RU2711544C1 - Biomaterial for making prostheses of heart valves and method of producing biomaterial - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine and discloses a biomaterial for the manufacture of heart valve prostheses and a method for preparing it. Biomaterial includes bovine pericardium coated with nanodiamonds of detonation synthesis, having positive electrokinetic potential in water, with content of nanodiamonds of 1.5 to 4 mg per gram of biological tissue, with average size of aggregates from 17 to 28 nm on surface of biomaterial.

EFFECT: biomaterial is characterized by high strength and wear resistance and is intended for improving mechanical properties of materials for making artificial heart valves.

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Description

Technical field

The invention relates to medicine, namely to the production of biological prostheses of heart valves, preferably based on bovine pericardium for cardiovascular surgery with improved mechanical properties.

State of the art

Xenogenic biological bovine pericardial heart valve prostheses are widely used in world cardiac surgery (Zilla, P., Brink, J., Human, P., & Bezuidenhout, D. (2008). Prosthetic heart valves: Catering for the few. Biomaterials, 29 (4), 385-406). To prevent a pronounced immune response and increase the mechanical strength of the biological tissue, the collagen matrix of the bioprosthesis is stabilized with glutaraldehyde (Manji, RA, Zhu, LF, Nijjar, N.K., Rayner, D.C., Korbutt, GS, Churchill, TA, Ross, DB (2006) Glutaraldehyde-Fixed Bioprosthetic Heart Valve Conduits Calcify and Fail From Xenograft Rejection. Circulation, 114 (4), 318-327), but it is the free aldehyde groups of glutaraldehyde that are not bound to the protein structure of the collagen matrix that are the centers of initiation of calcification (Sinha, P., Zurakowski, D., Susheel Kumar, T.K., Ne, D., Rossi, C, & Jonas, R.A. (2012). Effects of glutaraldehyde concentration, pretreatment time, and type of tissue (porcine versus bovine) on postimplantation calcification. The Journal of Thoracic and Cardiovascular Surgery, 143 (1), 224-227). It was proposed to use chitosan coatings to bind free aldehyde groups (Gallyamov, M.O., Chaschin, IS, Khokhlova, M.A., Grigorev, T.E., Bakuleva, NP, Lyutova, I G., Khokhlov, AR (2014 ). Collagen tissue treated with chitosan solutions in carbonic acid for improved biological prosthetic heart valves. Materials Science and Engineering: C, 37 (1), 127-140).

In particular, a prior art method for modifying the surface of a bovine pericardium with chitosan from water saturated with carbon dioxide at high pressure is known (Patent RU 2519219). The method allows to increase the efficiency and duration of functioning of the biological prosthesis of the heart valve in the body by eliminating the possibility of the formation of calcium precipitate on its surface, to improve elasticity, to strengthen antimicrobial properties. However, the material described in the prototype is characterized by low strength.

The problem of creating a new generation of biological prostheses with a significantly increased service life (of the order of several tens of years) is a non-trivial task, the solution of which will allow the installation of prosthetic heart valves, which, on the one hand, will best reproduce the work of their own human valve and will not require lifelong anticoagulant therapy , unlike mechanical prostheses, and on the other hand, will reduce the risk of the need for repeated operations to replace them, which is important for patients ntov all age groups, but especially for the elderly, for whom there is a high risk of complications in revision surgeries.

Biological tissue from a bovine pericardium for manufacturing biological heart valves and a method for treating bovine pericardium, which increase the biocompatibility of biological tissue and its service life (RU 2456023), are known in the prior art, while the abovementioned advantages are provided due to the physical plasma treatment of the tissue using plasma gas a method in which nitrogen, hydrogen, argon or, preferably, oxygen is used as the ionized gas. Before the plasma gas treatment, the fabric is dried, a biocompatible metal-containing coating is applied to the plasma-treated fabric, and after the plasma treatment, the dried implant is exposed to moisture. The metal-containing coating is applied by chemical vapor deposition or gas deposition in a plasma medium. The metal-containing coating is selected from the group consisting of Ti, Ta, Nb, Zr, Hf, Ir, Au, Pd, Pt, Ag and Cu.

