阅读说明：本技术 一种修复子宫内膜的丝素蛋白/功能化聚三亚甲基碳酸酯水凝胶及制备方法 (Silk fibroin/functionalized poly (trimethylene carbonate) hydrogel for repairing endometrium and preparation method thereof ) 是由 杨立群 于 2020-05-29 设计创作，主要内容包括：本发明属于生物医用材料与再生医学领域,具体涉及一种修复子宫内膜的丝素蛋白/功能化聚三亚甲基碳酸酯水凝胶及制备方法。该水凝胶包含丝素蛋白、功能化聚三亚甲基碳酸酯以及活细胞,丝素蛋白与功能化聚三亚甲基碳酸酯的质量比为5～1:1。本发明通过引入含有亲水链段且不含任何酯键、在降解过程中无酸性降解产物生成、且性能可控调节的功能化聚三亚甲基碳酸酯来提高水凝胶的生物相容性,并通过功能化聚三亚甲基碳酸酯的结构与组成、含量等因素来调控丝素蛋白水凝胶的成胶时间与成胶方式,并同时负载细胞/干细胞,对负载的细胞没有副作用,且对植入部位没有刺激性,进而达到修复子宫内膜的目的。(The invention belongs to the field of biomedical materials and regenerative medicine, and particularly relates to a silk fibroin/functionalized polytrimethylene carbonate hydrogel for repairing endometrium and a preparation method thereof. The hydrogel comprises silk fibroin, functionalized polytrimethylene carbonate and living cells, wherein the mass ratio of the silk fibroin to the functionalized polytrimethylene carbonate is 5-1: 1. The invention improves the biocompatibility of the hydrogel by introducing the functionalized polytrimethylene carbonate which contains hydrophilic chain segments, does not contain any ester bonds, does not generate acid degradation products in the degradation process and has controllable and adjustable performance, regulates the gelling time and the gelling mode of the silk fibroin hydrogel by factors such as the structure, the composition, the content and the like of the functionalized polytrimethylene carbonate, loads cells/stem cells at the same time, has no side effect on the loaded cells and has no irritation to the implantation part, thereby achieving the aim of repairing endometrium.)

1. The silk fibroin/functionalized polytrimethylene carbonate hydrogel for repairing endometrium is characterized by comprising silk fibroin, functionalized polytrimethylene carbonate and living cells, wherein the mass ratio of the silk fibroin to the functionalized polytrimethylene carbonate is 5-1: 1, and the mass of the living cells is 0.01-10% of that of the hydrogel.

2. The endometriotic silk fibroin/functionalized polytrimethylene carbonate hydrogel of claim 1, wherein the silk fibroin is tussah silk fibroin or mulberry silk fibroin or a mixture thereof.

3. An endometrial repair silk fibroin/functionalized polytrimethylene carbonate hydrogel according to claim 1, wherein the functionalized polytrimethylene carbonate has the general formula:

R₁＝-H，-CH₃，-OH，-CH₂OH，-CH₂CH₃，-CH₂CH₂CH₃，-(CH₂)₃CH₃，-CH₂OCH₃，-OCH₃，-OCH₂CH₂＝CH₂，-CH₂CH₂＝CH₂，-CH₂Cl，-CH₂Br，-CH₂N₃any one of the above;

R₂＝-H，-CH₃，-OH，-CH₂OH，-CH₂CH₃，-CH₂CH₂CH₃，-(CH₂)₃CH₃，-CH₂OCH₃，-OCH₃，-OCH₂CH₂＝CH₂，-CH₂CH₂＝CH₂，-CH₂Cl，-CH₂Br，-CH₂N₃any one of the above;

wherein: n is 10-400, m is 1-50, y1 is 1-1000, y2 is 1-1000, x 1: y1 is 10-1: 1, and x 2: y2 is 10-1: 1.

4. The endometriotic silk fibroin/functionalized polytrimethylene carbonate hydrogel of claim 4, wherein preferably, n is 20 to 50 or 80 to 150, m is 1 to 20; when n is 20-50, y1 or y2 is 10-100; when n is 80-150, y1 or y2 is 1-50.

