Patent Description:
Prostheses of heart valves may be manufactured from artificial materials - then they are known as mechanical prostheses, and from biological materials - then they are known as biological prostheses.

For patients with valvular defects in whom all methods of conservative treatment were applied, the only way of therapy is the implantation of heart valve prosthesis.

Problems related to the implantation of these protheses are, above all, degeneration of biological prosthesis and clotting on mechanical prosthesis. The valve built of BC is a solution for these problems, since, which was proved by the research conducted, no clotting takes place on its surface and this material is characterised by high durability in comparison to natural tissues.

Prostheses currently available on the market are very expensive, so, as a result, the choice is not only dependent on the biological qualities but also on the costs related to the implantation of various prostheses. The solution proposed by us provides the doctor and the patient with the possibility of application of a cheap and safe treatment.

The currently known and used valves possess multiple faults. They require an additional treatment, i.e., anti-clotting treatment, they are subject to degeneration and, therefore, a new material is being searched for.

The descriptions of inventions present inter alia an elastic stent of biologically joined heart valves which allows a concurrent implantation of biological prosthesis of aortic valve and mitral valve - <CIT>. <CIT> discloses an upgraded tricuspid mechanical valve, in which the casing of the valve has an articulated mechanism, what allows the spinning of cusps and their supporting. Another invention description <CIT> discloses a mechanical tricuspid heat valve.

The utility of BC in the healing of various wounds was already proven in the <NUM>. As regards wound dressing and superficial/surface application, BC is a non-toxic, non-irritating, hypoallergic, non-pyrogenic and biocompatible material.

BC may become the material used for manufacturing of durable, inner implants (vascular prostheses and heart valve prostheses), used especially in vascular surgery and cardio surgery.

Bacterial cellulose is a non-toxic, non-irritating, hypoallergic, non-pyrogenic and biocompatible material. It is characterised by a high mechanical strength.

There are many attempts to substitute the currently used cardio-vascular implants, made from zoonotic and artificial materials, with BC membrane prostheses.

A great value of BC is its impermeability even for the slightest blood cellular elements (the diameter of thrombocytes reaches the values of <NUM> - <NUM> and the size of the bio cellulose fibre net reaches from <NUM> to <NUM>). The surface of BC is therefore completely smooth for blood, which reduces the possibility of clotting. This attribute should also make the outgrow of BC through the own cells of the host organism impossible.

To date, the inventions concerning BC in situ modification methods were described, and this modification method is based on the application of chemical additives to the culture medium. In biomedicine, these inventions are applied most frequently as new generation dressing materials especially in hard to heal wounds and as a material substituting natural tissues, inter alia cartilage tissue.

The description of patent <CIT> presents a method for obtaining a modified BC membrane, by adding a culture medium of poly-amino-saccharides, especially chitosan, in a microcrystalline form, of an average molecular weight of <NUM> kDa and deacetylation degree of <NUM>-<NUM>%, in a quantity of <NUM>,<NUM>-<NUM> parts by weight. The BC membranes obtained according to the invention possess glucosamines in the chain, formed as a result of the degradation of poly-amino-saccharides or their derived substances. The composite material derived in this manner is characterised by bioactivity and biocompatibility desired for biomedical application and it is biodegradable.

The description of patent application <CIT> discloses a method for manufacturing a modified BC, through the application of carboxymethylcellulose (CMC) as an additive to the culture medium, used in the quantity of <NUM>% - <NUM>% w/v. The BC membranes created in the presence of CMC are characterised by higher dry and wet weight, as well as increased water absorbency in comparison to BC membranes synthetized without the addition of CMC.

In patent <CIT> a method of modification of BC membranes which consists in enriching a culture medium with <NUM>-<NUM>% CMC, and then, after the end of cultivation, oxidizing the membrane with <NUM>% solution of sodium periodate at a temperature of <NUM> for <NUM> hours is presented. The membranes obtained are characterised by increased water absorbency and a higher water retention capacity, as a result of which a thicker, better hydrated material is created.

