CN116254615A

CN116254615A - Preparation method of endometrium repair membrane and prepared endometrium repair membrane

Info

Publication number: CN116254615A
Application number: CN202310537891.3A
Authority: CN
Inventors: 王菲; 赵明达; 董佳桓; 唐月婷
Original assignee: Neo Modulus Suzhou Medical Sci Tech Co ltd
Current assignee: Neo Modulus Suzhou Medical Sci Tech Co ltd
Priority date: 2023-05-15
Filing date: 2023-05-15
Publication date: 2023-06-13
Anticipated expiration: 2043-05-15
Also published as: CN116254615B

Abstract

The invention discloses a preparation method of an endometrium repair membrane, which comprises the following steps: and delivering a spinning solution to a spinning electrode of the electrostatic spinning equipment for spinning to form the endometrium repair film, wherein the spinning current is controlled to be changed regularly within the spinning time period, the spinning current is controlled to be within the range of 0.01 mA-0.4 mA, and the time integral value of the spinning current is controlled to be within the range of 1C-9C. The endometrial repair film prepared by the method has uniform thickness and standard deviation (STDEV) of less than 0.02.

Description

Preparation method of endometrium repair membrane and prepared endometrium repair membrane

Technical Field

The invention relates to the field of medical biological materials, in particular to a preparation method of an endometrium repair membrane and the prepared endometrium repair membrane.

Background

According to research data, uterus-related lesions in women of childbearing age are important factors for infertility, wherein the infertility rate caused by uterine cavity problems is 7.8% -34.9%, and the infertility caused by oviduct, ovary, endocrine and other reasons can be solved by the current assisted reproduction technology, but infertility caused by uterine factors still cannot be perfectly solved. By taking intrauterine adhesion as an example, artificial abortion in China is carried out for 1300 thousands of times each year, and researches show that the incidence rate of intrauterine adhesion after abortion can reach 15% -40%, so that the postoperative pregnancy rate is low.

At present, main clinical means for solving the problem of fibrotic scar tissue formed in the damaged uterus repair process at home and abroad comprise the use of an intrauterine device, a uterine cavity supporting balloon and biological glue (comprising sodium hyaluronate, chitosan and the like) after adhesion separation operation, wherein the means mainly depend on physical barrier action to prevent adhesion from forming, but do not help functional regeneration of the damaged uterus tissue, and have the defects of ischemic necrosis of tissue caused by incarceration, infection and compression and too short residence time in uterine cavity to maintain the completion of tissue repair.

In recent years, with the development of tissue engineering, implants prepared by an electrostatic spinning method are also beginning to be applied to the field of uterine repair, and the prepared implants generally have good biocompatibility and can promote the wound repair of endometrium.

However, since a high voltage is required to pull the charged solution during the electrospinning process, the charged solution is usually pulled by a fixed voltage, and the viscosity, the degree of crosslinking, the voltage, etc. of the solution affect the morphology of the finally formed fibers, which results in uneven fiber diameter and uneven thickness of the resulting electrospun film, and it is particularly difficult to obtain a thin electrospun film having an even thickness and an even fiber diameter. In addition, the existing preparation method has poor repeatability, and under the condition of adopting the same spinning solution and spinning conditions when spinning for multiple times, it is difficult to ensure that the spinning film prepared each time has similar thickness and uniform thickness.

Disclosure of Invention

In view of the above problems of the prior art electrospun membranes for use in the repair of endometrium, the present invention aims to provide a method for preparing a thin electrospun membrane (i.e. endometrium repair membrane) having a uniform thickness.

In one aspect, the present invention provides a method for preparing an endometrium repair membrane, comprising: and delivering a spinning solution to a spinning electrode of the electrostatic spinning equipment for spinning to form the endometrium repair film, wherein the spinning current is controlled to be changed regularly within the spinning time period, the spinning current is controlled to be within the range of 0.01 mA-0.4 mA, and the time integral value of the spinning current is controlled to be within the range of 1C-9C.

In some embodiments, the spinning time period comprises at least one current constant period during which the spinning current remains constant.

In some embodiments, the spinning duration comprises at least one current linear variation period during which the spinning current varies linearly with time.

In some embodiments, the at least one current constant period comprises a first current constant period during which the spinning current is maintained at a first current constant value and a second current constant period during which the spinning current is maintained at a second current constant value, the second current constant value being greater than the first current constant value; the at least one current line change period includes a first current line change period, a start current of the first current line change period is a first current constant value, and an end current is a second current constant value. For example, the first current constant period takes 40 min-60 min, the second current constant period takes 40 min-120 min, the first current linear variation period takes 180 min-240 min, the first current constant value is 0.02 mA-0.2 mA, the second current constant value is 0.02 mA-0.4 mA, and the linear variation rate of the spinning current in the first current linear variation period is 0-0.1 mA/min.

