3D printing hydrogel stent and preparation method thereof

Abstract

The embodiment of the invention relates to a 3D hydrogel scaffold and a preparation method thereof.The 3D hydrogel scaffold comprises a dual-network structure, the dual-network structure comprises a PCLMA network structure formed by PCLMA photo-crosslinking and a network structure formed by crosslinking a first composite material and the PCLMA network structure, the first composite material comprises ColMA and EPLMA, and the second composite material comprises ColMA and EPLMA. The 3D hydrogel stent is good in biocompatibility with a human body, can enable the human body to recover an initial state after being implanted for a period of time, can play a role of a bile duct stent, and has a good bile duct management characteristic.

Description

Technical Field

[0001] The present application relates to the technical field of biomedical materials, and in particular, to a 3D hydrogel scaffold and a preparation method thereof. Background Art

[0002] Biliary tract injury refers to the destruction of the integrity and continuity of the bile duct structure and the bile-enteric pathway, resulting in a series of pathophysiological changes such as bile duct obstruction, bile leakage, jaundice, infection, systemic inflammatory response, and liver function damage. Failure to repair bile duct injury in a timely manner or improper selection of repair methods can cause bile duct stenosis. Introducing the bile duct through surgery can be used to relieve bile duct stenosis and repair damaged bile ducts. However, after the introduction of the stent bile duct, blockage and discomfort may occur in the human body after a period of time, which cannot play a good role as a bile duct substitute and also causes additional discomfort to the human body. Summary of the invention

[0003] The present application is provided to solve the above defects existing in the prior art. A 3D hydrogel scaffold and a preparation method thereof are needed, which have good biocompatibility with the human body, can restore the human body to its original state after being implanted for a period of time, can play the role of a bile duct stent, and have good bile duct biochemical properties.

[0004] In a first aspect of the present application, a 3D hydrogel scaffold is provided, wherein the 3D hydrogel scaffold comprises a dual network structure, wherein the dual network structure comprises a PCLMA network structure formed by photo-crosslinking of PCLMA (polycaprolactone maleimide [Poly (ε-caprolactone)-maleimide)) and a network structure formed by crosslinking a first composite material and the PCLMA network structure, wherein the first composite material comprises ColMA (type III collagen) and EPLMA (modified polylysine).

[0005] In some embodiments, the EPLMA is obtained by reacting polylysine with glycidyl methacrylate.

[0006] In some embodiments, the 3D hydrogel scaffold further comprises a second outer cross-linked network structure, wherein the second outer cross-linked network structure is obtained by cross-linking a second composite material and a base cross-linked network structure, and the composite material comprises ColMA, EPLMA and liposomes loaded with IL-6 (interleukin).

[0007] In some embodiments, the 3D hydrogel scaffold is loaded with passaged BMSCs (bone marrow mesenchymal stem cells).

[0008] In a second aspect of the present application, a method for preparing the 3D hydrogel scaffold described in any embodiment of the present application is provided, the preparation method comprising: Based on the 3D printer, under the first exposure condition, the PCLMA material undergoes a cross-linking reaction to obtain an initial PCLMA hydrogel; after the initial PCLMA hydrogel is washed, based on the 3D printer, under the second exposure condition, the initial PCLMA hydrogel undergoes a photo-cross-linking reaction to obtain an initial hydrogel scaffold; The initial hydrogel scaffold was immersed in a solution containing ColMA and EPLMA; The initial PCLMA hydrogel after immersion was subjected to a cross-linking reaction based on a 3D printer under the third exposure condition to obtain a 3D hydrogel scaffold with a dual network structure (PCLMA / / ColMA / EPLMA hydrogel scaffold).

[0009] In some embodiments, the PCLMA / / ColMA / EPLMA hydrogel scaffold is immersed in a solution containing IL-6@Lip, ColMA and EPLMA; the immersed PCLMA / / ColMA / EPLMA hydrogel scaffold undergoes a cross-linking reaction based on a 3D printer under a fourth exposure condition to obtain a 3D hydrogel scaffold (PCLMA / / ColMA / EPLMA / IL-6@Lip hydrogel scaffold).

[0010] In some embodiments, the first exposure condition includes: a light intensity of 10-15 mW / cm 2 ; The exposure time is 10-20s. In some embodiments, the second exposure condition includes: the light intensity is 10-15 mW / cm 2 ; The exposure time is 20-40s. In some embodiments, the third exposure condition includes: the light intensity is 10-15 mW / cm 2 ; The exposure time is 10-20s. In some embodiments, the fourth exposure condition includes: the light intensity is 10-15 mW / cm 2 ; Exposure time is 10-20s.

[0011] In some embodiments, in the solution containing ColMA and EPLMA, the mass fraction of ColMA is 5-15%; the mass fraction of EPLMA is 0.5-2%. The solvent of the solution containing ColMA and EPLMA is water or the like. Preferably, in the solution containing ColMA and EPLMA, the mass fraction of ColMA is 8-12%; the mass fraction of EPLMA is 0.5-1.2%.

[0012] In some embodiments, in the solution containing IL-6@Lip, ColMA and EPLMA, the mass fraction of IL-6@Lip is 15-25%, the mass fraction of ColMA is 5-15%; and the mass fraction of EPLMA is 0.5-2%.

[0013] In some embodiments, the initial PCLMA hydrogel is rinsed with a mixed solution of methanol and isopropanol. In some embodiments, the volume ratio of methanol to isopropanol in the mixed solution of methanol and isopropanol is 0.5-2:1; for example, 1:1, etc. The number of rinses can be 1 or more times, for example, 3-6 times, etc.

[0014] In some embodiments, the preparation method further includes: inoculating the BMSCs cell suspension on a PCLMA / / ColMA / EPLMA hydrogel scaffold or a PCLMA / / ColMA / EPLMA / IL-6@Lip hydrogel scaffold, and obtaining a 3D hydrogel scaffold loaded with cultured BMSCs cells after culturing.

[0015] The 3D hydrogel scaffold and the preparation method thereof provided in each embodiment of the present application, the 3D hydrogel scaffold includes a double network structure, the double network structure includes a base cross-linked network structure and a first outer cross-linked network structure cross-linked with the base cross-linked network structure, so that the double network structure has good physical and chemical characteristics, such as compression strength and compression modulus, so that it can play the supporting role of the bile duct stent; the material of the base cross-linked network structure is PCLMA, and the first outer cross-linked network structure is obtained by cross-linking a first composite material and PCLMA, and the composite material includes ColMA and EPLMA, has antibacterial properties, and has good biocompatibility with the human body. After being implanted for a period of time, it can restore the human body to its original state and can better play the role of a bile duct substitute. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In the drawings, which are not necessarily drawn to scale, the same reference numerals may describe similar parts in different views. The same reference numerals with letter suffixes or different letter suffixes may represent different instances of similar parts. The accompanying drawings generally illustrate various embodiments by way of example and not limitation, and together with the specification and claims, are used to illustrate the embodiments applied for. When appropriate, the same reference numerals are used throughout the drawings to refer to the same or similar parts. Such embodiments are illustrative and are not intended to be exhaustive or exclusive embodiments of the present apparatus or method.

