RU2657611C1 - Biocompatible nanomaterial for the laser restoration of the integrity of the dissected biological tissues - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to the field of laser medicine and, specifically to reconstructive surgery. Biocompatible nanomaterial for laser restoration of the integrity of dissected biological tissues is described, containing an aqueous dispersion basis of the albumin protein, carbon nanotubes and a medical dye – indocyanine, characterized in that carbon nanotubes are multilayered carbon nanotubes and further contains bovine collagen protein with the following component ratio, mass%: albumin of 15 to 20, multilayered carbon nanotubes of 0.02 to 0.2, medical dye – indocyanine – of 0.005 to 0.01, bovine collagen protein in a concentration of 0.3 to 3, distilled water – the rest.

EFFECT: nanomaterial for laser welding has a high efficiency achieved due to high tensile strength of the laser weld, long shelf life and low cost.

1 cl, 1 dwg, 1 tbl

Description

The invention relates to the field of laser medicine and, in particular, to reconstructive surgery. The invention is intended for laser restoration of the integrity of dissected biological tissues, that is, for laser welding.

Existing traditional methods for restoring tissue integrity using suture materials, staplers, staples, adhesive compositions and other means have several disadvantages [1]. For example, low strength and leakage of the joint (seam).

A new direction in restoring tissue integrity is laser welding. In this case, special biocompatible materials are used - bio-solders, and laser radiation. By laser welding, a strong connection of parts of biological tissues is formed, that is, a so-called laser seam (LS) is created. Laser welding of biological tissues has potential advantages in surgery: tightness of the joint, quick healing of wounds, short time of surgical intervention, low response to foreign bodies, low risk of aneurysm during operations on arteries, reduced likelihood of fibrosis or its complete absence, very low probability of development stenosis.

One of the main and important elements of laser welding is bio-solder. An aqueous dispersion of bovine serum albumin (BSA) is often used as a solder. In the process of laser welding, the liquid form of bio-solder evaporates and a strong BSA seam is formed at the junction of biological tissues.

It is known that bio-solder for laser welding based on an aqueous dispersion of BSA makes it possible to realize tensile strength LS ~ 0.05 kPa (pig skin) [2]. Using the same solder during welding on the dog’s intestines, a laser weld strength of ~ 0.43 kPa was achieved [3]. However, such strength values are unsatisfactory, since they are several orders of magnitude inferior to the strength of the joints obtained by traditional surgical methods [4].

Carbon nanotubes (CNTs) have unique properties: high strength, specific conductivity, thermal conductivity, optical transparency, etc.

Composite nanomaterials, which include CNTs in a small percentage (<2-3%), also acquire indicators that cannot be achieved in other cases. New or substantially improved properties are acquired by nanomaterials under the influence of laser radiation. For example, strong biocompatible nanomaterials are formed under the action of laser radiation on water-protein dispersions containing CNTs [5]. Inside the light (porous) bulk nanomaterial with a BSA matrix and a CNT filler, a carbon nanotube framework is formed under the action of laser radiation, which causes a significant increase in the strength of the resulting nanomaterial. In particular, the BSA / CNT nanomaterial containing less than 0.1 wt. % CNTs obtained using laser technology and nanotechnology have a strength several times higher than BSA. Moreover, in the BSA / CNT nanomaterial, cells can grow and multiply, which contributes to the regeneration of biological tissues [6].

Also, the aqueous dispersion of the composite BSA / CNT nanomaterial can serve as a bio-solder for laser welding of biological tissues. Under the action of laser radiation, a BSA / CNT bio-solder forms a nanotube skeleton in a protein matrix, similar to the formation of the same skeleton in a bulk BSA / CNT composite nanomaterial. Therefore, the obtained LS has the same strength properties as the bulk composite BSA / CNT nanomaterial. In laser welding, a bio-solder composed of BSA and multilayer CNTs (MWCNTs), that is, a bio-solder made of BSA / MWCNTs, allows producing LS with very high values of absolute (~ 1 MPa) and relative strength (25-30%) in vitro [5 ].

Known bio-solder used in the method of laser welding of biological tissues, containing various proteins, including BSA, acting as a binder, as well as fillers from MWNTs and surfactants [7]. Laser welding using the proposed aqueous dispersion of BSA / MWNT bio-solder requires a high radiation power (tens of watts) and the welding process takes a long time (several minutes).

