CN114225121A

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Publication number: CN114225121A
Application number: CN202111580594.4A
Authority: CN
Inventors: 孔海磊; 黄建祥; 曹阳; 张传文
Original assignee: Wuxi Zhongke Guangyuan Biomaterials Co Ltd
Current assignee: Wuxi Zhongke Guangyuan Biomaterials Co Ltd
Priority date: 2021-12-22
Filing date: 2021-12-22
Publication date: 2022-03-25
Anticipated expiration: 2041-12-22
Also published as: CN114225121B

Abstract

The invention belongs to the technical field of biomedical materials, and particularly relates to an endothelialization part absorbable anti-adhesion fibrous membrane with endothelialization promoting effect and used for repairing a pericardium in a cardiac surgery operation and a preparation method thereof. The PLGA is a degradable functional polymer organic compound, has good biocompatibility and no toxicity, is widely used in pharmaceutical and medical engineering, and the electrostatic spinning technology is a common method for preparing fibrous membranes, has wide application in the fields of tissue engineering, tissue repair, drug carriers and the like, and can prepare the fibrous membranes with different fiber diameters, pores and thicknesses through electrostatic spinning. The surface grafting is a simple method capable of fixing the bioactive material on the surface of the material, and the specific function of promoting endothelialization is added while the original property of the electrospun fiber membrane is kept. The fibrous membrane of the invention can be partially absorbed to reduce the implantation amount, reduce the reaction of inflammation and reduce the foreign body sensation of patients.

Description

Endothelialization part absorbable anti-adhesion fibrous membrane and preparation method thereof

Technical Field

The invention belongs to the technical field of biomedical materials, and particularly relates to an endothelialization part absorbable anti-adhesion fibrous membrane with endothelialization promoting effect and used for repairing a pericardium in a cardiac surgery operation and a preparation method thereof.

Background

With the development of cardiovascular surgery, the number of heart surgery cases at home and abroad is increasing, and the probability of secondary heart surgery is also increasing. If an adhesion condition occurs after cardiac surgery, the risk of re-surgery increases. The anti-adhesion membrane can effectively reduce the condition of postoperative adhesion in the operation, and researches show that the biological valve endothelialization can effectively prevent the formation of thrombus, so that the anti-adhesion membrane is subjected to endothelialization treatment, the adhesion of platelets can be prevented and reduced while the adhesion is ensured.

Most of products mainly used for preventing pericardium from adhesion at present only have an anti-adhesion function.

The adopted raw material is polytetrafluoroethylene, has no degradability, can effectively prevent poststernal adhesion after cardiac surgery, but can cause infection; in addition, the products of absorbable materials are mainly

And

the former is a PLA-PEG polymer film, and the latter is a PLA sheet, which can be used for heart surgery and reduce the risk of adhesion, but the preparation method is difficult and has the risk of rejection reaction.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides an endothelialization part absorbable anti-adhesion fiber membrane and a preparation method thereof, aiming at solving the technical problems that most of the existing products for preventing pericardium from adhesion only have an anti-adhesion function, possibly cause infection, or the products with the absorption function are difficult to prepare and have rejection reaction.

The invention provides a preparation method of an endothelialization part absorbable anti-adhesion fibrous membrane, which has the following specific technical scheme:

the preparation method of the endothelialization part absorbable anti-adhesion fibrous membrane comprises the following steps:

s1, adding the polyvinyl alcohol solution and the boric acid solution into the polytetrafluoroethylene emulsion, mixing, and uniformly stirring to obtain a first spinning solution;

s2, performing electrostatic spinning treatment on the first spinning solution in the step S1 to obtain a PVA/PTFE composite fiber membrane, and performing soaking in purified water, ultrasonic cleaning, washing and drying to obtain a fiber membrane base membrane;

s3, dissolving the polylactic acid-glycolic acid copolymer in a mixed solution of acetone and DMF, and uniformly stirring to obtain a second spinning solution;

