CN114108184B

CN114108184B - Degradable graphene composite electrostatic spinning fiber film, preparation method and application

Info

Publication number: CN114108184B
Application number: CN202111386861.4A
Authority: CN
Inventors: 相文佩; 肖先金; 晏亮
Original assignee: Hunan Jialin Stem Cell Technology Co ltd
Current assignee: Hunan Jialin Stem Cell Technology Co ltd
Priority date: 2021-11-22
Filing date: 2021-11-22
Publication date: 2023-08-01
Anticipated expiration: 2041-11-22
Also published as: CN114108184A

Abstract

The invention belongs to the fields of biomedical materials and tissue engineering, and discloses a novel technology for applying a biocompatible degradable polylactic acid/graphene composite fiber film to ovarian tissue transplantation. Carrying out surface modification on graphene oxide by using a polymer to obtain functionalized graphene oxide; then dispersing graphene oxide or functionalized graphene oxide in a solvent A by ultrasonic to obtain a solution A; adding a high molecular polymer into the solvent B, and stirring to obtain a transparent solution B; mixing the solution A and the solution B, stirring, and performing ultrasonic treatment to obtain an electrostatic spinning solution; and carrying out electrostatic spinning on the obtained electrostatic spinning solution. The graphene composite electrostatic spinning fiber film prepared by the invention has stable structure, no obvious defect on the surface of the fiber, controllable continuous single fiber and perfect application in various aspects such as medicine slow release, air filtration, tissue engineering and the like. The invention provides a clinical application of a degradable graphene composite nanofiber membrane in ovarian tissue cryopreservation-transplantation.

Description

Degradable graphene composite electrostatic spinning fiber film, preparation method and application

Technical Field

The invention belongs to the technical field of spinning, and particularly relates to a degradable graphene composite electrostatic spinning fiber film, a preparation method and application thereof.

Background

The current human fertility decline has become a global problem, infertility has become the third biggest disease in the 21 st century, and women in the new era are facing increasingly serious fertility disorders; women of childbearing age have an increased proportion of childbearing age. In addition, with the continuous development of tumor diagnosis and treatment technology, young cancer patients can survive for a long time after treatment, so that the preservation of fertility is one of the most popular topics in the current reproduction field. Fertility preservation refers to the use of surgical, pharmaceutical or laboratory techniques to assist females or males at risk of sterility, protecting and preserving their ability to produce genetic offspring. In other words, the method is that the ovum, embryo, ovary tissue and the like of the patient which cannot be pregnant or is required to be frozen at ultralow temperature temporarily are kept, and the patient is resuscitated when the pregnancy is required, so that the patient is helped to obtain the offspring of the blood parent. Cryopreservation of ovarian tissues is an effective fertility preservation method, doctors obtain the ovarian cortex of a patient through an operation method, store the ovarian cortex in liquid nitrogen after freezing, and resuscitate and transplant the ovarian cortex into the patient when the physical condition of the patient allows or has fertility requirements. With the development of refrigeration technology, at the end of the 20 th century, several studies have found that the ovarian group can recover normal follicular morphology after thawing. Thereafter, ovarian tissue cryopreservation transplantation techniques are beginning to be applied clinically. Ovarian tissue cryopreservation is suitable for protecting fertility and endocrine function of ovary of tumor and non-tumor disease patients, and the best indication is pre-pubertal tumor patients, tumor patients needing immediate radiotherapy and chemotherapy treatment, and patients suffering from hormone sensitive tumor. In clinic, laparoscope is adopted to obtain materials of ovarian tissues, and ovarian cortex is transplanted on the peritoneum of the outer side wall of an ovary during transplantation. Although ovarian tissue transplantation has achieved encouraging results, there have been over a hundred live fetuses reported to date, but there are still key problems: the transplanted ovary cortex is slowly regenerated, a great number of follicles are lost due to ischemia during the regeneration of the blood vessel, and the function of the ovary is not recovered well, so that the transplanted ovary is not viable finally. Thus, there is a need to establish new techniques and methods to improve and accelerate angiogenesis of transplanted ovarian tissue, promote survival of transplanted ovarian tissue, and reduce follicular loss.

