CN110787318A - Artificial ligament with function of immunological osteogenesis and preparation method thereof - Google Patents
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- A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
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- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
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- A61L2430/10—Materials or treatment for tissue regeneration for reconstruction of tendons or ligaments
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Abstract
The invention discloses an artificial ligament with an osteogenesis immunity function and a preparation method thereof, wherein the preparation method comprises the following steps: (1) putting the PET artificial ligament in an ultrasonic machine, cleaning and airing; (2) cutting the PET artificial ligament obtained in the step (1), and putting the cut PET artificial ligament into a beaker filled with a Tris-HCL solution; then adding 25mL of dopamine hydrochloride solution into the beaker, and reacting for 1-24h to obtain the PDA modified PET ligament; (3) putting the PET ligament modified by the PDA obtained in the step (2) into a 50mL centrifuge tube, and adding a CS-ADH solution to obtain the PET ligament modified by the CS-PDA; (4) slowly and uniformly dropwise adding 1-20ug of rhBMP-2 solution on the surface of the PET ligament obtained in the step (3), freezing overnight at-20 ℃, and freeze-drying in a sterile freeze dryer to obtain the product. The PET artificial ligament is adsorbed on PET through the self-polymerization reaction of dopamine, and then chondroitin sulfate is connected to the surface of PET fibers through the reaction of dopamine and chondroitin sulfate-ADH, so that the PET artificial ligament is endowed with an immune regulation function.
Description
Technical Field
The invention relates to the technical field of medicine, in particular to an artificial ligament with an osteogenesis immunity function and a preparation method thereof.
Background
At present, the existing PET artificial ligament has low biomechanical performance due to the healing of fibrous scars formed by the PET artificial ligament and host bones in the host body through immunological rejection reaction. In the prior art, a modification technology specially aiming at the immune function of the artificial ligament does not exist, so that the improvement of the biomechanical property by promoting the integration of a graft-bone interface through immune regulation is very important.
Disclosure of Invention
The invention provides an artificial ligament with an immune osteogenesis function and a preparation method thereof, aiming at solving the problems in the prior art.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a preparation method of an artificial ligament with an immune osteogenesis function, which is characterized in that chondroitin sulfate/dopamine is adopted to modify the artificial PET ligament so as to endow the PET artificial ligament with a proper immune regulation function, and graft-bone interface integration is promoted through immune regulation so as to improve biomechanical properties.
Further, the preparation method of the artificial ligament with the function of immunoosteogenesis comprises the following steps:
(1) putting a PET artificial ligament with a certain size in an ultrasonic machine, and ultrasonically cleaning the PET artificial ligament with 75% ethanol for 25 min; then washing the PET artificial ligament with deionized water; finally, naturally airing the PET artificial ligament for 24 hours at room temperature;
(2) cutting the PET artificial ligament obtained in the step (1), putting the cut PET artificial ligament into a 100mL beaker, adding 25mL of Tris-HCL solution into the beaker, and stirring by using a small rotor to fully mix the PET artificial ligament and the Tris-HCL solution; then adding 25mL of dopamine hydrochloride solution into a beaker, and reacting for 1-24 h; then taking out the PET artificial ligament and washing off redundant polydopamine on the surface of the PET artificial ligament by using deionized water; placing the PET artificial ligament in a vacuum drying oven at 37 ℃ for 1 day to obtain a PET ligament modified by PDA;
(3) putting the PDA modified PET artificial ligament obtained in the step (2) into a 50mL centrifuge tube, and then adding 10mL of CS-ADH solution with the concentration of 0.5-24 mg/mL; incubating the centrifuge tube in a constant temperature shaking box at 60rpm and 37 ℃ overnight; gently washing the incubated material product with PBS for 2 times, and drying in a vacuum drying oven at 37 ℃ for 24h to obtain the CS-PDA modified PET ligament;
(4) slowly and uniformly dripping 1-20ug of rhBMP-2 solution on the surface of the PET ligament modified by the CS-PDA obtained in the step (3); freezing the obtained material in a refrigerator at-20 deg.C overnight, and freeze drying in a sterile freeze dryer. Further, the coal gangue in the step (1) is made into coal gangue raw gas through a coal pressure gasification furnace.