In some embodiments, the at least one current constant period comprises a first current constant period during which the spinning current is maintained at a first current constant value; the at least one current line change period includes a first current line change period, a starting current of the first current line change period is a first current constant value, and a terminating current is greater than the starting current. For example, the first current constant period takes 40 min-60 min, the first current linear change period takes 180 min-240 min, the first current constant value is 0.02 mA-0.2 mA, the termination current is 0.02 mA-0.4 mA, and the linear change rate of the spinning current is 0-0.1 mA/min in the first current linear change period.

In some embodiments, the spinning current is controlled to be in the range of 0.02 mA-0.4 mA in the spinning duration, and the time integral value of the spinning current in the spinning duration is in the range of 2.6-6C.

In some embodiments, the endometrial repair film has a thickness of 0.01-10 mm and a standard deviation of thickness of less than 0.02.

In some embodiments, the spinning solution is formed as follows: and conveying the solution of the high polymer material through a first liquid supply pipe, conveying the cross-linking agent solution through a second liquid supply pipe, and mixing the solution of the high polymer material and the cross-linking agent solution at the downstream junction of the first liquid supply pipe and the second liquid supply pipe in real time to form the spinning solution.

In some embodiments, the method of making further comprises: the crosslinking degree of the endometrium repair film is controlled within the range of 30% -60%, so that the Young modulus of a single wire of the formed endometrium repair film is larger than 0.5kPa.

In some embodiments, the degree of crosslinking of the endometrial repair film is controlled by controlling the amount of the crosslinking agent.

In another aspect, the present invention provides an endometrial repair film prepared according to the above-described preparation method.

Compared with the prior art, the invention has the following beneficial technical effects:

according to the preparation method of the endometrium repair film, the spinning current is controlled to be changed according to rules, the spinning current is controlled to be in the range of 0.01 mA-0.4 mA in the spinning time period, the time integral value of the current of the spinning current in the spinning time period is controlled to be in the range of 1C-9C, the thickness of the endometrium repair film prepared by the method is uniform, and the standard deviation (STDEV) of the thickness of the prepared film is smaller than 0.02.

Furthermore, the preparation method provided by the invention has repeatability. Since the current is always controlled to change regularly, and the current value is controlled to be the same as the integral value when spinning is carried out each time, when thin films are prepared through multiple times of spinning, the standard deviation STDEV of the average thickness among the films is very small and is only about 0.01, and the defect of large film thickness fluctuation when spinning is carried out by adopting fixed spinning voltage in the prior art can be avoided.

In addition, the invention can further control the crosslinking degree of the membrane, and can also improve the elastic modulus of the membrane, so that the Young modulus of single fiber of the membrane can reach more than 0.5kPa, and the prepared endometrium repair membrane has good flexibility and compliance and soft texture.

Therefore, the method can prepare the thin electrostatic spinning film with uniform thickness and good flexibility and compliance, and when the electrostatic spinning film is used for repairing damaged endometrium, the degradation time in vivo is moderate, and the requirements of the endometrium repairing duration and female physiological cycle are met; the shape of the uterine cavity can be changed, the requirements of different uterine shapes can be fully met in the use process, and the uterine cavity can be fully attached to the inner wall of the uterus; can effectively avoid adhesion and scar re-formation, thereby recovering the normal physiological function of the uterus.

In addition, the fiber diameter range of the membrane is 100 nm-600 nm due to the control of the spinning current, and the fiber diameter range can be more suitable for the growth of endometrial cells.

In addition, the raw materials of the electrostatic spinning membrane are natural protein materials and artificial synthetic materials, including gelatin, collagen and related derivatives, artificially synthesized recombinant collagen and the like, and the prepared electrostatic spinning membrane (endometrium repair membrane) has a microscopic multilayer structure, is beneficial to cells to invade the nanofiber membrane from multiple dimensions and climb and grow along the fiber direction, realizes 3D three-dimensional proliferation of the cells, promotes wound repair, and shortens wound repair time.

Drawings

Fig. 1 is a graph showing the change of spinning current with time in the preparation methods of endometrium repair films according to examples 1, 2 and 3 of the present invention.

Fig. 2 is a graph showing the variation of spinning current with time in the preparation method of an endometrium repair film according to example 4 and example 5 of the present invention.

Fig. 3 is a scanning electron microscope image of an endometrial repair film sample 1-1 according to example 1 of the invention.

Fig. 4 is a scanning electron microscope image of an endometrial repair film sample 4-1 according to example 4 of the invention.

Fig. 5 is a scanning electron microscope image of an endometrial repair film sample 9-1 according to a comparative example of the invention.

FIG. 6 is a staining chart of tissue sections of KLE cell cultures using endometrium repair membrane samples 1-1 of example 1 of the present invention.

FIG. 7 is a staining chart of tissue sections of KLE cell cultures using endometrium repair membrane sample 6 of example 6 of the present invention.

FIG. 8 is a staining chart of tissue sections of KLE cell cultures using endometrium repair membrane sample 7 of example 6 of the invention.

FIG. 9 is a staining chart of a tissue section of the endometrium repair membrane sample 1-1 according to example 1 of the present invention 30 days after implantation into the uterus of a rabbit.

