CN114732832A

CN114732832A - Implantable article for preventing intrauterine adhesion

Info

Publication number: CN114732832A
Application number: CN202210363491.0A
Authority: CN
Inventors: 刘丹; 张慧星; 简鹏; 李刚; 胡明月; 金鹤; 元娟娟; 李锋
Original assignee: General Hospital of Ningxia Medical University
Current assignee: General Hospital of Ningxia Medical University
Priority date: 2022-04-08
Filing date: 2022-04-08
Publication date: 2022-07-12

Abstract

The invention relates to a cell and a biological material, in particular to an implantable article for preventing intrauterine adhesion. The present disclosure provides an implantable article for preventing intrauterine adhesion, which includes hydrogel and organoid, and is an injectable implant, wherein after the implantable article which is liquid at room temperature is injected into the uterine cavity, the liquid hydrogel can be shaped in accordance with the lacuna in the uterine cavity in the gradual solidification process at the body temperature, and in the uterine cavity, the implantable article is shaped into the shape of the uterine cavity, and the contact between wound surfaces in the uterine cavity is completely isolated. The shaping process of the temperature-sensitive hydrogel completely meets the individual requirements of different uterine cavity shapes of human bodies. In addition, the injection type avoids the peeling of the cells by the cervical canal and the uncertainty of the placement position when the uterine cavity is placed.

Description

Implantable article for preventing intrauterine adhesion

Technical Field

The invention relates to a cell and a biological material, in particular to an implantable article for preventing intrauterine adhesion.

Background

Intrauterine adhesion (IUA) is one of the important causes of intractable infertility, because it causes irreversible deep injury to the uterine cavity basal layer endometrium due to common factors such as abortion, uterine curettage, infection, etc., resulting in loss of stem cells therein, difficulty in regeneration of the functional layer endometrium, replacement by fibrotic scar, menstrual disorder caused by the non-renewable functional layer endometrium, and failure to provide an environment which is good in nutrition and suitable for fertilized egg planting and survival. At present, the traditional treatment of intrauterine adhesion comprises three parts of hysteroscopic adhesion separation (TCRA), postoperative intrauterine placement of a physical barrier to isolate a wound surface and prevent recurrence of adhesion, and estrogen to promote endometrial regeneration. However, the re-adhesion rate after moderate and severe intrauterine adhesion operation is as high as 62.5%, and the recovery of fertility is not broken through by 50%. The key bottleneck that cannot be broken through in treatment is that stem cells at the injury and scar parts are lost or have low quality, and the intima repair obstacle causes poor intimal thinning function. How to improve the repair and recover the fertility to the maximum extent is a key problem to be solved urgently.

Under the condition that the prior art cannot break through disease reproduction improvement, stem cell therapy is a very promising research hotspot in recent years, including spinal cord sources, menstrual blood sources, umbilical cord sources, fat sources and the like, and the stem cell therapy achieves a repair effect superior to that of traditional therapy after clinical application and becomes the most promising application direction for clinical transformation of cell therapy of fibrotic diseases. However, limitations and deficiencies of stem cell applications include: the tumorigenicity of differentiation into functional target cells (i.e., committed differentiation) and the ethical issues that may be involved lead to limited clinical use due to their instability of differentiation in vitro and the potential for deregulation during differentiation leading to tumorigenesis, etc. Therefore, how to apply stem cells with the maximum potential regeneration capacity, simultaneously perform directional induced differentiation on the stem cells, identify and eliminate tumorigenic results of the stem cells, and simultaneously combine a biophysical support material to form a cell patch of a composite device, so that the cell patch not only can effectively isolate a wound surface, but also can promote endometrial proliferation and repair, and the problem to be solved is urgently needed.

It has been reported that cells are loaded using a bio-carrier as a therapeutic agent, and for example, patent document CN105378062A discloses that a biomaterial prepared using a cell-containing and temperature-sensitive cell-stabilizing biomaterial is used as a therapeutic agent for kidney diseases, but this document only loads cells at a cell level, that is, using a cell-stabilizing biomaterial such as hydrogel or the like, and has not been studied at a level where hydrogel is directly loaded on organoids. Compared with cells, the organoid directly has better treatment effect. However, because there is a great difference between the organoid and the cell properties, there is a great technical obstacle to using biological materials to load organoids, and there is no report yet.