[0017] Figure 1 The structure and composition characterization diagrams of the liposome and hydrogel materials of Experimental Examples 1 to 4 of the present application are shown, wherein: Figure 1 A is the DLS particle size distribution histogram of liposomes without IL-6 loading; Figure 1 B is the DLS particle size distribution histogram of IL-6@Lip; Figure 1 C is a TEM image of liposomes without IL-6 loading; Figure 1 D is the TEM image of IL-6@Lip; Figure 1E is the Fourier transform infrared spectra of Col and ColMA; Figure 1 F is the Fourier transform infrared spectra of EPL and EPLMA; Figure 1 G is the 1H NMR spectra of Col and ColMA; Figure 1 H is the 1H NMR spectrum of EPL and EPLMA.

[0018] Figure 2 The physicochemical property characterization diagrams of the hydrogel coatings of Experimental Examples 5 to 7 of the present application are shown, wherein: Figure 2 A is the rheological time scanning curve of the hydrogel; Figure 2 B is the rheological frequency scanning curve of the hydrogel; Figure 2 C is the stress-strain curve of the hydrogel; Figure 2 D is the statistical graph of compression modulus of hydrogel; Figure 2 E is the appearance and SEM image of the bile duct stent.

[0019] Figure 3 The biocompatibility evaluation diagrams of Experimental Examples 9 and 10 of the present application are shown, wherein: Figure 3 A is the colony image of the agar plate after the bile duct stent was cultured with Escherichia coli and Staphylococcus aureus in vitro; Figure 3 B is the calculation of the in vitro antibacterial rate of bile duct stent against Escherichia coli and Staphylococcus aureus; Figure 3 C is a live-dead staining image of the bile duct stent after culture with Escherichia coli and Staphylococcus aureus; Figure 3 D is the SEM image of Escherichia coli and Staphylococcus aureus.

[0020] Figure 4 The biocompatibility evaluation diagrams of Experimental Examples 11 to 13 of the present application are shown, wherein: Figure 4 A is the cytotoxicity graph of different concentrations of IL-6 liposomes cultured with BMSCs for 24 hours; Figure 4 B is the cell status diagram observed under an optical microscope after BMSCs were cultured with liposomes of different concentrations for 24 hours; Figure 4 C is the cytotoxicity diagram detected by CCK-8 method after the two bile duct stent extracts were cultured with BMSCs for 24 hours; Figure 4 D is the cell status observed under an optical microscope after BMSCs were cultured with two bile duct stent extracts for 24 hours; Figure 4 E is the light microscopy image and live-dead cell staining image of BMSCs seeded on the surface of the hydrogel scaffold after culture for 1, 3, and 5 days.

[0021] Figure 5 The migration, differentiation and macrophage induction diagrams of cells in Experimental Examples 14 to 16 of the present application are shown, wherein: Figure 5A is the image of cell scratch migration after human umbilical vein endothelial cells (HUVEC) were cultured with bile duct stent extract for 48 hours; Figure 5 B is the quantitative statistical value of the change of HUVEC scratch area over time; Figure 5 C is the ratio of the positive proportions of cell markers CD206 and CD86 after co-culture of RAW264.7 cells with bile duct scaffolds; Figure 5 D is a representative flow cytometry image of the positive ratio of cell markers CD206 and CD86 after co-culture of RAW264.7 cells with bile duct scaffolds; Figure 5 E is the RT-qPCR quantitative statistical graph of differentiation biomarkers after BMSCs were co-cultured with bile duct stents in a bile acid environment for 7 days.

[0022] Figure 6 The in vivo transplantation evaluation diagram of the 3D printed artificial bile duct of Experimental Example 17 of the present application is shown, wherein: Figure 6 A is a schematic diagram of the bile duct experimental surgery process: Figure 6 B is a photo of the gallbladder 14 days after surgery; Figure 6 C is a gallbladder X-ray image 14 days after surgery; Figure 6 D is the H&E staining image of the gallbladder at the material transplantation site 14 days after surgery.

[0023] FIG. 7( a ) shows a result diagram of the first part of the evaluation indicators of Experimental Example 17 of the present application.

[0024] FIG7( b ) shows a result diagram of the second part of the evaluation indicators of Experimental Example 17 of the present application.

[0025] FIG. 7( c ) shows a result diagram of the third part of the evaluation indicators of Experimental Example 17 of the present application.

[0026] FIG. 7( d ) shows a result diagram of the fourth part of the evaluation indicators of Experimental Example 17 of the present application. DETAILED DESCRIPTION

[0027] In order to enable those skilled in the art to better understand the technical solution of the present application, the present application is described in detail below in conjunction with the accompanying drawings and specific implementation examples. The embodiments of the present application are further described in detail below in conjunction with the accompanying drawings and specific implementation examples, but are not intended to limit the present application.

[0028] The words "first", "second" and similar words used in this application do not indicate any order, quantity or importance, but are only used to distinguish. The words "include" or "comprise" and similar words mean that the elements before the word include the elements listed after the word, and do not exclude the possibility of including other elements.

[0029] According to an embodiment of the present application, a 3D hydrogel scaffold is provided, wherein the 3D hydrogel scaffold includes a dual network structure, wherein the dual network structure includes a PCLMA network structure formed by photo-crosslinking of PCLMA and a network structure formed by crosslinking a first composite material and the PCLMA network structure, wherein the first composite material includes ColMA and EPLMA, and the first composite material includes ColMA (type III collagen) and EPLMA (modified polylysine).

[0030] The PCLMA is photo-crosslinked to obtain a base crosslinked network structure, and the PCLMA in the base crosslinked network structure is crosslinked with the first composite material to form a double network structure.

[0031] PCLMA has good printing and processing characteristics, and the printed scaffolds are of high precision, which is suitable for printing various complex patterns. The 3D hydrogel scaffold (PCLMA / / ColMA / EPLMA hydrogel scaffold) has a dual network structure, good strength and compression modulus, and can provide good support. It has antibacterial properties and good biocompatibility with BMSCs. It can promote cell migration and also have a polarization regulating effect on the phenotype of macrophages. After implantation, it can keep rabbits alive and has a certain bile duct replacement effect.

[0032] In some embodiments, the EPLMA is obtained by reacting polylysine with glycidyl methacrylate.

[0033] In some embodiments, the 3D hydrogel scaffold further includes a second layer of cross-linked network structure, wherein the second layer of cross-linked network structure is obtained by cross-linking a second composite material and a PCLMA network structure, wherein the second composite material includes ColMA, EPLMA, and IL-6 (interleukin)-loaded liposomes (IL-6@Lip). PCLMA / / ColMA / EPLMA / IL-6@Lip hydrogel scaffold.

[0034] Compared with the previously obtained PCLMA / / ColMA / EPLMA hydrogel scaffold, the obtained PCLMA / / ColMA / EPLMA / IL-6@Lip hydrogel scaffold added IL-6-loaded liposomes and formed a new cross-linked network structure.

[0035] The PCLMA / / ColMA / EPLMA / IL-6@Lip hydrogel scaffold has good compression strength and compression modulus, and can provide good support. It is also antibacterial and has good biocompatibility with BMSCs. It can promote cell migration and has a polarization regulatory effect on the phenotype of macrophages. After implantation, it can keep rabbits alive, and there is no bile leakage or bile duct obstruction after a period of implantation.

[0036] In some embodiments, the 3D hydrogel scaffold is loaded with passaged BMSCs (bone marrow mesenchymal stem cells).

