CZ299687B6 - Process for preparing controlled layers of fibrin on solid surfaces - Google Patents

Abstract

The present invention relates to a process for preparing controlled layers of fibrin on solid surfaces for cultivation of cells wherein the preparation process is characterized in that fibrinogen, preferably in concentration of 2 to 200 Ág/ml is adsorbed by adsorption from a solution on a solid surface in the first step, so treated surface is then subjected in the second step to the action of a thrombin solution preferably exhibiting activity of 0.1 to 10 U/ml, whereupon in the third step the surface is treated with a solution containing fibrinogen, preferably in concentration of 200 Ág/ml or greater and a thrombin inhibitor antithrombin or antithrombin in combination with heparin or heparinized blood plasma, whereby the phase of the thrombin solution treatment and subsequent treatment with the solution containing fibrinogen and antithrombin can be carried out repeatedly. Said fibrinogen solution in the first step can be represented by anti-coagulated blood plasma.

Description

Process for the preparation of controlled fibrin layers on solid surfaces

Technical field

Surface modifications of materials with biologically active substances for the preparation of support structures suitable as matrices for cell culture in tissue engineering.

BACKGROUND OF THE INVENTION

Most mammalian cells survive and proliferate only when they are anchored to extracellular matrix (ECM) through the binding of their transmembrane glycoproteins (integrins) to ECM proteins, particularly fibrinectin, laminin, and various types of collagen [1], repair of damaged tissues and organs of artificial skeleton (so-called scaffclds) used for attachment of cells and control of their growth. For this purpose, biodegradable materials [2] of synthetic polymers (polylactides, polyglycolides, polylactones, polyanhydrides, polyurethanes) or natural polymers (collagen, chitosan, hyaluronic acid), which gradually disintegrate after the formation of new tissue or organ, are used. Polymeric skeletons supporting the formation of classes of zionic tissues should contain sufficiently large communicating pores with suitable surface properties to allow migration and subsequent cell proliferation along with easy diffusion of nutrients and tissue growth factors (non-woven polymer fiber networks, porous sponges and foams).

Modification of synthetic surfaces by proteins containing cell adhesion mediated peptide sequences such as gelatin, collagens, fibronectin and laminin is a recognized way of preparing cell culture support structures [1],

At the time of vessel wall injury, a cascade of molecular blood coagulation reactions is activated, leading to the formation of the proteolytic enzyme thrombin. Thrombin converts circin fibrinogen molecules in the blood into fibrin molecules whose spontaneous association leads to the formation of a fibrin fiber network. Thrombin further acts to convert factor XIII present in the blood to active factor XHIa, serving as a catalyst for the formation of covalent bonds between fibrin molecules that sterilize the resulting fibrin gel. In order to prevent the formation of fibrin gels further in the bloodstream at the site of the blood vessel injury, thrombin molecules inhibit their activity in the blood by antithrombin.

In surgical procedures where blood coagulation is to be suppressed, the effect of antithrombin is enhanced by the intravenous administration of heparin anticoagulants. It is known that during the formation of a fibrin network, thrombin molecules can bind to fibrin in such a way that their active center is further able to transform fibnogen into fibrin without being available for inhibition by antithrombin. Bound thrombin is also able to activate factor XIII approximately 10 times faster than thrombin in solution. The fibrin gel, which is primarily used to stop bleeding, provides a temporary support structure for the attachment of cells migrating to the wound site in the following healing process.

During healing, collagen and other intercellular matter components formed by the attached cells are incorporated into fibrin and the fibrin gel is gradually degraded by fibrinolysis. Fibrin can interact with cells both indirectly, ie due to its ability to bind intercellular matter proteins (ECM proteins), eg fibronectin [3] or vitronectin [4] and a number of growth factors, as well as directly by specific cell binding, eg connective tissue cells [ 5] and vascular endothelial cells [6], through integrin receptors [7]. Integrins are cell surface receptors capable of forming specific bonds with peptide sequences present in adhesive ECM glycoproteins such as fibronectin and laminin, as well as in fibrin and fibrinogen.

