In-vitro method for the production of a homologous stented tissue-engineered heart valve

Each year around 20,000 patients die from heart valve dysfunction alone in the US, and more than 60,000 patients are forced to replace one or more heart valves surgically because of an already recognized dysfunction. As a replacement for the heart valve, either mechanical or biological valve prostheses (xenografts) can be used; more rarely cryopreserved or glutaraldehyde-fixed homografts are used.

However, mechanical valve prostheses often lead to foreign body reactions with thromboembolic complications, which are favored by the flow conditions in the heart that are modified by the artificial heart valve. Therefore, lifelong anticoagulation of the affected patient is necessary, which leads to a permanently increased risk of bleeding. A further, often life-threatening complication in patients with a mechanical heart valve are infections.

The xenografts are usually swine flaps, which are treated with glutaraldehyde. Pork prostheses can be used with good results in elderly patients, but tend to degeneration after only 12 to 15 years, so they are generally not suitable for young people. Furthermore, swine flap prostheses have an increased risk of infection compared to the healthy heart. Moreover, pigs tend to be calcified, which is why they are unsuitable for use in children and young people who have an increased calcium metabolism. Finally, they also represent body-deficient tissue, which with a certain probability is recognized as foreign by the body's immune system and can thus trigger adversive immune processes.

The third possibility is homografts, ie heart valves that are isolated from human donors. Although homografts are relatively resistant to infections, they also represent body-deficient tissue, which with a certain probability causes immune reactions. In addition, homografts as well as porcine valve prostheses are prone to calcification and are therefore subject to considerable degeneration, which usually necessitates reoperation after 7 to 12 years. Homografts are also available to a very limited extent.

In addition to the already described disadvantages of the flap prostheses used hitherto as flap replacements, ie the triggering of immune reactions, the increased risk of infection, the risk of thromboembolic processes and the tendency to degenerate, all known flaps are common in that they consist of inorganic material or organic material Important properties of a living matrix, such as the ability to repair, reconfigure, or grow. It follows, among other things, that in the case of childish valve patients so far, regular reoperations had to be accepted. However, in addition to the risk inherent in any cardiac surgery, the risk of morbidity and mortality increases with each reoperation, as the previous operations cause considerable adhesions in the thorax.

There is therefore an urgent need for a heart valve replacement which avoids the disadvantages described above. For this purpose it has already been proposed to manufacture artificial heart valves by "tissue engineering". "Tissue Engineering" deals with the development of "biohybrid" implants, which grow in the body into tissues or even whole organ systems. Also, the production of biohybrid heart valves in the form of individual flap seals has already been described; The so-called "tissue engineering" made the so-called "heart valve" so far had the disadvantage that they had inadequate, insufficient connective tissue structures and therefore could not withstand the flow conditions prevailing in the heart after dissolution of the biodegradable carrier structure.

<patcit id="pcit0001" dnum="DE19919625"><text>DE 19919625</text></patcit> describes one <u><i>in vitro</i>procedure</u> For producing a homologous heart valve. The heart valve described there is based on a biodegradable carrier, which is incubated with homologous fibroblasts and / or myofibroblasts to form a connective tissue-like matrix and is then populated with endothelial cells. The connective tissue-like matrix is ​​then transferred into a bioreactor for tissue rash. This heart valve is optimally adapted to the conditions of the human body. In the<patcit id="pcit0002" dnum="DE19919625"><text>DE 19919625</text></patcit> Describes at the time of its implantation nearly completely from autologous cell material, which is then sewn into the recipient heart. A disadvantage of this heart valve may be that surgical implantation is technically difficult to perform. Moreover, a problem could arise when the suture is to be passed through the autologous, tissue-engineered tissue to be sewn in. Owing to the extremely high load which the heart valve is then subjected to in the human body, cracks could develop in the region of the seam.

The object of the invention is therefore to provide improved homologous heart valves as well as a process for their production.