From the description provided in patent application <CIT> we know a dressing obtained from BC, coated with nano silver, which provides polymer with antibacterial properties. The invention discloses the obtaining of cellulose fibres with the use of Acetobacter xylinum BPR <NUM> strain. After cleaning, the cellulose fibres are treated, in this order, with <NUM> sodium periodate, <NUM>% amino thiourea in acetic acid, <NUM>% silver protein in <NUM>% sodium borate and silver salt in ammonia water at a temperature of <NUM>. The nano silver molecules formed as a result of the series of reactions cover BC fibres.

In the description of patent <CIT>a method for the manufacturing of biomaterial with cartilage properties with the use of the BC obtained in the process of cultivation of G. xylinus bacteria on a stationary medium is disclosed. After obtaining and cleaning, the BC membrane is modelled into a spatial structure of a desired shape and subjected to modification consisting in treating it with <NUM>% aqueous sodium lye solution, rinsing in distilled water, <NUM>% aqueous acetic acid solution and repeated rinsing in distilled water until the cellulose material reaches pH <NUM> - <NUM>. The material obtained this way has a very high mechanic strength, corresponding to the natural cartilage tissue, is biocompatible with human body and is non-allergic.

For applications in cardio surgery and vascular surgery, the materials manufactured as described above are unsuitable because they may be characterised by reduced human biocompatibility, inadequate mechanical properties, inadequate morphology and accelerated biodegradability.

In the description of patent application <CIT> (<CIT>), a heart valve prosthesis built up on a frame stent with leaflets made of particularly treated BC is presented. The method of preparing cellulosic material described in patent application <CIT> consists in producing a molded body made of bacterial cellulose, including the steps of mechanically pressing parts of the parts of molded body at temperatures in the range of <NUM> ° C to <NUM> ° C and pressing in the range of <NUM> to <NUM> MPa by <NUM>-<NUM>, treatment of the produced material with a solution consisting of: <NUM> weight % to <NUM> weight % glycerol and <NUM> weight % to <NUM> weight % alcohol / water mixture and drying the treated molded body. This technique is different from the method described in the present application for modifying the cellulose film which consists in initial drying and additional final soaking of the prepared structure in endotoxin-free water. The process of additional soaking of previously dried cellulosic material is important and has a significant impact on its strength parameters.

In the description of patent application <CIT> (<CIT>), a heart valve prosthesis molded on an expanding frame is presented. The cellulose material used as a valve described in <CIT> is a composite system - cellulose material coupled to the frame of a defined structure. In addition, composite materials based on bacterial cellulose with the addition of silicone in a ratio of <NUM> : <NUM> (% by weight) have been described. Claim <NUM> describes a method of forming cellulosic material in a glass container by pouring a cellulose-based mixture.

In the description of patent application <CIT> a heart valve molded on a stent as well as a particular BC treatment are presented. The description in <CIT> relates to a method of modifying cellulosic material of plant origin in order to obtain a structure suitable for producing a heart valve. It is important whether the cellulose is of plant or bacterial origin because of the chemical and mechanical properties that the particular material possess.

The invention described in <CIT> relates generally to a new and improved heart valve tissue pattern and heart valve for semilunar heart valve reconstruction. The physical profile of the valve of the present invention is a characteristic defined in part by the length of the lobes. The unique two-dimensional trefoil tissue pattern of the invention can be made from any appropriate materials such as, for example, autologous tissue including, for example, pericardium, fascia lata, rectus sheath (the fibrous tissue enveloping the abdominal muscles). Additional acceptable materials include heterograft (bovine, porcine, or other animal tissue) pericardium, synthetic materials, bioengineered materials, or any other like or suitable materials having appropriate plasticity and characteristics, as will be appreciated by those skilled in the art.

The method of modification of BC film in order to use it as a material for the production of bio prostheses in the circulatory system, obtained during stationary cultivation with the use of G. xylinus strain is characterised, according to the invention, in that sterile BC membranes are dried to a constant mass at room temperature of not more than <NUM>, then soaked in sterile distilled water at room temperature for no more than <NUM> minutes and stored under sterile conditions.

A variation of the invention is the BC film dried to a constant mass at room temperature of no more than <NUM> and then exposed to UV-C radiation with a total power of 12W and a maximum emission of <NUM> for a period of no more than <NUM> minutes. After exposure, the films are soaked in sterile distilled water at room temperature for a period not exceeding <NUM> minutes and stored at sterile conditions.

