KR101075399B1 - Production method of heterograft - Google Patents

Abstract

The present invention relates to a method for producing a xenograft prosthesis, and more specifically, the method of the present invention after acellularizing a heterologous biological tissue, the tissue is glutaraldehyde solution containing ���olol solvent in the state of non-membrane pressure It is made to include the anti-toxin process to fix and remove the toxicity, xenograft prosthesis prepared thereby can be useful in heart valve transplantation or cardiovascular surgery.

Heart valve, pericardium, vascular substitute, acellularization, glutaraldehyde solution, antitoxin, xenograft, fixation free platelet pressure

Description

Production method of heterograft

The present invention relates to a method for producing a xenograft prosthesis which can be used in heart valve transplantation and cardiovascular surgery.

With advances in cardiac surgery, the need for artificial prosthetic tissues other than self tissues to correct congenital complex cardiac anomalies, heart valve disease, and macrovascular disease has increased, and various alternatives have been researched and developed. Alternatives from have been developed and used for a long time. Glutalaldehyde (GA) has been widely used as a fixative solution for use in the human body of heterologous tissues and pericardium.

Insufficiency of artificial tissue valves or prosthetic tissue prostheses inserted into the human body is denatured, degenerated, and eventually leads to functional loss and disappearance due to chemical, physical, and immunological reactions with the living body. It is the complete calcification in the body. Many studies have been conducted to reduce the degeneration and degeneration of such tissues from the past until now, but the results are still not satisfactory.

In particular, the presence of dead tissue cells that provide a source of calcification in the pericardium or valves of non-cellularized cows or pigs, and the continuous inclusion of heterologous immunity; The aldehyde group (-CHO) of the aldehyde reacts with calcium in the body to cause calcification, but since it is not properly treated, it causes valve failure as early calcification after implantation in the heart.

In addition, these heart valves and cardiovascular substitutes are not produced and sold in Korea, but are expensive and rely on imports from abroad, and there is no domestic research technology. Therefore, it is to develop a method of fixation of tissues essential and essential for the production of artificial tissue valves, valve conduits, and cardiovascular substitutes, and in particular, attempted an immunologically superior method, and also overcomes the disadvantages of the fixation method of glutaraldehyde alone. It is to. Although the invention of the glutaraldehyde fixation method makes it easier to store or handle xenograft valves or pericardium fragments, many challenges remain in immune response and durability, and thus it is not perfect. It is emerging.

Accordingly, the present inventors have completed the present invention by developing a fixed treatment method and an antitoxin method using a new cell-free and solvent, etc. to solve these problems.

It is an object of the present invention to provide a method for producing a xenograft prosthesis which can be used in heart valve transplantation and cardiovascular surgery, and a xenograft prosthesis produced thereby.

In order to solve the above problems, the present invention acellularize the biological tissues of animals other than humans, and fix the acellularized tissues in a high concentration of glutaraldehyde solution containing ethanol solvent, the toxicity of the tissues It provides a method for producing a xenograft prosthesis comprising the step of removing. The present invention, in particular, by using a mixture of anhydrous solvent at the time of glutaraldehyde fixation in comparison with the glutaraldehyde alone fixation method used in the conventional manufacturing of artificial tissue valve, valve conduit, cardiovascular substitute, etc. Induce cross-linking of elastic fibers (elastin) and apply biochemical reactions to tissues to improve mechanical properties and reduce long-term calcification after fixation of valves or fresh pericardial fragments. In another aspect, the present invention, by using a high concentration of glutaraldehyde in the fixation process by treating xenograft pericardium or valves to contribute to anticalcification, eliminating the toxicity of residual glutaraldehyde that may be derived from glutaraldehyde To include the process of doing so.

The present invention also provides a xenograft prosthesis produced by the above method.

Hereinafter, the present invention will be described in detail.

According to the first aspect of the present invention, the present invention is a cell-free tissues of animals other than humans, and after fixing the acellularized tissues to a high concentration of glutaraldehyde solution containing an ethanol solvent, toxicity of the tissues It provides a method for producing a xenograft prosthesis comprising the step of removing. Preferably, the biological tissue may be selected from pericardium, heart valve, pericardium or dura mat, more preferably biological tissue derived from a cow or a pig.

Look at each process in detail. First, the pig aorta and pulmonary valve conduit and bovine pericardium and pig pericardium (hereinafter referred to as 'treatment tissue'), which were collected as a relatively sterile state from the slaughterhouse, were kept in refrigerated saline containing antibiotics, and then refrigerated. After pretreatment in the manufacturing room, the treated tissues are acellularized. The acellularization is achieved by sequentially treating storage tissues, hypertonic buffers, and isotonic buffers in the treated tissue, and proceed to acellularization with rotation shaking during all of the acellularizations, preferably Rotate and shake more than 50-200 times per minute. In the treatment of the buffer solution, it may be possible to proceed to acellularization by some repeated treatment. Preferably, the acellularization is 20-36 hours in 3-8 ℃ storage buffer, 6-12 hours in 3-5 ℃ hypertonic buffer, 11-13 hours in isotonic buffer of 3-5 ℃ In the treatment of the storage buffer, more preferably, the biological tissue is treated with the storage buffer for 6-14 hours, followed by the treatment with the storage buffer containing the detergent for 14-24 hours. To lose. As an example, the acellularization was performed for 14-14 hours in a 4 ℃ storage solution in the case of heart valves, 14-24 in a solution mixed with a storage solution and 0.1%-1% SDS solution at a temperature of 3-8 ℃ Time-treated, followed by another 6-12 hours treatment at 4 ° C. hypertonic solution followed by 12 hours treatment at 4 ° C. isotonic solution. In the case of pericardium, it is acellularized using a storage solution containing low concentration of SDS, or using sodium deoxycholate. It is also possible to use 14 to 24 hours in 3-8 ℃ depotable solution containing deoxycholate, 6-12 hours in hypertonic solution and 12 hours in 4 ℃ isotonic solution.

This complete acellularization followed by fixation under zero-pressure fixation. In order to maintain the natural shape during the fixing, the same hydraulic pressure is applied from the upper side and the lower side of the valve by fixing it in the forming mold to fix the valve to prevent the pressure difference between the upper and lower sides of the valve. In other words, by fixing when the pressure, fatigue, and impact applied to the valve are in a zero state, the valve tissue, particularly fibrin, can be maintained in the most appropriate state, thereby maintaining an ideal state even for long-term exercise or exposure (see FIG. 11). In particular, in order to maintain proper diastolic expansion, the aortic valve is 14-20 mmHg and the pulmonary valve has 7-10 mmHg load pressure. pressure).

In addition, the fixation may be performed before or after the treatment of the glutaraldehyde solution containing 1-3% of the ethanol solvent in the concentration of 0.25-0.625% of the glutaraldehyde solution, or 1-3% containing the ethanol solvent. The glutalaldehyde solution at a concentration of 0.25-0.625% may be treated before and after the concentration of the glutaraldehyde solution. The ethanol solvent may be 70-80% ethanol or mixed ethanol, the mixed ethanol may be composed of 67.5-75% ethanol and 2.5-5% octanol or octanediol. As an example, a 0.5% glutaraldehyde solution, commercially available, was fixed for 3-5 days under zero-pressure fixation, followed by a high concentration (1-3%) glutaraldehyde solution and 70-80. The solution containing% mixed ethanol (67.5-75% ethanol + 2.5-5% octanol or octanediol) can be treated for 1-2 days, after which it can be treated for 5 days at low concentration (0.25%).