s4, performing electrostatic spinning treatment on the second spinning solution obtained in the step S3 on the fiber basement membrane obtained in the step S2 to obtain a fiber membrane, and performing vacuum pumping and hot pressing treatment to obtain a part of absorbable anti-adhesion fiber membrane;

s5, placing part of the absorbable anti-adhesion fibrous membrane in the step S4 into an aqueous solution containing a stabilizer, adding a cross-linking agent and a material for promoting endothelialization, standing for reaction, washing with ethanol, and drying in vacuum to obtain the endothelialized part of the absorbable anti-adhesion fibrous membrane.

In some embodiments, in step S1, the concentration of the polyvinyl alcohol solution is 5 to 20% (W/V), the concentration of the boric acid solution is 5 to 10% (W/V), the concentration of the polytetrafluoroethylene emulsion is 30 to 80% (W/V), and the mass ratio of the polytetrafluoroethylene, the polyvinyl alcohol, and the boric acid in the first spinning solution is (5 to 20): 1: (0 to 0.001). The polyvinyl alcohol solution (PVA) is obtained by dissolving PVA powder with ultrahigh molecular weight in water with the temperature of 30-50 ℃ and uniformly stirring; the average molecular weight of the ultra-high molecular weight PVA powder is 105-107 g/mol.

In certain embodiments, the parameters of electrospinning are as follows: spinning voltage is 25-40 kV, the distance from a spinning nozzle to a receiving roller is 15-25 cm, the capacity of a spinning injector is 10ml, the humidity of a spinning environment is 45-60%, and the temperature is 15-25 ℃; soaking in pure water for 2-5 hours, ultrasonic cleaning for 30-60 minutes, and washing in purified water for 5-10 minutes; the drying mode is vacuum drying, nitrogen with the content of 99.999% is used for drying, and the drying time is 5-8 hours.

In some embodiments, in step S3, the mass ratio of the polylactic acid-glycolic acid copolymer to the acetone to the DMF in the second spinning solution is (1-1.5): 1: (0.5 to 1.5).

In certain embodiments, in step S4, the parameters of electrospinning are as follows: spinning voltage is 25-40 kV, the distance from a spinning nozzle to a receiving roller is 15-25 cm, the capacity of a spinning injector is 10ml, the humidity of a spinning environment is 45-60%, and the temperature is 15-25 ℃; the vacuum pressure of the vacuum drying is 5-10 kPa, and the pressure maintaining time is 6-8 hours; the hot pressing time is 25-30 minutes, and the hot pressing temperature is 40-50 ℃.

In some embodiments, in step S5, the concentration of the aqueous solution of the stabilizer is 1-5% (W/V), and the stabilizer is a triblock copolymer of polyoxyethylene-polyoxypropylene-polyoxyethylene.

In certain embodiments, in step S5, the crosslinker is a bifunctional crosslinker at a concentration of 50mM, the crosslinker is a combination of carbodiimide and N-hydroxysuccinimide at a molar ratio of 1: 1.

in some embodiments, in step S5, the ethanol rinsing time is 5 to 10 minutes, nitrogen with a content of 99.999% is used, and the drying is performed in a vacuum drying oven with a vacuum degree of 5 to 10kPa and a drying time of 5 to 8 hours.

In certain embodiments, in step S5, the endothelialized material is a polypeptide-based material, an antibody-based material, a growth factor-based material, or a statin-based material.

Further, the polypeptide material is RGD, YIGSR, REDV, CAG or SVVYGLR; the antibody material is CD 34; the growth factor material is VEGF, EGF, FGF or PDGF; the statin medicine material is telmisartan.

The invention provides another technical scheme, namely an endothelialization part absorbable anti-adhesion fibrous membrane, and the endothelialization part absorbable anti-adhesion fibrous membrane prepared by the method.