According to the invention, <u><i>in vitro</i>procedure</u> Method for producing a homologous heart valve, comprising the following steps:<ul><li>Providing a biodegradable carrier (scaffold),</li><li>Colonizing the carrier with homologous fibroblasts and / or myofibroblasts to form a connective tissue matrix,</li><li>If necessary. Colonizing the connective tissue matrix with endothelial cells</li><li>Attaching the connective-tissue matrix to a non-difficult or difficult-to-dismount frame construction (stent)</li></ul>Wherein the Which is populated with endothelial cells, is introduced into a pulsatile flow chamber in which it can be exposed to rising flow rates before or after attachment to the frame structure, and the flow rate is increased continuously or discontinuously.

In an alternative method, a homologous heart valve is produced by<ul><li>Providing a biodegradable carrier (scaffold) rigidly connected to a non-degradable frame construction (stent)</li><li>Colonizing the carrier with homologous fibroblasts and / or myofibroblasts to form a connective tissue matrix,</li><li>If necessary. Colonizing the connective tissue matrix with endothelial cells</li><li>Introducing the frame construction with the associated connective-tissue matrix into a pulsatile flow chamber in which it can be exposed to rising flow rates,</li><li>Continuously or discontinuously increasing the flow rate.</li></ul>

By means of the methods according to the invention, homologous heart valves can be produced, which have all the advantages of the invention <patcit id="pcit0003" dnum="DE19919625"><text>DE19919625</text></patcit> And furthermore avoid the fact that, at the time of the implantation of the flap, a seam must be guided through the connective tissue structures of the heart valve. They are able to cope with the conditions of the body in the body and can simply be surgically implanted.

The methods for producing the heart valve according to the invention as well as the cardiac flap thus produced are explained in more detail below.

In the following description, the term "carrier" means an acellular structure which, as explained in more detail below, is formed either from synthetic fibers or from an acellular connective tissue framework. The term "matrix" refers to a connective tissue structure which, in addition to fibroblasts and myofibroblasts, contains typical components of an extracellular matrix, namely collagen, elastin and glycosaminoglycans. Matrix-denoted structures typically contain degraded carrier components or no carrier components at all.

A biodegradable carrier is initially provided for carrying out the process according to the invention. On the one hand, the carrier material should be stable for a certain period of time in order to allow sufficient colonization or penetration with fibroblasts and / or myofibroblasts and to achieve the formation of a connective-tissue matrix, on the other hand within a reasonable time which is typically less than time , Which involves the formation of the homologous valve prosthesis, can be solved as a whole. It is preferred that degradation commence after about 8 days; It should normally be completed in less than 3 months, preferably already after 4 to 6 weeks.

After formation of a solid connective-tissue matrix structure, whose degradable carrier does not yet have to be dissolved, With endothelial cells. After the colonization has taken place, the connective tissue matrix is ​​applied to a non-degradable frame construction. Alternatively, however, a carrier which is already firmly connected to a frame construction can be subjected to the colonization steps. The possible alternative method variants are described in more detail below.

In a variant of the method according to the invention, the biodegradable carrier (scaffold) is first colonized with homologous fibroblasts and / or myofibroblasts to form a connective-tissue matrix. Subsequently, the matrix is ​​added as required. With endothelial cells. According to the invention, the previously formed heart valve-analogous structure can now be introduced into a pulsatile flow chamber in a further process step for tissue maturation and optimization of the hemodynamic function in which it can be exposed to rising flow rates. By continuously or discontinuously increasing the flow rate, it is thereby adapted to the flow conditions in the human body. For further stabilization, the heart valve-like structure is attached to a biocompatible frame construction made of non-degradable or difficultly degradable material. Once again into the pulsatile flow chamber. In the event that, following attachment to the frame construction in the flow chamber, the flow conditions in the human heart are adapted, the first incubation in the pulsatile flow chamber may be omitted. Through these procedures, vital heart valve prostheses are obtained which are adapted to the flow conditions in the human body.