The known effect of fibrin on accelerating tissue regeneration is commonly used in surgery where fibrin-based preparations are used as tissue adhesives. In tissue engineering, fibrin is used in the form of encapsulated cell gels for the preparation of three-dimensional structures [8, 9]. However, unsuitable mechanical properties do not make it possible to prepare stable forms of such gels, e.g.

-1GB 299687 Heart valve flaps. Therefore, more stable shape structures formed by other materials, mostly polymers, are used in porous form (sponges, foams, fiber networks) that are filled with fibrin gel with encapsulated cells [10-12]. Basic advantages of fibrin gels over other gel-based gel formulations collagen, alginate, etc. is the possibility to use autologous fibrin prepared from the patient's own blood.

Encapsulation of cells within the fibrin gel, successful in some cases in connective tissue replacement, may not be optimal for other applications. For example, encapsulated neural stem cells. NE-4C cells degraded the surrounding tibrin gel and died in the formed cavities similarly as if they float freely in nutrient solution [13]. In applications where cell ingrowth into the porous skeleton is desired, the cells hardly penetrate the pores filled with fibrin gel. Upon incubation of the fibrin gel with a mesenchymal stromal cell suspension, the cells grew on the surface of the gel without significantly migrating into its interior, with the gel under the layer gradually degrading.

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All of the artificial fiber gel preparation processes reported in the available literature are based on a fibrin network formation mechanism derived from the natural blood coagulation process in which a cascade of molecular reactions triggered by injury leads to the formation of the proteolytic enzyme thrombin that converts fibrinogen molecules circulating in blood to fibrin. The spontaneous association of moles of fibrin leads to the formation of a fiber network. In order to prevent the formation of fibrin gels further in the bloodstream, the thrombin molecules formed at the site of the blood vessel injury are inhibited by antithrombin activity in the blood. In surgical procedures where blood coagulation is to be suppressed, the effect of antithrombin is enhanced by the intravenous administration of heparin anticoagulants.

Artificial fibrin gels are prepared by mixing a fibrinogen solution with a thrombin solution. In this way, the fibrin network always forms in its entire volume containing a solution of fibrinogen and thrombin, so that it also fills all the void space inside the porous material [14-15].

The process not yet described, which is the object of the present invention, makes it possible to form on the surface. synthetic materials, a thin layer of fibrin gel with a controlled amount of fibrin without simultaneously forming the gel in the surrounding solution. By this method, the pore surfaces in the porous scaffolds may be coated so that their unused inner volume remains available for cell migration and proliferation.

SUMMARY OF THE INVENTION

SUMMARY OF THE INVENTION The subject of the invention is to coat the surface of the materials with a fibrin gel layer by catalytic action of surface-bound thrombin in contact with a fibrinogen solution, which may be blood plasma stabilized by an antithrombin inhibitor that inhibits thrombin in solution but not surface bound thrombin. heparinized, or with heparinized blood plasma (fibrinogen solution).

In the first step of the procedure, fibrinogen is adsorbed to the surface from the fibrinogen solution or the surface is incubated with anticoagulated blood plasma. Fibrinogen adsorbs on virtually all types of surfaces. The treated surface is then treated with a thrombin solution in a second step, whereby the thrombin spontaneously binds. After rinsing the unbound thrombin, in the third step, the activated surface is treated with a solution containing fibrinogen and antithrombin, which inhibits in solution but not the surface-bound thrombin. Heparinized blood plasma can also be used as a solution naturally containing 2.0 to 4.0 mg fibrinogen / ml and 115 to 160 µg antithrombin / ml. By the catalytic effect of mobilized thrombin, fibrinogen from a solution near the surface is transformed into fibrin forming a fibrin gel on the surface.