The solution of the key problems provides a new treatment scheme for the ovarian tissue transplantation, and has important scientific significance and wide application value for the fertility preservation field and even the whole reproductive medicine.

The invention provides a polylactic acid/graphene oxide/stem cell composite nanofiber membrane which is used for supporting transplanted ovarian tissues, promoting survival of the transplanted ovarian tissues and improving the success rate of transplantation.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a polylactic acid/graphene oxide/stem cell composite electrospun fiber film which is used for supporting the implantation of ovarian tissues and promoting the survival of the ovarian tissues and improving the success rate of implantation.

The invention is realized in the following way:

the invention provides a preparation method of a Graphene (GO) composite electrostatic spinning fiber film, which comprises the following steps:

step 1, PEG-NH is used by EDC and other methods ₂ Surface modification is carried out on graphene oxide by polymers such as PEI and the like to obtain functionalized graphene oxide (F-GO); then dispersing graphene oxide or F-GO in a solvent A by ultrasonic to obtain a solution A;

the polymer further comprises: single arm PEG-NH ₂ Double arm PEG-NH ₂ Four-arm PEG-NH ₂ Six-arm PEG-NH ₂ with-NH-PEI, etc ₂ And a polymer with groups and good biocompatibility.

Step 2, adding a high molecular polymer with a certain molecular weight into the solvent B, and stirring for 1-12 hours at a certain temperature to obtain a transparent solution B;

step 3, mixing the solution A and the solution B according to a certain volume ratio, stirring for a plurality of hours, performing ultrasonic treatment for 10-30 min, and standing for 1-12 h to obtain an electrostatic spinning solution;

and 4, carrying out electrostatic spinning on the obtained electrostatic spinning solution (adopting a 10 mL injector) to obtain the degradable graphene composite electrospun fiber film.

Further, in the step 1, the solvent A is dimethylformamide DMF;

the modification of the graphene oxide can effectively improve the dispersibility of the graphene oxide in the solvent A, facilitate the subsequent mixing with the high-molecular polymer, and avoid phase delamination or agglomeration at high concentration.

Further, the volume ratio of the mass of graphene oxide (or F-GO) to the solvent A in the step 1 is (0.1-2.0 g): 1 mL;

further, in step 2, solvent B is dichloromethane DCM;

the mass ratio of the high polymer to the solvent B in the step 2 is (8.0-16.0 g): 100.0 g;

the high molecular polymer in the step 2 is poly L-lactic acid, poly D-lactic acid, poly L, D-lactic acid, poly L-lactic acid-glycolic acid copolymer, the number average molecular weight is 10 ten thousand-40 ten thousand;

further, the volume ratio of the solvent A to the solvent B is (0.5 to 1.0) mL): 1.0mL, the balance of the volume is supplemented with pure solvent A;

wherein, the ratio of the solvent A to the solvent B is proper, if the solvent A is too much, the volatilization is too slow, so that the solvent A and the solvent B are mutually stuck on the plate, and even the generated fiber can be redissolved; if the solvent B is too much, volatilization is too fast, so that a spinning nozzle is blocked to prevent spinning, and the fiber can not be completely split and thinned after being dried very fast, so that the diameter of the fiber is larger. Meanwhile, the different proportions of solvents can influence the viscosity, conductivity and other parameters of the spinning solution, and influence the morphology and performance of the fiber.

The mass ratio of graphene oxide (or F-GO) to the high molecular polymer is (0.05-8.0): 100;

the presence of graphene oxide or F-GO can improve the conductivity of the electrospinning solution, facilitate electrostatic atomization, crack micro jet flow and improve the motion behavior in an electric field; meanwhile, the interaction force between high molecular polymers can be enhanced, and the mechanical property is improved.