5. The endometrium repairing silk fibroin/functionalized polytrimethylene carbonate hydrogel of claim 1, wherein the living cells are one or more of bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, embryonic stem cells, menstrual blood derived mesenchymal stem cells, amniotic mesenchymal stem cells, placental mesenchymal stem cells, human early pregnancies periosteum mesenchymal stem cells, adipose mesenchymal stem cells, endometrial stem cells, induced pluripotent stem cells; or the living cells are mixed cells of one or more than one of bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, embryonic stem cells, menses-derived mesenchymal stem cells, amnion mesenchymal stem cells, placenta mesenchymal stem cells, human early pregnancy periosteum mesenchymal stem cells, adipose mesenchymal stem cells, endometrium stem cells and induced pluripotent stem cells and one or two of endometrium mesenchymal stem cells and endometrium epithelial cells.

6. A method for preparing the endometriotic silk fibroin/functionalized polytrimethylene carbonate hydrogel of claims 1-5, comprising the steps of:

(1) taking trimethylolethane as a raw material, performing ester exchange and pyrolysis decarboxylation to prepare oxetane, sequentially adding 1-3L of polyethylene glycol ether and 0.2-0.8 mL of concentrated sulfuric acid into a reaction bottle, heating to 100-150 ℃ under stirring, dropwise adding 300-500 mL of a solution of 80-120 g of oxetane in polyethylene glycol ether, and reacting for 5-7 hours after dropwise adding; cooling to room temperature, recovering excessive polyglycol ether under reduced pressure, and distilling under reduced pressure to obtain colorless liquid;

(2) sequentially adding 100-150 g of the colorless liquid prepared in the step (1), 10-30 mL of pyridine and 1-2L of anhydrous tetrahydrofuran into a reaction bottle, and stirring to dissolve the colorless liquid; heating to 50-70 ℃, dropwise adding 500-600 mL of anhydrous tetrahydrofuran solution containing 40-60 g of triphosgene, and reacting for 10-15 h after dropwise adding; filtering to remove hydrochloride, recovering anhydrous tetrahydrofuran from the filtrate, extracting the residue with dichloromethane, combining the extract solutions, and distilling under reduced pressure to obtain a carbonate monomer EOTMC containing a polyethylene glycol side chain;

(3) adding polyethylene glycol (PEG) and the carbonate monomer EOTMC containing the polyethylene glycol side chain prepared in the step (2) into a sealed glass polymerization tube according to a molar ratio of 1: 40-60, uniformly mixing, adding an anhydrous toluene solution of stannous octoate with the concentration of 0.2-0.3 mol/L by using an injector, wherein the molar ratio of the stannous octoate to a reactant PEG plus EOTMC is 1: 10-10000, vacuumizing and introducing nitrogen at room temperature, repeatedly replacing for 4-6 times, sealing the sealed glass polymerization tube, and stirring and reacting for 20-30 hours in an oil bath at 100-150 ℃; after the reaction is finished, dissolving the product in dichloromethane, precipitating and purifying for 2-4 times by using petroleum ether to obtain a block copolymer PEOTMC-PEG-PEOTMC containing carbonic ester with a polyethylene glycol side chain and polyethylene glycol, drying in vacuum to balance weight, sealing, and refrigerating for storage;

(4) adding the PEOTMC-PEG-PEOTMC prepared in the step (3) and trimethylene carbonate into a sealed glass polymerization tube according to a molar ratio of 1: 40-60, uniformly mixing, adding an anhydrous toluene solution of stannous octoate with the concentration of 0.2-0.3 mol/L by using an injector, wherein the molar ratio of the stannous octoate to a reactant PEOTMC-PEG-PEOTMC + trimethylene carbonate is 1: 100-10000, vacuumizing and introducing nitrogen at room temperature, repeatedly replacing for 4-6 times, sealing the sealed glass polymerization tube, and stirring and reacting in an oil bath at 100-150 ℃ for 20-30 hours; after the reaction is finished, dissolving the product in dichloromethane, precipitating and purifying for 2-4 times by using methanol to obtain a segmented copolymer PTMC-PEOTMC-PEG-PEOTMC-PTMC of PEOTMC and trimethylene carbonate, drying in vacuum to a constant weight, sealing, and refrigerating for storage;

