KR100739422B1 - Calcification-resistant heparinized acellular bioprosthetic tissue implant and preparation method thereof - Google Patents

Abstract

The present invention relates to a heparin-coupled anticalcification acellular tissue implants and a method for producing the same, and more specifically, (a) treating a biological tissue of an animal other than a human with an acellular biological tissue; (b) crosslinking the acellular biological tissues with a glutaraldehyde solution; And (c) binding heparin to the cross-linked acellular biological tissue.

The heparin-coupled anticalcification-free acellular tissue implant of the present invention is excellent in anticalcification function, durability in vivo, blood compatibility and tissue compatibility (tissue compatibility), artificial heart valves, tissue vessels and It may be usefully used as an acellular biological tissue implant such as a circulatory surgical patch, tissue regeneration membrane, and tissue filler.

Heparin, calculation-resistance, blood compatibility, tissue compatibility, artificial cardiac valve, surgical tissue patch, tissue regeneration membrane, tissue filler

Description

Calcification-resistant heparinized acellular bioprosthetic tissue implant and preparation method

[Industrial use]

The present invention relates to a heparin-coupled anticalcification acellular biological tissue implant and a method for preparing the same, and more particularly, anticalcification function, in vivo durability, blood compatibility and tissue compatibility (tissue compatibility) Heparin-conjugated anticalcifiable acellular tissues that can be used as acellular tissue implants such as artificial heart valves, tissue vessel and circulatory surgical patches, tissue regeneration membranes, and tissue fillers It relates to water and a method for producing the same.

[Prior art]

Bioprosthetic tissue implants are biological tissues that can be implanted into human tissues or organs damaged by diseases such as chemical treatment of human or animal tissues to remove immune characteristics and confer specific functions. Porcine aortic valves (porcine aortic valve) or periccardium (porcine pericardium), bovine pericardium (bovine pericardium) and dura mater (bovine or swine) are commonly used as animal tissues for the preparation of the biotissue implants. Being.

In order to clinically use the biological tissue of the animal, there is a need for a method capable of maintaining strength and flexibility in a state where the transplanted biological tissue does not induce an immune rejection reaction by the host. At present, a general method for preparing clinical tissues is to first collect biological tissues, wash them with Hanks Solution to remove soluble antigenic substances, and then chemically treat (modify) the glutaraldehyde buffer solution to the glycoproteins contained in the biological tissues. A method of stabilizing biological tissues by crosslinking amino groups of glutaraldehyde and reducing with sodium borohydride (NaBH ₄ ) (A. Carpentier, Biological Tissue in Heart Replacement, MI Ionescu et all. Eds), Butterworth, London, 1972).

Representative biological tissue implants prepared as described above include tissue tissue valves, surgical tissue patches, bioprosthetic vascular grafts, ligaments, ligament and tendon substitutes, and the like.

The commercially available artificial heart valves can be divided into mechanical valves and biological tissue valves. Mechanical valves are durable but require anticoagulants because they involve thrombogenesis. On the other hand, biological tissue valves have similar functions and morphologies to living organisms, and have a very low thrombus formation rate and thus do not require anticoagulants. Due to pathological calcification, there is a defect in the valve's flexibility after transplantation.

In addition, surgical tissue patches widely used as biological tissues are used to reinforce and replace defects in the heart, blood vessels and tissues damaged by congenital abnormalities, diseases, or accidents. Currently, synthetic patches and animal tissue patches are used. (xenograft tissue patch) is used. Among these, tissue patches of animals have excellent blood compatibility and tissue compatibility, but have a disadvantage in that durability is inferior due to calcification in vivo as in the biological tissue valve.

In addition, tissue vessels widely used as living tissues are used to replace damaged vessels together with artificial artificial vessels. In addition, it is used as a tissue replacement implant such as ligaments and tendons using animal tissues and can be applied for tissue regeneration, wound healing and drug delivery system.

