3d-printed cervical plug and preparation method

Abstract

A 3D-printed cervical plug. A 3D-printed cervical plug support is filled with a nanocomposite hydrogel. The nanocomposite hydrogel is a supramolecular nanocomposite formed by self-assembly of (-)-epigallocatechin-3-gallate and metal ions in a hydrogel.

Description

3D printed cervical plug and preparation method thereof TECHNICAL FIELD

[0001] The present application relates to the technical field of medical devices, in particular to a 3D printed cervical plug and a preparation method thereof. BACKGROUND

[0002] Uterine contractions caused by transcervical embryo transfer often result in the expulsion of the fertilized egg from the cervix, significantly reducing maternal-fetal contact and thus leading to a lower embryo implantation rate. Most of the strategies currently reported are to reduce uterine contractions through drug intervention to improve embryo implantation after transfer, for example, atosiban, indomethacin and nolasiban, and the use of biologically active macromolecules such as hyaluronic acid, human chorionic gonadotropin and progesterone to enhance endometrial receptivity. However, due to the lack of effective regulation of the physical interaction at the maternal-fetal interface, it is extremely challenging to strengthen embryo-endometrial contact after transfer to prevent embryo expulsion.

[0003] In view of this, the present application is proposed. SUMMARY

[0004] The present application aims to provide a 3D printed cervical plug and a preparation method thereof, which can prolong the residence time of the embryo in the uterus and promote embryo implantation.

[0005] To achieve the above-mentioned purpose, the first technical solution adopted by the present application is:

[0006] The 3D printed cervical plug is filled with a nano-composite hydrogel in a 3D printed cervical plug support; wherein the nano-composite hydrogel is a supramolecular nanocomposite formed by self-assembly of (-)-epigallocatechin-3-gallate and metal ions in the hydrogel.

[0007] Preferably, the metal ions are any one or more of zinc ions, copper ions, magnesium ions, iron ions, molybdenum ions, calcium ions and manganese ions.

[0008] Preferably, the mass ratio of (-)-epigallocatechin-3-gallate to metal ions is (2-4):1.

[0009] Preferably, the mass ratio of (-)-epigallocatechin-3-gallate to metal ions is 4:1.

[0010] Preferably, the mass-volume concentration of (-)-epigallocatechin-3-gallate is 5-20 mg / mL.

[0011] The second technical solution adopted by the present application is:

[0012] The preparation method of any of the above-mentioned 3D printed cervical plugs, the method comprising:

[0013] The 3D printing is adopted to prepare the cervical plug stent;

[0014] After the (-)-epigallocatechin-3-gallate is uniformly mixed with the metal ion solution, sodium alginate and hyaluronic acid are added to form a nanocomposite hydrogel;

[0015] The nanocomposite hydrogel is filled in the cervical plug stent and solidified.

[0016] Preferably, the nanocomposite hydrogel is filled in the cervical plug stent.

[0017] Preferably, the pH of the solution is adjusted to 7.0-8.0 before the sodium alginate and hyaluronic acid are added.

[0018] Preferably, the pH of the solution is adjusted to 7.4 before the sodium alginate and hyaluronic acid are added.

[0019] Compared with the prior art, the present application has the following beneficial effects:

[0020] (1) The 3D printing cervical plug provided by the present application can effectively prevent the embryo from falling out of the uterus. The retention rate of the fluorescent microspheres similar in size to the blastocyst in the uterine cavity within 1 hour is increased from 44.83% to 83.13%, and the number of microspheres entering the uterine cervix is decreased from 31.03% to 10.84%.

[0021] (2) The 3D printing cervical plug provided by the present application can improve the physiological function of the embryo: the 3D printing cervical plug does not affect the development of the embryo, and the blastocyst formation rate compared with the blank control is 77.55% vs. 76.8%. The RT-qPCR results show that the 3D printing cervical plug can improve the expression level of the mRNA related to the development and implantation of the embryo, and the relative expression amount of Fgfr1, OTX2, Eomes, CDX2, HOXA10 and CD44 is higher than that of the control group.

