A decellularized amniotic membrane material and its preparation method and its application in preparing ocular surface repair material

Abstract

The present invention discloses a decellularized amniotic membrane material, a preparation method thereof, and an application thereof in the preparation of an ocular surface repair material. The preparation method comprises the following steps: (1) taking a placenta, separating the amniotic membrane from the placenta, and then using a cell scraper to remove excess tissue on the surface of the amniotic membrane to obtain a chorionic amniotic membrane; (2) washing the chorionic amniotic membrane treated in step (1) to obtain a washed chorionic amniotic membrane; (3) placing the washed chorionic amniotic membrane in a decellularizing reagent for decellularization to obtain a decellularized amniotic membrane; (4) washing the decellularized amniotic membrane, and then placing it in an ethanol solution containing a natural extract for soaking treatment; (5) taking out the decellularized amniotic membrane treated in step (4), and then drying, shearing, and irradiating and sterilizing it to obtain the decellularized amniotic membrane material. Studies have shown that the decellularized amniotic membrane material prepared by the method of the present invention has a good effect of promoting cell proliferation.

Description

Technical Field

[0001] The present invention relates to the technical field of ophthalmic material preparation, and in particular to a decellularized amniotic membrane material and a preparation method thereof, and application thereof in the preparation of an ocular surface repair material. Background Art

[0002] The amniotic membrane is a thin membrane composed of a single layer of epithelial cells connected to each other. It is the innermost layer of the fetal membrane and is a translucent membrane that is connected to the amniotic membrane layer covering the placenta and umbilical cord. In ophthalmology clinics, the amniotic membrane can be used as a biomaterial to treat ocular surface injuries and has important application value.

[0003] For example, Chinese invention patent CN202210366231.9 discloses a preparation process for a decellularized freeze-dried amniotic membrane product. The process involves placental stripping and typing, chemical treatment of the amniotic membrane, cleaning, decellularization, re-cleaning, freeze-drying, cutting and shaping, inner packaging, irradiation sterilization, and outsourcing of the finished product. The process can effectively decellularize both the amniotic membrane types, with and without a chorionic layer. The process also fully takes into account the key points that the amniotic membrane is transparent, small in size, and difficult to change the fluid, as well as the difficulty in accurately controlling the size after freeze-drying. The production process is rationally planned to improve the yield of the amniotic membrane and promote the development of the amniotic membrane industry.

[0004] However, the inventors found in their research that the decellularized amniotic membrane material prepared by the above method has a weak effect in promoting cell proliferation. When it is used to repair ocular surface damage, the repair cycle is relatively long and needs to be further improved. Summary of the invention

[0005] In order to solve at least one of the technical problems existing in the prior art, the present invention provides a decellularized amniotic membrane material, a preparation method thereof, and an application thereof in the preparation of an ocular surface repair material.

[0006] The technical solution of the present invention to solve the above technical problems is as follows:

[0007] The present invention first provides a method for preparing a decellularized amniotic membrane material, which comprises the following steps:

[0008] (1) taking the placenta, separating the amniotic membrane from the placenta, and then removing the excess tissue on the surface of the amniotic membrane with a cell scraper to obtain the amniotic membrane without chorionic villi;

[0009] (2) washing the chorionic villus-free amniotic membrane after being treated in step (1) to obtain a washed chorionic villus-free amniotic membrane;

[0010] (3) placing the washed chorionic villus-free amniotic membrane into a decellularizing reagent for decellularization to obtain a decellularized amniotic membrane;

[0011] (4) washing the decellularized amniotic membrane and then soaking it in an ethanol solution containing a natural extract;

[0012] (5) Taking out the decellularized amniotic membrane treated in step (4), and then drying, shearing and irradiating and sterilizing it to obtain the decellularized amniotic membrane material.

[0013] The present invention provides a novel method for preparing a decellularized amniotic membrane material. Studies have shown that, in the preparation method of the present invention, the decellularized amniotic membrane material prepared by soaking the decellularized amniotic membrane in natural extracts has a good effect of promoting cell proliferation.

[0014] Preferably, the placenta described in step (1) is screened for pathogens before separation of the amniotic membrane.

[0015] Preferably, the cleaning in step (2) refers to cleaning with physiological saline containing penicillin.

[0016] Preferably, the concentration of penicillin in the penicillin-containing physiological saline is 0.1-0.3 mg / mL.

[0017] Most preferably, the concentration of penicillin in the penicillin-containing saline is 0.2 mg / mL.

[0018] Preferably, the decellularization reagent in step (3) refers to an aqueous solution containing Triton X-100 and sodium deoxycholate.