Further, in the step 4, the electrostatic spinning has a positive voltage of 15-25 kV, a negative voltage of 0-5 kV, a spinning speed of 0.05-0.5 mm/min, a distance between a spinning nozzle and a receiver of 15-25 cm, and an electrostatic spinning time of 1-16 h.

Finally, the graphene composite electrostatic spinning fiber film which is degradable, good in mechanical property and smooth in surface is prepared.

Secondly, preparing a degradable polylactic acid (PLLA)/graphene composite electrostatic spinning fiber film with high biocompatibility by using the method in the first step, which comprises the following steps:

and step 1, preparing graphene oxide nanosheets, namely synthesizing graphene oxide by taking natural graphite as a raw material through modified hammer Ma Fa, and then performing ultrasonic treatment. And then centrifuged at 12000 rpm for 30 minutes to remove any unoxidized graphite flakes or unoxidized graphite oxide to obtain a graphite oxide dispersion. The resulting supernatant was collected and then lyophilized for 2 days.

And 2, preparing a polylactic acid/polyethylene glycol composite spinning dispersion. Polylactic acid (PLLA) is completely dissolved in an organic solvent system to form a uniform transparent polymer solution. Meanwhile, graphene oxide is dispersed into the same organic solvent system. Subsequently, the graphene oxide suspension was slowly added to the PLLA solution prepared in advance to obtain PLLA/graphene oxide composite spinning dispersion containing different graphene oxide concentrations (0.5, 1.0, 4.0 wt%). Before use, all dispersions were homogenized in a water bath sonicator and then left to stand overnight.

And 3, preparing the PLLA/GO composite electrospun fiber film by adopting an electrospinning technology. The PLLA/GO complex dispersion was placed in a commercial glass syringe (10 ml) equipped with a steel needle (27 g). Electrospinning was performed with a high voltage power supply of 15 kV and a dispersion of PLLA/graphene oxide composite was fed into the needle tip using a syringe pump at a speed of 0.30 mm/min. The electrospun nanofibers were collected on a target drum 20 cm from the syringe tip at a drum speed of 120 rpm. All experiments were performed at 25℃and a relative humidity of about 50%. Finally, the samples were freeze-dried for 48 hours to remove any organic chemicals and then cut to the same gauge for use.

(II) construction of POF model mice. 8 weeks C57BL/6 female mice are selected to construct a POF mouse model, and the purchased mice are normally fed for 2-3 days after entering the center of SPF-class animals, and the weights of the mice are weighed and recorded. Randomly dividing mice into a POF group and a blank control group, weighing the mice in the POF group before daily injection of medicaments, weighing cisplatin with corresponding doses according to 1.5mg/kg, preparing normal saline, performing intraperitoneal injection, and continuously injecting for 10 days; mice in the placebo group were injected with an equal amount of physiological saline. After 1 month, 10% chloral hydrate was anesthetized by intraperitoneal injection, and ovarian tissue was obtained after cervical dislocation after blood was obtained from the outer canthus of the eye. Serum is taken to detect hormone levels such as FSH, AMH and the like by an ELISA kit, ovarian tissues are taken to be fixed, and the number of follicles is counted.

And (III) ovarian tissue transplantation. Adult normal 8-week female C57BL mice are selected, and after sacrifice by cervical dislocation, ovarian tissues are taken and sheared into equal-sized ovarian tissue blocks, and the ovarian tissue blocks are transplanted into the ovarian cysts of the POF mice in situ. POF mice (50 mg/kg) were anesthetized by intraperitoneal injection with 1% sodium pentobarbital under sterile conditions. After conventional sterilization, the skin and muscle were incised at the costal horn to fully expose the right oviduct and ovarian tissue of the mice. Under a stereoscopic microscope, a small opening is cut on the ovarian envelope by using microsurgery scissors, and the ovarian tissue coated by the PLLA/GO composite electrospun fiber film is sent under the ovarian envelope by using microsurgery forceps. Kneading the ovarian envelope small opening with heated micro forceps, suturing back incision, feeding normally, taking out the ovarian tissue after 1, 2 and 3 months after transplantation, and evaluating the survival condition of the transplanted ovarian tissue by various means such as microscopic observation, immunohistochemistry, molecular marker detection and the like.