Closest to the proposed invention is bio-solder containing an aqueous dispersion base of albumin protein (bovine serum albumin, BSA), single-walled carbon nanotubes (SWCNTs) and medical dye-indocyanine green (ICC), with the following ratio of components: albumin - 20-25 wt. %, single-walled carbon nanotubes - 0.02 ÷ 0.05 wt. %, green indocyanin - 0.01 wt. %, distilled water - the rest [8]. The proposed bio-solder contains SWCNTs, which, after preparation (within a few hours), quickly aggregate in the form of bundles or agglomerates that are partially decontaminated. Consequently, the aqueous dispersion of the BSA / SWCNT / ICZ composite nanomaterial becomes more transparent for laser radiation (laser radiation is not efficiently absorbed), therefore, it is necessary to increase the total absorbed radiation energy and the time of the laser welding process.

The objective of the invention is to increase the bond strength of dissected biological tissues, increase the storage time of the nanomaterial, as well as reduce its cost.

The indicated technical problem is solved in that a biocompatible nanomaterial containing an aqueous dispersion base — albumin protein, carbon nanotubes, a medical dye — Indocyanin green, additionally contains bovine collagen protein in a concentration in the range from 0.3 to 3 wt. % The essence of the invention lies in the fact that during laser welding under the influence of laser radiation on bio-solder, a strong structure (nanocomposite material) of the laser weld is formed during the evaporation of the liquid component of the material. At the same time, a framework of carbon nanotubes is formed in a nanostructured protein matrix. The addition of collagen to the protein composition along with other components increases the strength of the laser seam and reduces the cost of bio-solder, and the addition of MWCNTs increases its shelf life.

The formed nanocomposite skeleton is an analogue of a natural biological matrix in which the conditions of self-organization of cellular material of the restored biological tissue can be provided.

Laser welding of dissected biological tissues using the proposed invention is carried out by stepwise moving the laser beam along the surface of the biological tissue pretreated with bio solder from the proposed nanomaterial, or stepwise moving the pretreated biological tissue perpendicular to the direction of the laser beam.

Consider an example of bio-solder preparation and laser welding. The first stage is as follows:

- MWCNTs are added to distilled water at a concentration of 0.02-0.2 wt. % Next, the resulting solution is stirred using a magnetic stirrer for 15-30 minutes.

- The resulting solution is placed in an ultrasonic homogenizer at a power of 25 to 33 W, a temperature of ≤60 ° C and a homogenization time of 30 to 90 minutes.

- BSA is added portionwise to a homogenized aqueous solution of MWCNTs in a calculation of a concentration of 15-20 wt. %

- In an aqueous solution of BSA / MWCNT add collagen in the calculation of the concentration of 0.3-3 wt. %

- Indocyanin green is added to the resulting solution in the calculation of the concentration of 0.005-0.01 wt. %

- The resulting solution is stirred on a magnetic stirrer for 2-2.5 hours

At the second stage, bio-solder is applied to the area of dissected biological tissue. For this, microsyringes or a specialized biomaterial feeding (dosing) system are used. The solder consumption per weld length is approximately 1 ml / cm.

At the third stage, the edges of the dissected biological tissue are fixed as close as possible relative to each other. For this, tightening structures and / or surgical forceps are used.

In the fourth stage, a laser effect occurs on the area of the dissected biological tissue. For this, a laser radiation source with a wavelength in the range from 800 to 820 nm is used. The power of laser radiation is in the range of 2-3 watts. The exposure process is carried out by stepwise moving the laser beam along the surface of the biological tissue or stepwise moving the biological tissue perpendicular to the direction of the laser beam. The generation mode is continuous.

Table 1 shows the results of measurement (in vitro) of the tensile strength of the seam obtained by laser welding using the example of cartilaginous tissue of beef trachea. Here are the absolute tensile strength and relative tensile strength, defined as the ratio of LS strength to the strength of whole cartilage tissue. It is seen that with an increase in the concentration of collagen, the strength of the laser seam increases. This is due to the fact that collagen is a much stronger material than BSA; therefore, when it is partially replaced by collagen in an aqueous dispersion of bio-solder, the strength of the laser seam increases.

However, in BSA / MWCNT / ICZ / collagen bio-solder at collagen concentrations of more than 3 wt. % LS strength is deteriorating. In fact, collagen is a poorly soluble material in water, and at a high concentration (> 3 wt.%) In the dispersion accumulations of collagen fibers form. As a result, the dispersion loses homogeneity. It is not possible to homogenize it again using an ultrasonic (ultrasound) dispersant, because under the influence of the ultrasonic dispersant in the dispersion, protein denaturation occurs and bio-solder loses its important functional property: to promote cell multiplication, in addition, the uniformity of its structure is impaired and mechanical properties are deteriorated. In BSA / MWCNTs / ICZ / collagen at concentrations ≤0.3 wt. % of collagen, the effect of the latter is imperceptible due to a slight change in the total composition of proteins: BSA + collagen.

