A hydrogel material for genital reconstruction

Abstract

The application belongs to the field of biomedical materials. The application relates to a hydrogel material for genital plastic surgery and a preparation method thereof. The method uses sodium alginate, carboxymethyl chitosan and gelatin as main raw materials, and the hydrogel material is prepared through the following steps: blending with mesenchymal stem cell solution, freeze-drying, calcium ion cross-linking, zwitterionic copolymer, self-assembly of heparin calcium, tannic acid and heparin calcium. The hydrogel material for genital plastic surgery can effectively solve the problems of poor mechanical property and long plastic repair period of the material for genital plastic surgery in the prior art, has excellent antibacterial and anti-inflammatory effects, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to a hydrogel material for genital reconstruction and a preparation method thereof. The present application belongs to the field of biomedical materials. BACKGROUND

[0002] The progress of genital reconstruction technology is a boon to some male friends with congenital genital defects, which can help them regain confidence. For some patients with urogenital tract deformities, trauma, and penile malignancies, the current drug treatment effect is very limited, and the main method is still to use surgical prosthesis implantation, including flap or cartilage combined with artificial prosthesis to reconstruct the penis, etc. However, due to the lack of normal cavernosal tissue, the appearance and erectile function of most patients after penile surgery are still very poor. Therefore, the key to improving penile reconstruction surgery lies in the development of artificial synthetic materials that can replace natural cavernosal tissue. The structure characteristics, pro-angiogenic effect, and mechanical properties of the material all have specific requirements. It not only needs to meet the reticular tissue structure and special diastolic force of the cavernosal tissue, but also needs to have abundant vascular sinus generation, so as to truly realize the recovery of penile erectile function.

[0003] The commonly used materials at present are biocompatible materials such as silicone rubber, but due to the difference in stiffness, poor affinity and other heterogeneity challenges, it will cause discomfort in the early stage of implantation, and also damage the remaining cavernosal tissue and its function. In addition, the size of the prosthesis will also affect the comfort and functionality. The current products have the problems of poor customization, high price, etc.

[0004] In addition, due to the complexity of the genital structure, the formation of adipocytes, epithelial cells, and endothelial cells after implanting the material is slow, and the entire plastic repair cycle is too long, which seriously affects the treatment effect. Human mesenchymal stem cells (human mesenchymal stem cells) are a kind of cells that can self-renew and have multi-directional differentiation ability, which can differentiate into adipocytes, osteocytes, chondrocytes, etc. They can also differentiate into uroepithelial cells and vascular endothelial cells. At the same time, it has paracrine ability and plays a role in tissue repair and immune regulation. In recent years, mesenchymal stem cell-derived therapy has become one of the promising strategies for the treatment of neurodegenerative diseases.

[0005] Therefore, it is of great significance to develop an implant material with high compatibility and excellent compliance, as well as rapid repair, and a preparation method thereof. SUMMARY

[0006] The application aims at the problems of poor mechanical property of materials for genital plastic surgery and long plastic repair period in the prior art, and provides a preparation method of a novel hydrogel material for genital plastic surgery.

[0007] The application provides a preparation method of a zwitterionic copolymer, comprising the following steps.

[0008] S11, ring-opening reaction of lactic acid, carboxylated catechol derivative and epoxy acrylate is carried out to obtain an intermediate product;

[0009] S12, free radical polymerization of the intermediate product and a zwitterion is carried out to obtain a target product, i.e., the zwitterionic copolymer.

[0010] Further, the lactic acid, carboxylated catechol derivative and epoxy acrylate are added in a molar ratio of carboxyl group to epoxy group of 1:1.

[0011] The amount ratio of the lactic acid and the carboxylated catechol derivative is 0.3-0.5 mol:0.5-0.7 mol.

[0012] Further, the amount ratio of the intermediate product and the zwitterion is 0.4-0.5 mol:0.5-0.6 mol.

[0013] The zwitterion contains an acryloyloxy structure.

[0014] The application further provides a zwitterionic copolymer prepared by the preparation method.

[0015] Another object of the application is to provide a preparation method of a hydrogel material for genital plastic surgery, comprising the following steps.

[0016] S21, preparation of a mixed solution and freeze-drying, i.e.

[0017] Mesenchymal stem cell solution is added to the polymer-based mixed solution, and then the mixture is uniformly mixed and poured into a mold; and then freeze-drying is performed.

[0018] S22, ion cross-linking, i.e.

[0019] The substance obtained in the step S21 is placed in a calcium chloride solution, and then is left to stand for 30-40 min, and then the excess liquid is removed.

[0020] S23, self-assembly, i.e.

[0021] The material obtained in step S22 is placed in the amphoteric ion copolymer solution, the heparin calcium solution A, the tannic acid solution, and the heparin calcium solution B, respectively, and each process is allowed to stand for 40-60 min;

[0022] S24, freeze-drying, i.e.

[0023] The material obtained in step S23 is subjected to freeze-drying treatment to obtain a hydrogel material for genital reshaping.

[0024] Further, the high-molecular-based mixed solution is a sodium alginate solution, a carboxymethyl chitosan solution, and a gelatin solution mixed at a volume ratio of 1.0-2.0:1.0:1.0-2.0; and

[0025] The concentration of the sodium alginate solution is 0.02-0.05 g / mL;

[0026] The concentration of the carboxymethyl chitosan solution is 0.01-0.03 g / mL;

[0027] The concentration of the gelatin solution is 0.02-0.05 g / mL.

[0028] Further, the solute sodium alginate of the sodium alginate solution is added by high-molecular-weight sodium alginate and low-molecular-weight sodium alginate at a mass ratio of 0.6-0.8:0.2-0.4; and

[0029] The number-average molecular weight of the high-molecular-weight sodium alginate is 100-150 kDa;

[0030] The number-average molecular weight of the low-molecular-weight sodium alginate is 5-10 kDa.

[0031] Further, the volume ratio of the amount of the mesenchymal stem cell solution to the high-molecular-based mixed solution is 1.0:0.8-1.2;

[0032] The concentration of the mesenchymal stem cell solution is 10 9 -10 10 mg / mL.

[0033] Further, the concentration of the calcium chloride solution is 0.01-0.03 g / mL.