Disclosure of Invention

To solve the problems in the related art, the present disclosure provides an implantable article for preventing intrauterine adhesion.

In a first aspect, the present disclosure provides an implantable article for preventing intrauterine adhesions, including hydrogels and organoids.

In combination with the first aspect, the hydrogel is liquid at room temperature.

In combination with the first aspect, the hydrogel forms a coagulated hydrogel at body temperature, wherein the coagulated hydrogel has pores.

In combination with the first aspect, the organoid is loaded within the pores of the coagulated hydrogel.

In combination with the first aspect, the pores have a pore size of about 100 microns.

In combination with the first aspect, the hydrogel is a biodegradable temperature-sensitive hydrogel.

In combination with the first aspect, the hydrogel has a pH value that is the same as a physiological condition of a human.

In combination with the first aspect, the organoid is a micro-endometrial organ of endometrial glandular epithelial origin formed by directed differentiation.

In combination with the first aspect, the implant is injectable.

In a second aspect, the present disclosure also provides a method for preparing the aforementioned implant, the method comprising the steps of:

(1) dissociating the endometrioid organ from the matrigel, and adding an endometrioid organ culture medium to prepare an organoid suspension;

(2) placing the liquid hydrogel which is in accordance with the diameter of the micropores released by the organoid in a low-temperature environment to prepare organoid cell suspension, and fully and uniformly mixing the organoid cell suspension and the hydrogel to form the implantable object.

In combination with the second aspect, the low temperature environment is ice at 4 ℃, and preferably, the organoid cell suspension has a concentration of 15%; more preferably, 1mL of organoid cell suspension is mixed with 3mL of hydrogel.

In a third aspect, the present disclosure also provides a use of an implant according to the preceding description for the preparation of a medicament for the treatment of functional repair of the endometrium.

In combination with the third aspect, the functional repair process for treating endometrium is as follows:

firstly, separating, purifying and specially culturing endometrium organoids obtained from autologous endometrium of a patient;

dissociating the endometrioid organ from the matrigel, and adding 1mL of endometrioid organ culture medium to prepare an organoid suspension;

step three: placing liquid hydrogel which is in accordance with the diameter of micropores released by organoids on ice at 4 ℃, preparing the hydrogel with the concentration of 15%, and fully and uniformly mixing 1mL of organoid cell suspension and 3mL of hydrogel to form an implantable object;

step four: after various preparations before perfection of a patient with uterine cavity adhesion, performing hysteroscopy uterine cavity adhesion decomposition, and taking complete adhesion separation to recover normal shape of the uterine cavity and taking the condition that two fallopian tube openings can be seen simultaneously in the same visual field as a standard for stopping the operation;

step five: after residual blood and uterine swelling medium in the uterine cavity are absorbed completely, 4ml of implantable material prepared in the three steps is injected into the uterine cavity, a patient takes a head which is low and has a high hip, the liquid cell patch is kept for 20 minutes, the implantable material is solidified into solid with jelly texture at the temperature of the human body, and the organoid contained in the implantable material forms a composite device, so that a wound surface after adhesion separation can be effectively isolated, adhesion can be prevented from being formed after the wound surface is attached, meanwhile, the texture of jelly-like tissues is soft, compression can not be generated on blood vessels of the uterine muscle layer, and good blood supply in intimal repair can be guaranteed. Then, the hydrogel pores slowly release organoid, and collect to the wound surface after adhesion separation, so that functional repair of endometrium is promoted, and the effect of improving reproduction is achieved;

step six: observing the general condition of the patient after the operation, whether the patient has the adverse reactions such as allergic fever and the like, and managing and re-diagnosing according to the standard flow of the cell treatment after the intrauterine adhesion separation operation.

Defining:

unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The term "room temperature" as used in this disclosure is around 25 ℃.

The term "body temperature" as used in this disclosure is around 37 ℃.

