Method of processing animal or human biological tissue, eg swine or bovine pericardium or human cadaver mitral valves and properly processed biological tissue

FIELD: medicine.

SUBSTANCE: group of inventions refers to medicine. The group of inventions consists of two objects: a method of processing and a biological tissue. The method is characterised by taking the biological tissue stabilised by glutaric dialdehyde and presented by both animal and human biological tissue, e.g. swine or bovine pericardium or human cadaver mitral valves wherein physical plasma processing, particularly of collagen tissue is performed.

EFFECT: group of inventions provides higher biocompatibility of biological tissue, cell colonisation and prolonged service life.

9 cl


The invention relates to a method for processing biological tissue of animal or human origin, such as heart valves, pig or heart valves from bovine pericardium or heart valves of the human body and accordingly treated biological tissue.

Regarding the background of the invention it should be noted that the methods work by using pre-processed in different ways biological tissues of animal origin as collagen matrices, in particular, tissue engineering, are of great importance in various surgical areas for future improved therapies. The relevant fields of application it should be noted in cardiovascular surgery, but Orthopaedics and neurosurgery as well as possible applications.

When applying collagen matrix in cardiovascular surgery should be provided with good blood compatibility and mechanical strength. As an example in this connection we should mention heart valves of animal origin, such as valves, pig or valves from bovine pericardium, as well as biological implants vessels with small diameters and pumping cavity biological or mechanical injection pumps blood. When orthopedic surgical treatment is x special interest are the strong collagen matrix to replace cartilage, ligaments and tendons. Finally, in neurosurgery collagen tissue for closure of the skull, for example, after tumor operations, should also be considered as the scope of this invention.

Special problems underlying the invention should be clear from the example of replacement heart valves. So, when replacing heart valves, implanted in more than 200,000 patients per year worldwide, approximately 50% made of artificial mechanical heart valves, and 50% of the biological implants based on the heart valves of pigs and valves from bovine pericardium. When the implantation of mechanical heart valves in the aftercare required the introduction of drugs that prevent blood clotting to avoid embolism occurring in prosthetics. Thus, patients receiving such assistance, be "suffering from artificial hemophilia".

The difficulty with biological heart valves animal is that to achieve long-term stability, strength they should be treated with glutaraldehyde. Due to the presence of free aldehyde groups derived from glutaric dialdehyde, biological heart valves essentially have a toxic effect and are therefore unable to form colonies with to Adami. The formation of cell colonies should make these bioclean reliable for much longer. However, before the formation of cell colonies should be detoxification. Medical studies that were used binders available aldehyde groups, in this context known from the prior art. In this regard, provides links to the following literature sources: Gott JP, Chih P., Dorsey L., Jay J.L., Jett G.K., Schoen F.J., Girardot J. M., R. A. Guyton "Calcification of porcine valves: a successful new method of antimineralisation" ("Calcification (calcification) of the valves of pigs: a successful new way of antimonarchical") in Ann. Thorac. Surg. 1992; 53:207-216; M. Jones, Eidbo E.E., Hilbert S.L" Ferrans V.J., Clarck R.E. "Anticalcification treatments of bioprosthetic heart valves: in vivo studies in sheep" ("Anticarcinoma processing Bioprocessing heart valves: studies in sheep in vivo"in J. Cardiovasc. Surg. 1989; 4: 69-73; Grabenwõger M., J. Sider, Fitzal f, Zelenka C., U. Windberger, Grimm M. "Impact of glutaraldehyde on calcification of pericardial bioprosthetic heart valve material" ("the Effect of glutaraldehyde on the calcification of pericardial bioprotective material heart valves") in Ann. Thorac. Surg. 1996; 62:772-7; and, finally, C.L. Webb, Benedict J.J., Schoen FJ., Linden J.A., R.J. Levy "Inhibition of bioprosthetic valve calcification with aminodiphosphonate covalently bound material to residual aldehyde groups" ("Inhibition of calcification Bioprocessing valve by iminodiethanol, covalently bound substances with residual aldehyde groups") in Ann. Thorac. Surg. 988; 46:309-16.