[0034] Further, the concentration of the amphoteric ion solution is 0.01-0.02 g / mL;

[0035] The concentration of the heparin calcium solution A is 0.005-0.01 g / mL;

[0036] The concentration of the tannic acid solution is 0.005-0.01 g / mL; and

[0037] The concentration of the heparin calcium solution B is 0.01-0.02 g / mL.

[0038] Advantages of the present application:

[0039] (1) The present application provides a preparation method of a hydrogel material for genital plastic surgery, which uses sodium alginate, gelatin and carboxymethyl chitosan as main raw materials, is blended with mesenchymal stem cell solution, freeze-dried, and then cross-linked with calcium ions, and is prepared by self-assembly of zwitterionic copolymer, heparin calcium, tannic acid and heparin calcium. Firstly, the hydrogel with sodium alginate as the main component and gelatin and carboxymethyl chitosan as the auxiliary components has excellent mechanical properties, antibacterial properties, cell proliferation and differentiation promoting functions, etc.; secondly, the use of high and low molecular weight structures of sodium alginate ensures the mechanical strength while having excellent flexibility; thirdly, the blending of mesenchymal stem cell solution with sodium alginate, gelatin and carboxymethyl chitosan solution freeze-dried helps to keep the stem cell structure intact in the pores of the hydrogel and maintain biological activity, and reduces the plastic repair period; fourthly, the zwitterionic copolymer can further improve the mechanical properties of the hydrogel, and on the other hand, can improve the adsorption rate of heparin calcium and tannic acid, and has the functions of improving anti-inflammatory and antibacterial properties, preventing thrombosis, etc.; fifthly, heparin calcium has excellent antithrombotic effect and does not easily cause electrolyte disorder, and is adsorbed in the hydrogel, having a long-acting effect; sixthly, the tannic acid structure on the surface of the hydrogel has excellent cell adsorption properties and promotes healing.

[0040] (2) The present application provides a hydrogel material for genital plastic surgery, which uses self-made zwitterionic copolymer containing zwitterions, alcohol hydroxyl groups, catechol groups and ester groups. Firstly, the zwitterionic copolymer has excellent biocompatibility and water solubility; secondly, the zwitterions can provide electrostatic adsorption with gelatin and carboxymethyl chitosan, the alcohol hydroxyl groups can form hydrogen bonds with sodium alginate, gelatin and carboxymethyl chitosan to improve the mechanical properties; thirdly, the catechol structure can form a coordination with heparin calcium, and is compatible with the tannic acid structure, improving the mechanical properties. DETAILED DESCRIPTION

[0041] The present application will be described in detail below in conjunction with the examples. However, it should be understood that the following examples are only illustrative of the embodiments of the present application, and are not a limitation on the scope of the present application.

[0042] The purpose of the present application is to develop a genital plastic hydrogel material with excellent mechanical properties while improving the plastic repair effect. The idea is to combine sodium alginate, which has excellent biocompatibility and biodegradability, and can be cross-linked with calcium ions to form a stable three-dimensional hydrogel; gelatin, as a hydrolysis product of collagen, has good biocompatibility, biodegradability and low antigenicity, can promote cell proliferation and differentiation, help platelet coagulation, and has excellent mechanical properties; carboxymethyl chitosan has excellent biocompatibility and biodegradability, as well as good antibacterial and film-forming properties. As the main raw material, it loads mesenchymal stem cells with high differentiation capacity to promote the differentiation of genital tissue and improve the plastic repair effect; while the mechanical properties, cell adsorption, antibacterial and anti-inflammatory structure synergistically. The theoretical basis is as follows: first, the two molecular weights of sodium alginate are complementary to each other to improve the strength and toughness; then blend with gelatin and carboxymethyl chitosan; then mix evenly with mesenchymal stem cell solution, freeze-drying process, preliminary cross-linking, locking stem cells, improving efficacy; second, place the above-mentioned preliminary cross-linked material in a calcium chloride ion solution to form a cross-linked hydrogel; third, the surface of the hydrogel contains exposed amino and hydroxyl structures, which are hydrogen-bonded to the anions and hydroxyl groups in the zwitterionic copolymer; then place in calcium heparin, the zwitterionic copolymer and the coordination; then place in tannic acid, the calcium ions in the calcium heparin and the tannic acid are coordinated; finally, place in calcium heparin to form a final product rich in calcium heparin. After preliminary cross-linking by freeze-drying, multi-step cross-linking by calcium ions, and freeze-drying process, the mechanical properties can be effectively improved; at the same time, chitosan, zwitterionic copolymer, heparin calcium, tannic acid and other substances synergistically, have excellent antithrombotic and antibacterial functions, can promote rapid cell differentiation and improve plastic repair effect. The implementation of the present application is as follows:

[0043] The present application provides a preparation method of a zwitterionic copolymer, comprising the following steps:

[0044] S11, in the dark, lactic acid, carboxylated catechol derivative, epoxy acrylate, polymerization inhibitor, tetrabutylammonium bromide are added to tetrahydrofuran, heated to reflux for 18-24h; reduce to room temperature, vacuum distillation, take the concentrate to vacuum drying at 40℃ for 12h, get intermediate product.

[0045] The amount of lactic acid, carboxylated catechol derivative and epoxy acrylate is added in a molar ratio of 1:1 of total carboxyl group to epoxy group; and,

[0046] The amount of lactic acid, carboxylated catechol derivative is 0.3-0.5mol:0.5-0.7mol;

[0047] The epoxy acrylate, tetrabutylammonium bromide and tetrahydrofuran are used in a ratio of 0.1 mol:0.02 mol:100 mL.

[0048] The amount of the polymerization inhibitor is 0.2wt% of the epoxy acrylate; and the polymerization inhibitor in the following examples of the present application is p-hydroxyanisole.

[0049] The carboxylated catechol derivative can be gossypol, protocatechuic acid, gallic acid, dihydrocaffeic acid, danshensu, etc.; and gallic acid is preferred.

[0050] The epoxy acrylate can be glycidyl methacrylate or glycidyl (meth)acrylate, etc.; and glycidyl methacrylate is preferred.

[0051] S12, the intermediate product, the zwitterion and the photoinitiator are added into N,N-dimethylformamide, and then are irradiated under a UV lamp for 10-50 min; after the UV lamp is removed, vacuum distillation is performed; methanol is added into the concentrate to produce a precipitate; the precipitate is filtered and vacuum dried at 60℃ for 8 h to obtain the zwitterionic copolymer.