Due to the use of the modification method according to the invention, it is possible to obtain a biocompatible material with the properties comparable to the natural tissues building walls of blood vessels or valves, used in cardio surgery and vascular surgery for the production of bioimplants. In contrast to unmodified membranes, the material modified according to the invention is characterized by a water content similar to natural tissues and much higher mechanical strength. The material obtained according to the invention also has increased resistance to degradation processes in the human body.

The heart valve forming element is made of one sheet of biocompatible material, favourably polymer, favourably BC and is a flat, favourably biconnected figure, favourably with three axes of symmetry intersecting at one point, with a centrally made hole with three equal sides, not necessarily straight, forming a figure with the symmetry analogous to that of an equilateral triangle. The axes of the symmetry of the element and the hole coincide. The edges of the hole are the first internal sides of the first areas of the element, and the first areas of the element being essentially quadrilateral in shape. Between the first areas of the element there are, directly adjacent to them, second areas of the element, whose outer edges are favourably rounded convexly. The second sides of the first areas are generally perpendicular to the first sides of the first areas, with the ratio of the length of the first side of the first area to the second side of the first area being between π/<NUM> and 2π/<NUM>. The measure of the angles between the second and third sides of the first areas is 2π/<NUM> to 5π/<NUM>. The length of the outer sides of the first areas is at least equal to the length of the first inner sides of the first areas. The ratio of the radius of the circle coinciding with the centre of the symmetry of the hole and tangent to the outer side of the first area at mid-height to the length of the second side of the first area is between 2π/<NUM> and 5π/<NUM>.

The object of the invention is to provide a new heart valve and a new method of BC modification as the material which the valve is made of. Moreover, the invention also aims to use the new valve in cardiac surgery.

The object of the invention is an element for manufacturing a heart valve which contains:.

The element is preferably a bi-coherent figure.

The element where the axes of symmetry of the element and the hole overlap.

The element where the first areas meet the second areas on the outer side of the element and the element has strengthening knobs, which are favourably semi-circular.

The object of the invention is a method for producing the modified BC film defined above by using a G. xylinus strain, where:.

The method in which dried BC is exposed to UV-C radiation using lamps with a total power of 12W and maximum emission at <NUM>, for a period of <NUM> minutes, while maintaining sterility.

The method in which dried BC film is soaked in sterile distilled water at room temperature for up to <NUM> minutes and stored under refrigerated conditions while remaining sterile.

The method where, after exposure, it is soaked in sterile distilled water at room temperature for up to <NUM> minutes and stored under refrigerated conditions while remaining sterile.

The object of the invention is also an implantation kit, which contains the element specified above for use as a heart valve in cardio surgery.

The invention is illustrated by the following examples, which do not limit the scope thereof.

Sterilized BC films were laid on a flat surface and dried to constant weight at room temperature not exceeding <NUM> (convection drying, carried out in a dryer without forced circulation at 25ºC. The drying agent was atmospheric air). The films were then packed and sterilized in an autoclave at <NUM> for <NUM> minutes. Dried BC films were stored sterile.

BC films dried as in Example <NUM> were exposed to UV-C radiation with lamps with a total power of <NUM> W and maximum emission at <NUM>. Dried BC films were placed <NUM> away from the lamp and exposed for <NUM>. BC films were stored sterile.

BC films prepared as in Example <NUM> were soaked in sterile distilled water at room temperature for up to <NUM> minutes and stored under refrigerated conditions, while sterile.

A heart valve component is made of a single sheet of biocompatible material, for example BC, as shown in <FIG>.

The element for manufacturing a heart valve consisting of a flat sheet of BC with three axes of symmetry, having a centrally made hole <NUM> with three equal sides which form a figure with the symmetry of an equilateral triangle, such that.

The edges of the central hole <NUM>, which is an equilateral triangle, are the first sides A of the first areas I of the element. The first areas I of the element are similar in shape to a quadrilateral whose outer edge is convexly rounded. The arc of this rounding at its highest point is tangential to a circle having radius R equal to the length of the edge A, and of the centre coinciding with the centre of the central hole <NUM>. Between the first areas I of the element there are immediately adjacent second areas II of the element II whose outer edges are convexly rounded.

The second H sides of the first areas I are perpendicular to the edge A of the central hole <NUM>. The measure of the angle β between the second sides <NUM> is 2π/<NUM> and the relationship between the length of the edge A and the length of the second H side of the first area I is <MAT>.