[0037] There are two types of 3D hydrogel scaffolds loaded with passaged BMSCs cells, which are PCLMA / / ColMA / EPLMA / BMSCs and PCLMA / / ColMA / EPLMA / IL-6@Lip / BMSCs. These two 3D hydrogel scaffolds can promote the differentiation of BMSCs into single-tube epithelial cells under the induction of TUDCA. They also have good compressive strength and compression modulus, and can provide good support. They are also antibacterial and have good biocompatibility with BMSCs. They can promote cell migration and have a polarization regulatory effect on the phenotype of macrophages. After implantation, rabbits can survive, and there is no reaction of bile leakage or bile duct obstruction after a period of implantation. Moreover, there is no obvious inflammatory state after a period of implantation, and the bile duct mucosa is intact.

[0038] There was almost no significant difference between the PCLMA / / ColMA / EPLMA / IL-6@Lip / BMSCs and the initial value 7 days after implantation, indicating that long-term transplantation of bile duct stents would not affect the gallbladder.

[0039] In some embodiments, the liposome (IL-6@Lip) is a spherical structure. In some embodiments, the hydrated particle size of the liposome is 247 nm±26.43 nm, and the PDI value is 0.347.

[0040] According to an embodiment of the present application, a method for preparing the 3D hydrogel scaffold described in any embodiment of the present application is also provided, and the preparation method comprises: Based on the 3D printer, under the first exposure condition, the PCLMA material undergoes a cross-linking reaction to obtain an initial PCLMA hydrogel; after the initial PCLMA hydrogel is washed, based on the 3D printer, under the second exposure condition, the initial PCLMA hydrogel undergoes a photo-cross-linking reaction to obtain an initial hydrogel scaffold; The initial hydrogel scaffold was immersed in a solution containing ColMA and EPLMA; The initial PCLMA hydrogel after immersion undergoes a cross-linking reaction based on a 3D printer under the third exposure condition to obtain a PCLMA / / ColMA / EPLMA hydrogel scaffold with a double network structure.

[0041] In some embodiments, the PCLMA / / ColMA / EPLMA hydrogel scaffold is immersed in a solution containing liposomes, ColMA and EPLMA; the immersed PCLMA / / ColMA / EPLMA hydrogel scaffold undergoes a cross-linking reaction based on a 3D printer under a fourth exposure condition to obtain a PCLMA / / ColMA / EPLMA / IL-6@Lip hydrogel scaffold.

[0042] After the cross-linking reaction, the solid material can be washed with PBS to obtain a hydrogel scaffold. Through multiple cross-linking, a stable hydrogel scaffold with good compression strength and compression modulus is formed, which can improve the supporting effect after implantation and better play the role of bile duct substitute.

[0043] In some embodiments, the first exposure condition includes: a light intensity of 10-15 mW / cm 2 ; The exposure time is 10-20s. In some embodiments, the second exposure condition includes: the light intensity is 10-15 mW / cm 2 ; The exposure time is 20-40s. In some embodiments, the third exposure condition includes: the light intensity is 10-15 mW / cm 2 ; The exposure time is 10-20s. In some embodiments, the fourth exposure condition includes: the light intensity is 10-15 mW / cm 2 ; The exposure time is 10-20s. Photocrosslinking forms a scaffold with good support.

[0044] In some embodiments, the preparation method of ColMA is as follows: type I collagen (Col1) reacts with methacrylic anhydride at room temperature to obtain type III collagen (Col1MA). In some embodiments, the mass volume ratio of type I collagen to methacrylic anhydride is 0.8-1.2 g:1 mL. In some embodiments, the reaction time is 20-30 h. In some embodiments, after the type I collagen aqueous solution is mixed with methacrylic anhydride, type I collagen (Col1) reacts with methacrylic anhydride. The pH of the type I collagen aqueous solution is 6.8-7.2. The mass volume ratio of type I collagen to water in the type I collagen aqueous solution is 1 g:45-55 mL.

[0045] In some embodiments, the preparation method of EPLMA is: polylysine and glycidyl methacrylate react under heating conditions to obtain EPLMA. In some embodiments, the mass ratio of polylysine to glycidyl methacrylate is 7-9:1. In some embodiments, the polylysine solution is mixed with glycidyl methacrylate and reacted, and the mass volume ratio of polylysine to solvent dimethyl ammonium formate in the polylysine solution is 1g:4-5mL. In some embodiments, the heating conditions are: temperature 40-60°C, time 40-55h.

[0046] In some embodiments, the preparation method of liposome (IL-6@Lip) is as follows: after hydrogenated soy lecithin (HSPC) and cholesterol (CHO-HP) are mixed, an aqueous solution of growth factors is added, and the liposome (IL-6@Lip) is obtained by reacting at room temperature. In some embodiments, the mass volume ratio of HSPC, CHO-HP, and aqueous solution of growth factors is 1:2-3:1-1.5. In some embodiments, the reaction time is 10-40 minutes. Ultrasonic treatment can be performed during the reaction.

[0047] In some embodiments, in the solution containing ColMA and EPLMA, the mass fraction of ColMA is 5-15%; the mass fraction of EPLMA is 0.5-2%. Preferably, in the solution containing ColMA and EPLMA, the mass fraction of ColMA is 8-12%; the mass fraction of EPLMA is 0.5-1.2%.

[0048] In some embodiments, in the solution containing IL-6@Lip, ColMA and EPLMA, the mass fraction of IL-6@Lip is 15-25%, the mass fraction of ColMA is 5-15%; and the mass fraction of EPLMA is 0.5-2%.

[0049] In some embodiments, the initial PCLMA hydrogel is rinsed with a mixed solution of methanol and isopropanol. In some embodiments, the volume ratio of methanol to isopropanol in the mixed solution of methanol and isopropanol is 0.5-2:1; for example, 1:1, etc. The number of rinses can be 1 or more times, for example, 3-6 times, etc.

[0050] In some embodiments, the preparation method further comprises: inoculating a BMSCs cell suspension on a PCLMA / / ColMA / EPLMA hydrogel scaffold or a PCLMA / / ColMA / EPLMA / IL-6@Lip hydrogel scaffold, and obtaining a 3D hydrogel scaffold loaded with passaged BMSCs cells after culturing. The loading of BMSCs can improve the adaptability of the hydrogel scaffold after implantation.

[0051] Example 1 Pour the prepared PCLMA into the printer to print the hydrogel (light intensity: 13 mW / cm 2 , exposure time: 15 s, number of base layers: 1, base exposure time: 15 s), rinse the printed hydrogel 3-6 times with a methanol: isopropanol (v:v = 1:1) mixed solution, and photocrosslink again for 30 s (light intensity: 13 mW / cm 2), and the initial hydrogel scaffold after cross-linking was rinsed with ethanol. The obtained hydrogel scaffold was immersed in a solution containing 10% by mass fraction of Col1MA and 1% by mass fraction of EPLMA overnight and then photocross-linked (light intensity: 13 mW / cm 2 , exposure time: 15 s) to obtain a 3D printed hydrogel scaffold with a double network structure (PCLMA / / ColMA / EPLMA hydrogel scaffold). The preparation method of each raw material is as follows: Weigh 1 g of type I collagen (Col1) and dissolve it in 50 mL of distilled water. After it is fully dissolved, adjust the pH to 7, add 1 mL of methacrylic anhydride, react at room temperature for 24 h, dialyze against distilled water for 3-5 days (molecular weight cutoff: 3500), centrifuge at 10000 rpm for 5 min, filter through neutral filter paper, and freeze-dry at -80°C to obtain modified type III collagen (ColMA).

