KR20160028253A - Method of preparing heterograft using space-filler - Google Patents

Abstract

The present invention relates to a method for producing a heterogeneous transplant tissue by using a space filler, and to a heterogeneous transplant tissue produced by the same. The heterogeneous transplant tissue, produced by the method of the present invention, has the reduced generation of calcification, has less toxicity, and causes significantly reduced immune response when the heterogeneous transplant tissue is transplanted, and thus can be used as a biotissue for heterogeneous transplant which is effective and safe. The method for producing a heterogeneous transplant tissue includes a decellularization step, a space filler processing step, a biotissue fixation step, and a toxicity removal step.

Description

TECHNICAL FIELD The present invention relates to a method of preparing heterograft using space filler,

The present invention relates to a method of manufacturing a xenotransplantation tissue using a space filler and a xenotransplantation tissue produced thereby.

Research and development of patches in heart valves, valve conduits and cardiovascular prostheses by past methods, both at home and abroad, were at a standstill, but the concept of heterologous immunity, the development of tissue engineering and xenotransplantation, It is becoming active. There have been no studies on the development of heterogeneous tissues for cardiovascular systemic replacements such as xenografts, valve conduits, heterotopic pericardials, etc., although it has been the same in Korea in the past, and it has been tried by some biotechnology teams. In Korea, the prototype has not been tried yet.

Defects such as xenogenic tissue valves inserted into the human body or xenografts are often degenerated and degenerated due to chemical, physical, and immunological reactions with the body, resulting in loss of function and extinction. Is completely calcified in the body. Although there have been many studies to reduce the degeneration and degeneration of such tissues from the past to the present, there have been no satisfactory results yet.

These heterogeneous tissues have been used with glutaraldehyde fixed to maintain tissue mechanical stability, reduce heterophasic activity, and maintain sterility. However, it is known that free aldehyde groups, which do not participate in the cross-linking of cytosol, and free aldehyde groups, which are present in large amounts in the cell membrane after glutaraldehyde fixation to heterologous tissues, The immune response to the tissue antigen of the remaining cellular components is known to cause tissue calcification.

Treatment of glutaraldehyde fixed heterogeneous tissue with organic solvent removes the phospholipid, which is the material of calcification, and causes structural changes of collagen and elastin to reduce calcification in vivo, and short chain alcohol such as ethanol When used at high concentrations, anticarcinogenic effects appear, and long chain alcohols such as octanediol are structurally similar to phospholipids, which can reduce phospholipids more effectively by removing phospholipids.

In order to remove the free aldehyde group which does not participate in cross-linking of collagen after glutaldehyde fixation, detoxification treatment with amino acid is carried out and the amino group present in the amino acid is left free in glutaraldehyde fixed tissue Calcification can be prevented by bonding with an aldehyde group.

In addition, a freeze process is required to remove the immune response to the tissue antigen of the cellular component remaining in the heterogeneous tissue after glutaraldehyde fixation to reduce calcification.

Recently, the immune response between xenoantigen and xenoantibody on the cell surface of xenogeneic tissue has been suggested as a mechanism of calcification. This heterologous antibody is a natural antibody to galactose [alpha] (1,3) -galactose (alpha-GAL) present in all mammals except primates such as human, ape and some monkeys (Old World monkeys) It is known to cause hyperacute rejection of the human body. alpha GAL antigenic determinants are found on the cell surface of animals and are known to induce a strong immunological response to primates, although their frequency varies according to the species of mammals and also within the same species. Since the alpha-Gal antigenic determinant present in this heterologous tissue reacts with the patient's anti-gal antibody to cause an immune response and eventually induces calcification of the xenogeneic tissue, the alpha-Gal antigen Alpha-galactosidase can be used as an enzyme to remove the factor.

These heart valves and cardiovascular substitutes are not produced or sold domestically, but are highly dependent on imports from foreign countries and have no domestic research technology. Accordingly, the present inventors made extensive efforts to solve these problems, and as a result, they have completed the present invention by developing a method for double-fixing a xenograft using a space filler and removing toxins.

Application No. 10-2010-0063218

One aspect of the present invention is to provide a method for producing a xenograft tissue using a space filler.

One aspect of the present invention also relates to a xenotransplantation tissue produced by the method.

Cardiovascular systemic replacement with heterologous biopsy has been widely used because of its excellent hemodynamic characteristics, reduced side effects due to the use of anticoagulants, and excellent durability against infection. However, the degeneration due to calcification during long- And is particularly problematic in growing pediatric patients.

We used genipin or GA (glutaraldehyde) as a double crosslinking technique, which uses two crosslinking agents by adding carbodiimide, which is weakly toxic and fixes the tissue using natural crosslinking agent such as genipin, which is low in toxicity with GA (glutaraldehyde) Crosslinking can be increased while reducing the fixed concentration of.

After glutaraldehyde treatment, the phospholipid attached to the cell membrane of the xenogeneic tissue is involved in the initial stage of calcification by providing phosphorus through hydrolysis by alkaline phosphatase, To remove the phospholipids and to reduce the calcification in vivo by causing structural changes of collagen and elastin, heterotopic tissue fixed with glutaraldehyde was treated with organic solvent.

The aldehyde group remaining after cross-linking of collagen by glutaraldehyde is oxidized in the body to form an acid which participates in deposition of calcium in plasma or aldol condensate is formed by polymerization to participate in calcification. The amino acid was used to form a covalent Schiff base (Schiff base), which was neutralized.

Since the residual cells generated after crosslinking treatment of glutaraldehyde and the like cause calcification, a non-saturation process is applied to maximize the efficiency of cell removal while minimizing damage to heterogeneous tissues.

We can cut the end of the alpha gal epitope, 1,3 linkage, by completely removing the α-Gal epitope, a residual heterozygous α-Gal epithelium, Alpha-galactosidase was applied.

Free aldehyde groups that are not involved in the tissue collagen cross-linking of glutaraldehyde are known to cause cytotoxicity and calcification, which can be treated with space-filler to inactivate these free aldehyde groups. In addition, the space-filler forms a membrane that prevents the release of glutaraldehyde from the tissue. It fills the void space between the cell-removed site and the collagen after the freeze, thereby eliminating the calcium and phospholipids in the serum and producing hydroxylapatite crystals It also blocks platelet receptor sites present in collagen and prevents platelet aggregation or adhesion and increases hydrophilicity.

Free aldehyde groups that are not involved in the tissue collagen cross-linking of glutaraldehyde are known to cause cytotoxicity and calcification, which can be treated with space-filler to inactivate these free aldehyde groups. In addition, the space-filler forms a membrane that prevents the release of glutaraldehyde from the tissue. In particular, it clears the calcium and phospholipids in the serum by filling the vacancy space between the cell-removed site and the collagen after the saturation, It also blocks platelet receptor sites present in collagen and prevents platelet aggregation or adhesion and increases hydrophilicity. Therefore, space-filler treatments such as polyethylene glycol (PEG) and jeffamine, which are known as epoxy compounds, can improve the anticarcinogenic effect.

Therefore, in order to improve the anticarcinogenic effects of the conventional organic solvent treatment, antitoxinization, cell-freezing, and alpha-galactosidase treatment, a space-filler treatment such as polyethylene glycol (PEG) (Synergistic effect).

Therefore,

(A) decellularizing a biological tissue of a mammal;

(B) treating a space filler in the non-saturating biotissue;

(C) fixing the processed biomolecule to the space filler; And

(D) removing the toxicity of the fixed biotissue

The present invention also provides a method for producing a living tissue for xenotransplantation.