In an alternative variant of the method according to the invention, the biodegradable carrier (scaffold) can be firmly connected to the non-difficult or difficultly degradable frame construction (stent) before the colonization. In a further step, the carrier connected to the framework is then used to form a connective-tissue matrix with homologous fibroblasts and / or myofibroblasts and subsequently, With endothelial cells. For the tissue maturation and optimization of the hemodynamic function, the pre-formed heart valve-analogous structure is subsequently introduced into a pulsatile flow chamber in which it can be exposed to rising flow rates. By continuously or discontinuously increasing the flow rate, a vital heart valve prosthesis is also obtained which is able to withstand the flow conditions in the human body.

Overall, the following process variants are thus obtained:<ul><li>Variant 1:<ul><li>Providing a carrier without frame construction</li><li>Settle</li><li>Adapt in a pulsatile flow chamber</li><li>Fastening on a non-difficult or difficult to dismantle frame construction (stent)</li><li>If necessary. Afteradapting the "gestent" heart valve</li></ul></li><li>Variant 2:<ul><li>Providing a carrier without frame construction</li><li>Settle</li><li>Fastening on a non-difficult or difficult to dismantle frame construction (stent)</li><li>Adapt the "gestent" heart valve</li></ul></li><li>Variant 3:<ul><li>Providing a carrier on a non-difficult or difficult to dismantle frame construction</li><li>Settle</li><li>Adapt</li></ul></li></ul>

The carrier material is preferably a structure composed of polymer fibers, a porous polymer structure or an acellular biological tissue. Suitable synthetic polymers for this use include bioerodible polymers such as polyglycolic acid (PGA), polylactic acid (PLA) polyhydroxyalkanoate (PHA) and poly-4-hydroxybutyrate (P4HB), polycaprolactones (PLGA), polycarbonates, Polyanhydrides, polyamino acids, polyorthoesters, polyacetates, polycyanoacrylates and degradable polyurethanes and non-erodible polymers such as polyacrylates, ethylene vinyl acetate polymers and other substituted cellulose acetates, and derivatives thereof. Polyester is preferred.

Preferred biodegradable polymers selected from polymers of the following group: polyesters of hydroxycarboxy acids, polyanhydrides of dicarboxyesters, and copolymers of hydroxycarboxy acids and dicarboxyesters.

In a further embodiment the material consists of a synthetic polymer of at least one of the following monomers: glycolide, lactide, p-dioxanone, caprolactone, trimethylene carbonate, butyrolactone. In particular embodiments, the material is selected from a group consisting of polymers or copolymers of glycolic acid, lactic acid and sebacic acid. Polyglycolic acid polymers are preferred.

These polymers can be used either pure or in mixtures of two or more of the substances mentioned or mixtures of these substances with further biodegradable polymers. In a preferred embodiment, a copolymer of 85% PGA and 15% PLA is used.

In another particular embodiment, the carrier is made from a polyhydroxyalkanoate (PHA). PHA can be coated with a further non-degradable polymer. A preferred polyhydroxyalkanoate is degraded for this use<i>in vivo</i> Within less than 9 months, more preferably in less than 6 months, and most preferably in less than 3 months. A preferred composition of the polyhydroxyalkanoates includes 2- 3-, 4- or 5-hydroxyacids, eg, poly-4-hydroxybutyrates. Further, the composition may include a poly-4-hydroxybutyrate-co-3-hydroxybutyrate, as well as combinations thereof. Poly-4-hydroxybutyrate is most preferred.

In a further particular embodiment, the carrier consists of homopolymers and copolymers with any combination of the following monomers: 3-hydroxybutyrates, 3-hydroxyvalerate, 3-hydroxypropionate, 2-hydroxybutyrate, 4-hydroxybutyrate, 4-hydroxyvalerate, 3-hydroxyhexanoate, 3-hydroxyheptanoate , 3-hydroxyoctanoate, 3-hydroxynonanoate, 3-hydroxytridecanoate, 3-hydroxytetradecanoate, 3-hydroxypentadecanoate, 3-hydroxyhexadecanoate, 3-hydroxyheptadecanoate and 3-hydroxyoctadecanoate.

