RU2642259C2 - Tissue-engineering biodegradable vascular implant - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: made of biodegradable polymers by electrospinning with layer-by-layer introduction of biologically active molecules into the vascular wall: vascular endothelial growth factor (VEGF), fibroblast growth factor (bFGF) and chemoattractant molecule (SDF-1α). The polymer composition is a mixture of polyhydroxybutyrate-valerate and polycaprolactone (PGBV/PCL), taken in the ratio of 1:2. Electrospinning is performed with the participation of a solution containing a polymer composition with VEGF molecules, and then a solution of polymers with an aqueous fraction of biologically active molecules - bFGF and SDF-1α. The invention can be used in cardiovascular surgery for reconstructive operations on vessels.

EFFECT: tissue-engineering graft fabricated by this method corresponds to the layered structure of the vessel wall of a living organism while preserving the vitality and functional activity of biologically active components before the degradation of the polymer matrix.

3 cl, 1 tbl, 2 ex

Description

The invention relates to the field of medicine and tissue engineering and can be used in cardiovascular surgery when performing reconstructive operations on the vessels.

In recent years, research has been actively conducted for the needs of vascular surgery, aimed at creating tissue-engineered vascular grafts "ready to use" that can support cell viability and function in the body. The use of matrices, or so-called substrates, from natural or synthetic polymers has already led to significant success in tissue engineering of blood vessels. An important characteristic of such substrates is the porosity of the material, which promotes cell migration, signal transmission, nutrient delivery, and removal of metabolic products. In addition, the matrices used provide mechanical support to the cells forming the tissue of the future vessel.

To date, the most promising method is the manufacture of vascular matrices by electrospinning (electrospinning) from biocompatible bioresorbable polymer compositions. Electrospinning makes it possible to obtain nanofibers from a wide variety of materials (polymers, composites, semiconductors, metals, and even ceramics), forming highly porous vascular grafts of different diameters and with different strength characteristics [Hasan A, Memic A, Annabi N et al. Electrospun scaffolds for tissue engineering of vascular grafts. Acta Biomater .; 10, 11-25 (2014)].

It is known that a full-fledged tissue-engineered blood vessel should consist of three layers: functional endothelium; copper formed by smooth muscle cells; as well as adventitia formed by fibroblasts. At the same time, biologically active molecules are stimulators of the formation of new tissues in the polymer framework. In tissue engineering of blood vessels, growth factors have found their application, affecting the functions of fibroblasts, as well as endothelial and smooth muscle cells. Growth factors are able to regulate cell migration, their proliferation, differentiation, apoptosis and dedifferentiation. [Briggs T., Arinzeh T.L. Growth factor delivery from electrospun materials // J. Biomater. Tissue Eng. - 2011 .-- Vol. 1, No. 2. - P. 129-138.].

However, growth factors are unstable and have a short half-life as a result of proteolytic degradation, which makes their introduction into the systemic circulation ineffective and leads to the need for delivery to the tissue regeneration site.

A synthetic vascular prosthesis is known that contains biologically active molecules and extracellular matrix proteins, while polylactic acid, a copolymer of polylactic and polyglycoic acid, polycaprolactone and mixtures thereof are proposed to be used as structure-forming polymers (Appl. US 2010/0125330 A1: Int. Cl. A61F 2 / 82, B05D 7/00. Synthetic vascular prosthesis and method of preparation / Bronislava G. Belenkaya (US); Edward C. Kwok (US); appl.no. 12/620403, filed 11.17.2009; pub. Date 20.05. 2010.). When performing electrospinning, at least one additional ingredient, which can be any vascular growth factors, such as: TGF 1, PDGF-BB and VEGF, any natural component of the extracellular matrix, namely proteins for cell adhesion, proteoglycans, is incorporated into the polymer structure hyaluronic acid or peptides containing an amino acid sequence that stimulates cell adhesion (e.g., RGD).

The disadvantage of this technical solution is the method of manufacturing a prosthesis, in which there is no selective layered introduction of biologically active components (growth factors and extracellular matrix proteins) into the polymer matrix composition, which prevents the differentiated involvement of progenitor cells in the wall thickness of the polymer structure for subsequent modeling of the vascular wall, by structure close to natural.

