CN1775304A - Preparation method of biologically active artificial biological valve - Google Patents

Abstract

The invention relates to a preparation method of an artificial biological valve, belonging to the field of biological materials. The invention uses animal or human allogeneic heart valve as raw material, removes the cells of the animal or human allogeneic heart valve by chemical method, and obtains extracellular matrix material of the decellularized valve. Immerse this material in different concentrations and different proportions of proanthocyanidin solutions, shake and cross-link under appropriate temperature conditions, so as to obtain a non-toxic, non-shrinking artificial biological valve material, which is used for the preparation of clinical heart valve replacement, repair and regeneration . The method of the invention can adjust the crosslinking mechanical strength, enzymolysis resistance, crosslinking agent release, and tissue regeneration promotion ability by adjusting the concentration of the crosslinking agent proanthocyanidin solution, the crosslinking time, and the cleaning time. The process of the invention is simple and easy to implement, and is convenient to popularize.

Description

Preparation method of biologically active artificial biological valve

technical field

The invention relates to a preparation method of a biologically active artificial biological valve, which belongs to the field of biomedical engineering.

Background technique

Heart valve disease is a serious disease that endangers human health. At present, artificial heart valve replacement surgery is mainly used for heart valve patients [Julie R, Boris AN, Vacanti JP. Tissue engineering: a 21st century solution to surgical reconstruction. Annthorac surg, 2001 , 72: 577-591.]. Prosthetic heart valves include mechanical valves and biological valves, each of which has its own successes and shortcomings. The implanted life span of the mechanical valve can reach 25 years, so it is the most widely used artificial valve at present. However, mechanical valves have serious defects that are difficult to overcome, such as non-physiological, obstruction to normal blood flow, bleeding complications caused by life-long anticoagulant drugs after surgery, a high thrombosis rate of 5% per year, and inability to follow the heart rate of adolescents. Grow up and grow up and so on. These problems make the use of mechanical valves potentially multiple risk factors. It also makes the research hotspot of artificial heart valve turn to biological valve [Julie R, Boris AN, Vacanti JP.Tissue engineering: a 21st century solution to surgical reconstruction. Ann thorac surg, 2001, 72:577-591.].

A biological valve is a valve prosthesis processed from animal or human heart valves or pericardium with similar structure and function to the human body [Schmidt CE, Baier JM. Acellular vascular tissue: natural biomaterials for tissue repair and tissue engineering. Biomaterials, 2000, 21 (2000): 2215-2231.]. As far as biological valves are concerned, homograft valves have a high success rate in clinical application, but their sources are limited; although heterogeneous valves come from a wide range of sources, they are antigenic and must be cross-linked with glutaraldehyde before they can be used. The porcine aortic valve cross-linked by glutaraldehyde has certain strength and toughness, basically retains the collagen structure of the porcine heart valve, reduces immunogenicity, and largely avoids the disadvantage of lifelong anticoagulation after mechanical valve replacement . However, when this valve is directly implanted in the body, severe calcification often occurs, and the residual toxicity of glutaraldehyde itself after glutaraldehyde treatment and the residual immunogenicity of the valve tissue greatly reduce its working life in vivo[Schmidt CE, Baier JM. Acellular vascular tissue: natural biomaterials for tissue repair and tissue engineering. Biomaterials, 2000, 21(2000): 2215-2231.]. In addition, the decellularized artificial valve without any cross-linking is easy to infect certain pathogenic bacteria or viral diseases, such as Creutzfeldt-Jakob's disease and other disadvantages. Martin et al. and Walles et al. have detected and confirmed that porcine endogenous retroviruses can be expressed in human cells [Neuenschwander S, Hoerstrup SP. Heart valve tissue engineering. Transplant immunology, 2004, 12(3-4): 359-365 .]. Therefore, there is an urgent need for a new type of non-toxic, antibacterial, antiviral and anti-inflammatory cross-linking agent to replace glutaraldehyde cross-linked natural valve materials.