Hereinafter, the present invention will be described in detail.

The non-saturation process in step (A) will remove the cell, cell membrane and the like which are the greatest factors of the immune reaction while excluding the damage of the tissue as much as possible. Since residual cells generated during the treatment of glutaraldehyde and other crosslinking agents may cause calcification, it is necessary to maximize the efficiency by removing the cells as much as possible, and the toxic complications of the residual cytotoxic substances (protease, DNA, RNA) Should be minimized.

Thus, the freeze saturation is preferably

(a1) treating the storage buffer solution containing 0.1 to 1.5% (v / v) SDS (sodium dodecyl sulfate) at 3 to 5 DEG C for 12 to 36 hours;

(a2) treating the storage buffer solution comprising 0.1 to 2.0% (v / v) Triton X-100 (Triton X-100) at 3 to 5 ° C for 12 to 36 hours;

(a3) washing with distilled water and PBS (0.01 M, pH 7.4) at 3 to 5 ° C for 6 hours to 24 hours and 30 minutes to 2 hours, respectively.

In yet another embodiment,

(a1 ') treating the storage buffer solution containing 0.1 to 1.5% SDS (sodium dodecyl sulfate) at 3 to 5 DEG C for 12 to 36 hours;

(a2 ') storage buffer containing 0.1 to 2.0% Triton X-100 (Triton X-100) at 3 to 5 ° C for 12 to 36 hours;

(a3 ') treating a 0.1 to 0.8% SDS (sodium dodecylsulfate) solution at 3 to 5 DEG C for 12 to 36 hours; And

(a4 ') 0.1 to 2.0% Triton X-100 (Triton X-100) at 3 to 5 캜 for 12 to 36 hours.

In yet another embodiment,

(a1 ") for from 12 hours to 36 hours at a storage temperature of from 3 to 5 DEG C comprising from 0.1 to 1.5% SDS (sodium dodecyl sulfate);

(a2 ") treating the storage buffer solution containing 0.1 to 2.0% Triton X-100 at 3 to 5 DEG C for 12 to 36 hours; And

(a3 ") hypertonic buffer solution at 3-5 [deg.] C for 3-6 hours.

Each of the above steps may be repeated one or more times depending on the animal and tissue characteristics of the tissue.

The storage buffer, isotonic buffer solution and hypertonic buffer solution of the present invention are generally known to be isotonic with a salt concentration of 0.9% in physiological saline, It is defined as malfunction.

The living tissue may be a living tissue of a mammal other than a human, and the kind thereof is not limited. Examples of such mammals include primates, poultry, wild boars and animals, rabbits, goats, animals, dogs, and the like.

In addition, the kind of the biomedical tissue is not limited as long as it corresponds to a tissue capable of tissue transplantation. For example, it may be a blood vessel, a valve, a skin, a fascia, a cornea, a cartilage, a ligament, a amniotic membrane and the like, preferably a cardiovascular tissue, that is, a pericardium, a heart valve, a pericardium, a dura.

In the present invention, the space-filler in step (B) refers to a synthetic or natural resin capable of filling voids between cells where cells are removed after collagenization and collagen.

The space filler forms a membrane that prevents the release of glutaraldehyde from the tissue. The space filler removes calcium and phospholipids in the serum by filling the vacancy space between the collagen and the site where the cell is removed after the freeze, and the possibility of the formation of hydroxyapatite crystals It also blocks platelet receptor sites in collagen and prevents clumping or adhesion of platelets and increases hydrophilicity.

The space filler is preferably selected from the group consisting of polyethylene glycol (PEG), polyacrylamide (PAAm), polyethylene (PE), jeffamine, polyacetal (POM), polyacrylic (PC), polypropylene (PP), polyethylene terephthalate (PET), polybutylene terephthalide (PBT), polystyrene (PS), styrene- acrylonitrile (SAN), acrylonitrile-butadiene- , Polyamide-imide (PAI), polyarylate, polyurethane (PU), polyvinyl chloride (PVC), polyamide- (TPO), polypropylene / ethylene, propylene / EPDM), thermoplastic elastomers, polyaryl sulfone (PAS), polyether sulfone, polyether sulfone, polyether sulfone, polysulfone, polytetrafluoroethylene (PTFE) ), Polyethersulfone (PES), polyphenylene sulfide (PPS), polyurea (PU), may be natural rubber, synthetic rubber, epoxy, phenolic, polyester, polyamide, silicon, and combinations thereof. More preferably, it may be polyethylene glycol (PEG), polyacrylamide (PAAm), or jeffamine.

In the embodiment of the present invention, when the biotissue is bovine pericardial, the space filler can treat Jeffamine of 0.03 M to 0.06 M at room temperature for 12 hours to 36 hours.

When the living tissue is a bovine pericardial sac, the space filler may be treated with 0.03 M to 0.06 M Jeffamine at room temperature for 12 hours to 36 hours.

If the biotissue is a porcine valve or valve catheter, 25% to 50% (v / v) of PEG can be treated at room temperature for 12 to 36 hours.

In the present invention, the immobilization of step (C) may be carried out using glutaraldehyde, Genipin, carbodiimide or EDC / NHS (1- (3-dimethylaminopropyl) -3- ethyl carbodiimide / Nhydroxysuccinimide) as a fixative.

The concentration of the compound in the fixing solution is preferably 0.1 to 1.0%. It is preferable that the fixing liquid further contains an alcohol-based solvent. The alcoholic solvent may be ethanol, methanol, acetone, ethyl acetate, hexane, butanol, acetonitrile, 1,4-dioxane, isopropanol, It is preferable that the fixing of the step (2) is repeatedly performed one to three times.

In addition, in the method of removing alpha-Gal epitopes in the step (A) of the non-saturating step, an alpha-Gal epitope can be cleaved, - can be treated with galactosidase. This step can be performed by treating an isotonic solution containing 0.01 to 1.0 U / ml of? -Galactosidase.

In addition, the method of manufacturing a biotissue of the present invention may further include a step of removing toxicity. It is a mixture of L-lysine, glycine, chitosan, glutamic acid, arginine, heparin, homocysteic acid, sodium borohydride ) And sodium bisulfate, and the treatment of the compound may preferably be carried out at 4 to 37 占 폚 for 12 to 48 hours.

Particularly, when the biotissue is a heart valve of a pig,

(A) freezing the heart valve of pigs using 0.25% (v / v) SDS and 0.5% (v / v) TritonX-100;

(B) treating 0.1 units / mL of? -Galactosidase;

(C) treating 25% (v / v) PEG;

(D) 0.5% (v / v) glutaraldehyde;

(E) solvent treatment with 75% (v / v) ethanol and 5% (v / v) octanol; And

(F) a step of detoxifying with 0.1 M of glycine.

The xenotransplantation biotissues produced by the above-described method have a reduced incidence of calcification, are less toxic, and significantly reduce the immune response upon xenotransplantation, and thus can be used as an effective and safe xenotransplantation biotissue have.

Accordingly, one aspect of the present invention provides a xenograft for xenotransplantation produced by the above method. The biotissue can be used as a xenograft prosthesis for use in heart valves, surgical tissue patches, tissue vessels, ligaments, tendons, or porous supports for tissue engineering.

Since the xenotransplantation biotissue produced by the method of the present invention reduces the incidence of calcification, is less toxic, and significantly reduces the immune response upon xenotransplantation, the effective and safe xenotransplantation biopsy Can be utilized.