Further, the pH of the Tris-HCl solution in the step (2) is 8.5-9.0.
Further, the concentration of the dopamine hydrochloride solution in the step (2) is 4-5 mg/mL.
Further, the preparation method of the CS-ADH solution in the step (3) comprises the following steps: adding 300mg of CS into 40mL of PBS solution, adding 383.4mg of EDC and 230mg of NHS, mixing and stirring for 30min to activate carboxyl on a CS chain; then adding 700mgADH and continuously stirring at normal temperature overnight; dialyzing the reaction mixed solution by using a dialysis bag with the weight average molecular weight cutoff of 8kDa for 3 days, and changing water in a beaker every 8 hours; and finally freezing the residual dialysis solution overnight, and carrying out vacuum freeze drying for 3 days to obtain the product.
Further preferably, the pH of the PBS solution is 5.0-6.0.
Further preferably, the molecular weight ratio of COOH, EDC, NHS and ADH is 1: 2.5: 2.5: 5.
further preferably, the solvent of the CS-ADH solution in the step (3) is PBS.
Another aspect of the present invention is to provide an artificial ligament having an immunoosteogenesis function, which is prepared by any one of the above methods.
By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:
(1) in the prior art, in order to realize that the artificial ligament has an immune regulation function, the artificial ligament is realized by modifying a chondroitin sulfate coating, but the chondroitin sulfate cannot be directly connected with a PET ligament, so that the artificial ligament is adsorbed on the PET by adopting a self-polymerization reaction of dopamine, and then is connected with the surface of a PET fiber through a reaction of the dopamine and chondroitin sulfate-ADH, so that the PET artificial ligament is endowed with the immune regulation function;
(2) the BMP-2 is loaded on the surface of the CS through the natural affinity adsorption action of the CS and the BMP-2, the BMP-CS-PDA modified artificial ligament is prepared, and the graft-osseointegration action is further enhanced.
Drawings
FIG. 1 is a flow chart of the preparation of an artificial ligament with an immunoosteogenesis function according to the present invention;
FIG. 2 is a diagram of the molecular formula change process of an artificial ligament with an osteogenesis stimulating function according to the present invention.
Detailed Description
The present invention will be further described with reference to specific embodiments, but the present invention is not limited to the following embodiments.
Example 1
As shown in fig. 1, a method for preparing an artificial ligament with an immunoosteogenesis function includes the steps of:
(1) putting a PET artificial ligament with a certain size in an ultrasonic machine, and ultrasonically cleaning the PET artificial ligament with 75% ethanol for 25 min; then washing the PET artificial ligament with deionized water; finally, naturally airing the PET artificial ligament for 24 hours at room temperature;
(2) cutting the PET artificial ligament obtained in the step (1), putting the cut PET artificial ligament into a 100mL beaker, adding 25mL of Tris-HCL solution with the pH value of 8.5 into the beaker, and stirring by using a small rotor to fully mix the PET artificial ligament and the Tris-HCL solution; then adding 25mL of dopamine hydrochloride solution with the concentration of 4mg/mL into a beaker, and reacting for 1 h; then taking out the PET artificial ligament and washing off redundant polydopamine on the surface of the PET artificial ligament by using deionized water; placing the PET artificial ligament in a vacuum drying oven at 37 ℃ for 1 day to obtain a PET ligament modified by PDA;
(3) putting the PDA modified PET artificial ligament obtained in the step (2) into a 50mL centrifuge tube, and then adding 10mL of CS-ADH solution with the concentration of 0.5 mg/mL; incubating the centrifuge tube in a constant temperature shaking box at 60rpm and 37 ℃ overnight; gently washing the incubated material product with PBS for 2 times, and drying in a vacuum drying oven at 37 ℃ for 24h to obtain the CS-PDA modified PET ligament;
(4) slowly and uniformly dripping 1ug of rhBMP-2 solution on the surface of the PET ligament modified by the CS-PDA obtained in the step (3); freezing the obtained material in a refrigerator at-20 deg.C overnight, and freeze drying in a sterile freeze dryer.