[0052] The intermediate product, the zwitterion and N,N-dimethylformamide are used in a ratio of 0.4-0.5 mol:0.5-0.6 mol:500 mL.

[0053] The zwitterion contains an acryloyloxy structure; and can be 2-methacryloyloxyethylphosphocholine or methacryloyl ethyl sulfobetaine.

[0054] The amount of the photoinitiator is 1-5wt% of the total mass of the reactants.

[0055] The photoinitiator is a commonly used photoinitiator; and can be photoinitiator 1173, photoinitiator 184, photoinitiator 2959, photoinitiator 369 or photoinitiator TPO, etc.; and the photoinitiator in the following examples of the present application is photoinitiator 1173.

[0056] The light intensity of the UV lamp is 200-600 mW / cm 2 .

[0057] The present application also provides a preparation method of the hydrogel material for genital plastic surgery, which comprises the following steps:

[0058] S21, preparation of a mixed solution and freeze-drying, i.e.

[0059] The mesenchymal stem cell solution is added into the polymer-based mixed solution, and then is uniformly mixed and poured into a mold; and then is freeze-dried at-20℃ for 24 h.

[0060] The preparation method of the high polymer-based mixed solution is: preparing sodium alginate solution, carboxymethyl chitosan solution and gelatin solution respectively; the preparation method of the sodium alginate comprises the following steps: dissolving sodium alginate in deionized water containing a magnetic rotor, stirring for 2 hours at room temperature, and then filtering with 0.6 μm and 0.3 μm microporous filter membranes in sequence to be sterile, so as to obtain a solution with a concentration of 0.02-0.05 g / mL; the preparation method of the carboxymethyl chitosan comprises the following steps: dissolving carboxymethyl chitosan in deionized water, stirring until no particles are present, and then filtering with 0.6 μm and 0.3 μm microporous filter membranes in sequence to be sterile, so as to obtain a solution with a concentration of 0.01-0.03 g / mL; the preparation method of the gelatin comprises the following steps: dissolving gelatin in deionized water, heating in a water bath to 50 ℃, stirring until completely dissolved, and then filtering with 0.6 μm and 0.3 μm microporous filter membranes in sequence to be sterile, so as to obtain a solution with a concentration of 0.02-0.05 g / mL; mixing the sodium alginate solution, the carboxymethyl chitosan solution and the gelatin solution according to a volume ratio of 1.0-2.0:1.0:1.0-2.0 to obtain the high polymer-based mixed solution.

[0061] The solute sodium alginate of the sodium alginate solution is added by high molecular weight sodium alginate and low molecular weight sodium alginate according to a mass ratio of 0.6-0.8:0.2-0.4; and

[0062] The number average molecular weight of the high molecular weight sodium alginate is 100-150 kDa; which can be 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, or a range value composed of any two numerical values;

[0063] The number average molecular weight of the low molecular weight sodium alginate is 5-10 kDa; which can be 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, or a range value composed of any two numerical values.

[0064] The volume ratio of the mesenchymal stem cell solution to the high polymer-based mixed solution is 1.0:0.8-1.2;

[0065] The concentration of the mesenchymal stem cell solution is 10 9 -10 10 ML;

[0066] The mesenchymal stem cells can be bone marrow-derived mesenchymal stem cells, umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, urine-derived mesenchymal stem cells, etc.; and the mesenchymal stem cells in the following examples of the present application are umbilical cord mesenchymal stem cells.

[0067] The mesenchymal stem cell serum-free culture medium used in the application is purchased from Shanghai Xiaopeng Biological Technology Co., Ltd., and the product name is: human mesenchymal stem cell serum-free basic culture medium (Israel Biological Industries Co., Ltd., product number: 05-200-1A). It is a clinical grade mesenchymal stem cell culture medium, which is free of serum and animal components, and can maintain the typical characteristics of mesenchymal stem cells. It has obtained the American FDA clinical registration number (DMF NO: 29469).

[0068] The preparation method of the mesenchymal stem cell solution described in the following examples of the application is:

[0069] (1) Take 200 mL of sterile fat removed from the abdomen of a patient or liposuction. After the fat tissue is fully separated and cut, wash it with 0.25% chloramphenicol solution three times, and repeatedly wash it with 37°C phosphate buffer until the lower layer of the fat washing liquid is light red after standing. The fat tissue is digested with 0.1% type I collagenase (Sigma-Aldrich Co. LLC., MO, USA) for 30 min-1 h until it is milky, and the stromal vascular fraction is collected by centrifugation at 1200g for 10 min at 37°C. The precipitate is resuspended with a clinical grade mesenchymal stem cell serum-free medium, filtered through a 100 μm nylon filter, and then injected into a 10 cm culture dish at a volume of 10 mL per dish using a pipette. The culture dish is incubated in a 37°C incubator containing 5% humidified carbon dioxide;

[0070] (2) Mesenchymal stem cell subculture: the human adipose-derived mesenchymal stem cells of (1) are cultured in a clinical grade mesenchymal stem cell serum-free medium, and the culture medium is replaced every two days. When the cells reach 80-90% confluence, they are digested with 0.25% trypsin and subcultured at a ratio of 1:3. The P4 generation of adipose-derived mesenchymal stem cells is identified by osteogenic and adipogenic differentiation and surface antigen (CD29, CD44, CD73, CD105, CD34, CD45) identification, and is ready for use;

[0071] (3) Quantification: dilute the mesenchymal stem cells in phosphate buffer by 1000 times, and automatically analyze them in the Nanosight instrument according to Brownian motion and diffusion coefficient. The parameter settings are as follows: temperature 26.0±0.1℃, viscosity 0.870±0.002 cP, 25 frames / s, and test time 60 s.

[0072] S22, ion crosslinking, i.e.

[0073] Place the material obtained in step S21 in a calcium chloride solution and stand for 30-40 min, and remove the excess liquid;

[0074] The preparation method of the calcium chloride solution comprises the following steps: dissolving anhydrous calcium chloride powder in deionized water, stirring until no particles are present, and filtering with a microporous filter membrane with a pore size of 0.6 μm and 0.3 μm in sequence until sterile, to obtain a solution with a concentration of 0.01-0.03 g / mL.

