Tritium-Labeled Nanodiamonds as an Instrument to Analyze Bioprosthetic Valve Coatings: A Case of Using a Nanodiamond Containing Coating on a Pork Aorta.

Coatings with xenogenic materials, made of detonation nanodiamonds, provide additional strength and increase elasticity. A functionally developed surface of nanodiamonds makes it possible to apply antibiotics. Previous experiments show the stability of such coatings; however, studies on stability in the bloodstream and calcification of the material in natural conditions have yet to be conducted. Tritium-labeled nanodiamonds (negative and positive) were obtained by the tritium activation method and used to develop coatings for a pork aorta to analyze their stability in a pig's bloodstream using a radiotracer technique. A chitosan layer was applied from a solution of carbonic acid under high-pressure conditions to prevent calcification. The obtained materials were used to prepare a porcine conduit, which was surgically stitched inside the pig's aorta for four months. The aorta samples, including nanodiamond-coated and control samples, were analyzed for nanodiamond content and calcium, using the radiotracer and ICP-AES methods. A histological analysis of the materials was also performed. The obtained coatings illustrate a high in vivo stability and low levels of calcification for all types of nanodiamonds. Even though we did not use additional antibiotics in this case, the development of infection was not observed for negatively charged nanodiamonds, opening up prospects for their use in developing coatings.

Coating based on chitosan/vancomycin nanoparticles: Patterns of formation in a water-carbon dioxide biphase system and in vivo stability.

The patterns of formation of chitosan nanoparticles doped with vancomycin and coatings based on them in carbonate solutions have been investigated for the first time in this study. Using a technique of radioactive indicators, it was found that at a CO2 pressure of 30 MPa, the yield of the nanoparticles was ∼85 %, and a maximum antibiotic encapsulation efficiency of ∼30 % was achieved. By spectrophotometric and high-resolution microscopy, it was found that the coating of stabilized xenopericardial tissue of bioprosthetic heart valve, based on chitosan nanoparticles doped with vancomycin with a zeta potential |ζ| ∼20 mV completely covers collagen fibers by depositing about 60 nm nanoparticles onto them under direct deposition from carbonic acid at a pressure of 30 MPa CO2. The coating preserves the mechanical strength characteristics of collagen tissue and completely suppresses the growth of S. aureus pathogenic biofilm. This is consistent with the observed increase in antibiotic release of 15 % when the medium was acidified. Histological study demonstrated that the structure of pericardial tissues was not significantly altered by the deposition nanoparticles from carbonic acid. It was found that the rate of biodegradation of polymers and vancomycin in the coating differs by half (16 weeks for the rat model). A significantly lower degradation rate of antibiotics (∼50 % of vancomycin total remaining mass and ∼25 % of chitosan) was associated with its reliable encapsulation into nanoparticles.

Chitosan/hyaluronic acid polyanion bilayer applied from carbon acid as an advanced coating with intelligent antimicrobial properties for improved biological prosthetic heart valves.

The tightly bonded shielding coating on biomatrix significantly enhances the functionality of medical devices, bioprostheses in particular. In our work we have obtained a polyelectrolyte coating on a biomatrix by sequentially depositing chitosan and hyaluronic acid (HA) from solutions in carbonic acid under pressure. This approach makes it possible to obtain hybrid biomatrix with a firmly bonded polymer screen due to the electrostatic bonding of polyions. High-precision analysis using a tritium label shows a 3-fold increase in quantity of HA in carbonic acid under pressure compared to the conventional method. The presence of the chitosan layer increases the HA adsorption by 15-20 % due to electrostatic interaction of differently charged polymers. Antimicrobial results show the possibility of implementing an induced antimicrobial response, due to the lysis of the upper layer of the coating (HA) and the release of antimicrobial agents in the case of growth of pathogens on the bioprosthesis.

Structural and mechanical characteristics of collagen tissue coated with chitosan in a liquid CO2/water system at different pressures.

Chitosan coatings of biological heart-valve prostheses enhance their biocompatibility, resistance to pathogenic microflora and lifetime. Collagen tissues can be coated with chitosan in aqueous solution acidified, to make chitosan soluble, with H2CO3 formed from a coexisting liquid CO2 phase under pressure. The advantage of H2CO3 is that it can be easily removed after the coating procedure. This study assessed the effects of 6-50 MPa CO2 pressure during the coating procedure on the structure and mechanical properties of the resulting biocomposite matrices. The dependence of chitosan adsorption on CO2 pressure was bell-shaped, reaching a maximum adsorption of 0.8 mass % at 40 MPa. Tissue surface became highly porous upon pressure treatment. At 50 MPa, the pores merged to form furrows with lengths of several hundred micrometers, accompanied by collagen fibril reorganisation. Chitosan coating did not affect tissue tensile strength in the axial direction, but increased it by 75% in the radial direction in the tissue coated at 50 MPa pressure. Strain at break, a measure of elasticity, increased in both directions by up to 100% upon coating with chitosan. CO2 pressure of 30-50 MPa seems thus optimal in terms of chitosan incorporation and tissue mechanical properties.