However, such a living tissue implant can avoid the immune response by the host and maintain flexibility and strength, but has a disadvantage of poor durability due to calcification when used for a long time.

Calcification is a common problem in living tissues such as tissue heart valves, surgical tissue patches, bioprosthetic vascular grafts, ligaments, ligament and tendon substitutes. Calcification refers to the deposition of calcium compounds, such as calcium phosphate or calcium carbonate, such as hydroxyapatite, Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ ), into the material implanted in the body, The physical properties of the tissue material, in particular its flexibility, are inferior and eventually lead to destruction and degradation in vivo.

To date, the mechanism of calcification is not clear, but i) the side effects of biological tissue processing and the compounds used therein, in particular glutaraldehyde and unreacted residual glutaraldehyde, ii) alteration of the chemical structure of the tissue. And calcification occurs due to a complex cause such as the immune response of the human body (host), adsorption of proteins and circulating cells.

In particular, biologically untreated tissues have a high degree of decomposition in the body, but the fact that the calcification is greatly reduced is indicated that glutaraldehyde treatment itself is a one-dimensional dimension of calcification, and carbodiimide or polyethylene glycol diglycerides instead of glutaraldehyde. Crosslinking treatments with diepoxy compounds such as ridyl ethers have been studied (T. Okoshi et al., TASAIO, 35, 411, 1990), but further studies are required for in vivo stability and blood compatibility. In addition, calcium ions that cause calcification are positive ions, thus binding protamine (G.Golomb et al., J. Biomed. Mater. Res., 25, 85, 1991) or aluminum (CL Webb et al., TASAIO, 34, 855, 1988). A method of suppressing the adsorption of calcium by electrical repulsive force by introducing a cation such as, an iron compound (M. Bailwin et al., Trans. Soc. Biomat., 14,61, 1991) into the tissue in advance.

In addition, anionic polysaccharides such as chondroitin sulfate (GM Bernacca et al., Biomaterials, 13, 345, 1992), chitosan (J. Chanda et al., Biomaterials, 15, 465, 1994), acetylsalicylic acid (US Pat. No. 4,838,888), acrylic acid or acrylic esters (US Pat. No. 4,770,665), aminooleic acid (WO 89/06945) physiological calcification inhibitor diphosphonate (RJ Levy et al., Circulation, 81, 349, 1985), cleaning agents such as sodium dodecyl sulfate (RJ Levy et al., CRC Crit. Rev. Biocompat., 2, 147, 1986), sulfonated polyethylene oxides (Korean Patent Nos. 1996-3746 and 1998-66242) have been proposed.

However, the above methods are not easy to manufacture, and the proposed compounds have not been tested for in vivo stability and blood compatibility, requiring further research for clinical use.

In order to improve this, a biotissue implant (Korean Patent Laid-Open No. 2001-38908) has been proposed, in which heparin, a physiological antithrombogenic substance, is bound to a glutaraldehyde-modified biological tissue, and a glutaraldehyde modified Biotissue implants (Korean Patent Publication No. 2004-0070988) have been proposed that chemically binds arginine, one of the amino acid amino acids, to biological tissues. However, until now, acellular tissues have not been applied to biological tissues extracted from humans or animals. There was no introduction of biotissue implants that bind heparin, an antithrombogenic substance after treatment.

The present invention is to solve the above-mentioned calcification problem, the specific object of the present invention is excellent in anti-calcification function, in vivo durability, blood compatibility (blood compatibility) and angiogenesis function, artificial heart valve, circulatory surgical patch (Patch), tissue vessels, skin regeneration, etc. It provides a method for producing a heparin-coupled anti-calcifying acellular biological tissue implants that can be usefully used as acellular biological tissue implants.

It is also an object of the present invention to provide a heparin-bound anticalcifiable acellular biological tissue implant prepared by the above method.