The invention has the following beneficial effects: in the preparation method of the endothelialization part absorbable anti-adhesion fibrous membrane provided by the invention, PLGA is a degradable functional polymer organic compound, has good biocompatibility and no toxicity, and is widely used in pharmaceutical and medical engineering. The electrostatic spinning technology is a common method for manufacturing the fibrous membrane, has wide application in the fields of tissue engineering, tissue repair, drug carriers and the like, and can prepare the fibrous membrane with different fiber diameters, pores and thicknesses through electrostatic spinning. The surface grafting is a simple method capable of fixing the bioactive material on the surface of the material, and the specific function of promoting endothelialization is added while the original property of the electrospun fiber membrane is kept. The endothelialization part prepared by the invention can absorb the anti-adhesion fiber membrane, and the part can absorb the anti-adhesion fiber membrane, thereby reducing the implantation amount, reducing the reaction of inflammation and reducing the foreign body sensation of a patient.

Drawings

FIG. 1 is a flow chart of a preparation method of an endothelialization part absorbable anti-adhesion fiber membrane provided by the invention;

FIG. 2 is an electron microscope scanning image of the endothelialized partial absorbable anti-adhesion fibrous membrane of example 1 of the present invention;

FIG. 3 is an electron microscope scanning image of the endothelialized partial absorbable anti-adhesion fibrous membrane of example 2 of the present invention;

FIG. 4 is the scanning electron microscope image of the endothelial absorbable anti-adhesion fibrous membrane of example 3.

Detailed Description

In order that the objects, aspects and advantages of the present invention will become more apparent, the present invention will be further described in detail with reference to the following detailed description of preferred embodiments thereof, with reference to the accompanying drawings in which fig. 1 to 4 are shown.

Example 1

The preparation method of the endothelialization part absorbable anti-adhesion fibrous membrane provided by the embodiment has the following specific technical scheme:

(1) preparation of fiber film-based film

And preparing the fiber membrane base film by adopting an electrostatic spinning technology. 100g of polyvinyl alcohol powder is dissolved in water at 50 ℃ to prepare polyvinyl alcohol emulsion with the concentration of 20%, 500g of polytetrafluoroethylene and 0.1g of boric acid are respectively prepared into solutions with the concentrations of 50% and 10%, the three solutions are mixed, and the mixture is stirred for 5 hours at the temperature of 50 ℃ to obtain first spinning solution. And then injecting the spinning solution into a 10ml injector, and preparing the fiber membrane by using an electrostatic spinning method, wherein the spinning voltage is 30kV, the distance between a spinning nozzle and a collecting roller is 20cm, the spinning environment humidity is 45-60%, and the temperature is 15-25 ℃. And putting the obtained fiber membrane into an ultrasonic cleaning machine, soaking for 2 hours in purified water, then ultrasonically cleaning for 30 minutes, then washing for 5 minutes by using the purified water, finally putting into a vacuum drying oven, and drying for 5 hours by using nitrogen to obtain the fiber membrane basal membrane.

(2) Preparation of partially absorbable anti-adhesion fibrous membrane

And preparing a part of absorbable anti-adhesion fibrous membrane by adopting an electrostatic spinning technology. 100g of PLGA was dissolved in a mixed solution of 100g of acetone and 100g of DMF, and stirred at 50 ℃ for 5 hours to obtain a second spinning solution. And (2) filling the spinning solution into a 10ml injector, placing the fiber membrane substrate membrane on a collecting roller, enabling a spinning nozzle to be 20cm away from the collecting roller, spinning at the voltage of 30kV, the spinning environment humidity of 45-60% and the temperature of 15-25 ℃, spinning a layer of fiber membrane on the polytetrafluoroethylene fiber membrane substrate membrane, and obtaining the double-layer fiber membrane after spinning. And (3) vacuumizing the fiber membrane in a vacuum drying oven, wherein the vacuum pressure is 5kPa, and keeping the pressure for 8 hours. And (3) carrying out hot pressing on the vacuumized double-layer fiber membrane in an electric heating constant temperature incubator for 25 minutes at the hot pressing temperature of 45 ℃, and finally obtaining the partially absorbable anti-adhesion fiber membrane.