Fig. 10 is a photograph of the pre-operative endometrium status of a patient with intrauterine adhesion.

Fig. 11 is a photograph of endometrium status after implantation of endometrium repair film sample 1-1 according to example 1 of the present invention in a patient for 30 days.

FIG. 12 is a photograph of a section of electrospun membrane after implantation of endometrium repair membrane sample 1-1 according to example 1 of the present invention into a patient for 3 days.

Detailed Description

Various aspects of the invention are described in detail below with reference to the drawings and detailed description. It should be understood by those skilled in the art that the following exemplary embodiments are illustrative only and are not intended to limit the scope of the present invention in any way.

In the exemplary embodiment of the invention, spinning solution is spun into a fibrous membrane material through an electrostatic spinning device, in order to obtain an electrostatic spinning membrane with uniform thickness, in a spinning time period, a spinning current is controlled to change regularly, a time integral value of the spinning current in the spinning time period is adjusted in real time to be kept in a range of 1-9C, for example, a range of 2.6-6C, and the spinning current is controlled to be always in a range of 0.01-0.4 mA in real time.

In some specific embodiments, the control of the spinning current can be achieved by adjusting the spinning voltage, the spinning voltage is 45-60 kV, the total spinning time is 250-380 min, the spinning current is controlled to change along with time by adjusting the spinning voltage, the required time integral value of the spinning current is 1-9C (for example, 2.6-6C), and the current is ensured to be always in the range of 0.01-0.4 mA, for example, the range of 0.02-0.4 mA.

In some embodiments, the spinning time period can be divided into a plurality of time periods, and each time period is controlled so that the current changes regularly.

For example, the spinning duration comprises at least one current constant period during which the spinning current remains constant. The spinning duration may also include at least one current line variation period during which the spinning current varies linearly with time.

In some embodiments, the at least one current constant period comprises a first current constant period during which the spinning current is maintained at a first current constant value and a second current constant period during which the spinning current is maintained at a second current constant value; the at least one current linear change period includes a first current linear change period having a start current of a first current constant value and an end current of a second current constant value, the second current constant value being greater than the first current constant value. For example, the spinning duration is sequentially composed of a first current constant period, a first current linear change period and a second current constant period, wherein the first current constant value is in the range of 0.02-0.2 mA, the second current constant value is in the range of 0.02-0.4 mA, and the spinning current is increased from the first current constant value to the second current constant value at the increasing rate of 0.001mA/min (the increasing rate can be 0.001mA/min or any value in the range of 0-0.1 mA/min). The first current constant period occupies 40-60 min; the first current linear change period occupies 180-240 min; the second current constant period occupies 40-120 min.

In an alternative embodiment, the spinning duration may include only the first current constant period and the first current linear variation period. For example, the spinning period is composed of a first current constant period and a first current linear variation period in this order. Wherein the initial first current constant value is in the range of 0.02-0.2 mA, the second current constant value is in the range of 0.02-0.4 mA, and the spinning current is increased from the first current constant value to the termination current at the increasing rate of 0.001mA/min (the increasing rate can be 0.001mA/min or any value in the range of 0-0.1 mA/min). The first current constant period occupies 40-60 min; the first current change period takes 180-240 min.

In alternative embodiments, the spinning duration may be composed of a plurality of current constant periods and a plurality of current line variation periods in sequence, and the spinning duration may also include only the current constant periods or only the current line variation periods. The spinning current may be increased or decreased at different rates of change during the period of current line change.

The endometrium repair film (electrospun film) prepared according to the embodiment of the invention can be prepared to have a thickness of 0.01-10 mm, for example 0.20-0.25 mm, and a standard deviation of thickness (STDEV) of less than 0.02. Further, when the current change, the same current time integral value, and the same current value are repeatedly produced with the same law, the thickness standard deviation (STDEV) of the average thickness of the produced plurality of films is only 0.01.

The fiber diameter of the prepared endometrium repair film has a variation range of 100-500 nm, such as 200-400nm, and is a thin electrostatic spinning film with uniform thickness.

In order to improve the elastic modulus, the crosslinking degree of the membrane can be further controlled to be in a range of 10% -90% (for example, in a range of 30% -60%), so that the Young modulus of a single wire of the formed endometrium repair membrane is larger than 0.5kPa.

The spinning solution can be formed by mixing a high polymer material solution and a cross-linking agent solution in real time, wherein the high polymer material can be one or more of collagen, elastin, proteoglycan, gelatin and derivatives thereof. Specifically, the polymer material solution is conveyed through the first liquid supply pipe, and the preparation method of the polymer material solution is as follows: mixing one or more of purified water, acetic acid, ethyl acetate, PBS buffer solution and ethanol to prepare a dissolving solution, mixing one or more of collagen, elastin, proteoglycan, gelatin and derivatives thereof, adding the mixture into the prepared dissolving solution, mixing and dissolving structural protein and derivatives thereof and a mixed solvent in a mass ratio of 1:3-1:12, and stirring until the polymer material is completely dissolved. The cross-linking agent solution is conveyed through the second liquid supply pipe and is prepared by mixing one or more of glutaraldehyde water solution, glyoxal water solution, EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide), and the liquid flow rate ratio in the first liquid supply pipe and the second liquid supply pipe can be adjusted according to the cross-linking agent adding ratio. The first liquid supply pipe and the second liquid supply pipe are converged at the tail end, so that the polymer material solution and the cross-linking agent solution are uniformly mixed to form a spinning solution, and then the spinning solution is conveyed to a spinning electrode of the electrostatic spinning equipment for spinning.

