WO2024078253A1 - Procédé d'anti-calcification pour bioprothèse tissulaire, et bioprothèse tissulaire - Google Patents

Procédé d'anti-calcification pour bioprothèse tissulaire, et bioprothèse tissulaire Download PDF

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Publication number
WO2024078253A1
WO2024078253A1 PCT/CN2023/118847 CN2023118847W WO2024078253A1 WO 2024078253 A1 WO2024078253 A1 WO 2024078253A1 CN 2023118847 W CN2023118847 W CN 2023118847W WO 2024078253 A1 WO2024078253 A1 WO 2024078253A1
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Prior art keywords
reducing agent
bioprosthetic tissue
calcification
tissue
solution
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PCT/CN2023/118847
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English (en)
Chinese (zh)
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于世河
何海红
刘世红
陈国明
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上海微创心通医疗科技有限公司
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Publication of WO2024078253A1 publication Critical patent/WO2024078253A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials

Definitions

  • the present invention relates to the technical field of medical equipment, and in particular to an anti-calcification method of a biological prosthetic tissue and the biological prosthetic tissue.
  • animal-derived collagen fiber tissue materials commonly used in clinical bioprostheses such as bovine pericardium and small intestinal submucosa
  • chemical reagents such as glutaraldehyde and/or formaldehyde.
  • fixation methods usually result in residual aldehyde groups in bioprosthetic tissues, and a large number of studies have shown that bioprosthetic tissues fixed with formaldehyde/glutaraldehyde will cause calcification in vivo after implantation.
  • the anti-calcification treatment methods for bioprosthetic tissues reported in the prior art include: decellularization treatment, alcohol treatment, surfactant treatment, metal ion competition and other methods. Although the above methods have certain anti-calcification effects, they are not ideal in practical applications.
  • the decellularization method can remove most of the antigens and DNA in biological tissues, but it will make the biological tissues loose, resulting in a decrease in the mechanical properties of the bioprosthetic tissues.
  • the alcohol treatment method has a good short-term anti-calcification effect, but it cannot remove the residual aldehyde groups on the bioprosthetic tissues. Due to the presence of residual aldehyde groups, the long-term calcification risk of the bioprosthetic tissues is still very high.
  • the surfactant treatment method can remove phospholipids in the bioprosthetic tissues and have a certain anti-calcification effect, but the aldehyde group still exists as a calcification inducing factor, resulting in poor anti-calcification effect of the bioprosthetic tissues.
  • the metal ion competition method can combine with the existing binding sites of the bioprosthetic tissues to have a certain anti-calcification effect, but it also cannot completely solve the induction of long-term calcification of aldehyde groups.
  • the prior art discloses a method for treating biological prosthetic tissue with 2-aminooleic acid to prevent calcification of biological prosthetic tissue.
  • the bovine pericardium fixed with glutaraldehyde is placed in a sodium borate buffer solution (pH11) containing sodium 2-aminooleate and incubated overnight at standard temperature.
  • a sodium borate buffer solution pH11
  • In vivo implantation shows that the treated tissue has a significant effect on improving calcification compared with the untreated tissue.
  • Gott JP et al. conducted a valve replacement study using a young sheep model and found that while this method improves valve calcification, it is easy to cause tissue loosening, pericardial hairy hematoma, and other conditions.
  • the prior art also discloses a method for reducing calcification by capping bioprosthetic tissue.
  • the bioprosthetic tissue fixed with glutaraldehyde is rinsed with 20% ethanol, exposed to a capping solution containing ethanolamine and sodium borohydride at room temperature for 4 hours, and the excess capping solution is rinsed off.
  • the results of small animal model implantation show that compared with heat-fixed bioprosthetic tissue, capping can reduce the in vivo calcification of bioprosthetic tissue; however, the capping reagent used in this method is strongly alkaline. Under strong alkaline conditions, the reducing agent sodium borohydride is very likely to produce gas, which will fill the bioprosthetic tissue, destroy the internal fiber structure of the tissue, and reduce the tissue performance.
  • the prior art also discloses a double cross-linking process that can improve the anti-calcification effect of bioprosthetic tissue.
  • the bioprosthetic tissue is heat-fixed with glutaraldehyde, the fixed bioprosthetic tissue is cross-linked with Jeffamine and reduced with sodium borohydride, and then the bioprosthetic tissue is treated with a surfactant to preserve and sterilize the tissue.
  • the results of small animal model implantation show that compared with heat-fixed bioprosthetic tissue, the double cross-linking process can reduce the in vivo calcification of bioprosthetic tissue; however, the bioprosthetic tissue treated by this process is also strongly alkaline. Under strong alkaline conditions, the reducing agent sodium borohydride is very likely to produce gas, which will fill the bioprosthetic tissue, destroying the internal fiber structure of the tissue and reducing the tissue performance.
  • the object of the present invention is to provide an anti-calcification method for bioprosthetic tissue and bioprosthetic tissue, so as to solve the problems in the prior art that the anti-calcification method has an unsatisfactory anti-calcification effect and the tissue performance is reduced after the anti-calcification treatment.