[0019] Preferably, the content of Triton X-100 in the decellularization reagent is 0.1 to 1 g / mL; the content of sodium deoxycholate is 0.1 to 1 g / mL;

[0020] Most preferably, the content of Triton X-100 in the decellularization reagent is 0.5 g / mL; the content of sodium deoxycholate is 0.5 g / mL.

[0021] Preferably, the natural extract in the aqueous solution containing the natural extract in step (4) is prepared by the following method:

[0022] Saffron and ganoderma are crushed and then extracted with an organic solvent. After the extraction, the extract is taken and concentrated and dried to obtain a saffron-ganoderma organic solvent extract;

[0023] The natural extract is obtained by taking the saffron-Ganoderma lucidum organic solvent extract.

[0024] Preferably, the organic solvent is acetone.

[0025] Preferably, the weight ratio of saffron to ganoderma lucidum is 4-6:1-3.

[0026] Most preferably, the weight ratio of saffron to ganoderma is 5:2.

[0027] Preferably, the total weight ratio of the Chinese medicinal materials saffron and ganoderma lucidum to the organic solvent is 1 kg: 8 to 12 L.

[0028] Most preferably, the ratio of the total weight of the Chinese medicinal materials saffron and ganoderma lucidum to the amount of the organic solvent is 1 kg:10 L.

[0029] Preferably, the method for preparing the natural extract further comprises the following steps:

[0030] Take the saffron-Ganoderma lucidum organic solvent extract and apply it to a macroporous resin column; first use an ethanol aqueous solution with a volume fraction of 26-29% to elute and remove impurities; then use an ethanol aqueous solution with a volume fraction of 41-43% to elute, collect the eluate eluted with the ethanol aqueous solution with a volume fraction of 41-43%, and concentrate and dry to obtain the natural extract.

[0031] Most preferably, the method for preparing the substance for promoting tissue regeneration further comprises the following steps:

[0032] Take the saffron-Ganoderma lucidum organic solvent extract and apply it to a macroporous resin column; first elute it with a 28% by volume ethanol aqueous solution to remove impurities; then elute it with a 42% by volume ethanol aqueous solution, collect the eluate eluted with the 42% by volume ethanol aqueous solution, and concentrate and dry it to obtain the natural extract.

[0033] In the preparation method described in the present invention, the decellularized amniotic membrane is soaked in a natural extract obtained by further eluting the saffron-Ganoderma lucidum organic solvent extract through the above-mentioned macroporous resin elution conditions of the present invention. Compared with soaking in the saffron-Ganoderma lucidum organic solvent extract as the natural extract, the cell proliferation-promoting effect of the prepared decellularized amniotic membrane material can be greatly improved.

[0034] In addition, the inventors also found in the research that the macroporous resin elution conditions of the present invention are also very critical; the decellularized amniotic membrane must be soaked in a natural extract prepared by further subjecting the saffron-Ganoderma organic solvent extract to the above-mentioned macroporous resin elution conditions of the present invention, compared with soaking in the saffron-Ganoderma organic solvent extract as a natural extract, in order to further significantly improve the cell proliferation promoting effect of the prepared decellularized amniotic membrane material; however, soaking in a natural extract prepared by further subjecting the saffron-Ganoderma organic solvent extract to other macroporous resin elution conditions does not further significantly improve the cell proliferation promoting effect of the prepared decellularized amniotic membrane material.

[0035] Preferably, the mass fraction of the natural extract in the ethanol solution containing the natural extract in step (4) is 0.5-2%.

[0036] Most preferably, the mass fraction of the natural extract in the ethanol solution containing the natural extract in step (4) is 1%.

[0037] Preferably, the ethanol solution in the ethanol solution containing the natural extract in step (4) refers to an ethanol solution with a volume fraction of 75%.

[0038] Preferably, the soaking treatment time in step (4) is 3 to 6 hours.

[0039] Most preferably, the soaking treatment time in step (4) is 5 hours.

[0040] The present invention also provides a decellularized amniotic membrane material prepared by the above preparation method.

[0041] The present invention also provides a use of the above-mentioned decellularized amniotic membrane material in preparing an ocular surface repair material.

[0042] Beneficial effects: The present invention provides a new method for preparing decellularized amniotic membrane materials; studies have shown that in the preparation method of the present invention, the decellularized amniotic membrane material prepared by soaking the decellularized amniotic membrane in natural extracts has a good effect of promoting cell proliferation. Therefore, when the decellularized amniotic membrane material prepared by the present invention is used for repairing ocular surface damage, it helps to shorten the repair cycle and has important application value. DETAILED DESCRIPTION

[0043] The present invention is further explained below in conjunction with specific examples, but the examples do not limit the protection scope of the present invention.

