WO2008089708A1

WO2008089708A1 - Biomaterial based on nanofibrillar layers and method of preparation thereof

Info

Publication number: WO2008089708A1
Application number: PCT/CZ2008/000005
Authority: WO
Inventors: Petr Lesny; Eva Sykova; Jiri Michalek; Martin Pradny; Oldrich Jirsak; Lenka Martinova
Original assignee: Ustav Experimentalni Mediciny Av Cr, V.V.I.; Ustav Makromolekularni Chemie Av Cr, V.V.I.; Technicka Univerzita V Liberci
Priority date: 2007-01-23
Filing date: 2008-01-09
Publication date: 2008-07-31
Also published as: CZ200754A3; CZ300805B6

Abstract

The invention relates to a biomaterial based on nanofibrillar layers, which consists of at least one nanofibrillar layer and living cells firmly connected with this nanofibrillar layer, wherein the nanofibrillar layer is formed by synthetic polymers or co-polymers of monomers selected from the group comprising methacrylic acid esters, methacrylic acid amides, poly urethanes, polyvinyl alcohols and polymers derived from lactic acid and its derivatives, and to a method of preparation thereof.

Description

Biomaterial based on nanofϊbrillar layers and method of preparation thereof

Technical Field

The invention relates to biomaterial based on nanofϊbrillar layers and a method of preparation thereof.

Background Art

At present, preparation of various forms of solid materials, defined by the presence of pores, resp. of specific surface corresponding to structural unit dimensions in the order of nanometers to hundreds of nanometers, is at the cutting edge of material engineering. Nearly all important properties of such systems result from the extraordinarily large specific surface. While the porous materials based on polymers and the nanomaterials prepared by the method of organized supramolecular structures have been extensively studied for a long time, the fibers having the diameter in the order of tens to hundreds of nanometers receive an increased attention only in the last five years. From these fibers, fibrillar layers having good mechanical properties can be formed directly by a spinning process. The mechanical properties as well as the morphology are favourably influenced by the anisotropic character of the fibrillar layer. The pores in such layers possess a specific geometry, thanks to which the surfaces of the fibers are easily accessible.

Generally, nanofibers are described as fibers, the diameter of which is in the submicron region, i.e. up to 1000 run. They possess a number of exceptional properties, such as a large specific surface of the fibers, a big porosity of the fiber layer and a small diameter of the pores. With regard to cell cultures, the nanofibrillar layer structure is similar to the structure of extracellular matrix. In consistence with this fact are repeated observations of a higher adhesion of cells to nanofibers, compared with microfibers or layers of identical polymers [Schindler M, Ahmed I, Kamal J, Nur EKA, Grafe TH, Young Chung H, et al. A synthetic nanofibrillar matrix promotes in vivo-like organization and morphogenesis for cells in culture. Biomaterials, 2005 Oct;26(28):5624-31.; Rho KS, Jeong L, Lee G, Seo BM, Park YJ, Hong SD, et al. Electro spinning of collagen nanofibers: effects on the behavior of normal human keratinocytes and early-stage wound healing. Biomaterials. 2006 Mar;27(8): 1452-61.; Min BM, Lee G, Kim SH, Nam YS, Lee TS, Park WH. Electrospinning of silk fibroin nanofϊbers and its effect on the adhesion and spreading of normal human keratinocytes and fibroblasts in vitro. Biomaterials. 2004 Mar- Apr; 25(7-8): 1289-97].

The use of microfibers for the preparation of porous three-dimensional tissue repairs containing cells is known since 1993; this technology served first for the experimental preparation of joint cartilage implants [Robinson D, Efrat M, Mendes DG, Halperin N, Nevo Z. Implants composed of carbon fiber mesh and bone-marrow-derived, chondrocyte-enriched cultures for joint surface reconstruction. Bull Hosp Jt Dis. 1993 Spring: 53(l):75-82.]. This use of the microfibers is protected by patent [US 5 759 830, US 5 770 417]. The three-dimensional network of microfibers provides for the mechanical hardness of the material, maintains its three-dimensional shape and the mutual spatial arrangement of the cells. This method, however, is not applicable to non-woven nanofibrillar textiles, because at reaching the porosity necessary for the intercalation of the cells between the fibers (pores in the order of micrometers to tens of micrometers), such material would collide by its own weight.

The standard process for cultivating the cultures of tissue and stem cells is cultivation in monolayer; for this cultivation, special surface-treated materials are used, to which the cells adhere (tissue plastic, laminium or polylysin surface-treated glass, tissue plastic overgrown with inactivated fibroblasts etc.). Alternative cultivation methods include cultivation in the suspension or in materials based on gels, e.g. collagen, agarose or MatriGel®. However, all the hereinabove described cultivation methods have some disadvantages. The growth of cells in the monolayer culture is limited by their adhesion to the cultivation material, the medium can access only the apical layer; the cell properties change after achieving a confluent layer; compounds produced by the cells are released into the medium and do not stay in the vicinity of the cells. The advantage of the monolayer cultivation is - apart from the fact that it is a very well standardized process - also the possibility to observe individual cells through a microscope (Fig. 1). At the cultivation of cells on fibers or in macroporous materials the pores of which have the dimensions in the order of tens of micrometers, the cells behave similarly to the cells cultivated in monolayer - they grow on flat parts of the material surface (Fig. 2).