[0022] (3) In animal experiments, compared with the traditional embryo transfer, the implantation rate of the 3D printing cervical plug treatment is increased from 45% to 65%, and the implantation rate is increased by 1.4 times. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1A is a physical diagram of the 3D printing cervical plug stent in Example 1, and Figure 1B is a scanning electron microscope diagram of the nanocomposite hydrogel in Example 1;

[0024] Figure 2 is the biocompatibility of the 3D printing cervical plug in Example 1;

[0025] Figure 3A is the distribution of fluorescent microspheres in the mouse uterus of the 3D printing cervical plug in Example 1, and Figure 3B is the quantitative analysis of the number of fluorescent microspheres in the mouse uterus of the 3D printing cervical plug in Example 1;

[0026] Figure 4A shows the effect of the 3D-printed cervical plug on the formation rate of zygotes at different cleavage stages, and Figure 4B shows the effect of the 3D-printed cervical plug on the expression of embryo growth and development-related factors in the zygotes.

[0027] Figure 5 is a simulation diagram of the stress and strain of the cervix after the 3D-printed cervical plug is used to block the cervix in Example 1.

[0028] Figure 6 shows the effect of the 3D-printed cervical plug and the control group on the implantation promotion effect in embryo transfer in Example 2.

[0029] Figure 7 shows the effect of the 3D-printed cervical plug and the control group on the implantation promotion effect in embryo transfer in Example 3. DETAILED DESCRIPTION

[0030] To make the objectives, technical solutions and advantages of the present application clearer, further detailed description will be given below in conjunction with specific embodiments and drawings. It should be understood that these descriptions are only exemplary and are not intended to limit the scope of the present application. In addition, in the following description, the description of well-known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present application. If specific conditions are not specified in the examples, the conventional conditions or the conditions recommended by the manufacturer are used. If the manufacturer of the reagent or instrument is not specified, it is a conventional product that can be purchased on the market.

[0031] The first embodiment of the present application provides a 3D-printed cervical plug, which is filled with a nano-composite hydrogel in a 3D-printed cervical plug support. The nano-composite hydrogel is a supramolecular nano-composite formed by self-assembly of (-)-epigallocatechin-3-gallate and metal ions in the hydrogel.

[0032] Understandably, the nano-composite hydrogel can be filled in the 3D-printed cervical plug support by any method, for example, the filling can be achieved by injection. The filling amount of the nano-composite hydrogel is not particularly limited, and those skilled in the art can fill an appropriate amount of nano-composite hydrogel in the hollow part according to the size of the cervical plug support.

[0033] (-)-Epigallocatechin-3-gallate (EGCG) is derived from green tea polyphenols, which has strong antioxidant, anti-inflammatory and anticancer activities, and can protect reproductive cells and oocytes from oxidative stress damage and has a preventive effect on infertility. Metal ions such as zinc ions, copper ions, magnesium ions, iron ions, molybdenum ions, calcium ions and manganese ions are essential for the maintenance of normal reproductive function and embryonic development. The embodiment of the present application can promote embryo implantation by rapidly self-assembling supramolecular polyphenol nano-composites through coordination chelation of EGCG and these metal ions, and then compounding with the hydrogel.

[0034] The amount of EGCG and metal ions is determined according to the bicoordination of the two, for example, the mass ratio of EGCG and metal ions is generally (2-4): 1. In some more preferred embodiments, the mass ratio of EGCG and metal ions is 4: 1.

[0035] In addition, the mass volume concentration of EGCG can also be determined according to actual needs, for example, it can be 5-20 mg / mL.

[0036] The first embodiment of the present application provides a preparation method of 3D printed cervical plug, comprising:

[0037] The 3D printing is used to prepare the cervical plug stent;

[0038] After the (-)-epigallocatechin-3-gallate is uniformly mixed with the metal ion solution, sodium alginate and hyaluronic acid are added to form a nanocomposite hydrogel;

[0039] The nanocomposite hydrogel is filled in the cervical plug stent and solidified.

[0040] The present application first combines the clinical results in reproduction with 3D printing technology; it has been verified that there is an interaction force between the 3D printed cervical plug stent and the cervix, and the stress and strain make the CervPlug remain in the cervix without falling off. The present application makes the CervPlug have shape and biological functions at the same time through simple injection filling.