Figure 1 shows the results of DAPI (4 ', 6-Diamidino-2-phenylindole, dihydrochloride) (DAPI) on periarticular pericardium. DAPI staining showed a marked decrease in the DNA of the non-sensitized cortical capsule (Method 1). (a) fresh bovine pericardium; (b) Decellularized (decellated) bovine pericardium (fluorescence microscopy, original magnification x 200).
2 is a color change (1-12) after completing all of the tissue processing (method 1).
(a) EDC / NHS treated group changed to white, GA treated group changed to ivory-yellow, and genipin treated group changed to blue-black. (b) The space filler treatment group (acryl, 0.27 ± 0.05, Jeffamine, 0.34 ± 0.03 PEG, 0.29 ± 0.03) was thicker than the space filler treatment except GA group. (Each group N = 10)
Figure 3. In vitro results of cross-linking efficiency test (Method 1). (a) Thermal stability test. The shrinkage temperature of all treated groups was significantly higher than that of fresh small cysts (69.42 ℃, P <0.05). (b) Pronase test showed similar thermal stability.
Figure 4 is the result of cross-linking efficiency (method 3). (a) Thermal stability test (b) Pronase test.
Fig. 5 shows the result of the thermal stability test in the rat implantation model (Method 5).
6 shows the result of the thermal stability test in the wild-type mouse transplantation model (Method 5).
Fig. 7 shows the result of confirmation of the remaining mass after the decomposition and pronase test (method 4). The porcine valve showed a pronase stability that was lower than that of the space filler treated with bovine pericardium (p = 0.016, p = 0.008). Compared with the GA-fixed group, the decellularization and space filler treated groups showed a statistically significant decrease in pronase stability (p = 0.008, p = 0.008). The treatment of the space filler also showed a pronase stability that was reduced in the porcine valve compared to the GA fixed group (p = 0.008).
(A) + Space filler + GA, 5: Decell (B) + Space filler + GA, 2: Decell (A) + GA, GA.
6: simple GA fixation, 7: Decell (A) + GA, 8: Decell (A) + Space filler + GA in the pig valve.
Decell: decellularization, GA: glutaraldehyde
Fig. 8 is a diagram showing the result of pronase test in the rat transplantation model (Method 5).
FIG. 9 is a graph showing pronase test results in a wild-type mouse transplant model (method 5).
FIG. 10 shows contact cytotoxicity of porcine aortic smooth muscle cell and bovine pericardium (Method 1). The cytotoxicity of each tissue treatment method was confirmed. A phase contrast microscope image of cultured cells in contact with the pericardium (original magnification x40). Cells were labeled with Annexin V fluorescein isothiocyanate (FL1) and propidium iodide (FL2) and identified by flow cytometry.
Fig. 11 is a diagram showing the result of Calcium quantitation (Method 1) for the pericardial sac to be removed. The pericardial tissue was inserted into the rabbit muscle for 4, 8 weeks. The results of cytotoxicity were correlated with the degree of calcification. All values were expressed as mean ± SEM (ug / mL / mg, dry weight, n = 5).
FIG. 12 shows von Kossa staining (Method 1) results of the pericardial sacs. Tissue was inserted into the rabbit's muscle for 4-8 weeks. Especially in the GA-PEG treated group, calcification was very low (0.62 ± 0.1 μg / ml / mg). These results were observed with von Kossa staining. Light microscopic image of bovine pericardium (original magnification x40).
Figure 13. Von Kossa staining, hematoxylin and eosin staining, and immunofluorescence test for PEG-treated pericardium (Method 1). Tissue was inserted into the rabbit's muscle for 4-8 weeks. The infiltrated cells were identified as CD4 and macrophages (immunohistochemistry and immunofluorescence). An optical microscope image of the pericardium (original magnification x40, x100). Fluorescence microscope images of bovine pericardium (original magnification x40, x100).
Fig. 14 is a result of quantitative determination of calcium two months after subcutaneous implantation in rats (Method 2).
FIG. 15 shows the result of H & E staining (x100) 2 months after subcutaneous implantation in rats (Method 2). A; Group 1, B; Group 2, C; Group 3.
16. von Kossa staining (x100) 2 months after rat subcutaneous implantation (method 2). A; In the absence of calcification, B; When there is calcification. The darkly dyed part represents the calcified part.
FIG. 17 shows the results of quantitative determination of calcium after 2 months of intramuscular transplantation of rabbits (Method 3).
Fig. 18 shows the result of H & E staining 2 months after intramuscular transplantation of rabbits (Method 3).
Figure 19 is the result of von Kossa staining 2 months after intramuscular transplantation of rabbits (method 3).
Fig. 20 shows H & E staining results after tissue treatment in all groups (method 4). Many inflammatory cells were infiltrated in the simple GA fixed group. However, inflammatory cell infiltration decreased after deaerating and space filler treatment.
A through E: bovine pericardium, F through H: porcine valves
A: GA, B: Decellularization (A) + GA, C: Decellularization (A) + Space filler + GA, D: Decellularization (B) G: Decellularization (A) + GA, H: Decellularization (A) + Space filler + GA.
GA; Glutaraldehyde
FIG. 21 is a view showing content indices of calcium and phosphoric acid according to a tissue and a treatment method (Method 4). The bovine pericardium showed a higher calcium and phosphorus content index than the pig aorta and pulmonary valve in the GA - treated group.
Calcium and phosphorus content of porcine aortic valve was the lowest in GA group and GA and decellular and space filler groups after decellularization.
In the bovine pericardium, 1: simple GA fixation, 2: decell (A) + GA, 3: decell (B) + GA, 4: decell (A) + space filler + GA, GA.
6: simple GA fixation, 7: decell (A) + GA, 8: decell (A) + space filler + GA in the porcine valve.
Decell: decellularization, GA: glutaraldehyde
22 shows the calcium content of each pericardium in a rat implantation model (method 5).
FIG. 23 shows the calcium content of each pericardium in the wild-type mouse transplantation model (method 5).
Figure 24 shows the H & E stain of each pericardium in a rat implant model (method 5).
Figure 25 shows von Kossa stain results of each pericardium in a rat implant model (method 5).
26 is a view showing the shapes of the early type stent (D type; A, B) and deformed stents (M type; C, D). The diameter of the valve was in the range of 20 to 26 mm, and both ends of the stent were shaped like a trumpet, 4 mm wider than the diameter of the valve. The wall of the stent was partially covered with M type to reduce the overall valve stent diameter. The valve in the stent showed grossly good occlusion (B, D) (Method 6).
Fig. Fig. 1 shows a delivery catheter of a valve stent. The proximal portion of the delivery catheter contained a loading area of the valve stem with a 17.5 mm conical tapered tip (white arrows in A, B).
Dotted arrows in C, D, thick arrows in C, D), the valve stand is perfectly placed according to the self-expanding nature. The hook head (E) helps in a controlled deployment to the target site.
28 is a view showing the shape of a valve stent in the process. Single-plane C-arm fluoroscopy shows a good position of the valve stent prior to injection (arrow-head in A). After the successful implantation of the valve stent, the position of the valve stent was confirmed by transcranial echocardiography (white arrow in B) and C-arm biopsy (black arrow in C). RV: right ventricle.
FIG. 29 is a result of observing a plate stent 6 months after the implantation (method 6). Well-preserved implanted valve leaf. The right ventricular outflow tract shows a well-endothelialized inner surface of the stent wall and an outer surface of the stent in the central pulmonary artery (arrow head A).
(B) The transplanted valve leaf in a good location was preserved in a very good form without pulmonary stenosis or obstruction in the transthoracic echocardiography (C) (B).
FIG. 30 is a histological view of tissue valve leaflets at 10 months after transplantation in 11 sheep (Method 6). Hematoxylin and eosin staining (A) and Masson et al. In the trichrome staining (B, blue color), the collagen structure was well maintained.
In Von Kossa staining, there was no clear calcium staining (C, 3.92 ug / mg).