(5) dissolving the PTMC-PEOTMC-PEG-PEOTMC-PTMC block copolymer synthesized in the step (4) in dichloromethane to form a solution, wherein the concentration of the PTMC-PEOTMC-PEG-PEOTMC-PTMC block copolymer is 5-15 wt%, placing the solution in an ice bath, cooling to 0 ℃, and then sequentially dropwise adding triethylamine and methacryloyl chloride which are respectively 2-4 times of molar equivalent of the PTMC-PEOTMC-PEG-PEOTMC-PTMC block copolymer; stirring the reaction mixture at 0 ℃ for 3-5 h, standing at room temperature for 1-3 h, filtering to remove triethylamine hydrochloride, precipitating the filtrate with excessive petroleum ether to obtain functionalized polytrimethylene carbonate, and finally drying at 35-40 ℃ under a vacuum condition to constant weight;

(6) mixing 5-10 wt% of silk fibroin aqueous solution and functionalized poly-trimethylene carbonate in a mass ratio of 2-4: 1 to form a solution, adding 0.5-2 wt% of photoinitiator into the solution, pouring the solution into a culture dish, and adding 0.5-2 × 10 wt% of living cells⁶Adding cells/ml density into the mixture, and placing the mixture at the strength of 6-12 mW/cm²And irradiating under an ultraviolet lamp with the wavelength of 365nm for 4-6 min to form the hydrogel.

Technical Field

The invention belongs to the field of biomedical materials and regenerative medicine, and particularly relates to a silk fibroin/functionalized polytrimethylene carbonate hydrogel for repairing endometrium and a preparation method thereof.

Background

Infertility becomes the third most serious disease after cancer and cardiovascular diseases, and poses a significant threat to human reproduction and growth, and is one of the important social problems facing all countries in the world. At present, endometrial injury becomes one of the main causes of female secondary infertility, and how to improve the effect of comprehensive treatment and improve the pregnancy rate of patients becomes the key point of endometrial repair. Tissue engineering and regenerative medicine have become important therapeutic means for improving the health of the body since the 21 st century. The stem cells are widely regarded in the fields of tissue engineering and regenerative medicine, and a new chapter is opened for repairing endometrial injury. At present, most of transplantation modes for repairing endometrium by using stem cells are local in-situ injection or intravenous injection of uterus, and the phenomena of small survival quantity (in-situ injection in uterus) or low recruitment efficiency (intravenous injection) of damaged parts of the endometrium are easy to occur, so that the survival rate of the stem cells is low, and the successful conception rate after treatment is not ideal. Therefore, how to effectively gather the cells at the damaged part of the tissue to exert the effect is a problem which needs to be solved by applying stem cells to repair the damaged endometrium.

The biological material plays an important role in tissue injury repair, and can promote stem cells to be effectively gathered at a tissue injury part so as to improve the effect of stem cell treatment. A polymeric hydrogel is a polymer that swells in water and retains a large amount of water without dissolving. As the hydrogel polymer network is filled with a large amount of water, the whole material has the property of fluid, which is similar to the body tissue filled with a large amount of aqueous solution, and the soft and wet surface greatly reduces the stimulation of the material to the surrounding tissue, so that the hydrogel has good biocompatibility and has great development potential in the direction of tissue engineering and regenerative medicine. The silk fibroin is a pure natural protein, has good compatibility and biodegradability, and the degradation final product is amino acid, so the silk fibroin is very suitable for being used as a biological material. The silk fibroin aqueous solution can form hydrogel after being naturally placed, but the gel time is long, so that the wide application of the silk fibroin aqueous solution is limited.