After fixing to glutaraldehyde, residual glutaraldehyde is removed by removing one or more amino acids selected from lysine, glycine, borohydride, glutamic acid and arginine to remove free aldehyde group (-CHO) of glutaraldehyde. Eliminates the toxicity of aldehydes. At this time, the concentration of the added amino acid is 0.05 ~ 0.2 M, it is preferable to treat at 34 ~ 42 ℃ for 24 to 48 hours. In addition, due to the diamine bridge by lysine or glycine, not only has an anticalcification effect, but also the cross-link can be increased to increase the thickness and tension of the xenograft prosthesis. For example, after the fixation, immersed in lysine (0.05-0.2M L-lysine solution (acetic acid buffer, or PBS), pH 4.5 ~ 7.6), treated at 37 ° C. for 24-48 hours, or glycine (0.05-0.2M Soak Glysine with PBS solution for 24-48 hours at room temperature, or use Sodium borohydride solution (Sodium borohydride 0.08M in buffered glycine (0.2% HEPES, 0.2M glycine, 0.1M NAOH, pH 10.3) or buffered carbonate (0.1M) Incubate 24-48 hours at room temperature to remove reagents (aldehyde groups) that did not react to NaHCO ₃ , 0.1M Na ₂ CO ₃ , pH; 10.3) or completely saturated glutamic acid solution (saturated aqueous solution). solution of L-glutamic acid, or 0.05-0.1M L-glutamic acid, ph 3, room temperature) or 2% arginine (or 0.12M, L-arginine, in PBS phosphate buffered saline (PBS) at pH 11) Incubate at room temperature for 24 to 48 hours, using one of several antitoxin treatment methods. After the final disinfection, the solution is stored in 2% benzyl alcohol solution or 1% benzylkonium solution after the final disinfection.

In addition, according to a second aspect of the present invention, the present invention provides a xenograft prosthesis produced by the above method. In particular, xenograft prostheses produced by the above method from pig or bovine pericardium can be applied to cardiovascular surgery, such as replenishment of defective pericardium, closure and reinforcement of cardiovascular defects, and use as a prosthesis for complex cardiovascular surgery. Do. As a part of blood vessels or heart, it serves as a material to allow blood to flow in a desired direction without leakage. The xenograft prosthesis prepared according to the present invention is immunogenicity is removed by acellularization, and the tissue is processed. As a result, calcification does not occur, and durability and mobility are improved, and thus the function of the tissue to be transplanted is maintained for a long time.

As described above, according to the present invention, the acellularized tissues of animals other than humans are acellularized, and the acellularized tissues are fixed in a high concentration of glutaraldehyde solution containing an ethanol solvent, and then the toxicity of the tissues is removed. Provided are a method for producing a xenograft prosthesis comprising a step and an anticalcified and antitoxinized acellular xenograft prosthesis produced thereby.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Examining Structural Properties Between Tension and Thickness

In this example, pig aortic valves, pulmonary artery valves, pericardium and bovine pericardium are fixed in 0.25-0.3%, 0.5-0.625%, or 1, 2%, 3% of different glutalaldehyde solutions, and the respective tension and The thickness was measured and the fixation was performed in the state of no-membrane pressure using the apparatus and principle shown in FIG.

1-1 Preparation of Materials

In a slaughterhouse, under the cooperation of a veterinarian, the heart and pericardium are extracted from healthy slaughtered pigs, placed in a cold phosphate buffered solution (PBS, 0.1 M, pH 7.4) and transported in an ice box to a laboratory. Separate the pericardium, and store it for use in the experiment. The pericardium of a healthy slaughtered cow is also removed and carried in the same way.

1-2 hetero valves, fixation of the pericardium

Aortic valves, pulmonary valves, pericardium, and bovine pericardium of pigs transported to the laboratory refrigerated (4 ° C.) in the slaughterhouse are fixed immediately within at least 12 hours after slaughter.

① The group fixed with glutaraldehyde (GA) alone was immersed in 0.5% -0.625% or 1.5%, 3% buffered glutaraldehyde and fixed at 4C for 2-3 days, then 0.3% -0.25% buffered glutaraldehyde at room temperature. Fixed for 7 days at.

② The group using the solvent for anticalcification together with the fixation of glutaraldehyde was treated with 0.25% -0.3%, 0.5% -0.625% or 1-3% glutaraldehyde before and after fixing.

-80% ethanol, 80% mixed ethanol (75% ethanol + 5% octanol or 75% ethanol + 5% octanediol), fixed at 4 ° C and then fixed at 0.25% -0.3% glutalaldehyde at room temperature for 12 days or ,

-After fixation in 0.5% -0.625% glutaraldehyde for 5 days, put in shaker bath containing 80% ethanol or 75% ethanol + 5% octanol or 75% ethanol + 5% octanediol (PBS buffer, Ph 7.4) 2 After treatment for 3 days, soaked in 0.25% -0.3% glutaraldehyde at room temperature again and fixed for 7 days.

At this time, when the xenograft aortic valve and pulmonary valve are fixed, they are placed in the formation frame to maintain their natural shape, and the aortic valve is 14-20 mmHg and the pulmonary valve is 7-10 mmHg to maintain the proper diastolic spread. After applying the pressure (distension pressure), after confirming that the valve does not escape to the back was fixed to zero pressure (zero pressure). In this way, when the fixed valve pressure is fixed, the same hydraulic pressure is applied to the upper and lower sides of the valve, so that there is no pressure difference between the upper and lower valves, and thus the optimal valve can be fixed. In other words, by fixing when the pressure, fatigue, and impact applied to the valve are in a zero state, the valve tissue, in particular, the fibrin, can be maintained in the most appropriate state, thereby maintaining an ideal state even for long-term exercise or exposure (see FIG. 12). The fixed pericardial fragments were washed several times at room temperature with PBS solution, and then stored in PBS solution at 4 ° C.

1-3 Structural Characteristic Inspection

(1) physical activity test

Treated grafts were palpated, sutured and stretched directly with the examiner's hand. For the consistency of the examination, the criteria were evaluated by a single thoracic surgeon. The palpation measures the softness of the tissue by touching it, the suture describes the feeling of penetrating the tissue using 5-0 prolene (Ethicon, Johnson & Johnson), and the elongation measures the extent of stretching through the tissue. Measurement technology.

Promote the graft with no treatment to the next level based on 0 (soft = 1, slightly soft = 2, normal = 3, not soft = 4, slightly hard feeling = 5), suture (sleeping = 1, Slightly Entered = 2, Moderate = 3, Slightly Resistant = 4, Resistant = 5), Elongation (Slant = 1, Slightly Stretched = 2, Moderate = 3, Not Stretched = 4, Almost Stretched) None = 5), scores of the grafts were averaged and compared by low GA, high GA, and GA + Solvent fixation.

(2) examination of tension and elasticity of xenografts and pericardium;

The treated porcine aortic valve and porcine pulmonary artery valve were unfolded, and the longitudinal sections of 7.0 x 5.0 mm were taken in circumferential and radial directions, respectively, and the tensile strength and elasticity of 5 mm in width were measured. The mean value was made to represent the tensile strength and elasticity of the valve. Similarly, by measuring the tensile strength and elasticity of 5 mm wide by taking a rectangular section of 0.5 × 5 cm in all six directions that can be obtained by unfolding the treated porcine pericardium and bovine pericardium and varying the angle by 30 °. His average value was attempted to be representative of the tensile strength and elasticity of the pericardium. The tension was measured using Japan Tech & Manufacture, Digital Force Gauge, Model 5FGN, and automated materials testing system.The pericardium measured the load speed 100 mm / min and the valve load speed 6mm / min. It is expressed in mm.