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

&Lt; Example 1: Physical-mechanical evaluation of space-filler addition >

In this example, the physicomechanical evaluation was performed according to the addition of the space-filler.

1-1. Collection, processing and storage of materials

(1) Method 1 : The pericardium of cattle slaughtered at the slaughterhouse is collected under the cooperation of a veterinarian, and the blood is washed, stored in 4 ° C physiological saline, stored in a refrigerator box, and brought to the laboratory. The fresh pericardial slice is taken off and rinsed with physiological saline solution. Disinfect with 0.1% peracetic acid (PAA) at room temperature and wash with distilled water for 1 hour. For pericardiocentesis, treatment with 0.25% SDS in 1 L of distilled water (10 mM Tris, pH 8) for 24 h and treatment with 0.5% Triton X-100 in the same storage solution for 24 h. It is then rinsed with distilled water at 4 ° C for 12 hours. Finally, wash with phosphate-buffered solution (PBS, 0.01 M, pH 7.4) at 4 ° C for 1 hour.

This unspecific pericardial sac was treated with various space fillers (Jeffamine, pAAm, or PEG) and then treated with GA, 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide / Nhydroxysuccinimide (EDC / NHS) And then fixed.

(2) Method 2 : The microcapsules were treated with 0.1 U / mL of α-galactosidase in isotonic solution during de-saturation, and 50% polyethylene glycol (MW 1000) solution of 0.01 M PBS (pH 7.4) Were added to the tissue for one day [Table 2].

Groups Bovine pericardium One
2 GA fixed
Vein saturation ± alpha-galactosidase + GA fixation 3 Depletion saturation ± alpha-galactosidase + Space filler treatment + GA fixation

(3) Method 3 : After treatment with 0.1 U / mL of α-galactosidase in the isotonic solution, the 50% polyethylene glycol (MW 1000) solution of 0.01 M PBS Were added to the tissue at room temperature for one day (Table 3).

Groups Bovine pericardium One
2 Fresh bovine pericardium
GA fixed 3 Tax evasion saturation + GA fixed 4 Venus saturation + Space filler treatment + GA fixation 5 Taxane saturation + alpha-galactosidase + Space filler treatment + GA fixation

(4) Method 4 : Bovine pericardial tissue and porcine valve tissue were divided into three groups according to the tissue treatment method used arbitrarily.

Simple GA fixation treatment (GA fixed group)

Tax evasion (A) + GA fixing (tax evasion A group)

(A) + spacefiller + GA fixing method (Spacefiller A group)

Other simple deaerating methods (B) were applied to examine the tissue properties of the small cystic tissues.

Degassing saturation (B) + GA fixing method (degeneration B group)

(B) + spacefiller + GA fixing method (Spacefiller B group) [Table 4]

Experimental Method Tissue material Group Small intestine Simple GA Fix
Depletion saturation (A) + GA fixation
Depletion saturation (A) + Space filler treatment + GA fixation
Depletion saturation (B) + GA fixation
Depletion saturation (B) + Space filler treatment + GA fixation Pig valve Simple GA Fix
Depletion saturation (A) + GA fixation
Depletion saturation (A) + Space filler treatment + GA fixation

GA; glutaraldehyde

Decontamination of Bovine Pericardium and Pig Heart Valve (A) Method : All bovine pericardial and porcine heart valve tissues were sterilized in distilled water for 2 hours using 4% (v / v) ethanol 0.1% acetic acid (PAA). All tissues were then washed with distilled water for 30 minutes. 1.0% sodium dodecyl sulfate (SDS) and the stock solution were added at 4 ° C for 24 hours, and 1.0% Triton X-100 with the stock solution was added at 4 ° C for 24 hours. This solution was washed 4 to 5 times, then 0.5% SDS solution was added at 4 ° C for 24 hours, and 1.0% Triton X-100 solution was added at 4 ° C for 24 hours. After an additional wash was repeated twice, the tissue was washed with phosphate buffered saline (PBS) for 24 h at 4 ° C.

Degasification (B) of bovine pericardium (B) : In order to compare the mechanical and chemical characteristics of the pericardium in the pericardium, another deethanization method was planned. All bovine pericardial tissues were disinfected with 0.1% PAA in 4% (v / v) ethanol in distilled water for 2 hours at room temperature and induced to decrease bioburden and washed with distilled water for 30 minutes. The retentate solution with 0.25% SDS was added at 4 ° C for 24 hours, the tissue added with 0.5% Triton X-100 and the stock solution at 4 ° C for 24 hours, then washed with distilled water for 30 minutes, The hypertrophied solution was incubated at 4 ° C for 4 hours and then the tissues were washed with PBS for 24 hours at 4 ° C.

Spacefiller Treatment: A 25% solution of polyethylene glycol (MW 1000) in 0.01 M PBS (pH 7.4) was added to the tissue for one day at room temperature after degassing.

(5) Method 5 : Bovine pericardium was treated as follows.

GA (glutaraldehyde) group: Only GA was fixed.

Decell (Decellularization) + GA group: After freeze saturation, GA was fixed.

Decell + PEG (polyethylene glycol) + GA group: treated with PEG, a space filler after freeze-drying, and fixed with GA.

Decell + Jeffamine + GA group: treated with Jeffamine, a space filler, and immobilized by GA, after saturation.

Decellularization : A solution of 0.1% peracetic acid and 4% ethanol in distilled water was treated at room temperature for 2 hours and then washed for 30 minutes. After 24 hours of storage at 4 ° C in a solution containing 0.25% SDS (sodium dodecyl sulfate), the cells were treated with a stock solution containing 0.5% Triton X-100 for 24 hours and washed with distilled water. After 4 hours at 4 ° C, the cells were treated with a hypertonic solution for 4 hours and washed with PBS (phosphate buffered saline) for 24 hours.

PEG (polyethylene glycol) treatment: 25% PEG and 0.01% neomycin were mixed in 0.01 M PBS for 24 hours at room temperature. After that, the solution was treated with the solution at 4 ° C for 3 hours and washed with PBS solution at the same temperature for 24 hours.

Jeffamine treatment : 0.03M Jeffamine and 0.01% neomycine were added to 0.25 M MES (2-morpholinoethanesulfonic acid) and mixed at room temperature for 24 hours. After that, the solution was treated with the solution at 4 ° C for 3 hours and washed with PBS solution at the same temperature for 24 hours.

Fixation : treated with 0.5% glutaraldehyde at room temperature for 3 days and treated with 0.25% glutaraldehyde for 7 days. After fixation, they were cut into 5 mm diameter circular sections and stored in 4% propylene oxide solution.

(6) Method 6 : Porcine pericardium was perfused with 0.25% sodium dodecyl sulfate and 0.5% TritonX-100 and treated with 0.1 units / mL alpha-galactosidase to reduce immunogenicity. Space filler treatment with 0.5% glutaldehyde fixation, solvent treatment with 75% ethanol + 5% octanol and non - toxic digestion with 0.1M glycine.