The inhibitory function of antithrombin in the third step is to inhibit thrombin molecules that are partially released from the active surface. If the solutions are not stabilized in this way, they are formed

These include fibrin gels released by thrombin molecules released therefrom. The specific inhibition of thrombin in solution and not of surface-bound thrombin is that the antithrombin molecule does not have sufficient access to the active site of the surface-bound thrombin for spherical reasons. Antithrombin is still the only known thrombin inhibitor with such properties. Further growth of the fibrin layer in the event of further contact with fibrinogen, e.g., blood, can be blocked by treating the surface with a thrombin-bound inhibitor in a fourth step. Such known inhibitors are hirudin and D-phenylanyl-L-propyl-L-arginine chloromethyl ketone (PPACK).

The reduction in the amount of fibrin in the surface gel layer is accomplished by reducing the time the surface is in contact with the fibrinogen solution or by adding thrombin-bound inhibitors, as in the fourth step. The amount of bound fibrin is increased, in addition to the length of time the surface is in contact with the fibrinogen solution, so that after the third step the surface is re-treated with the thrombin solution. This binds additional thrombin to the surface layer of the fibrin gel, which can then further activate fibrinogen at the interface of the fibrinogen solution to the surface and further grow the fibrin network on the surface. These steps can be repeated several times.

The thrombin-coupled fibrin gel surface layer can be stabilized by treatment with a solution containing fibrin stabilizing factor XIII, including, e.g., blood plasma, wherein the bound thrombin activates the conversion of factor XIII to the active form of XHIa.

The fibrin gel surface layer can be further modified, eg, by binding ECM proteins using the interaction between fibrin and ECM proteins.

The main advantage of the process is the amount of surface coverage of the intricately structured materials, such as spongy or fibrous tissue support scaffolds, with a fibrin gel layer without filling the internal gaps or pores.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Surface coating of polystyrene film deposited on an optical element for infrared reflection spectroscopy (IR ATR). First, fibrinogen was adsorbed to the surface of polystyrene from a 2 µg fibrinogen / ml solution of 0.05 M Tris buffer, pH 7.4 containing 0.1 mM NaCl and 2.5 mM CaCl ₂ (TB). The adsorption time was 90 min at 24 ° C. Then the surface was thoroughly rinsed with TB. In the next step, a 2.5 ΝΙΉ U thrombin / tnl TB solution was brought to the surface and allowed to act for 10 min. The surface was rinsed with TB. Next, a solution of 200 µg fibrinogen / ml TB containing 0.5 IU antithrombin II / ml TB and 10 UJ heparin / ml was applied to the surface and allowed to react for 100 min. Subsequently, the surface-bound thrombin was inhibited by a solution containing 10 µM PPACK and 6 N 1 H U hirudin / ml TB for 15 min. The surface was rinsed with TB. The gel preparation was monitored in situ by measuring protein infrared absorption bands by IR ATR. There was no further gel increase on the resulting surface upon reintroduction of the fibrinogen solution (inhibition verification). Weight of fibrin on the surface determined from IR. the measurement of the dried resultant gel on the surface of the optical element was 2 µg fibrin / cm ² .

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Example 2

Covering of polyethylene terephthalate foil surface. First, fibrinogen was adsorbed on the film surface from a 2 µg fibrinogen / mol TB solution. The adsorption time was 90 min at temperature

Deň: 18 ° C. Thereafter, the surface was thoroughly rinsed with TB

2.5 ΝΊΗ For thrombin / ml, TB, leave for 10 min. The surface was rinsed with TB. Next, a solution of 200 µg fibrinogen / ml TB containing 0.5 IU antithrombin ΙΙΙ / ml TB and 10 IU heparin / ml was brought to the surface and allowed to react for 100 min and rinsed with TB. The surface weight of fibrin determined from IR ATR measurements of the dried gel on the film was 2.1 µg fibrin / cm ² .