[0052] Weigh 7.16 g of polylysine and dissolve it in 30 mL of dimethyl ammonium formate. After it is fully dissolved, add 0.852 g of glycidyl methacrylate and react at 50°C for 48 h. After the reaction is completed, add 10 times the amount of cold ether for precipitation. Add 30 mL of dimethyl ammonium formate to re-dissolve and then precipitate again with cold ether. Collect after vacuum drying.

[0053] Example 2 Weigh 7.4 mg HSPC and 16.5 mg CHO-HP, dissolve in 1 mL ethanol, and disperse by ultrasonic. After fully dissolved, inject into 9 mL of vigorously stirred growth factor aqueous solution (1 mg / ml). Stir for 15 min and then ultrasonicate for 15 min (power 30%, on 5 s, off 5 s) to obtain liposome solution.

[0054] The prepared liposome solution was dissolved in a solution containing 10% by mass of ColMA and 1% by mass of EPLMA to obtain a mixed solution. The mass fraction of liposomes in the mixed solution was 20%. The mixed solution was mixed with PCLMA / / ColMA / EPLMA. The liposomes, ColMA and EPLMA in the mixed solution were photocrosslinked with PCLMA (light intensity: 13 mW / cm 2 , exposure time: 15s) to form the second layer of network and obtain the 3D printed hydrogel scaffold (PCLMA / / Col1MA / EPLMA / IL-6@Lip hydrogel scaffold).

[0055] Example 3 The sterile 3D printed hydrogel scaffold PCLMA / / ColMA / EPLMA was washed with PBS and placed in a 48-well plate. BMSCs were digested with 0.05% trypsin and resuspended in α-MEM complete medium. The cell density was adjusted to 1 million / mL. At a density of 100,000 cells per scaffold, 100 μL of cell suspension was inoculated on the scaffold, and then placed in a cell culture incubator for 2 weeks. When the cells grew on the surface of the scaffold, PCLMA / / ColMA / EPLMA / BMSCs were obtained.

[0056] Example 4 The sterile 3D printed hydrogel scaffold PCLMA / / ColMA / EPLMA / IL-6@Lip was washed with PBS and placed in a 48-well plate. BMSCs were digested with 0.05% trypsin and resuspended in α-MEM complete medium. The cell density was adjusted to 1 million / mL. At a density of 100,000 cells per scaffold, 100 μL of cell suspension was inoculated on the scaffold, and then placed in a cell culture incubator for 2 weeks. When the cells grew on the surface of the scaffold, PCLMA / / ColMA / EPLMA / IL-6@Lip / BMSCs were obtained.

[0057] Experimental Example 1 The samples (liposomes without IL-6 loading and IL-6@Lip) were ultrasonicated for 30 seconds to make them dispersed evenly, and the particle size and potential were tested by laser nanoparticle size analyzer. The results are as follows: Figure 1 A (liposomes without IL-6 loading) and Figure 1 As shown in B (IL-6@Lip).

[0058] The particle size distribution of liposomes without IL-6 loading is as follows Figure 1 As shown in Figure A, the hydrated particle size of the liposomes without IL-6 loading is distributed at 214±2.79 nm, the PDI value is 0.342, and the dispersibility is good. After loading IL-6, the hydrated particle size of the IL-6@Lip liposomes is 247 nm±26.43 nm, the particle size increases slightly, the PDI value is 0.347, and the dispersibility of the nanoparticles is worse than that of the blank liposomes without IL-6 loading.

[0059] Experimental Example 2 The sample (liposomes without IL-6 and IL-6@Lip liposomes) solution was diluted to a certain concentration, ultrasonically dispersed, and the solution was dropped onto the copper grid with a 20 μL pipette. Then, it was negatively stained with 1% phosphotungstic acid and dropped onto the copper grid. After natural evaporation, the internal tissue morphology of the sample was observed using a transmission electron microscope. The test conditions were: 5 kV electron beam, and the results were as follows Figure 1 C (liposomes without IL-6 loading) and Figure 1D (IL-6@Lip).

[0060] The preparation method of liposomes not loaded with IL-6 includes: weighing 7.4 mg HSPC and 16.5 mg CHO-HP, dissolving them in 1 mL ethanol, and ultrasonically dispersing them to obtain a liposome solution not loaded with IL-6 after they are fully dissolved. The liposome solution not loaded with IL-6 is subjected to low-temperature drying to obtain liposomes not loaded with IL-6.

[0061] TEM images showed that both the liposomes without IL-6 loading and IL-6@Lip were spherical. After loading with IL-6, the size distribution of IL-6@Lip liposomes was not as uniform as that of the blank liposomes without IL-6 loading.

[0062] Experimental Example 3 8 mg of samples (including type I collagen Col, type III collagen ColMA, polylysine, and EPLMA involved in Example 1) were weighed and mixed with an appropriate amount of dried potassium bromide powder (mass ratio of about 5%) in an agate mortar. After grinding thoroughly to make the two evenly mixed, an appropriate amount of each ground sample powder was taken for tableting (vacuum pressure 20 mmHg, tableting for 5 min) to obtain sample slices. The scanning range was set to 4000-400 cm -1 , using Fourier transform infrared spectrometer to detect, the results obtained are as follows Figure 1 E and Figure 1 As shown in F.

[0063] Figure 1 E1541 cm -1 The absorption peaks at 1242 cm are the NH bending vibration of the amide II band and the CN stretching vibration of the amide group; -1 It is the NH stretching vibration deformation peak of the amide IⅡ band; it can be seen from the spectrum that ColMA is significantly enhanced in the corresponding characteristic absorption peak compared with Col, which is due to the newly formed amide bond in the reaction between Col and methacrylic anhydride, indicating that methacrylamide groups are introduced into the Col molecular chain. This shows that ColMA was successfully synthesized.

[0064] pass Figure 1 F can obtain the spectra of EPLMA and EPL. It can be seen from the spectra that EPLMA is successfully synthesized.

[0065] Experimental Example 4 8 mg of samples (including type I collagen Col, type III collagen, ColMA, polylysine, and EPLMA involved in Example 1) were weighed respectively, and deuterated heavy water was added to dissolve them. After ultrasonic dispersion, the solution was completely dissolved and clarified, and then placed in a clean NMR tube. The NMR structure was determined by using a NMR spectrometer at room temperature, and the spectrum was analyzed using MestReNova software. The results are as follows: Figure 1 G and Figure 1 As shown in H.

[0066] Figure 1 G is the H NMR spectra of Col and ColMA. Analysis of the NMR results shows that new acrylic acid proton peaks (=CH2) appear at 5.3 and 5.5 ppm, corresponding to the absorption peaks of hydrogen on the olefin bond of the methacrylamide bond, proving that the Col modification was successful. Figure 1 H is EPL, EPLMA 1 The H NMR spectrum showed that the hydrogen on the double bond was at 5.6 ppm and 6.0 ppm, while EPL did not contain any hydrogen on the double bond, indicating that EPLMA was successfully synthesized.

[0067] Experimental Example 5 Rheological measurements were performed on the samples (PCLMA / / ColMA hydrogel scaffold, PCLMA / / ColMA / EPLMA hydrogel scaffold of Example 1, PCLMA / / ColMA / EPLMA / IL-6@Lip hydrogel scaffold of Example 2) using a stainless steel parallel plate rotor with a diameter of 25 mm. G' characterizes the elastic modulus of the sample, and G" characterizes the viscous modulus of the sample. Dynamic strain scanning was performed from 0.1 to 10 rad / s at room temperature to determine the linear viscoelastic range of the hydrogel, and the storage modulus (G') and loss modulus (G") change curves were recorded. The results are shown in Figure 2. Figure 2 A and Figure 2 As shown in B.

