CN112843335A - Exosome-loaded PET artificial ligament and preparation method thereof - Google Patents
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- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims abstract description 19
- 229920001690 polydopamine Polymers 0.000 claims abstract description 16
- 239000000725 suspension Substances 0.000 claims abstract description 16
- 238000000034 method Methods 0.000 claims abstract description 12
- CTENFNNZBMHDDG-UHFFFAOYSA-N Dopamine hydrochloride Chemical compound Cl.NCCC1=CC=C(O)C(O)=C1 CTENFNNZBMHDDG-UHFFFAOYSA-N 0.000 claims abstract description 10
- 229960001149 dopamine hydrochloride Drugs 0.000 claims abstract description 10
- 238000006243 chemical reaction Methods 0.000 claims abstract description 7
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 18
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- 238000002791 soaking Methods 0.000 claims description 6
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- 101000934368 Homo sapiens CD63 antigen Proteins 0.000 claims description 3
- 101000839464 Leishmania braziliensis Heat shock 70 kDa protein Proteins 0.000 claims description 3
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- 229920000139 polyethylene terephthalate Polymers 0.000 description 68
- 239000005020 polyethylene terephthalate Substances 0.000 description 68
- 210000001264 anterior cruciate ligament Anatomy 0.000 description 2
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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Abstract
The invention discloses an exosome-loaded PET artificial ligament and a preparation method thereof, the method comprises the steps of placing a pretreated PET artificial ligament in a Tris-HCL solution, adding a dopamine hydrochloride solution, reacting, cleaning, drying, placing in an exosome suspension, reacting for 10-20min at 4-37 ℃, and obtaining the exosome-loaded PET artificial ligament after the reaction is finished. According to the invention, the functional specificity modification layer loaded with exosomes is prepared on the surface of the PET artificial ligament material, and the exosomes have positive charge property and can generate electrostatic interaction with Poly Dopamine (PDA) with negative charge, so that the constructed PDA/exosome modification layer can be stably combined with the PET artificial ligament, and the biological performance of the PET artificial ligament can be remarkably improved by the method; the exosome loaded on the surface of the material can regulate the synthesis of proteins targeting the interior of cells and start related cascade reactions through DNA, RNA, protein and the like contained in the exosome after being implanted into a body, so that the host integration of the graft is promoted.
Description
Technical Field
The invention relates to the technical field of biomedical materials, in particular to an exosome-loaded PET artificial ligament and a preparation method thereof.
Background
The polyethylene terephthalate (PET) artificial ligament which is commonly used in clinic has poor biological activity due to the hydrophobicity and chemical inertness of the material, and has the problems of synovitis, bone duct expansion and the like after being applied to anterior cruciate ligament reconstruction. Therefore, it is very important to improve the biological performance of PET ligament and promote the host integration process of artificial ligament.
Exosomes are important tools for intercellular signal transmission, and play an important role in promoting tissue repair and regeneration. It has been shown in the present study that exosomes can promote the healing process after tendon/ligament reconstruction. However, no method for exosomal modification specifically for artificial ligaments exists in the prior art.
Disclosure of Invention
The invention aims to provide an exosome-loaded PET artificial ligament and a preparation method thereof, aiming at the defects in the prior art.
In order to achieve the purpose, the invention adopts the technical scheme that:
the invention provides a preparation method of an exosome-loaded PET artificial ligament, which comprises the following steps:
step one, placing the PET artificial ligament in absolute ethyl alcohol for soaking for more than 30min, then carrying out ultrasonic treatment for 5-15min at normal temperature, cleaning with deionized water to remove the ethyl alcohol, and placing in a vacuum drying oven until the PET artificial ligament is completely dried;
step two, placing the PET artificial ligament processed in the step one in a Tris-HCL solution, stirring to enable the Tris-HCL solution to fully infiltrate the PET artificial ligament, then adding a dopamine hydrochloride solution with the same volume as the Tris-HCL solution, reacting for 18-36h, washing the PET artificial ligament with deionized water, and placing the PET artificial ligament in a vacuum drying oven until the PET artificial ligament is completely dried to obtain a poly-dopamine modified PET artificial ligament;
and step three, placing the polydopamine modified PET artificial ligament prepared in the step two into an exosome suspension, reacting for 10-20min at the temperature of 4-37 ℃, and obtaining the exosome-loaded PET artificial ligament after the reaction is finished.