Since the polymer solution and the crosslinking agent solution are mixed in real time, the crosslinking degree of the film can be controlled by controlling the content of the crosslinking agent. For example, the solution of polymeric material is a gelatin solution at a concentration of 16.7% and the cross-linking agent solution is a glyoxal solution at a concentration of 10% such that the cross-linking agent comprises 15% wt of the spinning solution.

In the electrostatic spinning process, spinning can comprise non-needle spinning, single-needle spinning and multi-needle spinning, the receiving device comprises a rotary collecting mandrel, a plane collecting plate and winding and unwinding, the distance range of a spinning nozzle to the receiving device is 50-600 mm, the flow rate range of spinning solution is 1-20 ml/hr, and the spinning current is controlled as described above.

In order to further increase the degree of crosslinking, the spun film may be subjected to a high temperature treatment and the residual solvent may be removed. The temperature range of the high-temperature treatment in the oven is 50-160 ℃, and the time range is 2-10 h.

The prepared electrostatic spinning membrane (endometrium repair membrane) can be sterilized by adopting a dry heat sterilization, high-pressure steam sterilization, hydrogen peroxide plasma sterilization, ethylene oxide sterilization or irradiation sterilization mode, or by adopting a gradient ethanol soaking and cleaning mode before the product is used.

Various aspects of the invention will be described in detail below in connection with particular embodiments:

[ example 1 ]

Example 1 provides a method for preparing an endometrium repair membrane comprising the steps of:

(i) 200ml of glacial acetic acid, 200ml of ethyl acetate and 100ml of water are prepared into a dissolution solution, 100g of gelatin is added into the dissolution solution, and the dissolution solution is stirred for 10 hours to be completely dissolved, so as to obtain a first solution.

(ii) 50ml of aqueous glyoxal solution was prepared, the concentration of glyoxal being 10%, to obtain a second solution.

(iii) The first solution is added into a first liquid supply pipe of the electrostatic spinning equipment, the second solution is added into a second liquid supply pipe, the liquid supply speed of the first liquid supply pipe is set to be 2ml/h, the liquid supply speed of the second liquid supply pipe is set to be 0.5ml/h, meanwhile, liquid supply is started, and the first solution and the second solution are uniformly mixed at the junction of the first liquid supply pipe and the second liquid supply pipe to form spinning solution.

(iv) And delivering the spinning solution to a spinning electrode of the electrostatic spinning equipment for spinning. Wherein the spinning temperature is 25 ℃, the humidity of the spinning space is 40% RH, the spinning working distance is 150mm, the rotation speed of the receiving roller is 80rpm, and the receiving area is 30 cm.300cm.

During the spinning period, the spinning voltage is adjusted so that the spinning current is controlled according to a curve A2 shown in fig. 1, specifically as follows:

the total spinning time is 330min, and the total spinning time sequentially comprises a first current constant period, a first current linear change period and a second current constant period. Wherein the initial current (i.e., the first current constant value) is set to 0.1mA, and the control current is kept unchanged at 0.1mA for a first current constant period of time, which lasts for 40min; then during a first current linear change period, the control current is increased to 0.32mA (stop current, namely a second current constant value) at a constant speed of 0.001mA/min along with time, and the period lasts for 220min; thereafter, the control current was kept constant at 0.32mA for a second current constant period of about 70 minutes. In the present embodiment, the time integrated value q=4c of the spinning current is controlled.

(v) And (3) obtaining a nanofiber membrane after spinning, putting the nanofiber membrane into an oven, and treating the nanofiber membrane at 150 ℃ for 6 hours to obtain the treated nanofiber membrane (namely the electrostatic spinning membrane for endometrial repair, also called endometrial repair membrane).

And repeatedly spinning for 9 times, wherein the first solution and the second solution are prepared by adopting the same formula and the same preparation mode in each spinning, and the same current control and the same spinning conditions are adopted, so that 10 film samples are obtained, and the number of the film samples is respectively 1-1 to 1-10.

The film samples prepared by the method of this example were all 30cm by 300cm in size, and the thickness was measured as follows.