In addition, research was conducted to achieve detoxification using citric acid. It was partly successful, as described by H. Gulbins, Goldemund A., Anderson I., U. Haas, Uhlig, A., Meiser C., Reichart B. "Preseeding with autologous fibroblasts improves endothelialisation of glutaraldehyde-fixed porcine aortic valves" ("Pre-seeding with autologous fibroblasts improves endothelialization linked with glutaraldehyde aortic valve of a pig") in J. Thorac. Cardiovasc. Surg. 2003; 125:592-601.

Achieved degree of detoxification in this case was only 20% to 30%.

When appropriate detoxification tissue associated with glutaraldehyde, and the corresponding ability to perceive the outer layer of the body's own tissue after implantation, the so-called "endothelialization allows you to reach for heart valve so that it continued to function in life without the introduction of drugs for inhibition of blood coagulation.

Another embodiment of the present invention are implants vessels of small diameter. Implants of this type are now usually made of plastic material, such as PTFE (polytetrafluoroethylene) or PET (polyethylene terephthalate). They have a relatively high rate of clogging, particularly when used as a vascular replacement for p is referencesa vessels of the legs, coronary shunting and peripheral dialysis shunts. The consequences of blockage of blood vessels in these areas prosthetics are severe, including a leg amputation, myocardial infarction, leading to death, or the need to repair shunt. There is also biological tissue of the small diameter of animal origin, linked with glutaraldehyde, in the form of a pig, bovine or goat donor vessels, can lead to a significant improvement in the case, if the fabric of this species can undergo detoxification and endothelialization, or be endothelialization in the blood stream. When applying muscle pumps you can also avoid thromboembolic complications, if the components that come into contact with blood, will be able to increase biocompatibility, for example, through appropriate measures detoxification.

Other applications of such biological tissue in the form of collagen matrices of animal origin can be durable and biocompatible associated with glutaraldehyde and subjected to detoxification of biological tissue for the treatment of osteoarthritis of the hip joint, knee joint and ankle joint. In addition, the application for closure of the skull after trauma or tumor operations using appropriately treated with glutaraldehyde, for example, is by detoxifying bovine pericardium as cerebrosides application may be exactly the same as applied, for example, in thoracic surgery to replace the chest wall or diaphragm, abdominal cavity surgery for replacement of the abdominal wall or ENT (ear, nose, throat) area to replace the eardrum.

Given the difficulties described in the description of the prior art, the invention is directed to a method of processing the collagen tissue of animal or human origin and correspondingly treated biological tissue, and the biocompatibility and durability of the fabric increases so that the use of tissue in the body can radically increase in the optimal case, up to the constant use.

The basic idea of the invention consists in the physical plasma processing biological tissue to solve these problems.

Studies have shown that physical plasma processing in which the gases are exposed to the excitement and success of the radicals, and which chemically neutralizes the toxicity obtained free aldehyde groups due to linking with glutaraldehyde, it is possible, therefore, to achieve significantly improved detoxification compared with the previous level as the first stage to improve implant properties of the tissue. The studies gave statedemocracy over 80%. Thus, closed endothelial surface may be subjected to colonization on surfaces in contact with the blood of the examined tissue.

Chemical process, mainly occurring during the plasma treatment can be briefly described using the example of the application during the process of gaseous oxygen. Thus, the components present in the plasma, namely, oxygen ions and excited oxygen to form carbon dioxide and water in the reaction with the hydrocarbons at the tissue surface. This reaction can thus be used to remove the aldehyde groups present on tissue surfaces, such aldehyde groups, which are formed when linking with glutaraldehyde, resulting in their detoxification.