1-2. Physical epidemiological assessment

(1) Tensile strength

The pericardium of the bovine collagen fibers intersect with each other in unsteady direction, and the distribution is not uniform in each region. Therefore, if one part is removed at random, the tensile strength of the pericardium can not be represented. Therefore, a 0.5 cm × 5 cm rectangular cross-section was taken for each of the six directions that could be obtained by unfolding the treated pericardium and varying the angle by 30 °, and measuring the tensile strength for the width of 5 mm, Of the tensile strength. The tensile strength was measured at a load speed of 100 mm / min by using Japan Tech & Manufacture (Mitutoyo, Japan), Digital Force Gauge (IMADA, Japan), Model 5FGN and automated materials testing system. mm). The thickness of the specimens was also measured several times in one sample with a caliper Mitutoyo Thickness Gauge (Digimatic 543-122-15, Mitutoyo, Japan) to see the relationship between the thickness of the pericardium and the tension.

(2) Elasticity test

The elongation was measured by measuring the length at the point where it was stretched in both directions and the degree of elongation was expressed in%.

(3) Permeability test

After the pressure was applied from one side of the pericardium with physiological saline for one hour, the amount of permeated physiological saline was measured and defined as the permeability, which was expressed as mL / hr · cm ² . The number of samples was 10 in each group.

(4) Compliance test

When the pressure was increased from 100 mmHg to 200 mmHg using physiological saline solution, the volume increased due to the increased pericardium was defined as the degree of purity, which was expressed as μL / mmHg · cm ² . The number of samples was 30 in each group.

1-3. result

(1) DAPI staining, color and thickness

In method 1, DAPI staining of the tissue was performed in order to confirm the freezing of the small cecum, and DAPI staining showed a marked decrease in the DNA of the uninfected small cysts (Fig. 1). NHS, GA, and genipin. The EDC / NHS treated group was white, the GA treated group was dark yellow, and the Genipin treated group was treated with space fillers (Jeffamine, acrylamide, PEG) The group changed to blue-black (Fig. 2a). After the space filler treatment, the overall color became clearer.

Space filler treated groups (acryl, 0.27 ± 0.05, Jeffamine, 0.34 ± 0.03 PEG, 0.29 ± 0.03) were thicker than before space filler treatment except for GA group [Fig.

(2) Tensile strength

(2-1) Method 1 Tensile strength and strain values for [Table 1] are shown in Table 5. GA, EDC / NHS, and genipin were all effective crosslinkers with good tensile strength.

(2-2) Tensile strength and strain values of the method 2 are shown in Table 6. In the PEG-treated group only, the statistical significance was lower than that of the control group.

The results of permeability test were compared with those of the control group. On the other hand, the permeability of group 3, which was treated with space filler after PEG treatment, showed no significant difference from the control group. Similar to the results of the permeability test, there was no significant difference in the group 3 treated with PEG as a space filler compared with the control group. However, in the group 2 treated with PEG alone and in the absence of PEG treatment, (Table 7).

(2-3) Tensile strength and strain values of Method 3 are shown in Table 9. As the thickness of the small cyst increased, the unit area of the cyst decreased, and it was thought that it affected the unit tension.

(2-4) The tensile strength and the elastic test results of the method 4 are shown in Table 10. In the tensile strength test, the deaerative group (9.1 ± 4.3, n = 12) showed a decrease in tensile strength compared to GA fixed group (13.8 ± 3.6, N = 12) (p = 0.005). However, there was no statistically significant difference (P = 0.195) when comparing the tensile strength after the spacefiller treatment (11.7 ± 3.6, N = 12) compared to the GA fixed group. In the elasticity test, an increase in elasticity was observed in the Spacefiller treatment group (87.8 ± 20.9, N = 12) compared to the other groups (GA fixed group, 69.1 ± 14.3, deceased group 70.6 ± 6.1, N = 12) P = 0.008, P = 0.004).

Tensile strength test and elasticity test of bovine pericardium N Thickness (mm) Tensile strength (MPa) Elasticity (%) GA 12 0.43 + 0.06 13.8 ± 3.6 70.6 ± 6.1 Decell (A) + GA 12 0.50 + - 0.05 9.1 ± 4.3 69.1 ± 14.3 Decell (A) + space filler + GA 12 0.52 + 0.04 11.7 ± 3.6 87.8 ± 20.9 Decell (B) + GA 12 0.37 + 0.04 12.0 + - 5.4 38.2 ± 6.2 Decell (B) + space filler + GA 12 0.36 + 0.04 9.8 ± 4.0 43.5 ± 11.4

Decell (A); Decellularization (A), Decell (B); decellularization (B), GA; glutaraldehyde, MPa = Kgf / 5 mm &lt; ^{2 &gt;}

Analysis of the tensile strength and elasticity of the two types of deaeration method showed that the deaerated (B) method (38.2 ± 6.2 versus 69.1 ± 14.3, n = 12, p <0.001) , Respectively.

(2-5) Mechanical test of method 5: There was no significant difference between the groups in both the rat experimental group and the wide-type mouse experimental group [Table 11].

The results of tensile strength test in rat implantation model. Groups
(Bovine Pericardium) Tensile Strength Sample Thickness (mm) Peak load (N) Ultimate Strength (Mpa) Elongation at ultimate strength (%) GA n = 12 0.39 + 0.02 24.26 + - 4.77 12.44 + - 2.60 55.92 + - 5.95 De + GA n = 12 0.39 ± 0.01 22.08 ± 1.60 11.24 + - 0.93 51.28 + - 8.28 De + PEG + GA n = 12 0.40 ± 0.01 22.89 ± 2.15 11.51 ± 0.99 55.49 + - 9.32 De + Jeffamine + GA n = 12 0.40 ± 0.01 21.39 ± 1.78 10.59 + - 0.85 52.41 + - 10.08

The results of tensile strength test in wide type mouse implantation model. Groups
(Bovine Pericardium) Tensile Strength Sample Thickness (mm) Peak load (N) Ultimate Strength (Mpa) Elongation at ultimate strength (%) GA n = 12 0.40 + 0.02 20.28 ± 2.31 10.16 + - 2.60 71.64 + - 14.45 De + GA n = 12 0.39 ± 0.01 21.28 ± 1.79 10.93 ± 1.05 57.64 + - 6.63 De + PEG + GA n = 12 0.41 + - 0.01 20.67 ± 2.69 10.27 + - 0.74 61.24 + - 10.67 De + Jeffamine + GA n = 12 0.41 + 0.02 21.25 + 1.68 10.59 + - 0.85 59.43 + - 8.95

&Lt; Example 2: Cross-linking evaluation of space-filler addition >

In this example, the efficiency of cross-linking with the addition of space-filler was evaluated.

2-1. cross-linking assessment

(1) Thermal stability test

The pericardium pieces fixed by different methods were cut into 8x30 mm sections, 5 per each group, and subjected to a constant tension of 95 g. Then, they were placed in distilled water at 55 ° C and the water temperature was raised at a rate of 2 ° C / min. The temperature at the time of rapid shrinkage, that is, the temperature at which the collagen denaturation of the tissue occurred, was compared with each other.