It has proven to be useful to use biodegradable carriers having a polymer density of about 40 to 120 mg / cm 3³ to use. Below 40 mg / cm³ The polymer tissue is too labile, above 1 20 mg / cm³ The tissue is too dense to allow the penetration of fibroblasts within a reasonable period of time. In preferred embodiments, the density of the biodegradable carrier is 50 to 80 mg / cm 3³, Particularly preferably 70 mg / cm 3³. In the present invention, a polymeric carrier of the company Albany International Research, Mensville, MA, with a density of approx. 70 mg / cm 3 was obtained with good results³ , And a polymeric carrier from TRANSOME INC., Palm Bay, FL. USA.

The fibers of the carrier can have a diameter of 6 to 20 μm, preferably 10 to 18 μm. However, tissues with other fiber thicknesses are also conceivable, which however, on the one hand, have to give the carrier a certain stability, on the other hand, must permit the colonization and penetration of the carrier with fibroblasts or myofibroblasts. For porous (spongy) polymer forms, pore sizes of 80-240 μm have proven to be favorable. The pores can be obtained by the so-called "salt-leaching" technique, which is known to a person skilled in the art.

Instead of a synthetic carrier, as described above, the use of an acellular connective tissue framework is conceivable. For example, a pig's flap could be converted into an immunologically neutral tissue (<nplcit id="ncit0001" npl-type="s"><text>Bader et al., Eur. J. Cardiothorac. Surg. 14, 279, 1998</text></nplcit>), Which could subsequently be colonized with homologous cells. Human heart valves can also be recolonized after neutralization.

The biodegradable carrier is first incubated with a fibroblast population. When using homologous fibroblasts and / or myofibroblasts, ie fibroblasts and / or myofibroblasts from a human, but not necessarily the patient, the same HLA typing should be considered. Fibroblast populations can be obtained, for example, from peripheral blood vessels, both arteries and veins. The radial arteries of the forearm are particularly suitable for this, which is available in most cases for the explantation of the forearm due to the arterial double supply of the arm. Alternatively, vascular cells can be obtained from blood vessels of the leg, eg the saphenous vein. Furthermore, the myofibroblasts and endothelial cells can be obtained from bone marrow precursor cells or from pluripotent stem cells or genetically manipulated cells.

The cells can be obtained, for example, from vessel fragments, in which the tissue fragments are first, as shown in FIG <nplcit id="ncit0002" npl-type="s"><text>Ignition et a /. (Eur, J. Cardiothorac Surg 13.160, 1998)</text></nplcit>), Fragmented in tissue fragments and incubated for about 1 to 2 weeks under normal cell culture conditions (37 ° C., 5%₂, 95% humidity) until the cells form a confluent cell layer on the bottom of the culture dish. They are then subjected to multiple passages in order to obtain a cell culture free of residual tissue material. After two to three passages, the mixed cell populations can be purified by incubating them with an endothelial cell-specific fluorescent marker (Dil-Ac-LDL, from Medical Technologies Inc., Stoughton, MA) and using flow cytometry (FACStar Plus, Becton Dickinson) Are separated. Fluorescence-labeled cells are endothelial cells, unlabelled cells are fibroblasts and myofibroblasts. These are cultured for a further two to three weeks and during this time are subjected to two to four passages to obtain a sufficient number of cells for the subsequent colonization of the carrier.

A purified as described or any other pure fibroblast / myofibroblast culture can now be used for the colonization of the polymer support. For this, the surface area of ​​the carrier is approximately 10⁵ Up to 6 x 10^8th Fibroblasts and / or myofibroblasts. In this case, "surface" is not the actual surface area of ​​the polymer, but the surface which can be seen in a plane when viewed from above in a plane. The fibroblasts are usually administered for 60 to 90 min. Time to attach to the wearer. The supernatant medium can then be removed and fibroblast suspension added once more. However, between the first and second addition of fibroblast suspension, 2 to 36 hours, preferably 24 hours, are normally allowed to elapse.

In a preferred embodiment of the method according to the invention, a further 3 to 14 times, particularly preferably 5 to 10 times, fibroblasts and / or myofibroblasts are added to the carrier or to the matrix which gradually forms after the initial fibroblast addition.