[0075] S23, self-assembly, i.e.

[0076] The material obtained in the step S22 is sequentially placed in an amphoteric copolymer solution, a calcium heparin solution A, a tannic acid solution, and a calcium heparin solution B, and each process is statically placed for 40-60 min.

[0077] The concentration of the amphoteric solution is 0.01-0.02 g / mL.

[0078] The concentration of the calcium heparin solution A is 0.005-0.01 g / mL.

[0079] The concentration of the tannic acid solution is 0.005-0.01 g / mL.

[0080] The concentration of the calcium heparin solution B is 0.01-0.02 g / mL.

[0081] S24, freeze-drying, i.e.

[0082] The material obtained in the step S23 is freeze-dried at-20℃ for 24 h to obtain a hydrogel material for genital shaping.

[0083] In the preparation method of the hydrogel material for genital shaping provided by the embodiment of the present application, sodium alginate, carboxymethyl chitosan, and gelatin solution are first blended with mesenchymal stem cell solution, then subjected to freeze-drying treatment, and then crosslinked with calcium ions, so that the cells can be fixed in the hydrogel to the maximum extent, and the shaping and repairing period is effectively reduced; in addition, the use of calcium chloride and calcium heparin in combination can achieve high antithrombotic performance, while reducing the material cost, which is conducive to the popularization and use.

[0084] In order to further understand the present application, a hydrogel material for genital shaping provided by the present application is described in detail below in combination with specific embodiments, and the protection scope of the present application is not limited by the following embodiments.

[0085] Embodiment 1

[0086] A preparation method of an amphoteric copolymer comprises the following steps:

[0087] S11, under light shielding, lactic acid, gallic acid, glycidyl methacrylate, polymerization inhibitor, tetrabutylammonium bromide were added into tetrahydrofuran, heated to reflux for 20 hours; reduced to room temperature, distilled under reduced pressure, and the concentrate was placed in vacuum drying at 40℃ for 12 hours to obtain the intermediate product.

[0088] The lactic acid, gallic acid and glycidyl methacrylate were added in a molar ratio of 1:1 of the total carboxyl group to the epoxy group; and,

[0089] The lactic acid and gallic acid were added in a ratio of 0.4mol:0.6mol;

[0090] The glycidyl methacrylate, tetrabutylammonium bromide, tetrahydrofuran were added in a ratio of 0.1mol:0.02mol:100mL;

[0091] The amount of the polymerization inhibitor was 0.2wt% of the glycidyl methacrylate.

[0092] Its infrared data are as follows: 3495cm -1 :-OH wide peak exists; 3011cm -1 , 1595cm -1 , 1498cm -1 : benzene ring exists; 1735cm -1 : -C=O exists; 1606cm -1 , 811cm -1 : -C=C- exists; 1265cm -1 , 893cm -1 , 825cm -1 : epoxy group does not exist.

[0093] S12, the intermediate product, 2-methacryloyloxyethylphosphocholine, photoinitiator 1173 were added into N,N-dimethylformamide, and placed under UV lamp for irradiation for 30 minutes; then the UV lamp was removed, distilled under reduced pressure, and methanol was added to the concentrate to produce a precipitate, which was filtered and dried at 60℃ under vacuum for 8 hours to obtain the zwitterionic copolymer

[0094] The intermediate product, 2-methacryloyloxyethylphosphocholine, N,N-dimethylformamide were added in a ratio of 0.45mol:0.55mol:500mL.

[0095] The amount of the photoinitiator 1173 was 3wt% of the total mass of the reactants;

[0096] The light intensity of the UV lamp was 400mW / cm 2 .

[0097] Its infrared data as follows: 3495cm -1 : -OH wide peak exists; 3011cm -1 , 1595cm -1 , 1498cm -1 : benzene ring exists; 1735cm -1 : -C=O exists; 1606cm -1 , 811cm -1 : -C=C- does not exist; 1187cm -1 : -P=O exists.

[0098] A preparation method of a hydrogel material for genital plastic surgery, comprising the following steps:

[0099] S21, preparing a mixed solution, freeze-drying, namely

[0100] Adding a mesenchymal stem cell solution to the polymer-based mixed solution, pouring into a mold after uniform mixing, and freeze-drying at-20℃ for 24h.

[0101] The polymer-based mixed solution is: a sodium alginate solution with a concentration of 0.04g / mL; a carboxymethyl chitosan solution with a concentration of 0.02g / mL; and a gelatin solution with a concentration of 0.04g / mL; mixing sodium alginate, carboxymethyl chitosan and gelatin solutions according to a volume ratio of 1.5:1.0:1.5 to obtain the polymer-based mixed solution;

[0102] The solute sodium alginate of the sodium alginate solution is added according to a mass ratio of 0.7:0.3 of high-molecular-weight sodium alginate and low-molecular-weight sodium alginate; and

[0103] The number-average molecular weight of the high-molecular-weight sodium alginate is 130kDa;

[0104] The number-average molecular weight of the low-molecular-weight sodium alginate is 8kDa;

[0105] The volume ratio of the amount of the mesenchymal stem cell solution to the polymer-based mixed solution is 1.0:1.0;

[0106] The concentration of the mesenchymal stem cell solution is 5×10 9 / mL.

[0107] S22, ion crosslinking, namely

[0108] Placing the substance obtained in the step S21 into a calcium chloride solution, standing for 30min, and removing excess liquid;

[0109] The concentration of the calcium chloride solution is 0.02g / mL.

[0110] Its infrared data as follows: 3486cm-1 : -OH wide peak exist; 3314 cm -1 : -NH- exist; 3011 cm -1 , 1595 cm -1 , 1498 cm -1 : benzene ring exist; 1711 cm -1 : -C=O weaken; 1698 cm -1 : -C=O (coordination with calcium) exist.

[0111] S23, self-assembly, i.e.

[0112] The material obtained in step S22 is placed in zwitterionic copolymer solution, heparin calcium solution A, tannic acid solution, heparin calcium solution B in turn, wherein each process is placed for 50 min.

[0113] The concentration of the zwitterionic solution is 0.015 g / mL;

[0114] The concentration of the heparin calcium solution A is 0.008 g / mL;

[0115] The concentration of the tannic acid solution is 0.008 g / mL; and

[0116] The concentration of the heparin calcium solution B is 0.015 g / mL.