In order to accelerate the formation of silk fibroin gel, the domestic patent publication No. CN102174203A adopts the blending with PLA-PEG-PLA to prepare the silk fibroin hydrogel, so that the preparation time of the silk fibroin hydrogel is controllable. However, the PLA component in the PLA-PEG-PLA copolymer structure is easy to generate acidic degradation products in the degradation process, so that the pH of an application part is reduced, and the activity of hydrogel-loaded drugs or cells is reduced. This method, although accelerating gelation of silk fibroin hydrogel, has a possibility of decreasing biocompatibility of silk fibroin hydrogel, and further improvement is required to obtain silk fibroin hydrogel that can be rapidly gelled and has good biocompatibility.

Disclosure of Invention

The invention aims to provide a silk fibroin/functionalized polytrimethylene carbonate hydrogel for repairing endometrium and a preparation method thereof, which solve the defect of overlong gelation time of silk fibroin hydrogel, obviously improve the defect of polyester blend, ensure that acid and acid degradation products are not generated in the degradation process of the hydrogel, always keep good biocompatibility of silk fibroin, do not reduce the activity of load cells, and have wide application prospect in the biomedical field of endometrial repair and the like.

In order to solve the technical problems, the following technical scheme is adopted:

the silk fibroin/functionalized polytrimethylene carbonate hydrogel for repairing endometrium comprises silk fibroin, functionalized polytrimethylene carbonate and living cells, wherein the mass ratio of the silk fibroin to the functionalized polytrimethylene carbonate is 5-1: 1, and the mass of the living cells is 0.01-10% of that of the hydrogel.

The silk fibroin/functionalized poly (trimethylene carbonate) hydrogel for repairing endometrium is tussah silk fibroin or mulberry silk fibroin or a mixture of the tussah silk fibroin and the mulberry silk fibroin.

The silk fibroin/functionalized polytrimethylene carbonate hydrogel for repairing endometrium has the following general formula:

R₁＝-H，-CH₃，-OH，-CH₂OH，-CH₂CH₃，-CH₂CH₂CH₃，-(CH₂)₃CH₃，-CH₂OCH₃，-OCH₃，-OCH₂CH₂＝CH₂，-CH₂CH₂＝CH₂，-CH₂Cl，-CH₂Br，-CH₂N₃any one of the above;

R₂＝-H，-CG₃，-OH，-CH₂OH，-CH₂CH₃，-CH₂CH₂CH₃，-(CH₂)₃CH₃，-CH₂OCH₃，-OCH₃，-OCH₂CH₂＝CH₂，-CH₂CH₂＝CH₂，-CH₂Cl，-CH₂Br，-CH₂N₃any one of the above;

wherein: n is 10-400, m is 1-50, y1 is 1-1000, y2 is 1-1000, x 1: y1 is 10-1: 1, and x 2: y2 is 10-1: 1.

The silk fibroin/functionalized polytrimethylene carbonate hydrogel for repairing endometrium is preferably selected from the group consisting of n being 20-50 or 80-150, and m being 1-20; when n is 20-50, y1 or y2 is 10-100; when n is 80-150, y1 or y2 is 1-50.

The silk fibroin/functionalized poly-trimethylene carbonate hydrogel for repairing endometrium is characterized in that the living cells are one or more than one of bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, embryonic stem cells, menses source mesenchymal stem cells, amnion mesenchymal stem cells, placenta mesenchymal stem cells, human early pregnancy periosteum mesenchymal stem cells, adipose mesenchymal stem cells, endometrium stem cells and induced pluripotent stem cells; or the living cells are mixed cells of one or more than one of bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, embryonic stem cells, menses-derived mesenchymal stem cells, amnion mesenchymal stem cells, placenta mesenchymal stem cells, human early pregnancy periosteum mesenchymal stem cells, adipose mesenchymal stem cells, endometrium stem cells and induced pluripotent stem cells and one or two of endometrium mesenchymal stem cells and endometrium epithelial cells.