Further, the exosome is an secretion derived from a human bone marrow mesenchymal stem cell, a human umbilical cord mesenchymal stem cell, a human adipose stem cell, a human tendon stem cell or a human peripheral blood stem cell, and contains marker proteins CD63 and HSP 70.
Further, the preparation method of the exosome suspension comprises the following steps:
step one, when the cell fusion degree of the human bone marrow mesenchymal stem cells, the human umbilical cord mesenchymal stem cells, the human adipose-derived stem cells, the human tendon stem cells or the human peripheral blood stem cells is 70-80%, washing with a phosphate buffer solution (1x), and then culturing in a fresh serum-free culture medium for 48-60 h to obtain a first supernatant;
centrifuging the first supernatant, and removing the precipitate to obtain a second supernatant; centrifuging the second supernatant, and removing the precipitate to obtain a third supernatant; centrifuging the third supernatant, and removing the precipitate to obtain a fourth supernatant; centrifuging the fourth supernatant, and removing the supernatant to obtain a first precipitate;
step three, after the first precipitate is resuspended by phosphate buffer solution (1x), centrifuging and discarding supernatant fluid to obtain a second precipitate, and repeatedly centrifuging and purifying to obtain a purified exosome;
step four, suspending the purified exosomes in phosphate buffer (1x) to prepare the exosome suspension.
Further preferably, the concentration of the exosome suspension is 20-300 μ g/ml.
Further, the pH of the Tris-HCl solution is 8.0-9.0.
Further, the concentration of the dopamine hydrochloride solution is 4-6 mg/mL.
Further, the temperature in the vacuum drying oven was 37 ℃.
The second aspect of the invention provides the exosome-loaded PET artificial ligament prepared by the preparation method.
By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:
according to the invention, the functional specificity modification layer loaded with exosomes is prepared on the surface of the PET artificial ligament material, and the exosomes have positive charge property and can generate electrostatic interaction with Poly Dopamine (PDA) with negative charge, so that the constructed PDA/exosome modification layer can be stably combined with the PET artificial ligament, and the biological performance of the PET artificial ligament can be remarkably improved by the method; the exosome loaded on the surface of the material can regulate the synthesis of proteins targeting the interior of cells and start related cascade reactions through DNA, RNA, protein and the like contained in the exosome after being implanted into a body, so that the host integration of the graft is promoted.
The preparation process is simple and easy to operate, does not need expensive and complicated equipment, has mild conditions and low cost, and has better application prospect.
Drawings
FIG. 1 is a transmission electron micrograph of exosomes derived from human mesenchymal stem cells.
Detailed Description
The invention provides a preparation method of an exosome-loaded PET artificial ligament.
1. Preparation of exosome suspension:
step one, when the cell fusion degree of human bone marrow mesenchymal stem cells, human umbilical cord mesenchymal stem cells, human adipose-derived stem cells, human tendon stem cells or human peripheral blood stem cells is 70-80%, washing with phosphate buffer solution (1x), and then culturing in a fresh serum-free culture medium for 48h to obtain a first supernatant;
centrifuging the first supernatant for 10min at the rotating speed of 300g, and then removing the precipitate to obtain a second supernatant; centrifuging the second supernatant at 2000g for 10min, and removing the precipitate to obtain a third supernatant; centrifuging the third supernatant at 10000g for 30min, and removing the precipitate to obtain a fourth supernatant; centrifuging the fourth supernatant at 120000g for 70min, and removing the supernatant to obtain a first precipitate;
step three, after the first precipitate is resuspended by phosphate buffer solution (1x), centrifuging at the rotating speed of 120000g for 70min, then discarding the supernatant to obtain a second precipitate, and repeating the operation of resuspending the phosphate buffer solution (1x) at the rotating speed of 120000g for 70min, centrifuging for 70min, then discarding the supernatant to obtain the precipitate, and obtaining a purified exosome after 1-3 times;
step four, suspending the purified exosomes in phosphate buffer (1x) to prepare an exosome suspension with the concentration of 20-300 mu g/ml, wherein the exosome suspension contains marker proteins CD63 and HSP 70.