In order to achieve the above object, the present invention

(a) treating biological tissues of animals other than humans with acellular biological tissues;

(b) crosslinking the acellular biological tissue with a glutaraldehyde solution; And

(c) binding heparin to the cross-linked acellular biological tissue;

It provides a method for producing anti-calcification acellular biological tissue implants comprising heparin-containing.

The present invention also provides a heparin-bound anticalcifiable acellular tissue implant prepared by the above method.

Hereinafter, the present invention will be described in more detail.

The present inventors have shown that when heparin is bound to biological tissues extracted from animals other than humans, and then heparin is bound, anti-calcification function and biomarkers are compared with those of hepatin-binding biological tissues which have not been treated with conventional acellular biological tissues. It has been found to be excellent in durability, blood compatibility and tissue compatibility and has come to complete the present invention.

Heparin-coupled anticalcification-free acellular tissue graft production method of the present invention comprises the steps of first, treating the non-human animal tissues with acellular biological tissue; Second, crosslinking the acellular biological tissues with a glutaraldehyde solution; And third, binding heparin to the cross-linked acellular biological tissue. Hereinafter, the three steps will be described in more detail.

First, non-human animal tissues are treated with acellular tissues. The biological tissues of animals other than humans that can be applied to this step are preferably biological tissues extracted from cattle, horses, pigs, and the like, particularly cardiac valve, pericardium, and dura mater. In the present invention, the biological tissue is treated as an acellular biological tissue by the method of hemolysis of the cells contained in the biological tissue and the removal of the intracellular components through chemical and enzymatic treatment before the biological tissue is fixed with the glutaraldehyde solution. . As a solution used in the cell hemolysis process, a storage tris buffer (pH 8.0) is used, and as a protease inhibitor, phenylmethyl-sulfonyl fluoride is preferable. Treating acellular biological tissue using a storage tris buffer solution containing the protease inhibitor, and comprising a protease inhibitor containing 1% octylphenoxypolyethoxyethanol (Triton X-100). Treat with stock Tris buffer solution and wash with Hank's solution. This step can be repeated several times depending on the acellular tissue used. Residual nucleic acids in biological tissues are treated with a stock Tris buffer solution containing a protease inhibitor containing 1% Triton X-100, while enzymes such as ribonuclease and deoxyribonuclease Degradation by treatment with. Thereafter, washing with Hank's solution and treatment with 1% SDS solution removes remaining components inside the acellular biological tissue.

The acellular tissues treated in this way can remove the remaining cells and apply them as collagen matrix to improve the problems of foreign body reactions and immune responses, which are disadvantages of cellular tissues, and increase the maximum tensile strength and the maximum elongation. Physical properties are also improved. As such, after treatment with acellular living tissue, staining may be performed using light microscopy, and the area of the cell where the cell exists is preferably not more than 0.1% of the total area.

Second, acellular living tissue is crosslinked with glutaraldehyde solution. The extracted acellular biological tissues need to be preserved and treated at stable pH conditions that do not damage the tissues. In the present invention, in accordance with the conventional method (Carpenter, etc.) was treated with an aqueous solution of glutaraldehyde in order to improve the mechanical properties of the acellular biological tissues, to reduce the degradation in the body, to remove the side effects caused by the immune response and sterilization. 0.05-1.5% is preferable and, as for the density | concentration of the aqueous solution of glutaraldehyde used for processing acellular biological tissue, 0.1-1.0% is more preferable. If the concentration of the glutaraldehyde aqueous solution is less than 0.05%, it is difficult to expect the crosslinking reaction effect of the above-mentioned glutaraldehyde solution treatment, and if it exceeds 1.5%, the crosslinking reaction is rapidly progressed and the glutaraldehyde is formed into the acellular biological tissue. There is a problem that can not be uniformly crosslinked.