The preparation method of the silk fibroin/functionalized polytrimethylene carbonate hydrogel for repairing endometrium comprises the following steps:

(1) taking trimethylolethane as a raw material, performing ester exchange and pyrolysis decarboxylation to prepare oxetane, sequentially adding 1-3L of polyethylene glycol ether and 0.2-0.8 mL of concentrated sulfuric acid into a reaction bottle, heating to 100-150 ℃ under stirring, dropwise adding 300-500 mL of a solution of 80-120 g of oxetane in polyethylene glycol ether, and reacting for 5-7 hours after dropwise adding; cooling to room temperature, recovering excessive polyglycol ether under reduced pressure, and distilling under reduced pressure to obtain colorless liquid;

(2) sequentially adding 100-150 g of the colorless liquid prepared in the step (1), 10-30 mL of pyridine and 1-2L of anhydrous tetrahydrofuran into a reaction bottle, and stirring to dissolve the colorless liquid; heating to 50-70 ℃, dropwise adding 500-600 mL of anhydrous tetrahydrofuran solution containing 40-60 g of triphosgene, and reacting for 10-15 h after dropwise adding; filtering to remove hydrochloride, recovering anhydrous tetrahydrofuran from the filtrate, extracting the residue with dichloromethane, combining the extract solutions, and distilling under reduced pressure to obtain a carbonate monomer EOTMC containing a polyethylene glycol side chain;

(3) adding polyethylene glycol (PEG) and the carbonic ester monomer EOTMC containing the polyethylene glycol side chain prepared in the step (2) into a sealed glass polymerization tube according to the molar ratio of 1: 40-60, uniformly mixing, adding an anhydrous toluene solution of stannous octoate with the concentration of 0.2-0.3 mol/L by using an injector, wherein the molar ratio of the stannous octoate to a reactant PEG and the EOTMC is 1: 10-10000, vacuumizing at room temperature, introducing nitrogen, repeatedly replacing for 4-6 times, sealing the sealed glass polymerization tube, and stirring and reacting for 20-30 hours in an oil bath at the temperature of 100-150 ℃; after the reaction is finished, dissolving the product in dichloromethane, precipitating and purifying for 2-4 times by using petroleum ether to obtain a block copolymer PEOTMC-PEG-PEOTMC containing carbonic ester with a polyethylene glycol side chain and polyethylene glycol, drying in vacuum to balance weight, sealing, and refrigerating for storage;

(4) adding the PEOTMC-PEG-PEOTMC prepared in the step (3) and trimethylene carbonate into a sealed glass polymerization tube according to the molar ratio of 1: 40-60, uniformly mixing, adding an anhydrous toluene solution of stannous octoate with the concentration of 0.2-0.3 mol/L by using an injector, wherein the molar ratio of the stannous octoate to a reactant PEOTMC-PEG-PEOTMC + trimethylene carbonate is 1: 100-10000, vacuumizing and introducing nitrogen at room temperature, repeatedly replacing for 4-6 times, sealing the sealed glass polymerization tube, and stirring and reacting in an oil bath at 100-150 ℃ for 20-30 hours; after the reaction is finished, dissolving the product in dichloromethane, precipitating and purifying for 2-4 times by using methanol to obtain a segmented copolymer PTMC-PEOTMC-PEG-PEOTMC-PTMC of PEOTMC and trimethylene carbonate, drying in vacuum to a constant weight, sealing, and refrigerating for storage;

(5) dissolving the PTMC-PEOTMC-PEG-PEOTMC-PTMC block copolymer synthesized in the step (4) in dichloromethane to form a solution, wherein the concentration of the PTMC-PEOTMC-PEG-PEOTMC-PTMC block copolymer is 5-15 wt%, placing the solution in an ice bath, cooling to 0 ℃, and then sequentially dropwise adding triethylamine and methacryloyl chloride which are respectively 2-4 times of molar equivalent of the PTMC-PEOTMC-PEG-PEOTMC-PTMC block copolymer; stirring the reaction mixture at 0 ℃ for 3-5 h, standing at room temperature for 1-3 h, filtering to remove triethylamine hydrochloride, precipitating the filtrate with excessive petroleum ether to obtain functionalized polytrimethylene carbonate, and finally drying at 35-40 ℃ under a vacuum condition to constant weight;

(6) mixing 5-10 wt% of silk fibroin aqueous solution and functionalized poly-trimethylene carbonate according to the mass ratio of 2-4: 1 to form a solution, adding 0.5-2 wt% of photoinitiator into the solution, pouring the solution into a culture dish, and adding 0.5-2 × 10 wt% of living cells⁶Adding cells/ml density into the mixture, and placing the mixture at the strength of 6-12 mW/cm²And irradiating under an ultraviolet lamp with the wavelength of 365nm for 4-6 min to form the hydrogel.