From the element created by a suitable, known anastomosis, for example stitching, a valve is formed such that the second areas of the second element bend outwards and form T-shaped fasteners in the tube in which the valve is to operate, as shown in <FIG> in a top view <MAT> <MAT> <MAT> <MAT>.

The element for manufacturing a heart valve as shown in <FIG> is made of a single flat sheet of BC with three axes of symmetry, having a centrally made hole <NUM> with three equal sides which form a figure with the symmetry of an equilateral triangle, such that.

The element for creating the heart valve as shown in <FIG> is manufactured as in Example <NUM>, but for an angle of β of 2π/<NUM>, the relationships between the radius of the circle R and the length of the edge A of the central hole <NUM> and the length of the other side H are: <MAT> <MAT> <MAT> <MAT> <MAT>.

From the element created by a suitable, known anastomosis, for example stitching, a valve is formed such that the second areas of element II are bent outwards and form T-shaped fasteners in the tube in which the valve is to operate, as shown in <FIG> in a top view.

The element as in Example <NUM> was cut out from BC obtained as in Examples <NUM>-<NUM>, additionally forming reinforcing cusps C. During the stitching process, the reinforcing cusps C are bent downwards, to the outer side of the valve, as schematically shown in <FIG>.

The suture method involves determining three points every <NUM> degrees, on the circumference of the circle in the tube in which the valve is to operate. This ring will be the place where the edges of the central opening of the element will be connected with the tube in which the valve will function. The tube may be made of artificial material or natural tissues.

The connection between the edges of the central opening and the ring may be made by continuous or interrupted surgical suture.

The areas of the element marked on <FIG> with number II constitute "T" shaped overlaps by connecting them with the wall of the tube according to the diagram in <FIG>. The height of this connection is determined by H, counted from the points of stitching of the corners of the central hole upwards on the wall of the tube, evenly located above the points of the corners.

In this way the lower level of the connection between the valve inside the tube's light and the element remains completely tight - impermeable to liquid, and the stitching of "T" shaped overlaps from the top will cause the liquid flowing from the top to the bottom in the tube to produce a tight adherence of the areas of the element numbered I in <FIG>. The liquid flowing inversely will cause the free opening of the areas I and thus the free flow of the liquid only in this direction.

Modifications of BC aiming at obtaining a material resistant to enzymes and selected pathogenic microorganisms and determining the influence of these modifications on other properties of the polymer.

Resistance of modified BC to in vitro degradation was investigated by determining the changes occurring in the polymer during incubation of samples at 37oC in a solution simulating physiological fluids in the absence and presence of Aspergillus fumigatus.

BC samples (measuring <NUM> x <NUM>) modified by drying at room temperature and soaking in water and by drying at room temperature, exposed to UV radiation and soaked in water were placed in sterile bottles of <NUM> filled with <NUM> of sterile SBF liquid. For the study of mechanical properties, modified BC strips (<NUM> x <NUM>) were placed in <NUM> of the solution in sterile bags. A sufficient amount of A. fumigatus liquid fungus culture was added to a part of the samples immersed in SBF solution so that the initial number was about <NUM> cfu per <NUM> of liquid. Degradation changes of BC samples were examined after <NUM>, <NUM>, <NUM>, <NUM> days from the beginning of incubation.

Determination of BC biodegradability degree:.

The results of the experiment were developed using the statistical program SigmaPlot <NUM> (SYSTAT Software, Germany), with ANOVA one-way analysis of variance for significance level p<<NUM>.

The wet mass of BC samples modified by drying at room temperature and soaking in water and then stored in sterile SBF fluid for <NUM>, <NUM> and <NUM> days increased twice, and after <NUM> days of incubation no statistically significant differences were observed. In the case of samples incubated in the presence of A. fumigatus, greater changes in wet mass were observed than in the case of BC samples stored under sterile conditions. After only <NUM> days of incubation, wet mass increased about <NUM>-fold, and after <NUM> days - <NUM>-fold.

Different results were obtained during storage of BC samples modified by drying at room temperature, UV radiation and soaking in water. In this case, incubation in sterile SBF fluid resulted in a decrease in the content of BC wet mass by approx. <NUM>% (<FIG>), and incubation in SBF fluid in the presence of A. fumigatus resulted in a decrease in this content by approx. <NUM>% only after <NUM> days of incubation.