(3) statistical method

Xenograft valves and pericardial tension were expressed as Kgf / width 5 mm and elasticity was expressed as the mean ± standard deviation of the ratio of increased length to initial length. In the thermal stability test, the temperature at the time of contraction of the pericardial section was denoted by C and each value was expressed by the mean ± standard deviation. Statistical differences between groups were verified by paired T-test and ANOVA test. In each experiment, the comparison between each group up to the high and low glutaraldehyde-fixed groups was performed by the Turkey b test. p <0.05 is considered significant.

1-4 results

(1) Physical activity test.

A subjective evaluation of the palpation, suture and elongation of xenografts by a thoracic surgeon was performed. Regardless of the type of graft, nothing was treated, and the evaluation was repeated for each fixation method based on 0. Therefore, the graft was divided into valves and pericardial groups, and the results are shown in Table 1 below. There was no significant difference in palpation between low concentration GA, high concentration GA, and GA + Solvent treatment. There was no difference between the low and high GA groups, but there was a significant difference in the valve (P = 0.018) and pericardial (P = 0.009) groups in the suture test. In the renal test, there was a significant difference between the low concentration and GA + Solvent groups in both valve (P = 0.000) and pericardium (P = 0.0329).

In other words, there was no statistically significant difference in facilitation findings, and the GA + Solvent group felt more firmly in the suture and kidney drawings than in the low concentration GA. Although subjective, the surgeon evaluated the extent to which the surgeon felt during the operation.

(2) Pressure-tension, pressure-elasticity comparison

In the directional pressure-tension and pressure-elasticity test, there was no statistically significant difference in pressure-tension and pressure-elasticity between the GA fixation group and the GA + Solvent fixation group in the pig aortic valve, and in the pulmonary valve of the pig. There was no statistically significant difference in pressure-tension and pressure-elasticity between GA and Sol + GA groups.

In the case of porcine pericardium and bovine pericardium, there was no statistically significant difference in pressure-tension and pressure-elasticity between GA and Sol + GA groups.

(3) Comparison between low and high glutaraldehyde groups

Low glutaraldehyde, high glutaraldehyde, and solvent addition were examined.

① There was no statistically significant difference in the tension and elasticity comparison between the low and high GA groups in both circumferential and radial directions (P> 0.05).

② In circumferential tension comparison, the pulmonary valve of pigs was not different between low and high GA groups, regardless of the presence or absence of solvent addition.However, in the circumferential elasticity comparison, there was no difference between solvent and no solvents (low GA and high GA: 50.90+). 23.54 vs 102.50 + 33.75, p = 0.028), elasticity at high GA fixation in the solvent added group (low concentration GA treatment + solvent group vs high concentration GA + Solvent treatment group: 62.89 + 39.88 vs 108.00 + 37.75, p = 0.001) Increased. In the radial tension comparison, there was no difference between the low and high GA groups, regardless of the addition of solvent, but in the radial elasticity comparison, the elasticity was increased in the high concentration of GA in the solvent-added group. (Low GA + solvent vs high GA + solvent: 84.39 + 26.20 vs 125.44 + 42.72, P = 0.011).

③ The bovine pericardium showed no statistically significant difference in tension comparison between low and high GA group (P> 0.05), regardless of the addition of solvent (P> 0.05). The elasticity was higher than that of the added group (low concentration GA vs low concentration GA + solvent: 34.26 + 15.29 vs 21.00 + 8.27, p = 0.000).

④ There was no statistically significant difference in tension comparison between the low and high GA groups in the pig pericardium (P> 0.05). However, in the comparison of elasticity, the group without solvent (low GA vs. high GA: 13.19 + 5.89 vs 21.66 + 3.23, p = 0.001) and the group with solvent (low GA + solvent vs. high GA + solvent: 13.66 + 6.02 vs 20.86 + 7.43 p = 0.000) all had increased elasticity in high concentration GA group.

As a result of this Example, there was no statistically significant difference in facilitation in physical activity test of cardiovascular tissue, and the GA + Solvent group felt more firmly in the suture and kidney drawings than in the low concentration GA. Although subjective, the surgeon assessed the extent to which the surgeon felt during the operation.

In addition, in the unidirectional pressure-tension and pressure-elasticity test, there was no statistically significant difference in pressure-tension and pressure-elasticity between the GA fixation group and the GA + Solvent fixation group in the pig aortic valve. In addition, there was no statistically significant difference in pressure-tension and pressure-elasticity between GA and GA + Solvent anchor groups. In the case of porcine pericardium and bovine pericardium, there was no statistically significant difference in pressure-tension and pressure-elasticity between GA and Sol + GA groups. The use of solvents during GA fixation did not result in any physical changes in the tissues that showed statistically significant differences. In conclusion, GA fixation improves tension and elasticity, but cross link is different along the structural fiber direction of valve.

Example 2: Histological Analysis According to a Fixation Method

In this example, the porcine pericardium, the aorta, the pulmonary artery valve, and the human pericardium were observed by optical and electron microscopy before and after fixation in a 0.25%-0.625% glutalaldehyde solution. Groups treated with porcine pericardium, aorta, and pulmonary artery valves 0.25%-0.625% glutaraldehyde before and after fixation with 70-80% mixed ethanol and acellularized with sodium dodecylsulfate (SDS). 0.25%-0.625% glutaraldehyde-fixed group, 70% -80% mixed ethanol-treated and then acellularized again 0.25%-0.625% glutaraldehyde-fixed group, acellularized and ethanol-treated again 0.25%- Each group was observed by optical and electron microscopy divided into groups of 0.625% glutaraldehyde fixation. In addition, the fixation was carried out in the state of no-film valve pressure.

2-1 Preparation and Fixing of Material

Immediately remove the pericardium, aorta and pulmonary valves from healthy slaughtered pigs and place them in cold phosphate buffered solution (PBS, 0.1M, pH 7.4, 4 ° C) in ice boxes for transport to the laboratory. Porcine pericardium, aortic valve, and pulmonary artery valve, which have been refrigerated in the slaughterhouse (4 ° C) to the laboratory, are fixed immediately within at least 12 hours after slaughter. Porcine pericardium, aortic valve, and pulmonary valve were removed from the slaughterhouse and fixed in 0.625% glutalaldehyde solution (PBS buffer, pH 7.4) for 2 days at 4 ° C, followed by an additional 7 days at room temperature. After fixing, divide the tissue into pieces of 1.5 cm in width and length. Tissue pieces that do not require further treatment are washed with PBS solution for 24 hours at room temperature and then stored in PBS solution at 4 ° C. Organizations requiring additional anticalcification should proceed to the next step.

2-2 Histological Examination

(1) Optical and electron microscopy

Optical microscopic examination was performed using extracted pericardium, aorta, and pulmonary valves. A 2 mm to 3 mm thick tissue was placed in Dubosqcbrasil solution for 1 hour, fixed in 10% formalin, and then paraffin-embedded. Hematoxylin-eosin, von Kossa, phosphotungstic acid hematoxylin, Movat pentachrome, and elastica-van Gieson staining were performed.

Electron microscopic examination was performed by 1 mm sized tissue, fixed in 4% glutaraldehyde-PBS (0.1 mol / L phosphate buffer, pH 7.4) solution, and then in 1% OsO4 (0.1 mol / L phosphate buffer, pH 7.4). Post fixation was performed for embedding into the Epon LX 112 fixative. Toluidine blue staining was performed on 1 um sections and observed under an optical microscope. After locating the appropriate tissue specimens, ultrathin sections of Reichert Ultracut E-ultramicrotome (Leica, Inc., Buffalo, NY) were used to stain Uranyl acetate and Lead citrate. The electron microscope JEM-100CX (Magnification; X100-X450,000, acceleration voltage: 120 kV, JEOL USA, Inc.) was observed.