The design idea of the invention is as follows: the biocompatibility of the hydrogel is improved by introducing the functionalized polytrimethylene carbonate which contains hydrophilic chain segments, does not contain any ester bonds, does not generate acid degradation products in the degradation process, and has controllable and adjustable performance, the gelation time and the gelation mode of the silk fibroin hydrogel are regulated and controlled by the factors such as the structure, the composition, the content and the like of the functionalized polytrimethylene carbonate, cells/stem cells are simultaneously loaded, no side effect is caused to the loaded cells, no irritation is caused to the implantation part, and the aim of repairing the endometrium is further fulfilled.

In addition, the invention selects specific functionalized polytrimethylene carbonate, which has the functions and effects that: the gelation mode and time of the silk fibroin hydrogel are regulated and controlled by the controllable adjustment of the self structure composition and performance, meanwhile, the biocompatibility of the silk fibroin hydrogel is improved by utilizing the advantages of the self non-ester bond structure and the generation of acidic degradation products, and the side effect of PLA, PLGA or other polyester chain segments caused by the acidic degradation products is avoided. The living cells are one or more of bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, embryonic stem cells, menses-derived mesenchymal stem cells, amnion mesenchymal stem cells, placenta mesenchymal stem cells, human early pregnancy periosteum mesenchymal stem cells, adipose mesenchymal stem cells, endometrium stem cells and induced pluripotent stem cells; or the living cells are mixed cells of one or more than one of bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, embryonic stem cells, menses-derived mesenchymal stem cells, amnion mesenchymal stem cells, placenta mesenchymal stem cells, human early pregnancy periosteum mesenchymal stem cells, adipose mesenchymal stem cells, endometrium stem cells and induced pluripotent stem cells and one or two of endometrium mesenchymal stem cells and endometrium epithelial cells. The functions and effects of the selection are as follows: the recruitment or induction of endometrioid cells and the utilization of paracrine action to promote the repair of endometrium.

Due to the adoption of the technical scheme, compared with the prior art, the invention has the following beneficial effects:

1. the method is simple and easy to implement, has rich raw material resources, and is favorable for realizing industrial production.

2. The functionalized polytrimethylene carbonate chain segment related in the preparation process of the invention does not contain ester bonds, has good biocompatibility, does not generate acid substances in the degradation process, does not generate side effects on loaded cells, and can obviously improve the biocompatibility of the silk fibroin hydrogel.

3. The performance of the functionalized polytrimethylene carbonate chain segment involved in the preparation process can be controllably adjusted, so that the performance of the prepared silk fibroin hydrogel is controllable.

4. The gel forming method of the silk fibroin hydrogel prepared by the invention is selective, namely the gel can be formed by depending on the temperature and can also be formed by ultraviolet light.

5. The silk fibroin hydrogel prepared by the method can quickly form gel, the gelation time is controllable, and the gel can be formed in 1-60 minutes at 25-39 ℃ or under an ultraviolet condition.

Drawings

FIG. 1 is a nuclear magnetic spectrum of a carbonate monomer having a polyethylene glycol side chain in example 1.

FIG. 2 is a photograph of the fibroin/functionalized polytrimethylene carbonate hydrogel of example 1.

FIG. 3 is a scanning electron micrograph of the fibroin/functionalized polytrimethylene carbonate hydrogel of example 1.

FIG. 4 shows the results of pregnancy experiments with the fibroin/functionalized polytrimethylene carbonate hydrogel of example 1.

Detailed Description

The invention will now be further illustrated by reference to specific examples: