WO2019090897A1 - 3d printed biological artificial ovary scaffold capable of activating primordial follicles, artificial ovary thereof, and use thereof - Google Patents

Abstract

A 3D printed biological scaffold capable of activating primordial follicles in ovarian cortex. By means of 3D printing technology, a primordial follicle activator and a hormone and a growth factor that are capable of promoting follicular development are mixed with a scaffold material for shaping by printing; then ovarian cortical fragments containing primordial follicles are implanted in the printed scaffold. After being implanted into a body, the cortical fragments can obtain better scaffold-based protection and richer nutrition supply, and the biological scaffold slowly degrades in the body to slowly release the follicle activator, thereby continuously activating the primordial follicles.

Description

3D printed artificial ovarian bioscaffold capable of activating primordial follicles and artificial ovary and use thereof Technical field

The present invention relates to the field of biotechnology, and in particular to the preparation of a 3D printed biological scaffold capable of activating primordial follicles in the ovarian cortex.

Background technique

With the rapid development of China's social economy, women's status is getting higher and higher, and at the same time, they are facing increasing work and psychological pressure. The reproductive age of women of childbearing age is continuously delayed. Coupled with the increasingly serious environmental pollution, various poisonous Harmful substances are getting closer and closer to life, and the combination of social and environmental factors has caused a large number of reproductive health problems. Since the opening of the second-child policy, there are more and more maternity women. Therefore, how to make the pregnancy process of super-age-age women with ovarian function gradually declining has become a research hotspot in recent years.

In addition to the pregnancy problems of women of childbearing age, in recent years, due to the fast pace of life and high work pressure, many women have a premature trend of entering the peri-menopause period, which has many adverse effects on physical and mental health, such as emotional instability and irritability. Various menopausal symptoms such as depression and memory loss, skin aging, obesity, osteoporosis, etc. How to adjust the physiological and psychological state of women in the aging period, delay aging, alleviate the symptoms of perimenopausal period, improve the quality of life after menopause, and it is more and more worthy of attention in a society where the population is aging.

(a) ovarian failure

With the increase of age, female ovarian function gradually declines to permanent menstruation, this physiological phenomenon is called menopause. When the follicles in the ovary are used up, or the remaining follicles lose their response to gonadotropins, the follicles do not develop and secrete estrogen, and do not stimulate endometrial growth, leading to menopause.

The most common treatment for menopause is hormone replacement/supplementation, which is supplemented with exogenous hormones when menopausal women are deficient in hormones. Although it has certain effects on improving menopausal syndrome, there are some drawbacks, such as: hormone therapy is not suitable for all people, patients with breast, uterine disease and diabetes, high blood pressure and other diseases need to be used with caution; hormone therapy The amount required by each person is different, it is difficult for doctors to give the most appropriate dose, and excessive hormones in the body can cause uncontrollable side effects such as endometrial cancer, ovarian cancer or breast cancer; exogenously administered hormones are artificial Synthetic, regardless of composition or proportion, can not replace the effect of its own secretion of hormones.

(2) Premature ovarian failure (POF)

The average natural menopausal age of women is around 50 years old. When this phenomenon occurs before the age of 40, it is called premature ovarian failure, which accounts for about 1% to 3% of women of childbearing age. Premature ovarian failure is mainly caused by premature depletion of primordial follicles in the ovary or inability of primord follicles to develop.

At present, assisted reproductive technologies such as IVF technology have become the key technology for the treatment of infertility. However, the key to IVF technology is that mothers must be provided with a high-quality mature egg. If the mother has no eggs, it can be discharged or ovulated. Low function, such as for patients with premature ovarian failure, then this technology can not help.

(three) polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is a common endocrine syndrome in women of childbearing age. The incidence rate is 6-10% of women of childbearing age. It is mainly characterized by menstrual thinning or amenorrhea, infertility, ovarian polycystic Sexual change, obesity, hairy, hyperandrogenism, etc. Treatment for patients with PCOS, including surgery, medication, and assisted reproductive technology.

Among them, PCOS patients use assisted reproductive technology, especially for PCOS patients who have ovulation but still not pregnant after 6 months of ovulation induction therapy, or multiple drug ovulation therapy and adjuvant therapy without ovulation and urgent treatment In pregnant patients, you can choose the assisted reproductive technology of embryo transfer, including in vitro fertilization (IVF) and in vitro maturation (IVM).

(four) ovarian cancer (Ovarian Cancer, OAC)

Ovarian cancer is a malignant tumor with high incidence in the ovary. In addition to interfering with the normal secretion of ovarian hormones, it also destroys most ovarian tissues, causing infertility and seriously endangering women's reproductive health.