(2) anticalcification and acellularization treatment

1) 80% Ethanol Kills

For 80% ethanol treatment, the tissue pieces were treated for 24 hours in two different ways, before and after fixation of 0.625% glutalaldehyde, and after each treatment, the physiological solution (PBS) was sufficiently washed (3 × 10 min) and the next step was performed. Was implemented.

The fixed or unfixed tissue pieces were placed in a shaker bath containing 4 ° C. 80% ethanol solution (PBS buffer, pH 7.4) for 24 hours at room temperature, and then stored in 4 ° C. PBS solution or unfixed tissue pieces were washed. After fixed 0.625% glutaraldehyde, and stored in 4 ℃ PBS solution.

2) Treatment after acellularization with sodium dodecyl sulfate (SDS)

Tissue pieces were treated for 14 hours at room temperature in Hypotonic buffer (Tris, 10 mmol / L; EDTA, Aprotinin, PH 8, Sigma Co., USA), then 24 hours in Hypotonic buffer containing 0.1-0.5% SDS, and then Isotonic buffer ( Tris, 50 mmol / L; EDTA, Aprotinin, PH 8, Sigma Co., USA), treated for 24 hours, acellularized, washed and stored in menstrual water, and acellularized. The group fixed in 0.625% glutaraldehyde fixative solution was further divided for 2 weeks, and stored at 4 ° C after washing with physiological water (PBS) for further examination.

3) Ethanol treatment and acellularization treatment using sodium dodecyl sulfate (SDS)

After treatment with 80% ethanol at room temperature for 24 hours, the cells were re-cellularized by the above-described method, and then fixed in 0.625% glutaraldehyde-fixed solution for 2 more weeks. The experiment was carried out in a group fixed in glutaraldehyde fixed solution for two more weeks.

2-4 results

(1) Comparison of porcine pericardium after glutaraldehyde fixation, ethanol and acellularization treatment (SDS)

Without any treatment, the human pericardium (0.36 ± 0.02 mm) appeared to be three times thicker than porcine pericardium (0.13 ± 0.05 mm), both of which had a serous layer, fibrosa, and epicardial connective tissue ( epicardial connective tissue). The mesentery layer formed a soft layer consisting of mesothelial cells. The fibrous layer made up the bulk of the pericardium and consisted of collagen, elastic fibers, nerves, blood vessels, and lymphatic vessels. The epicardial connective tissue was composed of loosely connected collagen and elastic fibers to form a rough layer (Fig. 1).

Porcine pericardium treated with 0.625% glutaraldehyde only, 80% ethanol treated before and after 0.625% glutalaldehyde fixation, and acellularized with sodium dodecylsulfate (SDS). 0.625% glutaraldehyde-fixed group, 80% ethanol-treated group after acellularization and then 0.625% glutaraldehyde-fixed group after acellularization, 0.625% glutalaldehyde-fixed group, and collagen maintained well It was. However, collagen fibers of the group treated with acellularization alone or without glutaraldehyde fixation after acellularization treatment were also loosely connected than other groups (FIG. 2).

(2) Comparison of porcine aortic valve and pulmonary artery valve after glutaraldehyde fixation, anticalcification and acellularization treatment

In the absence of any treatment, the porcine aortic valve and pulmonary artery valve consisted of three layers of lamina fibrosa, lamina spongiosa, and lamina ventricularis. Fibrous plate was composed of columnar collagen fibers, spongy plate was composed mainly of glycosaminoglycans (proteoglycan), and ventricular plate was composed of elastin with radial elastic fibers.

Porcine aortic and pulmonary artery valves treated with 0.625% glutaraldehyde only, 80% ethanol treated before and after 0.625% glutalaldehyde fixation, and acellularized with sodium dodecylsulfate (SDS) treatment. In the saturation process, 0.625% glutaraldehyde-fixed group, 80% ethanol-treated and acellularized again, and then 0.625% glutaraldehyde-fixed group, acellularized and ethanol-treated again, and 0.625% glutalaldehyde-fixed group again Collagen was well maintained. However, in the acellularization-only or first-treated group, collagen structure was loosely linked compared to other groups as in the pericardium (FIG. 3).

As a result, the xenograft porcine pericardium, aortic valve, and pulmonary valve prosthetic prosthesis did not show any significant difference in the electron microscopic structure of the collagen skeleton according to the fixation method under various conditions. There was no structural or mechanical loss due to tissue damage. However, microscopic degeneration of collagen tissues was observed in the electron microscope when the cells were acellularized with SDS before GA fixation.

Example 3: Optimization of Decellularization Conditions

In this embodiment, porcine aorta and pulmonary valves, aortic and pulmonary artery walls, and porcine pericardium vary in concentration, incubation time, temperature conditions, and osmotic pressure for solutes, and various decellularization solutions. And decellularization by culturing in a single step and several steps through the combination. After decellularization experiments, hematoxylin-eosin (H & E) staining was used to evaluate the degree of decellularization and preservation of extracellular matrix.

3-1 Preparation of Materials

The heart, pericardium and bovine pericardium of healthy pigs slaughtered in the slaughterhouse are collected with the help of a veterinarian, immediately washed with residual blood and stored in 4 ° C saline solution in a cold box and brought to the laboratory. In the laboratory, the collected fresh pig heart is removed to obtain aortic valve and pulmonary valve conduit, and the already collected pig pericardium section and bovine pericardium section are washed several times with physiological saline and combined antimicrobial agents (1cc / L, antibiotic & antimycotic) solution, Sigma) in a phosphate buffered saline (phosphate buffered saline, PBS pH 7.4) is refrigerated at 4 ℃ for about 1 to 2 hours and then decellularized.

3-2 Decellularization

(1) General Principles:

In general, bioburden reduction is attempted by complex antimicrobial agents (1 cc / L, antibiotic & antimycotic solution, Sigma, 100 U penicillin, 0.1 mg streptomycin, 0.25 g / mL amphotericin) before starting decellularization. After that, antibiotics were not used during the decellularization process. Decellularization experiments were incubated in a rotating shaker at a speed of at least 200 times per minute during each treatment, and when transferred to other solutions, physiological solutions (50) were used to minimize residual solvents or detergents. mL PBS) was washed three more times for 5-10 minutes.

Basically, the solution used for decellularization should be 15-20 times the sample tissue volume (weight). The hypotonic buffer, isotonic buffer and hypertonic buffer used in some experiments were prepared and used in the following compositions and pH.

Hypotonic buffer: Tris, 10 mmol / L; ethylendiamine tetraacetic acid, 0.1% -0.05%; aprotinin, 5-10 KIU / mL, neomycin trisulfate 50-100 mg / L; pH 8

Isotonic buffer: Tris, 50 mmol / L; NaCl, 0.15 mol / L; ethylendiamine tetraacetic acid, 0.1-0.05%; aprotinin, 5-10 KIU / mL, neomycin trisulfate 50-100 mg / L; pH 8

Hypertonic buffer: Tris, 100 mmol / L; NaCl, 0.3-0.6 mol / L; ethylendiamine tetraacetic acid, 0.1-0.05%; aprotinin, 5-10 KIU / mL, neomycin trisulfate 50-100 mg / L; pH 8

Other solutions used were physiological phosphate solution (PBS), distilled water, and saline, depending on the solvent and detergent used. After the decellularization process, the tissues were washed in saline and fixed in 10% formalin for biopsy and other necessary tests. The degree of decellularization and preservation of extracellular matrix were observed at 400 magnification with specimens prepared by hematoxylin-eosin (H & E) staining. To assess decellularization, H & E stained samples without decellularization were used as control specimens. In some decellularized tissues, alpha-Gal and DAPI staining may be used to determine the extent of xenoantigen clearance.