[ example 2 ]

The production method similar to that of example 1 was adopted in this example, except that the controlled current range and the current time integral value were different in the same spinning period. Specifically, in the spinning step (iv):

during the spinning period, the spinning voltage is adjusted so that the spinning current is controlled according to the curve A1 shown in fig. 1, specifically as follows:

the total spinning time is 330min, and the total spinning time sequentially comprises a first current constant period, a first current linear change period and a second current constant period. Wherein the initial current (i.e., the first current constant value) is set to 0.18mA, and the control current is kept constant at 0.18mA for a first current constant period of time, which lasts for 40min; then during a first current linear change period, the control current is increased to 0.4mA (stop current, namely a second current constant value) at a constant speed of 0.001mA/min along with time, and the period lasts for 220min; thereafter, the control current was kept constant at 0.4mA for a second current constant period of time, which was about 70min. In the present embodiment, the time integrated value q=6c of the spinning current is controlled.

And repeatedly spinning for 9 times, wherein the first solution and the second solution are prepared by adopting the same formula and the same preparation mode in each spinning, and the same current control and the same spinning conditions are adopted, so that 10 film samples are obtained, and the number of the film samples is respectively 2-1 to 2-10.

[ example 3 ]

during the spinning period, the spinning voltage is adjusted so that the spinning current is controlled according to a curve A3 shown in fig. 1, specifically as follows:

the total spinning time is 330min, and the total spinning time sequentially comprises a first current constant period, a first current linear change period and a second current constant period. Wherein, the initial current (namely the first current constant value) is 0.02mA, the control current is kept unchanged at 0.02mA in the first current constant period, and the period lasts for 40min; then the current is controlled to increase to 0.24mA (the termination current, i.e. the second current constant value) at a constant speed with time at a rate of increase of 0.001mA/min in a first current linear change period, and the period lasts for 220min; thereafter, the control current was kept constant at 0.24mA for a second current constant period of time, which was about 70min. In the present embodiment, the time integrated value q=2.6c of the spinning current is controlled.

And repeatedly spinning for 9 times, wherein the first solution and the second solution are prepared by adopting the same formula and the same preparation mode in each spinning, and the same current control and the same spinning conditions are adopted, so that 10 film samples are obtained, and the number of the film samples is respectively 3-1 to 3-10.

[ example 4 ]

In this example, a similar preparation method to example 1 was used, except that different spinning currents and spinning durations were used, but the current time integral values were all 4C. Specifically, in the spinning step (iv):

during the spinning period, the spinning voltage is adjusted so that the spinning current is controlled according to a curve B1 shown in fig. 2, specifically as follows:

the total spinning time is 250min, and the total spinning time sequentially comprises a first current constant period and a first current linear change period. The initial current is 0.18mA (namely a first current constant value), and the control current is kept unchanged at 0.18mA in a first current constant period, and the period lasts for 40min; the control current was then increased to 0.39mA (termination current) at a constant rate of increase of 0.001mA/min over time during the first current line change period, which lasts for 210min. In the present embodiment, the time integrated value q=4c of the spinning current is controlled.

And repeatedly spinning for 9 times, wherein the first solution and the second solution are prepared by adopting the same formula and the same preparation mode in each spinning, and the same current control and the same spinning conditions are adopted, so that 10 film samples are obtained, and the samples are respectively numbered as samples 4-1 to 4-10.

[ example 5 ]

during the spinning period, the spinning voltage is adjusted so that the spinning current is controlled according to a curve B3 shown in fig. 2, specifically as follows:

the total spinning time is 380min, and the total spinning time sequentially comprises a first current constant period, a first current linear change period and a second current constant period. Wherein, the initial current (namely the first current constant value) is 0.05mA, the control current is kept unchanged at 0.05mA in the first current constant period, and the period lasts for 40min; then the control current is increased to 0.27mA (stop current, namely a second current constant value) at a constant speed of 0.001mA/min along with time in a first current linear change period, and the period lasts for 220min; thereafter, the control current was kept constant at 0.27mA for a second current constant period of time, which was about 120min. In the present embodiment, the time integrated value q=4c of the spinning current is controlled.

And repeatedly spinning for 9 times, wherein the first solution and the second solution are prepared by adopting the same formula and the same preparation mode in each spinning, and the same current control and the same spinning conditions are adopted, so that 10 film samples are obtained, and the number of the film samples is respectively 5-1 to 5-10.

[ example 6 ]

In this example, a preparation method similar to that of example 1 was employed, except that glyoxal solutions of 5% and 20% concentration were respectively selected as the second solution (crosslinker solution).

In this example, the film number obtained by electrospinning using a glyoxal aqueous solution having a concentration of 5% as a crosslinking agent solution by the above method was sample 6, and the film number obtained by electrospinning using a glyoxal aqueous solution having a concentration of 20% as a crosslinking agent solution was sample 7.

The film samples prepared by the method of this example were 30cm by 300cm in size, thickness and other properties as described below.

[ comparative example ]

The comparative example was conducted by a production method similar to that of example 1 except that a fixed spinning voltage was used, and the spinning current and the current time integral value were not particularly controlled. Specifically, in the spinning step (iv):

the total spinning time is 330min, the spinning voltage is set to be 55kV, the voltage is kept fixed by observing the spinning voltage every ten minutes, and the current at the time is recorded.