2. Preparing the exosome-loaded PET artificial ligament:
step one, placing the PET artificial ligament in absolute ethyl alcohol for soaking for more than 30min, then carrying out ultrasonic treatment for 5-15min at normal temperature, washing for more than 3 times by using deionized water to remove the ethyl alcohol, and placing in a vacuum drying oven at 37 ℃ until the PET artificial ligament is completely dried;
step two, placing the PET artificial ligament processed in the step one in a Tris-HCL solution, stirring to enable the Tris-HCL solution to fully infiltrate the PET artificial ligament, then adding a dopamine hydrochloride solution with the same volume as the Tris-HCL solution, reacting for 18-36 hours, then washing the PET artificial ligament with deionized water, and placing the PET artificial ligament in a vacuum drying oven at 37 ℃ until the PET artificial ligament is completely dried to obtain the polydopamine modified PET artificial ligament;
and step three, placing the polydopamine modified PET artificial ligament prepared in the step two in the exosome suspension prepared in the method, reacting for 10-20min at 4-37 ℃, and obtaining the PET artificial ligament loaded with the exosome after the reaction is finished.
As a preferable example, the pH of the Tris-HCl solution is 8.0 to 9.0
As a preferable example, the concentration of the dopamine hydrochloride solution is 4-6 mg/mL.
The invention is further described with reference to the following drawings and specific examples, which are not intended to be limiting. It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.
Example 1
Preparation of an exosome-loaded PET artificial ligament:
step one, placing the PET artificial ligament in absolute ethyl alcohol for soaking for more than 30min, then carrying out ultrasonic treatment for 10min at normal temperature, washing for more than 3 times by using deionized water to remove the ethyl alcohol, and placing in a vacuum drying oven at 37 ℃ until the PET artificial ligament is completely dried;
step two, placing the PET artificial ligament processed in the step one in 50mL of Tris-HCL solution with the pH value of 8.0, stirring by using a small rotor to enable the Tris-HCL solution to fully infiltrate the PET artificial ligament, then adding 50mL of dopamine hydrochloride solution with the concentration of 5mg/mL, reacting for 26 hours, washing the PET artificial ligament by using deionized water, and placing the PET artificial ligament in a vacuum drying box at 37 ℃ until the PET artificial ligament is completely dried to obtain the polydopamine modified PET artificial ligament;
and step three, placing the polydopamine modified PET artificial ligament prepared in the step two in exosome suspension derived from human bone marrow mesenchymal stem cells of 50 microgram/ml, reacting for 20min at 4 ℃, and obtaining the PET artificial ligament loaded with exosomes required after the reaction is finished.
Example 2
Preparation of an exosome-loaded PET artificial ligament:
step one, placing the PET artificial ligament in absolute ethyl alcohol for soaking for more than 30min, then carrying out ultrasonic treatment for 10min at normal temperature, washing for more than 3 times by using deionized water to remove the ethyl alcohol, and placing in a vacuum drying oven at 37 ℃ until the PET artificial ligament is completely dried;
step two, placing the PET artificial ligament processed in the step one in 50mL of Tris-HCL solution with the pH value of 8.5, stirring by using a small rotor to enable the Tris-HCL solution to fully infiltrate the PET artificial ligament, then adding 50mL of dopamine hydrochloride solution with the concentration of 6mg/mL, reacting for 20 hours, washing the PET artificial ligament by using deionized water, and placing the PET artificial ligament in a vacuum drying box at 37 ℃ until the PET artificial ligament is completely dried to obtain the polydopamine modified PET artificial ligament;
and step three, placing the polydopamine modified PET artificial ligament prepared in the step two in exosome suspension derived from 100 mu g/ml human adipose-derived stem cells, reacting for 15min at the temperature of 20 ℃, and obtaining the PET artificial ligament loaded with the exosome required after the reaction is finished.
Example 3
Preparation of an exosome-loaded PET artificial ligament:
step one, placing the PET artificial ligament in absolute ethyl alcohol for soaking for more than 30min, then carrying out ultrasonic treatment for 10min at normal temperature, washing for more than 3 times by using deionized water to remove the ethyl alcohol, and placing in a vacuum drying oven at 37 ℃ until the PET artificial ligament is completely dried;
step two, placing the PET artificial ligament processed in the step one in 50mL of Tris-HCL solution with the pH value of 9.0, stirring by using a small rotor to enable the Tris-HCL solution to fully infiltrate the PET artificial ligament, then adding 50mL of dopamine hydrochloride solution with the concentration of 4mg/mL, reacting for 32 hours, washing the PET artificial ligament by using deionized water, and placing the PET artificial ligament in a vacuum drying box at 37 ℃ until the PET artificial ligament is completely dried to obtain the polydopamine modified PET artificial ligament;
and step three, placing the polydopamine modified PET artificial ligament prepared in the step two in exosome suspension derived from 30 microgram/ml human umbilical cord mesenchymal stem cells, reacting for 10min at 37 ℃, and obtaining the PET artificial ligament loaded with exosomes required after the reaction is finished.