(3) Grafting an endothelialization material on a fiber membrane base membrane:

putting part of absorbable anti-adhesion fiber membrane into a stabilizer (polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymer in the form of PEO-PPO-PEO) solution with the concentration of 5% (W/V), adding a crosslinking agent (carbodiimide and N-hydroxysuccinimide according to the molar ratio of 1: 1) with the concentration of 50mM and a material (REDV) for promoting endothelialization for 2 hours, washing with ethanol for 10 minutes, vacuumizing to 5kPa in a vacuum drying oven, and drying with nitrogen with the content of 99.999% for 5 hours to finally obtain the endothelialized part absorbable anti-adhesion fiber membrane.

This example also provides an endothelialized, partially absorbable anti-adhesion fibrous membrane prepared according to the above method, as shown in figure 2.

Example 2

(1) preparation of fiber film-based film

And preparing the fiber membrane base film by adopting an electrostatic spinning technology. 25g of polyvinyl alcohol powder is dissolved in water at 50 ℃ to prepare polyvinyl alcohol emulsion with the concentration of 5%, 300g of polytetrafluoroethylene and 0.05g of boric acid are respectively prepared into solutions with the concentrations of 30% and 5%, the three solutions are mixed, and the mixture is stirred for 5 hours at the temperature of 50 ℃ to obtain first spinning solution. And then injecting the spinning solution into a 10ml injector, and preparing the fiber membrane by using an electrostatic spinning method, wherein the spinning voltage is 25kV, the distance between a spinning nozzle and a collecting roller is 15cm, the spinning environment humidity is 45-60%, and the temperature is 15-25 ℃. And putting the obtained fiber membrane into an ultrasonic cleaning machine, soaking for 3 hours in purified water, then ultrasonically cleaning for 45 minutes, then washing for 7 minutes by using the purified water, finally putting into a vacuum drying oven, and drying for 7 hours by using nitrogen to obtain the fiber membrane basal membrane.

(2) Preparation of partially absorbable anti-adhesion fibrous membrane

And preparing a part of absorbable anti-adhesion fibrous membrane by adopting an electrostatic spinning technology. 120g of PLGA was dissolved in a mixed solution of 100g of acetone and 50g of DMF, and stirred at a temperature of 50 ℃ for 5 hours to obtain a second spinning solution. And (2) filling the spinning solution into a 10ml injector, placing the fiber membrane substrate membrane on a collecting roller, spinning a spinneret at a distance of 15cm from the collecting roller, spinning at a spinning voltage of 25kV and a spinning environment humidity of 45-60% at a temperature of 15-25 ℃, spinning a layer of fiber membrane on the polytetrafluoroethylene fiber membrane substrate membrane, and obtaining the double-layer fiber membrane after spinning. And (3) vacuumizing the fiber membrane in a vacuum drying oven, wherein the vacuum pressure is 6kPa, and keeping the pressure for 8 hours. And (3) carrying out hot pressing on the vacuumized double-layer fiber membrane in an electric heating constant temperature incubator for 27 minutes at the hot pressing temperature of 50 ℃, and finally obtaining the partially absorbable anti-adhesion fiber membrane.

(3) Grafting an endothelialization material on a fiber membrane base membrane:

putting a part of absorbable anti-adhesion fiber membrane into a stabilizer (polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymer in the form of PEO-PPO-PEO) solution with the concentration of 1% (W/V), adding a cross-linking agent (carbodiimide and N-hydroxysuccinimide according to the molar ratio of 1: 1) with the concentration of 50mM and a material for promoting endothelialization, reacting for 2 hours, washing with purified water for 5 minutes, vacuumizing to 8kPa in a vacuum drying oven, and drying with nitrogen with the content of 99.999% for 7 hours to finally obtain the endothelialized part absorbable anti-adhesion fiber membrane.