Known vascular graft made of bioresorbable polymer compositions with incorporated biologically active molecules in the thickness of the matrix wall (PCT / US 2009/030407; Int. O. A61L 27/14, A61F 2/06. Compositions and methods for promoting patency of vascular grafts / Breuer Chistopher (US), Kryiakides Themis (US), Ron J ason (US); assign Yale University (US) Fil. 08.01.2008; Pub. Date 07.16.2009). The main polymers used are polylactic and polyglycolic acids, polyurethanes, polycaprolactone, polyhydroxyalkanoates, polyanhydrides and their combinations, mixtures or their copolymers. To ensure tissue remodeling at the site of polymeric vascular grafts, it is proposed to use cytokines and chemokines, which help to attract cells into the implantation zone from the bloodstream or surrounding tissues. These included the monocytic chemoattractant protein MCP-1, IL-1β and granulocyte colony-stimulating factor G-CSF. In this case, the selected cytokines were preliminarily enclosed in microspheres or capsules and locally introduced into the vascular graft.

The disadvantage of the proposed approach is the method of incorporation of biologically active components in the composition of the graft - the dispersion method, which is not able to provide a long and uniform output of these substances into the graft location zone over time. In addition, the use of MCP-1, IL-1β, which are pro-inflammatory agents, can provoke excessive attraction of a monocytic-macrophage fraction to the vascular graft, which can stimulate the development of a local inflammatory reaction and neointima hyperplasia. Granulocyte colony-stimulating growth factor G-CSF stimulates the proliferation and differentiation of late progenitor cells into neutrophils, which are not only unable to form de novo tissue in place of the polymer scaffold, which is similar in structure to the natural tissue of the vascular wall, but can also stimulate the development of a local inflammatory reaction in graft location zone.

Closest to the claimed technical solution is a two-layer biodegradable vascular graft made by electrospinning and the introduction of biologically active substances into the polymer skeleton (Appl. US 2014/0309726 A1: Int. CI. A61F 2/06, A61L 27/20, A61L 27/20 54, A61L 27/50. Biodegradable vascular grafts / Yadong Wang (US); appl.no. 14/6365987, filed 06/16/2014; pub. Date 10/16/2014.). The presented vascular graft contains an inner layer made of a biodegradable polyester compound - polyglycerolsebacate (PGS), and an outer shell made of polycaprolactone (PCL) and / or polymers or copolymers of glycoic and lactic acid. To reduce the thrombogenic properties of the implant and increase its biocompatibility, the inner surface of the graft is covered with heparin, and the outer shell is impregnated with any biologically active components that promote tissue regeneration (stem cell growth factor (SCF), vascular endothelial growth factor (VEGF), transforming growth factor (TGF ), fibroblast growth factor (FGF), epidermal growth factor (EGF), platelet growth factor (PDGF), colony stimulating factor (CSF), insulin-dependent growth factor (IGF), chemoattractant mo ekulu (SDF), extracellular matrix proteins - collagen, elastin, fibronectin, laminin, etc.)..

The disadvantage of this method is the lack of selectivity of the introduction into the outer shell of biologically active components and extracellular matrix proteins. The feasibility of using the proposed abundance of growth factors and extracellular matrix proteins is doubtful, since it can lead to uncontrolled tissue regeneration at the site of biodegradable vascular graft and, as a result, undesirable morphological manifestations (mismatch of the normal morphological structure of the vascular wall of the newly formed hyperplasia against the background of similar tissue stimulation, tissue with impaired patency of the vascular graft, etc.). Also, there is no justification for the dose of administered substances, which is a very important aspect in stimulating the growth of new tissues and their formation.

The technical result of the invention is the creation of a biodegradable polymer tissue-engineered vascular graft corresponding to the layered structure of the vessel wall of a living organism while maintaining the functional activity of biologically active components up to a predetermined period of degradation of the polymer matrix.

The technical result is achieved due to the differentiated and layer-by-layer incorporation of biologically active molecules in a given concentration in the manufacture of a porous vascular implant by electrospinning.

Taking into account the mechanisms of angiogenesis and the stimulating effect of growth factors on certain progenitor cells, the most effective combinations of biologically active components were selected [Lee K., Silva EA, Mooney D.J. Growth factor delivery-based tissue engineering: general approaches and a review of recent developments // J. R. Soc. Interface - 2011. - No. 8. - P. 153-170.]. So, to stimulate endothelization of grafts, vascular endothelial growth factor (VEGF) was used, which acts as the main regulator of vascular development in embryogenesis (vasculogenesis), as well as their formation in the adult body (angiogenesis). Being a chemoattractant for VEGF endothelial progenitor cells, it attracts them from the bloodstream to recreate a monolayer of endothelial cells on the inner surface of biodegradable vascular implants. However, there is no need to incorporate VEGF over the entire thickness of the graft wall.