Example 3

Covering the surface of graphite. First, fibrinogen was adsorbed onto the surface of the graphite film from a 15 µg stream at a concentration of 2 µg fibrinogen / ml 0.05 M TB. The adsorption time was 90 min at 24 ° C. Then the surfaces were thoroughly rinsed with TB. Subsequently, a solution of 2.5 NIH U of thrombin / ml TB was applied to the surface, allowed to act for 10 min. The surface was rinsed with TB. A solution of 200 µg fibrinogen / ml TB containing 0.5 IU antithrombin ΙΙΙ / ml TB and 10 µl heparin / ml was then applied to the surface. After 60 min, the surface was rinsed with TB. After microscopic preparation, the fibrin network prepared on the graphite surface was imaged by transmission electron microscopy.

Example 4

Modification of fibrin gel on the gold surface by laminin and collagen I. First, fibrinogen was adsorbed to the gold surface of the SPR sensor from a 2 µg fibrinogen / ml TB solution, followed by a 2.5 NIH solution for thrombin / ml TB, allowed to act 10 min. The surface was rinsed with TB. A solution of 200 µg fibrinogen / ml TB containing 0.5 IU antithrombin ΙΙΙ / ml TB and 10 IU heparin / ml was then applied to the surface. After 60 min the surface was rinsed with TB,

Finally, a 50 µg laminin / ml solution was applied to the surface. After 100 min, an increase in the effective refractive index was observed. The increase in the refractive index caused by the binding of the aminine molecules represented 46% of the fibrin gel. *

When a 100 µg collagen I / ml TB solution was applied to the fibrin-coated surface, 35 the increase in refractive index due to collagen binding was 16% of the fibrin gel.

. Example 5

Formation of fibrin gel on the surface of quartz glass. The UV-VIS spectroscopy tube was filled with a fibrinogen solution of 2 µg fibrinogen / ml TB. After 90 min at 24 ° C, the cuvette was rinsed with TB. Next, the cuvette was filled with a solution of 2.5 NIH U thrombin / ml TB. After 10 min, the cuvette was rinsed with TB. Subsequently, the cuvette was filled with a solution of 200 µg fibrinogen / ml TB containing 0.5 IU antithrombin ΙΠ / ml TB and 10 IU heparin / ml. After 120 min, the cuvette was rinsed with TB. The increase in light scattering continuously measured by light passage through the cuvette confirmed the formation of a scattering fibrin network. The zero turbidity of the fibrinogen solution taken from the acid after gel formation confirmed that gel formation occurs only on the glass surface of the cuvette. The light scattering caused by the fibrin network mobilized on the cuvette surface did not change after the solutions were changed. When a fibrinogen solution containing no thrombin inhibitors was used, an increase in solution turbidity indicative of activation of fibrin formation in solution by thrombin released from the surface was observed.

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Example 6

Formation of fibrin gel from blood plasma. The UV-VIS spectroscopy tube was filled with fibrinogen solution at 2 µg fibrinogen / ml TB: After 90 minutes at 24 ° C, the tube was flushed with TB. Next, the cuvette was filled with human blood plasma inhibited by 10 IU heparin / ml. After 120 min, the cuvette was rinsed with TB. The increase in light scattering suggested formation of a fibrin network on the cuvette surface.

Example 7

Regulation of the amount of fibrin gel on the gold surface of a surface plasmon resonance (SPR) optical sensor. Fibrinogen was adsorbed to the surface from 2 µg fibrinogen / ml TB. The adsorption time was 90 min at 24 ° C. Then the surface was thoroughly rinsed with TB. Next, a solution of 2.5 NIH U thrombin / ml TB was added to the surface, allowed to act for 10 min. The surface was rinsed with TB. A solution of 200 µg fibrinogen / ml TB containing 0.5 IU antithrombin ΙΠ / ml TB and 10 IU heparin / ml was then applied to the surface and allowed to react for 120 min. The surface was rinsed again with TB. Subsequently, the surface-bound thrombin was inhibited with a solution containing 10 µm PACK and 6 ΝΪΗ U hirudin / ml TB for 15 min. The surface was rinsed again with TB. The in situ change in the effective refractive index relative to the (not linear) weight of the fibrin gel on the gold surface was 2.7 times the adsorbed mono. molecular layers of fibrinogen. These steps were repeated. The in situ measured optical response of the SPR sensor, proportional to the gel weight, was 4.5 times the adsorbed fibrinogen monomolecular layer. The steps were then repeated again. The in situ measured optical response of the SPR sensor, proportional to the gel weight, was 6.1 times the adsorbed fibrinogen monomolecular layer. The fibrin network was treated with a solution of 30 µg ΧΙΠ / ml TB.