[0117] Its infrared data are as follows (reflection infrared): 3489 cm -1 : -OH wide peak exist and strengthen; 3314 cm -1 : -NH- do not exist; 3011 cm -1 , 1595 cm -1 , 1498 cm -1 : benzene ring exist; 1711 cm -1 : -C=O weaken; 1698 cm -1 : -C=O (coordination with calcium) exist and strengthen.

[0118] S24, freeze-drying, i.e.

[0119] The material obtained in step S23 is freeze-dried at -20℃ for 24 h to obtain a hydrogel material for genital shaping.

[0120] Example 2

[0121] Other than example 1, the difference is that:

[0122] A preparation method of a zwitterionic copolymer, in step S11,

[0123] The amount ratio of lactic acid and gallic acid is 0.5 mol:0.5 mol.

[0124] Example 3

[0125] Other than Example 1, except that:

[0126] A preparation method of a zwitterionic copolymer, in the S11 step,

[0127] The amount ratio of the lactic acid, gallic acid is 0.3 mol:0.7 mol.

[0128] Example 4

[0129] Other than Example 1, except that:

[0130] A preparation method of a zwitterionic copolymer, in the S12 step,

[0131] The amount ratio of the intermediate product, zwitterion, N,N-dimethylformamide is 0.5 mol:0.5 mol:500 mL.

[0132] Example 5

[0133] Other than Example 1, except that:

[0134] A preparation method of a zwitterionic copolymer, in the S12 step,

[0135] The amount ratio of the intermediate product, zwitterion, N,N-dimethylformamide is 0.4 mol:0.6 mol:500 mL.

[0136] Example 6

[0137] Other than Example 1, except that:

[0138] A preparation method of a zwitterionic copolymer, comprising the following steps:

[0139] S11, in the dark, lactic acid, gallic acid, glycidyl methacrylate, polymerization inhibitor, tetrabutylammonium bromide are added to tetrahydrofuran, heated to reflux for 18h; reduce to room temperature, reduced pressure distillation, take the concentrate is placed in a vacuum at 40℃ drying 12h, to obtain the intermediate product.

[0140] The amount ratio of the lactic acid, gallic acid and glycidyl methacrylate is added according to the molar ratio of the total carboxyl group and epoxy group 1:1; and,

[0141] The amount ratio of the lactic acid, gallic acid is 0.4 mol:0.6 mol;

[0142] The amount ratio of the glycidyl methacrylate, tetrabutylammonium bromide, tetrahydrofuran is 0.1 mol:0.02 mol:100 mL;

[0143] The amount of the polymerization inhibitor is 0.2 wt% of the glycidyl methacrylate.

[0144] Its infrared data are as follows: 3495 cm -1 : -OH wide peak exists; 3011 cm -1 , 1595 cm -1 , 1498 cm -1 : benzene ring exists; 1735 cm -1 : -C=O exists; 1606 cm -1 , 811 cm -1 : -C=C- exists; 1265 cm -1 , 893 cm -1 , 825 cm -1 : epoxy group does not exist.

[0145] S12, the intermediate product, 2-methacryloyloxyethyl phosphocholine, a photoinitiator 1173 are added into N, N-dimethylformamide, and after being irradiated under a UV lamp for 10 min, the UV lamp is removed, vacuum distillation is carried out, methanol is added into the concentrate to produce a precipitate, filtration is carried out, the precipitate is taken, and vacuum drying is carried out at 60℃ for 8h to obtain the zwitterionic copolymer

[0146] The amount ratio of the intermediate product, 2-methacryloyloxyethyl phosphocholine, N, N-dimethylformamide is 0.45 mol: 0.55 mol: 500 mL.

[0147] The amount of the photoinitiator 1173 is 5 wt% of the total mass of the reactants;

[0148] The light intensity of the UV lamp is 600 mW / cm 2 .

[0149] Its infrared data are as follows: 3495 cm -1 : -OH wide peak exists; 3011 cm -1 , 1595 cm -1 , 1498 cm -1 : benzene ring exists; 1735 cm -1 : -C=O exists; 1606 cm -1 , 811 cm -1 : -C=C- does not exist; 1187 cm -1 : -P=O exists.

[0150] Example 7

[0151] Other than example 1, the difference is that:

[0152] A preparation method of a zwitterionic copolymer, comprising the following steps:

[0153] S11, in the dark, lactic acid, gallic acid, glycidyl methacrylate, polymerization inhibitor, tetrabutylammonium bromide are added into tetrahydrofuran, heated to reflux for 24h; reduce to room temperature, distillation under reduced pressure, take the concentrate to 40℃ under vacuum drying 12h, to obtain the intermediate product.

[0154] The lactic acid, gallic acid and glycidyl methacrylate are added in a total carboxyl to epoxy molar ratio of 1:1; and,

[0155] The lactic acid, gallic acid are added in a ratio of 0.4mol:0.6mol;

[0156] The glycidyl methacrylate, tetrabutylammonium bromide, tetrahydrofuran are added in a ratio of 0.1mol:0.02mol:100mL;

[0157] The amount of the polymerization inhibitor is 0.2wt% of the glycidyl methacrylate.

[0158] Its infrared data are as follows: 3495cm -1 :-OH wide peak exists; 3011cm -1 , 1595cm -1 , 1498cm -1 : benzene ring exists; 1735cm -1 :-C=O exists; 1606cm -1 , 811cm -1 :-C=C- exists; 1265cm -1 , 893cm -1 , 825cm -1 : epoxy group does not exist.

[0159] S12, the intermediate product, 2-methacryloyloxyethylphosphocholine, photoinitiator 1173 are added into N,N-dimethylformamide, placed under UV lamp radiation for 50min, remove the UV lamp, distillation under reduced pressure, add methanol to the concentrate, produce precipitation, filter, take the precipitate, vacuum drying at 60℃ for 8h, to obtain the zwitterionic copolymer

[0160] The intermediate product, 2-methacryloyloxyethylphosphocholine, N,N-dimethylformamide are added in a ratio of 0.4-0.5mol:0.5-0.6mol:500mL.

[0161] The amount of the photoinitiator 1173 is 1wt% of the total mass of the reactants;

[0162] The UV light intensity is 200 mW / cm 2 .