Moreover, 7.0-7.6 are preferable and, as for pH in the case of processing acellular biological tissue with glutaraldehyde aqueous solution, 7.1-7.5 are more preferable. For this purpose, it is preferable to treat the glutaraldehyde aqueous solution with an appropriate buffer solution. Conditions for the buffer solution should be excellent in stability for the treatment or storage of glutaraldehyde of the acellular biological tissues, preferably inert and excellent buffering capacity that does not interfere with the cross-linking reaction by glutaraldehyde, typically phosphate buffer Saline (phosphate buffered saline, PBS, pH 7.4, 0.01 ~ 0.02M) can be used.

Third, heparin is bound to the cross-linked acellular living tissue. The acellular biological tissue crosslinked with the glutaraldehyde solution is left unreacted glutaraldehyde in the biomaterial during the crosslinking and preservation process with the glutaraldehyde solution, and the unreacted glutaraldehyde is the calcification of the biomaterial. It is pointed out as the main cause of this. Therefore, methods for removing unreacted glutaraldehyde groups as suitable compounds containing amino groups have been reported to exhibit anticalcification effects, and chitosan (see J. Chanda et al., Biomaterials, 15, 465 (1994)), aminooleic acid (WO 89). / 06945), amino polyethylene oxide sulfonic acid (Kim Youngha et al., See Korean Patent 131046 and 181691) and the like are representative examples.

However, since the amino group-containing compounds for anticalcification are rarely used in implants in the body, it is necessary to verify the stability. In contrast, heparin used in the present invention is a polydisperse natural polysaccharide synthesized in the body and is a safe compound currently used as a representative anti-blood premise in the clinic. Heparin has a low molecular weight heparin of 2,000 to 5,000 and a high molecular heparin of 7,000 to 20,000 depending on the type, and low molecular heparin has been reported to have higher antithrombogenicity than polymer heparin (RD Rosengerg, Heparin: New Biochemical and Medical Aspects, Witt) Ed., Walter de Gruyter, Berle (1983).

The concentration of heparin used in the method of the present invention is preferably 0.05 to 20%, more preferably 0.1 to 10%. If the concentration of heparin is less than 0.05%, the concentration is too small to reduce the heparin treatment effect, if it exceeds 20% there is a problem that the expensive heparin treatment effect does not increase in proportion to economical.

Moreover, in this invention, what decompose | disassembled heparin by suitable methods, such as oxidation, can also be used as needed. For oxidation of heparin, see J. Chanda et al., Biomaterials, 15, 465 (1994).

Since the structure of heparin contains a considerable concentration of amino groups and carboxylic acid groups in addition to many sulfonic acid groups (antithrombogenic expression) in the polysaccharide skeleton, they can be reacted with aldehyde groups and combined with Schiff bases and acetal compounds, respectively (RD Rosenberg, Heparin: New biomedical and medical aspects, W.de Gruyter, Berlin (1983). The Schiff base produced during the heparin bonding process is preferably stabilized by reducing with sodium borohydride (NaBH ₄ ). The concentration of sodium borohydride used for the Schiff base reduction is preferably 0.001 N to 1 N, more preferably 0.005 N to 0.5 N. If the concentration of sodium borohydride is less than 0.001 N, the reduction reaction is not sufficient, if the concentration exceeds 1 N there is a problem that the reduction effect does not increase proportionally.

Anticalcified acellular biotissue implants prepared by the methods described above were subjected to appropriate treatments (PBS wash, amino acid solution wash, methanol treatment) as needed to treat surgical tissue patches, tissues in the heart valve, surgical / circulatory / dental fields. It can be used as a porous support for blood vessels, tissue tendons, ligaments and tissue engineering.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the present invention is not limited by these examples.