The main fibroblast growth factor (bFGF) affects a wide range of cells and tissues. It is a mitogen for fibroblasts, as a result of which it plays a significant role in tissue remodeling and regeneration processes. The angiogenic function of bFGF is the ability to stimulate dormant endothelial cells, causing them to proliferate and organize into tubular structures [Yun Y.-R. Fibroblast growth factors: biology, function, and application for tissue regeneration // J. Tissue Eng. - 2010 .-- Vol. 2010. - 218142.]. In addition, bFGF stimulates the proliferation of smooth muscle cells in the blood vessel wall and contributes to their survival [Kapo M.R. VEGF-A and FGF-2 synergistically promote neoangiogenesis through enhancement of endogenous PDGF-B-PDGFR signaling // Journal of Cell Science. - 2005. - No. 118. - P. 3759-3768.].

SDF-1 is not a growth factor, however, this chemoattractant molecule has pronounced angiogenic activity and helps to attract progenitor cells of mesenchymal origin (MMSC) into the wall of a future vessel. Isoforms of SDF-1α and SDF-1β inhibit apoptosis of endothelial cells, stimulate their proliferation and the formation of a capillary tube.

Thus, the last two biomolecules can help migrate into the thickness of the graft wall and proliferate in it fibroblasts and MMSCs - cells that form new tissue in place of the bioresorbable tubular skeleton.

Taking into account the peculiarities of the effect on various types of VEGF, bFGF cells and the chemoattractant SDF-1a molecule, we developed original technologies for the combined incorporation of these biomolecules into the polymer composition.

A mixture of polyhydroxybutyrate-valerate (PHBV) and polycaprolactone (PCL) in a 1: 2 ratio, dissolved in chloroform to obtain a 5% concentration of PHBV and 10% PCL, was used as polymers for fabricating the tissue engineering matrix.

The preliminary introduction of growth factors into the solution of the biodegradable polymer allows them to be encapsulated in a polymer fiber and isolated from the environment. In this case, the exit of growth factors from this type of matrix is carried out in the process of passive diffusion from pores penetrating the fiber, as well as as a result of biodegradation of the polymer.

Dilution of biologically active components (VEGF, bFGF, SDF-1a) was carried out with physiological saline (0.9% Na Cl) or phosphate-buffered saline. In this case, the final concentration of VEGF, bFGF and SDF-1a was from 12.5 to 500 ng per 1 ml of polymer solution, which corresponds to the effective effect of biologically active molecules with a bioresorption period of polymer fibers up to 3 years. The choice of this concentration is also due to the need to promptly stimulate cell migration and proliferation in the area of location of vascular grafts, taking into account the decrease in the reparative functions of the human body with age, especially against the background of existing chronic diseases.

The resulting polymer solutions in an organic solvent and an aqueous solution of biomolecules were mixed until a suspension was obtained. Being in a polar solvent - water or phosphate buffer, the growth factor molecules do not come into contact with a non-polar solvent and, therefore, do not undergo denaturation.

Two-phase electrospinning was carried out in the following mode: the voltage on the needle was from 15 to 23 kV, the feed rate of the polymer solution was from 0.3 to 1 ml / h, the rotation speed of the collector was from 100-250 rpm, the distance from the needle to the winding collector was 15 cm, needle size - from 22 to 27G. Under the selected mode, complete polymer fibers with a diameter of up to 500 nm were formed along with the formation of micron-sized filaments.

It was noted that the introduction of an aqueous phase into polymer matrices decreased the strength and elongation of the graft, but the stiffness of the product also decreased. There was no significant difference in the change in the physicomechanical properties of the vascular implant when using the fractional ratio of the polymer solution and the aqueous phase containing 1 or several differentiating factors in the range from 20: 1 to 40: 1, which made it possible to use polymer dilution data in the manufacture of grafts ( Table 1).

The method is as follows. 2 types of mixtures of a polymer solution on chloroform with biomolecules in a ratio of 20-40: 1 are preliminarily prepared.