The treatment of ovarian cancer is mainly surgery, supplemented by chemotherapy, radiation, and immunotherapy. Surgical treatment is the most effective treatment. In principle, the scope of surgery should be as far as possible to remove the primary tumor and all metastases. For those with fertility requirements, those who meet the conditions can maintain conservative fertility.

When most ovarian tissues are destroyed by tumors or other causes leading to follicular developmental disorders, there are still a large number of primordial follicles in the follicular cortex. Ovarian cortical tissue freezing is a common fertility protection measure today, how to make it in subsequent in vitro culture. Its activation, growth and ovulation are the current research hotspots.

Summary of the invention

At present, many studies have found many signaling pathways and targets related to primordial follicle activation, such as PTEN and PI3K-mTOR signaling pathway, LKB1-AMPK signaling pathway, etc. The present invention utilizes 3D printing technology to activate primordial follicle activators and promote follicles. The developing hormones and growth factors are mixed into the scaffold material, printed, and then transferred into the ovarian cortical fragments containing the original follicles. After transplanting back into the body, the cortical fragments can have better scaffold protection and richer nutrient supply. The bioscaffold is slow in the body. Degradation, sustained release of follicle activators, continuous activation of primordial follicles.

One aspect of the present invention provides a bioscaffold that mimics an artificial ovary, which is obtained by a 3D printing method, and the printing material used for 3D printing is a mixture of a biocompatible material, a primordial follicle activator, and a primordial follicle development promoter.

One aspect of the present invention provides an artificial ovary comprising a biological scaffold and a tissue fragment containing the primordial follicle embedded in the biological scaffold, wherein the bio scaffold is obtained by 3D printing, and the printed material is a biocompatible material, a primordial follicle A mixture of activators, follicle developmental enhancers.

In the technical solution of the present invention, the biocompatible material simulates a component contained in ovarian tissue selected from the group consisting of collagen I, sodium alginate, gelatin, agarose, Matrigel, hyaluronic acid, chitosan. One or a combination of any of several kinds of dextran. Preferred is a mixture of sodium alginate and gelatin, a mixture of sodium alginate and matrigel, a mixture of gelatin and matrigel, a mixture of collagen I and sodium alginate and gelatin, a mixture of collagen I and sodium alginate and matrigel, collagen I and a mixture of gelatin and matrigel.

In the technical solution of the present invention, the prepared biological scaffold is first UV-crosslinked and then cross-linked with CaCl ₂ . Can be soaked in CaCl ₂ for -20 ° C for short-term storage, or -80 ° C refrigerator for long-term storage, soaked in PBS or saline before use.

In the technical solution of the present invention, the hormone or growth factor for promoting follicular development is selected from the group consisting of follicle stimulating hormone (FSH), luteinizing hormone (LH), epidermal growth factor (EGF), and insulin-transferrin-selenium (ITS). One or a combination of several. Preferred are follicle stimulating hormone, a combination of follicle stimulating hormone and luteinizing hormone, a combination of follicle stimulating hormone and luteinizing hormone and epidermal growth factor, follicle stimulating hormone and luteinizing hormone and epidermal growth factor and insulin-transferrin- A combination of selenium. More preferably, the amount of follicle stimulating hormone (FSH) is 10-1000 mIU/ml, luteinizing hormone (LH) 1-100 mIU/ml, epidermal growth factor (EGF) 0.1-100 ng/ml, insulin-transferrin-selenium (ITS). Insulin-transferrin-selenium contains 1-100 ug/ml recombinant human insulin, 1-100 ug/ml human transferrin (essentially iron-free) and 0.1-100 ng/ml sodium selenite.

In the technical solution of the present invention, the primordial follicle activator comprises a signaling pathway agonist or inhibitor associated with primordial follicle activation, and a basal culture fluid. Wherein the relevant signaling pathway agonist or inhibitor is selected from the group consisting of a PTEN inhibitor, a PI3K signaling pathway activator, an AKT signaling pathway activator, a mTOR signaling pathway activator, or a combination of several.

In the technical solution of the present invention, the PTEN inhibitor is selected from the group consisting of SF1670 (0.5-100 uM), VO-Ohpic trihydrate (0.1-100 nM), pic bpv (0.1-100 nM), and phen type bpv (0.1-100 nM). Combination of species or multiples;

In the technical solution of the present invention, the PI3K signaling pathway agonist is selected from the group consisting of 1,3-diacyl quinic acid (0.1-100 uM), 740Y-P (PDGFR 740Y-P) (0.1-300 ug/ml) or A variety of combinations.