[0068] Figure 2 A and Figure 2 B is the relationship between the elastic modulus (G'), viscous modulus (G") of the hydrogel and time and frequency. When the elastic modulus is greater than the viscous modulus, the hydrogel is in a gel state. Figure 2 A and Figure 2 As shown in Figure B, after the hydrogel is gelled, with the increase of time and angular frequency, G' is always greater than G", indicating that the hydrogel can be stably in a gel state.

[0069] Experimental Example 6 600 μL of hydrogel (PCLMA / / ColMA hydrogel scaffold, PCLMA / / ColMA / EPLMA hydrogel scaffold, PCLMA / / ColMA / EPLMA / IL-6@Lip hydrogel scaffold) was prepared into a highly consistent cylindrical shape with a bottom diameter of 11 mm. The prepared hydrogel sample was then placed on the sample stage of the universal testing machine, and the height of the clamp was adjusted so that the upper and lower bottom surfaces of the hydrogel just contacted the clamp. The compression rate was then set to 0.05 mm / s, and the sample was compressed at a constant rate to 70% of the strain. The pressure and displacement values ​​were recorded to obtain Figure 2 C and Figure 2 The results are shown in D. The compression modulus was calculated according to the following formula: Where F is the recorded pressure value and S is the cross-sectional area of ​​the hydrogel sample.

[0070] Figure 2 C is the compressive stress-strain curve of the hydrogel. It can be seen from the figure that after adding EPLMA, the compressive strength of the hydrogel increased, and IL-6@Lip had little effect on the compressive strength of the PCLMA / / ColMA / EPLMA hydrogel scaffold. Figure 2 As shown in D, the maximum compression modulus of ColMA at a strain of 80% is 20.6±4.3 kPa, and the compression modulus of the PCLMA / / ColMA / EPLMA hydrogel scaffold after the addition of EPLMA increases to 31.1±2.4 kPa. The addition of liposomes has little effect on the compression modulus, and the compression modulus of PCLMA / / ColMA / EPLMA / IL-6@Lip is 35.5±5.4 kPa.

[0071] Experimental Example 7 The prepared hydrogel scaffolds (initial hydrogel scaffold, PCLMA / / ColMA hydrogel scaffold, PCLMA / / ColMA / EPLMA hydrogel scaffold, PCLMA / / ColMA / EPLMA / IL-6@Lip hydrogel scaffold) were dried, gold was sprayed on the hydrogel surface for 30 s, and the surface morphology of the 3D printed hydrogel was observed using a scanning electron microscope. The test conditions were: 5 kV electron beam, and the following was obtained: Figure 2 The results shown in E.

[0072] The preparation method of PCLMA / / ColMA hydrogel scaffold includes: pouring the prepared PCLMA into the printer to print the hydrogel into shape (light intensity: 13 mW / cm 2, exposure time: 15 s, number of base layers: 1, base exposure time: 15 s), rinse the printed hydrogel 3-6 times with a methanol: isopropanol (v:v = 1:1) mixed solution, and photocrosslink again for 30 s (light intensity: 13 mW / cm 2 ), and the initial hydrogel scaffold after cross-linking was rinsed with ethanol. The obtained hydrogel scaffold was immersed in a solution containing 10% mass fraction of Col1MA overnight and then photocross-linked (light intensity: 13 mW / cm 2 , exposure time: 15 s) to obtain a 3D printed hydrogel scaffold with a double network structure (PCLMA / / ColMA).

[0073] PCLMA has good 3D printing processing characteristics, and the printed scaffolds are high in precision and suitable for printing various complex patterns. We first printed a PCLMA circular porous scaffold and sprayed ColMA / EPLMA hydrogel on its outer surface to promote cell adhesion and growth. The PCLMA printed scaffolds observed by optical microscopy and SEM have high precision and uniform structure. After surface modification of ColMA / EPLMA hydrogel, the pore structure is filled.

[0074] Experimental Example 9-Antibacterial The bacterial suspension of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) was inoculated onto LB agar plates by streaking method, incubated upside down at 37°C for 24 hours, and single colonies were selected and inoculated into LB liquid culture medium, incubated in a constant temperature shaker at 37°C for 4 hours to obtain bacterial suspension for subsequent experiments.

[0075] A bile duct scaffold (PCLMA / / ColMA / EPLMA hydrogel scaffold in Example 1) was placed in a 24-well plate, and then 100 μL of 1×10 8 CFU / mL of bacterial solution was added to the bile duct stent. The blank control group used sterile saline as a control. 1 mL of sterile saline was added and the well plate was placed in a 37°C constant temperature shaker for 24 hours. After the culture was completed, the bacterial solution was diluted with saline in a gradient manner, and the 103-fold dilution was spread on the LB agar plate, inverted and cultured in a 37°C incubator for 24 hours, and the number of colonies was counted to obtain the following: Figure 3 B, and take photos of the agar plate to obtain the results shown in Figure 3 The results shown in A.

[0076] Transplant substitutes with antibacterial properties play an important role in avoiding bacterial infection after transplantation and leading to transplantation failure. Polylysine has strong broad-spectrum antibacterial properties. Figure 3A shows that the PCLMA / / ColMA / EPLMA bile duct stent has a very strong antibacterial effect on Escherichia coli, and a slightly weaker antibacterial effect on Staphylococcus aureus. The antibacterial rate against Escherichia coli reached 99.99%, while the antibacterial rate against Staphylococcus aureus was 86.04%. The main pathogenic bacteria of biliary tract infection are Gram-negative bacteria, so the PCLMA / / ColMA / EPLMA bile duct stent we prepared is effective in inhibiting biliary tract infection that may occur after transplantation.

[0077] Experimental Example 10-Antibacterial S. aureus and E. coli were revived and cultured to the logarithmic phase. The culture medium was removed by centrifugation and then resuspended in PBS. The bacterial concentration was adjusted to 1×10 8 CFU / mL. 100 μL of 1×10 8 CFU / mL of bacterial solution was added to the bile duct stent. Sterile saline was used as a control in the blank control group. 1 mL of sterile saline was added, and the well plate was placed in a 37°C constant temperature shaker for incubation for 24 hours. After the culture is completed, the bacterial solution was transferred to a 1 mL EP tube, and 3 μL of dead / live bacterial fluorescent staining reagent (Syto-9:PI = 1:1) was added. The staining reaction was carried out in the dark for 15 minutes. After the staining is completed, centrifuge at 5000 rpm for 20 minutes, discard the supernatant, and wash twice with PBS. Finally, the bacterial pellet was resuspended in PBS, 20 μL was added to the slide, covered with a coverslip, and observed under a fluorescence microscope. Syto-9 shows green fluorescence at an excitation / emission wavelength of 539 / 570-620 nm, and PI shows red fluorescence at an excitation / emission wavelength of 470 / 490-540 nm, as shown below. Figure 3 The results shown in C.