The stress at a break (σ) of non-incubated BC samples modified by drying at room temperature and soaking in water was about <NUM> MPa and the relative elongation at break (ε) about <NUM>%. In the case of samples modified by drying at room temperature, UV radiation and soaking in water, σ of the non-incubated membranes was about <NUM> MPa and ε about <NUM>%.

Storage both in sterile SBF fluid and in the presence of A. fumigatus deteriorated the mechanical strength of BC membranes modified by drying at room temperature and soaking in water. Already after <NUM> days of incubation in sterile SBF fluid a decrease in σ by about <NUM>% was observed, after <NUM> days by about <NUM>%, and after <NUM> and <NUM> days by about <NUM>%. Incubation in the presence of A. fumigatus for the period of <NUM> days caused a similar decrease in σ as in the case of fungus -free incubation by about <NUM>%, but after <NUM> and <NUM> days σ decreased significantly more, by <NUM> and <NUM>% respectively. ε of the samples did not change during the whole incubation period in sterile SBF fluid, whereas in the presence of A. fumigatus it decreased by about <NUM>% after <NUM> days of incubation and by about <NUM>% after <NUM> days. The values of σ and ε of BC samples dried at room temperature, exposed to UV radiation and soaked in water and then incubated in sterile SBF fluid did not change in comparison to non-incubated samples, except for ε samples stored for <NUM> days, the value of which increased by about <NUM>%. On the other hand, BC samples incubated in SBF fluid in the presence of A. fumigatus showed about <NUM> and <NUM>% lower σ value after <NUM> and <NUM> days of incubation respectively, in comparison to radiated and non-incubated samples. ε of these samples did not change, except for samples incubated for <NUM> days, which increased by about <NUM>%.

When analysing the diffractograms of all modified BC samples, <NUM> characteristic diffraction bands were observed at the reflective angle of 2θ <NUM>°, <NUM>° and <NUM>°. The incubation of the samples under the conditions simulating body fluids did not affect the position of diffraction lines but caused a change in the intensity of diffraction bands at the angles of reflection 2θ <NUM>° and <NUM>°. Additionally, it was noted that in the case of the diffractogram of BC dried at room temperature and soaked in water and then stored for <NUM> days in both sterile SBF and liquid SBF in the presence of A. fumigatus, the intensity of the diffraction line is increased at an angle of 2θ <NUM>°. The crystallinity degree (Cr. ) of all modified BC samples calculated on the basis of the formula of Segal et al. (<NUM>) was about <NUM>%.

Changes in BC crystallinity caused by biodegradation of samples modified by drying at room temperature and soaking in water, drying at room temperature, UV radiation and soaking in water were compared. The crystallinity degree of all tested samples differed only slightly. The exception was BC dried at room temperature and soaked in water and then kept for <NUM> days in the presence of fungus. In this case, a decrease in Cr. by about <NUM>% was observed in comparison with the non-incubated sample.

The decomposition temperature of BC that is not incubated, dried at room temperature, UV-radiated and soaked in water was approximately <NUM> lower than that of BC that is dried at room temperature and soaked in water. A lower decomposition temperature indicates lower thermal stability of the material, probably due to greater susceptibility to biodegradation of the material. SEM images of the surface of all BC samples modified and then incubated in sterile SBF fluid did not show any differences compared to the surface of non-incubated samples. Hardly visible, thin fibres can be observed on the sample surface. A more compact structure of BC samples incubated in sterile SBF fluid may be the reason for its increased mechanical strength in comparison with the samples incubated in SBF in the presence of A.

Obtaining BC with greater resistance to biodegradation characteristic for bio-prostheses of blood vessels and aortic valves.

Mechanical testing of BC and BC-based composite materials.

The conducted research allowed to choose BC with no worse tensile strength than natural tissues of the swine cardiovascular system. In order to achieve this, BC sheets of <NUM> x <NUM> in size were subjected to a tensile test. The reference material were natural tissues: aorta, aortic valve and pericardial sac fragments. Natural tissues were properly prepared and supplied by the Medical University of Gdansk. The tissues were divided into two groups. The first group consisted of tissues stored in physiological saline solution, from the moment the samples were delivered to the moment the tensile test was performed. However, the second group consisted of tissues additionally washed with <NUM>% glutaraldehyde for <NUM> minutes prior to the tensile test.