Among the conditions usually used for the cell growth of fibroblasts (eg, 5% CO₂, Incubation at 37 ° C., sterile medium), a solid connective tissue structure develops after approximately one to three weeks. In a preferred embodiment, this structure is subsequently incubated with a pure endothelial cell suspension. Like the fibroblasts, the endothelial cells can be enriched by FACS and subsequently expanded in several passages (preferably 3). It is also preferred for endothelial cells to carry out the colonization with in each case about 10⁵ up to 5x10^8th Endothelial cells several times, eg 3 to 14 times. In preferred embodiments, the colonization with endothelial cells is repeated 5 to 10 times. Between two colonization steps should be at least 60 minutes, but preferably 2 to 24 hours. However, the endothelial cell colonization step is optional.

The cells used for the colonization of the carrier are preferably human cells. However, it is particularly preferred to use autologous fibroblasts and / or myofibroblasts as well as, Endothelial cells. For this purpose, tissue, for example from one of its vessels, is taken from the patient whose heart valve is to be replaced. As already mentioned above, the arteria radialis and the saphenous vein or bone marrow are indicated. The use of the autologous cells for the construction of the heart valve has the essential advantage that the valve after implantation into the patient does not represent a body-deficient tissue and thus immune reactions against the artificial heart valve seem virtually impossible.

Approx. 14 days after the optional addition of the endothelial cells, a tissue with a superficial single cell layer of endothelial cells and a connective-tissue basic structure can be detected histologically and immunohistochemically.

In a preferred embodiment, the connective-tissue matrix is ​​in the form of a heart valve and is provided with a broad connective-tissue edge, the so-called seam ring, which is fixed on a circular frame construction. An example of such a heart valve with a seam ring is shown in FIG.

In a further preferred embodiment, the connective-tissue matrix is ​​in the form of a band or a ring. This embodiment requires a seam ring which is provided with a triangular support structure as a frame construction. The B and / or the ring is then routed around this three-peaked structure. An example of this embodiment is shown in FIG. The diameter of the frame construction is individually selectable and depends on the anatomical requirements of the patient.

The frame construction (stent) according to the invention can be constructed from different materials. In order to give the newly produced tissue the longest possible life and strength, the material should be constructed from a non-degradable biocompatible material or alternatively from a difficultly degradable biocompatible material, eg carbon, PTFE, dacron, metal, PHA, Hydroxbutyrate (P3HB). "Hard to digest" means an abbaudauer of more than one year.

The attachment of the connective-tissue matrix to the frame construction can be effected by means of conventional sewing technique. In one embodiment, the matrix can be fixed to the frame construction by means of fibrin glue. Particularly preferably, the binding of the connective-tissue matrix to the carrier is effected by conventional sewing technique in conjunction with fibrin glue. The shape of the individual heart flap can also be stabilized either by sewing or by gluing with fibrin glue, in which the edges surrounding the tip tips in the direction of the center of the circle are sewn or glued.

According to the invention, in a further method step, the pre-formed heart valve-like structure can now be introduced into a pulsatile flow chamber in which it can be exposed to rising flow rates. It has been found that the formation of a flow-resistant connective-tissue matrix can be achieved by a slow adaptation of the flow rates.

For carrying out the process according to the invention, for example, <patcit id="pcit0004" dnum="DE19919625"><text>DE19919625</text></patcit> Described bioreactor.

In one embodiment of the invention, flow rates between 5 ml / min. And 8000 ml / min., Preferably between 30 ml / min. And 5,000 ml / min, more preferably 50 ml / min. To 2000 ml / min. The data refer to the flow through the valve prosthesis. As an initial flow rate, flow rates of 50 to 100 ml / min. As appropriate. These flow rates, for example, are sent through the heart valve with a pulse frequency of 5 to 10 pulses per minute. The flow rate is then adjusted continuously or discontinuously to up to 5000 ml / min. Increased. At the same time, the pulse rate is increased to 180 pulses / min. raised. The specified data are the limits that are not normally exceeded.