The spinning was repeated 9 times, each of which was prepared into a first solution and a second solution using the same formulation and preparation method, and the same voltage control (even though the voltage was kept at a constant voltage of 55 kV) and the same spinning conditions were used, thereby obtaining 10 film samples in this comparative example, numbered as samples 8-1 to 8-10, respectively.

Through observing the current value, the current is irregular in 10 times of spinning, the time integral value of the current is different in each experiment, the current is irregularly changed in the range of 2-6C, and the time integral values of the currents of samples 8-1-8-10 are respectively as follows: q1=5c, q2=3c, q3=5c, q4=5c, q5=4c, q6=2c, q7=4c, q8=3c, q9=6c, q10=2c. The current range and the integrated value corresponding to each sample are specifically shown in table 1.

The film samples prepared by the method of this comparative example were all 30cm x 300cm in size and the thickness was measured as follows.

The following thickness measurement and scanning electron microscope observation were performed on the electrospun film samples prepared in the exemplary examples and comparative examples of the present invention:

1. thickness measurement

The samples were subjected to thickness measurement and the thickness average (Avg) and thickness standard deviation (STDEV) of the samples were calculated in order. Specifically, the measurement and calculation are performed as follows:

take sample 1-1 as an example. Sample 1-1 was an electrospun membrane (endometrium repair membrane) 30cm x 300cm, and thickness measurement was performed at 1 position selected every 3cm in the width direction at the midpoint in the length direction of sample 1-1, 10 points were measured in total, and thickness measurement values of 10 points were: 0.200mm, 0.215mm, 0.227mm, 0.224mm, 0.217mm, 0.208mm, 0.203mm, 0.208mm, 0.222mm, 0.229mm. The average thickness of sample 1-1 was thus calculated to be 0.215mm.

The standard deviation of thickness (STDEV) of sample 1-1 was calculated as in equation (1):

(1)

in the method, in the process of the invention,

is the standard deviation of thickness (STDEV),>

for the thickness measurement of each point of the sample, +.>

For the thickness average of the sample, +.>

Is the number of samples.

From this calculation, the standard deviation of thickness (STDEV) of the individual films of sample 1-1 was 0.010.

The thicknesses of samples 1-2 to 1-10 were measured according to the above-described methods, and the standard deviation (STDEV) of the thickness of each film was calculated, as shown in Table 1 below.

As can be seen from the above calculation, the average thickness Avg of samples 1-1 to 1-10 is 0.215mm, 0.204mm, 0.232mm, 0.225mm, 0.229mm, 0.207mm, 0.220mm, 0.221mm, 0.225mm, 0.220mm, respectively.

Calculating a thickness standard deviation (STDEV) of 10 film thicknesses of samples 1-1 to 1-10 according to the formula (1), wherein,

thickness standard deviation (STDEV) of 10 films, < >>

For each ofAverage thickness Avg,/of the stretch film>

Is the average value of the thickness of ten films, +.>

Is the number of samples. The thickness standard deviation (STDEV) of the 10 film thicknesses was calculated to be 0.010.

The rest samples are measured and calculated in the same way as samples 1-1 to 1-10. The specific results are shown in Table 1.

TABLE 1 Current integration values, spinning time, thickness standard deviation data for samples 1-10

/>

In table 1, the thickness dispersion is represented by the thickness standard deviation STDEV, and the smaller the STDEV, the more concentrated the data distribution and the more uniform the thickness.

As can be seen from table 1, by controlling the current variation during spinning so that the current is in the range of 0.01 to 0.4ma and the current time integral value is in the range of 1c to 9c, and by regularly varying the current (including, for example, at least one current constant period and at least one current linear variation period), a thin electrospun film having a uniform thickness can be produced. Also, the film thickness increases as the current time integrated value increases, and the film thicknesses having equal integrated values are close. Specifically, the STDEV values of the individual films of samples 1 to 5 are all in the range of 0.006 to 0.013 and are all smaller than 0.02, so that the preparation method can prepare a thin electrospun film (endometrium repair film) with uniform thickness.

Meanwhile, the preparation method has repeatability, and the thin electrostatic spinning film with uniform thickness can be still prepared during multiple spinning. Taking samples 1-1 to 1-10 as an example, when spinning for multiple times, after the control current is changed regularly (for example, the control current comprises 2 current constant time periods and 1 current linear change time period), the STDEV of the multiple films with the same current time integral value and current value is very small, and the STDEV of the average thickness of 10 films of samples 1-1 to 1-10 is only 0.010.

In contrast, when the electrostatic spinning film is prepared by adopting the fixed voltage method, the current changes irregularly, the current time integral value also changes irregularly, and the STDEV value of a single film is larger than 0.02.