  • the present invention provides an anti-calcification method for a bioprosthetic tissue, comprising:
  • the bioprosthetic tissue that has been at least partially cross-linked and fixed in a degreasing solution for degreasing treatment, wherein the degreasing solution includes an alcohol solution containing a surfactant;
  • the defatted bioprosthesis tissue is placed in a solution containing a reducing agent for reduction treatment.
  • the anti-calcification method of the bioprosthetic tissue comprises:
  • step S2 placing the bioprosthesis tissue cleaned in step S1 into a degreasing solution for degreasing treatment
  • step S3 placing the bioprosthesis tissue after the defatting treatment in step S2 into a solution containing a reducing agent for reduction treatment;
  • step S4 Cleaning the bioprosthesis tissue after the reduction treatment in step S3.
  • tissue source of the bioprosthetic tissue is selected from bovine pericardium, porcine pericardium, porcine tissue valve, blood vessel, skin, small intestinal submucosa, meninges, bladder lining, ligament or tendon.
  • the cleaning solution used to clean the bioprosthetic tissue in step S1 is sterile physiological saline or phosphate buffer solution, wherein the concentration of phosphate in the phosphate buffer solution may be 0.05-0.2 mol/L, and the pH value may be 6.8-8.6.
  • the mass concentration of the surfactant is 0.1%-10%.
  • the mass concentration of alcohol is 10%-90%.
  • the degreasing solution is a phosphate buffer solution containing a surfactant and an alcohol, the concentration of the phosphate buffer solution is 0.05-0.2 mol/L, and the pH value of the solution is 6-8.
  • step S2 after the bioprosthesis tissue is degreased, a phosphate buffer solution with a pH value of 6.8-8.6 and a concentration of 0.05-0.2 mol/L or sterile physiological saline is used to clean the degreased bioprosthesis tissue.
  • the alcohol is selected from one or a combination of methanol, ethanol, isopropanol, ethylene glycol, propylene glycol, glycerol and sorbitol.
  • the surfactant is selected from one or a combination of non-ionic surfactants, ionic surfactants and zwitterionic surfactants.
  • nonionic surfactant is selected from one or a combination of TritonX-100, Tween, glucosides, methylglucosamine, N-dodecylglucosamine, glycosyllithocholic acid ester amphiphilic molecules, glycoenzymes, digitonin, alkylglucosamide and cocoyl monoethanolamide;
  • the ionic surfactant is selected from one or a combination of sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, sodium deoxycholate, sodium cholate, sarcosine, sodium dodecyl polyoxyethylene ether sulfate and glucose lipid quaternary ammonium salt;
  • the zwitterionic surfactant is selected from 3-(3-(cholamidopropyl)dimethylamino)propanesulfonic acid inner salt, One or a combination of sulfobetaines, carboxylic acid betaines, oleyl sulfate ester type imidazoline, dodecylaminopropionic acid and amino acid surfactants.
  • the mass concentration of the reducing agent in the solution containing the reducing agent is 0.01%-10%.
  • the mass concentration of the reducing agent in the solution containing the reducing agent is 0.02%-5%.
  • the mass concentration of the reducing agent in the solution containing the reducing agent is 0.02%-2%.
  • the solution containing the reducing agent is a phosphate buffer containing the reducing agent
  • the concentration of the phosphate buffer is greater than or equal to 0.05 mol/L
  • the pH value is 3.8-10.0.
  • the solution containing the reducing agent is a phosphate buffer containing the reducing agent
  • the concentration of the phosphate buffer is 0.05-0.5 mol/L
  • the pH value is 6.8-8.6.
  • the reduction treatment is performed once or multiple times.
  • step S3 when the bioprosthetic tissue is subjected to reduction treatment in step S3, the reduction treatment is performed 2-6 times.
  • the reducing agent is selected from borohydrides and/or boranes.
  • the reducing agent is selected from one or a combination of sodium borohydride, sodium cyanoborohydride, sodium triacetoxyborohydride, zinc borohydride, sodium triacetylborohydride, lithium borohydride, diborane, dimethylaminoborane and tert-butylamine borane.
  • the reducing agent is selected from reducing metals and/or hydrides thereof.
  • the reducing agent is selected from one or a combination of sodium aluminum hydride, dimethoxyethoxyaluminum hydride, diisobutylaluminum hydride, zinc powder and magnesium powder.
  • the reducing agent is selected from a sulfur-containing reducing agent or triethylsilane.
  • the sulfur-containing reducing agent is selected from sodium dithionite and/or sodium bisulfite.
  • step S4 the bioprosthetic tissue after reduction treatment is cleaned with a phosphate buffer solution or an alcohol solution, wherein the pH value of the phosphate buffer solution is 6.8-8.6, the concentration of phosphate can be 0.05-0.2 mol/L, and the volume fraction of alcohol in the alcohol solution can be 10%-50%.
  • the present invention also provides a bioprosthetic tissue, using the bioprosthetic tissue as described above. It is prepared by calcification method.
  • the anti-calcification method of bioprosthetic tissue provided by the present invention has the following advantages:
  • the present invention innovatively adopts a degreasing process combined with a reducing agent reduction method to perform anti-calcification treatment on biological prosthetic tissues, wherein the surfactant used in the degreasing process has lipophilic and hydrophilic properties, alcohol can dissolve small molecular lipids such as phospholipids in the material, and ultimately better removes the lipids contained in the biological material, and the reduction process can reduce the Schiff bases and residual aldehyde groups generated in the fixation process, play a role in stabilizing chemical bonds, are not easy to hydrolyze, and are safer to implant in the body.
  • the combination of the two-step method significantly reduces the phospholipid content and residual aldehyde groups of the final material, has a better anti-calcification effect, and the two-step reactions are independent of each other and easy to implement in terms of process.
  • the selected degreasing reagent has good water solubility, is easy to penetrate, can effectively dissolve phospholipids in tissues, has the advantages of good anti-calcification effect, and has little damage to tissues.
  • the reduction treatment in the present invention uses a low concentration of reducing agent, and the bioprosthetic tissue can be subjected to multiple reduction treatments.
  • the concentration of the reducing agent is reduced (the reducing agent is relatively active and easily generates gas), which is beneficial to reducing the gas generation rate, thereby protecting the structure of the bioprosthetic tissue from being damaged by the gas, with little impact and damage to the structure of the bioprosthetic tissue, making the reduction treatment process more gentle; on the other hand, performing multiple reduction treatments also ensures the reliability of the effect of the reduction treatment.
  • the anti-calcification treatment method of the present invention has strong process adaptability and little effect on the performance of bioprosthetic tissue.
  • the treated bioprosthetic tissue has a wider range of applications and can be dehydrated and dried, EO sterilized, or liquid stored and liquid sterilized.
  • the tissue can be used to manufacture dry films.
  • FIG1 is a schematic flow chart of an anti-calcification method for a bioprosthetic tissue provided by an embodiment of the present invention
  • FIG. 2 is a comparison of the experimental results of calcification performance evaluation of each embodiment group and a control group in the embodiments of the present invention.
  • the prior art adds biomaterials to an anti-calcification solution for treatment, and the reaction conditions are complex, which affects the performance of the obtained biomaterials.
  • the purpose of the present invention is to provide an anti-calcification method for bioprosthetic tissues to solve the problems that the anti-calcification effect of the anti-calcification method in the prior art is not ideal and the tissue performance is reduced after the anti-calcification treatment.
  • the anti-calcification treatment method adopted by the present invention can protect the residual aldehyde groups of the biomaterials and is easy to implement in terms of technology.
  • the present invention provides an anti-calcification method for bioprosthetic tissue, the steps of which mainly include: placing the bioprosthetic tissue that has been at least partially cross-linked and fixed in a degreasing solution for degreasing treatment, wherein the degreasing solution includes an alcohol solution containing a surfactant; and then placing the degreased bioprosthetic tissue in a solution containing a reducing agent for reduction treatment.
  • a two-step treatment method wherein the surfactant used in the degreasing process has lipophilic and hydrophilic properties, the alcohol can dissolve small molecular lipids such as phospholipids in the material, and finally the lipids contained in the biological material can be better removed, and the reduction process can reduce the Schiff base and residual aldehyde groups generated in the fixation process, which plays a role in stabilizing chemical bonds, and finally the phospholipid content and residual aldehyde groups of the material are reduced, which has a better anti-calcification effect and is easy to achieve in terms of process.
  • the anti-calcification method of a bioprosthetic tissue may further include the following steps:
  • step S2 placing the bioprosthesis tissue cleaned in step S1 into a degreasing solution for degreasing treatment
  • step S3 placing the bioprosthesis tissue after the defatting treatment in step S2 into a solution containing a reducing agent for reduction treatment;
  • step S4 Cleaning the bioprosthesis tissue after the reduction treatment in step S3.
  • the tissue source of the bioprosthetic tissue can be bovine pericardium, porcine pericardium, porcine tissue valve, blood vessel, skin, small intestinal submucosa, meninges, bladder lining, ligaments and tendons, etc.
  • the cross-linked fixed bioprosthetic tissue can be fixed with glutaraldehyde commonly used in the industry.
  • the fixed bioprosthetic tissue is cleaned with a cleaning solution to fully clean the free glutaraldehyde in the bioprosthetic tissue.
  • sterile physiological saline or PBS buffer (phosphate buffer solution) with a pH value of 6.8-8.6 and a concentration of 0.05-0.2 mol/L can be used for cleaning.
  • PBS buffer phosphate buffer solution
  • concentration of 0.05-0.2 mol/L can be used for cleaning.
  • it can be repeatedly cleaned 3-5 times at room temperature, and each cleaning is 3-5 minutes to fully clean the free glutaraldehyde or formaldehyde in the bioprosthetic tissue to ensure the
  • the purpose of immersing the cleaned bioprosthetic tissue in a degreasing solution for degreasing is to dissolve the oil and phospholipids in the bioprosthetic tissue, thereby reducing the phospholipid content, thereby achieving the anti-calcification effect of the bioprosthetic tissue.
  • the degreasing solution of the present invention can be an alcohol solution containing a surfactant, and can be configured with a phosphate buffer, wherein the mass concentration of the surfactant can be 0.1%-10% (W/V), the mass concentration of the alcohol is 10%-90% (W/V), the concentration of the phosphate buffer is 0.05-0.2 mol/L, and the pH value of the solution is 6-8.
  • the reaction container When the bioprosthetic tissue is immersed in the degreasing solution for degreasing, the reaction container can be placed in a constant temperature shaking shaker, the temperature is set to 15-50°C, the rotation speed is 50-200rpm, and the constant temperature oscillation is 2-96h.
  • the degreasing bioprosthetic tissue can be cleaned before proceeding to the subsequent steps.
  • a phosphate buffer solution with a pH value of 6.8-8.6 and a concentration of 0.05-0.2 mol/L or sterile saline can be used for cleaning 3-5 times, each time for 3-5 minutes, to clean off the degreasing solution remaining on the tissue.
  • the alcohol in the above-mentioned degreasing solution can be selected from monohydric alcohol and/or polyhydric alcohol, specifically can be selected from one or a combination of methanol, ethanol, isopropanol, ethylene glycol, propylene glycol, glycerol and sorbitol.
  • the surfactant in the degreasing solution can be selected from one or more combinations of non-ionic surfactants, ionic surfactants and zwitterionic surfactants.
  • the non-ionic surfactant is selected from one or more combinations of TritonX-100, Tween, glucosides, methylglucosamine, N-dodecylglucosamine, glycosyllithocholic acid ester amphiphilic molecules (GLC-1, GLC-2 and GLC-3), glycoenzyme (GDN), digitonin, alkylglucosamide and cocoyl monoethanolamide
  • the ionic surfactant is selected from one or more combinations of TritonX-100, Tween, glucosides, methylglucosamine, N-dodecylglucosamine, glycosyllithocholic acid ester amphiphilic molecules (GLC-1, GLC-2 and GLC-3), glycoenzyme (GDN), digitonin, alkylglu
  • the next step is to reduce the bioprosthetic tissue using a reducing agent, and reduce the Schiff base and residual aldehyde groups, carboxyl groups, etc. generated during cross-linking fixation using the reducing agent, thereby stabilizing the chemical bonds.
  • the reducing agent can be selected from borohydrides and/or boranes, specifically selected from one or more combinations of sodium borohydride, sodium cyanoborohydride, sodium triacetoxyborohydride, zinc borohydride, sodium triacetylborohydride, lithium borohydride, diborane (borane), dimethylaminoborane and tert-butylamine borane.
  • the reducing agent can also be selected from reducing metals and/or their hydrides, specifically, can be selected from one or a combination of sodium aluminum hydride, dimethoxyethoxyaluminum hydride, diisobutylaluminum hydride, zinc powder and magnesium powder.
  • the reducing agent in step S3 of the present invention can also be selected from a sulfur-containing reducing agent or triethylsilane.
  • the sulfur-containing reducing agent can be selected from sodium dithionite and/or sodium bisulfite.
  • the mass concentration of the reducing agent is 0.01%-10% (W/V), preferably 0.02%-5%, and more preferably 0.02%-2%.
  • the present invention preferably uses a low concentration of reducing agent to perform reduction treatment on the bioprosthesis, which can avoid the high concentration of reducing agent that easily generates gas, prevent the gas from filling the bioprosthesis tissue, destroy the internal fiber structure of the tissue, and reduce the tissue performance.
  • the solution containing the reducing agent of the present invention is configured with a phosphate buffer, which can further reduce the generation of reducing agent gas, and the concentration of the phosphate buffer is greater than or equal to 0.05 mol/L, and the pH value is 3.8-10.0.
  • the concentration of the phosphate buffer is 0.05-0.5 mol/L, and the pH value is preferably 6.8-8.6.
  • the present invention uses a relatively low concentration of reducing agent to reduce the bioprosthetic tissue, Original, reducing the generation of reducing agent gas, in order to achieve the effect of fully reducing the Schiff base and residual aldehyde groups, carboxyl groups, etc. in the bioprosthetic tissue, the present invention performs one or more reduction treatments when using the reducing agent solution to perform reduction treatment on the bioprosthetic tissue. That is, after completing a reduction treatment of the bioprosthetic tissue using the reducing agent solution, a new reducing agent solution is replaced, and the bioprosthetic tissue is repeatedly reduced once again, and this is repeated multiple times, and multiple reduction treatments are performed to achieve full reduction of the bioprosthetic tissue.
  • the present invention performs 2-6 reduction treatments when using the reducing agent solution to perform reduction treatment on the bioprosthetic tissue, that is, in the above-mentioned constant temperature oscillation experiment, a new reducing agent solvent is replaced and oscillated 2-6 times under the same conditions.
  • step S4 is performed to clean the bioprosthetic tissue.
  • a phosphate buffer solution with a pH value of 6.8-8.6 and a concentration of 0.05-0.2 mol/L or an alcohol solution with a volume fraction of 10%-50% can be used for cleaning 3-5 times, each time for 3-5 minutes, to clean off the reducing agent remaining on the tissue.
  • the bioprosthetic tissue after the bioprosthetic tissue is cleaned, it can be stored or subsequently processed, such as dried, sterilized, etc.
  • the processed bioprosthetic tissue can also be used to manufacture a dry film.
  • the present invention also provides a biological prosthetic tissue, which is prepared by being treated with the anti-calcification method of the biological prosthetic tissue as described above.
  • the anti-calcification method of bioprosthetic tissue provided in this embodiment 1 comprises the following steps:
  • Degreasing treatment The cleaned bovine pericardial tissue was placed in a degreasing solution for degreasing treatment.
  • the degreasing solution was a 0.1 mol/L PBS buffer solution containing 20% (W/V) isopropanol and 0.5% (W/V) sodium dodecyl sulfate, with a pH value of 7.40 ⁇ 0.1.
  • the reaction container was placed on a constant temperature shaker and the speed was set at 60 rpm, constant temperature shaking at 25°C for 24 h, and then washed 5 times with 0.1 mol/L sterile PBS buffer solution, each washing for 5 min;
  • the defatted bovine pericardial tissue was placed in a reducing agent solution.
  • the reducing agent solvent was a 0.1 mol/L PBS buffer solution containing 0.1% (W/V) tert-butylamine borane, with a pH value of 7.2.
  • the reaction container was placed on a constant temperature shaker, the speed was set to 60 rpm, and the shake was carried out at room temperature for 4 hours.
  • the reduced bovine pericardial tissue is cleaned with a 20% ethanol solution for 5 times, each time for 5 minutes, to obtain the anti-calcification treated bovine pericardial tissue.
  • the anti-calcification method of bioprosthetic tissue provided in this embodiment 2 comprises the following steps:
  • Degreasing treatment The cleaned bovine pericardial tissue was placed in a degreasing solution for degreasing treatment.
  • the degreasing solution was a 0.2 mol/L PBS buffer solution containing 60% (V/V) ethylene glycol and 2.0% (W/V) Tween, with a pH value of 7.2 ⁇ 0.1.
  • the reaction container was placed on a constant temperature shaker, set to a speed of 60 rpm, and kept at 25°C for 24 hours. After completion, it was washed 5 times with a 0.05 mol/L sterile PBS buffer solution, each washing for 5 minutes;
  • the defatted bovine pericardial tissue was placed in a reducing agent solution.
  • the reducing agent solvent was a 0.05 mol/L PBS buffer solution containing 0.05% (W/V) sodium dithionite, with a pH value of 4.6.
  • the reaction container was placed on a constant temperature shaker, the speed was set to 60 rpm, and the shake was carried out at room temperature for 4 hours.
  • the reduced bovine pericardial tissue is cleaned with sterile saline for 5 times, each time for 5 minutes, to obtain the anti-calcification treated bovine pericardial tissue.
  • the anti-calcification method of bioprosthetic tissue provided in this embodiment 3 comprises the following steps:
  • Degreasing treatment The cleaned bovine pericardial tissue was placed in a degreasing solution for degreasing treatment.
  • the degreasing solution was a 0.1 mol/L PBS buffer solution containing 30% (V/V) ethanol, 20% (V/V) glycerol and 5.0% (W/V) sodium dodecyl sulfate, with a pH value of 8.0 ⁇ 0.1.
  • the reaction container was placed on a constant temperature shaker, set to a speed of 60 rpm, and kept at 25°C for 24 hours. After completion, it was washed 5 times with a 0.2 mol/L sterile PBS buffer solution, each washing for 5 minutes;
  • the defatted bovine pericardial tissue was placed in a reducing agent solution.
  • the reducing agent solvent was a 0.3 mol/L PBS buffer solution containing 0.05% (W/V) triethylsilane, with a pH value of 7.8.
  • the reaction container was placed on a constant temperature shaker, the speed was set to 60 rpm, and the shake was carried out at room temperature for 4 hours.
  • the reduced bovine pericardial tissue is cleaned and washed 5 times with a 20% ethanol solution, each time for 5 minutes, to obtain the anti-calcification treated bovine pericardial tissue.
  • the anti-calcification method of bioprosthetic tissue provided in this embodiment 4 comprises the following steps:
  • Degreasing treatment The cleaned bovine pericardial tissue was placed in a degreasing solution for degreasing treatment.
  • the degreasing solution was a 0.1 mol/L PBS buffer solution containing 50% (V/V) ethanol and 10.0% (W/V) sodium deoxycholate, with a pH value of 7.6 ⁇ 0.1.
  • the reaction container was placed on a constant temperature shaker, set to a speed of 60 rpm, and kept at 25°C for 24 hours. After completion, it was washed 5 times with a 0.1 mol/L sterile PBS buffer solution, each washing for 5 minutes;
  • Reduction treatment The defatted bovine pericardial tissue was placed in a reducing agent solution, the reducing agent solvent being a 0.5 mol/L PBS buffer solution containing 0.05% (W/V) sodium triacetylborohydride, pH The value was 8.6, the reaction container was placed on a constant temperature shaker, the speed was set to 60 rpm, and the shaking was carried out at room temperature for 4 hours.
  • the reducing agent solvent being a 0.5 mol/L PBS buffer solution containing 0.05% (W/V) sodium triacetylborohydride, pH The value was 8.6
  • the reaction container was placed on a constant temperature shaker, the speed was set to 60 rpm, and the shaking was carried out at room temperature for 4 hours.
  • the reduced bovine pericardial tissue is cleaned and washed 5 times with a 20% ethanol solution, each time for 5 minutes, to obtain the anti-calcification treated bovine pericardial tissue.
  • the anti-calcification method of bioprosthetic tissue provided in this embodiment 5 comprises the following steps:
  • Degreasing treatment The cleaned bovine pericardial tissue was placed in a degreasing solution for degreasing treatment.
  • the degreasing solution was a 0.1 mol/L PBS buffer solution containing 60% (V/V) ethanol and 4.0% (W/V) carboxylic acid betaine, with a pH value of 7.40 ⁇ 0.1.
  • the reaction container was placed on a constant temperature shaker, set to a speed of 60 rpm, and kept at 25°C for 24 hours. After completion, it was washed 5 times with a 0.1 mol/L sterile PBS buffer solution, each washing for 5 minutes;
  • the defatted bovine pericardial tissue was placed in a reducing agent solution.
  • the reducing agent solvent was a 0.1 mol/L PBS buffer solution containing 0.05% (W/V) diisobutylaluminum hydride, with a pH value of 7.4.
  • the reaction container was placed on a constant temperature shaker, the speed was set to 60 rpm, and the shake was carried out at room temperature for 4 hours.
  • the reduced bovine pericardial tissue is cleaned with sterile saline for 5 times, each time for 5 minutes, to obtain the anti-calcification treated bovine pericardial tissue.
  • the anti-calcification method of bioprosthetic tissue provided in this embodiment 6 comprises the following steps:
  • Degreasing treatment Put the cleaned bovine pericardial tissue into the degreasing solution for degreasing treatment.
  • the lipid solution is: 0.1 mol/L PBS buffer solution containing 70% (V/V) sorbitol and 3.0% (W/V) carboxylic acid betaine, with a pH value of 8.5 ⁇ 0.1.
  • the reaction container is placed on a constant temperature shaker, set to a speed of 60 rpm, and shaken at a constant temperature of 25°C for 24 hours. After completion, it is washed 5 times with 0.2 mol/L sterile PBS buffer solution, each washing for 5 minutes;
  • the defatted bovine pericardial tissue was placed in a reducing agent solution.
  • the reducing agent solvent was a 0.5 mol/L PBS buffer solution containing 0.05% (W/V) sodium cyanoborohydride, with a pH value of 9.6.
  • the reaction container was placed on a constant temperature shaker, the speed was set to 60 rpm, and the shaking was carried out at room temperature for 4 hours.
  • the reduced bovine pericardial tissue is cleaned with sterile physiological saline for 5 times, each time for 5 minutes, to obtain the anti-calcification treated bovine pericardial tissue.
  • the anti-calcification method of bioprosthetic tissue provided in this embodiment 7 comprises the following steps:
  • Degreasing treatment The cleaned bovine pericardial tissue was placed in a degreasing solution for degreasing treatment.
  • the degreasing solution was a 0.1 mol/L PBS buffer solution containing 50% (V/V) ethanol, 1.0% (W/V) Tween and 1% (W/V) sodium dodecyl sulfate, with a pH value of 7.40 ⁇ 0.1.
  • the reaction container was placed on a constant temperature shaker, set to a speed of 60 rpm, and kept at 25°C for 24 hours. After completion, it was washed 5 times with a 0.1 mol/L sterile PBS buffer solution, each washing for 5 minutes;
  • the defatted bovine pericardial tissue was placed in a reducing agent solution.
  • the reducing agent solvent was a 0.1 mol/L PBS buffer solution containing 5% (W/V) tert-butylamine borane, with a pH value of 7.4.
  • the reaction container was placed on a constant temperature shaker, the speed was set to 80 rpm, and the shake was carried out at room temperature for 10 hours.
  • the reduced bovine pericardial tissue is cleaned with sterile saline for 5 times, each time for 5 minutes, to obtain the anti-calcification treated bovine pericardial tissue.
  • the anti-calcification method of bioprosthetic tissue provided in this embodiment 8 comprises the following steps:
  • Degreasing treatment The cleaned bovine pericardial tissue was placed in a degreasing solution for degreasing treatment.
  • the degreasing solution was a 0.1 mol/L PBS buffer solution containing 50% (V/V) ethanol, 2.0% (W/V) Tween, 1% (W/V) sodium dodecyl polyoxyethylene ether sulfate, and 1.5% dodecyl aminopropionic acid, with a pH value of 7.40 ⁇ 0.1.
  • the reaction container was placed on a constant temperature shaker, set to a speed of 60 rpm, and kept at 25°C for 24 hours. After completion, it was washed 5 times with a 0.1 mol/L sterile PBS buffer solution, each washing for 5 minutes;
  • the defatted bovine pericardial tissue was placed in a reducing agent solution.
  • the reducing agent solvent was a 0.3 mol/L PBS buffer solution containing 8% (W/V) diisobutylaluminum hydride, with a pH value of 8.1.
  • the reaction container was placed on a constant temperature shaker, the speed was set to 100 rpm, and the shake was carried out at room temperature for 28 hours.
  • the reduced bovine pericardial tissue is cleaned with sterile saline for 5 times, each time for 5 minutes, to obtain the anti-calcification treated bovine pericardial tissue.
  • the present invention also sets up three control group experiments, and the control group and the bioprosthetic tissues obtained by the scheme of the embodiments of the present invention are tested for performance indicators.
  • the specific technical schemes of the three control groups are as follows.
  • Liquid sterilization can be achieved by storing the bovine pericardium in glutaraldehyde storage solution.
  • the treatment plan for the bioprosthetic tissue in control group 2 was as follows:
  • Degreasing treatment The cleaned bovine pericardial tissue is placed in a degreasing solution for degreasing treatment.
  • the degreasing solution is: a 0.1 mol/L PBS buffer solution containing 60% (V/V) ethylene glycol and 2.0% (W/V) Tween, with a pH value of 7.40 ⁇ 0.1.
  • the reaction container is placed on a constant temperature shaker, set the speed to 60 rpm, and shake at a constant temperature of 25°C for 24 hours. After completion, it is washed 5 times with 0.1 mol/L sterile PBS buffer solution, each washing for 5 minutes.
  • the treatment plan for the bioprosthetic tissue in the control group 3 was as follows:
  • Reduction treatment Prepare 1L of 0.1mol/L PBS buffer solution containing 0.44% (W/V) sodium borohydride, put the washed cross-linked and fixed bovine pericardial tissue into the prepared reducing agent solution, put the reaction container on a constant temperature shaker, set the speed to 60rpm, and shake at room temperature for 4h. Wash the reduced bovine pericardial tissue, wash it 5 times with 0.1mol/L sterile PBS buffer solution, each time for 5min, to obtain the treated bovine pericardial tissue.
  • bioprosthetic tissues obtained in Examples 1-8 of the present invention and Control Groups 1-3 were tested for performance indicators, and comparative tests were performed from two aspects: mechanical stretching and calcification performance evaluation.
  • the bovine pericardial tissues obtained from the treatment of each embodiment group and the control group were taken to prepare pericardial samples.
  • the length of each sample was 50 mm and the width was 5 mm.
  • the test gauge length was set to 25 mm and the tensile rate was set to 100 mm/min. The tensile test was performed until the sample broke and the test time was recorded.
  • the mechanical tensile comparison is shown in Table 1 below.
  • Examples 1-8 of the present invention adopt a two-step treatment method, including a degreasing step and a reduction step, and the reaction conditions of the two steps can be controlled separately.
  • the mechanical properties of the pericardial material obtained are good, which are equivalent to the mechanical properties of the pericardial material obtained by the traditional process treatment method.
  • the two-step reactions are independent of each other, which is easier to implement in terms of process.
  • the calcification performance evaluation experiment selected a rat subcutaneous implantation animal model, and the tissue samples obtained from the control group and the example group were implanted into 3-week-old Wistar rats that had just been weaned using conventional surgical methods.
  • the tissue samples were taken out 8 weeks after implantation and dried in a constant temperature oven at 80°C for 48 hours to constant weight.
  • the dried samples were tested for calcium content using a flame-atomic absorption spectrophotometer, and the test results are shown in Figure 2.
  • the anti-calcification method of bioprosthetic tissue provided by the present invention has the following advantages:
  • the present invention innovatively adopts a degreasing process combined with a reducing agent reduction method to carry out anti-calcification treatment on biological prosthetic tissues, wherein the surfactant used in the degreasing process has lipophilic and hydrophilic properties.
  • Alcohol can dissolve small molecule lipids such as phospholipids in the material, and ultimately better remove the lipids contained in the biomaterial.
  • the reduction process can reduce the Schiff bases and residual aldehyde groups produced in the fixation process, which plays a role in stabilizing chemical bonds. It is not easy to hydrolyze and is safer to implant in the body.
  • the combination of the two-step method significantly reduces the phospholipid content and residual aldehyde groups in the final material, has a better anti-calcification effect, and the two-step reactions are independent of each other, which is easy to implement in terms of process.
  • the selected degreasing reagent has good water solubility, is easy to penetrate, can effectively dissolve phospholipids in tissues, has the advantages of good anti-calcification effect, and has little damage to tissues.
  • the reduction treatment in the present invention uses a low concentration of reducing agent, and the bioprosthetic tissue can be subjected to multiple reduction treatments.
  • the concentration of the reducing agent is reduced (the reducing agent is relatively active and easily generates gas), which is beneficial to reducing the gas generation rate, thereby protecting the structure of the bioprosthetic tissue from being damaged by the gas, with little impact and damage to the structure of the bioprosthetic tissue, making the reduction treatment process more gentle; on the other hand, performing multiple reduction treatments also ensures the reliability of the effect of the reduction treatment.
  • the anti-calcification treatment method of the present invention has strong process adaptability and little effect on the performance of bioprosthetic tissue.
  • the treated bioprosthetic tissue has a wider range of applications and can be dehydrated and dried, EO sterilized, or liquid stored and liquid sterilized.
  • the tissue can be used to manufacture dry films.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Transplantation (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Dermatology (AREA)
  • Medicinal Chemistry (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • General Health & Medical Sciences (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
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  • Chemical Kinetics & Catalysis (AREA)
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Abstract

L'invention concerne un procédé d'anti-calcification pour une bioprothèse tissulaire, ainsi qu'une bioprothèse tissulaire. Le procédé d'anti-calcification pour une bioprothèse tissulaire consiste à : placer une bioprothèse tissulaire au moins en partie fixée par réticulation dans une solution de dégraissage en vue d'un traitement de dégraissage, la solution de dégraissage comprenant une solution d'alcool contenant un tensioactif ; et placer la bioprothèse tissulaire dégraissée dans une solution contenant un agent réducteur en vue d'un traitement de réduction. Le procédé d'anti-calcification pour la bioprothèse tissulaire a un bon effet, et peut réduire la dégradation des performances d'un tissu provoquée par un traitement d'anti-calcification.
PCT/CN2023/118847 2022-10-12 2023-09-14 Procédé d'anti-calcification pour bioprothèse tissulaire, et bioprothèse tissulaire WO2024078253A1 (fr)

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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4885005A (en) * 1982-11-12 1989-12-05 Baxter International Inc. Surfactant treatment of implantable biological tissue to inhibit calcification
US5215541A (en) * 1982-11-12 1993-06-01 Baxter International Inc. Surfactant treatment of implantable biological tissue to inhibit calcification
WO2004006974A1 (fr) * 2002-07-16 2004-01-22 Edwards Lifesciences Corporation Attenuation renforcee de la calcification de materiaux biologiques et reduction accrue de leur teneur en phospholipides
CN101184516A (zh) * 2005-03-25 2008-05-21 爱德华兹生命科学公司 对生物假体组织进行处理以缓解植入后钙化
WO2013102048A2 (fr) * 2011-12-30 2013-07-04 University Of Miami Matrice de régénération tissulaire
CN110917398A (zh) * 2019-12-09 2020-03-27 沛嘉医疗科技(苏州)有限公司 生物材料的抗氧化方法及生物材料
WO2021211344A1 (fr) * 2020-04-15 2021-10-21 Vitae LLC Procédé de traitement de tissu à base de collagène pour des dispositifs bioprothétiques
CN114652893A (zh) * 2020-12-23 2022-06-24 上海微创心通医疗科技有限公司 生物假体组织的处理方法和生物假体心脏瓣膜

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4885005A (en) * 1982-11-12 1989-12-05 Baxter International Inc. Surfactant treatment of implantable biological tissue to inhibit calcification
US5215541A (en) * 1982-11-12 1993-06-01 Baxter International Inc. Surfactant treatment of implantable biological tissue to inhibit calcification
WO2004006974A1 (fr) * 2002-07-16 2004-01-22 Edwards Lifesciences Corporation Attenuation renforcee de la calcification de materiaux biologiques et reduction accrue de leur teneur en phospholipides
CN101184516A (zh) * 2005-03-25 2008-05-21 爱德华兹生命科学公司 对生物假体组织进行处理以缓解植入后钙化
WO2013102048A2 (fr) * 2011-12-30 2013-07-04 University Of Miami Matrice de régénération tissulaire
CN110917398A (zh) * 2019-12-09 2020-03-27 沛嘉医疗科技(苏州)有限公司 生物材料的抗氧化方法及生物材料
WO2021211344A1 (fr) * 2020-04-15 2021-10-21 Vitae LLC Procédé de traitement de tissu à base de collagène pour des dispositifs bioprothétiques
CN114652893A (zh) * 2020-12-23 2022-06-24 上海微创心通医疗科技有限公司 生物假体组织的处理方法和生物假体心脏瓣膜

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