The cells of animal valves are thoroughly extracted by detergent, enzyme digestion and ultrasonic cleaning, and the natural three-dimensional scaffold structure composed of collagen, elastin, fibronectin and laminin is retained. Since the scaffold structure is an extracellular matrix, which contains various functional proteins such as glycoproteins and proteoglycans, it has good biocompatibility. In addition, this stent maintains the natural shape of the valve, unlike a polymer material stent that needs to be shaped according to hemodynamics [Schmidt CE, Baier JM. Acellular vascular tissue: natural biomaterials for tissue repair and tissue engineering. Biomaterials, 2000, 21( 2000): 2215-2231.]. However, the artificial valve SYNERGRAFT ^TM produced by SYNERGRAFT Company without any cross-linking fixation process, after clinical application, not only could not regenerate the valve endothelium in situ in vivo, but caused a strong immune inflammatory reaction, resulting in many cases Tragedy of patients dying within 1-2 years after surgery [Simon P., Kasimir MT, Seebacher G. et al. Early failure of the tissue engineered porcine heartvalve SYNERGRAFT ^TM in pediatric patients. European Journal of Cardio-thoracic Surgery. 23(2003): 1002-1006.]. It seems that the application of natural decellularized valve materials also requires a novel non-toxic, antibacterial, antiviral and anti-inflammatory crosslinking agent for its crosslinking treatment.

In order to overcome the above-mentioned defects, the cross-linking and fixing process is an important treatment method. People have found a variety of cross-linking agents to cross-link animal valve stents, such as polyepoxide (PC), glycerol, α-aminooleic acid ( AOA), NO-ReactTM, cyanimide, carbodiimide (1-ethyl-3 (-3 dimethyl aminopropyl) carbodiimide hydrochloride, EDC), adipyl dichlorid, cyclohexane diisocyanate (hexamethylene diisocyanate, HMDC), alginic acid Azide (alginate azide) and sodium alginate (sodium alginate), etc. [Julie R, Boris AN, Vacanti JP. Tissue engineering: a 21st century solution to surgical reconstruction. Annthorac surg, 2001, 72: 577-591., Schmidt CE , Baier JM. Acellular vascular issue: natural biomaterials for tissue repair and tissue engineering. Biomaterials, 2000, 21 (2000): 2215-2231.], but due to its cross-linking effect, the toxicity of the cross-linking agent itself, the cross-linking The performance of the resulting biological prosthesis is still unsatisfactory. Therefore, it is still necessary to find a more suitable cross-linking agent, so that the cross-linked valve has appropriate mechanical properties, strong stability and durability, and low toxicity, which is conducive to the ingrowth of valve constituent cells, and can be antibacterial, Antiviral, anti-inflammatory, etc.

Proanthocyanidins are a class of plant secondary metabolites that widely exist in fruits, vegetables, petals, seeds and bark, and are also important components of red wine. They are used as food, natural antioxidants and medicines to scavenge free radicals, Protects cardiovascular and cerebrovascular, has antibacterial, antiviral, anti-inflammatory, anticancer and other activities, its metabolic pathway in the body is clear, and has no toxicity [Packer L., Rimbach, and Virgili F.Antioxidant activity and biological properties of a procyanidin-rich extract from pine (Pinus Maritima) bark, pycnogenol. Free Radical Biology & Medicine, 27(5/6): 704-724, 1999.]. Although Nimni et al. proved that proanthocyanidin can be used to cross-link bovine tendon and pericardium [Bo Han, Jason Jaurequi, Bao Wei Tang, Marcel E.Nimni. Proanthocyanidin: A natural crosslinking reagent for stabilizing collagen matrices. J Biomed Mater Res. 65A: 118 -124, 2003.], but the use of proanthocyanidins to cross-link decellularized heart valves to prepare biological valves or prepare tissue engineering heart valve scaffold materials has not been reported. This constitutes the idea of the present invention.

Contents of the invention

The purpose of the present invention is to provide a method for preparing a biologically active artificial biological valve. The present invention aims to provide a biologically active biological valve by using proanthocyanidins as a novel cross-linking agent to cross-link the decellularized heart valve. It has the advantages of excellent mechanical properties, stability, durability and biocompatibility, and no toxicity, so as to meet the needs of the development and production of a new generation of biological valves.

The present invention uses animal heart valves as raw materials, removes the cells of the animals or heart valves by chemical methods, obtains the decellularized extracellular matrix of the valves as a cross-linking object, and immerses the extracellular matrix of the valves in different concentrations and different proportions of proanthocyanidin solutions , under different temperature conditions, shaking and cross-linking for a certain period of time, a valve material with a maximum tensile strength of more than 8.0Mpa, which is significantly higher than that of fresh pig valve or pig valve cross-linked with glutaraldehyde, can be obtained. High, strong resistance to enzymatic hydrolysis, and the release of cross-linking agents is non-toxic. By adjusting the concentration of cross-linking agent proanthocyanidins, cross-linking temperature, shaking speed, cross-linking time and preservation method after cross-linking, the mechanical strength, enzymolysis resistance, cross-linking agent release speed, and tissue regeneration promotion of the valve or stent can be adjusted. ability etc. The cross-linking agent proanthocyanidins used in the present invention can also be all artificial or natural materials containing collagen components. The decellularized valve material obtained by cross-linking can be directly used as an artificial biological valve, or as a tissue engineered heart valve scaffold, so as to be used for further preparation or construction of heart valves, clinical replacement, repair and regeneration. Proanthocyanidins themselves not only have cross-linking effect, but also have the functions of scavenging free radicals, anti-oxidation, anti-cancer, anti-inflammation, cardiovascular and cerebrovascular protection and promoting tissue regeneration. By adopting the method provided by the present invention, products with different characteristics can be prepared according to different degrees of tissue damage and different requirements for biological valves or tissue engineering heart valve brackets during repair, so as to meet the needs of clinical applications.

The preparation method of the biologically active artificial biological valve of the present invention includes pre-cross-linking treatment, cross-linking process and post-cross-linking treatment and preservation. Now it is described as follows:

1 Cross-linking pre-treatment process

The purpose of this process is to obtain fully cleaned decellularized heart valves, including two steps:

1) Heart valve raw material collection and decellularization

The invention takes the decellularized pig heart valve or the decellularized human allogeneic heart valve as the crosslinking object. In the slaughterhouse, the aortic valve or pulmonary valve in the heart of the freshly slaughtered pig is cut out together with the surrounding blood vessels, and placed in a phosphate buffer solution without Ca ²⁺ and Mg ²⁺ ions (that is, D-Hanks solution, the formula is shown in Table 1). Take it back within 1 hour at 4°C, immediately cut off the valve leaflet carefully, wash it with D-Hanks solution, and decellularize according to the following method:

The decellularization solution is divided into A solution and B solution, both of which are prepared with D-Hanks solution. The valve is decellularized in two steps, the first step is using liquid A, and the second step is using liquid B. In each step, add 2-20ml to each valve. The formulas of liquid A and liquid B are as follows:

The decellularization solution A can be any one of the following three solution formulations (1), (2) and (3).

Formula (1) contains 0.05% or 0.1% trypsin and 0.02% ethylenediamine tetraacetic acid disodium salt (EDTA).

Formula (2) contains 0.05% or 0.1% trypsin, 0.5% non-ionic detergent polyethylene glycol octyl phenyl ether (4-(1,1,3,3-Tetramethylbutyl) phenyl-polyethylene glycol, trade name Triton X-100), 0.5% sodium deoxycholate and 0.02% EDTA.

Formulation (3) contained 0.5% Triton X-100, 0.5% sodium deoxycholate and 0.02% EDTA.

The trypsin and the like contained in the above three formulas are all mass percentages, that is, the mass numbers contained in every 100ml.

The formula of decellularization solution B is a mixed solution of 20 μg/ml nuclease (RNase) and 0.2 mg/ml deoxynuclease (DNase) prepared in D-Hanks solution.

Table 1. Formula of phosphate buffer solution without Ca ²⁺ and Mg ²⁺ ions (ie D-Hanks solution). Element Dosage(g/L) KClKH ₂ PO ₄ NaClNaHCO ₃ Na ₂ HPO ₄ 7H ₂ O phenol red 0.440.068.000.350.090 or 0.01

The conditions required for the decellularization of animal or human allograft heart valves for the preparation of artificial biological valves: the temperature is 1-40°C, preferably 20-37°C; the shaking speed of the shaking table is 0-300rpm (including no shaking), preferably 120-240rpm; air humidity is 30%-95%, preferably 50%-95%; _CO2 concentration is 0.3%-5%, preferably 0.3% or 5%; time is 10 minutes-100 hours, preferably 1-48 hours.

2) Pre-treatment of cross-linking

After the above-mentioned decellularization treatment, pre-cross-linking treatment was performed as follows: each valve was soaked in 5-10 ml of D-Hanks solution for cleaning. The conditions required for cleaning: the shaking speed of the shaking table is 0-300rpm (including no shaking), preferably 120-240rpm; the time is 0.5-480 hours, 48-240 hours; the D-Hanks solution is replaced every day.

2 Cross-linking process

The decellularized heart valve that has been fully cleaned by the above method is soaked in 1-20ml of 0.01-20mg/ml (mass-volume ratio) proanthocyanidin D-Hanks liquid solution prepared by each valve to make the decellularized heart valve Cross-linking; the preferred ratio is that each valve is soaked in 1-10ml of D-Hanks solution of 0.06-10mg/ml (mass-volume ratio) proanthocyanidins. The temperature for crosslinking the decellularized heart valve is 1-40°C, preferably 20-37°C. The cross-linking is completed under the condition of shaking on a shaker, and the shaking speed is preferably 10-300 rpm, preferably 60-120 rpm. The crosslinking time is from 1 minute to 240 hours, preferably from 0.5 to 48 hours.

3 Handling and storage after cross-linking

For the cross-linked decellularized animal heart valves, after cross-linking, add 2-20ml of D-Hanks solution to each valve to soak the valve, and wash for 0-480 hours (including no cleaning), preferably the cleaning time is 5 minutes- 1 hour. Washing is completed at 1-40°C, preferably at a temperature of 20-37°C, on a shaking table at 0-300rpm (including no shaking), preferably at a shaking speed of 60-120rpm. According to this method, the artificial biological valve material or the tissue engineering heart valve support with the above-mentioned characteristics can be prepared. Store in D-Hanks solution at 1-10°C until use. The best storage temperature is 4°C.

The performance of the proanthocyanidin cross-linking method used in the present invention to prepare the artificial biovalve with bioactivity is evaluated by the following four methods.

1 Scanning electron microscope observation

The decellularized heart valve prepared by the above method was fixed with 2.5% glutaraldehyde, then dehydrated with 50%, 80%, 95% and 100% ethanol, and dried with 6-methyldisilanzane (HMDS). Electron microscope was used to observe the morphology of valve extracellular matrix after decellularization and whether the decellularization was comprehensive.

2 Determination of maximum tensile strength

Cut the leaflets cross-linked with different concentrations of proanthocyanidins prepared by the present invention into strips with a width of 4 mm and a length of 25 mm along the fiber direction. Cut one piece from each leaflet, 6 pieces for each concentration. The test is carried out on a SHIMADZU universal mechanical testing machine with a tensile rate of 2mm/min.

3 Cross-linking stability detection

3.1 Mechanical stability test

Soak the petal leaves cross-linked with 10 mg/ml proanthocyanidins prepared by the present invention in D-Hanks solution, and replace the D-Hanks solution every day. After different immersion times, the leaflets were taken out, and the maximum tensile strength was measured according to the method described in 2.2.

3.2 Detection of resistance to enzymatic hydrolysis

Soak the leaflets cross-linked with different concentrations of proanthocyanidins prepared by the present invention in the type 2 collagenase solution prepared with D-Hanks solution at a concentration of 1000 U/ml, and enzymatically digest 1 , 3, 7 and 24 hours, measure the leaf weight (Wt) of each concentration of proanthocyanidins cross-linking, and calculate the percentage of reduction of the leaflets of each concentration of proanthocyanidins cross-linking relative to the original weight (W ₀ ) (i.e. enzymatic digestion Rate, ΔW%).

ΔW%＝(W ₀ -Wt)/W ₀ ×100

The larger the ΔW% value, the smaller the resistance to enzymatic hydrolysis.

3.3 Determination of release rate of crosslinking agent after crosslinking

Soak the lobes cross-linked with 10 mg/ml proanthocyanidins prepared by the present invention in D-Hanks solution, store them in 37°C and 5% _CO2 environment, replace the D-Hanks solution every day, and make the cross-linking agent proanthocyanidins freed. Measure the absorbance of the replaced D-Hanks solution containing the released proanthocyanidins with a spectrophotometer, and calculate the released concentration according to the standard curve. Specific method: Take 1ml of the proanthocyanidin sample to be tested, add 3ml of 12% vanillin methanol solution, and then add 6ml of 1.2M HCl methanol solution to mix. After 25 minutes, divide the mixed solution into 4 equal parts and measure the absorbance at a wavelength of 500nm.

4 Detection of cross-linking agents on proliferation potential and toxicity of valve interstitial cells

4.1 Valve mesenchymal cell culture

Take fresh calf heart aortic valve leaflets less than 24 hours old, scrape off the endothelial cells, cut into small pieces of ^1-2mm2 , and prepare type II with a concentration of 2mg/ml in serum-free M199 culture medium Collagenase solution was digested for 1 hour at 37°C and 120rpm shaking, and the obtained cells were cultured in M199 culture medium containing 20% fetal bovine serum at 37°C and 5% _CO2 , and the medium was changed every 2-3 days. Cells of passage 2-4 were used for experiments.

4.2 Detection of Proanthocyanidins' Inhibition of Cell Proliferation Potential and Cytotoxicity

The valvular interstitial cells were seeded in a 96-well plate at a density of 1000 cells per well, and after 1 day, they were replaced with the above-mentioned culture medium containing different concentrations of proanthocyanidins and cultured for 6 days, and the cell viability was detected by the MTT method. The specific method is that after the culture expires, the culture is washed, and 100 μl/well of serum-free cell culture medium containing 0.5 mg/ml MTT is added, and the culture is continued for 4 hours under normal conditions, the culture medium is discarded, and 100 μl of dimethyl ethylene The sulfone fully dissolves the purple substance, detects the absorbance value at a wavelength of 490nm on a spectrophotometer, and calculates the number of cells based on the standard curve. The higher the activity, the more the number of cells, and the smaller the inhibition of cell proliferation potential; the higher the concentration of proanthocyanidins to maintain the number of original cells, the lower the toxicity.

The present invention uses proanthocyanidin as a cross-linking agent to cross-link the decellularized heart valve to directly prepare a biological valve or prepare a tissue-engineered heart valve stent. Compared with the cross-linking effect of glutaraldehyde, it has mechanical properties, stability, durability and biocompatibility Excellent antibacterial properties, no toxicity, etc., plus the antibacterial, antiviral, anti-inflammatory, anticancer, and cardiovascular and cerebrovascular protection activities of proanthocyanidins themselves, the biological valve material directly prepared by the process technology of the present invention has excellent application prospects . Simultaneously, the process of the present invention is simple and easy to implement and popularize.

In summary, the bioprosthetic valve of the present invention, which has mechanical strength, anti-enzymolysis ability, cross-linking agent release speed, tissue regeneration promotion ability, etc. Has unique advantages.

Description of drawings

Fig. 1 is a scanning electron micrograph of a porcine heart aortic valve after being decellularized and cleaned by the method of the present invention.

Fig. 2 is an optical microscope photo of the decellularized valve cross-linked by the cross-linking agent proanthocyanidin used in the present invention.

Fig. 3 is the decellularized valve (concentration is 2.5mg/ml, 5mg/ml, 10mg/ml to 20mg/ml) crosslinked with glutaraldehyde (6.25mg/ml) crosslinked by crosslinking agent proanthocyanidin used in the present invention Comparison of maximum tensile strength of decellularized valves, non-crosslinked decellularized valves (0 mg/ml), and fresh valves. "*" in the figure means p<0.05, there is a significant difference between groups

Fig. 4 shows the change of the maximum tensile strength of the decellularized valve cross-linked by proanthocyanidins used in the present invention at a concentration of 10 mg/ml after soaking in D-Hanks solution for different days.

Fig. 5 is the decellularized valve crosslinked with different concentrations (2.5mg/ml, 5mg/ml, 10mg/ml and 20mg/ml) of proanthocyanidins used in the present invention and the decellularized valve (6.25mg/ml) crosslinked by glutaraldehyde , and non-crosslinked decellularized valves (0 mg/ml) at different times (1, 2, 4 and 24 hours) of the anti-collagenase degradation ability comparison graph.

Fig. 6 shows the release process of the proanthocyanidin cross-linking agent when the decellularized valve cross-linked with proanthocyanidin used in the present invention is soaked in D-Hanks solution for 45 days. The upper right corner of the figure is the proanthocyanidin absorbance-concentration standard curve.

Fig. 7 shows the procyanidin cross-linking agent used in the present invention to inhibit the proliferation potential of valve interstitial cells and the results of toxicity tests. "*" in the figure indicates that p<0.05, there is a significant difference between groups.

Figure 8 shows the decellularized valves in which proanthocyanidins used in the present invention are cross-linked for 240 hours by adding 20ml per valve, at a concentration of 0.0625mg/ml, at 4°C, and shaking at 120rpm

Figure 9 shows the decellularized valves of proanthocyanidins used in the present invention at a concentration of 5 mg/ml, added to each valve in 20 ml, 37° C., and shaken at 120 rpm for 0.5, 1, 3 and 7 hours (in sequence 2, 3, 4, 5, valve 1 in the figure is an uncrosslinked decellularized valve).

Detailed ways

The following specific examples are introduced to further illustrate the substantive characteristics and remarkable progress of the present invention, but the present invention is by no means limited to the examples.

Example 1

The preparation method of the decellularized porcine aortic valve is prepared according to the aforementioned method. Figure 1 shows that the porcine aortic valve cells and their components have been eluted, and the extracellular matrix remains intact. Add 2ml of D-Hanks solution to each valve to soak the valve, shake and wash at 240rpm at 37°C for 0.5 hours. Then add 10 mg/ml proanthocyanidin D-Hanks solution at a ratio of 2 ml to each valve for soaking, shake at 120 rpm, and cross-link at 37° C. for 48 hours. Add 2ml of D-Hanks solution to each valve to soak the valve, shake and wash at 37°C and 120rpm for 240 hours, and change the solution every 24 hours. The artificial biovalve valve material of the present invention that makes like this, do above-mentioned performance evaluation, among Fig. 2, 1 is the decellularized valve of 6.25mg/ml glutaraldehyde cross-linking, has obvious shrinkage, and among the figure 2, 3, 4 and 5 are decellularized valves cross-linked by the cross-linking agent proanthocyanidin used in the present invention at a concentration of 2.5 mg/ml, 5 mg/ml, 10 mg/ml to 20 mg/ml, which are soft and smooth without shrinkage after cross-linking. Figure 3 shows that the maximum tensile strength of proanthocyanidin-crosslinked acellular valve is 11MPa, which is better than that of glutaraldehyde-crosslinked decellularized valve and also better than that of non-crosslinked decellularized valve. Figure 4 shows that the maximum tensile strength of the decellularized valve after proanthocyanidin cross-linking is similar to that at the end of cross-linking when soaked in D-Hanks solution for 45 days, without decreasing. It shows that the cross-linking agent proanthocyanidin used in the present invention has good stability to the cross-linking of the decellularized valve. Fig. 5 shows that the decellularized valves cross-linked with different concentrations of proanthocyanidins and the acellular valves cross-linked with glutaraldehyde of the present invention have strong resistance to enzymatic hydrolysis. Compared with the non-crosslinked acellular valve, the degradation rate of the decellularized valve cross-linked by the two cross-linking agents was very low, indicating that the constituent molecules of the decellularized valve were cross-linked and modified by proanthocyanidin and glutaraldehyde, respectively. The enzymatic degradation rate of proanthocyanidins was only 6.94%, which indicated that proanthocyanidins had a good cross-linking effect and the effect was good. Figure 6 shows that the release of proanthocyanidins from the decellularized valves cross-linked by proanthocyanidins was mainly concentrated in the first 4 days, and the concentration decreased from 234.6 μg/ml to 3.2 μg/ml. This may be caused by a small amount of excess proanthocyanidins left over from the crosslinking process remaining after washing. After 4 days, the release amount was very small, which may be the reason for the good stability of mechanical strength after crosslinking. Figure 7 shows that the cross-linking agent proanthocyanidins used in the present invention does not inhibit the proliferation potential of valve interstitial cells when the concentration is below 125 μg/ml, and even promotes it. When the concentration is greater than 250 μg/ml, the number of cells is less than the initial inoculation number, It is toxic; and glutaraldehyde does not inhibit the proliferation potential of valve interstitial cells until the concentration is 0.0313 μg/ml, and it has been significantly inhibited when the concentration is 0.313 μg/ml. When the concentration is greater than 2.5 μg/ml, the number of cells Less than the initial inoculation number, showing toxicity. The results showed that proanthocyanidins inhibited the proliferation potential of valve interstitial cells by 40000 times less than glutaraldehyde (125:0.0313), and their toxicity was 100 times less (250:2.5). The release amount of the residual cross-linking agent proanthocyanidins within 4 days is below its toxicity range, without any toxicity.