Example 8

Surface coating of poly (2-hydroxyethyl) methacrylate hydrogel (PHEMA). The UVVIS spectroscopy cuvette, whose inner walls were coated with PHEMA film, was filled with a fibrinogen solution of 2 µg fibrinogen / ml TB. After 90 minutes at 24 ° C, the cuvette was rinsed with TB, and the cuvette was filled with a solution of 2.5 NIH U thrombin / ml TB. After 10 minutes, the cuvette was rinsed with TB. As a result, the cuvette was filled with a solution of 200 µg fibrinogen / ml TB containing 0.5 IU antithrombin ΙΠ / ml TB and 10 IU heparin / ml. After 120 minutes, the cuvette was rinsed with TB. The increase in light scattering continuously measured by light passage through the cuvette confirmed the formation of a scattering fibrin network.

Example 9. Surface coating of polylactide fibers. A tablet prepared by compressing a 60 µm thick block of polylactide fibers was placed in a solution of 50 µg fibrinogen / ml TB. After 90 minutes at 24 ° C, the tablet was rinsed with TB. Then, the tablet was incubated with a solution of 2.5 NIH U thrombin / ml TB. After 10 minutes, the tablet was rinsed with TB. Subsequently, the tablet was incubated with a solution of 200 µg fibrinogen / ml TB containing 0.5 IU antithrombin ΙΠ / ml TB and 10 IU heparin / ml. After 120 minutes, the tablet was rinsed with TB.

After staining the fibrin in the fluorescein block, the confocal microscope showed the presence of a gel layer on the surface of the individual fibers and the maintenance of the void between the fibers. After incubating an analogous tablet with a fibrinogen solution in which fibrin network formation was normally activated by the addition of thrombin, the space between the fibers was completely filled with the fibrin network.

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CZ 299687 Bo

Example 10

Growth of bovine vascular endothelial cells (Line CPAE ATCC CCL-209, Rockville, MA, USA) on a fibrin gel coated polystyrene surface. Fibrinogen was adsorbed to the surface of culture polystyrene dishes from a solution of 2 µg of fibrinogen / ml TB. The adsorption time was 90 minutes at 24 ° C. Then the surface was thoroughly rinsed with TB. Next, a solution of 2.5 NIH U thrombin / ml TB was applied to the surface, allowed to act for 10 minutes. The surface was rinsed with TB. Subsequently, a solution of 200 µg fibrinogen / ml TB containing 0.5 IU antithrombin 111 / ml TB and 10 IU heparin / ml was applied to the surface and allowed to react for 120 minutes and rinsed with TB. The treated surface was incubated with a suspension of bovine vascular endothelial cells in culture medium. Endothelial cells were seeded at a concentration of 17,000 cells / cm ² . After seven days, the number of viable cells on the surface of the untreated culture dish was 1.77 times

10 ⁵ / cm ² , while on a fiber gel surface 3.03 x 10 ⁵ / cm 2.