[0044] Example 1 Preparation method of decellularized amniotic membrane material

[0045] (1) obtaining an isolated human placenta after pathogen screening, separating the amnion from the placenta, and then removing excess tissue on the surface of the amnion with a cell scraper to obtain a chorionic amnion;

[0046] (2) washing the chorionic villus-free amniotic membrane treated in step (1) to obtain a washed chorionic villus-free amniotic membrane; the washing refers to washing with a penicillin-containing saline solution having a penicillin concentration of 0.2 mg / mL;

[0047] (3) placing the cleaned chorionic villus-free amniotic membrane in a decellularization reagent for decellularization oscillation treatment for 40 minutes to obtain a decellularized amniotic membrane; the decellularization reagent refers to an aqueous solution containing Triton X-100 and sodium deoxycholate, wherein the content of Triton X-100 is 0.5 g / mL; the content of sodium deoxycholate is 0.5 g / mL;

[0048] (4) washing the decellularized amniotic membrane, and then soaking it in an ethanol solution containing a natural extract for 5 hours; wherein the mass fraction of the natural extract in the ethanol solution containing the natural extract is 1%, and the ethanol solution refers to an ethanol solution with a volume fraction of 75%;

[0049] (5) taking out the decellularized amniotic membrane treated in step (4), and then drying, shearing and irradiating and sterilizing it to obtain the decellularized amniotic membrane material;

[0050] Wherein, the natural extract described in step (4) is prepared by the following method:

[0051] Saffron and ganoderma lucidum are crushed and refluxed with an organic solvent at 56° C. for 1 hour. After the extraction, the extract is taken and concentrated and dried to obtain a saffron-ganoderma lucidum organic solvent extract; wherein the weight ratio of saffron to ganoderma lucidum is 5:2; the total weight ratio of saffron and ganoderma lucidum to the amount of the organic solvent is 1 kg:10 L; the organic solvent is acetone;

[0052] The natural extract is obtained by taking the saffron-Ganoderma lucidum organic solvent extract.

[0053] Example 2 Preparation method of decellularized amniotic membrane material

[0054] (1) obtaining an isolated human placenta after pathogen screening, separating the amnion from the placenta, and then removing excess tissue on the surface of the amnion with a cell scraper to obtain a chorionic amnion;

[0055] (2) washing the chorionic villus-free amniotic membrane treated in step (1) to obtain a washed chorionic villus-free amniotic membrane; the washing refers to washing with a penicillin-containing saline solution having a penicillin concentration of 0.2 mg / mL;

[0056] (3) placing the cleaned chorionic villus-free amniotic membrane in a decellularization reagent for decellularization oscillation treatment for 40 minutes to obtain a decellularized amniotic membrane; the decellularization reagent refers to an aqueous solution containing Triton X-100 and sodium deoxycholate, wherein the content of Triton X-100 is 0.5 g / mL; the content of sodium deoxycholate is 0.5 g / mL;

[0057] (4) washing the decellularized amniotic membrane, and then soaking it in an ethanol solution containing a natural extract for 5 hours; wherein the mass fraction of the natural extract in the ethanol solution containing the natural extract is 1%, and the ethanol solution refers to an ethanol solution with a volume fraction of 75%;

[0058] (5) taking out the decellularized amniotic membrane treated in step (4), and then drying, shearing and irradiating and sterilizing it to obtain the decellularized amniotic membrane material;

[0059] Wherein, the natural extract described in step (4) is prepared by the following method:

[0060] (1) Saffron and Ganoderma lucidum are crushed and refluxed with an organic solvent at 56° C. for 1 hour. After the extraction, the extract is taken and concentrated and dried to obtain a saffron-Ganoderma lucidum organic solvent extract; wherein the weight ratio of saffron to Ganoderma lucidum is 5:2; the total weight ratio of saffron and Ganoderma lucidum to the organic solvent is 1 kg:10 L; and the organic solvent is acetone;

[0061] (2) Take the saffron-Ganoderma lucidum organic solvent extract and apply it to an HP-20 macroporous resin column; first use 5 times the column volume of 28% ethanol aqueous solution to elute and remove impurities; then use 5 times the column volume of 42% ethanol aqueous solution to elute, collect the eluate eluted with 42% ethanol aqueous solution, and concentrate and dry to obtain the natural extract.