This example also provides an endothelialized, partially absorbable anti-adhesion fibrous membrane prepared according to the above method, as shown in figure 3.

Example 3

(1) preparation of fiber film-based film

And preparing the fiber membrane base film by adopting an electrostatic spinning technology. 50g of polyvinyl alcohol powder is dissolved in water at 50 ℃ to prepare polyvinyl alcohol emulsion with the concentration of 10%, 800g of polytetrafluoroethylene and 0.08g of boric acid are respectively prepared into solutions with the concentrations of 80% and 8%, the three solutions are mixed, and the mixture is stirred for 5 hours at the temperature of 50 ℃ to obtain first spinning solution. And then injecting the spinning solution into a 10ml injector, and preparing the fiber membrane by using an electrostatic spinning method, wherein the spinning voltage is 40kV, the distance between a spinning nozzle and a collecting roller is 25cm, the spinning environment humidity is 45-60%, and the temperature is 15-25 ℃. And putting the obtained fiber membrane into an ultrasonic cleaning machine, soaking the fiber membrane in purified water for 5 hours, then ultrasonically cleaning the fiber membrane for 60 minutes, then flushing the fiber membrane for 10 minutes by using ethanol, finally putting the fiber membrane into a vacuum drying oven, and drying the fiber membrane for 8 hours by using nitrogen to obtain the fiber membrane-based membrane.

(2) Preparation of partially absorbable anti-adhesion fibrous membrane

And preparing a part of absorbable anti-adhesion fibrous membrane by adopting an electrostatic spinning technology. 150g of PLGA was dissolved in a mixed solution of 100g of acetone and 150g of DMF, and stirred at 50 ℃ for 5 hours to obtain a second spinning solution. And (2) filling the spinning solution into a 10ml injector, placing the fiber membrane substrate membrane on a collecting roller, enabling a spinning nozzle to be 25cm away from the collecting roller, spinning at the voltage of 40kV, the spinning environment humidity of 45-60% and the temperature of 15-25 ℃, spinning a layer of fiber membrane on the polytetrafluoroethylene fiber membrane substrate membrane, and obtaining the double-layer fiber membrane after spinning. And (3) vacuumizing the fiber membrane in a vacuum drying oven, wherein the vacuum pressure is 10kPa, and keeping the pressure for 6 hours. And (3) carrying out hot pressing on the vacuumized double-layer fiber membrane in an electric heating constant temperature incubator for 30 minutes at the hot pressing temperature of 40 ℃, and finally obtaining the partially absorbable anti-adhesion fiber membrane.

(3) Grafting an endothelialization material on a fiber membrane base membrane:

putting a part of absorbable anti-adhesion fiber membrane into a stabilizer (polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymer in the form of PEO-PPO-PEO) solution with the concentration of 5% (W/V), adding a cross-linking agent (carbodiimide and N-hydroxysuccinimide according to the molar ratio of 1: 1) with the concentration of 50mM and a material for promoting endothelialization into the absorbable anti-adhesion fiber membrane for reaction for 2 hours, flushing the absorbable anti-adhesion fiber membrane with ethanol for 7 minutes, vacuumizing the absorbable anti-adhesion fiber membrane to 10kPa in a vacuum drying oven, and drying the absorbable anti-adhesion fiber membrane for 6 hours by using nitrogen with the content of 99.999 percent to finally obtain the endothelialization part absorbable anti-adhesion fiber membrane.

This example also provides an endothelialized, partially absorbable anti-adhesion fibrous membrane prepared according to the above method, as shown in figure 4.

As shown in fig. 2-3, different fiber membranes of different fiber diameters, pores and thicknesses can be prepared with different voltage and spinning height.

The above description is only for the purpose of illustrating preferred embodiments of the present invention and is not to be construed as limiting the invention, and the present invention is not limited to the above examples, and those skilled in the art should also be able to make various changes, modifications, additions or substitutions within the spirit and scope of the present invention.