The mixture No. 1 includes from 20 to 40 parts of a solution of PGBV / PCL in chloroform and 1 part of VEGF in phosphate-buffered saline or 0.9% NaCl solution in a concentration of 1 to 20 μg per 1 ml of an aqueous solution, which is equivalent to the final concentration from 12.5 to 500 ng per 1 ml of polymer solution;

Mixture No. 2 consists of 20-40 parts of a polymer solution in chloroform and 1 part of an aqueous composition containing a mixture of bFGF and SDF-1a from 1 to 20 μg of each differentiation factor per 1 ml of an aqueous solution, which is equivalent to a final concentration of 12.5 to 500 ng of each differentiating factor per 1 ml of polymer solution, depending on the ratio of the growth factor solution to the polymer solution used.

After the mixtures are prepared, the vascular implant is electroformed using mixture No. 1 for 1/3 of the total graft production time, and the remaining 2/3 of the time is performed by two-phase electrospinning with mixture No. 2.

The advantages of the proposed method for incorporating differentiating factors into a biodegradable vascular implant is not only taking into account the specificity of the effect of incorporated bioactive molecules in the context of the layered formation of the future vascular wall based on a temporary biodegradable tubular scaffold, but also a sufficient concentration of VEGF, bFGF and SDF-1a activity of the final product after its implantation in the body, despite the long periods of bioresorption (up to 3 years).

Below are examples of the implementation of the proposed method.

Example 1. To incorporate VEGF onto the inner surface of a biodegradable vascular graft, a mixture of 1 is prepared, for which a sample of dry polymers based on polyhydroxybutyrate valerate (PHBV) and polycaprolactone (PCL) is calculated at the rate of 0.5 g PHBV (with the inclusion of 8% oxyvalerate) and 1.0 g of PCL, 10.0 ml of chloroform was added as solvent. To prepare an aqueous solution of VEGF, 20 μg of growth factor is diluted in 0.5 ml of physiological saline (0.9% NaCl) and added to the finished polymer solution PHBV / PCL. We get the ratio of the polymer solution to the aqueous phase 20: 1. Mixing the ingredients is performed on a magnetic stirrer. The final concentration of VEGF per 1 ml of the obtained polymer solution is 500 ng.

At the same time, mixture 2 is prepared for incorporating bFGF and SDF-1α. Again, 10.0 ml of polymer PHBV / PCL solution is prepared previously (description above). For an aqueous solution containing bFGF and SDF-1α, 20 μg of each differentiation factor is taken and added to 0.5 ml of physiological saline (0.9% NaCl). Then two prepared solutions (mixture 1 and mixture 2) are mixed on a magnetic stirrer. The resulting ratio of polymer solution to aqueous phase will be 20: 1. The final concentrations of bFGF and SDF-1α per 1 ml of the obtained polymer solution are 500 ng.

The process of two-phase electrospinning is carried out in the following order:

First, 1/3 of the entire production time of a small diameter vascular graft (1 hour) is electrospinning using mixture 1 in the following mode: the voltage on the needle is 23 kV, the feed rate of the polymer solution is 0.3 ml / h, the rotation speed of the collector is 200 rpm, the distance from the needle to the winding collector is 15 cm, the size of the needle is 27G.

After that, 2/3 of the entire time of manufacture of a vascular graft of small diameter (2 hours) is electrospinning using mixture 2 in the following mode: the voltage on the needle is 23 kV, the feed rate of the polymer solution is 0.3 ml / h, the rotation speed of the collector - 200 rpm, the distance from the needle to the winding collector is 15 cm, the needle size is 27G.

Example 2. To incorporate VEGF on the inner surface of a biodegradable vascular graft, a mixture of 1 is prepared, for which a weighed sample of dry polymers based on polyhydroxybutyrate valerate (PHBV) and polycaprolactone (PCL) is calculated at the rate of 1.0 g of PHBV (with the inclusion of 8% oxyvalerate) and 2.0 g of PCL, 20.0 ml of chloroform was added as solvent. To prepare an aqueous VEGF solution, 10 μg of growth factor is diluted in 0.5 ml of physiological saline (0.9% NaCl) and added to the finished polymer solution PHBV / PCL. We get the ratio of the polymer solution to the aqueous phase 40: 1. Mixing the ingredients is performed on a magnetic stirrer. The final concentration of VEGF per 1 ml of the obtained polymer solution is 125 ng.