[0062] Comparative Example 1 Preparation method of decellularized amniotic membrane material

[0063] (1) obtaining an isolated human placenta after pathogen screening, separating the amnion from the placenta, and then removing excess tissue on the surface of the amnion with a cell scraper to obtain a chorionic amnion;

[0064] (2) washing the chorionic villus-free amniotic membrane treated in step (1) to obtain a washed chorionic villus-free amniotic membrane; the washing refers to washing with a penicillin-containing saline solution having a penicillin concentration of 0.2 mg / mL;

[0065] (3) placing the cleaned chorionic villus-free amniotic membrane in a decellularization reagent for decellularization oscillation treatment for 40 minutes to obtain a decellularized amniotic membrane; the decellularization reagent refers to an aqueous solution containing Triton X-100 and sodium deoxycholate, wherein the content of Triton X-100 is 0.5 g / mL; the content of sodium deoxycholate is 0.5 g / mL;

[0066] (4) washing the decellularized amniotic membrane, and then soaking it in an ethanol solution containing a natural extract for 5 hours; wherein the mass fraction of the natural extract in the ethanol solution containing the natural extract is 1%, and the ethanol solution refers to an ethanol solution with a volume fraction of 75%;

[0067] (5) taking out the decellularized amniotic membrane treated in step (4), and then drying, shearing and irradiating and sterilizing it to obtain the decellularized amniotic membrane material;

[0068] Wherein, the natural extract described in step (4) is prepared by the following method:

[0069] (1) Saffron and Ganoderma lucidum are crushed and refluxed with an organic solvent at 56° C. for 1 hour. After the extraction, the extract is taken and concentrated and dried to obtain a saffron-Ganoderma lucidum organic solvent extract; wherein the weight ratio of saffron to Ganoderma lucidum is 5:2; the total weight ratio of saffron and Ganoderma lucidum to the organic solvent is 1 kg:10 L; and the organic solvent is acetone;

[0070] (2) Take the saffron-Ganoderma lucidum organic solvent extract and apply it to an HP-20 macroporous resin column; first use 5 times the column volume of 10% ethanol aqueous solution to elute and remove impurities; then use 5 times the column volume of 30% ethanol aqueous solution to elute, collect the eluate eluted with 30% ethanol aqueous solution, and concentrate and dry to obtain the natural extract.

[0071] Comparative Example 2 Preparation method of decellularized amniotic membrane material

[0072] (1) obtaining an isolated human placenta after pathogen screening, separating the amnion from the placenta, and then removing excess tissue on the surface of the amnion with a cell scraper to obtain a chorionic amnion;

[0073] (2) washing the chorionic villus-free amniotic membrane treated in step (1) to obtain a washed chorionic villus-free amniotic membrane; the washing refers to washing with a penicillin-containing saline solution having a penicillin concentration of 0.2 mg / mL;

[0074] (3) placing the cleaned chorionic villus-free amniotic membrane in a decellularization reagent for decellularization oscillation treatment for 40 minutes to obtain a decellularized amniotic membrane; the decellularization reagent refers to an aqueous solution containing Triton X-100 and sodium deoxycholate, wherein the content of Triton X-100 is 0.5 g / mL; the content of sodium deoxycholate is 0.5 g / mL;

[0075] (4) washing the decellularized amniotic membrane, and then soaking it in an ethanol solution containing a natural extract for 5 hours; wherein the mass fraction of the natural extract in the ethanol solution containing the natural extract is 1%, and the ethanol solution refers to an ethanol solution with a volume fraction of 75%;

[0076] (5) taking out the decellularized amniotic membrane treated in step (4), and then drying, shearing and irradiating and sterilizing it to obtain the decellularized amniotic membrane material;

[0077] Wherein, the natural extract described in step (4) is prepared by the following method:

[0078] (1) Saffron and Ganoderma lucidum are crushed and refluxed with an organic solvent at 56° C. for 1 hour. After the extraction, the extract is taken and concentrated and dried to obtain a saffron-Ganoderma lucidum organic solvent extract; wherein the weight ratio of saffron to Ganoderma lucidum is 5:2; the total weight ratio of saffron and Ganoderma lucidum to the organic solvent is 1 kg:10 L; and the organic solvent is acetone;

[0079] (2) Take the saffron-Ganoderma lucidum organic solvent extract and apply it to an HP-20 macroporous resin column; first use 5 times the column volume of 40% ethanol aqueous solution to elute and remove impurities; then use 5 times the column volume of 60% ethanol aqueous solution to elute, collect the eluate eluted with 60% ethanol aqueous solution, and concentrate and dry to obtain the natural extract.

[0080] The proliferation rates of epidermal cells promoted by the decellularized amniotic membrane materials prepared in Examples 1-2 and Comparative Examples 1-2 are shown in Table 1.