Claims

1. The preparation method of the endothelialization part absorbable anti-adhesion fibrous membrane is characterized by comprising the following steps:

2. The method for preparing the endothelialization partially absorbable anti-adhesion fiber membrane according to claim 1, wherein in step S1, the concentration of the polyvinyl alcohol solution is 5-20% (W/V), the concentration of the boric acid solution is 5-10% (W/V), the concentration of the polytetrafluoroethylene emulsion is 30-80% (W/V), and the mass ratio of the polytetrafluoroethylene, the polyvinyl alcohol and the boric acid in the first spinning solution is (5-20): 1: (0 to 0.001).

3. The method for preparing the endothelialized partially absorbable anti-adhesion fibrous membrane according to claim 1, wherein in step S2, the parameters of electrospinning are as follows: spinning voltage is 25-40 kV, the distance from a spinning nozzle to a receiving roller is 15-25 cm, the capacity of a spinning injector is 10ml, the humidity of a spinning environment is 45-60%, and the temperature is 15-25 ℃; soaking in pure water for 2-5 hours, ultrasonic cleaning for 30-60 minutes, and washing in purified water for 5-10 minutes; the drying mode is vacuum drying, nitrogen with the content of 99.999% is used for drying, and the drying time is 5-8 hours.

4. The method for preparing the endothelialization partially absorbable anti-adhesion fiber membrane according to claim 1, wherein in step S3, the mass ratio of the polylactic acid-glycolic acid copolymer, the acetone and the DMF in the second spinning solution is (1-1.5): 1: (0.5 to 1.5).

5. The method for preparing the endothelialized partially absorbable anti-adhesion fibrous membrane according to claim 1, wherein in step S4, the parameters of electrospinning are as follows: spinning voltage is 25-40 kV, the distance from a spinning nozzle to a receiving roller is 15-25 cm, the capacity of a spinning injector is 10ml, the humidity of a spinning environment is 45-60%, and the temperature is 15-25 ℃; the vacuum pressure of the vacuum drying is 5-10 kPa, and the pressure maintaining time is 5-8 hours; the hot pressing time is 25-30 minutes, and the hot pressing temperature is 40-50 ℃.

6. The method for preparing the endothelialized partially absorbable anti-adhesion fiber membrane according to claim 1, wherein in step S5, the concentration of the aqueous solution of the stabilizer is 1-5% (W/V), and the stabilizer is a triblock copolymer of polyoxyethylene-polyoxypropylene-polyoxyethylene; the cross-linking agent is a bifunctional cross-linking agent, the concentration of the cross-linking agent is 50mM, the cross-linking agent is a composition of carbodiimide and N-hydroxysuccinimide, and the molar ratio of the carbodiimide to the N-hydroxysuccinimide is 1: 1.

7. the method for preparing the endothelialized partially absorbable anti-adhesion fibrous membrane according to claim 1, wherein in step S5, ethanol washing is performed for 5-10 minutes, nitrogen with a content of 99.999% is used, and drying is performed in a vacuum drying oven with a vacuum degree of 5-10 kPa and a drying time of 5-8 hours.

8. The method for preparing the endothelialized partially absorbable anti-adhesion fibrous membrane of claim 1, wherein in step S5, the endothelialized material is a polypeptide material, an antibody material, a growth factor material or a statin drug material.

9. The method for preparing the endothelialized partially absorbable anti-adhesion fibrous membrane of claim 8, wherein the polypeptide material is RGD, YIGSR, REDV, CAG, or SVVYGLR; the antibody material is CD 34; the growth factor material is VEGF, EGF, FGF or PDGF; the statin medicine material is telmisartan.

10. An endothelialized partially absorbable anti-adhesion fibrous membrane, characterized in that the endothelialized partially absorbable anti-adhesion fibrous membrane produced by the method according to any one of claims 1 to 9.