[0041] Since the cervix is not completely closed, the shape and size of the cervix can be obtained through three-dimensional color ultrasound, and then the cervical plug stent is designed and 3D printed according to the results, and the size is different for different people. The 3D printed cervical plug stent can be combined with the size of the cervix to prevent the embryo from falling off after transplantation. The specific method of the 3D printed cervical plug stent is the commonly used 3D printing method for those skilled in the art, without special limitation. Exemplarily, it can include the following steps: a) three-dimensional color ultrasound is used to collect cervical data and size; b) the cervical plug is designed according to the collected cervical data; c) polylactic acid PLA is used as raw material, and the 3D printer is used to print according to the designed cervical plug, and the printed cervical plug stent is finely sanded with sandpaper to avoid damage to the cervix. The printed cervical plug stent is in the form of a screw thread, hollow, and the thickness, diameter, bolt length and total length can be adjusted according to actual conditions.

[0042] The preparation method of the nanocomposite hydrogel is that EGCG and metal ions are self-assembled in the hydrogel. In order to promote the coordination of EGCG and metal ions, the pH of the solution is adjusted to 7.0-8.0 before adding sodium alginate and hyaluronic acid.

[0043] In some preferred embodiments, a crosslinking agent can also be added to facilitate the formation of the hydrogel. The crosslinking agent includes D-glucose lactone, calcium carbonate, and the like, and the specific amount is adjusted according to the actual needs.

[0044] To make the technical solutions of the present application clearer, the following describes the 3D printed cervical plug and its performance through a plurality of specific embodiments.

[0045] The 3D printer used in the embodiments of the present application is Zortrax M200 Plus (Warmińsko-mazurskie, Polish), and the raw material is PLA. Example 1

[0046] 1.1 Preparation of 3D printed cervical plug (CervPlug):

[0047] The 3D printing is used to make a customized cervical plug support: according to the cervical size (0.63 cm and 2.06 cm), the printed cervical plug support is in a threaded shape, hollow and has a thickness of 0.5 mm, a diameter of 0.63 cm, a bolt part length of 2.06 cm, a total length of 2.26 cm, and a round table diameter of 0.8 cm at both ends, which is slightly wider than the middle diameter.

[0048] The one-pot method is used to prepare the hydrogel in situ: 100 μL of EGCG solution (10 mg / mL), 50 μL of ZnSO4ꞏ7H2O (5 mg / mL), and 100 μL of distilled water are mixed uniformly; the pH of the solution is adjusted to 7.4; then 200 μL of sodium alginate (45 mg / mL), 200 μL of hyaluronic acid (45 mg / mL), 100 μL of CaCO3 (45 mg / mL), and 50 μL of D-glucose lactone (150 mg / mL) are added to form an EGCG-Zn Ⅱ nanocomposite hydrogel. 1 mL of the mixture is injected into the 3D printed cervical plug support using a 1 mL syringe, and then the model is placed at 37 °C to allow the mixture to solidify, thereby assembling the 3D printed cervical plug CervPlug.

[0049] The actual picture of the 3D printed cervical plug prepared in this example is shown in Fig. 1A, which has a hollow part for filling the nanocomposite hydrogel, and the scanning electron microscope image of the nanocomposite hydrogel is shown in Fig. 1B.

[0050] 1.2 Biocompatibility test of CervPlug

[0051] The PLA, EGCG-Zn IINanocomplexes integrated hydrogel and CervPlug were immersed in 10 mL of DMEM medium for 48 h (PLA, EGCG-Zn II nanocomplexes integrated hydrogel and CervPlug experimental groups), and DMEM medium without any treatment was used as the control group (Control). Cell viability was evaluated by CCK-8 method. 5 x 10 3 NIH-3T3 cells were seeded in 96-well plates. Then, the above treated DMEM was co-cultured with the cells seeded in 96-well plates for 6, 12, 24, 36 and 48 hours, respectively. Then, 10 μL of CCK-8 reagent was added to each 100 μL, and incubated at 37°C for 1 h in a 5% CO2 incubator. The absorbance (OD value) at 450 nm was measured by a microplate reader. The cell viability of the Control group was recorded as 100%, and the cell viability of other experimental groups was compared with the Control group. The experimental results are shown in Figure 2, which shows that CervPlug has good biological safety.