The invention further aims to provide the degradable graphene composite electrostatic spinning fiber film prepared by the preparation method of the degradable graphene composite electrostatic spinning fiber film.

The invention further aims at providing a medicine slow-release film prepared by using the degradable graphene composite electrostatic spinning fiber film.

The invention further aims to provide an air filtering membrane prepared by using the degradable graphene composite electrostatic spinning fiber film.

By combining all the technical schemes, the invention has the advantages and positive effects that: the solvent used in the invention has low cost, no pollution and low toxicity, and the abandoned product has no adverse effect on the environment basically; the polylactic acid/graphene composite electrospinning fiber films with different sizes can be obtained by changing the structure of a spray head, controlling experimental parameters and the like; the polylactic acid/graphene composite electrospun fiber film prepared by the invention has stable structure, no obvious defect on the surface of the fiber, controllable continuous single fiber and obviously improved mechanical property, and can be used for improving the application of the polylactic acid/graphene composite electrospun fiber film in the aspects of medicine slow release, air filtration, tissue engineering and the like.

The degradable polylactic acid/graphene coincidence electrospinning fiber film provided by the invention has a remarkable effect in the ovarian tissue cryopreservation-transplantation technology. Experimental results show that the PLLA/GO composite electrospun fiber membrane can promote the establishment of blood supply connection between the transplanted ovary and the in-situ ovary and the regeneration of blood vessels at the transplanted site, and the novel material has the advantages of small volume, stable physical property, convenient operation, easy fusion, strong operability in operation, great improvement of the survival quality of the ovary after the ovarian transplantation operation and great application prospect in clinic.

Effects and advantages obtained by combining experimental or experimental data with the comparison of the prior art: compared with the prior art, the method disclosed by the invention is environment-friendly, and can be used for efficiently preparing the degradable graphene composite electrostatic spinning fiber film which is good in biocompatibility and strong in mechanical property and can be used for remarkably improving and accelerating vascularization of transplanted ovarian tissues.

Table 1 is a comparison of mechanical properties of the degradable graphene composite electrospun fiber films prepared in the examples of the present invention.

Drawings

For a clearer description of the technical solutions of the embodiments of the present application, the following description will briefly explain the drawings needed in the embodiments of the present application, and it is obvious that the drawings described below are only some embodiments of the present application, and that other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art;

FIG. 1 is a flow chart of a preparation method of a degradable graphene composite electrospun fiber film provided by an embodiment of the invention;

fig. 2 is an SEM image of the degradable graphene composite electrospun fiber film prepared in embodiment 1 provided in the example of the present invention;

fig. 3 is an SEM image of the degradable graphene composite electrospun fiber film prepared in embodiment 2 provided in the example of the present invention;

fig. 4 is an SEM image of a degradable graphene composite electrospun fiber film prepared according to embodiment 3 provided in the example of the present invention;

fig. 5 is an SEM image of a degradable graphene composite electrospun fiber film prepared according to embodiment 4 provided in the example of the present invention;

fig. 6 is a digital picture of the degradable graphene composite electrospun fiber film prepared in embodiment 3 provided in the example of the present invention;

FIG. 7 is a Raman spectrum of the degradable graphene composite electrospun fiber film prepared in embodiments 3 and 4 provided in the examples of the present invention;

fig. 8 is a mechanical property diagram of the degradable graphene composite electrospun fiber film prepared in embodiment 1 provided in the example of the present invention;