In the publication, after the implantable material which is liquid at room temperature is injected into the uterine cavity, the liquid hydrogel can be shaped to conform to the lacuna in the process of gradual solidification at the body temperature, and the implantable material is shaped to the shape of the uterine cavity in the uterine cavity, so that the contact among wound surfaces in the uterine cavity is completely isolated. The shaping process of the temperature-sensitive hydrogel completely meets the individual requirements of different uterine cavity shapes of human bodies. In addition, the injection type avoids the peeling of the cells by the cervical canal and the uncertainty of the placement position when the uterine cavity is placed.

In the present disclosure, the hydrogel after coagulation is jelly-like and the hardness does not cause pressure on the blood vessels and the intima of the uterine muscle wall. The solidified hydrogel has pores with the diameter of about 100 microns, and is a proper pore size for organoid escape. The implantable material has controllable biological degradability, is matched with the growth speed of a new endometrium, and has no toxic or side effect on a human body in about 20 to 30 days at present. In addition, in the process of gradual degradation of the hydrogel (about 20 days), the organoids can be slowly and uniformly released and recruited to the damaged part for continuous repair; achieving the purpose of improving reproduction. The hydrogel can be of the type consisting of a temperature-sensitive extracellular matrix.

In the present disclosure, organoids are prepared for patient autologous endometrial origin, without immune rejection.

In the present disclosure, organoids are slowly released from hydrogel pores and recruited to the wound surface for repair after detachment of adhesions.

Drawings

Other features, objects, and advantages of the present disclosure will become more apparent from the following detailed description of non-limiting embodiments when taken in conjunction with the accompanying drawings. In the drawings:

FIG. 1 is HE staining after implantable implantation;

FIG. 2 shows the GFP green fluorescent endometrioid organ culture;

FIG. 3 shows the GFP cell patch in situ;

FIG. 4 shows that the left uterus of the left mouse is not provided with implantable articles for intrauterine adhesion, and the right uterus is provided with implantable articles after molding, so that pregnancy can be seen.

Detailed Description

Hereinafter, exemplary embodiments of the present disclosure will be described in detail with reference to the accompanying drawings so that those skilled in the art can easily implement them. Also, for the sake of clarity, parts not relevant to the description of the exemplary embodiments are omitted in the drawings.

In the present disclosure, it is to be understood that terms such as "including" or "having," etc., are intended to indicate the presence of the disclosed features, numbers, steps, behaviors, components, parts, or combinations thereof, and are not intended to preclude the possibility that one or more other features, numbers, steps, behaviors, components, parts, or combinations thereof may be present or added.

It should be further noted that the embodiments and features of the embodiments in the present disclosure may be combined with each other without conflict. The present disclosure will be described in detail below with reference to the accompanying drawings in conjunction with embodiments.

Example 1:

preparation of endometrioid organ:

step 1: repeatedly washing the obtained mouse endometrial tissue in normal saline containing 2% (v/v) double antibody for several times;

step 2, adding the washed tissue into HBSS buffer solution, and processing the tissue into tissue fragments of 1-2 mm3 within 15 min;

step 3, adding a digestive juice I into the tissue fragments, digesting for 1-3 h at a constant temperature of 37 ℃, adding an HBSS buffer solution to stop digestion, and centrifuging to retain precipitates;

step 4, adding a digestive juice II into the precipitate obtained in the step 3, digesting at the constant temperature of 37 ℃ for 8-12 min, adding an HBSS buffer solution to stop digestion, centrifuging and retaining the precipitate;

step 5, adding an HBSS buffer solution into the sediment obtained in the step 4 to resuspend the sediment, filtering by using a 100-micron filter screen and centrifuging to obtain an endometrial epithelial cell sediment;

step 6, adding erythrocyte lysate into the endometrial epithelial cell sediment for cracking, then adding HBSS buffer solution to stop cracking, and obtaining cell sediment after centrifugation;

step 7, adding a precooled Advanced DMEM/F12 culture medium into the cell sediment, uniformly mixing, adding matrigel, uniformly mixing, inoculating the mixture into a culture dish after forming gel drops, standing, inverting the mixture at a constant temperature of 37 ℃ for a period of time after solidification, adding amplification culture medium, performing amplification culture for 5-7 days under the conditions of 37 ℃ and 5% CO2, and replacing the amplification culture medium every 2-3 days;

and 8, adding a differentiation medium into the system after the amplification culture, and continuously culturing for 13-18 d, wherein the differentiation medium is replaced once every other day to obtain the endometrioid organ.