[0163] Its infrared data are as follows: 3495 cm -1 : -OH wide peak exists; 3011 cm -1 , 1595 cm -1 , 1498 cm -1 : benzene ring exists; 1735 cm -1 : -C=O exists; 1606 cm -1 , 811 cm -1 : -C=C- does not exist; 1187 cm -1 : -P=O exists.

[0164] Example 8

[0165] Other than Example 1, the difference is that:

[0166] A preparation method of a zwitterionic copolymer, in the S12 step,

[0167] The 2-methacryloyloxyethylphosphocholine is replaced by methacryloyl ethyl sulfobetaine.

[0168] Example 9

[0169] Other than Example 1, the difference is that:

[0170] A preparation method of a hydrogel material for genital plastic surgery, comprising the following steps:

[0171] S21, preparing a mixed solution, freeze-drying, that is,

[0172] The mesenchymal stem cell solution is added to the polymer-based mixed solution, and after uniform mixing, it is poured into a mold; freeze-drying at -20℃ for 24h.

[0173] The polymer-based mixed solution is: the sodium alginate solution has a concentration of 0.05g / mL; the carboxymethyl chitosan solution has a concentration of 0.03g / mL; the gelatin solution has a concentration of 0.05g / mL; the sodium alginate, carboxymethyl chitosan, and gelatin solutions are mixed in a volume ratio of 2.0:1.0:1.0 to obtain the polymer-based mixed solution;

[0174] The solute sodium alginate of the sodium alginate solution is added in a mass ratio of 0.6:0.4 of high molecular weight sodium alginate and low molecular weight sodium alginate; and

[0175] The number average molecular weight of the high molecular weight sodium alginate is 150kDa;

[0176] The number average molecular weight of the low molecular weight sodium alginate is 5kDa;

[0177] The volume ratio of the mesenchymal stem cell solution to the polymer base mixed solution is 1.0:0.8;

[0178] The concentration of the mesenchymal stem cell solution is 1×10 9 individual / mL.

[0179] S22, ion crosslinking, i.e.

[0180] The substance obtained in step S21 is placed in a calcium chloride solution, and is left to stand for 40 min, and the excess liquid is removed.

[0181] The concentration of the calcium chloride solution is 0.03 g / mL.

[0182] The infrared data thereof are as follows: 3486 cm -1 : -OH wide peak exists; 3314 cm -1 : -NH- exists; 3011 cm -1 , 1595 cm -1 , 1498 cm -1 : benzene ring exists; 1711 cm -1 : -C=O weakens; 1698 cm -1 : -C=O (coordinates with calcium) exists.

[0183] S23, self-assembly, i.e.

[0184] The substance obtained in step S22 is placed in a zwitterion copolymer solution, a heparin calcium solution A, a tannic acid solution, and a heparin calcium solution B in sequence, and is left to stand for 40 min in each process.

[0185] The concentration of the zwitterion solution is 0.02 g / mL.

[0186] The concentration of the heparin calcium solution A is 0.01 g / mL.

[0187] The concentration of the tannic acid solution is 0.01 g / mL; and

[0188] The concentration of the heparin calcium solution B is 0.02 g / mL.

[0189] The infrared data thereof are as follows (reflection infrared): 3489 cm -1 : -OH wide peak exists and enhances; 3314 cm -1 : -NH- does not exist; 3011 cm -1 , 1595 cm -1 , 1498 cm -1 : benzene ring exists; 1711 cm -1 : -C=O weakens; 1698 cm -1: -C=O (coordinated with calcium) exists and is enhanced.

[0190] S24, freeze-drying, i.e.

[0191] The material obtained in step S23 is freeze-dried at -20℃ for 24h to obtain the hydrogel material for genital reconstruction.

[0192] Example 10

[0193] Other than Example 1, except that:

[0194] A method for preparing a hydrogel material for genital reconstruction, comprising the following steps:

[0195] S21, preparation of mixed solution, freeze-drying, i.e.

[0196] The mesenchymal stem cell solution is added to the polymer-based mixed solution, and after uniform mixing, poured into a mold; freeze-dried at -20℃ for 24h.

[0197] The polymer-based mixed solution is: a solution with a sodium alginate solution concentration of 0.02g / mL; a solution with a carboxymethyl chitosan solution concentration of 0.01g / mL; a solution with a gelatin solution concentration of 0.02g / mL; the sodium alginate, carboxymethyl chitosan and gelatin solutions are mixed in a volume ratio of 2.0:1.0:2.0 to obtain the polymer-based mixed solution;

[0198] The solute sodium alginate of the sodium alginate solution is added by high molecular weight sodium alginate and low molecular weight sodium alginate in a mass ratio of 0.8:0.2; and

[0199] The number average molecular weight of the high molecular weight sodium alginate is 100kDa;

[0200] The number average molecular weight of the low molecular weight sodium alginate is 10kDa;

[0201] The volume ratio of the amount of mesenchymal stem cell solution to the polymer-based mixed solution is 1.0:1.2;

[0202] The concentration of the mesenchymal stem cell solution is 1×10 10 / mL.

[0203] S22, ionic crosslinking, i.e.

[0204] The material obtained in step S21 is placed in a calcium chloride solution, and left to stand for 30min, and the excess liquid is removed;

[0205] The concentration of the calcium chloride solution is 0.01g / mL.

[0206] Its infrared data are as follows: 3486cm -1: -OH wide peak exists; 3314 cm -1 : -NH- exists; 3011 cm -1 , 1595 cm -1 , 1498 cm -1 : benzene ring exists; 1711 cm -1 : -C=O weakens; 1698 cm -1 : -C=O (coordinates with calcium) exists.

[0207] S23, self-assembly, i.e.

[0208] The material obtained in the step S22 is placed in the zwitterionic copolymer solution, the heparin calcium solution A, the tannic acid solution, and the heparin calcium solution B in sequence, wherein each process is placed for 60 min.

[0209] The concentration of the zwitterionic solution is 0.01 g / mL;

[0210] The concentration of the heparin calcium solution A is 0.005 g / mL;

[0211] The concentration of the tannic acid solution is 0.005 g / mL; and

[0212] The concentration of the heparin calcium solution B is 0.01 g / mL.

[0213] The infrared data thereof are as follows (reflection infrared): 3489 cm -1 : -OH wide peak exists and strengthens; 3314 cm -1 : -NH- does not exist; 3011 cm -1 , 1595 cm -1 , 1498 cm -1 : benzene ring exists; 1711 cm -1 : -C=O weakens; 1698 cm -1 : -C=O (coordinates with calcium) exists and strengthens.

[0214] S24, freeze-drying, i.e.

[0215] The material obtained in the step S23 is freeze-dried at -20℃ for 24 h to obtain the hydrogel material for genital shaping.

[0216] Example 11

[0217] Other than Example 1, the difference is that:

[0218] A preparation method of a hydrogel material for genital shaping, in the step S21,

[0219] The sodium alginate, carboxymethyl chitosan, and gelatin solution are mixed in a volume ratio of 1.0:1.0:1.0.

[0220] The hydrogel material for genital plastic surgery in the following comparative examples is compared with Example 1:

[0221] Comparative Example 1

[0222] Other than Example 1, the difference is that:

[0223] A method for preparing a hydrogel material for genital plastic surgery, in the S23 step,

[0224] The material obtained in the S22 step is sequentially placed in a heparin calcium solution A, a tannic acid solution, and a heparin calcium solution B.

[0225] Comparative Example 2

[0226] Other than Example 1, the difference is that:

[0227] A method for preparing a zwitterionic copolymer, in the S12 step,

[0228] The amount ratio of the intermediate product, 2-methacryloyloxyethyl phosphorylcholine, and N,N-dimethylformamide is 0 mol:0.55 mol:500 mL; that is, no intermediate product is added.

[0229] Comparative Example 3

[0230] Other than Example 1, the difference is that:

[0231] A method for preparing a zwitterionic copolymer, in the S12 step,

[0232] The amount ratio of the intermediate product, 2-methacryloyloxyethyl phosphorylcholine, and N,N-dimethylformamide is 0.45 mol:0 mol:500 mL; that is, no 2-methacryloyloxyethyl phosphorylcholine is added.

[0233] Comparative Example 4

[0234] Other than Example 1, the difference is that:

[0235] A method for preparing a hydrogel material for genital plastic surgery, in the S21 step,

[0236] The sodium alginate, carboxymethyl chitosan, and gelatin solution are mixed at a volume ratio of 1.5:0:1.5; that is, no carboxymethyl chitosan solution is added.

[0237] Comparative Example 5

[0238] Other than Example 1, the difference is that:

[0239] A method for preparing a hydrogel material for genital plastic surgery, in the S21 step,

[0240] The sodium alginate, carboxymethyl chitosan, and gelatin solution are mixed at a volume ratio of 1.5:1.0:0; that is, no gelatin solution is added.

[0241] Comparative Example 6

[0242] Other than Example 1, except that:

[0243] A method for preparing a genital reshaping hydrogel material, in the S21 step,

[0244] The solute sodium alginate of the sodium alginate solution is added at a mass ratio of 0.7:0 of high-molecular-weight sodium alginate and low-molecular-weight sodium alginate; that is, no low-molecular-weight sodium alginate is added.

[0245] Comparative Example 7

[0246] Other than Example 1, except that:

[0247] A method for preparing a genital reshaping hydrogel material, in the S21 step,

[0248] The solute sodium alginate of the sodium alginate solution is added at a mass ratio of 0:0.3 of high-molecular-weight sodium alginate and low-molecular-weight sodium alginate; that is, no high-molecular-weight sodium alginate is added.

[0249] Comparative Example 8

[0250] Other than Example 1, except that:

[0251] A method for preparing a genital reshaping hydrogel material, in the S21 step,

[0252] The volume ratio of the mesenchymal stem cell solution to the high-molecular-weight base mixture is 0:1.0; that is, no mesenchymal stem cell solution is added.

[0253] Comparative Example 9

[0254] Other than Example 1, except that:

[0255] A method for preparing a genital reshaping hydrogel material, in the S21 step,

[0256] There is no freeze-drying operation step.

[0257] Comparative Example 10

[0258] Other than Example 1, except that:

[0259] A method for preparing a genital reshaping hydrogel material,

[0260] There is no calcium chloride solution ion crosslinking step.

[0261] Comparative Example 11

[0262] Other than Example 1, except that:

[0263] A method for preparing a genital plastic hydrogel material, in the S23 step,

[0264] The material obtained in the S22 step is sequentially placed in a zwitterionic copolymer solution, a tannic acid solution, and a heparin calcium solution B.

[0265] Comparative Example 12

[0266] Other than Example 1, except that:

[0267] A method for preparing a genital plastic hydrogel material, in the S23 step,

[0268] The material obtained in the S22 step is sequentially placed in a zwitterionic copolymer solution, a heparin calcium solution A, and a heparin calcium solution B.

[0269] Comparative Example 13

[0270] Other than Example 1, except that:

[0271] A method for preparing a genital plastic hydrogel material, in the S23 step,

[0272] The material obtained in the S22 step is sequentially placed in a zwitterionic copolymer solution, a heparin calcium solution A, and a tannic acid solution.

[0273] Comparative Example 14

[0274] Other than Example 1, except that:

[0275] The heparin calcium solution is replaced by a heparin sodium solution.

[0276] The physical properties of the genital plastic hydrogel materials prepared in the examples and comparative examples of the present application are measured, and the results are shown in Table 1.

[0277] Table 1: Physical test properties of each example

[0278]

[0279]

[0280] First, as can be seen from Table 1 Examples 1-11, the genital plastic hydrogel material of the present application has excellent mechanical properties, cell compatibility, plastic repair effect, and antibacterial properties, etc.