(2) In the present embodiment, decellularization was performed by using a multi-step method that undergoes several steps of decellularization.

That is, 6-14 hours in a storage solution at 4 ℃ to perform decellularization, 14-24 hours in a storage solution of 8-10 ° C mixed with 0.1-1.0% SDS solution, and then broken again After treatment for 6-12 hours in the aqueous solution, it was treated for 12 hours in an isotonic solution at 4 ℃. In all processes, decellularization was performed with rotation shaking more than 200 times.

In particular, the heart valves were treated for 6 hours in a 4 ° C. storage solution, and treated at 8 ° C. for 14 hours in a mixture of a storage solution and 0.1% -1% SDS solution, and then treated for 6 hours in a 4 ° C. hypertonic solution. Then, decellularization was performed by performing treatment for 12 hours in an isotonic solution at 4 ° C.

In the case of pericardium, the acellularization of the heart valve is performed in the same manner at a low concentration of SDS solution, or in a storage solution mixed with washing solution using another detergent (deoxycholate, etc.) for 6-14 hours, 0.5-2% sodium 14-24 hours in 3-8 ° C storage solution containing deoxycholate, 6-12 hours in hypertonic solution, and 12 hours in 4 ° C isotonic solution.

3-3 results

The results of the above example are shown in FIG. Figure 4a is a result of H & E staining of the porcine aortic valve, pulmonary valve and bovine pericardium without the decellularization, the blue dots and the like shows a state that is not decellularized. Figure 4b is treated for 6-14 hours in a stock solution at 4 ℃, 14-24 hours in a 8-10 ° C stock solution mixed with 0.1-1.0% SDS solution, and then again 6- in hypertonic solution After 12 hours of treatment, the results of H & E staining of aortic valves, porcine pericardium, porcine pulmonary artery valves and bovine pericardium were shown for 12 hours in isotonic solution at 4 ° C. Decellularization can be confirmed that the nucleus was removed.

SDS is one of the ionic detergents that has a very strong decellular effect, which removes acidic phospholipids from cells, and these SDSs have highly ionic, amphipathic ligands, which are hydrophobic domains. Hydrophobic domains bind to proteins to expose hydrophilic domains, creating a lot of negative charges, making the proteins hydrophilic, and binding to water to increase edema of tissues and break hydrogen bonds. By reducing the safety of collagen. In addition, elastin is primarily hydrophobic, so when exposed to SDS, elastin loses its hydrophobicity and is exposed to aqueous environments. SDS is known to be able to completely remove cellular proteins such as vimentin and remaining nuclear material compared to other detergents, but SDS tends to destroy the native tissue structure, reducing glycoaminoglycan (GAG) or collagen structure. You can weaken (integrity).

Therefore, since decellularization by SDS alone is toxic and incomplete in long-term exposure, in the present embodiment, as described above, by treating the tissue with the storage solution before SDS exposure, the low concentration by treating the storage solution with the SDS is mixed. The decellularization using SDS and osmotic pressure was optimized. By pretreatment of tissue in a storage solution, the cells are expanded by osmotic pressure, the cell membranes are broken, and the cells are destroyed to decellularize. At this time, the efficiency of decellularization can be increased by enzymatic or chemical treatment to remove cell debris. The most commonly used solution is 10 mM tris buffer solution, which is mixed with chelating agent EDTA (ethylenediaminetetraacetic acid) to weaken the binding between cells and extracellular matrix, allowing them to escape well and leaks when cells are destroyed. Protease inhibitors such as phenylmethylsulfonylfluoride, aprotinin and leupeptin can be added to prevent the activity of various enzymes in cells. In addition, treatment at low temperature (4 ° C.) may impair the activation or function of various enzymes or cleaning agents, but the exposure to decellularization may cause relatively long exposure to enzymatic and chemical decellularization. It is believed that it is possible to maintain the effect and in particular to minimize damage to the extracellular matrix.

In this embodiment, the tissue is treated in a storage solution mixed with a storage solution and SDS, and then treated in a hypertonic solution to remove cytotoxic substances including SDS using osmotic pressure. It can relieve edema of tissues, rearrange collagen arrangement in tissues, normalize osmotic pressure of tissues by treating tissues in low temperature isotonic solution, and remove extracellular matrix that can occur during decellularization process. To protect.

In conclusion, decellularization is slightly different from tissue to tissue to achieve optimal decellularization, but the SDS concentration is 0.1-2% as described above, and the total time to incubate in storage buffer solution should be 20 to 36 hours. Degradation may be achieved by sequentially treating the hypotonic solution, the hypertonic solution, and the isotonic solution. At this time, it is thought to be treated for at least 6-12 hours in hypertonic solution and at least 12 hours in isotonic solution.

Example 4 Fatigue Durability Test for Various Fixing Methods

In the present embodiment, the prepared tissue was fixed or decellularized, and the tissue permeability and purity were measured before and after the fatigue test, and the degree of visible change of the tissue structure was confirmed by histological examination through an optical microscope.

4-1 Preparation of Materials

In a slaughterhouse, with the help of a veterinarian, the slaughterer thrives healthy cows or pigs and immediately removes bovine, swine pericardium, porcine aorta, and pulmonary artery valves by aseptic method and put them in a cold phosphate buffered solution (PBS, 0.1M, pH 7.4, 4 ℃) in an ice box. Take it to the lab.

4-2 Decellularization and Fixation

Fresh heterologous pericardium was soaked in hypotonic buffer for 12 hours, washed and then soaked in hypotonic buffer containing 0.25% SDS for 16 hours, and then soaked in isotonic solution for 8 hours. Anti-proteinase or anti-fibrinase was added to the solution used for acellularization, and the whole process was carried out in a rotating shaker at 4 ° C in a refrigerated state. Since then, it has been fixed. As a control, untreated neoplastic heterologous pericardium was immediately fixed.

Glutaraldehyde and a solvent were used for fixation. Glutaraldehyde 0.2%, 0.25%, 0.5%, 0.6%, 0.625%, 1%, 2%, 3% solution and the like were used, and the solvent was ethanol 60%, 80%, 95% solution or 67.5% A mixed solution of -75% ethanol and 2.5-5% octanol, or 2.5-5% octanediole was used. The temperature of the fixation was 4 ° C., room temperature or high temperature (37-45 ° C.).

For storage, 0.25% or 0.3% glutaraldehyde was used, and pericardium fragments stored at room temperature for 12 days were used for the experiment. Initially, 0.3% glutaraldehyde solution was used, but later, 0.25% was used to unify the method.

4-3 Inspection

(1) Permeability inspection

After decellularization, fixation, storage, etc., the pericardium fragments were treated in one direction using a saline solution at 100 mmHg for 1 hour, and the amount of saline penetrating the pericardial sac was measured. The results are expressed as the volume of normal saline (cc / hrcm 2) penetrating pericardial fragments of the same area for 1 hour. The difference in permeability seems to be related to the extent of permanent stretch of the collagen fiber bundle and the occurrence of gaps.

(2) compliance check

After decellularization, fixation, and storage, the pericardium fragments were treated with physiological saline, and hydrostatic pressures of 100, 120, 140, 160, 180, and 200 mmHg, respectively, were applied on one side to measure the deformed volume of the pericardium. While the pressure is increased from 100 mmHg to 200 mmHg, the deformed volume is defined as the value of purity, and divided into unit areas in order to allow comparison even when the pericardium fragments are different, and the unit is μL / mmHgcm ² . The change in purity is related to the degree of collagen fiber bundle degeneration, relaxation and damage.