The application of metal coatings to biological tissues using pulsed laser spraying is described in US 2012/0221099 Al (prototype). The possibility of coating amorphous and diamond-like carbon on biological tissue using an appropriate medium is also described there. Using this approach, biological tissue is placed in a special vacuum chamber for pulsed laser spraying, where there is a metal target that is irradiated with a laser beam. In the case of applying carbon coatings, the process is two-stage and includes an additional stage of applying a hydrocarbon layer using a plasma process. As the process continues, the hydrogen content in the plasma polymer decreases until carbon is present in the form of diamond. This method of applying metal coatings to biological tissues is quite complex and requires special equipment. The application text does not contain data on the thickness of the applied coating, which are important in determining the mechanical properties of the material and the stability of the coating on the surface of the biological tissue.

Disclosure of Invention

The technical problem solved by the invention is the creation of biomaterial for the manufacture of prosthetic heart valves based on bovine pericardium and a method for producing biomaterial devoid of the disadvantages of the above analogues, and characterized by high strength and wear resistance.

The technical result, to which the claimed invention is directed, is to obtain biomaterial with controlled mechanical properties while maintaining elasticity, biocompatibility and antimicrobial properties while simplifying the method for its preparation.

The technical result is achieved by applying to the surface of the bovine pericardium (pericardium of the biological prosthesis of the heart valve) a nanodiamond coating (film) from 1.5 to 4 mg of nanodiamonds per gram of bovine pericardium tissue, with an average aggregate size of nanodiamonds from 17 to 28 nm, thereby increasing the strength of the material , compared to an unmodified sample. The application of the nanodiamond film is carried out from an aqueous suspension with a concentration of nanodiamonds of 1-1.5 g / l and the size of nanodiamond aggregates from 50 to 100 nm on the pericardial surface of the biological prosthesis of the heart valve. Detonation synthesis nanodiamonds are used (see, for example, Ultrananocrystalline Diamond. Synthesis, Properties, and Applications. 2006. Ed. O.A. Shenderova, D.M. Gruen), which have a positive value of the electrokinetic potential in water. Thus obtained material has a tensile strength of 9-10 MPa, an initial Young's modulus of about 1 MPa and a final Young's modulus of about 30 MPa.

In one embodiment, a layer of chitosan in an amount of 20 mg / g may be applied to the surface of the nanodiamond.

The technical result is also achieved through the implementation of a method for producing biometrics, including:

- preparation of an aqueous suspension of detonation synthesis nanodiamonds with a nanodiamonds concentration of 1-1.5 g / l from nanodiamonds powder by ultrasonic treatment, followed by evaporation of the suspension to a dry residue and resuspension in water with a nanodiamonds concentration of 1-1.5 g / l using ultrasonic processing;

- placement of bovine pericardium in suspension, stirring for 7-8 hours, keeping at 4 ° C for 10-12 hours;

- extraction of bovine pericardium from suspension, washing with physiological saline;

- storage in physiological saline at 4 ° C.

Ultrasonic processing is carried out for 2-3 hours at an ultrasonic power of 110 W and an operating frequency of 35 kHz. Mixing a suspension of nanodiamonds with a bovine pericardium placed in it is carried out on an orbital shaker at a speed of 190 rpm at room temperature of 20-25 ° C. If it is necessary to obtain biomaterial with a chitosan coating, the latter is applied to a nanodiamond from a solution of 1 g / l in 0.01 N hydrochloric acid, for which a suspension of nanodiamonds is mixed with a solution of chitosan, the resulting suspension is incubated for 48 hours at room temperature (20-25 ° C), then centrifuged for 30 minutes at 13400 rpm, the precipitate is decanted, washed with water and suspended in water to a concentration of 1-1.5 g / L.

The thickness of the deposited nanodiamond film was determined using the method of radioactive indicators. The obtained value is optimal for obtaining the claimed mechanical properties because it provides a complete uniform stable coating of the surface of the biological tissue.

Specified in the claimed method, the point values of the parameters during the implementation of the invention may have an allowable deviation of no more than 10%.

Brief Description of the Drawings

The invention is illustrated by drawings, where in FIG. 1 shows micrographs of the surface of a matrix coated with nanodiamonds: smooth side (left), rough side (right); in FIG. 2 - dependence of the strain on the stress of the matrix modified by nanodiamonds and the control sample; in FIG. 3 - micrographs of the surface of the matrix coated with chitosan-modified nanodiamonds: smooth side (left), rough side (right); in FIG. 4 - dependence of the strain on the stress of the matrix modified by a nanodiamond with adsorbed chitosan and a control sample.