In the embodiment of the present invention, the AKT signaling pathway agonist is selected from the group consisting of 1,3-diacyl quinic acid (0.1-100 uM), deoxyandrographolide (0.1-100 mM), and SC79 (0.1-10 ug/ml). A combination of one or more.

In the present invention, the mTOR signaling pathway agonist is selected from the group consisting of MHY1485 (0.1-10 uM), PA (0.1-200 uM), PRO (0.1-100 uM), or a combination thereof.

In the technical solution of the present invention, the basal culture solution comprises a culture solution for culturing the primordial follicle, preferably comprising a medium, an antibiotic, a carbon source, a protease inhibitor, preferably comprising αMEM, FBS/BSA, sodium pyruvate, penicillin. Streptomycin. More preferably, αMEM is used as a mother liquor containing 10% FBS, 5.5 mg/ml sodium pyruvate, 100 IU/ml penicillin, and 100 ug/ml streptomycin.

In the technical solution of the present invention, the biocompatible stent has a void, and the diameter of the void is 1.5 to 3 times, preferably 2-2.5 times, the size of the ovarian cortex.

In the technical solution of the present invention, the 3D printing size of the biocompatible stent is: pore size (R): 100 μm-800 μm, preferably 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm. , 600 μm, 650 μm, 700 μm, 750 μm; line stacking angle: 0°-180°, preferably 5°, 10°, 15°, 20°, 30°, 40°, 50°, 60°, 70°, 80 °, 90°, 100°, 110°, 120°, 130°, 140°, 150°, 160°, 170°, 175°.

In the technical solution of the present invention, the shape of the biological scaffold may be various shapes such as a circle, an ellipse, a rectangle, a square, an irregular shape, and the like. A communication structure having a certain porosity inside.

In a solution of the invention, the tissue fragments comprising the primordial follicles are activated for soaking for 0.1-24 hours, preferably 0.5-6 hours, prior to implantation into the bioscaffold using the primordial follicle activator.

In a solution of the invention, the tissue fragments comprising the primordial follicle are selected from ovarian tissue fragments comprising primordial follicles, more preferably ovarian cortical fragments comprising primordial follicles.

Another aspect of the invention provides a method of preparing an artificial ovary comprising the steps of:

The biocompatible scaffold is printed by 3D printing, and the biocompatible scaffold is printed with a biocompatible material and a mixture of primordial follicle activator and primordial follicle development promoter.

Another aspect of the invention provides a method of preparing an artificial ovary comprising the steps of:

1) Printing the biocompatible stent by 3D printing, and printing the biocompatible stent material as a biocompatible material and a mixture of primordial follicle activator and primordial follicle development promoter;

2) soaking the ovarian cortical fragments containing the primordial follicles in a mixture of the primordial follicle activator and the basal medium;

3) Inject the activated ovarian cortical fragments onto the 3D printed biocompatible scaffold obtained in step 1) to obtain an artificial ovary.

Another aspect of the invention provides the use of the artificial ovary of the invention as a drug screening, follicular research model.

Another aspect of the invention provides the use of the artificial ovary of the invention for treating follicular dysplasia diseases.

In the technical solution of the present invention, the follicular development disorder disease is selected from the group consisting of ovarian failure, premature ovarian failure, polycystic ovary syndrome, ovarian cancer, and ovarian injury caused by trauma.

Another aspect of the invention provides a method of treating a follicular developmental disorder comprising the step of transplanting an artificial ovary of the invention into a subject.

Another aspect of the invention provides the use of the artificial ovary-like bioscaffold of the invention for the preparation of an artificial ovary.

Another aspect of the present invention provides the use of the artificial ovary-like biological scaffold of the present invention for preparing a medical device simulating an artificial ovary.

In the technical solution of the present invention, a biocompatible scaffold containing a primordial follicle activator and a hormone or growth factor for promoting follicular development is produced by 3D printing, and the frozen fresh ovarian cortical fragments containing primordial follicles are different in different concentrations. After incubation for 0.5 to 6 hours in the basal medium of the combined primordial follicle activator, it is transferred into the interior of the biocompatible scaffold and then transplanted back into the body.

In the technical solution of the present invention, the egg cell, the follicle, the primordial follicle, the tissue containing the primordial follicle, for example, the ovarian cortex including the follicle, and the like are non-human cells, or the egg cells, follicles, and primordial follicles used in the present invention. Tissues containing primordial follicles are commercially available.