(3) fatigue test

The equipment was designed for the experiment. It is a device that can repeatedly apply pulsating pressure in one direction at short intervals to a circular pericardial flap of 25 mm in diameter, and to 0 to 180 mmHg to reproduce the case where the pericardial flap is implanted in vivo and used as a valve leaf. The pulsating pressure can be reproduced. A total of 15 million pressure stresses are applied seven times per second.

(4) optical microscopy

The pericardium fragments before and after each experiment and stored in the 10% formalin solution were immediately sent to the pathology department, paraffin embedded tissues were cut into 2 ~ 4㎛ sections and subjected to hematoxylin-eosin staining.

4-4 results

(1) Measurement of permeability and purity of fresh right pericardium

Permeability and purity were measured using fresh right pericardium without any treatment as a basis for comparison of the basic experimental results (Table 2). The untreated fresh right pericardium showed relatively high permeability and purity compared to the fixed xenograft, indicating poor mechanical durability.

(2) Experimental group with difference in solvent mixing or solvent concentration

There were eight samples in this group (Table 3, 1.-8.). Microscopic observation showed no particular histological difference between each sample before and after the fatigue experiment (FIG. 5A). However, in the permeability and purity test, the mixture of ethanol solvent and fixation showed better properties than the case where no solvent was mixed, that is, the permeability and purity remained low before and after the fatigue test (Table 3). Moreover, even when the solvent was mixed at the time of fixation, when the concentration of the solvent was high, a better result was shown. In the end, we found that fixing with the highest possible concentration of solvent yielded better results. Further experiments showed that the best results were obtained when the concentration of ethanol was about 80%. When octanediol and octanol were added in addition to ethanol as a solvent, the ethanol concentration was not significantly affected. Fixation using ethanol and butanol as solvents was advantageous in terms of maintaining permeability and purity, but it was difficult due to the subjective discomfort when the surgeon performed the experimental surgical operation using the sample. Finally, 80% ethanol was mixed with 0.6% glutaraldehyde solution and fixed for 3 days, and then soaked in 0.3% glutaraldehyde solution for 12 days to maintain permeability and purity. There was also very little change (Table 3).

(3) Experiment group with difference in the use of high concentration glutalaldehyde

There were four samples in this group. The concentration of fixation glutaraldehyde is generally classified into low concentration (less than 1%) and high concentration (about 2 to 3%), and thus the present inventors have instead of the conventional fixation method using concentration glutaraldehyde below 1%. 2- 3% high concentration Permanent fragments were fixed using glutaraldehyde and a solvent, and then permeability, purity and histological characteristics were checked before and after the fatigue test. As a result, the group using 2-3% glutalaldehyde was not inferior to the group using 0.5% glutalaldehyde in the permeability, purity and histological characteristics before and after the fatigue test. Histologically, no significant difference was found before and after the fatigue test (FIG. 5B).

(4) Experiment group with difference in decellularization and solvent use

There were four samples in this group. Decellularization traditionally uses a solution of sodium dodecylsulfate (SDS), and recently other substances. In this experiment, 0.1-0.5% SDS solution was mainly used, and permeability, purity, Histological characteristics were confirmed. As a result, the samples subjected to decellularization had much higher permeability and higher purity even before the fatigue test (Table 3). After the fatigue test, it was thought that the difference between the decellularized group and the unexecuted group was greater, which deteriorated the mechanical properties of the pericardium. In addition, the decellularized pericardium fragments had already lost the density of collagen fiber bundles, sometimes gaps were observed before the fatigue test, and this phenomenon was intensified after the fatigue test. It was confirmed that the fragmentation (fracture), damage and gap (gap) is more severe (Fig. 5c)

In conclusion, the following conclusions were obtained in the present embodiment. First, the xenograft has better mechanical durability when it is fixed by using a solvent such as ethanol than when it is fixed by using glutaraldehyde alone. Second, when glutaraldehyde and ethanol are mixed and used for fixing, the best mechanical and physical durability is obtained when the concentration of ethanol is about 70-80%. Third, when using high concentrations (2-3%) of glutaraldehyde, which is not currently used to reduce the toxicity of glutaraldehyde, the result is similar to that of low concentration (less than 1%) glutaraldehyde. It has a degree of mechanical and physical durability. Fourth, decellularization is considered to be an essential process for reducing the immunogenicity of xenografts. However, decellularized right pericardium shows increased permeability and purity, resulting in poor mechanical and physicochemical properties. The purity increased further, and collagen fibers were severely damaged in histological examination, resulting in poor mechanical and physical durability.

Example 5: Antitoxin Treatment

5-1 Preparation of Materials

From the slaughterhouse pigs (3 months-4 months old), the pericardium is immediately extracted and placed in PBS solution (0.1M, pH 7.4) in an ice box and transported to the laboratory. The experimental group was a total of 4 groups (Group 1: 0.625% GA, Group 2: 0.625% GA + 80% Ethanol, Group 3: 0.625% GA + L-lysine, Group 4: 0.625% GA + 80% Ethanol + L-lysine) The number of pericardial sections in each group was eight. In addition, since glycine is nonpolar neutral and lysine is polar basic, but both NH ₃ groups work, anti-toxin treatment may be performed using glycine instead of lysine. Other amino acids such as borohydride, glutamic acid, homocysteic acid can be treated to antitoxin.

5-2 Fixing and Pretreatment

(1) Fixation: Soak the porcine pericardium in 0.625% glutalaldehyde (PBS buffer, pH 7.4) and fix it at 4 ° C for 2 days, and then fix it further at room temperature for 7 days. After fixation, the pericardium is divided into pericardium of 1.5 cm in size. The pericardium fragments that do not require further treatment are washed with PBS solution for 24 hours at room temperature, and then transplanted into rats. Pericardial fragments requiring additional anticalcification proceed to the next step.

(2) Pretreatment of Ethanol: The pericardium fragments treated with 0.625% glutalaldehyde fixation are treated in a shaker bath containing 80% ethanol (PBS buffer, pH 7.4) for 24 hours at room temperature. The pericardium fragments that do not require further treatment are washed with PBS solution at room temperature for 24 hours and then transplanted into rats.

(3) Pretreatment of L-lysine: Pericardial fragments treated with 0.625% glutaraldehyde fixation or additional ethanol treatment were washed with PBS solution, and then washed with 0.1M L-lysine solution (acetic acid buffer, 0.5M, pH 7.6). Soak is treated for 48 hours at 37 ℃. After washing for 24 hours at room temperature with PBS solution, and stored in PBS solution of 4 ℃ until transplanted to the rat.

(4) Other amino acids can be treated to antitoxin. Soak glycerine (0.05-0.2M Glysine with PBS solution) for 24-48 hours at room temperature, or use Sodium borohydride solution (Sodium borohydride 0.08M in buffered glycine (0.2% HEPES, 0.2M glycine, 0.1M NAOH, pH 10.3) Or incubate at room temperature for 24-48 hours to remove reagents (aldehyde groups) that did not react with buffered carbonate (0.1 M NaHCO 3, 0.1 M Na 2 CO 3, pH; 10.3), or a fully saturated glutamic acid solution ( saturated aqueous solution of L-glutamic acid, or Incubate at 0.05-0.1M L-glutamic acid, ph 3, room temperature) or 2% arginine (0.12M, L-arginine in PBS phosphate buffered saline (PBS) at pH 11) at room temperature for 24-48 hours. . After the antitoxin treatment, the final disinfection in the multi-synthetic disinfectant solution is stored in 2% benzyl alcohol solution or 1% benzylkonium solution.