Industrial applicability

The solution of the invention can be used in medicine to prepare and modify support structures suitable as a matrix for tissue culture cells, [1] Yang, S. F .; Du, Z.H .; Leong K.F .; Chua, C.-K. Tissue Eng. 7 (2001) 679 and Tissue Eng. 8 (2002) I [2] P and Agunatillake, R Adhikari, European Cells and Materials 5 (2003) 1-16 [3] Makogenenko E, Tsurupa G, Ingham, K. Medved L: Interaction of fibrion (ogen) with fibrinectin : Further characterization and localization of the fibronectin-binding site. Biochemistr (2002) 7907-7913. Preissmen RT, Jenne D: Vitronectins. A new molecular connection in haemostasis. Tbromb. Haemostas. 66 (1991) 189-194 [5] Reach A, Odrljin, T., Francis CW: Binding of Fibroblast Growth Factor to Fibrinogen and Fibrin. J, Biot Chem. 273 (1998) 7554-7559 [6] Abha Sahi and Charles W. Francis, Vascular endothelial growth factor binds to fibrinogen and fibrin and stimulates endothelial cell proliferation, Blood 96 (2002) 945-953 [7] Cheresh DA, Berliner SA, Vicente V, Ruggerí ZM: Recognition of Discinitive Adhesive Sites on Fibrinogen by Related Integrates on Platelets and Endothelial Cells, Cell 58 (1989), 945-953, [8] JP Stegemann and R.M. Nerem, Phenotype modulation in vascular tissue engineering using biochemical and mechanical stimulation, Ann Biochem Eng 31 (2003) 391-402 [9] S.Jockenhoevel, G. Zund, S.P. Hoerstrup, K. Chalabi, J.S. Sachweh and L. Demircan et al.> Fibrin Gel - Advantages of New Scaffold in Cardiovasdular Tissue Engineering, Eur J. Cardiothorac Sug 19 (2001) 424-430 [10] G.A. Ammer, T.SA. Mahmoed and R. Langer, A. Biodegradable Composite Scaffold for Cell Transplantation, J. Orthop Res 20 (2002) 16-20 [11] Anita Mola, Marjolein I. van Lieshout, Christa G. Dam-de Veena, Stefan Neuenschwanderb, Simon P. Hoerstrupb, Frank Pt Baaijensa and Carlijn V.C. Bouten, Fibrin as a cell carrier in cardiovascular tissue engineering applications, Biomaterials 26 (2005) 311345 3 1 21 [12] Hokugo, Akishige; Takamoto, Tomoakt; Tabata, Yasuhiko Preparation of hybrid scaffold from fibrin and biodegradable polymer fiber. Biomaterials 27 (2006) 61-67 [13] Riedel T., Brynda E., Lesny P., Jendelova P., Sykova E., Dyr J. E., Houska M. Threedimensional fibrin scaffolds, 43rd Microsymposium, Polymer Biomaterials: Biomimetic and

Bioanalogous Systems', Prague 2004, PC46

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[14] Ronfard V. (Isitis SA; Episource S. A. Switz.) Fibrin Cell Support and Methods of Use, PCT Int. Appl. WO 2004022696 A2 18 Mar 2004, [15] Hokugo, Akishige ,; Takamoto, Tomoaki; Tabata, Yasuhiko. Preparation of hybrid scaffold from fibrin and biodegradable polymer fiber, Biomaterials Π (2006) 61-67

Claims (5)

PATENT CLAIMS. A method for the preparation of regulated fibrin layers on solid cell culture surfaces, comprising, in a first step, adsorbing fibrinogen to a solid surface from a fibrinogen solution preferably at a concentration of 2 to 200 pg / ml, and treating the treated surface with solution A thrombin, preferably having an OO activity of up to 10 IU / ml, characterized in that the surface is then treated with a solution containing fibrinogen, preferably at a concentration of 200 µg / ml or higher, and antithrombin III, or heparinized blood plasma. 2. The method of claim 1 wherein the fibrinogen solution in the first step is Anticoagulated blood plasma. Method according to claim 1 or 2, characterized in that the antithrombin III has an O 2 concentration of up to 10 IU / ml. 25 Method according to claim 1 or 2 or 3, characterized in that the treatment with the thrombin solution and the subsequent treatment with the solution containing fibrinogen and antithrombin III is carried out repeatedly. Method according to claim 4, characterized in that heparin is added to decanthrombin ΠΓ, 30 preferably at a concentration of 1 to 100 IU / ml.