Non-woven nanofibrillar textile layers prepared by an electrospinning method can be used for the cultivation of cells in monolayer, similarly to e.g. tissue plastic; first experiments with cell cultures on nanofibrillar textiles were published in 2002 [Li WJ, Laurencin CT, Caterson EJ, Tuan RS, Ko FK. Electrospun nanofibrous structure: a novel scaffold for tissue engineering. J Biomed Mater Res. 2002 Jun 15; 60(4):613- 21.]. A cell cultivation system on nanofibrillar layers is protected by a patent [US 6 790 455]. A prospective beneficial use of the nanofibrillar layers in biomedicine has led to filing a cumulative patent application [PCTΛJS2004/029765], in which, however, the use of nanofibers for the construction of tissue repairs is not mentioned.

Many authors have attempted to prepare three-dimensional tissue repairs using nanofibers. Patented was the use of nanofibrillar layer as a substrate on which the smooth muscle cells are cultivated and subsequently further cell layers are produced on these cells, the layers being separated by a biomaterial (e.g., based on polymer) [US 6 428 802]. Published was also the preparation of a tissue repair by means of cell cultivation on parallel layers formed by nanofibrillar networks and substrate. These layers can be separated from each other by an arbitrary biomaterial [WO 2005/047493]. However, the formation of a homogeneous tissue by cross connection of the cell-containing layers is not described in this document.

Biomaterials based on 2-hydroxyethylmethacrylate (HEMA) copolymers were already repeatedly prepared. These copolymers are highly biocompatible and the cell adhesion to their surfaces depends on their electrical charge [Lesny P, Pradny M, Jendelova P, Michalek J, Vacik J, Sykova E. Macroporous hydrogels based on 2-hydroxyethyl methacrylate. Part 4: Growth of rat bone marrow stromal cells in three-dimensional hydrogels with positive and negative surface charges and in polyelectrolyte complexes. Journal of Materials Science, 2006 Sep; 17(9):829-33.]. Disclosure of Invention

Object of the present invention is a biomaterial based on nanofibrillar layers, consisting of at least one nanofibrillar layer and living cells firmly connected with said nanofibrillar layer, wherein the nanofibrillar layer is formed by synthetic polymers or copolymers of monomers selected from the group comprising methacrylic acid esters, methacrylic acid amides, polyurethanes, polyvinyl alcohols and polymers derived from lactic acid and its derivatives.

It is an aspect of the invention that the nanofibrillar layer is non- woven.

In a preferred embodiment, the monomers are selected from the group comprising 2- hydroxyethyl methacrylate, 2-ethoxyethyl methacrylate and 2-hydroxypropyl methacrylamide.

It is an aspect of the invention that the cells are selected from the group comprising the cells of connective tissue, epithelial cells, parenchymal organ cells and mesenchymal stem cells derived from bone marrow or adipose tissue. In a preferred embodiment, the cells are selected from the group comprising chondrocytes, fibroblasts, hepatocytes and mesenchymal stem cells derived from bone marrow or adipose tissue.

The biomaterial according to the present invention is suitable for the construction of two- and three-dimensional structures, useful for tissue engineering.

It is an aspect of the invention that the biomaterial according to the present invention consists of at least two nanofibrillar layers covered on both sides confiuently by living cells, whereas these layers are interconnected by the growth of the cells.

Another aspect of the present invention is the biomaterial according to the present invention, consisting of one nanofibrillar layer overgrown with living cells on one side, wherein the cells are functionally polarized. The ability of the polymer to form fibers, i.e. spinning, is affected by a plurality of process and material parameters, such as the electrical field intensity, electrical conductivity, viscosity, molar weight, surface tension, polymer concentration, solvent, dielectrical properties of the polymer solution, hydrophility/hydrophobicity, polymerization degree and branching degree of the polymer or the embodiment of the experiment. The properties of the spun material are affected not only by the chemical composition of the polymer, but also by the spinning parameters. By a change in the above-mentioned parameters, not only the spinning process, but also the structure of the resulting layer and the fineness of the fibers can be affected to a certain extent. The optimum process and system parameters must be determined separately for each polymer solution spun by the electrostatic spinning method, because the parameters are transferable only to a very limited degree.

The method of preparation of the synthetic polymer or copolymer nanofibers in accordance with the present invention can be the electrostatic spinning method ,,Nanospider", in which the fibers are formed by the action of electrostatic field from a thin layer of a polymer solution, carried by a pivoted cylinder, which forms a positively charged electrode, and the fibers are deposited onto a collector, which forms a counter electrode [CZ 294274 (B6), WO 2005/024101].

The contact with the extracellular matrix molecules and the adhesion to them are for most cells very important factors, which are the condition for their adhesion and growth in the organism. The cell cultivation in two-dimensional cultivation systems does not reflect the natural environment in the organism. The structure of the nanofibrillar layer is compared to the structure of basal membrane.