Example 2

The preparation method of the decellularized porcine heart aortic valve was prepared according to the above method, adding 2ml of D-Hanks solution to each valve to soak the valve, and shaking and cleaning at 240rpm at 37°C for 0.5 hours. Then add 0.0625mg/ml proanthocyanidin D-Hanks solution at a ratio of 20ml to each valve for soaking, and cross-link at 4°C for 120 hours at a shaking speed of 120rpm. Then add 2ml of D-Hanks solution to each valve to soak the valve, shake and wash at 240rpm at 37°C for 24 hours. The artificial biological valve material of the present invention can be produced, its maximum tensile strength is 8.8MPa, the enzymatic degradation rate is 14.69%, and the cross-linking agent can basically be released within 1 day without any toxicity. Valves 1 and 2 in Figure 8 show no shrinkage after cross-linking, good stability, and can be stored in D-Hanks solution at 4°C or 37°C for at least 45 days.

Example 3:

The preparation method of the decellularized porcine heart aortic valve is prepared according to the above method, adding 8ml of D-Hanks solution to each valve to soak the valve, and cleaning at 37°C for 96 hours under the condition of standing still (0rpm). Then add 0.625 mg/ml proanthocyanidin D-Hanks solution to soak in 10 ml per valve, cross-link at 4° C. for 144 hours at a shaking speed of 120 rpm. Add 8ml of D-Hanks solution to each valve to soak the valve, and wash it manually at 4°C for 10 minutes. The bioprosthetic valve material of the present invention obtained in this way has a maximum tensile strength of 11.6 MPa, an enzymatic degradation rate of 9.24%, and a cross-linking agent that can be basically released within 1 day without any toxicity. Valves 3 and 4 in Fig. 8 show no shrinkage after cross-linking, good stability, and can be stored in D-Hanks solution at 4°C or 37°C for at least 45 days.

Example 4:

The preparation method of the decellularized porcine heart aortic valve was prepared and cleaned according to the method in Example 3. Then add 2ml of 10mg/ml proanthocyanidins in D-Hanks solution for each valve to soak, and cross-link at 37°C for 3 minutes at a shaking speed of 120rpm. Add 2ml of D-Hanks solution to each valve to soak the valve, shake and wash at 120rpm at 37°C for 1 hour. The artificial biological valve material of the present invention can also be produced, the maximum tensile strength is 9.4Mpa, the enzymatic degradation rate is 8.98%, and the cross-linking agent can be basically released within 1 day without any toxicity. The valve 6 in Fig. 8 shows that after the cross-linking is completed, it does not shrink, has good stability, and can be stored in D-Hanks solution at 4°C or 37°C for at least 45 days.

Example 5:

The preparation method of the decellularized porcine heart aortic valve was prepared and cleaned according to the method in Example 3. Then add 5 mg/ml proanthocyanidin D-Hanks solution at a ratio of 20 ml to each valve for soaking, and cross-link at 37° C. for 0.5, 1, 3 and 7 hours at a shaking speed of 120 rpm. Carry out cleaning after cross-linking by the method of embodiment 3 again. The artificial biological valve material of the present invention can also be produced, the maximum tensile strength is 9.4Mpa, the enzymatic degradation rate is 8.89%, and the cross-linking agent can be basically released within 1 day without any toxicity. Figure 9 shows that after the cross-linking is completed, there is no shrinkage, and the stability is good, and it can be stored in D-Hanks solution at 4°C or 37°C for at least 45 days.