With an increase in the concentration of MWCNTs and ICZ in the dispersion of bio-solder, the strength of the laser weld is correlated. For example, in the case of an aqueous dispersion of bio-solder in the composition of 20 wt. % BSA / 0.2 wt. % MWCNTs / 0.01 wt. % ICZ / 3 wt. % collagen, for LH the absolute tensile strength of ~ 1.77 MPa is realized, the relative tensile strength of ~ 52% with the absolute tensile strength of ~ 3.4 MPa for continuous cartilage of beef trachea. It can be seen that this figure is 30% higher in comparison with the relative tensile strength of the laser seam achieved in the prototype.

In FIG. Figure 1 shows a typical laser weld pattern obtained with a JEOL JSM-6010 scanning electron microscope. It can be seen that the seam looks like a uniform strip connecting the two parts of the fabric. It is continuous, hermetically connects tissues and has a width of about 35-40 microns. In some cases, the width of the laser seam reached 80 μm.

Thus, in the case of adding collagen to the biosolders of the BSA / MWCNT / ICZ composition during laser welding, the mechanical tensile strength of the laser seam increases, that is, high bond strength of biological tissues is ensured.

Important advantages of bio-solder made by the proposed method, relative to the known materials and prototype are [3, 4, 6, 7]:

- its matrix consists of biological materials (albumin protein, collagen) and of fillers in the form of multilayer carbon nanotubes and the medical dye Indocyanin green, and, in general, the nanomaterial is biocompatible;

- the degree of safety of the proposed bio-solder-nanomaterial is higher, since it includes MWCNTs, and the bio-solder-nanomaterial considered in the prototype contains SWCNTs, and its degree of safety is lower due to the high activity of SWCNTs;

- with the composition of 20 wt. % BSA / 0.2 wt. % MWCNTs / 0.01 wt. % ICZ / 3 wt. % collagen for cartilage tissue realized the relative strength of the laser suture ~ 52%, which is higher than that achieved in the prototype (~ 30%);

- when storing the proposed BSA / MWCNT / ICZ / collagen bio-solder for one week at room temperature (20-25 ° С) or in a household refrigerator (4 ° С) for one month its properties practically do not change, while bio-solder , considered in the prototype, significantly loses its properties when stored at room temperature for more than 2 hours;

- in the proposed bio-solder, the proportion of BSA was reduced by replacing it with collagen, and SWCNTs were replaced by MWCNTs, which significantly reduces the cost of bio-solder, considered in the prototype, due to the high cost of BSA by more than an order of magnitude unlike collagen, and also because of the high cost of SWCNTs relative to MWCNT;

- the laser seam formed using the proposed bio-solder is homogeneous and airtight, has a small width of ~ 35-80 microns, and after reunification (healing) of the tissue parts is practically not noticeable.

The advantage of bio-solder for laser welding obtained by the proposed method is its high efficiency, achieved due to the high tensile strength of the laser weld, a high degree of safety of carbon nanotubes, a long shelf life and low cost of nanomaterial.

Thus, the problem posed in this patent application has been solved. A method for the preparation of bio-solder for laser welding based on the biological material of albumin, collagen and filler from multilayer carbon nanotubes and the medical dye Indocyanin green is proposed. Bio-solder, which is a nanomaterial, is biocompatible.

Information sources

1. Lasers in surgery. Ed. O.K. Skobelkin. M .: Medicine, 1989 .-- 256 p.

2. Forer B., Vasilyev T., Brosh T., et al. Lasers in Surgery and Medicine, vol. 9999, 1 (2005).

3. Simhon D., Halpern M., Brosh T., and et al. Annals of surgery, vol. 245 (2), 206-213 (2007).

4. Hench L., Jones D. Biomaterials, artificial organs, and tissue engineering. M .: Technosphere. 2007 .-- 304 s.

5. Gerasimenko A.Yu., Ichkitidze L. P., Podgaetsky V. M., Ponomareva O. V., Selishchev S. V. // News of universities. Electronics, 2010. No. 4. S. 33-41.

6. Bobrinetskiy I., Gerasimenko A., Ichkitidze L. et al. American Journal of Tissue Engineering and Stem Cell, 1 (1), 27-38 (2014).

7. RF patent No. 2425700.

8. RF application for invention No. 2016150636, decision on the grant of a patent dated 09.21.2017 (prototype).

Claims (2)

A biocompatible nanomaterial for laser restoration of the integrity of dissected biological tissues, containing an aqueous dispersion base of albumin protein, carbon nanotubes and a medical dye, Indocyanine, characterized in that multilayer carbon nanotubes are used as carbon nanotubes and additionally contains bovine collagen protein in the following ratio of components, wt. %: albumen from 15 to 20 multilayer carbon nanotubes from 0.02 to 0.2 medical dye - indocyanin from 0.005 to 0.01 collagen protein in concentration from 0.3 to 3 distilled water rest

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