Cells strongly adhere to the nanofibrillar layers of the biomaterial according to the present invention formed by the polymers with nanofibrillar structure, even if they do not display any affinity to the polymers themselves - without the nanofibrillar structure. After the inoculation of the cells on the nanofibrillar layer, in several days or weeks (depending on the cell concentration) the cells densely overgrow the nanofibrillar layer and their extremities ingrow also between the fibers. If the nanofibrillar layer is sufficiently thin (units to tens of micrometers), the growth of the cells reaches to the other side of the layer; from the layer thickness ca 100 micrometers the cells do not reach to the other side.

The cultivation of cells on the nanofibrillar layers has many advantages in comparison with their cultivation in monolayer, in suspension or in gel. During the cultivation on nanofibers - in contrast to the cultivation in monolayer -, the medium can access both the apical and the basal side of the cells. If the nanofibrillar textile is during the cultivation placed in an interface of two media differing in either physical (temperature) or chemical (concentration of growth factors, composition of the medium) properties in such a way that one medium is in contact with the apical side of the cells and the other medium is in contact with the basal side of the cells, the difference in these properties on the apical and basal sides of the cell can induce its functional polarization (namely a different concentration of the receptors on the membranes of the apical and the basal part of the cell). The functional polarization of the cells can be further exploited, e.g. in the construction of detoxication bioreactors containing hepatocytes, in which the physiological placement of a hepatocyte between the capillary and billiary stream is simulated [Ostrovidov S, Jiang J, Sakai Y, Fujii T. Membrane-based PDMS microbioreactor for perfused 3D primary rat hepatocyte cultures. Biomed Microdevices, 2004 Dec; 6(4):279-87.]. A further advantage of the cell cultures on nanofibrillar layers is the possibility of the double-side growth of the cells on the nanofibrillar layer. The cells growing on the nanofibrillar layer maintain their ability of growth in space, which is demonstrated by interconnection of two layers containing cells by their growth when the layers are brought close to each other.

Object of the present invention is a method of preparation of the biomaterial of the present invention, wherein a synthetic polymer or copolymer of monomers selected from the group comprising methacrylic acid esters, methacrylic acid amides, polyurethanes, polyvinyl alcohol and polymers derived from lactic acid and its derivatives is prepared and the thus prepared synthetic polymer or copolymer is spun by an electrostatic spinning method (electrospinning), subsequently on the spun layer of the synthetic polymer or copolymer prepared by the electrostatic spinning method, cells are inoculated and cultivated under standard conditions for tissue culture cultivation until a confluent layer of cells is achieved. The standard conditions for tissue culture cultivation comprise the use of a combination of a medium suitable for the given type of cells, foetal calf serum or a replacement thereof, antibiotics, optionally growth factors, and the cultivation proceeds in incubators at 37 °C and 5 % CO₂, and the cultivation medium is changed every 2-3 days.

Preferably, the monomers are selected from the group comprising 2-hydroxyethyl methacrylate, 2-ethoxyethyl methacrylate and 2-hydroxypropyl methacrylamide.

It is an aspect of the method according to the invention that the cells are selected from the group comprising the cells of connective tissue, epithelial cells, parenchymal organ cells and mesenchymal stem cells derived from bone marrow or adipose tissue. In a preferred embodiment, the cells are selected from the group comprising chondrocytes, fibroblasts, hepatocytes and mesenchymal stem cells derived from bone marrow or adipose tissue.

Another aspect of the present invention is the method of preparation of the biomaterial according to the present invention, consisting of at least two nanofibrillar layers interconnected by the growth of the cells, wherein a synthetic polymer or copolymer of monomers selected from the group comprising methacrylic acid esters, methacrylic acid amides, polyurethanes, polyvinyl alcohols and polymers derived from lactic acid and its derivatives is prepared, the thus prepared synthetic polymer or copolymer is spun by an electrostatic spinning method, subsequently on the layer of the synthetic polymer or copolymer spun by the electrostatic spinning method, the cells are inoculated and cultivated under standard conditions for tissue culture cultivation until a confluent coverage of the nanofibrillar layer is achieved, and then at least two layers of the spun synthetic polymer or copolymer, overgrown with the cells, are manually or automatically stacked onto each other and onto this system of layers, pressure of 5 to 20 g/cm³ is applied and the system is cultivated under standard conditions for tissue culture cultivation for the period of 1 to 4 weeks.

Preferably, the monomers are selected from the group comprising 2-hydroxyethyl methacrylate, 2-ethoxyethyl methacrylate and 2-hydroxypropyl methacrylamide. It is an aspect of this embodiment of the invention that the cells are selected from the group comprising the cells of connective tissue, epithelial cells, parenchymal organ cells and mesenchymal stem cells derived from bone marrow or adipose tissue. In a preferred embodiment, the cells are selected from the group comprising chondrocytes, fibroblasts, hepatocytes and mesenchymal stem cells derived from bone marrow or adipose tissue.