[0052] 1.3 CervPlug plugging effect test

[0053] Model experiment using ICR mouse uterus: Fluorescent microspheres with a diameter of 100 μm similar to the size of blastocysts were injected into the mouse uterus through the cervix, with an injection volume of 100 uL. The distribution of fluorescent microspheres in the uterus at different times was observed using a live imaging instrument. The results are shown in Figure 3A. CervPlug was used to block the cervix, and the Control group was not treated. After injecting fluorescent microspheres into the mouse uterus and oscillating in vitro for 1 h, the number of fluorescent microspheres in the uterus of the two groups was quantitatively analyzed. The results are shown in Figure 3B. In the Control group, the fluorescent microspheres in the uterine cavity were 44.83%, in the cervix were 31.03%, and in the others were 24.14%. In the CervPlug group, the fluorescent microspheres in the uterine cavity were 83.13%, in the cervix were 10.84%, and in the others were 6.02%.

[0054] 1.4 CervPlug effect on embryo growth and development test

[0055] An experiment group and a control group Control were set up. The experiment group was the 3D printed CervPlug in 1.1, and the control group was the 3D printed CervPlug stent in 1.1 filled with hydrogel without adding EGCG and zinc ions. The experiment group and the control group were respectively soaked in the embryo culture medium KSOM for 48 h. The KSOM culture medium treated by CervPlug was made into 8 droplets of 40 uL in a 35 mm embryo culture dish, and 10-20 fertilized eggs were cultured in each droplet to observe the growth and development of the embryos. As shown in FIG. 4A, CervPlug does not affect embryo development, and the blastocyst formation rates of the experiment group and the control group are 77.55% and 76.8%, respectively. The embryos treated by CervPlug and Control were collected and subjected to RT-qPCR to determine the potential effects of CervPlug on embryos, and the expression levels of embryo growth and development and implantation-related factors were obtained. The results are shown in FIG. 4B. The expression levels of growth factor receptor 1 (Fgfr1) in the CervPlug treatment and normal culture groups were 3.22 and 1.01, respectively. The expression levels of orthodontic homeobox 2 (OTX2) were 1.63 and 1.01, respectively. The expression levels of ectoderm protein (Eomes) were 1.95 and 1.01, respectively. The expression levels of caudal type homeobox 2 (CDX2) were 1.73 and 1.01, respectively. The expression levels of implantation homeobox A10 (HOXA10) were 3.68 and 1.02, respectively. The expression levels of cell surface adhesion receptor cluster of differentiation 44 (CD44) were 1.63 and 1.00, respectively.

[0056] 1.5 Verification of the interaction between CervPlug and the cervix

[0057] ANSYS Workbench was used to simulate the stress distribution of CervPlug blocking the cervix in 1.1. The Young's modulus of PLA is 277.7 MPa, and the Poisson's ratio is 0.36, while the Young's modulus of the cervix is 0.03878 MPa, and the Poisson's ratio is 0.4 (49, 50). The results are shown in FIG. 5. The friction coefficient between the CervPlug and the cervix is 0.1. When the cervix is blocked, it will cause slight deformation of the cervix. The maximum deformation caused by CervPlug on the upper end of the cervix is 1.02 mm under a maximum stress of about 8 Pa. The results show that the blockage of the cervix causes changes in the stress of the cervix, leading to deformation of the cervix. During the slight deformation of the cervix, a stress of 0-7.1 Pa is observed, indicating that there is an interaction between the cervix and CervPlug. Example 2

[0058] The 3D-printed CervPlug in Example 1.1 was adjusted only according to the mouse cervix, and the hydrogel preparation was the same as in Example 1.1, to obtain the CervPlug of the present example. The customized CervPlug was made by 3D printing: the size of the CervPlug was adjusted according to the size of the mouse cervix, and the hollow of the printed CervPlug was in a threaded shape, with a thickness of 0.3 mm, a threaded diameter of 1.5 mm, a corresponding length of 6 mm, a total length of 8 mm, and a circular table diameter of 1.8 mm.