Beneficial effect

1. The invention adopts an active substance mixed with hormones, growth factors and the like in the stent, which can be slowly released. After the cortical fragments are injected into the 3D printing stent and transplanted back into the body, the cortical fragments can have better stent protection and richer. Nutritional supply. The bioscaffold slowly degrades in the body, releasing the follicle activator and continuously activating the primordial follicle.

2, due to the addition of follicle activator in the stent, which greatly shortens the activation time of the original follicle in vitro, providing a new research tool to reveal the mechanism of primordial follicle activation.

3. Since the activation time of the original follicle in vitro and in vitro is shortened, the removal-activation-implantation procedure can be completed in the same operation, and the patient waiting time is reduced on the basis of avoiding the second operation, and the clinical treatment of premature ovarian failure and the auxiliary Reproductive technology offers new options.

4. After using the artificial ovary of the invention, the follicle activates and secretes a large amount of hormone regulating body secretion, and provides a new research direction for improving the menopausal syndrome and delaying aging.

Detailed ways

In order to more clearly understand the present invention, the present invention will now be further described with reference to the following examples. The examples are for illustrative purposes only and are not intended to limit the invention in any way. In the examples, each of the original reagent materials is commercially available, and the experimental methods not specifying the specific conditions are conventional methods and conventional conditions well known in the art, or in accordance with the conditions recommended by the instrument manufacturer.

The ITS commercial product used in the present invention contains 1.0 mg/ml recombinant human insulin, 0.55 mg/ml human transferrin (substantially iron-free) and 0.5 μg/ml sodium selenite.

Example 1 Design and preparation of 3D printed biological scaffold

(1) Combining computer aided design (CAD), using three-dimensional printing technology to construct a biological scaffold, the shape of the 3D bio scaffold is selected as a circle, a rectangle, a square, an ellipse or an irregular shape, and the internal structure is connected, Pore; pore size (R): 400 μm; line stacking angle: 30-120 degrees;

(2) Preparing a 3D printed biological scaffold printing material comprising a mixture of a biocompatible material, a primordial follicle activator, and a primordial follicle development promoter; the biocompatible material is 3%-5% sodium alginate and 8%- The mass ratio of 10% gelatin, sodium alginate and gelatin is 1:1, and it gels at room temperature after mixing. 740Y-P 10ug/ml, PA 8uM, PRO 2uM, FSH 100mIU, LH 10mIU, EGF 5ng/ml, ITS (used after 100-fold dilution) were added to the biocompatible material.

(3) printing the ovarian stent by 3D printing;

(4) The prepared scaffold is first UV-crosslinked for 10 minutes, then cross-linked with CaCl ₂ , soaked in CaCl ₂ for -20 ° C for short-term storage, or -80 ° C for long-term storage in the refrigerator, soaked in PBS or saline before use. .

Example 2 Design and preparation of 3D printed biological scaffold

The stent prepared in Example 2 was the same as the method of Example 1, except that the formulation of the printing material in the step (2) was carried out, and the formulation of the printing material was carried out in the proportions listed in the following table.

Example 2 Acquisition of primordial follicles

Female mice of 3 days old were sacrificed by cervical dislocation. After disinfecting the skin, the ovaries were aseptically removed from the back and placed in the separation medium (L-15+10% FBS+100 IU/ml penicillin+100 ug/ml chain). Mycin). The attached tissue around the ovary was removed under a stereoscopic microscope and washed 3 times in the separation solution. The ovary is then dosed with insulin under the microscope, and the mouse ovary is divided into tissue fragments containing primordial follicles.

Example 3 Activation of primordial follicles

Activators are agonists or inhibitors of the signaling pathways associated with primordial follicle activation and are of the prior art and are commercially available.

After cryopreservation or fresh ovarian cortex containing a large number of primordial follicles, after 0.5 to 6 hours of incubation in a basal medium containing different combinations of follicle activators, wash the saline three times, remove the foreign matter, and then transfer to print. A good bioscaffold was transplanted into the kidney membrane of adult female rats with bilateral ovarian ablation.

The basic broth formulation of the follicle activator is:

Example 4 Preparation and effect of 3D printed biological scaffold and artificial ovary

(1) Preserving the 3D printed biological scaffold prepared in Example 2 for pre-treatment of the biological scaffold: the spare ovarian bioscaffold stored at -80 ° C was taken out, placed in a UV station in a clean bench, at room temperature for 20 minutes, and then in physiological saline. Incubate for 30 minutes, wash and sterilize simultaneously.

(2) Three days old neonatal mouse ovarian cortical fragments were placed in the basal medium No. 1 of Example 2 for 1-6 hours to activate a large number of primordial follicles in the cortex in vitro.