In preferred embodiments, the flow rate is increased to 2,000 ml / min while the pulse frequency is increased to 70 to 100, preferably 80 pulses / min. Is raised. The loading of the stabilizing heart valve is thus adapted to almost physiological conditions. It has proven to be advantageous, but not necessary, to increase the flow rate and the pulse frequency in each case after approximately 24 to 48 hours. For example, a flow rate of 50 to 100 ml / min and a pulse rate of 5 to 10 pulses / min. On day 1 of the stay in the pulsatile river chamber, on day 3 an increase to 300 ml / min at 20 to 25 pulses / min, on day 5 to 700 ml / min. And 35 to 45 pulses / min, on day 7 to 1000 ml / min and 50 to 60 pulses / min, on day 9 to 1300 ml / min and 70 to 80 pulses / min, on day 11 to 1500 ml / min. And about 100 pulses / min, on day 13 to 1750 ml / min and about 120 pulses / min, and on day 1 5 to 2,000 ml / min and 140 pulses / min. Depending on the available time, size of the valve, size and age of the patient etc., however, a much slower increase in flow rates, pulse rate, or increase in flow rates and pulse rates can be useful.

In one embodiment of the invention, the systemic pressures prevailing in the pulsatile flow chamber are set to 10 to 2 40 mmHg. Preference is given to systemic pressures of 60 to 140, particularly preferably systemic pressures of 80 to 120 mm Hg.

The homologous or autologous heart valve produced by means of the method according to the invention has substantial advantages compared with the conventional mechanical and biological heart valves. Thus, in its preferred embodiment, the heart valve according to the invention consists of autologous tissue, ie tissue of the heart valve operation, as well as a further stabilizing biocompatible material, which is used as a frame construction. This prevents a foreign body reaction of the flap receiver on the implant. The risk of infection in recipients of a cardiac valve according to the invention is thus substantially reduced. Anticoagulation therapy is not required; Thus eliminating the risk of hemorrhagic complications. However, the most compelling advantage by far the heart valve according to the invention is the Tatsac hey, that it represents a living tissue and is thus capable of implantation to permanent regeneration and repair. Furthermore, in its preferred embodiment, the heart valve according to the invention combines the advantages of a fully autologous heart valve prosthesis and the very good hemodynamic functions of synthetic heart valve prostheses. Ultimately, in the case of cardiac valves according to the invention, the use of the biocompatible frame construction means that significantly degenerative changes and / or dysfunctions are to be expected during prolonged use, which significantly increases the life of the heart valve and thus significantly reduces the risk of reoperation.

The cardiac valve according to the invention contains a connective tissue structure which, in addition to fibroblasts and myofibroblasts, essentially contains components of a normal extracellular matrix, namely collagen, elastin and glycosaminoglycans. The flaps according to the invention thus have a proportion of collagen (26-60%), elastin (2-15%) and glycosaminoglycans corresponding to the native flap or the native flap seal. This connective tissue structure, built up on a biodegradable carrier (scaffold) and inhabited with endothelial cells, is further stabilized by a biocompatible frame construction. The attachment of the connective tissue structure to the biocompatible framework is done as described above. Such a heart valve prosthesis combines the advantages of an autologous heart valve prosthesis and the very good surgical implantability and function of synthetic heart valve prostheses.

It was shown that the heart valves according to the invention can withstand flow rates of more than 2000 ml / min, corresponding to the flow conditions prevailing in an adult human heart. Thus, according to the invention, an autologous heart valve can be provided, which is unconditionally suitable for implantation in children as well as adult patients.

The following figures and examples illustrate the invention.

Figure 1 shows a polymer made from a polymer. Carrier (see star) with seam ring (see arrow) after colonization with fibroblasts / myofibroblasts and endothelial cells.

Figure 2 shows a tubular populated matrix from which rings of 3.5 cm width can be cut around the frame construction shown in Figure 3.

FIG. 3 shows a schematic representation of the frame construction from FIG. 3 with the populated matrix guided around the three-pole support structure.