FIG. 9 is a graph showing that the graphene composite nanofiber membrane promotes the fusion of co-transplanted ovarian tissue and premature senility ovary of a model mouse, and the fusion surface has obvious vascular implantation and good blood supply effect compared with a control group and an ovary combined polylactic acid nanofiber membrane transplantation group;

in the figure: in-situ ovaries shown by oval black dotted lines with large diameters, transplanted ovaries shown by oval dotted lines with small diameters, and arrows indicate transplanted fusion surface neovascular;

fig. 10 is a schematic diagram showing that, compared with a control group and an ovarian combined polylactic acid nanofiber membrane transplantation group, the graphene composite nanofiber membrane provided by the embodiment of the invention has the advantages that the number of follicles of an overall transplanted ovary is increased by promoting the survival of the transplanted ovary, and the survival quality of the transplanted ovary is better;

fig. 11 is a columnar schematic diagram showing that the graphene composite nanofiber membrane can promote survival of transplanted ovaries so as to increase the number of follicles of the whole transplanted ovaries, and the survival quality of the transplanted ovaries is better compared with a control group and an ovarian combined polylactic acid nanofiber membrane transplantation group.

Detailed Description

The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. The present invention will be described in detail with reference to the accompanying drawings.

As shown in fig. 1, the invention firstly provides a preparation method of a degradable graphene composite electrostatic spinning fiber film, which comprises the following steps:

s101, PEG-NH is used by EDC and other methods ₂ Surface modification is carried out on graphene oxide by polymers such as PEI and the like to obtain functionalized graphene oxide (F-GO); and then dispersing graphene oxide or F-GO in the solvent A by ultrasonic to obtain a solution A.

S102, adding a high molecular polymer with a certain molecular weight into the solvent B, and stirring for 1-12 hours at a certain temperature to obtain a transparent solution B.

S103, mixing the solution A and the solution B according to a certain volume ratio, stirring for a plurality of hours, performing ultrasonic treatment for 10-30 min, and standing for 1-12 h to obtain the electrostatic spinning solution.

And S104, carrying out electrostatic spinning on the obtained electrostatic spinning solution (adopting a 10 mL injector) to obtain the degradable graphene composite electrospun fiber film.

Preferably, in step S101, solvent a is DMF;

Preferably, the volume ratio of the mass of graphene oxide (or F-GO) to the solvent a in step S101 is (0.1-2.0 g): 1 mL;

preferably, solvent B in step S102 is DCM.

Preferably, the mass ratio of the polymer to the solvent B in step S102 is (8.0 to 16.0 g): 100.0 g.

Preferably, the high molecular polymer in step S102 is poly L-lactic acid, poly D-lactic acid, poly L, D-lactic acid, poly L-lactic acid-glycolic acid copolymer, the number average molecular weight of which is 10-40 ten thousand.

Preferably, the volume ratio of the solvent a to the solvent B in step S103 is (0.5 to 1.0 mL): 1.0 The mL, the difference volume was made up with pure solvent A.

Wherein, the ratio of the solvent A to the solvent B is proper, if the solvent A is too much, the volatilization is too slow, so that the solvent A and the solvent B are mutually stuck on the plate, and even the generated fiber can be redissolved; if the solvent B is too much, volatilization is too fast, so that a spinning nozzle is blocked to prevent spinning, and the fiber is quickly dried and cannot be completely split and thinned, so that the diameter of the fiber is large. Meanwhile, the different proportions of solvents can influence the viscosity, conductivity and other parameters of the spinning solution, and influence the morphology and performance of the fiber.

Preferably, in step S103, the mass ratio of graphene oxide (or F-GO) to the high molecular polymer is (0.05-8.0): 100.

Preferably, in the step S104, the electrostatic spinning has a positive voltage of 15-25 kV, a negative voltage of 0-5 kV, a spinning speed of 0.05-0.5 mm/min, a distance between a spinning nozzle and a receiver of 15-25 cm, and an electrostatic spinning time of 1-16 h. Finally, the graphene composite electrostatic spinning fiber film which is degradable, good in mechanical property and smooth in surface is prepared.

The preparation method of the graphene composite electrostatic spinning fiber film is applied to preparation of degradable fiber films with high biocompatibility.