(3) The cultured cortical fragments are injected into the 3D printed biological scaffold.

(4) The bioscaffold containing cortical fragments was transplanted into the bilateral ovariectomized adult female rats (8-10 weeks) under the renal capsule, and the surgical procedure was greater than or equal to 6.

(5) The female rats were sacrificed on the 5th, 10th, and 15th day after the transplantation, and the stents were removed. Paraffin-embedded and 5um tissue sections were taken: H&E staining to observe the follicular developmental morphology; immunohistochemical staining of PCNA to detect cell proliferation; TUNNEL Apoptosis was detected.

The results showed that with the increase of the number of days, the results of the 12 groups of experiments showed the state of primordial follicle activation and development. It can be seen from the photos that some of the primordial follicles successfully activated and developed into the preantral follicle stage, and the developing follicular granulosa cells proliferated. . It is indicated that the scaffold and artificial ovary of the present invention successfully activates the primordial follicle of the mouse in vivo and promotes its development, and can provide an alternative for ovarian disease caused by loss or damage.

Claims (10)

1. A biological scaffold simulating an artificial ovary obtained by 3D printing. The printing material used for 3D printing is a mixture of biocompatible materials, primordial follicle activators, and primordial follicle development promoters.
2. An artificial ovary comprising a biological scaffold and a tissue fragment containing the primordial follicle embedded in the biological scaffold, wherein the bio scaffold is obtained by 3D printing, and the printed material is biocompatible material, primordial follicle activator, follicular development promoting a mixture of agents.
3. The artificial ovary-simulated biological scaffold according to claim 1 or the artificial ovary according to claim 2, wherein the hormone or growth factor that promotes follicular development is selected from the group consisting of follicle stimulating hormone (FSH), luteinizing hormone (LH), and epidermal cell growth. Combination of one or more of factor (EGF), insulin-transferrin-selenium (ITS); preferably a combination of follicle stimulating hormone, follicle stimulating hormone and luteinizing hormone, follicle stimulating hormone and luteinizing hormone and epidermis A combination of cell growth factors, follicle stimulating hormone and luteinizing hormone and a combination of epidermal growth factor and insulin-transferrin-selenium.
4. The artificial ovary-simulated biological scaffold according to claim 1 or the artificial ovary according to claim 2, wherein the primordial follicle activator comprises a signaling pathway agonist or inhibitor associated with primordial follicle activation, and a basal medium; wherein, The signaling pathway agonist or inhibitor is selected from the group consisting of a PTEN inhibitor, a PI3K signaling pathway activator, an AKT signaling pathway activator, a mTOR signaling pathway activator, or a combination of several;

   Preferably, the PTEN inhibitor is selected from the group consisting of one or more of SF1670, VO-Ohpic trihydrate, pic-type bpv (0.1-100 nM), phen-type bpv; the PI3K signaling pathway agonist is selected from the group consisting of 1,3-two coffee a combination of one or more of acyl quinic acid, 740Y-P; an AKT signaling pathway agonist selected from the group consisting of 1,3-diacyl quinic acid, deoxyandrographolide, a combination of one or more of SC79; mTOR The signaling pathway agonist is selected from a combination of one or more of MHY1485, PA, PRO.
5. The artificial ovary of claim 2, wherein the tissue fragments comprising the primordial follicle are selected from ovarian tissue fragments comprising primordial follicles, more preferably ovarian cortical fragments comprising primordial follicles.
6. A method of preparing an artificial ovary, comprising the steps of:

   The biocompatible scaffold is printed by 3D printing, and the biocompatible scaffold is printed with a biocompatible material as well as a mixture of primordial follicle activator and primordial follicle developmental promoter.
7. A method of preparing an artificial ovary, comprising the steps of:

   1) Printing the biocompatible stent by 3D printing, and printing the biocompatible stent material as a biocompatible material and a mixture of primordial follicle activator and primordial follicle development promoter;

   2) soaking the ovarian cortical fragments containing the primordial follicles in a mixture of the primordial follicle activator and the basal medium;

   3) Inject the activated ovarian cortical fragments onto the 3D printed biocompatible scaffold obtained in step 1) to obtain an artificial ovary.
8. Use of the artificial ovary of claim 2 as a drug screening, follicular research model.
9. The artificial ovary according to claim 2, wherein the follicular dysplasia is selected from the group consisting of ovarian failure, ovarian failure, polycystic ovary syndrome, ovarian cancer, and ovarian disease caused by trauma damage.
10. The use of the artificial ovary-like biological scaffold of claim 1 for the preparation of an artificial ovary.