[0081] Table 1. Proliferation rate of epidermal cells promoted by acellular amniotic membrane materials

[0082]

[0083] It can be seen from the experimental results in Table 1 that the decellularized amniotic membrane material prepared in Example 1 has a proliferation rate of epidermal cells of 260%. This indicates that in the preparation method described in the present invention, the decellularized amniotic membrane material prepared by soaking the decellularized amniotic membrane with the natural extract of the present invention has a better effect in promoting cell proliferation.

[0084] It can be seen from the experimental results in Table 1 that the decellularized amniotic membrane material prepared in Example 2 has a proliferation rate of epidermal cells of 540%, which is significantly higher than that of the decellularized amniotic membrane material prepared in Example 1. This shows that in the preparation method described in the present invention, the decellularized amniotic membrane is soaked in a natural extract obtained by further eluting the saffron-Ganoderma lucidum organic solvent extract under the above-mentioned macroporous resin elution conditions of the present invention. Compared with soaking in the saffron-Ganoderma lucidum organic solvent extract as a natural extract, the cell proliferation promoting effect of the prepared decellularized amniotic membrane material can be greatly improved.

[0085] It can be seen from the experimental results in Table 1 that the decellularized amniotic membrane material prepared in Comparative Example 1 has an improved rate of promoting the proliferation of epidermal cells compared with that in Example 1, but the improvement is not large; the decellularized amniotic membrane material prepared in Comparative Example 2 has not only not improved the rate of promoting the proliferation of epidermal cells compared with that in Example 1, but has decreased; this shows that: the macroporous resin elution conditions of the present invention are also very critical; the decellularized amniotic membrane must be soaked in a natural extract prepared by further subjecting the saffron-Ganoderma lucidum organic solvent extract to the above-mentioned macroporous resin elution conditions of the present invention, compared with soaking in the saffron-Ganoderma lucidum organic solvent extract as a natural extract, in order to further significantly improve the cell proliferation promoting effect of the prepared decellularized amniotic membrane material; however, soaking in a natural extract prepared by further subjecting the saffron-Ganoderma lucidum organic solvent extract to other macroporous resin elution conditions cannot further significantly improve the cell proliferation promoting effect of the prepared decellularized amniotic membrane material.

Claims

1. A method for preparing a cell-free amniotic membrane material, characterized in that: The following steps are included: (1) Take the placenta, separate the amniotic membrane from the placenta, and then use a cell scraper to remove the excess The remaining tissue is the amnion without chorionic villi; (2) washing the chorionic amniotic membrane treated in step (1) to obtain washed chorionic amniotic membrane; (3) placing the washed chorionic villus-free amniotic membrane into a decellularizing reagent for decellularization to obtain a decellularized amniotic membrane; (4) washing the decellularized amniotic membrane and then soaking it in an ethanol solution containing natural extracts; (5) taking out the decellularized amniotic membrane treated in step (4), and drying, shearing and irradiating and sterilizing the membrane to obtain the decellularized amniotic membrane material; The natural extract in the aqueous solution containing the natural extract in step (4) is prepared by the following method: Saffron and Ganoderma lucidum are crushed and extracted with an organic solvent, and after the extraction is completed, an extract is taken, concentrated and dried to obtain a saffron-Ganoderma lucidum organic solvent extract; The natural extract is obtained by taking the saffron-Ganoderma lucidum organic solvent extract; The mass fraction of the natural extract in the ethanol solution containing the natural extract in step (4) is 0.5-2%.

2. The method for preparing the decellularized amniotic membrane material according to claim 1, characterized in that: The placenta described in step (1) is screened for pathogens before separation of the amniotic membrane.

3. The method for preparing the decellularized amniotic membrane material according to claim 1, characterized in that: The cleaning described in step (2) refers to cleaning with physiological saline containing penicillin.

4. The method for preparing the decellularized amniotic membrane material according to claim 3, characterized in that: The concentration of penicillin in the penicillin-containing physiological saline is 0.1-0.3 mg / mL.

5. The method for preparing the decellularized amniotic membrane material according to claim 1, characterized in that: The decellularization reagent described in step (3) refers to an aqueous solution containing Triton X-100 and sodium deoxycholate.

6. The method for preparing the decellularized amniotic membrane material according to claim 5, characterized in that: The content of Triton X-100 in the decellularization reagent is 0.1~1g / mL; the content of sodium deoxycholate is 0.1~1g / mL.

7. The method for preparing the decellularized amniotic membrane material according to claim 1, characterized in that: The mass fraction of the natural extract in the ethanol solution containing the natural extract in step (4) is 1%.

8. The decellularized amniotic membrane material prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the decellularized amniotic membrane material according to claim 8 in preparing ocular surface repair materials.

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