5-3 Inspection

(1) Purpald inspection, thickness inspection

One pericardial piece in each group was examined for microstructure by optical and electron microscopy without implantation into rats. A aldehyde residue evaluation is performed to determine whether the detoxification has been properly performed in the pericardium treated with glutalaldehyde detoxification with L-lysine. To do this, the pericardium was washed with saline, soaked in 0.171M Purpald in 1M NaOH solution for 15 minutes, and then exposed to air for qualitative analysis of the time and color of the pericardium fragments. The same test is performed on the pericardium without detoxification, and the results are compared.

The average value of the pericardium of the treated pericardial pericardium was determined by unfolding the treated pericardial pericardium and taking a 0.5 x 5 cm rectangular section in all six directions that can be obtained by varying the angle by 30 °. Attempts were made to be representative of tensile strength. The tension was measured at a load speed of 100 mm / min using Japan Tech & Manufacture, Digital Force Gauge, Model 5FGN, and automated materials testing system. In addition, the caliper Mitutoyo Thickness Gauge (Digimatic 543-122-15, Mitutoyo, Japan) was used to measure the thickness of the pericardium, and the average thickness was measured three times in one sample.

(2) rat transplantation model

After anesthetizing the rats with Zoletil (0.2cc IP) and Rompun (0.1cc IP), four pouches were made in the subcutaneous tissue of the dorsal area. Suture. Eight weeks after pericardial implantation, the experiment was terminated and rats were anesthetized using CO2 gas. The pouch made at the time of implantation is peeled off and the pericardial fragments are removed. Each extracted pericardial piece is divided into three and used for analyzing the results. The life span of rats is about 2-3 years, and it is divided into 3 weeks of gestation and 3 to 14 weeks of growth, and adulthood after 14 weeks of age. It is known that calcification proceeds faster at younger age. In the example, mice at 3 weeks of age were used.

(3) Vonkossa Dyeing

Optical microscopic examination was performed by cutting the extracted pericardium into 2-3 mm thick tissue, placing it in Dubosqc-brasil solution for 1 hour, fixing it in 10% formalin, and then paraffin embedding tissue and cutting it into 2-4um sections to cut hematoxylin-eosin and vonkossa. Stain for histologic findings and the extent of calcium deposition.

(4) calcium determination

Calcium quantification was performed to determine the degree of calcification of the pericardium of each group. To do this, the pericardium is washed with saline, dried in a hot dryer for at least 24 hours, and then weighed. Add 3 ml of 1M HCl solution in a glass tube, warm in a 75oC dry oven (Fisher Scientific) and wait at least 24 hours for complete dissolution. The solution is transferred to an e-tube and evaporated for more than 12 hours using an automatic environmental speedvac system. Once pellets are made, calcium content is quantified using Automatic Chemistry I.S.E (HITACHI) equipment in 1 ml of PBS (phosphate buffered solution).

(5) statistical method

Statistical processing was performed using Microsoft Excel and SPSS 14.0K, and all statistics were expressed as mean ± standard deviation. The mean comparison between groups was verified by Student T-test, statistical difference between each group was verified by ANOVA and post-hoc test (Turkey test) and p <0.05 is considered meaningful.

5-4 Results

(1) tension inspection

 Measurements were made using five pericardium fragments in each group. The group treated with L-lysine after 0.625% glutalaldehyde fixation was the strongest at 5mm of 1.20 ± 0.30 Kg, and the group treated with both 80% Ethanol and L-lysine after fixation with 0.625% glutalaldehyde / 5mm at 0.90 ± 0.22 Kg , The group treated with 80% ethanol after 0.625% glutalaldehyde fixation was the weakest at 5mm in 0.85 ± 0.36 Kg, and the group treated with 0.625% glutalaldehyde fixation at 0.53 ± 0.34 Kg / 5mm (Fig. 6). There was a statistically significant difference between the groups (p = 0.035).

(2) thickness

Measurements were made using five pericardium fragments in each group. L-lysine treated group was thickest with 0.18 ± 0.02 mm after 0.625% glutalaldehyde fixation, and 0.13 ± 0.03mm, 0.625% for 80% ethanol treated group after 0.625% glutaraldehyde fixation. The group treated with both 80% Ethanol and L-lysine after glutaraldehyde fixation was the thinnest with 0.12 ± 0.01mm and 0.625% glutalaldehyde fixation, respectively, 0.10 ± 0.02mm. There was a statistically significant difference between each group (p <0.01) (FIG. 7).

(3) Purpald inspection

L-lysine-treated group after 0.625% glutalaldehyde fixation and 80% ethanol and L-lysine-treated group after 0.625% glutaraldehyde fixation showed weak purple color and 0.625% glutalaldehyde fixation group. And dark purple in 80% Ethanol treated group after 0.625% glutaraldehyde fixation (FIG. 8).

(4) Vonkossa Dyeing

 The pericardium was composed of the mesenteric layer, fibrous layer, and epicardial connective tissue. The fibrous layer made up the bulk of the pericardium and consisted of collagen, elastic fibers, nerves, blood vessels, and lymphatic vessels. Calcium was mainly deposited in epicardial connective tissue, and some invaded into the fibrous layer. In the group treated with only 0.625% glutaraldehyde fixation, calcium was deposited almost all over the layer, followed by 80% Ethanol treatment after 0.625% glutaraldehyde fixation, and L-lysine treatment after 0.625% glutaraldehyde fixation, 0.625% In the group treated with 80% Ethanol and L-lysine after glutaraldehyde fixation, three layers were relatively well maintained, and calcium deposition was observed mainly in the epicardial connective tissue (FIG. 9).

(5) Calcium Determination

Four isolated pericardial fragments were used for each group. The 80% ethanol treated group was the smallest at 13.6 ± 10.0 ug / mg after the 0.625% glutalaldehyde fixation and the 15.3 ± 1.0 ug / mg, 0.625% glue at the L-lysine treated group after the 0.625% glutalaldehyde fixation. 80% Ethanol and L-lysine treated group after dealdehyde fixation was the highest in the group treated with 16.1 ± 11.1 ug / mg and 0.625% glutalaldehyde fixation (51.2 ± 8.5 ug / mg). 80% Ethanol-treated group (p = 0.008) after 0.625% glutalaldehyde fixation, and L-lysine-treated group after 0.625% glutaraldehyde fixation (p = 0.002). ), And the group treated with 80% Ethanol and L-lysine after fixation with 0.625% glutalaldehyde (p = 0.012) showed a statistically significant decrease in calcium deposits (FIG. 10).

As described above, after anti-calcification of the porcine pericardium fixed with 0.625% glutalaldehyde with 80% Ethanol, L-lysine, the thickness and tension of each xenograft were measured and subcutaneous tissue of the rat was also measured. The effects of ethanol, L-lysine, and ethanol and L-lysine were only significantly lower in calcium deposits than in the GA-only group. There was no. In addition, the L-lysine treated group showed better results than the ethanol treated group in the thickness and tension tests before transplantation. This is interpreted as increased cross-link due to the effect of additional diamine bridge. In the Purpald test results, the ethanol-only group had a dark purple color similar to the GA fixation-only group, whereas the L-lysine-treated group or the ethanol- and L-lysine-treated group had a light purple color. This shows that L-lysine treatment effectively eliminated GA's free aldehyde group (-CHO). Eventually, in addition to GA free aldehyde groups, various reactions work on the calcification of xenografts, and it is preferable to treat Ethanol and L-lysine with different mechanisms of action in order to suppress them. In addition, the porcine pericardial prosthetic prosthesis did not show any significant difference in the microscopic structure of the collagen skeleton according to the fixation method under various conditions, so that there was no structural and mechanical loss due to tissue damage during various tissue processing using GA. Was thought to be.