[0059] The above CervPlug was used as the experimental group to block the cervix of the pseudopregnant female mouse, and the following control groups were set up: ① No EGCG and zinc ion group: 3D-printed CervPlug filled with hydrogel without added EGCG and zinc ions; ② EGCG group: 3D-printed CervPlug filled with hydrogel with only an equal amount of EGCG; ③ Zinc ion group: 3D-printed CervPlug filled with hydrogel with only an equal amount of zinc ions. Subsequently, the in vitro cultured blastocysts were subjected to embryo transfer experiment, and since the mice have bilateral uteri, 16 blastocysts were implanted into each mouse uterus, with 8 in each side. Each group of experiments was set up with 5 replicates, and 2 days after implantation, 1% Chicago blue 6B dye 100 μL was injected into the tail vein. The mice were sacrificed after 3 minutes, the uterus was removed, and the number of implantation sites was observed and recorded for statistical analysis. As shown in Figure 6, the implantation rates of the no EGCG and zinc ion group, the EGCG group, the zinc ion group, and the 3D-printed CervPlug experimental group were 42.5%, 50%, 47%, and 65%, respectively.

[0060] Example 3 CervPlug promotes embryo implantation

[0061] The 3D-printed CervPlug in Example 1.1 was adjusted only according to the mouse cervix, and the hydrogel preparation was the same as in Example 1.1, to obtain the CervPlug of the present example. The customized CervPlug was made by 3D printing: the size of the CervPlug was adjusted according to the size of the mouse cervix, and the hollow of the printed CervPlug was in a threaded shape, with a thickness of 0.3 mm, a threaded diameter of 1.5 mm, a corresponding length of 6 mm, a total length of 8 mm, and a circular table diameter of 1.8 mm.

[0062] The cervix of pseudopregnant female mice was blocked with the CervPlug described above, and the Control group was only physically stimulated. Subsequently, in vitro cultured blastocysts were used for embryo transfer experiments. Since the mice have a bilateral uterus, 16 blastocysts were implanted in each mouse, 8 in each side of the uterus. Each experiment was set up with 5 replicates, and 2 days after implantation, 1% Chicago blue 6B dye 100 μL was injected into the tail vein. After 3 minutes, the mice were sacrificed, and the uterus was removed, observed, and the number of implantation sites was recorded and statistically analyzed. As shown in Figure 7, the embryo implantation rate increased from 45% to 65% after using the CervPlug, an increase of 1.4 times.

[0063] Finally, it should be noted that the above examples are only representative of the present application. Obviously, the technical solutions of the present application are not limited to the above examples, and there can be many variations. All variations that can be directly derived or inferred by those of ordinary skill in the art from the content of the present application should be considered as falling within the protection scope of the claims of the present application.

Claims

1. A 3D printed cervical plug, characterized in that, The application discloses a 3D printing cervical plug stent filled with nano-composite hydrogel; wherein the nano-composite hydrogel is a supramolecular nano-composite formed by self-assembly of (-)-epigallocatechin-3-gallate and metal ions in the hydrogel.

2. The 3D printed pessary of claim 1, wherein, The metal ions are any one or more of zinc ions, copper ions, magnesium ions, iron ions, molybdenum ions, calcium ions and manganese ions.

3. The 3D printed pessary of claim 1, wherein, The mass ratio of the (-)-epigallocatechin-3-gallate to the metal ions is (2-4):

1.

4. The 3D printed pessary of claim 3, wherein, The mass ratio of the (-)-epigallocatechin-3-gallate to the metal ions is 4:

1.

5. The 3D printed pessary of claim 3, wherein, The mass-volume concentration of the (-)-epigallocatechin-3-gallate is 5-20 mg / mL.

6. The method of claim 1-5 for the preparation of 3D printed cervical plugs, characterized in that, The method comprises the following steps: The cervical plug stent is prepared by 3D printing; The (-)-epigallocatechin-3-gallate and the metal ion solution are uniformly mixed, and then sodium alginate and hyaluronic acid are added to form the nano-composite hydrogel; The nano-composite hydrogel is filled in the cervical plug stent and solidified.

7. The production method according to claim 6, wherein The solution pH is adjusted to 7.0-8.0 before the sodium alginate and the hyaluronic acid are added.

8. The production method according to claim 7, wherein The solution pH is adjusted to 7.4 before the sodium alginate and the hyaluronic acid are added.

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