The implementation of the invention

The present invention is illustrated by examples that do not limit the essence of the proposed solution, while clearly demonstrate the possibility of achieving the claimed technical result. In particular, the possibility of implementing the invention has been demonstrated when using a bovine pericardium as a collagen matrix, and it is also possible to use tissues of other animals used to make valves.

The preparation of nanodiamonds is carried out as follows. A suspension of nanodiamonds is prepared, for which a powder of nanodiamonds is suspended in water (a concentration of suspension nanodiamonds of 5 g / l to achieve the best separation of aggregates) using ultrasonic treatment for 2-3 hours at an ultrasonic power of 110 W (operating frequency 35 kHz). Then the suspension is kept at 4 ° C for 12-24 hours to precipitate large aggregates. After that, the supernatant suspension is removed, dried using a rotary evaporator, the amount of solid phase is determined gravimetrically and resuspended in deionized water to a final concentration of 1-1.5 g / l using ultrasonic treatment under similar conditions (for 2-3 hours, power ultrasound 110 W, operating frequency 35 kHz). The resulting suspension is characterized by an average aggregate size not exceeding 100 nm, according to the data of dynamic light scattering and electrokinetic potential from +20 to 40 mV. Two-stage ultrasonic treatment allows one to obtain a suspension of nanodiamonds with water with the above characteristics without using an immersion ultrasound source, which, firstly, deteriorates upon contact with the nanodiamonds and, secondly, its particles contaminate the resulting suspension.

To further improve the mechanical properties of the obtained biomaterial, a layer of chitosan is applied to the nanodiamond from a solution of 1 g / l in 0.01 n hydrochloric acid. A suspension of nanodiamonds is mixed with a solution of chitosan. The suspension thus obtained is incubated for 48-72 hours at room temperature (20-25 ° C), then centrifuged until the nanodiamond phase is completely precipitated (30 minutes at 13,400 rpm), the precipitate is decanted, washed with water and suspended in water until the concentration of the solid phase is 1-1.5 g / l. The resulting suspension is characterized by an average aggregate size not exceeding 100 nm, according to the data of dynamic light scattering and electrokinetic potential from +35 to +45 mV.

The pericardium of the biological prosthesis of the heart valve is placed in an aqueous suspension of nanodiamonds or nanodiamonds with a chitosan coating, mixed on an orbital shaker at a speed of 190 rpm at room temperature (20-25 ° C) for 7-8 hours, then kept at 4 ° C for 10-12 hours. Then the biomaterial is removed from the suspension and washed with saline. The resulting product is stored in physiological saline. Since a nanodiamond and a nanodiamond with a chitosan coating have a positive electrokinetic potential, the resulting film uniformly covers both the smooth and rough surfaces of the pericardium of the biological prosthesis of the heart valve and is retained on its surface due to electrostatic interaction. The obtained biomaterial is characterized by an initial Young's modulus of about 1 MPa and a final Young's modulus of about 30 MPa, as well as the starting material, while the tensile strength increases to 9-10 MPa compared to 6 for the starting material.

Example 1. The pericardium of a biological prosthesis of the heart valve in the form of a matrix with an area of 30 cm ² and a thickness of 0.4-0.5 mm, pre-treated with 0.625% glutaraldehyde solution and several times thoroughly washed with sterile physiological saline with a 6-fold change of solution at a rate of 500 -550 ml per 100 g of pericardium was placed in an aqueous suspension of nanodiamonds with a concentration of 1 mg / ml. To determine the amount of nanodiamond on the surface, tritium-labeled nanodiamonds were used. The suspensions were mixed on an orbital shaker at room temperature (20-25 ° C) for 7 hours, then the suspension with the matrix was kept at 4 ° C for 12 hours. Then the matrix was removed from the suspension and washed with saline. The resulting product was stored in physiological saline. In FIG. 1 shows a micrograph of a matrix with a deposited nanodiamond.

The size of aggregates on the matrix surface averaged 17 nm. To determine the number of nanodiamonds on the matrix surface, it was decomposed with nitric acid and the radioactivity was measured. It was found that the total number of nanodiamonds on the matrix surface is 4 mg per gram matrix.