As the demand for prosthetics continues to increase as cardiac surgery continues, prosthetics and prosthetic specimens from other animals differ in degree, but lack of mid- and long-term surgical outcomes following calcification after intracardiac transplantation. It remains a problem. Accordingly, the method for producing xenograft prostheses according to the present invention is expected to produce and commercialize prostheses that are more excellent in preventing and mitigating the effects of calcification and cytotoxicity than conventional prostheses, in particular, heart valve disease or heart. It may be applied to the manufacture of fully implantable heart valves, heart valve conduits, and vascular substitutes for use in vascular surgery.

In particular, the clinical use of artificial tissue grafts in the cardiovascular system can be divided into xenografts pericardium and valves. Xenograft pericardium has many uses, such as supplementation of a simple defective pericardium, closure or reinforcement of a cardiovascular defect site, or the like as a prosthesis for complex cardiovascular surgery. As part of the blood vessel or heart, it serves as a material that allows blood to flow in a desired direction without leakage. In this case, it is expected to improve the immunogenicity so that calcification does not occur for a long time, and the operability during surgery is improved by improving the physical characteristics. In addition, graft valves are intended to be applied to valve diseases, especially during cardiovascular surgery. In particular, they overcome the drawbacks of avoiding use in children and young people because of durability problems. It is expected that a reduced, long-term hemodynamically improved model can be produced. Xenograft conduits, which are considered to be the most widely used, are the valves used for right ventricular-pulmonary artery conduits in patients with congenital heart malformations who have undergone rasterial surgery. The valves used in the existing right ventricle-pulmonary artery catheter are not satisfactory in durability and require reoperation (replacement) due to the destruction of the valve structure or the deterioration of mobility within a few years. The xenograft valve fixed by the method developed in the present invention is expected to be able to live without problems for a longer period of time with a single operation because less calcification occurs and the valve inherent function is maintained for a long time.

Figure 1 is a histological examination of human pericardium and porcine pericardium stained with alkaline toluidine blue when no treatment. However, above: optical micrograph (AC: porcine pericardium, DF: human pericardium, collagen fibers are well maintained, human pericardium is twice as thick as porcine pericardium), bottom: electron micrograph (AC: porcine pericardium, DF: Human pericardium, collagen fibers are well maintained and human pericardium is three times as thick as pig pericardium.)

Figure 2 is a photograph showing the collagen pattern of porcine pericardium after glutaraldehyde fixation, anticalcification (ethanol), acellularization treatment (SDS). Collagen fibers were well maintained except for D, E, G. A: 0.625% GA, B: ethanol + 0.625% GA fixed, C: 0.625% GA + ethanol, D: acellularized (0.25% SDS) treated, E: acellularized (0.25% SDS) treated + 0.625% fixed , F: ethanol + acellularization (0.25% SDS) treatment + 0.625% GA, (G) acellularization (0.25% SDS) treatment + ethanol + 0.625% GA.

Figure 3 is a photograph showing the collagen pattern of porcine aortic valve, pulmonary artery valve after glutaraldehyde fixation, anticalcification, acellularization treatment. Collagen fibers were well maintained except for E, F and H. A: Porcine valve with no treatment, B: 0.625% GA fixation, C: Ethanol treatment + 0.625% GA fixation, D: 0.625% GA fixation + ethanol treatment, E: Acellularization (0.25% SDS) treatment, F : Acellularization (0.25% SDS) treatment + 0.625% GA fixation, G: ethanol treatment + acellularization (0.25% SDS) treatment + 0.625% GA fixation, H: acellularization (0.25% SDS) treatment + ethanol treatment + 0.625% GA fixed.

FIG. 4A is a histological picture of porcine aortic valve (top left), pulmonary artery valve (top right), and bovine pericardium (bottom left) unmagnified (magnification: 400x).

4B is a histological picture after H & E staining of decellularized aortic valve (top left), porcine pericardium (top right), pulmonary artery valve (bottom left), and bovine pericardium (bottom right) (magnification: 400 ×).

Figure 5a is a histological picture of the right pericardium before (BF) and after fatigue experiment (AF) according to the solvent mixing (1-4) or the difference in solvent concentration (5-8) (magnification: 100 times).

Figure 5b is a histological picture of the right pericardium before and after the fatigue experiment, according to the difference in the concentration of glutaraldehyde (magnification: 100 times).

FIG. 5C is a histological picture of the right pericardium before and after one fatigue experiment, according to the difference in decellularization and solvent use (80% ethanol) (magnification: 100-fold).

Figure 6 shows the results of the tension test of the pericardial fragments treated with lysine, ethanol.

Figure 7 shows the thickness test results of the pericardial piece according to lysine, ethanol treatment.

Figure 8 shows the Purpaid test results according to lysine, ethanol treatment.

9 shows the results of Vonkosa staining of the pericardium following treatment with lysine and ethanol.

Figure 10 shows the results of quantification of calcium in the pericardium following treatment with lysine and ethanol.

11 shows a picture and photograph illustrating the fixation of a non-dural valve pressure state.

Claims (13)

The acellularized tissues of animals other than humans were acellularized, and the acellularized tissues were fixed in a high concentration glutalaldehyde solution containing ethanol solvent at zero-pressure fixation, and then the toxicity of the tissues was determined. Method for producing a xenograft prosthesis comprising the step of removing. The method of claim 1, wherein the biological tissue is selected from pericardium, heart valve, pericardium, or dura mater. 3. The method according to claim 2, wherein said acellularization is achieved by sequentially treating storage tissue, hypertonic buffer and isotonic buffer in said biological tissue, and rotating shaking during said treatment. The method of claim 3, wherein the acellularization is 20-36 hours in 3-8 ℃ storage buffer, 6 ~ 12 hours in hypertonic buffer of 3 ~ 5 ℃, 11 in isotonic buffer of 3 ~ 5 ℃ Processing for ~ 13 hours. 5. The method of claim 4, wherein the treatment of the storage buffer consists of treating the biological tissue with the storage buffer for 6-14 hours and then treating the storage tissue with the detergent for 14-24 hours. How to. delete The method of claim 1, wherein the fixation is treated with a 0.25-0.625% glutaraldehyde solution before or after the 1-3% glutaraldehyde solution containing the ethanol solvent, or the ethanol solvent is contained. And treating the glutaraldehyde solution at a concentration of 0.25-0.625% before and after the treatment of the glutaraldehyde solution at a concentration of 1-3%. 8. The method of claim 7, wherein the ethanol solvent is 70-80% ethanol or mixed ethanol. 9. The method of claim 8, wherein the mixed ethanol consists of 67.5-75% ethanol and 2.5-5% octanol or octanediol. The method of claim 1, wherein the removal of the toxicity is performed by adding one or more amino acids selected from lysine, glycine, borohydride, glutamic acid and arginine after the fixation. The method of claim 10, wherein the concentration of the added amino acid is 0.05 ~ 0.2 M, characterized in that the treatment for 24 to 48 hours at 34 ~ 42 ℃. Anticalcified and anti-toxinized acellular xenograft prostheses produced by the method of any one of claims 1 to 5 and 7 to 11. The xenograft prosthesis of claim 12, wherein the xenograft prosthesis is used in a heart valve, surgical tissue patch, tissue vessel, ligament, tendon and porous support for tissue engineering.

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