(2) Resistance towards protease digestion

The pericardium is rinsed with distilled water and dried at 70 ° C for 24 hours and weight 1 is measured. To the 0.5 mg / mL Pronase ^® solution buffered with 0.01 M HEPES (pH 7.4), 0.1 M glycine and 0.01 M CaCl ₂ are added and the pericardium is added and reacted at 50 ° C for 24 hours. After washing with distilled water, dry at 70 ° C for 24 hours and measure weight (weight 2). The resistance to the protease is expressed as the residual weight% (= weight 2 / weight 1 x 100).

2-2. Crosslinking efficiency result [Figure 3]

(1) Thermal Stability Results

(1-1) In the method 1, as a result of heat-induced denaturation of collagen, there was no problem in terms of cross-link thermal stability as a result of the treatment conditions.

The shrinkage temperature of all treated groups was significantly higher than that of fresh small cysts (69.42 ℃, p <0.05). GA-treated group showed the highest shrinkage temperature at 86.28 ℃, 84.23 ℃, 84.44 ℃ and 86.14 ℃, respectively. The shrinkage temperatures of the Genipin-treated group were 82.28 ° C, 79.82 ° C, 79.70 ° C and 79.20 ° C, respectively, and the thermal stability was slightly higher than the shrinkage temperatures of 74.72 ° C, 81.68 ° C, 77.74 ° C and 76.87 ° C

In the EDC / NHS-treated group, the space filler increased the thermal stability and increased to 81.68 ° C in the Jeffamine group. In the GA-treated group and the Genipin-treated group, the space filler had no significant effect on the thermal stability (Fig. 3a).

(1-2) The shrinkage temperature at which collagen denaturation occurred in Method 3 was not significantly different between the control group treated with no-saturation and the group treated with space-filler PEG and α-galactosidase (Fig. 4a).

(1-3) In Method 4, Spacefiller A group was statistically (84.4 ± 0.2, N = 5) compared to the other groups (GA group: 83.5 ± 0.4, N = 5, (P = 0.007). However, we did not find any significant difference between the methods of de-saturation (group B: 83.6 ± 0.64, n = 5, space filler group B: 83.8 ± 0.6, n = 5) (p = 0.910 and p = 0.104).

(1-4) Method 5, there was no significant difference between the groups in both the rat experimental group and the wide-type mouse experimental group (Fig. 5) (Fig. 6).

(2) Pronase test results

(2-1) In Method 1, this result was similar to the thermal stability. GA-treated group and Genipin-treated group had higher protease resistance than fresh corticosteroid, and Genipin-treated group was much less effective than GA-treated group. In the EDC / NHS-treated group, the protease resistance was very low in the absence of the space filler, and more than 90% of the protease resistance was increased in the case of space filler treatment. The GA-treated group remained 89.3% -95.4% after the enzymatic degradation, showing the most effective cross-linking regardless of space filler treatment (Fig. 3b).

(2-2) In method 3, the resistance to pronase was lower in all treated groups than in the control group. There was no significant difference according to the method of processing the space filler (Fig. 4B).

(2-3) Analysis according to treatment method in method 4: In the GA-fixed group, there was a statistically significant difference between bovine pericardium (71.7 ± 8.1, N = 5) and porcine valve (78.2 ± 4.2, N = 10) (P = 0.059). There was no statistically significant difference (P = 0.161) between the small cysts (61.0 ± 13.1, N = 5) and porcine valves (74.5 ± 17.8, N = 10) However, pig heart valve (58.4 ± 6.1, n = 10) showed a decrease in proinflammatory stability compared to the small intestine (83.3 ± 4.5, n = 5) (P <0.001).

Analysis according to tissue type in method 4: There was no statistical difference according to the tissue preparation method (P = 0.09) in bovine pericardium. However, the decrease in proinflammatory stability of the pigs with Spacefiller A group (58.4 ± 6.1, N = 10) compared to the GA-fixed group (78.2 ± 4.2, N = 10) and the deteriorated group A (74.5 ± 17.8, N = 10) (P < 0.001, P = 0.003) (Fig. 7).

(2-4) In Method 5, there was no significant difference between the groups in the rat experimental group and the wide-type mouse experimental group (Fig. 8) (Fig. 9).

&Lt; Example 3: Evaluation of contact cytotoxicity of space-filler addition >

In this example, contact cytotoxicity due to space-filler addition was evaluated.

3-1. Contact cytotoxicity assessment

Smooth muscle cells of pigs are planted in 6-well. DMSO was treated with cells as a control, and cultured at 37 ° C in 5% CO ₂ in air for 48 hours.

The morphology of the cells was observed under a microscope and the cells attached to the plate were washed with cold PBS. Then add 1X binding buffer. 5 μl of the above solution, 5 μl of FITC, 5 μl of Annexin V and propidium iodide (PI), vortexed, and incubated for 15 minutes in a dark room at 25 ° C. Then, 400 μl of 1 × binding buffer was added to the tube and analyzed by FACS within 1 hour.

3-2 Contact cytotoxicity results [Figure 10]

In the method 1, 75.3% (none), 71.3% (Jeffamine), 84.7% (Acryl) and 82.8% (PEG) cells survived in the EDC / NHS-treated group. The EDC / NHS- treatment method was the most toxic.

In the GA-treated group, 76.8% (none), 87.3% (Jeffamine), 88.5% (Acryl) and 90.3% (PEG) cells survived and the space filler treatment increased cell survival by about 10%.

88.8% (none), 84.9% (Jeffamine), 88.3% (Acryl) and 91.8% (PEG) cells survived in the Genipin treatment group and were not toxic in the genipin group regardless of space filler treatment.

Example 4: Evaluation of in-vivo calcification of space-filler addition &gt;

In this example, in-vivo calcification with the addition of space-filler was evaluated.

4-1 Small animal experiment

(1) rabbit intramuscular implantation in Method 1 : Male New Zealand white rabbits were used to perform all surgical procedures aseptically. Each rabbit hindlimb skin was incised, a tissue section was inserted between the muscle layers, and after 4-8 weeks of extraction, calcium was quantitated.

(2) Rat subcutaneous implantation in method 2 : 10 male Sprague-Dawley rats at 4 weeks of age were used. Rat weight was measured before, during, 2 weeks, 1 month, and 2 months after surgery, because there was a possibility of difference in degree of calcification according to body weight of Rat. To evaluate the effect of each treatment on the degree of calcification, quantitative measurement of calcium was carried out after grafting of the bovine pericardial flap to the rats. All surgical procedures proceeded aseptically.

(3) Method 3 rabbit intramuscular implantation : Male New Zealand white rabbits were used to perform all surgical procedures aseptically. Tissue was transplanted to the legs and back, and after 2 months, the transplanted tissues were removed. The tissues of the rabbits attached to the tissue were detached and removed, and calcium was quantified.

(4) In method 4, anesthesia was performed by intraperitoneal injection using rat subcutaneous implantation : zolazepam + Tiletamine (0.2 CC, zolethyl, Virbac, France) and xylazine (0.2 CC, Rumpun, Bayer, Germany). After shaving the mice, I made four subcutaneous pockets on the back. Each treated tissue disk (diameter 5 mm) was poured into the pouch and the wound was sutured using a 4-0 monofilament suture. Antibiotics including cefazoline (20 mg / kg, Yuhan, Korea) were used for 3 days after transplantation. Two months after the experiment, CO2 gas was inhaled and sacrificed, and graft tissue samples were collected.