(Example 1)

Acellular Treatment and Glutaaldehyde Crosslinking Treatment

The pericardium extracted from the cows was preserved in Hanks' solution, and additional blood and tissues were removed with saline, and then the thick fat layer attached to the pericardium was removed from the sterilization chamber, followed by phenylmethyl-sulfonyl fluoride (0.35 mg). Cells were lysed by stirring at a constant rate at 4 ° C. for 24 hours in a solution of Hypotonic tris buffer (pH 8.0). In the next step, a stock solution containing a protease inhibitor containing 1% octylphenoxypolyethoxyethanol (Triton X-100) was treated at 4 ° C. for 24 hours and washed with Hank solution. In the third step, the sample was treated with a ribonuclease solution and a deoxyribonuclease mixed solution at 37 ° C. for 3 hours, followed by 1% octylphenoxypolyethoxyethanol (Triton X-100). The stored tris buffer solution containing the protease inhibitor containing was treated for 48 hours at 4 ℃. Finally it was washed in Hank solution, treated with 1% SDS solution and stored in Hank solution. The treated pericardial tissues were then washed several times with PBS, soaked in 0.625% glutaraldehyde solution for 3 days, and then washed again with PBS.

Heparin Immobilization in Acellular Tissues

The resulting acellular tissue was reacted for 2 days at 4 ° C. in a buffer solution (pH 11.0) in which 2.5% heparin (US Sigma, molecular weight 4,000, activity 100 unit / mg) was dissolved. Heparin-immobilized biological tissue was washed several times with 0.01 M PBS and then reduced in 0.01 N NaBH ₄ solution at 4 ° C. for 16 hours.

(Comparative Example 1)

The pericardium extracted from cattle was preserved in Hanks' solution, the thick fat layer attached to the pericardium was removed from the sterilization chamber and maintained in the cow for 24 hours to remove soluble protein. The treated biological tissues were washed with PBS solution and soaked in 0.4% glutaraldehyde solution for 10 hours, followed by 2.5% low molecular weight heparin (Sigma, USA, molecular weight 4,000, activity 100 unit / mg) buffer solution (pH 11.0). Reaction at 4 ° C. for 2 days. The heparin immobilized biological tissue was washed several times with PBS, and then reduced in 0.01 N NaBH ₄ solution at 4 ° C. for 16 hours.

(Comparative Example 2)

The pericardium extracted from cattle was preserved in Hank's solution, the thick fat layer attached to the pericardium was removed, and then stored in a solution of Hypotonic tris buffer (pH 8.0) containing phenylmethyl-sulfonyl fluoride for 24 hours. Cells were lysed by treatment at ° C. In the next step, the stock solution containing the protease inhibitor containing 1% octylphenoxypolyethoxyethanol (Triton X-100) was treated at 4 ° C. for 24 hours and washed with Hank solution. In the third step, the sample was treated with a ribonuclease solution and a deoxyribonuclease mixed solution at 37 ° C. for 3 hours, followed by 1% octylphenoxypolyethoxyethanol (Triton X-100). The storage tris buffer solution containing the protease inhibitor containing the was treated for 24 hours at 4 ℃. Finally it was washed in Hank solution, treated with 1% SDS solution and stored in Hank solution. The treated pericardial tissues were then washed several times with PBS and then treated for 10 hours in 0.4% glutaraldehyde solution. It was then reacted for 2 days at 4 ° C. in 2% arginine solution (pH 11.0). Arginine-fixed pericardial tissue was washed several times with PBS, and then reduced in 0.01 N sodium borohydride solution at 4 ° C. for 16 hours.

(Property evaluation)

Physical and chemical properties of the acellular biological tissue grafts of the present invention were measured through shrinkage temperature, mechanical tensile strength and elongation, degradability using collagenase, and calcification by animal subcutaneous muscle transplantation experiments. It was.

Shrinkage temperature was measured by differential scanning calorimeter (DSC, DSC, TA Instruments, DSC 2010) at a temperature of 2 ℃ / min, the higher the shrinkage temperature means that the stability of the acellular biological tissue.