Yet another aspect of the present invention is the method of preparation of the biomaterial according to the present invention, containing functionally polarized cells, wherein a synthetic polymer or copolymer of monomers selected from the group comprising methacrylic acid esters, methacrylic acid amides, polyurethanes, polyvinyl alcohols and polymers derived from lactic acid and its derivatives is prepared, the thus prepared synthetic polymer or copolymer is spun by an electrostatic spinning method, subsequently on the layer of the synthetic polymer or copolymer, spun by the electrostatic spinning method, the cells are inoculated and cultivated under standard conditions for tissue culture cultivation, whereby the layer of the spun synthetic polymer or copolymer is in the course of the cell cultivation process placed in the interface of two environments differing in their physical or chemical properties. Preferably, the monomers are selected from the group comprising 2-hydroxyethyl methacrylate, 2-ethoxyethyl methacrylate and 2-hydroxypropyl methacrylamide.

It is an aspect of this embodiment of the invention that the cells are selected from the group comprising the cells of connective tissue, epithelial cells, parenchymal organ cells and mesenchymal stem cells derived from bone marrow or adipose tissue. In a preferred embodiment, the cells are selected from the group comprising chondrocytes, fibroblasts, hepatocytes and mesenchymal stem cells derived from bone marrow or adipose tissue.

The whole process of the invention thus consists in an integral process comprising the preparation and characterization of the polymer, the spinning process, seeding of the nanofibers by a suitable cell culture, the cultivation of the cells on the nanofϊbrillar layer and the formation of the resulting material into a form suitable for the desired application in tissue engineering or a conventional treatment process. Biomedical applications

In the field of biomedical applications, it is necessary to take into account from several points of view the quality of the material used. The first aspect is the resulting utility physical and chemical properties, such as mechanical (rigidity, hardness, tenacity, elasticity), swelling (equilibrium water content), transport (permeability), optical (refractive index) and surface (harshness, wettability, contact angle) properties. Further important aspects are physical and chemical structure of the material, which substantially affect the material biotolerance in the application in a given environment. It is the required interaction of the material with a living tissue, i.e. cell culture, further the chemical stability of the material, resp. its controlled biodegradation. The requirements for the biomaterial properties are often contradictory and it is necessary to look for a compromise between them. Hence, for many applications, only a limited selection of suitable chemical structures exists.

Another problem in bioapplications is the necessary long-term tests of the effects and particularly the possible undesirable effects of the materials used. From all the mentioned reasons results a natural tendency to test for new applications the well- established and thus already for a long time used materials that are anticipated to comply with the requirements in the testing of toxicity, resp. tolerance. In the field of hydrogels, such a material is the crosslinked poly(2-hydroxyethyl methacrylate) and some of its copolymers or poly(2-hydroxypropyl methacrylamide).

The biomaterial according to the present invention can be used in tissue engineering e.g. as surface carrier of cells, producing growth factors: if the nanofibrillar layer is prepared from a non-degradable material and overgrown with the cells on one side, the cells release growth factors that can penetrate through the nanofibrillar layer. If the thus prepared material is applied on a target place, the effect of the growth factors is achieved without direct contact of the target place with the cells. Example of use: dermal covers overgrown with allogenic fibroblasts, which after applying to the wound release healing-enhancing factors.

The biomaterial according to the present invention can be used also as a polarized cell culture carrier: if the nanofibrillar layer is overgrown with cells on one side and placed in an interface of two environments with a different media compositions, a polarized cell culture is formed, in which the cells on the apical and on the basal side possess different surface properties. Example of use: hepatal bioreactor.

Another use of the biomaterial according to the present invention can be the construction of three-dimensional organ repairs: by stacking the nanofibrillar layers overgrown with cells and loading a uniform pressure on them, it is possible to form three-dimensional implants. By a suitable combination of the parameters - pressure, layer thickness, percentage of the surface covered by the cells, number of layers and cell types in individual layers -, the formation of relatively complex tissues can be achieved. Example: by a combination of the layers overgrown with hepatocytes and the layers overgrown with endothelial cells, a tissue repair with a similar distribution of cells as in hepatic tissue can be prepared.

Brief Description of Drawings

Fig. 1 shows human stromal cells of bone marrow growing in monolayer on a tissue culture plastic. The black line represents the scale = 100 μm.

Fig. 2 shows human chondrocytes (arrow) growing on the surface of a polylactide fiber having the thickness of 50 micrometers. The black line represents the scale =

50 μm.

Fig. 3 represents the scheme of the electrospinning machine. The reference numbers used are: 1 - metal cylinder, positively charged electrode, 2 - spun layer of the polymer solution, 3 - polymer solution reservoir, 4 - textile substrate (support material), 5 - direction of nanofiber formation, 6 - negatively charged earthed electrode, 7 - exhaustion of air and vapours.

Fig. 4 shows the structure of the nanofibrillar layer displayed by electron microscope

AQUASEM.

Fig. 5 shows the growth of human chondrocytes stained by immunofluorescence CFDA-SE (carboxyfluorescein diacetate, succinimidyl ester) on the nanofibrillar layer; (A) from the upper side, whole cells are visible (arrow), (B) from the lower side, only their extremities are visible. The white line represents the scale = 100 μm.

Fig. 6 shows the formation of tissue in the nanofibrillar implant prepared by rolling the nanofibres into a roll in the spinal cord of a laboratory sewer-rat and insertion lengthwise the longitudinal axis of the spinal cord ( A,C,E,G,I) or perpendicularly to the longitudinal axis of the spinal cord (B,D,F,H,J,K,L).