(5) In method 5, rat and wide type mouse subcutaneous implantation transplantation: rat and wide type mouse were selected as experimental animals. Ten rats were assigned to each experimental group and 40 rats and 40 wide type mice were used for the experiment. Four skin incisions were made on the back of the general anesthetized animal. Four treated small intestine fragments were implanted subcutaneously through each incision and the skin was sutured.

Harvesting: The rat was transplanted subcutaneously at 8 weeks and wide type mouse at 12 weeks.

4-2 Calcium analysis

The extracted tissue is washed with physiological saline, dried at 90 ° C for 24 hours, and then weighed. Then hydrolyzed with 5N HCl solution and dried. The amount of calcium and inorganic phosphate was determined by measuring the absorbance with HITACHI 7070.

4-3 in-vivo calcification results

(1) In method 1, the intramuscular tissues of rabbits were inserted for 4 to 8 weeks to analyze in vivo calcification. 4 weeks were insufficient to evaluate in vivo calcification (Fig. 11). Almost no calcification was observed in the Genipin-treated group (less than 1 μg / ml / mg) (FIG. 11 b). In the EDC / NHS or GA treatment group, the calcification due to the space filler treatment effect was reduced (p <0.05), and this biotite pattern was similar to the cytotoxicity analysis. In the GA-PEG group, calcification was particularly low (0.62 ± 0.1 μg / ml / mg). von Kossa staining showed similar results (Fig. 12). In the case of high calcification, more cell infiltration was observed in the tissues (hematoxylin and eosin stain) (Fig. 13). These cells were identified as CD4 antibody positive (abcam, ab61131; Sigma-Aldrich, A0545) and macrophages (immunohistochemistry and immunofluorescence [Fig. These results suggest that invasion of immune cells by cytotoxicity induces tissue calcification.

(2) In method 2, tissues were transplanted subcutaneously in rats and cultured for 2 months to measure calcium. [Table 13] [Fig. 14]. As a result, no saturation and PEG treatment showed a slight decrease in the degree of calcification compared to the control group, but did not show a statistically significant reduction.

After H & E staining, it was observed at a magnification of 100 times under a microscope. As a result, in the control group, the tissue was more compact, less damaged, and the cellular components were observed. However, in the remaining two groups, The density of the tissue was slightly less than that of the control group, and no significant difference was observed [Fig. 15]. In addition, after von Kossa staining for calcium-black staining, the degree of calcium deposition was observed by a microscope, and it was confirmed that the calcified portion appeared as a black stain (Fig. 16).

(3) In Method 3, the back and the undersurface of the rabbit were compared with each other (Fig. 17). In general, PEG and α-galactosidase treatment lowered the degree of calcification rather than saturation. In comparison with the pulmonary artery, small calcification was observed in the small intestine of the small intestine and the pulmonary artery in the small intestine, and the space filler treated group in the back and torso region was significantly lower than that in the non - saturated group.

After H & E staining, they were observed under a microscope. The degree of deposition in the leg was higher than that of the Rabbit. In both the small intestine and the porcine pulmonary artery, calcification and fibrocytic infiltration were more common in the pulmonary artery than in the pericardium (Fig. 18).

Similarly, the extent of calcium deposition by microscopy was confirmed by von Kossa staining (Fig. 19).

(4) Microscopic examination in Method 4 - Infiltration of inflammatory cells was confirmed by H & E staining. In a simple GA-fixed group, a large amount of inflammatory cells infiltrated the tissue. However, infiltration of inflammatory cells decreased after depletion and spacefiller treatment. In addition, calcium deposition was observed in all groups, and relatively low calcium deposition was observed in porcine valves by Von Kossa staining (Fig. 20).

Calcium analysis

In the method 4, the calcium content of the bovine pericardium (93.5 ± 47.6, N = 17) (p <0.001), the heart valve of the pig (55.25 ± 17.2 ㎍ / mg, Respectively. The phosphoric acid content of the small cysts (88.4 ± 64.8 ㎍ / ㎎, N = 17) was also higher than that of pig heart valves (52.2 ± 12.6 ㎍ / ㎎, N = 42) (P = 0.035). Calcium content index (63.3 ± 10.6 ㎍ / ㎎, N = 23) was higher in the hypotensive group than in the pig heart valve (44.1 ± 5.8, N = 44) (p <0.001). The phosphorus content of pig heart valve (37.3 ± 8.6 ㎍ / ㎎, N = 44) was lower than that of the small intestine (78.1 ± 19.0 ㎍ / ㎎, N = 23) (p <0.001).

The calcium content index in the Spacefiller group was lower in the pig heart valve (58.5 ± 25.8 ㎍ / ㎎, N = 46) than in the small cyst group (71.2 ± 13.3 ㎍ / ㎎, N = 27) (p <0.001). However, there was no significant difference in the phosphate content index between the two groups (bovine pericardium: 67.4 ± 17.5 ㎍ / ㎎, N = 27, pig heart valve group: 63.5 ± 34.5 ㎍ / ㎎, 0.596).

Analysis of the tissue material in Method 4: The calcium content index was the highest in the simple GA fixed group (93.5 ± 47.6, n = 17) in the small intestine group compared with the other groups (deaerated saturation group A: 63.3 ± 10.6, n = 23, space filler group A: 72.4 ± 30.9, n = 22) (p = 0.002, p = 0.027, respectively). In addition, in the simple GA fixed group (88.4 ± 64.8, n = 17), the phosphorus content indexes were different from those of the other groups (degeneration A group: 78.1 ± 19.0, n = 23, space filler A group: 44.8 ± 21.3, n = 22) p = 0.396, p = 0.001), respectively. Calcium content index was higher in the hypereuterated B group (71.3 ± 13.3, n = 27) than in the A group (63.3 ± 10.6, n = 23) when analyzed by deaeration and saturation A and B methods (p = 0.024). However, no statistically significant difference was observed between the two groups when the spacefiller was added (74.5 ± 19.8, n = 23 versus 72.4 ± 30.9, p = 0.741). The hypothetical B group (67.4 ± 17.5, n = 27) showed a lower phosphorus content index than the A group (78.1 ± 19.0, n = 23) (p = 0.043). However, there was a statistically significant higher phosphorus content index (p <0.001) compared to the Spacefiller B group (72.5 ± 20.1, n = 23) in the deteriorated saturated A group (44.8 ± 21.3, n = 22).

(44.1 ± 5.8, n = 44) were significantly lower in the other group (simple GA group: 55.2 ± 17.2, n = 42, p = 0.006, space filler group: 58.5 ± 25.8, n = 46, p <0.001), respectively. The phosphorus content index was also significantly lower in the depletion group A (37.3 ± 8.6, n = 44) than in the other groups (simple GA group: 52.2 ± 12.6, n = 42, p = 0.002, space filler A: 63.5 ± 34.5, p = 0.017) (Fig. 21).

(5) Calcium determination in method 5 : The calcium content of the tissue treated with PEG and the tissue treated with Jeffamine was significantly lower in the rat than in the other experimental groups (p <0.001). In wild type mice, the calcium content of PEG-treated tissues was significantly lower than that of the other experimental groups (p <0.001). Comparing PEG with Jeffamine, PEG significantly reduced tissue calcium deposition compared to Jeffamine (p = 0.001) (Fig. 22) [Fig. 23].

Tissue staining in method 5 : Infiltration of inflammatory cells in the space-treated tissue of H & E staining was small. The von Kossa staining showed less dyeing of the space-treated tissue [Fig. 24] (Fig. 25).