Preferred conditions for the implementation of gas-plasma method described in paragraphs 2 and 3 of the claims, where it is preferable to use oxygen as the excited gas. However, it can also be applied nitrogen, hydrogen and argon. Energy supply upon receipt of the plasma is preferably carried out using high frequency electromagnetic fields, in particular, the microwave field.

The above gas-plasma techniques are usually carried out with the introduction of a gas that is ionized, rolled back, i.e. sparse processing chamber. Also, who's the one to carry out plasma treatment, atmospheric plasma, using the plasma jet with the excited reactive gas at atmospheric conditions. The plasma jet is directed onto the surface of the implant, thereby local treatment. It happens so that oxygen ions present in the reactive gas reacts with the hydrocarbons tissue surface and form carbon dioxide and water.

In contrast, when plasma processing at atmospheric conditions there is no need for pre-conditioning the drying fabric and therefore can directly handle even a damp cloth in its original condition, when processing biological tissue above-mentioned plasma gas by the method of in-camera processing to ensure detoxification is very important that before plasma treatment is usually water-containing biological tissue exposed to drying, in particular the effects of vacuum and temperature.

Therefore, the biological tissue during processing survive in the complete absence of water.

Subjected to plasma processing tissue for use in implantation moistened by placing it, for example, in the liquid. So it regains its original consistency and proper execution, for example, becomes permanently flexible and durable for a long time, n is the sample, heart valve of a pig.

Particularly preferred variant of the method according to the invention provided in paragraphs 8-10, according to which the fabric is subjected to plasma processing, is supplied biocompatible metal-containing coating. This coverage is an optional biocompatible component on the surfaces of the implants, which for a long time promotes cell growth.

The preferred method of applying a metal-containing coating is the method of PACVD (plasma-assisted chemical vapor deposition - chemical vapour deposition or gases in a plasma environment, that the coating of the surfaces of plastic materials in artificial medical implants have achieved convincing results in terms of biocompatibility thus treated surfaces. This metallsoderjasimi coating selected from the group of metals consisting of Ti, Ta, Nb, Zr, Hf, Ir, Au, Pd, Pt, Ag and Cu. In this regard, it was proved that titanium is particularly good, and for a long time it was used as a particularly biocompatible material in many kinds of applications of implants. Also mentioned coating materials - silver and copper - can additionally or exclusively be introduced into the coating as antibacterial agents.

Paragraphs 11-14 of the claims relate to biological the first tissues of the animal or human origin, which can be used as an implant in the human or animal. According to this invention, at least the surface which can come into contact with the body, is subjected to plasma processing in accordance with the invention for its detoxification. As described above, the improved biocompatibility by using biocompatible metal-containing coating on biological tissue. Preferably, when the biological tissue is a heart valve prosthesis of the vessel in contact with the blood of the surface mechanical or biomechanical injection pumps blood, closed box for cranial hole or replacing cartilage, bone, tendons, diaphragm, chest wall, the wall of the abdominal cavity or the eardrum.

The invention is described in more detail in the following embodiment.

As an example, the collagen tissue of animal origin used heart valve of a pig, which should be used as implants in the human body.

He is preparing, is released from cells and is fixed, i.e. linked with glutaraldehyde after removal from the animal donor to stabilize the usual way. In this case, the valve is placed in a solution of glutaraldehyde concentration from 0.1 to 0.4% and strengthened in the flowing solution at the bottom is their pressures from 3 to 6 mm Hg within 24-48 hours.

Thus acquired heart valve pigs then slowly dried in vacuum and under the influence of temperature and thus fully dehydrated.

Then perform plasma processing of a heart valve of a pig in the processing chamber. For this purpose, the processing chamber is fully pumped (vacuumized) and then there is introduced oxygen. The plasma is heated through supply of high frequency electromagnetic fields, such as 40 kHz or 13.56 MHz, or upon excitation with microwaves. Gaseous oxygen is present in the processing chamber, is excited and converted into radicals under the related power supply.