Example 2
As shown in fig. 1, a method for preparing an artificial ligament with an immunoosteogenesis function includes the steps of:
(1) putting a PET artificial ligament with a certain size in an ultrasonic machine, and ultrasonically cleaning the PET artificial ligament with 75% ethanol for 25 min; then washing the PET artificial ligament with deionized water; finally, naturally airing the PET artificial ligament for 24 hours at room temperature;
(2) cutting the PET artificial ligament obtained in the step (1), putting the cut PET artificial ligament into a 100mL beaker, adding 25mL of Tris-HCL solution with the pH value of 8.7 into the beaker, and stirring by using a small rotor to fully mix the PET artificial ligament and the Tris-HCL solution; then adding 25mL of dopamine hydrochloride solution with the concentration of 4mg/mL into the beaker, and reacting for 12 h; then taking out the PET artificial ligament and washing off redundant polydopamine on the surface of the PET artificial ligament by using deionized water; placing the PET artificial ligament in a vacuum drying oven at 37 ℃ for 1 day to obtain a PET ligament modified by PDA;
(3) putting the PDA modified PET artificial ligament obtained in the step (2) into a 50mL centrifuge tube, and then adding 10mL of CS-ADH solution with the concentration of 6 mg/mL; incubating the centrifuge tube in a constant temperature shaking box at 60rpm and 37 ℃ overnight; gently washing the incubated material product with PBS for 2 times, and drying in a vacuum drying oven at 37 ℃ for 24h to obtain the CS-PDA modified PET ligament;
(4) slowly and uniformly dripping 10ug of rhBMP-2 solution on the surface of the PET ligament modified by the CS-PDA obtained in the step (3); freezing the obtained material in a refrigerator at-20 deg.C overnight, and freeze drying in a sterile freeze dryer.
Example 3
As shown in fig. 1, a method for preparing an artificial ligament with an immunoosteogenesis function includes the steps of:
(1) putting a PET artificial ligament with a certain size in an ultrasonic machine, and ultrasonically cleaning the PET artificial ligament with 75% ethanol for 25 min; then washing the PET artificial ligament with deionized water; finally, naturally airing the PET artificial ligament for 24 hours at room temperature;
(2) cutting the PET artificial ligament obtained in the step (1), putting the cut PET artificial ligament into a 100mL beaker, adding 25mL of Tris-HCL solution with the pH value of 9.0 into the beaker, and stirring by using a small rotor to fully mix the PET artificial ligament and the Tris-HCL solution; then adding 25mL of dopamine hydrochloride solution with the concentration of 4mg/mL into a beaker, and reacting for 24 h; then taking out the PET artificial ligament and washing off redundant polydopamine on the surface of the PET artificial ligament by using deionized water; placing the PET artificial ligament in a vacuum drying oven at 37 ℃ for 1 day to obtain a PET ligament modified by PDA;
(3) putting the PDA modified PET artificial ligament obtained in the step (2) into a 50mL centrifuge tube, and then adding 10mL of CS-ADH solution with the concentration of 24 mg/mL; incubating the centrifuge tube in a constant temperature shaking box at 60rpm and 37 ℃ overnight; gently washing the incubated material product with PBS for 2 times, and drying in a vacuum drying oven at 37 ℃ for 24h to obtain the CS-PDA modified PET ligament;
(4) slowly and uniformly dripping 20ug of rhBMP-2 solution on the surface of the PET ligament modified by the CS-PDA obtained in the step (3); freezing the obtained material in a refrigerator at-20 deg.C overnight, and freeze drying in a sterile freeze dryer.
The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.