Claims (8)

1. the preparation method of the artificial bio-prosthetic valve valve of a biologically active, comprise crosslinked pre-treatment, crosslinked and crosslinked post processing and preservation, it is characterized in that adopting procyanidin as the crosslinked cell cardiac valve that takes off of connection agent, its method is to be soaked in 1-20ml by every valve, containing procyanidin is the D-Hanks liquor of 0.01-20mg/ml, and it is crosslinked to do to take off the cell cardiac valve; Crosslinkedly take off the valvular temperature 1-40 of cell ℃, crosslinkedly finish under shaking table shakes condition, its speed of shaking is 10-300rpm, and crosslinking time is 1 minute-240 hours; Described D-Hanks is no Ca ⁺, Mg ⁺Ionic phosphate buffer solution, it consists of KCl 0.4g/L, KH ₂PO ₄40.06g/L, NaCl 8.00g/L, NaHCO ₃0.35g/L, Na ₂HPO ₄7H ₂O 0.09g/L, phenol red 0 or 0.01g/L.

2. press the preparation method of the artificial bio-prosthetic valve valve of the described biologically active of claim 1, it is characterized in that being soaked in the no Ca that procyanidin is 0.06-10mg/ml that contains of 1-10ml by every valve ⁺, Mg ⁺Ionic phosphate buffer solution.

3. press the preparation method of the artificial bio-prosthetic valve valve of the described biologically active of claim 1, it is characterized in that crosslinked pre-treatment comprises the collection of cardiac valve raw material and handles and pre-treatment with taking off cell, step is:

(a) clean:

Aortic valve or cusps of pulmonary valve are cut together with peripheral vessels in the pig heart that the slaughterhouse will just have been slaughtered, and place no Ca ²⁺, Mg ²⁺Take back in following 1 hour in 4 ℃ in the ionic phosphate buffer solution, and the clip valve leaflets, clean up with D-Hanks liquid;

(b) take off cell:

Taking off Cell sap and divide A liquid and B liquid, is basigamy system with D-Hanks liquid all.Valve takes off cell in two steps, first step A liquid, and second step was used B liquid; Each step all adds the ratio adding of 2-20ml in every valve; Take off Cell sap A liquid choosing any one kind of them from following three kinds of solution formulas:

Prescription 1 contains 0.05% or 0.1% trypsin, 0.02% disodiumedetate;

Prescription 2 contains 0.05% or 0.1% trypsin, 0.5% nonionic detergent Triton X-100,0.5% NaTDC and 0.02% disodiumedetate;

Prescription 3 contains 0.5% Triton X-100,0.5% NaTDC and 0.02% disodiumedetate;

Percent is mass number contained among every 100ml in the prescription 1,2 and 3.

Take off Cell sap B formula of liquid and be the nuclease of 20 μ g/ml of D-Hanks liquid preparation and the desoxyribose enzyme mixed liquor of 0.2mg/ml;

Cell free PROCESS FOR TREATMENT condition is that temperature is 1-40 ℃, and the shaking table speed of shaking is 0-300rpm, and air humidity is 30%-95%, CO ₂Concentration is 0.3%-5%, and the time is 10 minutes-100 hours.

4. press the preparation method of the artificial bio-prosthetic valve valve of claim 1 or 3 described biologically actives, it is characterized in that taking off cell PROCESS FOR TREATMENT temperature is 20-37 ℃, and shaking table shakes temperature 120-240rpm, and air themperature is 50-95%, and the time is 1-48 hour.

5. press the preparation method of the artificial bio-prosthetic valve valve of the described biologically active of claim 1, it is characterized in that the crosslinked solution soaking valve that adds the ratio adding D-Hanks of 2-20ml later with every valve, cleaned 0-480 hour, cleaning is to finish under 0-300rpm shakes condition at 0-40 ℃, shaking table.

6. press the preparation method of the artificial bio-prosthetic valve valve of claim 1 or 5 described biologically actives, it is characterized in that crosslinked back scavenging period is 5 minutes-1 hour, cleaning temperature 20-37 ℃, shaking table speed is 60-120rpm.

7. by the preparation method of the artificial bio-prosthetic valve valve of the described biologically active of claim 1, it is characterized in that cleaning in the D-Hanks solution that the back valve is kept at 1-10 ℃.

8. by the preparation method of the artificial bio-prosthetic valve valve of claim 1 or 7 described biologically actives, it is characterized in that cleaning in the D-Hanks solution that the back valve is kept at 4 ℃.

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