In addition, the tensile strength was measured by a tensile tester (Instron, Model 8511), 50 kg load cell (load cell). In addition, collagen degradability was about 1 mg of tissue was digested at 37 ℃ for 36 hours using a collagen degrading enzyme (Clostridium histolyticum Type II, activity 357 units / mg, Sigma, USA) solution and the remaining amount was dried and quantified (Bak Gi-dong, Biomaterials, etc.) , 18, 47, 1997).

In addition, the animal subcutaneous muscle transplantation experiments were carried out a patch of acellular tissue tissue (size 1 cm ㅧ 1.5 cm) to the subcutaneous muscle between the back skin and muscles of the rat (Sprague-Dawley rat, 5 weeks old male, weight 150 ~ 180g) After 8 weeks, samples taken out and hydrolyzed with 6N hydrochloric acid solution were performed by calcium quantification using an inductively coupled plasma (ICP, Jovin Yvon, 138 Ultrace).

The amount of calcium deposited was also expressed as the amount of calcium per microgram of dried tissue (mg). In the circulatory transplantation calcification experiment, dogs (Korean dogs, 25-30 kg) were used to extract the tissue patch prepared in the spindle form after 8 weeks of implantation into the aorta, and the amount of calcium deposited was measured by the same method (Pak Ki-dong et al., Biomaterilas, 18, 47, 1997).

Table 1 summarizes the results of the experiment in Example 1 and Comparative Examples 1 and 2.

division Shrinkage Temperature (℃) Collagen Residual Residue (%) Tensile strength (㎏ / ㎣) Elongation at break (%) Amount of calcium deposited (μg / mg) Example 1 90 92 1.96 41 5.9 Comparative Example 1 86 90 1.41 34 6.5 Comparative Example 2 86 91 1.52 34 17.0

As shown in Table 1, in the case of a biological tissue implant (Example 1) in which heparin-coupled biological tissues after treatment of non-human biological tissues with acellular biological tissues were not treated with acellular biological tissues, Compared with (Comparative Example 1) and a tissue-free tissue implant with arginine after treatment with acellular biological tissue (Comparative Example 2), physical properties (shrinkage temperature, collagen decomposition residue, maximum tensile strength, maximum elongation and amount of calcium deposited) are superior. And it was found. Therefore, the tissue-enhanced graft implanted with heparin after treatment of acellular tissue prepared according to the method of the present invention has excellent anti-calcification, durability in vivo, blood compatibility and tissue compatibility, Cardiac valves, tissue vessels and circulatory surgical patches (patches), tissue regeneration membranes and tissue fillers, such as a cell can be found to be useful for implantation.

Heparin-conjugated anticalcification-free acellular tissue implants of the present invention are excellent in anticalcification function, durability in vivo, blood compatibility and tissue compatibility (artificial heart valve, tissue vessels and circulatory system) Surgical patches, tissue rejuvenation membranes and tissue fillers, such as acellular implants can be usefully used.

Claims (14)

(a) treating a living tissue of a non-human animal selected from the heart valve, the pericardium, or the dura mater with acellular biological tissue; (b) crosslinking the acellular biological tissues with a glutaraldehyde solution at a pH of 7.0 to 7.6, 0.05 to 1.5 volume percent; (c) binding heparin to the crosslinked acellular biological tissue; And (d) binding the heparin to the acellular biological tissue and then reducing the Schiff base with 0.01 to 1 N sodium borohydride (NaBH ₄ ) Heparin-coupled anti-calcification acellular biological tissue comprising a manufacturing method comprising a. delete delete delete delete The method of claim 1, The heparin has a low molecular weight heparin molecular weight of 2,000 to 5,000, characterized in that the anticalcification acellular biological tissue implant manufacturing method. The method of claim 1, The heparin has a molecular weight of 7,000 to 20,000 heparin-coupled, characterized in that the heparin-coupled anticalcification acellular biological tissue implant manufacturing method. The method of claim 1, And wherein said heparin is partially oxidized. delete delete delete delete delete delete