The material is: Native BC, Modified BC and Composite BC-Polyvinyl alcohol, BC-hyaluronic acid.

The tensile test was carried out on an INSTRON model <NUM> testing machine with a single-axis tensile velocity of <NUM>/s. The distance between the jaws for BC stretching was <NUM>.

Studies show that thermally modified BC, i.e. dried "s" and then soaked, has the best tensile strength of all bio-nanocellulose materials and has a strength of <NUM> MPa. Native (homogeneous) BC and BC-based composite materials have a tensile strength of about <NUM> MPa, which is lower than that of natural materials. The value of approx. <NUM> MPa corresponding to thermal modified BC allows for further strength testing (tear test, fatigue test) on a selected BC material, which may be a potential material used in cardio surgery and vascular surgery.

The susceptibility of BC to in vitro degradation in simulated body fluid (SBF at <NUM>) was determined. Various methods of monitoring degradation changes were applied, e.g. determination of dry and wet mass changes, determination of biomaterial hydrolysis products using liquid chromatography and thermal stability of BC. The surface of biomaterial was evaluated by scanning electron microscopy (SEM), and the development of microorganisms deliberately introduced into the environment in which BC was incubated was also monitored.

During the storage of BC samples in sterile SBF fluid and PSB buffer no changes in dry matter were found for the whole <NUM>-month storage period. Furthermore, in the presence of S. aureus bacteria and C. albicans yeast, the dry matter of BC samples remained at a similar level. Significant decrease in BC dry matter was observed only in the samples incubated for <NUM> months in the presence of A. fumigatus fungus - BC dry matter decreased by <NUM>%. In the case of wet mass, it was found that it significantly increased after the second month of storage, regardless of the conditions (PBS buffer, SBF liquid in the presence and absence of microorganisms).

Moreover, in the presence of S. aureus bacteria and C. albicans yeast, the dry matter of BC samples remained at a similar level. Significant decrease in BC dry matter was observed only in the samples incubated for <NUM> months in the presence of A. fumigatus fungus - BC dry matter decreased by <NUM>%. In the case of wet mass, it was found that it significantly increased after the second month of storage, regardless of the conditions used (PBS buffer, SBF liquid in the presence and absence of microorganisms).

It was also shown that during the storage of BC samples, the growth of all tested microorganisms occurred. After <NUM> month, the number of S. albicans and A. fumigatus cells increased from about <NUM><NUM> to <NUM><NUM> cfu/cm<NUM> and remained at the same level for up to <NUM> months, which indicates that they were in the stationary phase of growth. The growth of microorganisms and wet BC mass indicates that degradation changes occur in this material, although its dry mass does not change significantly (except for the samples containing A. fumigatus fungi). It was also found that during the storage of samples on the BC surface only A. fumigatus fungi formed a biofilm on the surface. The concentration of saccharides, which are the products of BC hydrolysis, in the post-incubation fluids was so low that they could not be detected by thin layer chromatography (TLC). After a <NUM>-fold concentration of these fluids, both from samples with and without S. aureus bacteria and C. albicans yeasts, no BC hydrolysis products were found. However, they were present (though in small amounts) in a concentrated post-incubation fluid after only one month of BC treatment with A. fumigatus fungi. When analysing the obtained results, it can be stated, however, that the investigation of the presence of cellulose hydrolysis products in post-incubation fluids is not a good method to determine the degree of polymer biodegradation, because microorganisms can metabolize simple sugars, disaccharides and oligosaccharides produced in this process.

The storage of samples in PBS buffer and sterile SBF fluid for the period of <NUM> and <NUM> months resulted in a decrease in BC decomposition temperature by about <NUM>, which indicates degradation processes and reduction of water adsorbed on its surface by half on average. Similar changes were observed in the samples stored in the presence of S. aureus bacteria and C. albicans yeasts. However, the samples stored in the presence of A. fumigatus fungus for <NUM> months had thermal stability decreased by approx. The analysis of thermograms of the samples incubated in SBF fluid for <NUM> months in the presence and absence of microorganisms also showed an additional effect indicating the loss of chemically combined water.

Microscopic observations showed that the morphology of BC film surface after incubation in simulation fluids for the period of <NUM>-<NUM> months did not change significantly, it only became more porous.

The description of the tests that have been performed to assess the properties of BC.

The aim of the task was to assess the biomechanical properties of BC material in vitro.

For each test, single-axis tear tests were performed using Tytron <NUM> Microforce Testing System (MTS) with a <NUM> N force transducer. Percentage strain was measured using a video extensometer (Messphysik). For a proper strain analysis, appropriate markers were used to determine the L0 and L1 parameters of the specimen during breaking test. Before each test, the extensometer was calibrated with the use of standards provided by the manufacturer.

For proper measurement, efforts were taken to maintain the width to length ratio of the specimen <NUM>:<NUM>. Each time prior to the test, the thickness of the specimen was measured, which was an input to automatically obtain the cross-sectional area value and thus automatically recalculate the elasticity. In the case of tensile tests, the sample was preloaded with <NUM> N and the breaking test started from this value each time the test started.

The elasticity, percentage of real strain and breaking energy were measured. Taking into account the anisotropic character of BC material, the tests were performed in two conventional peripheral and axial directions. Prior to the tests, the tested material was weighed each time; it resulted from the fact that BC has strong hydrophilic properties, which allowed to obtain information on the extent to which the biomechanical parameters could be influenced by the degree of hydration of the sample.

Viscoelastic properties of BC samples were also tested by hysteresis. Prior to the test, the specimens were preloaded to <NUM> N, then stretched to <NUM>% strain and maintained for <NUM> seconds and then returned to their initial values. The hysteresis value was measured as the difference between the input and output energy.

Dynamic fatigue test was performed at <NUM> in an environmental chamber filled with DMEM/F12 medium supplemented with <NUM>% serum and antibiotics. Similarly to the tensile tests, BC samples were weighed before and after the completion of the test. The test was performed at <NUM> amplitude at <NUM> at <NUM>,<NUM> cycles. At the end of the fatigue test, the hysteresis of the specimen was determined each time.

The elasticity, strain and breaking energy differed depending on the direction of the specimen arrangement (<FIG>,<FIG>).

Hysteresis tests indicate that BC has poor viscoelastic properties. Due to the anisotropic character of the BC samples, the stiffness of the material was observed in a dynamic fatigue test (<FIG>).

Biomechanical properties indicate that the studied BC material has much higher stiffness in relation to human and swine tissues (pulmonary and aortic valves). The BC material has high hydrophilicity.

Features of the obtained valve:
For the material which is flat, flaccid and of the thickness between <NUM> and <NUM> micm as well as impenetrable for the morphotic components of blood:.

The study was performed on fresh heparinized swine blood obtained during traditional slaughter of animals in a slaughterhouse. A total of <NUM> experimental sessions were conducted - <NUM> without BC (control) and <NUM> with the use of BC (experimental). At half-hour intervals, activated coagulation time (ACT), total haemoglobin (Hb), free haemoglobin (fHb), erythrocyte count (RBC) and haematocrit (HTC) were examined.

ACT monitoring was an element of the methodology applied, i.e. the Schima test. This parameter, which is an indicator of the tendency of blood to coagulate, determines the moment of termination of the experiment and is not useful in itself for the assessment of the degree of haemolysis or thrombogenicity of the tested material. HCT, RBC and Hb changed slightly during the study and did not exceed the norms given in the literature for swine. fHb in plasma is closely correlated with the haemolysis process in the circulating medium which was studied. During the studies (both control and with the use of BC) it was subject to a gradual, systematic increase.

The comparison of fHb growth dynamics during control and experimental studies allowed to make a conclusion concerning the influence of BC on haemolysis. Since it is affected by both blood parameters (haematocrit, initial concentration of free haemoglobin) as well as pumping time, the obtained data required standardization. For this purpose, IH index (Free Haemoglobin Index) was used to determine an increase in plasma free haemoglobin content in mg/l of pumped blood. This is particularly important due to differences in the duration of individual trials. IH was calculated with the formula: <MAT>.

The mean (M±SD) haemoglobin index for control samples was equal to: IH = <NUM>±<NUM>, and for experimental IH = <NUM>±<NUM>, while the mean (M±SD) circulating medium flow time (M±SD) for control samples T = <NUM>±<NUM>. , and for experimental T = <NUM>±<NUM>. At the same time the differences between these mean pairs (control/experiment) turned out to be statistically insignificant (p><NUM>).

It is worth noting that despite a slightly longer time of pumping blood, a contact with BC resulted in a lower haemolysis index than for control samples.

Thrombogenicity testing according to the Schima protocol is limited to the assessment of the extent to which the surface of the tested material is covered by thrombi. Macroscopic evaluation of BC fragments after the completed experiments in most cases showed the presence of relatively small number of red thrombi, especially on the surface in direct contact with flowing blood. The number of observed thrombi were even lower on the "opposite" surfaces of scraps loosely adjacent to the walls of flow channels of the equipment.

The Schima BC test does not show a significant haemolytic activity and its thrombogenicity seems to be insignificant.

BC is characterized by low adhesiveness. Lack of proper adhesion of cells affects the generation of necrotic processes. Surface modification of BC with the use of natural proteins of intracellular matrix significantly improves the adhesiveness of cells and growth characteristic, what may have a significance in the case of clinical application of BC.

All implants of the valves were manufactured according to the protocol. During the first week after the implantation of the valves (first post-operative week), no adverse events were reported. All three sheep kept their normal appearance, general health and were not feverish. The recovery occurred in so-called normal status.

Then the sheep underwent a planned indirect ECG (within the period of <NUM> months). The sheep maintained a very good clinical condition. All echocardiograms show that the valves are in a good clinical condition. <NUM> months after the operation, the sheep underwent echocardiography again with further maintenance of good health condition. The echocardiography was performed inside the thorax cavity and the explant. Good elasticity and valve cusp movement were stated. Distinctively blocked or calcified valve cusp were not noted.

In the results of the sheep's blood tests, no significant abnormalities were detected. No signs of blood cells damage, haemolysis or infection.

Lack of significant insufficiency. Good movement of the cusps of the valve, good elasticity.

Scans reveal new structural defects in the valve cusps. There is a presence of minimum calcification, mainly in sheep no. The lowest calcification is seen in sheep no. Due to the hypertrophy of new tissues, there is a slight thickening of the wall and cusps both from the side of the tunica intima (inner) and also from the tunica externa (outermost).

The median calcium content is only <NUM>µg / mg tissue, between <NUM> and <NUM> (IQR <NUM>-<NUM>). The above graph shows the mean +/- SE and <NUM>% Cl.

The valve functions properly in the pulmonary position, more than <NUM> months after the pulmonary valve replacement surgery.

No surgical failure was noted. No damage to the material occurred. Lack of evidence of blood or platelet damage.

Low gradients and minor insufficiency were observed.

Only slight, temporary external calcifications connected with the suture were observed.

Good general clinical condition of animals.

Claim 1:
An element for manufacturing a heart valve comprising of a flat sheet of BC with three axes of symmetry, having a centrally made hole with three equal sides which form a figure with the symmetry of an equilateral triangle, characterised in that
- edges of the hole (<NUM>) are inner sides (A) of first areas (I) of the element,
- the first areas (I) of the element are quadrilateral,
- second areas (II) of the element are placed between the first areas (I) of the element adjacent thereto,
- the second areas (II) of the element are circle slices having a central angle (β) which is from 2π/<NUM> to 5π/<NUM> and radii equal to the lengths of edges of second sides (H) of the first areas (I) of the element,
- the edges of the second sides (H) of the first areas (I) of the element are perpendicular to the inner sides (A) of the first areas (I) of the element,
- the length of outer sides (A') of the first areas (I) of the element is equal to the length of the first inner sides (A) of the first areas (I) of the element,
- the ratio of the length of a radius (R) of a circle with its centre coinciding with a centre of symmetry (O) of the central hole (<NUM>) and tangent to the outer sides (A') of the first areas (I) of the element, at mid-height length, to the length of the second sides (H) of the first areas (I) of the element is between 2π/<NUM> and 5π/<NUM>,
- the ratio of the length of the first inner sides (A) of the first areas (I) of the element to the second sides (H) of the first areas (I) of the element is between π/<NUM> and 2π/<NUM>.