Embodiment case 1:

dispersing graphene oxide into DMF, mixing with DCM solution of polylactic acid, stirring for several hours, performing ultrasonic treatment for 10-30 min, standing for 1-12 h to obtain electrostatic spinning solution, and finally performing electrostatic spinning (using a 10 mL injector) to obtain the degradable graphene composite electrospun fiber film.

In the step 1, the volume ratio of the mass of graphene oxide (or F-GO) to the solvent A is 1:1 mL.

The mass ratio of the high polymer to the solvent B in the step 2 is 10 g:100 g.

In the step 2, the high molecular polymer is poly L-lactic acid, and the number average molecular weight is 30 ten thousand.

Wherein, the volume ratio of the solvent A to the solvent B is 1 mL:1 mL.

Wherein, the mass ratio of graphene oxide (or F-GO) to high molecular polymer is 0.5:100.

in the step 4, the electrostatic spinning has positive voltage of 16 kV, negative voltage of 2 kV, spinning speed of 0.01 mm/min, distance between a spinning nozzle and a receiver of 20 cm, and electrostatic spinning time of 6-12 h.

Embodiment case 2:

this embodiment differs from embodiment 1 in that:

the volume ratio of graphene oxide (or F-GO) to solvent A in step 1 is 0.5:1 mL.

The mass ratio of the high polymer to the solvent B in the step 2 is 12 g:100 g.

In the step 2, the high molecular polymer is poly D-lactic acid, and the number average molecular weight is 30 ten thousand.

Wherein, the volume ratio of the solvent A to the solvent B is 0.5mL:1 mL.

Wherein, the mass ratio of graphene oxide (or F-GO) to the high molecular polymer is 1.0:100.

in the step 4, the electrostatic spinning has a positive voltage of 20 kV, a negative voltage of 1 kV, a spinning speed of 0.02 mm/min, a distance between a spinning nozzle and a receiver of 15 cm, and an electrostatic spinning time of 4-8 h.

Embodiment 3:

this embodiment differs from embodiment 1 or embodiment 2 in that:

The mass ratio of the high polymer to the solvent B in the step 2 is 8 g:100 g.

In the step 2, the high molecular polymer is poly D-lactic acid, and the number average molecular weight is 40 ten thousand.

Wherein, the volume ratio of the solvent A to the solvent B is 1 mL:1 mL.

Wherein, the mass ratio of graphene oxide (or F-GO) to high molecular polymer is 0.1:100.

in the step 4, the electrostatic spinning has a positive voltage of 18 kV, a negative voltage of 0.5kV, a spinning speed of 0.1 mm/min, a distance between a spinning nozzle and a receiver of 21 cm, and an electrostatic spinning time of 3-6 h.

Embodiment 4:

this embodiment differs from embodiment 1, embodiment 2 or embodiment 3 in that

In the step 1, the volume ratio of the mass of graphene oxide (or F-GO) to the solvent A is 2:1 mL.

The mass ratio of the high polymer to the solvent B in the step 2 is 15 g:100 g.

In the step 2, the high molecular polymer is poly D-lactic acid, and the number average molecular weight is 25 ten thousand.

Wherein, the volume ratio of the solvent A to the solvent B is 0.8 mL:1 mL.

Wherein, the mass ratio of graphene oxide (or F-GO) to high molecular polymer is 6:100.

in the step 4, the electrostatic spinning has a positive voltage of 25 kV, a negative voltage of 0.5kV, a spinning speed of 0.04 mm/min, a distance between a spinning nozzle and a receiver of 25 cm and an electrostatic spinning time of 4-8 h.

In the present invention, fig. 2 is an SEM image of the degradable graphene composite electrospun fiber film prepared in embodiment 1.

Fig. 3 is an SEM image of the degradable graphene composite electrospun fiber film prepared in embodiment 2.

Fig. 4 is an SEM image of the degradable graphene composite electrospun fiber film prepared in embodiment 3.

Fig. 5 is an SEM image of the degradable graphene composite electrospun fiber film prepared in embodiment 4.

Fig. 6 is a digital picture of the degradable graphene composite electrospun fiber film prepared in embodiment 3.