A,B - transparent staining by hematoxylin-eosin, the rectangles mark the cut-outs with high and low density of the cells which are further shown in a larger scale. The scale = 500 μm.

C,D - details of ingrowth of tissue with a high density of cells, staining by hematoxylin-eosin. Scale = 100 μm.

E,F - details of ingrowth of tissue with a low density of cells, staining by hematoxylin-eosin. Scale = 100 μm. G,H - transparent immunohistochemical staining by antibody against NF 160; the rectangles mark the cut-outs that are further shown in a larger scale. Scale = 500 μm.

I - NF 160 in the implant inserted perpendicularly to the longitudinal axis of the spinal cord, positive dendrits of neural cells grow alongside the edge of the implant. Scale =

100 μm. J₅K - NF 160 positive dendrits of neural cells ingrowing into the implant inserted lengthwise the longitudinal axis of the spinal cord. Scale = 100 μm.

L - NF 160 positive dendrits of neural cells ingrowing into the implant inserted lengthwise the longitudinal axis of the spinal cord. Scale = 50 μm.

Fig. 7 shows the growth of sewer-rat's stromal cells of bone marrow stained immunofluorescently by phalloidin (cell cytoskeleton) and DAPI (cell nuclei) on the nanofibrillal layer in various magnifications.

Fig. 8 shows the biomaterial formed by the growth of human chondrocytes on the nanofibrillar layers which were brought close to each other and a uniform pressure was applied thereon; stained by hematoxylin-eosin. A — full picture, B - detail of figure A, on which the homogeneity of the tissue is visible; scale = 500 μm.

Fig. 9 shows human bone marrow stromal cells stained by phalloidin (cell cytoskeleton) and DAPI (cell nuclei) growing between two layers of nanofibrillar textile. Fig. 10 shows the growth of sewer-rat's bone marrow stromal cells on the nanofibrillar layer - reconstruction of display in a confocal microscope, taken by software Amira®. The cell cytoskeleton was stained by phalloidin. A - centre of the nanofibrillar layer with confluently growing cells, B - side view, marked is the position of the nanofibrillar layer (light) and overgrowing extremities of the cells (dark), C - spot, where the nanofibrillar layer was crinkled: the cells copy the shape of the layer, D - edge of the nanofibrillar layer where the cells grow isolated. Fig. 11 shows the growth of the sewer-rat's mesenchymal cells derived from adipose tissue on the nanofibrillar layer, stained by phalloidin. A - full picture, scale = 500 μm. B - detail, scale = 100 μm.

Fig. 12 shows the growth of human chondrocytes in a part of sample onto which pressure has been applied and in a part of the same sample onto which pressure has not been applied. A - during the growth in the part onto which pressure has been applied, the individual nanofibrillar layers were interconnected by the growth of the cells; also the extracellular matrix deposition occurred. Staining for collagen type II. Scale = 100 μm. B - during the growth in the part onto which pressure has not been applied, neither the interconnection of the layers nor the extracellular matrix deposition occurred. Stained by hematoxylin-eosin. Scale = 100 μm.

Examples

Example 1

Copolymer of 2-hydroxy ethyl methacrylate (25.5 g) with 2-ethoxyethyl methacrylate

(60 g) was prepared by oxidation - reduction radical polymerization of the monomers by treatment with ammonium persulfate (0.5 g) and sodium metabisulfite (0.5 g) in water-ethanol solution (380 g of ethanol, 17O g of water) at 23 °C for the period of 7 days. Then, the resulting copolymer was precipitated in water (3 1), dried and dissolved in ethanol to reach the concentration of 16.6 %. The copolymer molar weight was 6.78 . 10⁵ g/mol, intrinsic viscosity 27.1.

Example 2

Polymer of 2-ethoxyethyl methacrylate was prepared by oxidation - reduction radical polymerization of the monomer (85.5 g) by treatment with ammonium persulfate (0.5 g) and sodium metabisulfite (0.5 g) in water-ethanol solution (380 g of ethanol, 17O g of water) at 23 °C for the period of 7 days. Then, the resulting polymer was precipitated in water (3 1), dried and dissolved in absolute ethanol to reach the concentration of 16.1 %. The polymer molar weight was 6.78 . 10⁵ g/mol, intrinsic viscosity 26.9. Example 3

Poly(2-hydroxypropyl methacrylamide) was prepared by precipitation polymerization of 10 g if the monomer by treatment with 0.1 g azobis(isobutyronitrile) at 60 ⁰C for the period of 8 h in 40 g acetone, subsequent washing of the polymer by acetone and drying. The polymer molar weight was 8.2 . 10⁵ g/mol.

Example 4

Solution of the copolymer of Example 1 was spun by the electrostatic spinning method (electrospinning) on a laboratory model of instrument for the Nanospider technology. The scheme of the instrument is shown in Fig. 3 and was described in detail in the art [CZ 294274 (B6), WO 2005024101]. The spinning was performed at the electric tension of 25 kV.