[0078] After live-dead staining of Escherichia coli and Staphylococcus aureus cultured with the bile duct stent (PCLMA / / ColMA / EPLMA), the bacteria in the control group all showed green, representing live bacteria. However, most of the bacteria treated with the bile duct stent were red, representing dead bacteria. Figure 3 C It can be obtained that after PCLMA / / ColMA / EPLMA was mixed with Escherichia coli, fewer Escherichia coli survived and more Escherichia coli died. After PCLMA / / ColMA / EPLMA was mixed with Staphylococcus aureus, fewer Staphylococcus aureus survived and more Staphylococcus aureus died. This shows that PCLMA / / ColMA / EPLMA has good inhibitory effects on both Escherichia coli and Staphylococcus aureus.

[0079] The morphology of the Escherichia coli and Staphylococcus aureus involved in the above-mentioned Experimental Examples 9 and 10 was observed by scanning electron microscopy as follows, and the results are as follows: Figure 3As shown in D.

[0080] ① Obtaining bacteria: Take 1 mL of the cultured bacterial liquid and put it into a 1.5 mL centrifuge tube. Centrifuge it at 3000-5000 rpm for 20 minutes, remove the supernatant and keep the precipitate. Disperse it in PBS buffer to remove small molecular impurities such as bacterial culture medium and proteins secreted by bacteria, and extract relatively pure bacteria. This step can be centrifuged multiple times according to the characteristics of the sample.

[0081] ② Bacterial fixation: Take the bacterial precipitate obtained by centrifugation in the previous step and disperse it with 1 mL of 2.5% glutaraldehyde. Oscillate appropriately to make the bacteria fully dispersed in the glutaraldehyde solution and fix it for 2-3 hours.

[0082] ③ Sample cleaning: Centrifuge the bacteria fixed in the previous step at 3000-5000 rpm to obtain the precipitate, disperse it with PBS, and wash it by centrifugation. PBS can be used for multiple washes (usually 3-4 times) to remove the residual organic fixative. Finally, wash it by centrifugation with deionized water and disperse it in deionized water.

[0083] ④ Sample dropping and optimization: Take 2.5-5 μL of the bacteria dispersed in deionized water in the previous step, drop it on the single crystal silicon wafer, let it air dry naturally, cover it with a flat plate or other things during drying to prevent dust contamination, stick the naturally dried sample on the copper table with conductive glue, spray gold for 90-120 seconds, and take a picture.

[0084] Experimental Example 11-Liposome Cytotoxicity After rat BMSCs cells were digested and resuspended, the cells were diluted and the cell suspension was added to the cell counting plate to calculate the number of cells. According to the amount of 10,000 cells / well, they were added to a 96-well plate and cultured overnight in a carbon dioxide cell culture incubator at 5% CO2 and 37°C. After 24 h, IL-6@Lip solution (concentration gradient: 200, 400, 600, 800, 1000 μg / mL) was added to the 96-well plate at 100 μL / well, and 6 parallel wells were made for each material. After culturing for 24 h±2 h, the cell culture plate was removed, the culture medium in the cell culture plate was aspirated, the cells were washed with PBS, and the cell status was recorded under an optical microscope. The results are shown in the figure. Figure 4 As shown in B. Add 100 μL of CCK-8 solution diluted with DMEM basal medium at a volume ratio of 1:10 to each well. At the same time, add 100 μL of CCK-8 working solution to the cell-free well as a negative control. Incubate for 0.5-1 h in a 5% CO2, 37°C environment. Take out the cell culture plate and measure the OD value at a wavelength of 450 nm with an ELISA reader. The results are shown in Figure 4 As shown in A.

[0085] Cell viability = ×100%.

[0086] pass Figure 4 A shows that liposomes (IL-6@Lip) with a concentration of less than 600 μg / mL are basically non-toxic to rat BMSCs cells, and liposomes (IL-6@Lip) with a concentration of 600 μg / mL to 1000 μg / mL are slightly toxic to rat BMSCs cells; Figure 4 B It can be seen that liposomes (IL-6@Lip) have good compatibility with rat BMSCs cells.

[0087] Experimental Example 12-Scaffold Cytotoxicity BMSCs cells were seeded into 96-well plates at a number of 10,000 cells per well and cultured overnight to allow the cells to adhere to the wall. BMSCs were then co-cultured with 0.01 g of sterile bile duct stents (including: PCLMA / / ColMA / EPLMA hydrogel scaffolds prepared in Example 1 and PCLMA / / ColMA / EPLMA / IL-6@Lip hydrogel scaffolds prepared in Example 2) for 24 hours. After the scaffolds were removed, the cells were washed with PBS and photographed under an optical microscope to record the cell status. The results are shown in Figure 2. Figure 4 As shown in C.

[0088] Add 100 μL of CCK-8 solution diluted with DMEM basal medium at a volume ratio of 1:10 to each well. At the same time, add 100 μL of CCK-8 working solution to the cell-free well as a negative control. Incubate for 0.5-1 h in a 5% CO2, 37°C environment. Take out the cell culture plate and measure the OD value at a wavelength of 450 nm with an ELISA reader. The results are as follows: Figure 4 As shown in D.

[0089] Cell viability = ×100%.

[0090] Figure 4 PCLMA / / ColMA / EPLMA and PCLMA / / ColMA / EPLMA / IL-6@Lip in C were basically non-toxic to rat BMSCs cells; Figure 4 D It can be seen that PCLMA / / ColMA / EPLMA and PCLMA / / ColMA / EPLMA / IL-6@Lip have good compatibility with rat BMSCs.

[0091] Experimental Example 13-Cytotoxicity Two sterile 3D printed hydrogel scaffolds were selected: the PCLMA / / ColMA / EPLMA hydrogel scaffold prepared in Example 1 and the PCLMA / / ColMA / EPLMA / IL-6@Lip hydrogel scaffold prepared in Example 2, and the live / dead staining experiment of cytotoxicity was performed respectively, and the steps were as follows: After the scaffold was washed with PBS, it was placed in a 48-well plate. BMSCs were digested with 0.05% trypsin, resuspended in α-MEM complete medium, and the cell density was adjusted to 1 million / mL. At a density of 100,000 cells per scaffold, 100 μL of cell suspension was inoculated on the scaffold, and then placed in a cell culture incubator for 1 hour. After 1 hour of culture, the 48-well plate was removed, and α-MEM complete medium was added to completely immerse the scaffold. After culturing in a 37°C constant temperature carbon dioxide incubator (containing 5% CO2) for 1, 3, and 5 days, it was removed. The culture medium was discarded, and the cells were washed three times with PBS. The cells were incubated with live-dead staining solution (2 µM calceinAM, 8 µM PI) at room temperature for 20 minutes. The cells were washed once with PBS. After adding the anti-fluorescence quencher, images were collected using a fluorescence microscope to analyze the live and dead cells. Calcein AM labeled live cells emit green fluorescence, and propidium iodide (PI) labeled dead cells emit red fluorescence. The results are shown in Figure 2. Figure 4 As shown in E.

[0092] Figure 4 In E, during the 5-day culture period of PCLMA / / ColMA / EPLMA and PCLMA / / ColMA / EPLMA / IL-6@Lip, most of the BMSCs cells were in a living state, and the number of dead cells did not increase significantly with the extension of the culture time, indicating that PCLMA / / ColMA / EPLMA and PCLMA / / ColMA / EPLMA / IL-6@Lip were both compatible with BMSCs cells.

[0093] Experimental Example 14 The hydrogel was extracted with DMEM complete medium at a mass ratio of 1:10 at 37°C for 24 hours, and the PCLMA / / ColMA / EPLMA hydrogel scaffold prepared in Example 1 and the PCLMA / / ColMA / EPLMA / IL-6@Lip hydrogel scaffold prepared in Example 2 were obtained. Human umbilical vein endothelial cells (HUVEC) were seeded on a 48-well plate at a cell density of 30,000 / well and cultured for 24 hours until a fusion layer was formed. A scratch was formed with a 20 μL pipette tip. Then the hydrogel extract was added and cultured continuously. The blank control group was HUVEC alone. The scratch images at different times (0 h, 4 h, 24 h, 30 h, 48 h) were collected, and the scratch area was calculated using ImageJ software.

[0094] The scratch image results are as follows Figure 5 As shown in A, through Figure 5 A can be obtained that when BMSCs were cultured with PCLMA / / ColMA / EPLMA and PCLMA / / ColMA / EPLMA / IL-6@Lip 3D printed scaffold extracts, the scratch area was smaller than that of the control group after 24 and 30 hours of culture. This may be because the extracts of recombinant collagen and polylysine played a certain role in cell migration.

[0095] The quantitative statistics of the scratch area are as follows: Figure 5 As shown in B. From the area change trend in the figure, it can be seen that the hydrogel scaffold and IL-6 promoted the migration of BMSCs to a certain extent. The reason for the lack of significant changes may be that the extract was only incubated with the culture medium for 24 hours during the preparation process, and the release amount of recombinant collagen, polylysine, and IL-6, which mainly promote cell migration, was low.

[0096] Experimental Example 15 RAW264.7 cells (mouse mononuclear macrophage cells) were seeded into 6-well plates at a density of 1×106 per well. After overnight culture, they were induced with 1 μg / mL lipopolysaccharide (LPS) overnight, and then the hydrogel extracts of different groups were replaced and cultured in an incubator for 24 hours. RAW264.7 cells were scraped with a cell scraper, centrifuged at 1200 rpm for 5 minutes, and resuspended in PBS (100 μL). Then, RAW264.7 cells were incubated with anti-CD86 and anti-CD206. The expression of anti-CD86 (M1 macrophage marker) and CD206 (M2 macrophage marker) was detected by flow cytometry, and the blank control group was RAW264.7 cells alone.

[0097] The results are as follows Figure 5 C and Figure 5 As shown in D. Figure 5 C is the ratio of M2 macrophages to M1 macrophages cultured with bile duct stents after LPS induction. After culture with bile duct stents, the proportion of M2 macrophages increased significantly. Figure 5 D is a representative flow cytometric graph. The ratio of M2 macrophages marked by CD206 and M1 macrophages marked by CD86 is Figure 5 C shows the same trend.

[0098] Experimental Example 16 The sterile 3D printed hydrogel scaffolds were washed with PBS and placed in a 48-well plate. BMSCs were digested with 0.05% trypsin and resuspended in α-MEM complete medium. The cell density was adjusted to 1 million / mL. At a density of 100,000 cells per scaffold, 100 μL of cell suspension was inoculated on the scaffold, and then placed in a cell culture incubator for 2 weeks. When the cells grew on the surface of the scaffold, PCLMA / / ColMA / EPLMA / BMSCs hydrogel scaffolds and PCLMA / / ColMA / EPLMA / IL-6@Lip / BMSCs hydrogel scaffolds were obtained. After 2 weeks of culture, BMSCs were induced to differentiate into cholangiocytes. The control group was cultured with α-MEM complete medium, and the induced group was induced with α-MEM supplemented with 2 μM tauroursodeoxycholic acid (TUDCA) for 14 days. Total RNA of cells was extracted according to the operation steps of RNA purification kit. After measuring the concentration, 1 μg of total RNA was taken for cDNA synthesis using RT-PCR kit. qPCR was performed using Bio-Rad CFX96 system (Bio-Rad, USA). Data analysis was performed using Bio-Rad CFX Manager software (Bio-Rad, USA). The expression of GAPDH was used as the internal reference, and the relative expression of the target gene was calculated as 2 −ΔCT (ΔCT=ΔCT target gene-ΔCTGAPDH), which was normalized by the relative expression of the target gene in the control group (uninduced). The expression of bile duct epithelial cell differentiation markers (CK7, CK19, AQP1, AE2, CX43) was detected by RT-qPCR. The primer sequences of RT-qPCR are shown in Table 1.

[0099] Table 1 RT-qPCR primer sequences The results are as follows Figure 5 As shown in E, Figure 5 In E, BMSCs were digested with 0.05% trypsin and resuspended in α-MEM complete medium as blank group a; b and c were the results of the uninduced control group, b corresponding to PCLMA / / ColMA / EPLMA, c corresponding to PCLMA / / ColMA / EPLMA / IL-6@Lip; d and e were the results after TUDCA-induced differentiation treatment, d corresponding to PCLMA / / ColMA / EPLMA / BMSCs hydrogel scaffold after TUDCA induction, and e corresponding to PCLMA / / ColMA / EPLMA / IL-6@Lip / BMSCs hydrogel scaffold after TUDCA induction. Figure 5E It can be obtained that TUDCA can promote the differentiation of BMSCs into bile duct epithelial cells. First, BMSCs were inoculated on the surface of 3D-printed PCLMA / / ColMA / EPLMA and PCLMA / / ColMA / EPLMA / IL-6 scaffolds to allow the cells to grow on the scaffold surface. Then, TUDCA was used to induce BMSCs differentiation. The induced BMSCs on the scaffolds were subjected to RT-qPCR detection of bile duct epithelial cell markers. The results showed that after TUDCA induction, the BMSCs inoculated on the scaffolds could differentiate into single-tube epithelial cells, and the addition of IL-6 had no significant effect on the differentiation efficiency.

[0100] Experimental Example 17 The animal experiments were approved by the Animal Care and Use Committee (ACUC) of the Affiliated Hospital of Xiangya School of Medicine, Central South University, Haikou, and followed relevant laws and regulations. The animal experiment ethical review approval number is ZY-IRB-FOM-063. The experimental animals were adult New Zealand white rabbits with an average weight of 3.5 kg. The experimental animals were anesthetized by injection of propofol and xylazine hydrochloride injection through the ear vein, and lidocaine solution was used for local anesthesia in the abdominal incision. After standard laparotomy, the gallbladder was exposed, bile was aspirated with a syringe, and a longitudinal incision was made in the gallbladder. A hydrogel bile duct stent with a diameter of 4 mm was placed in the gallbladder cavity, sutured with 6-0 absorbable sutures, and the surgical site was covered with the greater omentum to maintain blood supply. All animals were killed 2 weeks later. After standard laparotomy, the gallbladder was exposed, iohexol was injected into the end of the gallbladder for X-ray contrast, and the gallbladder tissue was fixed with 4% paraformaldehyde for more than 24 hours, and H&E staining was performed according to standard procedures.