Fig. 7 is a raman spectrum of the degradable graphene composite electrospun fiber film prepared in embodiments 3 and 4.

Fig. 8 is a mechanical property diagram of the degradable graphene composite electrostatic spinning fiber film prepared in embodiment 1.

The invention is further described below in connection with specific application examples.

Application example

According to the invention, animal experiments show that the graphene composite nanofiber membrane can promote angiogenesis and fusion between in-situ ovaries and transplanted ovaries of mice with premature ovarian failure.

The invention carries out ovarian in-situ transplantation on single-side ovaries of an ovarian premature senility model mouse, which is divided into three groups, namely an ovarian transplantation group (contrast), an ovarian combined polylactic acid nanofiber membrane transplantation group and an ovarian combined graphene composite nanofiber membrane transplantation group, and experimental results show that:

compared with the control group and the ovarian combined polylactic acid nanofiber membrane transplantation group, the graphene composite nanofiber membrane promotes the fusion of co-transplanted ovarian tissue and premature senility ovary of a premature senility model mouse, the fusion surface has obvious vascular implantation, and the blood supply of the fusion surface is good (figure 9).

Compared with the control group and the ovarian combined polylactic acid nanofiber membrane transplantation group, the graphene composite nanofiber membrane promotes the survival of transplanted ovaries, so that the number of follicles of the whole transplanted ovaries is increased, and the survival quality of the transplanted ovaries is better (fig. 10 and 11).

Based on the fact that animal experiments find that the graphene composite nanofiber membrane can promote tissue and blood vessel regeneration, the graphene composite nanofiber membrane is firstly applied to transplantation after clinical cryopreserved ovary resuscitation, the ovarian tissue recovered after freezing is wrapped in the graphene composite nanofiber membrane, and the graphene composite nanofiber membrane is transplanted according to an operation scheme.

According to the consensus of ovarian tissue cryopreservation and transplantation specialists, the flow of ovarian tissue cryopreservation and transplantation is as follows;

(1) Strictly screening whether the ovarian tissue cryopreservation and transplantation conditions are met according to the ovarian tissue cryopreservation indication.

(2) Ovarian tissue is frozen.

(3) And judging the time and the indication of the ovarian tissue transplantation.

(4) Cryopreserved ovaries were resuscitated and transplanted.

The simulated application scheme of the ovarian transplantation operation is as follows:

female, 43 years old, pregnant 1 produced 0. Differentiation of stage IIb G2 endogenous forms in cervical squamous cell carcinoma is diagnosed and radiation and chemotherapy are required. The ovarian tissue cryopreservation prior to chemotherapy is determined by the desire to preserve fertility and endocrine function of the ovaries. The "autologous ovarian in situ transplantation operation" is to be performed in compliance with the ovarian tissue transplantation indication period after chemotherapy.

(4.1) resuscitation of ovarian tissue: and taking out 8 ovarian cortex slices from the thawed frozen stock solution, rapidly and sequentially placing the ovarian cortex slices into the resuscitation liquid with different concentration gradients, placing the ovarian cortex slices into the resuscitation liquid with each concentration for 15 min, fully washing, placing the tissue slices into a sterile sample bottle containing the resuscitation liquid, and rapidly delivering the tissues to an operating room within 5 min after resuscitation.

(4.2) transplantation of ovarian tissue: the patient is placed in supine position and operated under general anesthesia.

(4.3) intraoperative: a little ascites in the pelvic cavity is light yellow, the uterine is at the rear position, the size is normal, the appearance is not obvious abnormal, the left accessory is hung on the left upper abdominal wall, the left ovary is 2 cm multiplied by 3 cm multiplied by 3 cm, the appearance is not obvious abnormal, the right ovary is 1 cm multiplied by 1 cm multiplied by 1.5 cm, and the left accessory is hard and clings to the right pelvic wall. The inside of the pelvis is smoother, and no obvious adhesion is seen. A few fibrotic scar changes can be seen at the bilateral pelvic inlet. The operator decides to select the tissue graft below the scar and between the ureters on both sides after the discussion, and the tissue is soft and has no scar and good blood circulation. The left ovary suspension suture is cut off, and the ovary is still received back to the original position and fixed. And cutting off the peritoneum between the left sacral ligament and the left ureter, expanding the incision to 2 cm diameters, and taking 4 pieces of recovered ovarian tissues, wrapping a graphene composite nanofiber film on the surface, uniformly paving the film in the lower peritoneal wound surface, and suturing the lower peritoneal wound surface. The right processing method is the same as the left processing method.