Example 5 Polyurethane having the molecular weight 2000 (linear polycarbonate diol and alifatic isophoron-diisocyanate) (Larithane LS 1086 from the company Novotex, Italy) was spun from 15 weight % solution in dimethylformamide with 1 weight % tetraethylammonium bromide (referenced to polyurethane) by the electrostatic spinning method (electrospinning) on a laboratory model of instrument for the Nanospider technology. The spinning was performed at the electric tension of 25 kV.

Example 6

Polyvinyl alcohol having the hydrolysis degree of 80±8 % (Sloviol R - Chemicke zavody Novaky, Slovakia) was spun from 12 weight % aqueous solution together with glyoxal (3 weight %) and trihydrogenphosphoric acid (4.5 weight %) by the electrostatic spinning method (electrospinning) on a laboratory model of instrument for the Nanospider technology. The spinning was performed at the electric tension of

25 kV.

Subsequently, the nanofibrillar layer was heated to 140 °C for the period of 3 minutes, whereby crosslinking occurred and thereby the fibers were stabilized against dissolving in water.

Example 7 Copolymer of lactic acid and glycolic acid (type 7525DL HIGH IV from the company Lakeshore Biomaterials Birmingham, AL) was spun from 15 weight % solution in dichloromethane and acetone (4:1) by the electrostatic spinning method (electrospinning) on a laboratory model of instrument for the Nanospider technology. The spinning was performed at the electric tension of 25 kV.

Example 8

16.6 % ethanolic solution of the polymer of Example 1, the conductivity of which was adjusted to 260 μS/cm by the addition of saturated solution of sodium chloride, was spun by the electrostatic spinning method (electrospinning) on a laboratory model of instrument for the Nanospider technology. The surface tension of the polymer solution was 27.07 mN/m, the electric tension during the spinning process was 30 kV. The resulting nanofibrillar structure is shown in Fig. 4 (photograph from the microscope AQUASEM at a large magnification).

Example 9

16.1 % aqueous-ethanolic solution (66.3 % ethanol) of the polymer of Example 2, the conductivity of which was adjusted to 260 μS/cm by the addition of saturated solution of sodium chloride, was spun by the electrostatic spinning method (electrospinning) on a laboratory model of instrument for the Nanospider technology. The surface^" tension of the polymer solution was 26.9 mN/m, the electric tension during the spinning process was 34 kV.

Example 10 16.8 % aqueous solution of the polymer of Example 3, the conductivity of which was adjusted to 270 μS/cm by the addition of saturated solution of sodium chloride, was spun by the electrostatic spinning method (electrospinning) on a laboratory model of instrument for the Nanospider technology. The surface tension of the polymer solution was 26.2 mN/m, the electric tension during the spinning process was 31 kV.

Example 11

The nanofibrillar layer of Examples 1 and 4, having the area of 5 mm and thickness of 50 μm was dipped for one hour into a suspension of chondrocytes, marked by plasmatic staining CFDA, having the concentration of I xIO⁶ cells/ml and cultivated for 2 days under standard conditions for tissue culture cultivation (medium DMEM/F12 1:1, 10% FCS, penicillin+streptomycin, incubator with 5 % CO₂, 37 ⁰C). After this period, the nanofibrillar layer was densely covered by chondrocytes on both sides. Fig. 5 shows the growth of the cells on the upper (A) and the lower (B) side of the nanofibrillar layer.

Example 12

Onto a nanofibrillar layer prepared according to Examples 1 and 4 having the area of 5 mm² and the thickness of 50 μm, sewer-rat olfactory glial cells (OEG) were inoculated; the layer was rolled into a roll of the thickness of 1.5 mm and the length of 1 mm and inserted into the spinal cord of a laboratory sewer-rat. After 2 weeks, the cells of connective tissue and the blood vessels massively grew into the nanofibrillar roll implant. In the surrounding tissue, no inflammatory infiltration occurred. From proximal and distal surroundings of the implant (roll), the axis of which was identical with the axis of the spinal cord, also the fibers of neural cells grew through the implants, while in the implants that were perpendicular to the axis of the spinal cord, the fibers of neural cells grew through the implant only minimally (Fig. 6).

Example 13

Onto a nanofibrillar layer prepared according to Examples 1 and 4 having the area of 5 mm² and the thickness of 50 μm, sewer-rat stromal cells of bone marrow were inoculated in the concentration of I xIO⁴ / cm³. The cells were stained with phalloidin (red) and the nuclei were stained with DAPI (blue). In seven days, both cells cultures overgrew (comparatively to each other) the nanofibrillal layer comparatively to the growth on standard polystyrene adjusted for the growth of tissue cultures (Fig. 7).

Example 14

Five single nanofibrillar layers prepared according to Examples 1 and 4, having the area of 5 mm² and the thickness of 50 μm, were dipped for 1 hour into a suspension of human chondrocytes having the concentration of I xIO⁶ cells/ml and cultivated for 2 days under standard conditions for tissue culture cultivation (medium DMEM/F12 1 :1, 10% FCS, penicillin + streptomycin; incubator 5 % CO₂ and 37 °C) so that the chondrocytes have overgrown both sides of each layer of the non-woven nanofϊbrillar textile. The layers were then stacked onto each other, a uniform pressure was applied on them (lOg/cm²) and they were cultivated for the following three weeks under the same standard conditions as in the preceding cultivation. After this period, a sparse mesenchymal tissue that grew through all the nanofibrillar layers (Fig. 8) was formed in the material.