Claims (8)
1. A preparation method of an artificial ligament with an immunoosteogenesis function is characterized by comprising the following steps:
(1) putting a PET artificial ligament with a certain size in an ultrasonic machine, and ultrasonically cleaning the PET artificial ligament with 75% ethanol for 25 min; then washing the PET artificial ligament with deionized water; finally, naturally airing the PET artificial ligament for 24 hours at room temperature;
(2) cutting the PET artificial ligament obtained in the step (1), putting the cut PET artificial ligament into a 100mL beaker, adding 25mL of Tris-HCL solution into the beaker, and stirring by using a small rotor to fully mix the PET artificial ligament and the Tris-HCL solution; then adding 25mL of dopamine hydrochloride solution into a beaker, and reacting for 1-24 h; then taking out the PET artificial ligament and washing off redundant polydopamine on the surface of the PET artificial ligament by using deionized water; placing the PET artificial ligament in a vacuum drying oven at 37 ℃ for 1 day to obtain a PET ligament modified by PDA;
(3) putting the PDA modified PET artificial ligament obtained in the step (2) into a 50mL centrifuge tube, and then adding 10mL of CS-ADH solution with the concentration of 0.5-24 mg/mL; incubating the centrifuge tube in a constant temperature shaking box at 60rpm and 37 ℃ overnight; gently washing the incubated material product with PBS for 2 times, and drying in a vacuum drying oven at 37 ℃ for 24h to obtain the CS-PDA modified PET ligament;
(4) slowly and uniformly dripping 1-20ug of rhBMP-2 solution on the surface of the PET ligament modified by the CS-PDA obtained in the step (3); freezing the obtained material in a refrigerator at-20 deg.C overnight, and freeze drying in a sterile freeze dryer.
2. The method for preparing an artificial ligament having an immunoosteogenesis function according to claim 1, wherein the Tris-HCl solution of the step (2) has a pH of 8.5 to 9.0.
3. The method for preparing an artificial ligament having an osteogenesis stimulating function according to claim 1, wherein the concentration of the dopamine hydrochloride solution in the step (2) is 4-5 mg/mL.
4. The method for preparing an artificial ligament having an immunoosteogenesis function according to claim 1, wherein the CS-ADH solution in the step (3) is prepared by: first, 300mg of CS was added to a 40ml PBS solution, followed by 383.4mg of EDC and 230mg of NHS, and the mixture was stirred for 30min to activate the carboxyl groups on the CS chains; then adding 700mgADH and continuously stirring at normal temperature overnight; dialyzing the reaction mixed solution by using a dialysis bag with the weight average molecular weight cutoff of 8kDa for 3 days, and changing water in a beaker every 8 hours; and finally freezing the residual dialysis solution overnight, and carrying out vacuum freeze drying for 3 days to obtain the product.
5. The method for preparing an artificial ligament having an osteogenesis stimulating function according to claim 4, wherein the pH of the PBS solution is 5.0-6.0.
6. The method for preparing artificial ligament with immunoosteogenesis function according to claim 4, wherein the molecular weight of COOH, EDC, NHS and ADH is 1: 2.5: 2.5: the ratio of 5 is.
7. The method for preparing an artificial ligament having an osteogenesis stimulating function according to claim 1, wherein the solvent of the CS-ADH solution in the step (3) is PBS.
8. An artificial ligament having an immunoosteogenic function, produced by the method according to any of claims 1 to 7.
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Cited By (4)
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| CN111760069A (en) * | 2020-07-19 | 2020-10-13 | 复旦大学 | Silver-modified artificial ligament with broad-spectrum antibacterial property and preparation method thereof |
| CN111790005A (en) * | 2020-07-16 | 2020-10-20 | 复旦大学 | Polydopamine-modified artificial ligament and modification method thereof |
| CN112843335A (en) * | 2021-01-20 | 2021-05-28 | 上海市第六人民医院 | Exosome-loaded PET artificial ligament and preparation method thereof |
| CN114366853A (en) * | 2022-01-20 | 2022-04-19 | 华东理工大学 | High bone-inducing active dental implant coating and preparation method thereof |
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Cited By (4)
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| CN111790005A (en) * | 2020-07-16 | 2020-10-20 | 复旦大学 | Polydopamine-modified artificial ligament and modification method thereof |
| CN111760069A (en) * | 2020-07-19 | 2020-10-13 | 复旦大学 | Silver-modified artificial ligament with broad-spectrum antibacterial property and preparation method thereof |
| CN112843335A (en) * | 2021-01-20 | 2021-05-28 | 上海市第六人民医院 | Exosome-loaded PET artificial ligament and preparation method thereof |
| CN114366853A (en) * | 2022-01-20 | 2022-04-19 | 华东理工大学 | High bone-inducing active dental implant coating and preparation method thereof |
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