[0101] Since the wall of the rabbit bile duct is very weak, it cannot be sutured surgically. The gallbladder has a similar structure to the bile duct, and the wall of the gallbladder is tougher and can be sutured. Therefore, we used the gallbladder as a substitute to evaluate the support of the hydrogel scaffold and the patency of bile. Figure 6 As shown in A, I: preoperative skin preparation, II: opening the abdominal cavity, III: exposing the liver, IV: implanting the material, V: suturing the material to the gallbladder wall, VI: closing the abdominal cavity. Two weeks after the PCLMA / / ColMA / EPLMA-based stent was implanted in the gallbladder, all rabbits survived. Through X-ray imaging and anatomical observation, PCLMA / / ColMA / EPLMA had bile duct stenosis, and the other groups had no evidence of bile leakage or bile duct obstruction ( Figure 6 B& Figure 6 C), indicating that stent implantation would not induce inflammation of the bile acid environment and lead to bile duct obstruction. Subsequently, histopathological evaluation was performed and H&E staining of the gallbladder at the site of injury was performed. The results are as follows Figure 6D. The gallbladder mucosa and muscle layer were structurally intact, and no signs of gallstones or cholecystitis were found. In the PCLMA / / ColMA / EPLMA / BMSCs and PCLMA / / ColMA / EPLMA / IL-6@Lip / BMSCs hydrogel scaffold groups, the biliary mucosa was intact, while the biliary mucosa was thickened in the hydrogel scaffold group without BMSCs. And there was a significant inflammatory state in the PCLMA / / ColMA / EPLMA hydrogel scaffold group. These results indicate that the PCLMA / / ColMA / / EPLMA / IL-6@Lip / BMSCs hydrogel scaffold is safe and can be used as a bile duct substitute.

[0102] Whole blood cell and serum liver function were evaluated before surgery and on days 3, 7, and 14 after surgery, respectively. The evaluation indicators included aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TBIL), direct bilirubin (DBIL), albumin (ALB), alkaline phosphatase (ALP), γ-glutamyltransferase (γ-GT), and total bile acid (TBA) levels; (B) white blood cell count (WBC), lymphocyte count (Lymph), monocyte count (Mon), neutrophil count (Gran), lymphocyte percentage (Lymph%), monocyte percentage (Mon%), and neutrophil percentage (Gran%) to investigate whether the bile duct substitute (PCLMA / / ColMA / EPLMA / IL-6@Lip / BMSCs scaffold) induced an inflammatory response in vivo and whether liver function was abnormal. The results are shown in Figures 7(a), 7(b), 7(c), and 7(d). a), Figure 7 (b), Figure 7 (c) and Figure 7 (d), from left to right, they are defined as purple, blue, green, yellow and red, and each color corresponds to the blank group, PCLMA / / ColMA / EPLMA hydrogel scaffold group, PCLMA / / ColMA / EPLMA / IL-6 hydrogel scaffold group, PCLMA / / ColMA / EPLMA / BMSCs hydrogel scaffold group, and PCLMA / / ColMA / EPLMA / IL-6@Lip / BMSCs hydrogel scaffold group. It can be seen from Figure 7 (a), Figure 7 (b), Figure 7 (c) and Figure 7 (d) that the liver function of each group was affected to varying degrees 3 days after surgery and recovered to the initial level after 7 days. In addition, the number of white blood cells increased significantly three days after surgery, which may be related to postoperative foreign body reaction and abdominal wound healing. There was almost no significant difference from the initial value after 7 days. This shows that long-term transplantation of bile duct stents does not affect the gallbladder.

[0103] In addition, although exemplary embodiments have been described herein, the scope includes any and all embodiments based on the present application with equivalent elements, modifications, omissions, combinations (e.g., various embodiments intersecting schemes), adaptations or changes. The elements in the claims are to be interpreted broadly based on the language adopted in the claims, and are not limited to the examples described in this specification or during the practice of this application, and the examples are to be interpreted as non-exclusive. Therefore, this specification and examples are intended to be considered as examples only, and the true scope and spirit are indicated by the following claims and the full scope of their equivalents.

[0104] The above description is intended to be illustrative rather than restrictive. For example, the above examples (or one or more schemes thereof) may be used in combination with each other. For example, a person of ordinary skill in the art may use other embodiments when reading the above description. In addition, in the above-mentioned specific embodiments, various features may be grouped together to simplify the present application. This should not be interpreted as an intention that a feature of an application that does not require protection is necessary for any claim. On the contrary, the subject matter of the present application may be less than all the features of an embodiment of a particular application. Thus, the following claims are incorporated herein in the specific embodiments as examples or embodiments, wherein each claim is independently a separate embodiment, and it is considered that these embodiments may be combined with each other in various combinations or arrangements. The scope of the present invention should be determined with reference to the appended claims and the full scope of equivalent forms granted by these claims.

[0105] The above embodiments are only exemplary embodiments of the present application and are not intended to limit the present invention. The protection scope of the present invention is defined by the claims. Those skilled in the art may make various modifications or equivalent substitutions to the present invention within the essence and protection scope of the present application, and such modifications or equivalent substitutions shall also be deemed to fall within the protection scope of the present invention.

Claims

1. A 3D hydrogel scaffold, characterized in that: The 3D hydrogel scaffold includes a double network structure, which includes a PCLMA network structure formed by photo-crosslinking of PCLMA and a network structure formed by cross-linking a first composite material and a PCLMA network structure, wherein the first composite material includes ColMA and EPLMA.

2. The 3D hydrogel scaffold according to claim 1, characterized in that: The EPLMA is obtained by reacting polylysine with glycidyl methacrylate.

3. The 3D hydrogel scaffold according to claim 1, characterized in that The 3D hydrogel scaffold also includes a second layer of cross-linked network structure, which is obtained by cross-linking a second composite material and a PCLMA network structure, and the second composite material includes ColMA, EPLMA and IL-6-loaded liposomes.

4. The 3D hydrogel scaffold according to claim 1, characterized in that The 3D hydrogel scaffold is loaded with passaged BMSCs cells.

5. A method for preparing the 3D hydrogel scaffold according to any one of claims 1 to 4, characterized in that: The preparation method comprises: Based on the 3D printer, under the first exposure condition, the PCLMA material undergoes a cross-linking reaction to obtain an initial PCLMA hydrogel; after the initial PCLMA hydrogel is washed, based on the 3D printer, under the second exposure condition, the initial PCLMA hydrogel undergoes a photo-cross-linking reaction to obtain an initial hydrogel scaffold; The initial hydrogel scaffold was immersed in a solution containing ColMA and EPLMA; The initial PCLMA hydrogel after immersion is based on a 3D printer, and under the third exposure condition, a cross-linking reaction occurs to obtain a 3D hydrogel scaffold with a double network structure.

6. The preparation method according to claim 5, characterized in that: The PCLMA / / ColMA / EPLMA hydrogel scaffold is immersed in a solution containing IL-6@Lip, ColMA and EPLMA; the immersed PCLMA / / ColMA / EPLMA hydrogel scaffold undergoes a cross-linking reaction based on a 3D printer under the fourth exposure condition to obtain a 3D hydrogel scaffold.

7. The preparation method according to claim 5, characterized in that: In the solution containing ColMA and EPLMA, the mass fraction of ColMA is 5-15%; the mass fraction of EPLMA is 0.5-2%.

8. The preparation method according to claim 6, characterized in that: In the solution containing IL-6@Lip, ColMA and EPLMA, the mass fraction of IL-6@Lip is 15-25%, the mass fraction of ColMA is 5-15%; and the mass fraction of EPLMA is 0.5-2%.

9. The preparation method according to claim 5, characterized in that The first exposure condition includes: light intensity of 10-15mW / cm 2 ; Exposure time is 10-20s.

10. The preparation method according to claim 6, characterized in that: The preparation method further comprises: inoculating a BMSCs cell suspension on a PCLMA / / ColMA / EPLMA hydrogel scaffold or a PCLMA / / ColMA / EPLMA / IL-6@Lip hydrogel scaffold, and obtaining a 3D hydrogel scaffold loaded with passaged BMSCs cells after culturing.

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