Example 15

Five single nanofibrillar layers prepared according to Examples 1 and 4, having the area of 5 mm² and the thickness of 50 μm, were dipped for 1 hour into a suspension of human stromal bone marrow cells having the concentration of l ^χ10⁶ cells/ml and cultivated for the period of 2 days under standard conditions for tissue culture cultivation (medium DMEM/F12 1 :1, 10% FCS, penicillin + streptomycin; incubator 5 % CO₂ and 37 °C) so that the cells have overgrown both sides of each layer of non- woven nanofibrillar textile. The layers were then stacked onto each other, a uniform pressure was applied on them (10 g/cm²) and they were cultivated for the following two weeks under the same standard conditions as in the preceding cultivation. After this period, the cells have interconnected the single layers of the nanofibrillar textile (Fig. 9).

Example 16

Onto a nanofibrillar layer prepared according to Examples 1 and 4 having the area of 1 cm² and the thickness of 50 μm, stromal cells of bone marrow and mesenchymal cells derived from adipose tissue were inoculated in the concentration of 1 ^χlθ⁵ / cm³. After 3 days of cultivation under standard conditions for tissue culture cultivation, the cells have overgrown the whole nanofibrillar layer; the display in a confocal microscope after phalloidin staining has shown that the cells overgrew confluently the nanofibrillar layer (Fig. 10A) and extended their extremities into the nanofibrillar layer (Fig. 10B). In the areas in which the nanofibrillar layer was crinkled, the cells have overgrown its surface (Fig. 10C); the cells have grown also on the edge of the nanofibrillar layer, where the concentration of the cells was lowest (Fig. 10D).

Example 17 The same experiment as that of example 16 was repeated with a nanofibrillar layer prepared according to Examples 3 and 10 having the area of 1 cm² and the thickness of 50 μm. The 3D display in a confocal microscope after staining the cells with phalloidin has shown a similar growth as in the preceding example (Fig. 10E); in the areas on the edge, isolated cells were growing on the layer surface (Fig. 10F).

Example 18

On nanofibrillar layers prepared according to Examples 2 and 9 (Fig. 1 IA) and 3 and 10 (Fig. HB), mesenchymal cells derived from adipose tissue of a laboratory sewer- rat were cultivated under standard conditions for tissue culture cultivation for 4 days. After that, the cells were stained with phalloidin. The cells have overgrown confluently the nanofibrillar layer.

Example 19 10 single nanofibrillar layers prepared according to Examples 1 and 4 having the area of 10 mm and the thickness of 50 μm were dipped for 1 h into a suspension of human chondrocytes having the concentration of I xIO⁶ cells/ml and cultivated for the period of 2 days under standard conditions for tissue culture cultivation (medium DMEM/F12 1 :1, 10% FCS, penicillin + streptomycin; incubator 5 % CO2 and 37 °C) so that the chondrocytes have overgrown both sides of each layer of non-woven nanofibrillar textile. The layers were then stacked onto each other, an uneven pressure was applied (0-20 g/cm ) onto them, and they were cultivated for the following three weeks under the same standard conditions as in the previous cultivation. After this period, a sparse mesenchymal tissue that grew through all the nanofibrillar layers was formed and the deposition of extracellular matrix occurred in the parts of the material onto which the pressure higher than 5 g/cm² was applied (Fig. 12A); in the parts of the material onto which the pressure was not applied, the layer interconnection did not occur (Fig. 12B).

Example 20

Onto a nanofibrillar layer prepared according to Examples 1 and 4 having the area of 1 cm² the primoculture cells of olfactoric glia of laboratory sewer-rat were inoculated. This layer was placed in half-height of a test tube filled with cultivation solution (medium DMEM/F12 1 :1, 10% FCS, penicilin+streptomycin). The test tube was placed into an incubator (37 °C, 5% CO₂). After two hours, a piece of filtration paper containing 2 μg of NT3 factor and 2 μg of a dye (phenol red) was placed on the surface of the cultivation solution. For the period of 4 hours, the test tube was left on one place and there was no manipulation with it. After that period, the dye gradient was visible in the test tube, having the maximum at the surface of the solution and the minimum on the bottom of the test tube. The nanofϊbrillar layer containing the cells was fixed, stained with fluorescent antibodies for p75 receptor and the intensities of the staining of the cell membranes on the side facing the source of NT3 factor and on the averse side were determined. With the aid of picture analysis, the ratio of the fluorescence intensities of the cell membranes was determined to be 2.3: 1 that shows the functional polarization - a different distribution of p75 receptors on the cell membranes facing the source of the growth factor NT3 and those on the averse side.

Claims

1. A biomaterial based on nanofibrillar layers, characterized in that it consists of at least one nanofibrillar layer and living cells firmly connected with said nanofibrillar layer, wherein the nanofibrillar layer is formed by synthetic polymers or copolymers of monomers selected from the group comprising methacrylic acid esters, methacrylic acid amides, polyurethanes, polyvinyl alcohols and polymers derived from lactic acid and its derivatives.