Chitosan coatings with enhanced biostability in vivo.

In this article, we study the stability of chitosan coatings applied on glutaraldehyde-stabilized bovine pericardium when exposed to biodegradation in vivo in the course of model subcutaneous tests on rats. The coatings were deposited from carbonic acid solutions, that is, H2 O saturated with CO2 at high pressure. Histological sections of treated pericardium samples demonstrated that the structure of pericardial connective tissues was not significantly altered by the coating application method. It was revealed that the dynamics of biodegradation depended on the total mass of chitosan applied as well as on the DDA of chitosan used. As long as the amount of chitosan did not exceed a certain threshold limit, no detectable degradation occurred within the time of the tests (12 weeks for the rat model). For higher chitosan amounts, we detected a ∼20% reduction of the mass after the in vivo exposition. The presumed mechanism of such behavior is discussed. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 270-277, 2018.

Collagen tissue treated with chitosan solution in H2O/CO2 mixtures: Influence of clathrates hydrates on the structure and mechanical properties.

A mixture of water/carbon dioxide is a "green" perspective solvent from the viewpoint of biomedical applications. Clathrate hydrates are formed this solvent under certain conditions and a very interesting question is the impact of clathrates hydrates on the structure and properties of bovine pericardium, which is used in biomedicine, in particular as a main part of biological heart valve prostheses. The aim of the present work is to investigate the influence of clathrates on the structure and mechanical properties of the collagen tissue treated with chitosan in H2O/CO2 mixtures under pressure 3.0-3.5MPa and temperatures 2-4°C. It was first found that the clathrate hydrates in this media due to the strong fluctuations "bomb" collagen tissue of bovine pericardium, which is manifested in the appearance of numerous small gaps (pores) with mean size of 225±25nm and large pores with size of 1-3µ on the surface and within collagen matrices. High porosity leads to averaging characteristics of the organization structure in tissues with different orientation of the collagen fibers. As a result, the mechanical properties of the collagen tissue with a different orientation of the collagen fibrils become similar, which is quite different from their original properties. The structural changes caused by the influence of the environment clathrate hydrates led to a significant decrease of the tensile strength (30-47% in total, p<0.05) and initial elastic moduli (74-83%, p<0.05). However, the final elastic moduli and the maximum tensile virtually unchanged compared to the control. Nevertheless, it was found that the direct deposition of chitosan from the H2O/CO2 mixtures with clathrate improve the mechanical-strength properties of the porous matrices. We believe that these improved mechanical properties are achieved due to particularly deep and uniform impregnation of the collagen matrix with chitosan from its pressurized solutions in H2O/CO2 mixtures.

Collagen tissue treated with chitosan solutions in carbonic acid for improved biological prosthetic heart valves.

Calcification of bovine pericardium dramatically shortens typical lifetimes of biological prosthetic heart valves and thus precludes their choice for younger patients. The aim of the present work is to demonstrate that the calcification is to be mitigated by means of treatment of bovine pericardium in solutions of chitosan in carbonic acid, i.e. water saturated with carbon dioxide at high pressure. This acidic aqueous fluid unusually combines antimicrobial properties with absolute biocompatibility as far as at normal pressure it decomposes spontaneously and completely into H2O and CO2. Yet, at high pressures it can protonate and dissolve chitosan materials with different degrees of acetylation (in the range of 16-33%, at least) without any further pretreatment. Even exposure of the bovine pericardium in pure carbonic acid solution without chitosan already favours certain reduction in calcification, somewhat improved mechanical properties, complete biocompatibility and evident antimicrobial activity of the treated collagen tissue. The reason may be due to high extraction ability of this peculiar compressed fluidic mixture. Moreover, exposure of the bovine pericardium in solutions of chitosan in carbonic acid introduces even better mechanical properties and highly pronounced antimicrobial activity of the modified collagen tissue against adherence and biofilm formation of relevant Gram-positive and Gram-negative strains. Yet, the most important achievement is the detected dramatic reduction in calcification for such modified collagen tissues in spite of the fact that the amount of the thus introduced chitosan is rather small (typically ca. 1wt.%), which has been reliably detected using original tritium labelling method. We believe that these improved properties are achieved due to particularly deep and uniform impregnation of the collagen matrix with chitosan from its pressurised solutions in carbonic acid.