Preparation and transplantation of implantable articles:

step 1, dissociating the obtained endometrioid organs from matrigel, and adding 200ul of endometrioid organ culture medium to prepare a cell suspension;

step 2, placing the hydrogel on ice at 4 ℃, preparing the hydrogel with the concentration of 15%, and fully and uniformly mixing 200ul of cell suspension and 150ul of hydrogel for later use to form an implantable object;

step 3, establishing a uterine cavity adhesion mouse model, exposing uterine horns on two sides, injecting the uterine horns to the damaged part of the uterus of the mouse by using a 20ul pipette implantable object, wherein the volumes of two sides of the uterine horns are 40ul respectively;

and 4, step 4: ligating two sides of the injury part, and releasing the ligation band after 30min, wherein the implantable object forms a jelly-like composite device suitable for the shape of uterine cavity, and is tightly supported and isolated at the injury part.

And 5: and observing the repair and reproductive improvement fate of the endometrium of the mouse.

As can be seen from FIG. 1, the number of endometrial glands in each group of visual fields of the transplanted organoid is gradually increased along with the lapse of time, and when 4 weeks after transplantation, the number of the endometrial glands in the transplanted organoid is obviously higher than that in the control group.

As can be seen from the attached figure 2, the endometrium obtained by the GFP mouse can form GFP endometrium organoids under the culture of the conditioned medium, is in a green fluorescence imaging mode, has the diameter of more than 100um, and has the functions of proliferation and differentiation.

As can be seen in fig. 3, in vivo bioluminescent imaging shows that the hydrogel scaffold can retain the organoid at the wound site for a longer period of time.

As can be seen from FIG. 4, the uterus of mice on the IUA model side after organoid transplantation treatment was successfully pregnant, while the uterus of mice on the IUA model side without organoid transplantation treatment was not pregnant.

The foregoing description is only exemplary of the preferred embodiments of the disclosure and is illustrative of the principles of the technology employed. It will be appreciated by those skilled in the art that the scope of the invention in the present disclosure is not limited to the specific combination of the above-mentioned features, but also encompasses other embodiments in which any combination of the above-mentioned features or their equivalents is possible without departing from the inventive concept. For example, the above features and (but not limited to) the features disclosed in this disclosure having similar functions are replaced with each other to form the technical solution.

Claims

1. An implantable article for preventing intrauterine adhesions, comprising a hydrogel and an organoid.

2. An implantable article according to claim 1, said hydrogel being liquid at room temperature.

3. An implantable article according to claim 1 or 2, said hydrogel forming a solidified hydrogel at body temperature, wherein said solidified hydrogel has pores;

preferably, the organoid is loaded within the pores of the coagulated hydrogel;

more preferably, the pores have a pore size of about 100 microns.

4. An implantable article according to any one of the preceding claims, said hydrogel being a biodegradable temperature-sensitive hydrogel.

5. An implantable article according to any of the preceding claims, said hydrogel having a pH value that is the same as a physiological condition of a human.

6. An implantable article according to any of the preceding claims, said organoids being of endometrial glandular epithelial origin, formed by directed differentiation of micro-endometrial organs.

7. The implant of any preceding claim, which is injectable.

8. Method for the preparation of an implant according to any one of claims 1 to 7, comprising the steps of:

9. The method of claim 8, wherein the low temperature environment is at 4 ℃ on ice, preferably the organoid cell suspension is at a concentration of 15%; more preferably, 1mL of organoid cell suspension is mixed with 3mL of hydrogel.

10. Use of an implant according to any one of claims 1-7 in the manufacture of a medicament for the treatment of functional repair of the endometrium.