<Example 5: Evaluation of safety and efficacy of preclinical experiment using large-animal test for heterogeneous tissue with space-filler>

In this example, a heterogeneous tissue treated with a space-filler was attached to a nitinol-wire based self-expandable valved-stent, and the stent was inserted through a sheep vein (femoral vein, jugular vein) The safety and efficacy of the mid - term outcome over 6 months were evaluated in pre - clinical studies using large animals.

5-1. Fabrication and insertion of stent valves

Nitinol-wire based self-expandable valved-stent was fabricated on its own [Table 14] [Figure 26]. The pig pericardium was used for the valve leaf, and the pericardium of pig was free of 0.25% sodium dodecyl sulfate and 0.5% TritonX-100, and 0.1 units / mL alpha-galactosidase treatment was performed to reduce immunogenicity. The space filler treatment with 25% polyethylene glycol, 0.5% glutaldehyde fixation, solvent treatment with 75% ethanol + 5% octanol and non-toxic digestion with 0.1 M glycine ).

For large animal experiments, 12 sheep were used and 18 Fr. The delivery catheter was used to insert the stent valve through the femoral vein or jugular vein [Fig. 27]. A stent valve was placed by controlling the catheter handle and hook block using fluoroscopy (Fig. 28A) and echocardiography (Fig. 27).

Of valved-stent in pulmonic position Type D type M type Can be folded in the longitudinal axis No folding in the longitudinal axis Wire thickness 0.008 inch (0.2 mm) 0.010 inch (0.25 mm) Delivery system 18Fr 18Fr Valve wall Full covered Partially covered Diameter
x Total length 20 mm x 24 mm
22 mm x 25 mm
24 mm x 28 mm
26 mm x 33 mm 20 mm x 30 mm
22 mm x 33 mm
24 mm x 36 mm
26 mm x 38 mm Radial force 0.17 to 0.20 kgf 0.40 to 0.5 kgf

5-2. Safety and efficacy results of preclinical experiments using large animal experiments on heterogeneous tissues with space-filler

The overall results of jugular vein stent valve insertion in method 6 are described in Table 15. [ The weight of the sheep was 43.9 kg. In 12 sheep, stent valves (24 mm in diameter in 7 sheep and 26 mm in diameter in 5 sheep) were successfully inserted through the femoral vein (8) and the jugular vein (4). Eight sheep were inserted in a good location (Fig. 28B) (Fig. 28C), two sheep were inserted into the main pulmonary artery, and two sheep were inserted into the right ventricular outflow tract. Transthoracic Doppler echocardiography performed immediately after insertion showed good valve function without pulmonary embolization or reflux.

Three months after insertion of the stenting valve, the inner wall of the stent wall and the outer surface of the stent wall of the main pulmonary artery were well intimalized in the third volume of Table 15 (estimated to be the cause of death) 29A].

After 6 months of follow - up, 8 sacrifices were made (stent valve in 6 wells, good pulmonary valve position, 1 in the main pulmonary artery, 1 in the right ventricular outflow tract). Transthoracic echocardiography performed immediately prior to sacrifice showed no pulmonic valve regurgitation in five, minor pulmonic valve regurgitation in two, and mild pulmonic valve regurgitation in one. In 8 cardiac catheters, the systolic pressure difference between the right ventricle and the main pulmonary artery was 6 ± 3.8 mm Hg (range: 1 ~ 11) and the mean right ventricular systolic pressure was 22.9 ± 3.2 mm Hg (range: 18 ~ 26).

At the time of sacrifice, the shape and function of the inserted valve was well maintained in 5 out of 6 sheep with good stent valve position. After 6 months of insertion, good echocardiographic findings at the pulmonary valve position and visual findings with well-preserved valvular pattern were seen (FIG. 29B) [FIG. 29C].

Two valves inserted into the right ventricular outflow tract and two valves inserted into the main pulmonary artery did not function as valve valves attached to the stent wall.

The collagen structure was well maintained (Hematoxylin & Eosin and Masson &amp; Trichrome staining) in all the valve lobes extracted from the normal sacrifice biopsy [Fig. 30A] (Fig. 30B), without endometriosis and calcification.

In the eleventh amniotic valve of Table 15 inserted over 10 months, the calcium quantification was low (3.92 ug / mg) and was similar to the calcium quantification of the 6-month-old heterotopic valve [Table 15].

The overall results of a jugular vein pulmonary valve stent implantation.

* From the final transthoracic echocardiography, † From cardiac catheterization at the time of sacrification, ‡ From autopsy findings.

FV: femoral vein, JV: jugular vein, PR: pulmonary regurgitation, PA: pulmonary artery, RVOT: right ventricular outflow tract,

Claims (14)

A method for producing a xenograft for xenotransplantation comprising the steps of:
(A) decellularizing a biological tissue of a mammal;
(B) treating a space filler in the non-saturating biotissue;
(C) fixing the processed biospecimen to the space filler; And
(D) removing the toxicity of the fixed biotissue. The method according to claim 1, wherein the mammal is a mammal other than a human. The method according to claim 1, wherein the living tissue is selected from a pericardium, a heart valve, a pericardium, a dural sac, a blood vessel, and a blood vessel wall. The method of claim 1, wherein the space filler is selected from the group consisting of polyethylene glycol (PEG), polyacrylamide (PAAm), jeffamine, and combinations thereof. Gt; The method according to claim 1,
Wherein when the living tissue is a bovine pericardium, the space filler is treated with 0.03M to 0.06M of Jeffamine at room temperature for 12 hours to 36 hours. The method according to claim 1,
Wherein when the living tissue is a bovine pericardial, 25 to 50% of PEG is treated at room temperature for 12 to 36 hours. The method according to claim 1,
Wherein the biotissue is a porcine valve plate or a valve membrane conduit, wherein 25 to 50% of PEG is treated at room temperature for 12 to 36 hours. The method according to claim 1,
Wherein the immobilization of step (C) is performed using at least one selected from the group consisting of glutaraldehyde, Genipin and EDC / NHS (1- (3-dimethylaminopropyl) -3-ethyl carbodiimide / Nhydroxysuccinimide) Wherein the treatment solution is treated with a fixative solution containing a compound. The method according to claim 1,
Further comprising the step of treating the isotonic solution containing 0.01 to 1.0 U / ml of? -Galactosidase in the step (A). The method according to claim 1,
(D) L-lysine, glycine, chitosan, glutamic acid, arginine, heparin, homocysteic acid, sodium borohydride sodium borohydride, and sodium bisulfate. The method according to claim 1, wherein the detoxification step further comprises a detoxification step of treating a compound selected from the group consisting of sodium borohydride and sodium bisulfate. The method according to claim 10, wherein the toxic removal is performed at 4 to 37 캜 for 12 to 48 hours. The method according to claim 1,
When the biotissue is a heart valve of a pig,
(A) freezing the heart valve of pigs with 0.25% SDS and 0.5% TritonX-100;
(B) treating 0.1 units / mL of? -Galactosidase;
(C) treating 25% PEG;
(D) immobilizing with 0.5% glutaraldehyde;
(E) treating the solvent with 75% ethanol and 5% octanol; And
(F) a step of detoxifying with 0.1 M of glycine. A biomedical tissue for xenotransplantation produced by the method according to any one of claims 1 to 12. The xenotransplantation biopsy of claim 13, wherein the xenotransplantation biopsy is a xenograft prosthesis for use in heart valves, surgical tissue patches, tissue vessels, ligaments, tendons, or porous supports for tissue engineering.

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