This plasma gas acts on hydrocarbon group, CxHyon the surface fortified glutaraldehyde implant in accordance with the following reaction equation:

CxHy+(x+y/4)O2→ x CO2+y/2 H2O

As you can see, the hydrocarbons on the surface of the implant, such as, for example, acetaldehyde, turn into relatively harmless chemical compounds, carbon dioxide and water, which can easily be removed from the surface of the implant.

The above plasma processing next continues to be used for the deposition of metal-containing coating on the surface of the implant. For this purpose, the gaseous precursor is fed into the chamber for coating and split under the influence of plasma energy in its atomic components. Thus obtained ions are deposited on the surface. Usually as a metal-containing coating using PACVD method is mainly applied to titanium. Furthermore, the method itself is described in detail in EP 0897997 B1, using as example the coating on the substrate of a plastic material.

The pressure in the reactor for pretreatment and the coating is from 0.1 to 1030 mbar. When applying plasma ideally the pressure was more than 50 mbar. For pre-treatment working gas (e.g. oxygen) is introduced into the reactor at a volumetric rate of gas 0,04 N/min After stabilization of the final pressure to approximately 1 mbar, capacitive plasma input occurs at a power of 20 watts for 60 seconds. Then the gas supply is stopped and the reaction chamber is completely pumped out. For subsequent coating over the previous Ti[N(CH3)2]4directed carrier gas (hydrogen) in the bulk gas velocity of 0.09 N/min and introduced into the chamber for coating. The duration of the coating is approximately 300 seconds when applied plasma power of 20 watts. Further, the gas supply stops again, and the camera for applying the coating is ventilated.

After this vacuum plasma saturation, and applying cover the Oia heart valves of pigs, them again placed in the liquid, so that they, due to the fluid flow, again acquired its original consistency.

1. The method of processing biological tissue of animal or human origin, such as swine, bovine pericardium or heart valves of the human body, to increase the biocompatibility, including physical plasma processing tissue using a plasma gas method, in which the ionized gas is used, nitrogen, hydrogen, argon or, preferably, oxygen, characterized in that
before the plasma processing gas method the fabric is dried,
- the fabric is subjected to plasma processing, are biocompatible metallsoderjasimi floor,
after plasma treatment the dried implant is subjected to the action of moisture.

2. The method according to claim 1, in which the energy required for plasma is carried out using high frequency electromagnetic fields, in particular microwave field.

3. The method according to claim 1, wherein the plasma treatment is carried out with atmospheric plasma using the plasma jet at atmospheric conditions.

4. The method according to claim 1, wherein the drying is performed under vacuum and temperature.

5. The method according to claim 1, in which metallsoderjasimi coating is applied using a method of chemical deposition of vapors or gases in a plasma the environment.

6. The method according to any one of claims 1 to 5, in which metallsoderjasimi coating selected from the group consisting of Ti, TA, Nb, Zr, Hf, Ir, Au, Pd, Pt, Ag and Cu.

7. Biological tissue of animal or human origin for use as an implant in the human or animal obtained by the method according to any one of claims 1 to 6, in which at least the surface, which can be in contact with the body, after drying, the fabric is subjected to plasma treatment for detoxification, the surface subjected to plasma processing, is covered with a biocompatible metal-containing coating, and the fabric is exposed to moisture.

8. Biological tissue according to claim 7, in which metallsoderjasimi coating selected from the group consisting of Ti, TA, Nb, Zr, Hf, Ir, Au, Pd, Pt, Ag and Cu.

9. Biological tissue according to any one of claims 7 or 8, having a configuration in the form of a heart valve prosthesis of the vessel in contact with the blood of the surface mechanical or biomechanical injection pumps blood, closed insertion cranial hole or replacing cartilage, bone, tendons, diaphragm, chest wall, the wall of the abdominal cavity or the eardrum.


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