At the same time, mixture 2 is prepared for incorporating bFGF and SDF-1α. Again, 20.0 ml of polymer PHBV / PCL solution (previously described) is pre-prepared. For an aqueous solution containing bFGF and SDF-1α, 10 μg of each differentiation factor is taken and added to 0.5 ml of physiological saline (0.9% NaCl). Then two prepared solutions (mixture 1 and mixture 2) are mixed on a magnetic stirrer. The resulting ratio of polymer solution to aqueous phase will be 40: 1. The final concentrations of bFGF and SDF-1α per 1 ml of the obtained polymer solution are 125 ng.

The process of two-phase electrospinning is carried out in the following order:

First, 1/3 of the entire production time of a small diameter vascular graft (1 hour) is electrospinning using mixture 1 in the following mode: the voltage on the needle is 23 kV, the feed rate of the polymer solution is 0.3 ml / h, the rotation speed of the collector is 200 rpm, the distance from the needle to the winding collector is 15 cm, the size of the needle is 27G.

After that, 2/3 of the entire time of manufacture of a vascular graft of small diameter (2 hours) is electrospinning using mixture 2 in the following mode: the voltage on the needle is 23 kV, the feed rate of the polymer solution is 0.3 ml / h, the rotation speed of the collector - 200 rpm, the distance from the needle to the winding collector is 15 cm, the needle size is 27G.

Thus, the resulting vascular graft has a porous wall structure and consists of two interconnected layers (inner and outer), each of which contains the corresponding components necessary for complete tissue regeneration at the site of the temporary bioresorbable tubular skeleton and the formation of its own vessel.

In in vivo experiments (pericardial implantation of polymer matrices based on PHBV / PCL, PHBV / PCL + VEGF, PHBV / PCL + SFD-1a, PHBV / PCL + bFGF for 3 months), it was found that implantation of empty and biofunctionalized polymer matrices was not caused a local inflammatory reaction. After 3 months of implantation, active neoangiogenesis was noted in the matrices with VEGF and adjacent tissues. The thickness of the PHBV / PCL + bFGF matrices was significantly populated by fibroblasts and surrounded by the most pronounced connective tissue capsule. In matrices with incorporated SDF-la, active infiltration by cells synthesizing the extracellular matrix and neoangiogenesis with the formation of larger blood vessels relative to all the studied samples were observed. Thus, the incorporated molecules after release from the matrix showed biological activity in the surrounding tissues throughout the experiment.

The obtained results confirm the biological activity of the incorporated molecules in the biodegradable graft for layer-by-layer formation in situ (in place) of de novo tissue (newly formed).

Claims (3)

1. Tissue-engineered biodegradable vascular implant, consisting of a polymer matrix made of biodegradable polymers based on a mixture of polyhydroxybutyrate-valerate (PHBV) and polycaprolactone (PCL) by electrospinning, which includes the layered administration of biologically active molecules such as vascular endothelial growth factor V (growth factor V) fibroblast growth factor (bFGF) and chemoattractant molecule (SDF-1α), characterized in that endothelial growth factor (VEGF) is incorporated into the inner layer of the implant by mixing an aqueous solution containing VEGF molecules in a concentration of 1 μg to 20 μg per 1 ml of solution, with a polymer composition based on PHBV / PCL to a ratio of parts in the range from 20: 1 to 40: 1, and performing electrospinning with the resulting composition for 1/3 of total time of electroforming of the graft; and fibroblast growth factor (bFGF) and a chemoattractant molecule (SDF-1a) are incorporated into the outer layer of the implant, for which the polymer composition based on PHBV / PCL is mixed with the aqueous fraction of biologically active molecules bFGF and SDF-1a, where 1 ml of aqueous solution accounts for from 1 μg to 20 μg of each of the differentiation molecules, and continue electrospinning for 2/3 of the remaining time of electrospinning. 2. Tissue-engineered vascular implant according to claim 1, characterized in that the composition of the polymer composition includes a mixture of polyhydroxybutyrate valerate (PHBV) and polycaprolactone (PCL) in a 1: 2 ratio, dissolved in chloroform to a concentration of 5% PHBV and 10% PCL. 3. Tissue-engineered vascular implant according to claim 1, characterized in that the final concentration of each of the differentiating molecules per 1 ml of polymer solution is from 12.5 to 500 ng.