In FIG. Figure 2 shows the strain dependence of stress for a material modified with a nanodiamond as compared to a control sample — a collagen matrix modified with glutaraldehyde.

As follows from FIG. 2 nanodiamond coating gives the collagen matrix extra strength. If the values of the initial and final Young's moduli are the same for both samples and are 1 MPa and 20 MPa, respectively, then the tensile strength of the nanoamalc coated sample increases to 9 MPa compared to 6 MPa for the untreated sample.

Example 2. In an aqueous suspension of chitosan-coated nanodiamonds with a nanoparticle concentration of 1 mg / ml, collagen matrices processed as described in Example 1 were placed. Tritium-labeled nanodiamonds were used to determine the amount of nanodiamonds on the surface. The suspensions were mixed on an orbital shaker at room temperature (20-25 ° C) for 7 hours, then the suspension with the matrix was kept at 4 ° C for 12 hours. Then the matrix was removed from the suspension and washed with saline. The resulting product was stored in physiological saline. In FIG. Figure 3 shows a micrograph of a matrix with a deposited nanodiamond.

The size of aggregates on the surface of the matrices averaged 28 nm. To determine the number of nanodiamonds on the matrix surface, it was decomposed with nitric acid and the radioactivity was measured. It was found that the total number of nanodiamonds on the matrix surface is 1.5 mg per gram matrix.

In FIG. Figure 4 shows the strain dependence of stress for a material modified with a nanodiamond modified with chitosan, compared with a control sample — a collagen matrix modified with glutaraldehyde.

As follows from FIG. 4 nanodiamond coating gives the collagen matrix extra strength. If the values of the initial and final Young's moduli are the same for both samples and are 1 MPa and 20 MPa, respectively, then the tensile strength of the nanoamalz coated sample increases to 10 MPa compared to 6 MPa for the untreated sample.

Varying the mixing time of the suspension of nanodiamonds and bovine pericardium and incubation of the suspension at 4 ° C in the range of parameters given in the part "Implementation of the invention" leads to similar results in terms of the mechanical properties of the product.

Claims (10)

1. Biomaterial for the manufacture of prosthetic heart valves, including bovine pericardium coated with detonation synthesis nanodiamonds with positive electrokinetic potential in water, with a nanodiamond content of 1.5 to 4 mg per gram of biological tissue, with an average aggregate size of 17 to 28 nm per biomaterial surface. 2. The biomaterial according to claim 1, characterized in that it has a tensile strength of 9-10 MPa, an initial Young's modulus of about 1 MPa, and a final Young's modulus of about 30 MPa. 3. The biomaterial according to claim 1, characterized in that a layer of chitosan in an amount of 20 mg / g is deposited on the surface of the nanodiamond. 4. The method of obtaining biomaterial according to claim 1, including - preparation of an aqueous suspension of detonation synthesis nanodiamonds with a nanodiamonds concentration of 1-1.5 g / l from nanodiamonds powder by ultrasonic treatment, followed by evaporation of the suspension to a dry residue and repeated resuspension in water with a nanodiamonds concentration of 1-1.5 g / l s using ultrasonic treatment; - placement of bovine pericardium in suspension, stirring for 7-8 hours, keeping at 4 ° C for 10-12 hours; - extraction of bovine pericardium from suspension, washing it with physiological saline, followed by storage in physiological saline. 5. The method according to p. 4, characterized in that the ultrasonic treatment is carried out for 2-3 hours at an ultrasonic power of 110 W and an operating frequency of 35 kHz. 6. The method according to p. 4, characterized in that the mixing of the suspension of nanodiamonds with the bovine pericardium placed in it is carried out on an orbital shaker at a speed of 190 revolutions / min at room temperature of 20-25 ° C. 7. The method according to claim 4, characterized in that chitosan is applied to the nanodiamond from a solution of 1 g / l in 0.01 N hydrochloric acid, for which a suspension of nanodiamonds is mixed with a solution of chitosan, the resulting suspension is incubated for 48 hours at room temperature (20 -25 ° C), then centrifuged for 30 minutes at 13400 rpm, the precipitate is decanted, washed with water and suspended in water to a concentration of 1-1.5 g / L.

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