Verification example
The PET artificial ligament loaded with exosome (PET-exos group) and the untreated PET artificial ligament (contrast group) are applied to the reconstruction of rabbit anterior cruciate ligament, and the excessive inflammatory reaction is prompted to occur after the operation and the host integration is poor after the operation for 12 weeks and the synovial membrane of the knee joint in the contrast group is excessively proliferated. In the PET-exos group the graft was covered by ligament-like tissue and the regenerated tissue was white and more shiny, indicating better host integration.
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.
Claims (8)
1. A preparation method of an exosome-loaded PET artificial ligament is characterized by comprising the following steps:
step one, placing the PET artificial ligament in absolute ethyl alcohol for soaking for more than 30min, then carrying out ultrasonic treatment for 5-15min at normal temperature, cleaning with deionized water to remove the ethyl alcohol, and placing in a vacuum drying oven until the PET artificial ligament is completely dried;
step two, placing the PET artificial ligament processed in the step one in a Tris-HCL solution, stirring to enable the Tris-HCL solution to fully infiltrate the PET artificial ligament, then adding a dopamine hydrochloride solution with the same volume as the Tris-HCL solution, reacting for 18-36h, washing the PET artificial ligament with deionized water, and placing the PET artificial ligament in a vacuum drying oven until the PET artificial ligament is completely dried to obtain a poly-dopamine modified PET artificial ligament;
and step three, placing the polydopamine modified PET artificial ligament prepared in the step two into an exosome suspension, reacting for 10-20min at the temperature of 4-37 ℃, and obtaining the exosome-loaded PET artificial ligament after the reaction is finished.
2. The method according to claim 1, wherein the exosome is an exudate derived from a human bone marrow mesenchymal stem cell, a human umbilical cord mesenchymal stem cell, a human adipose stem cell, a human tendon stem cell or a human peripheral blood stem cell, and contains marker proteins CD63 and HSP 70.
3. The method for preparing a suspension of exosomes according to claim 2, comprising the steps of:
step one, when the cell fusion degree of the human bone marrow mesenchymal stem cells, the human umbilical cord mesenchymal stem cells, the human adipose-derived stem cells, the human tendon stem cells or the human peripheral blood stem cells is 70-80%, washing with a phosphate buffer solution, and then culturing in a fresh serum-free culture medium for 48-60 h to obtain a first supernatant;
centrifuging the first supernatant, and removing the precipitate to obtain a second supernatant; centrifuging the second supernatant, and removing the precipitate to obtain a third supernatant; centrifuging the third supernatant, and removing the precipitate to obtain a fourth supernatant; centrifuging the fourth supernatant, and removing the supernatant to obtain a first precipitate;
step three, after the first precipitate is resuspended by phosphate buffer solution, centrifuging and discarding supernatant fluid to obtain a second precipitate, and repeatedly centrifuging and purifying to obtain a purified exosome;
and step four, suspending the purified exosomes in a phosphate buffer solution to prepare the exosome suspension.
4. The method of claim 3, wherein the exosome suspension is at a concentration of 20-300 μ g/ml.
5. The method of claim 1, wherein the Tris-HCL solution has a pH of 8.0 to 9.0.
6. The method of claim 1, wherein the concentration of the dopamine hydrochloride solution is 4-6 mg/mL.
7. The method according to claim 1, wherein the temperature in the vacuum drying oven is 37 ℃.
8. An exosome-loaded PET artificial ligament prepared by the preparation method according to any one of claims 1 to 7.
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| CN115501387A (en) * | 2022-09-23 | 2022-12-23 | 山东第一医科大学(山东省医学科学院) | Titanium implant capable of slowly releasing trace elements and exosomes and preparation method thereof |
| CN115957378A (en) * | 2023-02-08 | 2023-04-14 | 上海交通大学医学院附属瑞金医院 | Bone repair composition and preparation method and application thereof |
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| CN110295142A (en) * | 2019-07-11 | 2019-10-01 | 中国医学科学院北京协和医院 | Promote the mesenchymal stem cell excretion body and its preparation method and application of angiogenesis |
| CN110787318A (en) * | 2019-11-12 | 2020-02-14 | 上海市第六人民医院 | Artificial ligament with function of immunological osteogenesis and preparation method thereof |
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