titano-organic structures on the surface of the nanograined Ti implants. Subsequent coincubation of the MC3T3-E1 mouse osteoblasts on the microtopographic/nanotopographic surface of the implants resulted in enhanced osteogenic cell differentiation (the production of alkaline phosphatase, osteopontin, and osteocalcin). In vivo assessment of the osseointegrative properties of the microtopographically/nanotopographically coated implants in a model of below-knee amputation in New Zealand rabbits demonstrated enhanced new bone formation in the zone of the bone-implant contact (as measured by X-ray study) and increased osseointegration strength (removal torque measurements). CONCLUSION: The fabrication of the hierarchical microtopographic/nanotopographic coatings on the nanograined Ti implants significantly improves the osseointegrative properties of the intraosseous Ti implants. This effect could be employed in both translational and clinical studies in orthopedic and reconstructive surgery.

Enhanced Osseointegrative Properties of Ultra-Fine-Grained Titanium Implants Modified by Chemical Etching and Atomic Layer Deposition.

An integrated approach combining severe plastic deformation (SPD), chemical etching (CE), and atomic layer deposition (ALD) was used to produce titanium implants with enhanced osseointegration. The relationship between morphology, topography, surface composition, and bioactivity of ultra-fine-grained (UFG) titanium modified by CE and ALD was studied in detail. The topography and morphology have been studied by means of atomic force microscopy, scanning electron microscopy, and the spectral ellipsometry. The composition and structure have been determined by X-ray fluorescence analysis, X-ray diffraction, and X-ray photoelectron spectroscopy. The wettability of the surfaces was examined by the contact angle measurement. The bioactivity and biocompatibility of the samples were studied in vitro and in vivo. CE of UFG titanium in basic (NH4OH/H2O2) or acidic (H2SO4/H2O2) piranha solution significantly enhances the surface roughness and leads to microstructures, nanostructures, and hierarchical micro-/nanostructures on the surfaces. In vitro results demonstrate deterioration of adhesion, proliferation, and differentiation of MC3T3-E1 osteoblasts cell for CE samples as compared to the non-treated ones. Atomic layer deposition of crystalline titanium oxide onto the CE samples increased hydrophilicity, changed the surface composition, and enhanced significantly in vitro characteristics. In vivo experiments demonstrated non-toxicity of the implants. Etching in basic piranha solution with subsequent ALD significantly improved implant osseointegration as compared with the non-modified samples.

Application of the allogenic mesenchymal stem cells in the therapy of the bladder tuberculosis.

Urogenital tuberculosis (TB) often leads to contraction of the bladder, a reduction of the urinary reservoir capacity, and, in the latest stage, to real microcystitis up to full obliteration. Bladder TB Stage 4 is unsuitable for conservative therapy, and cystectomy with subsequent enteroplasty is indicated. In this study, using a model of bladder TB in New Zealand rabbits, the therapeutic efficacy of the interstitial injection of autologous bone-derived mesenchymal stem cells (MSCs) combined with standard anti-TB treatment in the restoration of the bladder function was demonstrated. For analysis of the MSC distribution in tissues, the latter were labelled with superparamagnetic iron oxide nanoparticles. In vitro studies demonstrated the high intracellular incorporation of nanoparticles and the absence of cytotoxicity on MSC viability and proliferation. A single-dose administration of MSCs into the bladder mucosal layer significantly reduced the wall deformation and inflammation and hindered the development of fibrosis, which was proven by the subsequent histological assay. Confocal microscopy studies of the bladder cryosections confirmed the presence of superparamagnetic iron oxide nanoparticle-labelled MSCs in different bladder layers of the treated animals, thus indicating the role of stem cells in bladder regeneration.

Bioactive Coating with Two-Layer Hierarchy of Relief Obtained by Sol-Gel Method with Shock Drying and Osteoblast Response of Its Structure.

In this work, we analyze the efficiency of the modification of the implant surface. This modification was reached by the formation of a two-level relief hierarchy by means of a sol-gel approach that included dip coating with subsequent shock drying. Using this method, we fabricated a nanoporous layer with micron-sized defects on the nanotitanium surface. The present work continues an earlier study by our group, wherein the effect of osteoblast-like cell adhesion acceleration was found. In the present paper, we give the results of more detailed evaluation of coating efficiency. Specifically, cytological analysis was performed that included the study of the marker levels of osteoblast-like cell differentiation. We found a significant increase in the activity of alkaline phosphatase at the initial incubation stage. This is very important for implantation, since such an effect assists the decrease in the induction time of implant engraftment. Moreover, osteopontin expression remains high for long expositions. This indicates a prolonged osteogenic effect in the coating. The results suggest the acceleration of the pre-implant area mineralization and, correspondingly, the potential use of the developed coatings for bone implantation.

Experimental bladder regeneration using a poly-l-lactide/silk fibroin scaffold seeded with nanoparticle-labeled allogenic bone marrow stromal cells.

In the present study, a poly-l-lactide/silk fibroin (PL-SF) bilayer scaffold seeded with allogenic bone marrow stromal cells (BMSCs) was investigated as a potential approach for bladder tissue engineering in a model of partial bladder wall cystectomy in rabbits. The inner porous layer of the scaffold produced from silk fibroin was designed to promote cell proliferation and the outer layer produced from poly-l-lactic acid to serve as a waterproof barrier. To compare the feasibility and efficacy of BMSC application in the reconstruction of bladder defects, 12 adult male rabbits were divided into experimental and control groups (six animals each) that received a scaffold seeded with BMSCs or an acellular one, respectively. For BMSC tracking in the graft in in vivo studies using magnetic resonance imaging, cells were labeled with superparamagnetic iron oxide nanoparticles. In vitro studies demonstrated high intracellular incorporation of nanoparticles and the absence of a toxic influence on BMSC viability and proliferation. Following implantation of the graft with BMSCs into the bladder, we observed integration of the scaffold with surrounding bladder tissues (as detected by magnetic resonance imaging). During the follow-up period of 12 weeks, labeled BMSCs resided in the implanted scaffold. The functional activity of the reconstructed bladder was confirmed by electromyography. Subsequent histological assay demonstrated enhanced biointegrative properties of the PL-SF scaffold with cells in comparison to the control graft, as related to complete regeneration of the smooth muscle and urothelium tissues in the implant. Confocal microscopy studies confirmed the presence of the superparamagnetic iron oxide nanoparticle-labeled BMSCs in newly formed bladder layers, thus indicating the role of stem cells in bladder regeneration. The results of this study demonstrate that application of a PL-SF scaffold seeded with allogenic BMSCs can enhance biointegration of the graft in vivo and support bladder tissue regeneration and function.