2. The biomaterial according to claim 1, characterized in that the nanofibrillar laye is non-woven.

3. The biomaterial according to claim 1 or 2, characterized in that the monomers are selected from the group comprising 2-hydroxyethyl methacrylate, 2-ethoxyethyl methacrylate and 2-hydroxypropyl methacrylamide.

4. The biomaterial according to any of claims 1 to 3, characterized in that the cells are selected from the group comprising the cells of connective tissue, epithelial cells, parenchymal organ cells and mesenchymal stem cells derived from bone marrow or adipose tissue.

5. The biomaterial according to claim 4, characterized in that the cells are selected from the group comprising chondrocytes, fibroblasts, hepatocytes and mesenchymal stem cells derived from bone marrow or adipose tissue.

6. The biomaterial according to any of claims 1 to 5, characterized in that it consists of at least two nanofibrillar layers covered on both sides confluently by living cells, whereas these layers are interconnected by the growth of the cells.

7. The biomaterial according to any of claims 1 to 5, characterized in that it consists of one nanofibrillar layer covered with living cells on one side, wherein the cells are functionally polarized.

8. A method of preparation of the biomaterial according to claim 1, characterized in that a synthetic polymer or copolymer of monomers selected from the group comprising methacrylic acid esters, methacrylic acid amides, polyurethanes, polyvinyl alcohol and polymers derived from lactic acid and its derivatives is prepared and the thus prepared synthetic polymer or copolymer is spun by an electrostatic spinning method (electrospinning), subsequently on the spun layer of the synthetic polymer or copolymer, prepared by the electrostatic spinning method, cells are inoculated and cultivated under standard conditions for tissue culture cultivation until a confluent layer of cells is achieved.

9. The method according to claim 8, characterized in that the monomers are selected from the group comprising 2-hydroxyethyl methacrylate, 2-ethoxyethyl methacrylate and 2-hydroxypropyl methacrylamide.

10. The method according to claim 8 or 9, characterized in that the cells are selected from the group comprising the cells of connective tissue, epithelial cells, parenchymal organ cells and mesenchymal stem cells derived from bone marrow or adipose tissue.

11. The method according to claim 10, characterized in that the cells are selected from the group comprising chondrocytes, fibroblasts, hepatocytes and mesenchymal stem cells derived from bone marrow or adipose tissue.

12. The method of preparation of the biomaterial of claim 6, characterized in that a synthetic polymer or copolymer of monomers selected from the group comprising methacrylic acid esters, methacrylic acid amides, polyurethanes, polyvinyl alcohols and polymers derived from lactic acid and its derivatives is prepared, the thus prepared synthetic polymer or copolymer is spun by an electrostatic spinning method, subsequently on the layer of the synthetic polymer or copolymer spun by the electrostatic spinning method, the cells are inoculated and cultivated under standard conditions for tissue culture cultivation until a confluent coverage of the nanofibrillar layer is achieved, and then at least two layers of the spun synthetic polymer or copolymer, overgrown with the cells, are manually or automatically stacked onto each other and onto this system of layers, pressure of 5 to 20 g/cm³ is applied and the system is cultivated under standard conditions for tissue culture cultivation for the period of 1 to 4 weeks.

13. The method according to claim 12, characterized in that the monomers are selected from the group comprising 2-hydroxyethyl methacrylate, 2-ethoxyethyl methacrylate and 2-hydroxypropyl methacrylamide.

14. The method according to claim 12 or 13, characterized in that the cells are selected from the group comprising the cells of connective tissue, epithelial cells, parenchymal organ cells and mesenchymal stem cells derived from bone marrow or adipose tissue.

15. The method according to claim 14, characterized in that the cells are selected from the group comprising chondrocytes, fibroblasts, hepatocytes and mesenchymal stem cells derived from bone marrow or adipose tissue.

16. The method of preparation of the biomaterial of claim 7, characterized in that a synthetic polymer or copolymer of monomers selected from the group comprising methacrylic acid esters, methacrylic acid amides, polyurethanes, polyvinyl alcohols and polymers derived from lactic acid and its derivatives is prepared, the thus prepared synthetic polymer or copolymer is spun by an electrostatic spinning method, subsequently on the layer of the synthetic polymer or copolymer spun by the electrostatic spinning method, the cells are inoculated and cultivated under standard conditions for tissue culture cultivation, whereby the layer of the spun synthetic polymer or copolymer is in the course of the cell cultivation process placed on the interface of two environments differing in their physical or chemical properties.

17. The method according to claim 16, characterized in that the monomers are selected from the group comprising 2-hydroxyethyl methacrylate, 2-ethoxyethyl methacrylate and 2-hydroxypropyl methacrylamide.

18. The method according to claim 16 or 17, characterized in that the cells are selected from the group comprising the cells of connective tissue, epithelial cells, parenchymal organ cells and mesenchymal stem cells derived from bone marrow or adipose tissue.

19. The method according to claim 18, characterized in that the cells are selected from the group comprising chondrocytes, fibroblasts, hepatocytes and mesenchymal stem cells derived from bone marrow or adipose tissue.