When the static spinning film is prepared by adopting a fixed voltage method, the current and the current time integral value of each spinning are different, and the thickness difference of each film is larger when the spinning is performed for a plurality of times. As shown in Table 1, the average thickness of samples 8-1 to 8-10 (10 films) was 0.263mm, and the STDEV of 10 films was 0.163, i.e., the thickness of 10 films was randomly distributed in the range of 0.071 to 0.533mm, and the thickness deviation value was nearly 20 times that of the multiple films STDEV of the samples of the present invention, which were subjected to the current change control.

Therefore, the invention can avoid the defects of large film thickness fluctuation and poor repeatability when spinning by adopting fixed spinning voltage by controlling the current and the current time integral value and enabling the current to change according to a rule for electrostatic spinning, and the spinning method used by the invention has repeatability, and can still ensure that the thin electrostatic spinning film with uniform thickness is prepared when spinning is carried out for many times.

2. Scanning electron microscope observation

And (3) carrying out scanning electron microscope observation on the film samples of the examples 1-5 to observe the fiber diameters, wherein the fiber diameters are uniformly distributed and are distributed in a narrow range of 200-400nm, an electron microscope image of the sample 1-1 is shown in fig. 3, and an electron microscope image of the sample 4-1 is shown in fig. 4.

The film sample of the comparative example is observed by a scanning electron microscope, and the fiber diameter distribution of the observed film sample of the comparative example is wide and is between 100 and 900nm, wherein the electron microscope diagram of the sample 9-1 is shown in fig. 5, and the phenomenon of mutual adhesion and doubling between fibers (such as a circle mark in fig. 5) can be seen.

Therefore, the fiber diameter range of the prepared electrostatic spinning film can be narrower in distribution and is between 100nm and 500nm (for example, between 200nm and 400 nm) by controlling the spinning current and the integral value within a specific range and controlling the current to change regularly, and the fiber diameter range can be more suitable for the growth of endometrial cells.

The following tests for crosslinking degree, mechanical properties and KLE cell culture experiments were performed on the film samples prepared in the exemplary examples 1 and 6 of the present invention:

1. crosslinking degree test

The crosslinking degree was measured for samples 1-1 and samples 6 and 7, and the absorbance at 346nm was measured using an ultraviolet spectrophotometer (UV 1600), and the crosslinking degree was calculated according to formulas (2) and (3).

（2）

（3）

Wherein: n is the amount of lysine side chain ɛ -amino group (mol/g), A is the absorbance value, 1.46×10 ⁴ The molar absorption coefficient of the trinitrobenzene derivative, b is the optical path (cm), and X is the sample weight (g).

As a result of detection and calculation, the crosslinking degrees of samples 1 to 1 and samples 6 and 7 were 45%, 20% and 78%, respectively.

It can be seen that the degree of crosslinking of the electrospun film is affected by the content of the crosslinking agent, since the gelatin solution and the crosslinking agent solution are mixed in real time, the greater the content of the crosslinking agent, the higher the degree of crosslinking of the electrospun film.

2. Mechanical property test

The mechanical properties of the individual nanofibers of samples 1-1, 6 and 7 were tested using a nanoindenter (stinffness: 5.15N/m; tip radius:28.5 μm).

As a result of the examination, the Young's modulus of the single fiber of sample 1-1 was about 1.02kPa, the Young's modulus of the single fiber of sample 6 was about 0.56kPa, and the Young's modulus of the single fiber of sample 7 was about 2.05kPa. It follows that the higher the degree of crosslinking, the greater the Young's modulus of the filaments, and proper crosslinking can enhance the mechanical properties of the fibers.

3. KLE cell culture experiments

Samples 1-1, samples 6 and 7 were subjected to KLE cell culture experiments.

Specifically, samples were sterilized using ethylene oxide, cut into 1cm x 1cm squares, seeded with KLE cells, cultured for 7 days, fixed and HE section stained to observe the growth of cells on samples 1-1, 6 and 7.

FIG. 6 is a staining chart of a tissue section of film sample 1-1, FIG. 7 is a staining chart of a tissue section of film sample 6, and FIG. 8 is a staining chart of a tissue section of film sample 7.

As shown in fig. 6 to 8, sample 1-1 had a remarkable proliferation promoting effect on KLE cells. As a result, the electrospun film (sample 1-1) having a crosslinking degree of 45% was found to have the best effect of promoting cell proliferation.

According to the test, the Young's modulus of a single wire of the electrospun membrane can be improved by further controlling the crosslinking degree of the electrospun membrane, and the cell proliferation is facilitated when the crosslinking degree is controlled within a certain range, so that the prepared electrospun membrane has good flexibility and compliance, soft texture and is beneficial to repairing endometrium.

The following in vivo degradation experiments and clinical experiments were performed on the electrospun film samples prepared in the exemplary embodiments of the present invention:

1. in vivo degradation experiment of rabbit

Sample 1-1 was implanted into rabbits and the degradation of the spinning membrane and endometrial repair were observed.

Specifically, sample 1-1 was implanted into the uterus of a rabbit, and after 30 days, the uterus tissue of the rabbit was subjected to HE section staining, degradation of the spinning membrane in the uterus of the rabbit was observed, and repair was confirmed by observing the integrity of the endometrium.