[0281] Secondly, from the comparison of Example 1 and Comparative Examples 1-3, it can be observed that the genital shaping hydrogel material of the present application uses self-made zwitterionic copolymer, has a compatibilization effect, can effectively improve the mechanical properties; at the same time, the quaternary ammonium salt structure also has excellent synergistic antibacterial effect; the catechol structure has the function of synergistically adsorbing cells and improving the shaping repair effect; from the comparison of Example 1 and Comparative Examples 4-7, it can be observed that the hydrogel of the present application uses sodium alginate, carboxymethyl chitosan and gelatin as main raw materials, wherein the use of high and low molecular weight sodium alginate has better flexibility, carboxymethyl chitosan has excellent antibacterial property, and gelatin has the function of promoting cell proliferation and differentiation, and the mutual synergistic effect of each component has excellent mechanical properties and multifunctional properties such as antibacterial and cell proliferation and differentiation promotion; from the comparison of Example 1 and Comparative Example 8, it can be observed that stem cells have differentiation, can effectively promote rapid wound healing and play a function; from the comparison of Example 1 and Comparative Examples 1, 9-14, it can be observed that the layer-by-layer self-assembly of zwitterionic copolymer, heparin calcium A, tannic acid and heparin calcium B has excellent mechanical improvement effect, and the outer layer of tannic acid and heparin calcium has the functions of antithrombotic, antibacterial and cell adsorption, which synergistically improves the rehabilitation effect and effectively reduces the shaping repair period; in addition, the crosslinking density is improved after freeze-drying and calcium ion multi-step addition, which further improves the mechanical properties; thirdly, compared with heparin sodium, heparin calcium has more excellent mechanical properties and shaping repair effect.

[0282] In summary, the genital shaping hydrogel material of the present application has excellent mechanical properties, shaping repair effect, antibacterial property and cell compatibility.

[0283] The test method is as follows:

[0284] (1) OD value / absorbance: place the hydrogel to be tested in a 37℃ incubator to promote gel formation, carefully add culture medium after 30 min. Use CCK-8 to detect the change of cell proliferation activity on the 3rd day.

[0285] (2) Mechanical properties: use a universal mechanical testing machine to test the tensile strength of the sample, the sample specification is 10.0mm wide, 1.0mm thick and 40mm long, the tensile rate is 100mm / min at room temperature, and the test data is the average value of 5 parallel samples.

[0286] (3) Shaping repair period: use white rabbits for testing and verification, 10 white rabbits are randomly used for each example, and the average value is tested, the specific process is as follows:

[0287] The New Zealand big white rabbits are selected, the rabbits are anesthetized with 3% sodium pentobarbital at a dose of 1ml / kg, and after conventional skin preparation, disinfection and towel laying, the rubber ring is wrapped around the root of the rabbit sponge body, the rubber ring is clamped with an artery clamp, a 5mm longitudinal incision is made on the right penile sponge body, the water gel material to be detected is quickly transplanted to the penile sponge body defect and sutured. One month after the operation, the repaired male rabbits are caged with normal female rabbits, and the time length between caging and the date of birth of the new baby rabbits is recorded.

[0288] (4) Antibacterial property: according to GB4789.2-2010 "National food safety standard food microbiological examination determination of total number of colonies", Escherichia coli antibacterial inhibition experiment is carried out.

[0289] With the above ideal embodiments according to the present application as the inspiration, through the above description, relevant staff can make various changes and modifications without deviating from the technical idea of the present application. The technical scope of the present application is not limited to the content in the specification, and must be determined according to the scope of claims.

Claims

1. A method for preparing a zwitterionic copolymer, characterized in that: The following steps are involved: S11, subjecting lactic acid, a carboxylated catechol derivative and epoxy acrylate to a ring-opening reaction to obtain an intermediate product; S12, subjecting the intermediate product and the zwitterion to free radical polymerization to obtain a target product, i.e., a zwitterionic copolymer; The carboxylated catechol derivative is gallic acid; The zwitterions are 2-methacryloyloxyethyl phosphorylcholine and methacryloylethyl sulfobetaine.

2. The preparation method according to claim 1, characterized in that The lactic acid, carboxylated catechol derivative and epoxy acrylate are added in a molar ratio of the total amount of carboxyl groups to the epoxy groups of 1:1; and The usage ratio of the lactic acid and the carboxylated catechol derivative is 0.3-0.5 mol: 0.5-0.7 mol.

3. The preparation method according to claim 1, characterized in that The usage ratio of the intermediate product to the zwitterion is 0.4-0.5 mol:0.5-0.6 mol.

4. A zwitterionic copolymer, characterized in that Prepared by the preparation method according to any one of claims 1 to 3.

5. A method for preparing a hydrogel material for genital plastic surgery, characterized in that: The following steps are involved: S21, prepare the mixed solution and freeze-dry it. Add the mesenchymal stem cell solution to the polymer base mixture, mix well, and pour into a mold; freeze-dry; S22, ionic cross-linking, i.e. The substance obtained in step S21 is placed in a calcium chloride solution, allowed to stand for 30-40 minutes, and excess liquid is removed; S23, self-assembly, i.e. The material obtained in step S22 is placed in the solution prepared from the zwitterionic copolymer of claim 1, heparin calcium solution A, tannic acid solution, and heparin calcium solution B in sequence; wherein each process is allowed to stand for 40-60 minutes; S24, freeze-dried, i.e. The material obtained in step S23 is freeze-dried to obtain a hydrogel material for genital plastic surgery; The polymer-based mixed solution is a mixture of sodium alginate solution, carboxymethyl chitosan solution, and gelatin solution in a volume ratio of 1.0-2.0:1.0:1.0-2.0; The sodium alginate solute of the sodium alginate solution is prepared by adding high molecular weight sodium alginate and low molecular weight sodium alginate in a mass ratio of 0.6-0.8:0.2-0.4; and The number average molecular weight of the high molecular weight sodium alginate is 100-150 kDa; The number average molecular weight of the low molecular weight sodium alginate is 5-10 kDa.

6. The preparation method according to claim 5, characterized in that The concentration of the sodium alginate solution is 0.02-0.05 g / mL; The concentration of the carboxymethyl chitosan solution is 0.01-0.03 g / mL; The concentration of the gelatin solution is 0.02-0.05 g / mL.

7. The preparation method according to claim 5 or 6, characterized in that: The volume ratio of the mesenchymal stem cell solution to the polymer base mixed solution is 1.0:0.8-1.2; The concentration of the mesenchymal stem cell solution is 10 9 -10 10 pieces / mL.

8. The preparation method according to claim 5, characterized in that The concentration of the calcium chloride solution is 0.01-0.03 g / mL.

9. The preparation method according to claim 5, characterized in that The concentration of the zwitterion solution is 0.01-0.02 g / mL; The concentration of the heparin calcium solution A is 0.005-0.01 g / mL; The concentration of the tannic acid solution is 0.005-0.01 g / mL; and The concentration of the heparin calcium solution B is 0.01-0.02 g / mL.

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