Reagent combination and method for improving calcification resistance of bovine pericardial valve material
A bovine pericardium and valve technology, applied in the field of reagent combination to improve the anti-calcification performance of bovine pericardial valve materials, can solve problems such as insufficient glutaraldehyde cross-linking, and achieve the goal of improving anti-calcification performance, maintaining mechanical properties, and improving durability Effect
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[0042] In the preparation method of the present invention, its key points are the combined use sequence of glutaraldehyde, neomycin, naringenin, magnesium chloride, at first remove cell component, remove most of antigen and phospholipid; Then apply neomycin (- NH 2 donor) to cross-link carboxyl groups (-COOH) to stabilize glycosaminoglycans; glutaraldehyde (-CHO) is then used to cross-link amino groups (-NH 2 ), ensure the mechanical properties, and block the antigen and immobilize neomycin; then use glutathione to block the residual aldehyde group (-CHO) to remove the residual toxicity of glutaraldehyde cross-linking; then add polyphenol cross-linking to stabilize elastin (Cross-linked -OH, -C=O, -NH 2 ), reduce immune and inflammatory responses; finally use metal ions to stabilize elastin and block the calcium ion binding sites of polyphenols, and at the same time change the surface charge properties of the material to competitively inhibit calcium ion deposition; through t...
Embodiment 1
[0051] Embodiment 1 Early stage treatment
[0052] Take out the pericardium from the cattle (about 400-500Kg in body weight) in the slaughterhouse, separate the fat and outer membrane within 30 minutes of warm ischemia time, and use sterile PBS solution containing antibiotics (penicillin and streptomycin, the concentration is 100U / ml) ) washed several times, immersed in 0.1% bromogeramine for 30 minutes, rinsed the inner and outer sides of the pericardium with sterile PBS containing antibiotics for 10 minutes×3 times, and stored at a low temperature of 4°C.
Embodiment 2
[0053] Example 2 Decellularization
[0054] Step 1: Treat 0.1% bromogeramine at 37°C for 30 minutes, stirring once every 10 minutes;
[0055] Step 2: Rinse with 0.25% TritonX-100 at 37°C and 120 rpm for 48 hours, changing the solution every 12 hours;
[0056] Step 3: 3u / ml DNase-I / 0.03mg / ml RNase-A, 37°C, 120rpm, shaking and drifting for 24h, changing the medium every 12h;
[0057] Step 4: Rinse with distilled water for 48 hours, and change the solution every 8 hours;
[0058] Step 5: Rinse with PBS for 24 hours, and store after changing the medium.
[0059] Through the above steps, the cellular components are removed, and most of the antigens and phospholipids are removed.
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