The experimental result shows that the sample 1-1 is completely degraded (as shown in figure 9), the outline of the endometrium is complete, and the repairing effect is good.

From this, it can be seen that sample 1-1 (thickness of 0.215.+ -. 0.010 mm) is suitable for endometrial repair, taking into account the length of endometrial repair and the physiological cycle requirements of females.

2. Clinical trials

Patients with intrauterine adhesions and fertility requirements were screened for clinical trials, and the intrauterine adhesions were isolated by hysteroscopic surgery as shown in fig. 10 for preoperative intrauterine symptoms.

Sample 1-1 (electrospun film with thickness of 0.215.+ -. 0.010 mm) was selected for clinical trials on patients:

sterilizing the sample 1-1 by using ethylene oxide, and feeding 2 films with the length of 4cm multiplied by 4cm into the separated uterine cavity by using a conveying device; taking out one membrane slice after 3 days, carrying out MASSN staining, and observing by a microscope, wherein a microscopic slice photo is shown in fig. 12, the area A is gelatin fiber, the area B is gelatin fiber enriched with cells, and a large number of cells in uterine cavity blood can be enriched by the gelatin fiber membrane; after 30 days, the hysteroscope is reviewed, the state of the endometrium of the uterine cavity of the patient is shown in fig. 11, the complete uterine cavity of the patient can be seen, adhesion disappears, and the endometrium gland opening (as indicated by the arrow in fig. 11) is dense and obvious.

Therefore, the electrostatic spinning membrane prepared by the method can effectively treat and repair damaged endometrium, avoid adhesion and scar re-formation, and restore normal physiological function of uterus.

The present invention has been described in detail with reference to specific embodiments thereof, which are merely illustrative, and not intended to limit the scope of the invention, and those skilled in the art can make various modifications, changes or substitutions without departing from the spirit and scope of the invention. Accordingly, various equivalent modifications are intended to be included within the scope of this invention.

Claims

1. A method of preparing an endometrium repair membrane comprising: delivering a spinning solution to a spinning electrode of an electrostatic spinning device for spinning to form an endometrium repair film, wherein the preparation method comprises the following steps: and controlling the spinning current to change regularly within the spinning time length, controlling the spinning current to be in the range of 0.01 mA-0.4 mA, and controlling the time integral value of the spinning current to be in the range of 1-9C.

2. The method of claim 1, wherein the spinning time period comprises at least one current constant period during which the spinning current remains constant.

3. The method of claim 2, wherein the spinning time period comprises at least one current ramp period during which the spinning current varies linearly with time.

4. The method of manufacturing according to claim 3, wherein the at least one current constant period includes a first current constant period in which the spinning current is maintained at a first current constant value and a second current constant period in which the spinning current is maintained at a second current constant value, the second current constant value being greater than the first current constant value;

the at least one current line change period includes a first current line change period, a start current of the first current line change period is a first current constant value, and an end current is a second current constant value.

5. The method of claim 4, wherein the first constant current period is 40min to 60min, the second constant current period is 40min to 120min, the first current change period is 180min to 240min, the first constant current value is 0.02ma to 0.2ma, the second constant current value is 0.02ma to 0.4ma, and the linear change rate of the spinning current is 0 to 0.1ma/min during the first current change period.

6. The method of manufacturing according to claim 3, wherein the at least one current constant period includes a first current constant period during which the spinning current is maintained at a first current constant value;

the at least one current line change period includes a first current line change period, a starting current of the first current line change period is a first current constant value, and a terminating current is greater than the starting current.

7. The method of claim 6, wherein the first constant current period is 40min to 60min, the first current linear variation period is 180min to 240min, the first constant current value is 0.02ma to 0.2ma, the termination current is 0.02ma to 0.4ma, and the linear variation rate of the spinning current is 0 to 0.1ma/min during the first current linear variation period.

8. The production method according to claim 1, wherein the spinning current is controlled so as to be in a range of 0.02ma to 0.4ma for a spinning period, and a time integral value of the spinning current for the spinning period is in a range of 2.6c to 6c.

9. The method of claim 1, wherein the endometrial repair film has a thickness of 0.01-10 mm and a standard deviation of thickness of less than 0.02.

10. The method of claim 1, wherein the spinning solution is formed by: and conveying the solution of the high polymer material through a first liquid supply pipe, conveying the cross-linking agent solution through a second liquid supply pipe, and mixing the solution of the high polymer material and the cross-linking agent solution at the downstream junction of the first liquid supply pipe and the second liquid supply pipe in real time to form the spinning solution.

11. The method of manufacturing of claim 10, further comprising: the crosslinking degree of the endometrium repair film is controlled within the range of 30% -60%, so that the Young modulus of a single wire of the formed endometrium repair film is larger than 0.5kPa.

12. The method of claim 11, wherein the degree of crosslinking of the endometrial repair film is controlled by controlling the amount of the crosslinking agent.

13. An endometrium repair membrane produced by the production method according to any one of claims 1 to 12.