Example 1 Production of Folding Conduit (Tubes) Vehicles

A non-woven polyglycolic acid polymer (fiber diameter: 12-15 pms, polymer density: 70 mg / ml, Albany International Research, Mansfield MA, USA) is used to produce the lance-carrying conduit support. The polymer is cut in such a way that it forms a tube with a diameter of 19 mm. Three triangular sails are inserted into this conduit. This carrier can be used to produce 3-leaf flaps, ie pulmonary, aortic and tricuspid valves. 2 sails are inserted for mitral valves.

EXAMPLE 2 Production of a three-leafed heart valve prosthesis stabilized by a frame construction

A three-lobe cantilevered Condiut carrier is sterilized and inserted into medium (DM EM, GIBCO BRL-Life Technologies) for 24 hours to soak the polymer surface. Thereafter, the flap-shaped carrier is populated with 4 million fibroblasts per square meter surface every six minutes. Furthermore, the colonized carrier is incubated for 2 weeks (5% CO₂, 37 ° C, 95% humidity). The medium is changed every four days under sterile conditions. Subsequently, endothelial cells are applied to the populated flap-shaped support (3-4 million endothelial cells per square surface, 6 colonies every 90 minutes). After a further 2 weeks, the resulting tissue is placed over a prepared biologically compatible frame construction and connected to the frame construction by a seam. Subsequently, the entire structure is introduced into the flow chamber of the bioreactor under sterile cautery and installed here in the flow position. The bioreactor is now filled with medium and placed in the cell incubator. After the pump has been connected to the outside of the incubator via the compressed air hose, minimal pulsatile flows are started (50 ml / min). In 2-day increments, the flow rate and pulse rate are increased to 100 ml / min (pulse 10), 300 ml (pulse 25), 700 ml (pulse 35), 1000 ml (pulse 60) for a further 4 days. Subsequently (after 14 days), the tissue now formed is removed under sterile conditions and stored for biochemical, histological and mechanical analysis.

Example 3 Production of three-lobe-shaped carriers stabilized by a multi-lobe frame construction:

For the manufacture of the heart valve-shaped carrier 1-2 mm thick, non-woven co-polymer of polyglycolic acid (PGA) and polyhydroxyalkanoate (PHA) (fiber diameter 12-15 μm, polymer density 70 mg / ml) is used and cut so that a 3.5 Cm wide and 8.0 cm long band. This tape is bonded to the endpoints using resorbable suture and is additionally welded to the overlapping zones under application of heat (60-70 ° C). The resulting ring is placed over a triangular frame construction (Dacron) and connected to it by means of seam and using fibrin glue. Subsequently the individual Herzklappensegel were formed over the frame construct again under application of heat into the heart flap-typical, belly three-gel form.

EXAMPLE 4 Production of a three-leaf heart valve prosthesis, which is stabilized by a three-ply frame construction

A three-leaf carrier, stabilized by a three-ply frame construction, was sterilized and inserted into medium (DM, EM, GIBCO BRL-Life Technologies) for 24 hours to soak the polymer surface. Thereupon, the flap-shaped carrier is populated with 4 million fibroblasts per square meter surface every six minutes. Furthermore, the colonized carrier is incubated for 2 weeks (5% CO₂, 37 ° C, 95% humidity). The medium is changed every 4 days under sterile conditions. Subsequently, endothelial cells are applied to the populated flap-shaped support (3-4 million endothelial cells per square surface of squares, &amp; settlements every 90 minutes). Afterwards, the entire construction was introduced into the flow chamber of the bioreactor under sterile cautery and installed here in the flow position. The bioreactor is now filled with medium and placed in the cell incubator. After the connection to the pump outside the incubator has been established via the compressed air hose, minimum pulsatile flows are started (50 ml / min.). In 2-day increments, the flow rate and pulse rate increased to 100 ml / min. (Pulse 10), 300 ml (pulse 25, 700 ml (pulse 35), 1000 ml (pulse 60) for a total of another 4 days, and after 14 days the tissue is removed under sterile conditions and subjected to biochemical histological examination And mechanical analysis.