(4.4) the patient returns to the ward safely after operation, and the general situation is good, and no complications occur.

In the present invention, fig. 11 is a columnar schematic diagram showing that the graphene composite nanofiber membrane promotes survival of transplanted ovaries so as to increase the number of follicles of the whole ovaries after transplantation, and the survival quality of transplanted ovaries is better, compared with a control group and an ovarian combined polylactic acid nanofiber membrane transplantation group.

The foregoing is merely illustrative of specific embodiments of the present invention, and the scope of the invention is not limited thereto, but any modifications, equivalents, improvements and alternatives falling within the spirit and principles of the present invention will be apparent to those skilled in the art within the scope of the present invention.

Claims

1. The preparation method of the degradable graphene composite electrostatic spinning fiber film is used for preparing a frozen revived ovarian tissue wrapping film and is characterized by comprising the following steps of:

step one, EDC method is adopted to pass through PEG-NH ₂ Graphene oxide with PEI PolymerSurface modification is carried out to obtain functionalized graphene oxide; then dispersing the functionalized graphene oxide in a solvent A by ultrasonic to obtain a solution A; the volume ratio of the functionalized graphene oxide to the solvent A is (0.1-2.0 g): 1 mL;

step two, adding high molecular polymer poly L lactic acid or poly D lactic acid into the solvent B, and stirring for 1-12 hours at a certain temperature to obtain transparent solution B; the mass ratio of the high molecular polymer to the solvent B is (8.0-16.0. 16.0 g): 100.0 g;

step three, mixing the solution A and the solution B according to a certain volume ratio, stirring for a plurality of hours, performing ultrasonic treatment for 10-30 min, and standing for 1-12 h to obtain an electrostatic spinning solution;

step four, carrying out electrostatic spinning on the obtained electrostatic spinning solution to obtain a degradable graphene composite electrospun fiber film;

in the first step, the solvent A is DMF;

in the first step, PEG-NH ₂ Is one-armed PEG-NH _2、 Double-arm PEG-NH _2、 Four-arm PEG-NH ₂ Or six-arm PEG-NH ₂ ；

The mass ratio of the functionalized graphene oxide F-GO to the high molecular polymer is (0.05-8.0): 100;

the volume ratio of the solvent A to the solvent B is (0.5-1.0 mL): 1.0mL, the difference volume was supplemented with pure solvent A.

2. The method for preparing the degradable graphene composite electrospun fiber film according to claim 1, wherein in the second step, the solvent B is DCM;

in the second step, the molecular weight of the high molecular polymer is 10-40 ten thousand.

3. The method for preparing the degradable graphene composite electrostatic spinning fiber film according to claim 1, wherein in the fourth step, the positive voltage of electrostatic spinning is 15-25 kV, the negative voltage is 0-5 kV, the spinning speed is 0.05-0.5 mm/min, the distance between a spinning nozzle and a receiver is 15-25 cm, and the electrostatic spinning time is 1-16 h.

4. A degradable graphene composite electrospun fiber film prepared by the preparation method of the degradable graphene composite electrospun fiber film according to any one of claims 1-3.

5. Use of the degradable graphene composite electrostatic spinning fiber film according to claim 4 for preparing a revived ovarian tissue wrapping film after freezing.

6. A drug-eluting membrane prepared by using the degradable graphene composite electrospun fiber film of claim 4.

7. An air filtration membrane prepared from the degradable graphene composite electrostatic spinning fiber film of claim 4.